Receptor Knockout Research Articles

Aryl hydrocarbon receptor (AhR)-mediated immune responses to degeneration of the retinal pigment epithelium

Injuries to the retinal pigment epithelium (RPE) trigger immune responses, orchestrating interactions within the innate and adaptive immune systems in the outer retina and choroid. We previously reported that interleukin 17 (IL-17) is a pivotal signaling molecule originating from choroidal γδ T cells, exerting protective effects by mediating functional connections between the RPE and subretinal microglia. In this current study, we generated mice with aryl hydrocarbon receptor (AhR) knockout specifically in IL-17-producing cells. These animals had deficiency in IL-17 production from γδ T cells, and exhibited increased sensitivity to both acute and chronic insults targeting the RPE. These findings imply that IL-17 plays a crucial role as a signaling cytokine in preserving the homeostasis of the outer retina and choroid.

The role of placental kisspeptin in trophoblast invasion and migration: an assessment in Kiss1r knockout mice, BeWo cell lines and human term placenta.

Context There is mounting evidence implicating kisspeptin signalling in placental development and function. Aims This study aimed to elucidate kisspeptin's role in trophoblast invasion and migration using three experimental models. Methods First, we examined the mouse fetus and placenta in a kisspeptin receptor (Kiss1r) knockout (KO) model. Fetal/placental weights and gene expression (quantitative polymerase chain reaction) were assessed. Second, we determined kisspeptin effects on a human trophoblast (BeWo) cell line in vitro . Third, we examined KISS1 and KISS1R gene expression in human placenta from term and pre-term pregnancies. Key results No difference was found in fetal or placental weight between Kiss1r KO and wildtype mice. However, expression of the trophoblast invasion marker, Mmp2 mRNA, was greater in the placental labyrinth zone of Kiss1r KO mice. BeWo cell models of villus cytotrophoblast and syncytiotrophoblast cells exhibited kisspeptin protein expression, with greater expression in syncytiotrophoblast, consistent with KISS1 mRNA. Kisspeptin treatment inhibited the migratory potential of cytotrophoblast-like cells. Finally, while no difference was seen in KISS1 and KISS1R mRNA between term and pre-term placentas, we saw a difference in the relative expression of each gene pre-term. We also observed a positive correlation between KISS1 expression and maternal body mass index. Conclusions Our results indicate that kisspeptin may inhibit trophoblast invasion. Implications Further investigation is required to clarify specific regulatory mechanisms.

Oxytocin receptors in the nucleus accumbens shell are necessary for the onset of maternal behavior.

In rodents, oxytocin (Oxt) contributes to the onset of maternal care by shifting the perception of pups from aversive to attractive. Both Oxt receptor knockout (Oxtr -/-) and forebrain-specific Oxtr knockout (FB/FB) dams abandon their first litters, likely due to a failure of the brain to 'switch' to a more maternal state. Whether this behavioral shift is neurochemically similar in virgin females, who can display maternal behaviors when repeatedly exposed to pups, or what neuroanatomical substrate is critical for the onset of maternal care remains unknown. To understand similarities and differences in Oxtr signaling in virgin pup-sensitized Oxtr FB/FB as opposed to post-parturient Oxtr -/- and Oxtr FB/FB dams, maternal behavior (pup-sensitized females only) and immediate early gene activation were assessed. Pup-sensitized Oxtr FB/FB females retrieved pups faster on day one of testing and had reduced c-Fos expression in the dorsal lateral septum as compared to virgin pup-sensitized Oxtr +/+ females. This differs from what was observed in post-parturient Oxtr -/- and Oxtr FB/FB dams, where increased c-Fos expression was observed in the nucleus accumbens (NAcc) shell. Based on these data, we then disrupted Oxtr signaling in the NAcc shell or the posterior paraventricular thalamus (pPVT) (control region) of female Oxtr floxed mice using a Cre recombinase expressing adeno-associated virus. Knockout of the Oxtr only in the NAcc shell prevented the onset of maternal care post-parturient females. Our data suggest that a pup-sensitized brain may differ from a post-parturient brain and that Oxtr signaling in the NAcc shell is critical to the onset of maternal behavior.

