Use Of Mouse Models Research Articles

A novel mouse model for developmental and epileptic encephalopathy by Purkinje cell-specific deletion of Scn1b.

Loss of function variants of SCN1B are associated with a range of developmental and epileptic encephalopathies (DEEs), including Dravet syndrome. These DEEs feature a wide range of severe neurological disabilities, including changes to social, motor, mood, sleep, and cognitive function which are notoriously difficult to treat, and high rates of early mortality. While the symptomology of SCN1B -associated DEEs indicates broad changes in neural function, most research has focused on epilepsy-related brain structures and function. Mechanistic studies of SCN1B / Scn1b have delineated diverse roles in development and adult maintenance of neural function, via cell adhesion, ion channel regulation, and other intra- and extra-cellular actions. However, use of mouse models is limited as knockout of Scn1b , globally and even in some cell-specific models (e.g., Parvalbumin+ interneuron-specific knockout) in adult mice, leads to severe and progressive epilepsy, health deterioration, and 100% mortality within weeks. Here, we report findings of a novel transgenic mouse line in which Scn1b was specifically deleted in cerebellar Purkinje cells. Unlike most existing models, these mice did not show failure to thrive or early mortality. However, we quantified marked decrements to Purkinje cell physiology as well as motor, social, and cognitive dysfunction. Our data indicate that cerebellar Purkinje cells are an important node for dysfunction and neural disabilities in SCN1B -related DEEs, and combined with previous work identify this as a potentially vital site for understanding mechanisms of DEEs and developing therapies that can treat these disorders holistically.

Abstract IA013: Stromal and immune plasticity shape the metastatic microenvironment

Abstract Mortality from cancer is almost exclusively due to metastasis to distant organs. Therefore, understanding the biology of tumor metastasis is the most significant challenge in cancer research today. The tumor microenvironment is central in supporting tumor growth. However, the role of the metastatic microenvironment in the multistage process of metastasis is largely unresolved. Since each metastatic microenvironment exerts specific functions that support or oppose colonization by disseminated tumor, understanding distinct organ-specific mechanisms that enable metastatic growth is of crucial importance. Utilizing mouse models of breast cancer metastasis to lungs, brain and bone, combined with clinical data from patients with metastatic disease, we uncover the mechanisms that underlie the early events of metastasis to provide the mechanistic basis for therapeutic targeting to reduce metastatic relapse. Our findings reveal organ-specific co-evolution of stromal and immune cells at distinct metastatic niches, that lead to the formation of an immunosuppressive microenvironment, permissive to metastatic growth. Analysis of human metastasis reveal that these pathways are also operative in human disease. We show that genetic and/or pharmacological targeting of specific nodes in the interactions between stromal and immune cells results in attenuated metastasis and improved survival. Our findings imply that combinatorial therapeutic strategies to target the crosstalk between stromal and immune cells at the metastatic microenvironment may be a promising treatment strategy to inhibit metastatic relapse. Citation Format: Neta Erez. Stromal and immune plasticity shape the metastatic microenvironment [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr IA013.

Model-informed drug development for antimicrobials: translational pharmacokinetic-pharmacodynamic modelling of apramycin to facilitate prediction of efficacious dose in complicated urinary tract infections.

The use of mouse models of complicated urinary tract infection (cUTI) has usually been limited to a single timepoint assessment of bacterial burden. Based on longitudinal in vitro and in vivo data, we developed a pharmacokinetic-pharmacodynamic (PKPD) model to assess the efficacy of apramycin, a broad-spectrum aminoglycoside antibiotic, in mouse models of cUTI. Two Escherichia coli strains were studied (EN591 and ATCC 700336). Apramycin exposure-effect relationships were established with in vitro time-kill data at pH 6 and pH 7.4 and in mice with cUTI. Immunocompetent mice were treated with apramycin (1.5-30 mg/kg) starting 24 h post-infection. Kidney and bladder tissue were collected 6-96 h post-infection for cfu determination. A PKPD model integrating all data was developed and simulations were performed to predict bacterial burden in humans. Treatment with apramycin reduced the bacterial load in kidneys and bladder tissue up to 4.3-log compared with vehicle control. In vitro and in vivo tissue time-course efficacy data were integrated into the PKPD model, showing 76%-98% reduction of bacterial net growth and 3- to 145-fold increase in apramycin potency in vivo compared with in vitro. Simulations suggested that an 11 mg/kg daily dose would be sufficient to achieve bacterial stasis in kidneys and bladder in humans. PKPD modelling with in vitro and in vivo PK and PD data enabled simultaneous evaluation of the different components that influence drug effect, an approach that had not yet been evaluated for antibiotics in the cUTI model and that has potential to enhance model-informed drug development of antibiotics.

