SARS-CoV-2 infection activates inflammatory macrophages in vascular immune organoids.

SARS-CoV-2 provokes devastating tissue damage by cytokine release syndrome and leads to multi-organ failure. Modeling the process of immune cell activation and subsequent tissue damage is a significant task. Organoids from human tissues advanced our understanding of SARS-CoV-2 infection mechanisms though, they are missing crucial components: immune cells and endothelial cells. This study aims to generate organoids with these components. We established vascular immune organoids from human pluripotent stem cells and examined the effect of SARS-CoV-2 infection. We demonstrated that infections activated inflammatory macrophages. Notably, the upregulation of interferon signaling supports macrophages' role in cytokine release syndrome. We propose vascular immune organoids are a useful platform to model and discover factors that ameliorate SARS-CoV-2-mediated cytokine release syndrome.

The secreted protein Amuc_1409 from Akkermansia muciniphila improves gut health through intestinal stem cell regulation.

Akkermansia muciniphila has received great attention because of its beneficial roles in gut health by regulating gut immunity, promoting intestinal epithelial development, and improving barrier integrity. However, A. muciniphila-derived functional molecules regulating gut health are not well understood. Microbiome-secreted proteins act as key arbitrators of host-microbiome crosstalk through interactions with host cells in the gut and are important for understanding host-microbiome relationships. Herein, we report the biological function of Amuc_1409, a previously uncharacterised A. muciniphila-secreted protein. Amuc_1409 increased intestinal stem cell (ISC) proliferation and regeneration in ex vivo intestinal organoids and in vivo models of radiation- or chemotherapeutic drug-induced intestinal injury and natural aging with male mice. Mechanistically, Amuc_1409 promoted E-cadherin/ß-catenin complex dissociation via interaction with E-cadherin, resulting in the activation of Wnt/ß-catenin signaling. Our results demonstrate that Amuc_1409 plays a crucial role in intestinal homeostasis by regulating ISC activity in an E-cadherin-dependent manner and is a promising biomolecule for improving and maintaining gut health.

Mucosal TLR5 activation controls healthspan and longevity.

Addressing age-related immunological defects through therapeutic interventions is essential for healthy aging, as the immune system plays a crucial role in controlling infections, malignancies, and in supporting tissue homeostasis and repair. In our study, we show that stimulating toll-like receptor 5 (TLR5) via mucosal delivery of a flagellin-containing fusion protein effectively extends the lifespan and enhances the healthspan of mice of both sexes. This enhancement in healthspan is evidenced by diminished hair loss and ocular lens opacity, increased bone mineral density, improved stem cell activity, delayed thymic involution, heightened cognitive capacity, and the prevention of pulmonary lung fibrosis. Additionally, this fusion protein boosts intestinal mucosal integrity by augmenting the surface expression of TLR5 in a certain subset of dendritic cells and increasing interleukin-22 (IL-22) secretion. In this work, we present observations that underscore the benefits of TLR5-dependent stimulation in the mucosal compartment, suggesting a viable strategy for enhancing longevity and healthspan.

Radiation induces acute and subacute vascular regression in a three-dimensional microvasculature model.

Radiation treatment is one of the most frequently used therapies in patients with cancer, employed in approximately half of all patients. However, the use of radiation therapy is limited by acute or chronic adverse effects and the failure to consider the tumor microenvironment. Blood vessels substantially contribute to radiation responses in both normal and tumor tissues. The present study employed a three-dimensional (3D) microvasculature-on-a-chip that mimics physiological blood vessels to determine the effect of radiation on blood vessels. This model represents radiation-induced pathophysiological effects on blood vessels in terms of cellular damage and structural and functional changes. DNA double-strand breaks (DSBs), apoptosis, and cell viability indicate cellular damage. Radiation-induced damage leads to a reduction in vascular structures, such as vascular area, branch length, branch number, junction number, and branch diameter; this phenomenon occurs in the mature vascular network and during neovascularization. Additionally, vasculature regression was demonstrated by staining the basement membrane and microfilaments. Radiation exposure could increase the blockage and permeability of the vascular network, indicating that radiation alters the function of blood vessels. Radiation suppressed blood vessel recovery and induced a loss of angiogenic ability, resulting in a network of irradiated vessels that failed to recover, deteriorating gradually. These findings demonstrate that this model is valuable for assessing radiation-induced vascular dysfunction and acute and chronic effects and can potentially improve radiotherapy efficiency.

The Effects of Manual Acupuncture on Mitochondrial Fusion and Fission Gene Expression in Rat Spleen.

Background: A significant amount of research has been conducted to establish the validity of acupuncture, and it has been demonstrated through animal disease model studies that acupuncture influences mitochondrial changes. However, to more accurately examine the mechanisms of acupuncture treatment effectiveness in pathological models, it is crucial to investigate changes in disease-free animals. Among various hypotheses regarding the effects of acupuncture on the body, we focused on the result that acupuncture stimulation is related to mitochondria. Objectives: We examined the effects of acupuncture mitochondrial fission and fusionrelated mediators in disease-free Sprague Dawley (SD) rats' spleen meridian acupoints. Methods: SD rats were divided into control, SP1, SP2, SP3, SP5, and SP9 acupuncture groups. Acupuncture was performed at each point for 10 minutes daily for four days. Peroxisome proliferator-activated receptor-gamma coactivator 1-α (PGC-1α) and fission protein 1 (Fis1) levels were evaluated using quantitative real-time polymerase chain reaction (qRT-PCR), while dynamin-related protein 1 (DRP1), optic atrophy-1 (OPA1), mitofusin-1 (MFN1), and mitofusin-2 (MFN2) levels were assessed via western blotting. Mitochondria protein concentrations and NADH dehydrogenase activity in spleen tissues were measured using enzyme-linked immunosorbent assay (ELISA). Results: PGC-1α expression decreased in the SP1 (p < 0.01), SP5 (p < 0.05), and SP9 (p < 0.05) groups, while Fis1 expression increased in the SP1 (p < 0.01), SP5 (p < 0.01), and SP9 (p < 0.05) groups. DRP1, OPA1, MFN1, and MFN2 levels exhibited no significant changes. Mitochondrial protein concentrations decreased in the SP2 (p < 0.01), SP3 (p < 0.01), SP5 (p < 0.01), and SP9 (p < 0.01) groups, while NADH dehydrogenase activity decreased in the SP2 (p < 0.05) and SP9 (p < 0.05) groups. Conclusion: Acupuncture at the SP9 acupoint influenced the mitochondrial fission pathway by modulating PGC-1α and Fis1 mediators in the rat spleen under non-disease conditions.