Subconjunctival Administration of an Adeno-Associated Virus Expressing Stanniocalcin-1 Provides Sustained Intraocular Pressure Reduction in Mice

PurposeTo investigate subconjunctival administration of a single-stranded, adeno-associated virus, serotype 2, engineered to express Stanniocalcin-1 with a FLAG tag (ssAAV2-STC-1-FLAG) as a novel sustained intraocular pressure (IOP) lowering agent with a reduced ocular surface side effect profile. DesignIn vivo pre-clinical investigation in mice SubjectsC57BL/6J, DBA/2J, FP receptor knockout mice MethodsNormotensive C57BL/6J mice were treated with a subconjunctival injection of ssAAV2-STC-1-FLAG (2 μL; 6 x 109 viral genomes [VGs]) in one eye and the same volume and concentration of ssAAV2-GFP or the same volume of PBS in the fellow eye. Ocular hypertensive DBA/2J mice were subconjunctivally injected with 6 x 109 VGs of ssAAV2-STC-1-FLAG or ssAAV2-GFP. Steroid-mediated ocular hypertension was induced in C57BL/6J mice with weekly injections of dexamethasone into the conjunctival fornix, and mice were then injected subconjunctivally with 6 x 109 VGs of ssAAV2-STC-1-FLAG or ssAAV2-GFP. FP receptor knockout mice were injected subconjunctivally with 6 x 109 VGs of ssAAV2-STC-1-FLAG or PBS. An identical vector was constructed without the FLAG tag (ssAAV2-STC-1) and evaluated in normotensive C57BL/6J mice. IOP was assessed using the Tonolab tonometer for all experiments. Tumor necrosis factor alpha (TNFα), a marker of ocular surface inflammation, was compared between subconjunctivally delivered ssAAV2-STC-1 and other treatments including daily topical latanoprost. Main outcome measuresIOP assessment ResultsSubconjunctival delivery of ssAAV2-STC-1-FLAG significantly reduced IOP for 10 weeks post injection in normotensive mice. Maximal IOP reduction was seen at week 2 post injection (17.4%; 17.1 ± 0.8 vs 14.1 ± 0.8 mmHg, P<0.001). After the IOP-lowering effect had waned, a second injection restored the ocular hypotensive effect. Subconjunctivally delivered ssAAV2-STC-1-FLAG lowered IOP in DBA/2J mice (16.9%; 17.8 ± 2.0 vs 14.8 ± 0.9 mmHg, P<0.001) and steroid-mediated ocular hypertensive mice (19.4%; 18.7 ±0.9 vs 15.1 ± 0.5 mmHg, P<0.001) over the experimental period. This construct also reduced IOP to a similar extent in wild type (15.9%) and FP receptor knockout (15.7%) mice compared to the fellow eye. A related construct also lowered IOP without the FLAG tag in a similar manner. Reduction in conjunctival TNFα was seen when comparing subconjunctivally delivered ssAAV2-STC-1-FLAG to daily topical latanoprost. ConclusionsSubconjunctival delivery of the STC-1 transgene with a vector system may represent a novel treatment strategy for sustained IOP reduction and improved ocular tolerability that also avoids the daily dosing requirements of currently available medications.

An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene editing platforms that rely on nuclease activity, such as Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point™ base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared to the conventional CRISPR-Cas9 system. Site-specific knock-in of a chimeric antigen receptor (CAR) and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

IFN-γ-responsiveness of lymphatic endothelial cells inhibits melanoma lymphatic dissemination via AMPK-mediated metabolic control