Striatum-enriched protein, arginase 2 localizes to medium spiny neurons and controls striatal metabolic profile

Arginase 2 (Arg2) is the predominant arginase isoenzyme in the brain, however its distribution appears to be limited to selected, region-specific subpopulations of cells. Although striatum is highly enriched with Arg2, precise localization and function of striatal Arg2 have never been studied. Here, we confirm that Arg2 is the only arginase isoenzyme in the striatum, and, using genetic model of total Arg2 loss, we show that Arg2 in this region is fully responsible for arginase catalytic activity, and its loss doesn't induce compensatory activation of Arg1. We exhibit that Arg2 is present in medium spiny neurons (MSNs), striatum-specific projecting neurons, where it localizes in soma and neuronal processes, and is absent in astrocytes or microglia. Finally, analysis of NMR spectroscopy-measured metabolic profiles of striata of Arg2-null mice enabled to recognize two metabolites (NADH and malonic acid) to be significantly altered compared to control animals. Multivariate comparison of the data using orthogonal projections to latent structures discriminant analysis, allowed for discrimination between control and Arg2-null mice and identified metabolites that contributed the most to this between-group dissimilarity. Our study reveals for the first time the localization of Arg2 in MSNs and demonstrates significant role of this enzyme in regulating striatal metabolism. These findings may be especially interesting in the context of Huntington's disease (HD), a disorder that specifically affects MSNs and in which, with the use of mouse models, the onset of pathological phenotypes was recently shown to be preceded by progressive impairment of striatal Arg2, a phenomenon of an unknown significance for disease pathogenesis.

Mechanisms of HIF1A-mediated immune evasion in gastric cancer and the impact on therapy resistance

BackgroundThe high prevalence and detrimental effects on patient outcomes make gastric cancer (GC) a significant health issue that persists internationally. Existing treatment modalities exhibit limited efficacy, prompting the exploration of immune checkpoint inhibitors as a novel therapeutic approach. However, resistance to immunotherapy poses a significant challenge in GC management, necessitating a profound grasp of the intrinsic molecular pathways.MethodsThis study focuses on investigating the immunosuppressive mechanisms of quiescent cancer cells (QCCs) in GC, particularly their resistance to T-cell-mediated immune responses. Utilizing mouse models, gene editing techniques, and transcriptome sequencing, we aim to elucidate the interactions between QCCs, immune cells, and key regulatory factors like HIF1A. Functional enrichment analysis will further underscore the role of glycolysis-related genes in mediating immunosuppression by QCCs.ResultsThe cancer cells that survived GC treated with T-cell therapy lost their proliferative ability. QCCs, as the main resistance force to immunotherapy, exhibit stronger resistance to CD8+ T-cell attack and possess higher cancer-initiating potential. Single-cell sequencing analysis revealed that the microenvironment in the QCCs region harbors more M2-type tumor-associated macrophages and fewer T cells. This microenvironment in the QCCs region leads to the downregulation of T-cell immune activation and alters macrophage metabolic function. Transcriptome sequencing of QCCs identified upregulated genes related to chemo-resistance, hypoxia, and glycolysis. In vitro cell experiments illustrated that HIF1A promotes the transcription of glycolysis-related genes, and silencing HIF1A in QCCs enhances T-cell proliferation and activation in co-culture systems, induces apoptosis in QCCs, and increases QCCs' sensitivity to immune checkpoint inhibitors. In vivo, animal experiments showed that silencing HIF1A in QCCs can inhibit GC growth and metastasis.ConclusionUnraveling the molecular mechanisms by which QCCs resist T-cell-mediated immune responses through immunosuppression holds promising implications for refining treatment strategies and enhancing patient outcomes in GC. By delineating these intricate interactions, this study contributes crucial insights into precision medicine and improved therapeutic outcomes in GC management.