The integrity of the lymphatic system is critical for preventing the dissemination of tumor cells, such as melanoma, to distant parts of the body. IFN-γ is well studied as a negative regulator for lymphangiogenesis, which is strongly associated with cancer metastasis. However, the exact mechanisms underlying this process remain unclear. In the present study, we investigated whether IFN-γ signaling in lymphatic endothelial cells (LECs) affects tumor cell dissemination by regulating the barrier function of tumor-associated lymphatic vessels. Using LEC-specific IFN-γ receptor (IFN-γR) knockout mice, we found that the loss of IFN-γR in LECs increased the dissemination of melanoma cells into the draining lymph nodes. Notably, IFN-γ signaling in LECs inhibited trans-lymphatic endothelial cell migration of melanoma cells, indicating its regulation of lymphatic barrier function. Further investigations revealed that IFN-γ upregulated the expression of the tight junction protein Claudin-3 in LECs, while knockdown of Claudin-3 in LECs abolished IFN-γ-induced inhibition of trans-lymphatic endothelial migration activity. Mechanistically, IFN-γ inhibits AMPK signaling activation, which is involved in the regulation of fatty acid metabolism. Modulating fatty acid metabolism and AMPK activation in LECs also affected the lymphatic dissemination of melanoma cells, further confirming that this process is involved in IFN-γ-induced regulation of lymphatic barrier function. These results provide novel insights into how IFN-γ modulates tight junctions in LECs, inhibiting the dissemination of melanoma cells via the lymphatic vessels.

Testosterone and Erythrocyte Lifespan.

Endogenous and exogenous androgens increase circulating erythrocytes and hemoglobin but their effects on erythrocyte lifespan is not known. To investigate androgen effects on immature and mature erythrocyte lifespan in humans and mice using novel non-radioactive minimally invasive methods. Human erythrocyte lifespan was estimated using alveolar carbon monoxide concentration and blood hemoglobin in Levitt's formula in hypogonadal or transgender men before and up to 18 weeks after commencing testosterone (T) treatment. Erythrocyte lifespan was estimated in androgen receptor (AR) knockout and wild-type mice after T or dihydrotestosterone (DHT) treatment of intact females or orchidectomized males using in vivo biotin labelling of erythrocyte surface epitopes for reticulocytes (Ter119+CD71+) and two markers of erythrocytes (CD45-, Ter119+CD71-) monitoring their blood disappearance rate by flow cytometry. Before treatment, hypogonadal and transgender men had marked reduction in erythrocyte lifespan compared with controls. T treatment increased erythrocyte lifespan at 6 weeks but returned to pre-treatment levels at 18 weeks while serum T and blood hemoglobin were increased by T treatment remaining elevated at 18-weeks. In mice T and DHT treatment had higher erythrocyte (but not reticulocyte) lifespan but neither orchidectomy nor AR inactivation significantly influenced erythrocyte or reticulocyte lifespan. We conclude that hypogonadal men have reduced erythrocyte lifespan and acute androgen-induced increase in circulating erythrocyte lifespan may contribute to the well-known erythropoietic effects of androgens, but longer-term effects require further investigation to determine how much they contribute to androgen-induced increases in circulating hemoglobin.

Inflammatory & Apoptotic Factor Fluctuations Associated with Japanese Encephalitis Virus Infection in Transgenic IFNAR1-/- Mice.

Japanese encephalitis virus (JEV) is an orthoflavivirus that causes Japanese encephalitis, a mosquito-borne viral infection that primarily affects humans and animals. JEV is a major cause of encephalitis in many parts of Asia, particularly in rural and agricultural areas. In this study, we used the IFNAR1-/- mice model to investigate alterations in cytokine and apoptotic factor levels in IFNAR1-/- mice upon JEV infection. A 5-week-adult female C57BL/6 IFN-α/β receptor knockout (IFNAR1-/-) transgenic mice were intramuscularly inoculated with several viral titers and monitored within 10 dpi. The weight changes and survival rates were evaluated during the study period. Gene expression analysis was performed using RT-qPCR, targeting genes related to specific cytokines and apoptotic factors, to identify the inflammatory factors fluctuations associated with JEV strain KBPV-VR-27 infection in IFNAR1-/- mice. The expression of cytokine genes was enhanced in IFNAR1-/- mice infected with JEV KBPV-VR-27. Notably, a significant induction of cytokines, such as IL-13, IL-17α, IFN-β, and IFN-γ, was observed in the brain, while upregulation of IL-6, IFN-β, and IFN-γ was exhibited in the lung. In addition, among the targeted apoptotic factors, only significant induction of Bak was observed in the brain. We also found that the spleen exhibited a higher viral load compared to the brain and lungs. In conclusion, the findings of this study shed light on the varying viral loads across targeted organs, with the brain exhibiting a lower viral load but pronounced expression of targeted pro-inflammatory cytokines in IFNAR1-/- mice.