Langerhans cells and skin immune diseases.

Langerhans cells (LCs) are the key antigen-presenting cells in the epidermis in normal conditions and respond differentially to environmental and/or endogenous stimuli, exerting either proinflammatory or anti-inflammatory effects. Current knowledge about LCs mainly originates from studies utilizing mouse models, whereas with the development of single-cell techniques, there has been significant progress for human LCs, which has updated our understanding of the phenotype, ontogeny, differentiation regulation, and function of LCs. In this review, we delineated the progress of human LCs and summarized LCs' function in inflammatory skin diseases, providing new ideas for precise regulation of LC function in the prevention and treatment of skin diseases.

Dense but not alpha granules of platelets are required for insulin secretion from pancreatic β cells

ObjectivesPlatelets, originally described for their role in blood coagulation, are now also recognized as key players in modulating inflammation, tissue regeneration, angiogenesis, and carcinogenesis. Recent evidence suggests that platelets also influence insulin secretion from pancreatic β cells. The multifaceted functions of platelets are mediated by the factors stored in their alpha granules (AGs) and dense granules (DGs). AGs primarily contain proteins, while DGs are rich in small molecules, and both types of granules are released during blood coagulation. Specific components stored in AGs and DGs are implicated in various inflammatory, regenerative, and tumorigenic processes. However, the relative contributions of AGs and DGs to the regulation of pancreatic β cell function have not been previously explored. MethodsIn this study, we utilized mouse models deficient in AG content (neurobeachin-like 2 (Nbeal2) -deficient mice) and models with defective DG release (Unc13d-deficiency in bone marrow-derived cells) to investigate the impact of platelet granules on insulin secretion from pancreatic β cells. ResultsOur findings indicate that AG deficiency has little to no effect on pancreatic β cell function and glucose homeostasis. Conversely, mice with defective DG release exhibited glucose intolerance and reduced insulin secretion. Furthermore, Unc13d-deficiency in hematopoietic stem cells led to a reduction in adipose tissue gain in obese mice. ConclusionsObtained data suggest that DGs, but not AGs, mediate the influence of platelets on pancreatic β cells, thereby modulating glucose metabolism.

436 Regulation of brain-placental axis in mouse versus pig

Abstract Emerging evidence suggests that the placenta has key roles in the development of mammalian brain. The brain-placental axis refers to the molecular and cellular events by which the placenta programs the fetal brain development during reproduction and leaves a lasting influence on the health and disease of the brain later in life. Though most studies on the brain-placental axis have utilized mouse models, the pig, Sus scrofa, is now increasingly used as a large animal model to study brain diseases in humans. However, no study has been performed to understand the regulation of the brain-placental axis in a comparative manner between mouse and pig. In the present work, we performed a comparative analysis of single-nuclei gene expression patterns in the placenta and fetal brain between mouse and pig. The placenta and fetal brain cells expressing the common orthologous genes and the species-specific genes were identified in both species. The analysis showed that a relatively fewer number of cell clusters expressed genes in a correlated manner between the placenta and fetal brain of pig relative to that of mouse. The results further showed that specific genes related to visual perception were expressed in specific cell types of the fetal brain of both mouse and pig. These genes were expressed in a coordinated manner with a set of genes expressed in specific cell types of the placenta. Further analysis showed that these placental expressed genes were significantly differentially regulated (P < 0.05, fold changes > 2) in the term-placenta between nocturnal and diurnal animals. Significant enrichment (P < 0.05) of specific binding motifs of known transcription factors, whose functions are related to the specification of retinal cells, was observed in the immediate upstream region of these placenta genes. Phylogenetic analysis of these genes among 29 diverse mammalian species differing in placenta type and activity (nocturnal or diurnal) was performed. The Bayes Traits discrete trait correlation test using the inferred phylogenies and the known trait data identified genes associated with the correlated evolution (Log Bayes Factor > 2) of placenta with animal activity (nocturnal or diurnal). The functional association of these genes with visual perception was evident from differential expression in the ocular cells between pig and mouse. Together, the findings of this study show that the regulation of the brain-placental axis plays a role in the evolution of visual perception function of brain to shape the nocturnal or diurnal activity of placental animals. However, factors other than the brain-placental axis are also known to influence temporal changes in nocturnal or diurnal activity of specific animals. Thus, future studies, potentially applying experimental evolution approaches, are needed to dissect the mechanisms of nocturnal bottleneck in placental animals.