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.

Non-Invasive Detection of Early-Stage Fatty Liver Disease via an On-Skin Impedance Sensor and Attention-Based Deep Learning.

Early-stage nonalcoholic fatty liver disease (NAFLD) is a silent condition, with most cases going undiagnosed, potentially progressing to liver cirrhosis and cancer. A non-invasive and cost-effective detection method for early-stage NAFLD detection is a public health priority but challenging. In this study, an adhesive, soft on-skin sensor with low electrode-skin contact impedance for early-stage NAFLD detection is fabricated. A method is developed to synthesize platinum nanoparticles and reduced graphene quantum dots onto the on-skin sensor to reduce electrode-skin contact impedance by increasing double-layer capacitance, thereby enhancing detection accuracy. Furthermore, an attention-based deep learning algorithm is introduced to differentiate impedance signals associated with early-stage NAFLD in high-fat-diet-fed low-density lipoprotein receptor knockout (Ldlr-/-) mice compared to healthy controls. The integration of an adhesive, soft on-skin sensor with low electrode-skin contact impedance and the attention-based deep learning algorithm significantly enhances the detection accuracy for early-stage NAFLD, achieving a rate above 97.5% with an area under the receiver operating characteristic curve (AUC) of 1.0. The findings present a non-invasive approach for early-stage NAFLD detection and display a strategy for improved early detection through on-skin electronics and deep learning.

Dietary sugar kelp (Saccharina latissima) consumption did not attenuate atherosclerosis in low-density lipoprotein receptor knockout mice.

We previously demonstrated the beneficial effects of U.S.-grown sugar kelp (Saccharina latissima), a brown seaweed, on reducing serum triglycerides (TG) and total cholesterol (TC) and protecting against inflammation and fibrosis in the adipose tissue of diet-induced obesity mice. In this current study, we aimed to explore whether the dietary consumption of sugar kelp can prevent atherosclerosis using low-density lipoprotein receptor knockout (Ldlr KO) mice fed an atherogenic diet. Eight-week-old male Ldlr KO mice were fed either an atherogenic high-fat/high-cholesterol control (HF/HC) diet or a HF/HC diet supplemented with 6% (w/w) sugar kelp (HF/HC-SK) for 16 weeks. Consumption of sugar kelp significantly increased the body weight gain without altering fat mass and lean mass. Also, there were no significant differences in energy expenditure and physical activities between the groups. The two groups did not show significant differences in serum and hepatic TG and TC levels or the hepatic expression of genes involved in cholesterol and lipid metabolism. Although serum alanine aminotransferase (ALT) activity did not differ significantly between the two groups, there were significant increases in the expression of macrophage markers, including adhesion G protein-coupled receptor E1 and cluster of differentiation 68, as well as tumor necrosis factor alpha in the HF/HC-SK group compared to the HF/HC mice. The consumption of sugar kelp did not elicit a significant effect on the development of aortic lesions. Moreover, lipopolysaccharide-stimulated splenocytes isolated from HF/HC-SK-fed mice showed no significant changes in the mRNA levels of pro-inflammatory genes compared with those from the HF/HC mice. In summary, the consumption of dietary sugar kelp did not elicit anti-atherogenic and hepatoprotective effects in Ldlr KO mice.

Targeting Adenosine A2b Receptor Promotes Penile Rehabilitation of Refractory Erectile Dysfunction.