A Method to Study Migration and Invasion of Mouse Intestinal Organoids

Colorectal cancer (CRC) is the third most common cancer worldwide and it is the second leading cause of cancer death. In CRC, as in most cancers, the formation of metastasis through the migration and invasion of cancer cells to distant organs is associated with a dismal prognosis. The study of the mechanisms associated with cancer, and, in particular, CRC, changed in the last decade due to the introduction of organoids. These represent a step forward in terms of complexity from cell lines and allowed the use of mouse models in cancer research to be limited. Although organoids faithfully model the cellular complexity of CRC, current protocols do not allow for the use of organoids in some crucial processes of metastasis, such as migration and invasion. In this study, a method to study migration and invasion using mouse intestinal organoids in vitro is presented. This protocol provides researchers with the opportunity to investigate the migratory behavior of organoid lines and study the impact of distinct mutations on the migratory and invasive capacity of cancer cells.

How do teeth erupt?

The development of normal occlusion requires eruptive migration of teeth from their developmental position in the jaw into a functional position within the oral cavity. This process involves significant and coordinated movement in an axial direction and appropriate eruption through the gingival tissues. The mechanisms regulating these developmental events are poorly understood, and teeth retain eruptive potential throughout their lifespan. In recent years, the use of mouse models has helped to elucidate some of the underlying molecular and biological mechanisms of mammalian tooth eruption. Here, we outline our current understanding of tooth eruption mechanisms and discuss their relevance in terms of known human disorders of tooth eruption.

The universe is asymmetric, the mouse brain too.

Hemispheric brain asymmetry is a basic organizational principle of the human brain and has been implicated in various psychiatric conditions, including autism spectrum disorder. Brain asymmetry is not a uniquely human feature and is observed in other species such as the mouse. Yet, asymmetry patterns are generally nuanced, and substantial sample sizes are required to detect these patterns. In this pre-registered study, we use a mouse dataset from the Province of Ontario Neurodevelopmental Network, which comprises structural MRI data from over 2000 mice, including genetic models for autism spectrum disorder, to reveal the scope and magnitude of hemispheric asymmetry in the mouse. Our findings demonstrate the presence of robust hemispheric asymmetry in the mouse brain, such as larger right hemispheric volumes towards the anterior pole and larger left hemispheric volumes toward the posterior pole, opposite to what has been shown in humans. This suggests the existence of species-specific traits. Further clustering analysis identified distinct asymmetry patterns in autism spectrum disorder models, a phenomenon that is also seen in atypically developing participants. Our study shows potential for the use of mouse models to understand the biological bases of typical and atypical brain asymmetry but also warrants caution as asymmetry patterns seem to differ between humans and mice.

Chimeric Viruses Enable Study of Antibody Responses to Human Rotaviruses in Mice.