The mechanisms of adenosine and specific adenosine receptor subtypes in promoting penile rehabilitation remain unclear. Single-cell RNA sequencing of human corpus cavernosum, adenosine deaminase (ADA) and adenosine receptors knock-out mice (ADA-/-, A1-/-, A2a-/-, A2b-/-, and A3-/-), and primary corpus cavernosum smooth muscle cellsare used to determine receptor subtypes responsible for adenosine-induced erection. Three rat models are established to characterize refractory erectile dysfunction (ED): age-related ED, bilateral cavernous nerve crush related ED (BCNC), and diabetes mellitus-induced ED. In single-cell RNA sequencing data, the corpus cavernosum of ED patients show a decrease in adenosine A1, A2a and A2b receptors. In vivo, A2b receptor knock-out abolishes adenosine-induced erection but not that of A1, A2a, or A3 receptor. Under hypoxic conditions in vitro, activating the A2b receptor increases HIF-1α and decreases PDE5 expression. In refractory ED models, activating the A2b receptor with Bay 60-6583 improves erectile function and down-regulates HIF-1α and TGF-β. Administering Dipyridamole (40mgKg-1) to BCNC rats improve penile adenosine levels and erectile function. Our study reveals that the A2b receptor mediates adenosine-induced penile erection. Activating the A2b receptor promotes penile rehabilitation of refractory ED by alleviating hypoxia and fibrosis.

Mu-opioid receptor knockout on Foxp2-expressing neurons reduces aversion-resistant alcohol drinking.

Mu-opioid receptors (MORs) in the amygdala and striatum are important in addictive and rewarding behaviors. The transcription factor Foxp2 is a genetic marker of intercalated (ITC) cells in the amygdala and a subset of striatal medium spiny neurons (MSNs), both of which express MORs in wild-type mice and are neuronal subpopulations of potential relevance to alcohol-drinking behaviors. For the current series of studies, we characterized the behavior of mice with genetic deletion of the MOR gene Oprm1 in Foxp2-expressing neurons (Foxp2-Cre/Oprm1fl/fl). Male and female Foxp2-Cre/Oprm1fl/fl mice were generated and heterozygous Cre+ (knockout) and homozygous Cre- (control) animals were tested for aversion-resistant alcohol consumption using an intermittent access (IA) task, operant responding for a sucrose reward, conditioned place aversion (CPA) to morphine withdrawal, and locomotor sensitization to morphine. The results demonstrate that deletion of MOR on Foxp2-expressing neurons renders mice more sensitive to quinine-adulterated ethanol (EtOH). Mice with the deletion (vs. Cre- controls) also consumed less alcohol during the final sessions of the IA task, responded less for sucrose under an FR3 schedule, and were less active at baseline and following morphine injection. Foxp2-MOR deletion did not impair the ability to learn to respond for reward or develop a conditioned aversion to morphine withdrawal. Together, these investigations demonstrate that Foxp2-expressing neurons may be involved in escalation of alcohol consumption and the development of compulsive-like alcohol drinking.

The P2X7 Hypothesis of Central Post-Stroke Pain.

The present study examined how P2X7 receptor knockout (KO) modulates central post-stroke pain (CPSP) induced by lesions of the ventrobasal complex (VBC) of the thalamus in behaviors, molecular levels, and electrical recording tests. Following the experimental procedure, the wild-type and P2X7 receptor KO mice were injected with 10 mU/0.2 μL type IV collagenase in the VBC of the thalamus to induce an animal model of stroke-like thalamic hemorrhage. Behavioral data showed that the CPSP group induced thermal and mechanical pain. The P2X7 receptor KO group showed reduced thermal and mechanical pain responses compared to the CPSP group. Molecular assessments revealed that the CPSP group had lower expression of NeuN and KCC2 and higher expression of GFAP, IBA1, and BDNF. The P2X7 KO group showed lower expression of GFAP, IBA1, and BDNF but nonsignificant differences in KCC2 expression than the CPSP group. The expression of NKCC1, GABAa receptor, and TrkB did not differ significantly between the control, CPSP, and P2X7 receptor KO groups. Muscimol, a GABAa agonist, application increased multiunit numbers for monitoring many neurons and [Cl-] outflux in the cytosol in the CPSP group, while P2X7 receptor KO reduced multiunit activity and increased [Cl-] influx compared to the CPSP group. P2X4 receptor expression was significantly decreased in the 100 kDa but not the 50 kDa site in the P2X7 receptor KO group. Altogether, the P2X7 hypothesis of CPSP was proposed, wherein P2X7 receptor KO altered the CPSP pain responses, numbers of astrocytes and microglia, CSD amplitude of the anterior cingulate cortex and the medial dorsal thalamus, BDNF expression, [Cl-] influx, and P2X4 expression in 100 kDa with P2X7 receptors. The present findings have implications for the clinical treatment of CPSP symptoms.

FXR deletion attenuates intestinal barrier dysfunction in murine acute intestinal inflammation.

Accumulating literature suggests that the farnesoid-X receptor (FXR), a nuclear bile acid receptor best known for its role in bile acid homeostasis, is also a potent context-dependent regulator of inflammation. FXR may thus be relevant to several intestinal disease states including inflammatory bowel disease, necrotizing enterocolitis, and sepsis. In this study, we tested the effects of FXR deletion on acute murine intestinal inflammation. We found that FXR knockout (KO) mice were protected from intestinal injury and barrier dysfunction induced by lipopolysaccharide (LPS) injection, dithizone (DI)/Klebsiella, and cecal ligation/puncture models. In the LPS model, RNA sequencing and qPCR analysis showed that this protection correlated with substantial reduction in LPS-induced proinflammatory gene expression, including lower tissue levels of Il1a, Il1b, and Tnf. Examining functional effects on the epithelium, we found that LPS-induced tight junctional disruption as assessed by internalization of ZO-1 and occludin was ameliorated in FXR KO animals. Taken together, these data suggest a role for FXR in the intestinal barrier during inflammatory injury.NEW & NOTEWORTHY Intestinal barrier failure is a hallmark in gut-origin sepsis. We demonstrate that the intestinal barriers of farnesoid-X receptor (FXR) knockout (KO) animals are protected from inflammatory insult using multiple models of acute intestinal inflammation. This protection is due to decreased inflammatory cytokine production and maintenance of tight junctional architecture seen within the KO animals. This is the first report of FXR deletion being protective to the intestinal barrier.

Pathogenic differences of cynomolgus macaques after Taï Forest virus infection depend on the viral stock propagation.

Taï Forest virus (TAFV) is a negative-sense RNA virus in the Filoviridae family. TAFV has caused only a single human infection, but several disease outbreaks in chimpanzees have been linked to this virus. Limited research has been done on this human-pathogenic virus. We sought to establish an animal model to assess TAFV disease progression and pathogenicity at our facility. We had access to two different viral stock preparations from different institutions, both originating from the single human case. Type I interferon receptor knockout mice were inoculated with TAFV stock 1 or stock 2 by the intraperitoneal route. Inoculation resulted in 100% survival with no disease regardless of viral stock preparation or infectious dose. Next, cynomolgus macaques were inoculated with TAFV stock 1 or stock 2. Inoculation with TAFV stock 1 resulted in 100% survival and robust TAFV glycoprotein-specific IgG responses including neutralizing antibodies. In contrast, macaques infected with TAFV stock 2 developed disease and were euthanized 8-11 days after infection exhibiting viremia, thrombocytopenia, and increased inflammatory mediators identified by transcriptional analysis. Histopathologic analysis of tissue samples collected at necropsy confirmed classic filovirus disease in numerous organs. Genomic differences in both stock preparations were mapped to several viral genes which may have contributed to disease severity. Taken together, we demonstrate that infection with the two TAFV stocks resulted in no disease in mice and opposing disease phenotypes in cynomolgus macaques, highlighting the impact of viral stock propagation on pathogenicity in animal models.