The leading cause of gastroenteritis in children under the age of five is rotavirus infection, accounting for 37% of diarrhoeal deaths in infants and young children globally. Oral rotavirus vaccines have been widely incorporated into national immunisation programs, but whilst these vaccines have excellent efficacy in high-income countries, they protect less than 50% of vaccinated individuals in low- and middle-income countries. In order to facilitate the development of improved vaccine strategies, a greater understanding of the immune response to existing vaccines is urgently needed. However, the use of mouse models to study immune responses to human rotavirus strains is currently limited as rotaviruses are highly species-specific and replication of human rotaviruses is minimal in mice. To enable characterisation of immune responses to human rotavirus in mice, we have generated chimeric viruses that combat the issue of rotavirus host range restriction. Using reverse genetics, the rotavirus outer capsid proteins (VP4 and VP7) from either human or murine rotavirus strains were encoded in a murine rotavirus backbone. Neonatal mice were infected with chimeric viruses and monitored daily for development of diarrhoea. Stool samples were collected to quantify viral shedding, and antibody responses were comprehensively evaluated. We demonstrated that chimeric rotaviruses were able to efficiently replicate in mice. Moreover, the chimeric rotavirus containing human rotavirus outer capsid proteins elicited a robust antibody response to human rotavirus antigens, whilst the control chimeric murine rotavirus did not. This chimeric human rotavirus therefore provides a new strategy for studying human-rotavirus-specific immunity to the outer capsid, and could be used to investigate factors causing variability in rotavirus vaccine efficacy. This small animal platform therefore has the potential to test the efficacy of new vaccines and antibody-based therapeutics.

Autocrine insulin-like growth factor 2 signaling as a potential target in the associated development of pulmonary emphysema and cancer in smokers

BackgroundTobacco smoking causes pulmonary inflammation, resulting in emphysema, an independent risk factor for lung cancer. Induction of insulin-like growth factor 2 (IGF2) in response to lung injury by tobacco carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and polycyclic aromatic hydrocarbon benzo[a]pyrene in combination (NB), is critical for the proliferation of alveolar type 2 cells (AT2s) for lung repair. However, persistent IGF2 overexpression during NB-induced severe injury results in hyperproliferation of AT2s without coordinated AT2-to-AT1 differentiation, disrupting alveolar repair, which leads to the concurrent development of emphysema and lung cancer. The current study aims to verify the role of IGF2 signaling in the associated development of emphysema and cancer and develop effective pharmaceuticals for the diseases using animal models that recapitulate the characteristics of these chronic diseases.MethodsThe pathogenesis of pulmonary emphysema and cancer was analyzed by lung function testing, histological evaluation, in situ zymography, dihydroethidium staining, and immunofluorescence and immunohistochemistry analyses utilizing mouse models of emphysema and cancer established by moderate exposure to NB for up to seven months.ResultsModerate NB exposure induced IGF2 expression in AT2s during the development of pulmonary emphysema and lung cancer in mice. Using AT2-specific insulin receptor knockout mice, we verified the causative role of sustained IGF2 signaling activation in AT2s in emphysema development. IGF2-targeting strategies, including voltage-dependent calcium channel blocker (CCB) and a neutralizing antibody, significantly suppressed the NB-induced development of emphysema and lung cancer. A publicly available database revealed an inverse correlation between the use of calcium channel blockers and a COPD diagnosis.ConclusionsOur work confirms sustained IGF2 signaling activation in AT2s couples impaired lung repair to the concurrent development of emphysema and cancer in mice. Additionally, CCB and IGF2-specific neutralizing antibodies are effective pharmaceuticals for the two diseases.

Investigating Snake-Venom-Induced Dermonecrosis and Inflammation Using an Ex Vivo Human Skin Model.