Short-chain fatty acids mitigate Methamphetamine-induced hepatic injuries in a Sigma-1 receptor-dependent manner

Methamphetamine (Meth) is a potent psychostimulant with well-established hepatotoxicity. Gut microbiota-derived short-chain fatty acids (SCFAs) have been reported to yield beneficial effects on the liver. In this study, we aim to further reveal the mechanisms of Meth-induced hepatic injuries and investigate the potential protective effects of SCFAs. Herein, mice were intraperitoneally injected with 15 mg/kg Meth to induce hepatic injuries. The composition of fecal microbiota and SCFAs was profiled using 16 S rRNA sequencing and Gas Chromatography/Mass Spectrometry (GC/MS) analysis, respectively. Subsequently, SCFAs supplementation was performed to evaluate the protective effects against hepatic injuries. Additionally, Sigma-1 receptor knockout (S1R-/-) mice and fluvoxamine (Flu), an agonist of S1R, were introduced to investigate the mechanisms underlying the protective effects of SCFAs. Our results showed that Meth activated S1R and induced hepatic autophagy, inflammation, and oxidative stress by stimulating the MAPK/ERK pathway. Meanwhile, Meth disrupted SCFAs product-related microbiota, leading to a reduction in fecal SCFAs (especially Acetic acid and Propanoic acid). Accompanied by the optimization of gut microbiota, SCFAs supplementation normalized S1R expression and ameliorated Meth-induced hepatic injuries by repressing the MAPK/ERK pathway. Effectively, S1R knockout repressed Meth-induced activation of the MAPK/ERK pathway and further ameliorated hepatic injuries. Finally, the overexpression of S1R stimulated the MAPK/ERK pathway and yielded comparable adverse phenotypes to Meth administration. These findings suggest that Meth-induced hepatic injuries relied on the activation of S1R, which could be alleviated by SCFAs supplementation. Our study confirms the crucial role of S1R in Meth-induced hepatic injuries for the first time and provides a potential preemptive therapy.

Effect of placental circulating T cells expressing CD16 on multiple hematological and solid tumor cancers through combination with various monoclonal antibodies.

2554 Background: PT-CD16VS is an allogeneic cell therapy derived from human postpartum placental circulating T (PT) cells that are genetically modified to express CD16 and endogenous T cell receptor (TCR) knockout. PT-CD16VS cells can be combined with various monoclonal antibodies to engage in anti-tumor antibody-dependent cellular cytotoxicity (ADCC) against diverse cancers with a “universal receptor” approach. Here we report the characterization and preclinical evaluation of PT-CD16VS in combination with monoclonal antibodies against both hematological and solid tumor cancers. Methods: PT cells were activated, transduced with a lentiviral vector containing a construct expressing a high affinity CD16 variant, CD16VS, and transfected to knock out the TCR. In vitro,functional activity of PT-CD16VS cells in combination with monoclonal antibodies was assessed in cytotoxicity, cytokine release, and proliferation assays. Invivo, PT-CD16VS was combined with Rituximab in a Raji xenograft model in NSG mice, and with Trastuzumab in a subcutaneous NCI-N87 xenograft model in NSG mice in which tumor volume was measured and tumor samples were evaluated for PT-CD16VS infiltration. Results: In vitro, PT-CD16VS exhibited potent ADCC and cytokine release when combined with Rituximab against CD20+ Burkitt's lymphoma (Daudi and Raji), with Trastuzumab against HER2+ gastric carcinoma (NCI-N87), with Trastuzumab or Avelumab against HER2+/PDL-1+ non-small cell lung carcinoma (NCI-H1975) and Trastuzumab-resistant metastatic mammary adenocarcinoma (JIMT-1), and with Cetuximab against EGFR+ triple negative metastatic mammary adenocarcinoma (MDA-MB-231). Moreover, examples of PT-CD16VS antigen-specific proliferation were demonstrated against Raji and NCI-N87 when cells were combined with Rituximab and Trastuzumab, respectively. In vivo, in combination with Rituximab in the Raji mouse model, PT-CD16VS exhibited significant survival benefit compared to vehicle and Rituximab alone treated groups. In the subcutaneous NCI-N87 solid tumor model, PT-CD16VS with Trastuzumab demonstrated significant reduction in tumor volume compared to vehicle, Trastuzumab, and Enhertu groups. In addition, PT-CD16VS infiltration into the tumor was shown to be Trastuzumab dependent, with infiltrating cells expressing high levels of CD16 and Ki67. Conclusions: Our results show that PT-CD16VS have potent in vitro and in vivo ADCC activity, and a single drug product has the potential to be combined with various monoclonal antibodies to target multiple cancers across hematological and solid tumor indications. This “universal receptor” with antibody-dependent targeting approach, together with the benefits of an allogeneic cell platform, may democratize accessibility of such therapies for patients.

Liver-specific glucocorticoid action in alcoholic liver disease: Study of glucocorticoid receptor knockout and knockin mice

Background and aimGlucocorticoids are the only first-line drugs for severe alcoholic hepatitis (AH), with limited efficacy and various side effects on extrahepatic tissues. Liver-targeting glucocorticoid therapy may have multiple advantages over systemic glucocorticoid for AH. The aim of this study was to determine the role of hepatocellular glucocorticoid receptor (GR) in alcohol-associated steatosis (AS) and AH. Materials and methodsAS was induced by a high-fat diet plus binge alcohol in adult male and female mice with liver-specific knockout (LKO) and heterozygote of GR. AH was induced by chronic-plus-binge in middle-aged male mice with liver-specific knockin of GR. Changes in hepatic mRNA and protein expression were determined by quantitative real-time polymerase chain reaction and Western blot. ResultsGR-LKO aggravated steatosis and decreased hepatic expression and circulating levels of albumin in both genders of AS mice but only increased markers of liver injury in male AS mice. Marked steatosis in GR-LKO mice was associated with induction of lipogenic genes and down-regulation of bile acid synthetic genes. Hepatic protein levels of GR, hepatocyte nuclear factor 4 alpha, and phosphorylated signal transducer and activator of transcription 3 were gene-dosage-dependently decreased, whereas that of lipogenic ATP citrate lyase was increased in male GR heterozygote and LKO mice. Interestingly, hepatic expression of estrogen receptor alpha (ERα) was induced, and the essential estrogen-inactivating enzyme sulfotransferase 1e1 was gene-dosage-dependently down-regulated in GR heterozygote and knockout AS mice, which was associated with induction of ERα-target genes. Liver-specific knockin of GR protected against liver injury and steatohepatitis in middle-aged AH mice. ConclusionsHepatic GR deficiency plays a crucial role in the pathogenesis of AS induced by high-fat diet plus binge, and liver-specific overexpression/activation of GR protects against chronic-plus-binge-induced AH in middle-aged mice. Hepatocellular GR is important for protection against AS and AH.

Peripheral cytokine interleukin-10 alleviates perihematomal edema after intracerebral hemorrhage via interleukin-10 receptor/JAK1/STAT3 signaling.

The extent of perihematomal edema following intracerebral hemorrhage (ICH) significantly impacts patient prognosis, and disruption of the blood-brain barrier (BBB) exacerbates perihematomal edema. However, the role of peripheral IL-10 in mitigating BBB disruption through pathways that link peripheral and central nervous system signals remains poorly understood. Recombinant IL-10 was administered to ICH model mice via caudal vein injection, an IL-10-inhibiting adeno-associated virus and an IL-10 receptor knockout plasmid were delivered intraventricularly, and neurobehavioral deficits, perihematomal edema, BBB disruption, and the expression of JAK1 and STAT3 were evaluated. Our study demonstrated that the peripheral cytokine IL-10 mitigated BBB breakdown, perihematomal edema, and neurobehavioral deficits after ICH and that IL-10 deficiency reversed these effects, likely through the IL-10R/JAK1/STAT3 signaling pathway. Peripheral IL-10 has the potential to reduce BBB damage and perihematomal edema following ICH and improve patient prognosis.