Snakebite envenoming is a neglected tropical disease that causes >100,000 deaths and >400,000 cases of morbidity annually. Despite the use of mouse models, severe local envenoming, defined by morbidity-causing local tissue necrosis, remains poorly understood, and human-tissue responses are ill-defined. Here, for the first time, an ex vivo, non-perfused human skin model was used to investigate temporal histopathological and immunological changes following subcutaneous injections of venoms from medically important African vipers (Echis ocellatus and Bitis arietans) and cobras (Naja nigricollis and N. haje). Histological analysis of venom-injected ex vivo human skin biopsies revealed morphological changes in the epidermis (ballooning degeneration, erosion, and ulceration) comparable to clinical signs of local envenoming. Immunostaining of these biopsies confirmed cell apoptosis consistent with the onset of necrosis. RNA sequencing, multiplex bead arrays, and ELISAs demonstrated that venom-injected human skin biopsies exhibited higher rates of transcription and expression of chemokines (CXCL5, MIP1-ALPHA, RANTES, MCP-1, and MIG), cytokines (IL-1β, IL-1RA, G-CSF/CSF-3, and GM-CSF), and growth factors (VEGF-A, FGF, and HGF) in comparison to non-injected biopsies. To investigate the efficacy of antivenom, SAIMR Echis monovalent or SAIMR polyvalent antivenom was injected one hour following E. ocellatus or N. nigricollis venom treatment, respectively, and although antivenom did not prevent venom-induced dermal tissue damage, it did reduce all pro-inflammatory chemokines, cytokines, and growth factors to normal levels after 48 h. This ex vivo skin model could be useful for studies evaluating the progression of local envenoming and the efficacy of snakebite treatments.

Role of gut microbiota in the pathogenesis of castration-resistant prostate cancer: a comprehensive study using sequencing and animal models.

CRPC remains a significant challenge in prostate cancer research. We aimed to elucidate the role of gut microbiota and its specific mechanisms in CRPC using a multidisciplinary approach. We analyzed 16S rRNA sequencing data from mouse fecal samples, revealing substantial differences in gut microbiota composition between CRPC and castration-sensitive prostate cancer mice, particularly in Firmicutes and Bacteroidetes. Functional analysis suggested different bacteria may influence CRPC via the α-linolenic acid metabolism pathway. In vivo, experiments utilizing mouse models and fecal microbiota transplantation (FMT) demonstrated that FMT from healthy control mice could decelerate tumor growth in CRPC mice, reduce TNF-α levels, and inhibit the activation of the TLR4/MyD88/NF-κB signaling pathway. Transcriptome sequencing identified crucial genes and pathways, with rescue experiments confirming the gut microbiota's role in modulating CRPC progression through the TLR4/MyD88/NF-κB pathway. The activation of this pathway by TNF-α has been corroborated by in vitro cell experiments, indicating its role in promoting prostate cancer cell proliferation, migration, and invasion while inhibiting apoptosis. Gut microbiota dysbiosis may promote CRPC development through TNF-α activation of the TLR4/MyD88/NF-κB signaling pathway, potentially linked to α-linolenic acid metabolism.

Platelet-mediated circulating tumor cell evasion from natural killer cell killing via immune checkpoint CD155-TIGIT.

e14552 Background: Circulating tumor cells (CTCs) are precursors of cancer metastasis. However, how CTCs evade immunosurveillance during hematogenous dissemination remains unclear. The interaction between platelets and CTCs is the most extensively studied of all blood cells, and it is well known that CTCs are surrounded by platelets, which form microplots on their surface and play a crucial role in cancer metastasis. The precise mechanisms platelets employ to contribute to CTC immune evasion have not been fully elucidated. Our previous single-cell RNA sequencing data indicated high expression of platelet-related genes in CTCs. Here, our study demonstrated that platelet adhesion to CTCs is prevalent in multiple cancers and is indispensable for CTCs metastasis. Methods: CTC isolation was performed by our self-established ChimeraX-i120 platform. CTC-platelet adhesions were identified by single-cell RNA sequencing and multiplex immunofluorescence of blood samples from multiple cancer types. The cytotoxicity of natural killer (NK) cells was assessed by the reduction in tumor cell numbers measured using the xCELLigence system. In vitro and ex vivo co-culture systems were established by using platelets and NK cells isolated from the peripheral blood of patients for direct or indirect co-culturing with cell lines and patient-derived organoids (PDOs). The effects of platelets and NK cells on metastasis and immune responses were investigated in vivo by utilizing mouse models. Results: Clinically, CTC-platelet aggregates were associated with significantly shorter progression-free survival and overall survival in hepatocellular carcinoma patients. In vitro, ex vivo, and in vivo assays demonstrated direct platelet adhesions gifted cancer cells with an evasive ability from NK cell killing by upregulating inhibitory checkpoint CD155, therefore facilitating distant metastasis. Mechanistically, CD155 was transcriptionally regulated by the FAK/JNK/c-Jun cascade in a platelet contact-dependent manner. Further competition assays and cytotoxicity experiments revealed that CD155 on CTCs inhibited NK cell cytotoxicity only by engaging with immune receptor TIGIT, but not CD96 and DNAM1, another two receptors for CD155. Interrupting the CD155-TIGIT interactions with a TIGIT antibody restored NK cell immunosurveillance on CTCs and markedly attenuated tumor metastasis. Conclusions: Our results demonstrated CTC evasion from NK cell-mediated innate immunosurveillance mainly via immune checkpoint CD155-TIGIT, potentially offering an immunotherapeutic strategy for eradicating CTCs.

“Niclosamide: A potential antipruritic agent by modulating serotonin pathway through metabotropic glutamate receptors (mGluRs)”

Pruritus is an uncomfortable sensation induced by various pruritogens, including serotonin. Serotonin, acting as an inflammatory mediator, can activate a histamine-independent pathway. Consequently, many anti-pruritus medications, such as antihistamines, are not effective in adequately relieving patient symptoms. Niclosamide, an anthelmintic drug, has recently demonstrated an affinity for Metabotropic glutamate receptors (mGluRs). mGluRs are a group of receptors activated by glutamate, and they are involved in regulating neuronal excitability. In this study, we utilized mouse models of serotonergic itch and administered different doses of Niclosamide to examine the expression of mGluR1, mGluR5, and 5-HT2. The administration of 5 mg/kg Niclosamide successfully suppressed pruritus in the mice. Additionally, the levels of mGluR1, mGluR5, 5-HT2, and TRPV1 were significantly reduced. These findings suggest that Niclosamide holds promise as a potential antipruritic drug.

IC100, a humanized therapeutic monoclonal anti-ASC antibody alleviates oxygen-induced retinopathy in mice

BackgroundRetinopathy of prematurity (ROP), which often presents with bronchopulmonary dysplasia (BPD), is among the most common morbidities affecting extremely premature infants and is a leading cause of severe vision impairment in children worldwide. Activations of the inflammasome cascade and microglia have been implicated in playing a role in the development of both ROP and BPD. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is pivotal in inflammasome assembly. Utilizing mouse models of both oxygen-induced retinopathy (OIR) and BPD, this study was designed to test the hypothesis that hyperoxia induces ASC speck formation, which leads to microglial activation and retinopathy, and that inhibition of ASC speck formation by a humanized monoclonal antibody, IC100, directed against ASC, will ameliorate microglial activation and abnormal retinal vascular formation.MethodsWe first tested ASC speck formation in the retina of ASC-citrine reporter mice expressing ASC fusion protein with a C-terminal citrine (fluorescent GFP isoform) using a BPD model that causes both lung and eye injury by exposing newborn mice to room air (RA) or 85% O2 from postnatal day (P) 1 to P14. The retinas were dissected on P14 and retinal flat mounts were used to detect vascular endothelium with AF-594-conjugated isolectin B4 (IB4) and citrine-tagged ASC specks. To assess the effects of IC100 on an OIR model, newborn ASC citrine reporter mice and wildtype mice (C57BL/6 J) were exposed to RA from P1 to P6, then 75% O2 from P7 to P11, and then to RA from P12 to P18. At P12 mice were randomized to the following groups: RA with placebo PBS (RA-PBS), O2 with PBS (O2-PBS), O2 + IC100 intravitreal injection (O2-IC100-IVT), and O2 + IC100 intraperitoneal injection (O2-IC100-IP). Retinal vascularization was evaluated by flat mount staining with IB4. Microglial activation was detected by immunofluorescence staining for allograft inflammatory factor 1 (AIF-1) and CD206. Retinal structure was analyzed on H&E-stained sections, and function was analyzed by pattern electroretinography (PERG). RNA-sequencing (RNA-seq) of the retinas was performed to determine the transcriptional effects of IC100 treatment in OIR.ResultsASC specks were significantly increased in the retinas by hyperoxia exposure and colocalized with the abnormal vasculature in both BPD and OIR models, and this was associated with increased microglial activation. Treatment with IC100-IVT or IC100-IP significantly reduced vaso-obliteration and intravitreal neovascularization. IC100-IVT treatment also reduced retinal microglial activation, restored retinal structure, and improved retinal function. RNA-seq showed that IC100 treatment corrected the induction of genes associated with angiogenesis, leukocyte migration, and VEGF signaling caused by O2. IC100 also corrected the suppression of genes associated with cell junction assembly, neuron projection, and neuron recognition caused by O2.ConclusionThese data demonstrate the crucial role of ASC in the pathogenesis of OIR and the efficacy of a humanized therapeutic anti-ASC antibody in treating OIR mice. Thus, this anti-ASC antibody may potentially be considered in diseases associated with oxygen stresses and retinopathy, such as ROP.

A p21-ATD mouse model for monitoring and eliminating senescent cells and its application in liver regeneration post injury

Cellular senescence associates with pathological aging and tissue dysfunctions. Studies utilizing mouse models for cell lineage tracings have emphasized the importance of senescence heterogeneity in different organs and cell types. Here, we constructed a p21- [Akaluc - tdTomato - Diphtheria Toxin Receptor (DTR)] (ATD) mouse model to specifically study the undefined mechanism for p21-expressing senescent cells in the aged and liver injury animals. The successful expressions of these genes enabled in vitro flow cytometric sorting, in vivo tracing and eliminating of p21-expressing senescent cells. During the natural aging process, p21-expressing cells were found in various tissues of p21-ATD mice. Eliminating p21-expressing cells in the aged p21-ATD mice recovered their multiple biological functions. p21-ATD/Fah-/- mice, bred from p21-ATD mice and fumarylacetoacetate hydrolase (Fah)-/- mice of liver injury, showed that the majority of their senescent hepatocytes were the phenotype of p21+ rather than p16+. Furthermore, eliminating the p21-expressing hepatocytes significantly promoted the engraftment of grafted hepatocytes and facilitated the liver repopulation, resulting in the significant recovery from liver injury. Our p21-ATD mouse model serves as an optimal model for studying the pattern and function of p21-expressing senescent cells under the physical and pathological conditions during aging.

Lymphatic-Dependent Modulation of the Sensitization and Elicitation Phases of Contact Hypersensitivity

Allergic contact dermatitis is a common inflammatory skin disease comprising 2 phases. During sensitization, immune cells are activated by exposure to various allergens, whereas repeated antigen exposure induces local inflammation during elicitation. In this study, we utilized mouse models lacking lymphatics in different skin regions to characterize the role of lymphatics separately in the 2 phases, using contact hypersensitivity as a model of human allergic inflammatory skin diseases. Lymphatic-deficient mice exhibited no major difference to single antigen exposure compared to controls. However, mice lacking lymphatics in both phases displayed reduced inflammation after repeated antigen exposure. Similarly, diminished immune response was observed in mice lacking lymphatics only in sensitization, whereas the absence of lymphatics only in the elicitation phase resulted in a more pronounced inflammatory immune response. This exaggerated inflammation is driven by neutrophils impacting regulatory T cell number. Collectively, our results demonstrate that skin lymphatics play an important but distinct role in the 2 phases of contact hypersensitivity. During sensitization, lymphatics contribute to the development of the antigen-specific immunization, whereas in elicitation, they moderate the inflammatory response and leukocyte infiltration in a neutrophil-dependent manner. These findings underscore the need for novel therapeutic strategies targeting the lymphatics in the context of allergic skin diseases.