Modeling of three-dimensional innervated epidermal like-layer in a microfluidic chip-based coculture system.

Reconstruction of skin equivalents with physiologically relevant cellular and matrix architecture is indispensable for basic research and industrial applications. As skin-nerve crosstalk is increasingly recognized as a major element of skin physiological pathology, the development of reliable in vitro models to evaluate the selective communication between epidermal keratinocytes and sensory neurons is being demanded. In this study, we present a three-dimensional innervated epidermal keratinocyte layer as a sensory neuron-epidermal keratinocyte co-culture model on a microfluidic chip using the slope-based air-liquid interfacing culture and spatial compartmentalization. Our co-culture model recapitulates a more organized basal-suprabasal stratification, enhanced barrier function, and physiologically relevant anatomical innervation and demonstrated the feasibility of in situ imaging and functional analysis in a cell-type-specific manner, thereby improving the structural and functional limitations of previous coculture models. This system has the potential as an improved surrogate model and platform for biomedical and pharmaceutical research.

Analyzing angiogenesis on a chip using deep learning-based image processing.

Angiogenesis, the formation of new blood vessels from existing vessels, has been associated with more than 70 diseases. Although numerous studies have established angiogenesis models, only a few indicators can be used to analyze angiogenic structures. In the present study, we developed an image-processing pipeline based on deep learning to analyze and quantify angiogenesis. We utilized several image-processing algorithms to quantify angiogenesis, including a deep learning-based cell nuclear segmentation algorithm and image skeletonization. This method could quantify and measure changes in blood vessels in response to biochemical gradients using 16 indicators, including length, width, number, and nuclear distribution. Moreover, this procedure is highly efficient for the three-dimensional quantitative analysis of angiogenesis and can be applied to diverse angiogenesis investigations.

Hepatocyte DAX1 Deletion Exacerbates Inflammatory Liver Injury by Inducing the Recruitment of CD4+ and CD8+ T Cells through NF-κB p65 Signaling Pathway in Mice.

Fulminant hepatitis is characterized by rapid and massive immune-mediated liver injury. Dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX1; NR0B1) represses the transcription of various genes. Here, we determine whether DAX1 serves as a regulator of inflammatory liver injury induced by concanavalin A (ConA). C57BL/6J (WT), myeloid cell-specific Dax1 knockout (MKO), and hepatocyte-specific Dax1 knockout (LKO) mice received single intravenous administration of ConA. Histopathological changes in liver and plasma alanine aminotransferase and aspartate aminotransferase levels in Dax1 MKO mice were comparable with those in WT mice following ConA administration. Unlike Dax1 MKO mice, Dax1 LKO mice were greatly susceptible to ConA-induced liver injury, which was accompanied by enhanced infiltration of immune cells, particularly CD4+ and CD8+ T cells, in the liver. Factors related to T-cell recruitment, including chemokines and adhesion molecules, significantly increased following enhanced and prolonged phosphorylation of NF-κB p65 in the liver of ConA-administered Dax1 LKO mice. This is the first study to demonstrate that hepatocyte-specific DAX1 deficiency exacerbates inflammatory liver injury via NF-κB p65 activation, thereby causing T-cell infiltration by modulating inflammatory chemokines and adhesion molecules. Our results suggest DAX1 as a therapeutic target for fulminant hepatitis treatment.

Hepatocyte-Specific Deficiency of DAX-1 Protects Mice from Acetaminophen-Induced Hepatotoxicity by Activating NRF2 Signaling.

Acetaminophen (APAP) is a widely used analgesic and antipyretic drug, but its overdose can cause acute liver failure. The dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome, gene 1 (DAX-1, NR0B1), is an orphan nuclear receptor that acts as a transcriptional co-repressor of various genes. In this study, we identified the role of DAX-1 in APAP-induced liver injury using hepatocyte-specific Dax-1 knockout (Dax-1 LKO) mice. Mouse primary hepatocytes were used as a comparative in vitro study. APAP overdose led to decreased plasma alanine aminotransferase and aspartate aminotransferase levels in Dax-1 LKO mice compared to C57BL/6J (WT) controls, accompanied by reduced liver necrosis. The expression of the genes encoding the enzymes catalyzing glutathione (GSH) synthesis and metabolism and antioxidant enzymes was increased in the livers of APAP-treated Dax-1 LKO mice. The rapid recovery of GSH levels in the mitochondrial fraction of APAP-treated Dax-1 LKO mice led to reduced reactive oxygen species levels, resulting in the inhibition of the prolonged JNK activation. The hepatocyte-specific DAX-1 deficiency increased the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2) compared with WT controls after APAP administration. These results indicate that DAX-1 deficiency in hepatocytes protects against APAP-induced liver injury by Nrf2-regulated antioxidant defense.

The Role of DNA Methylation in Stroke Recovery.

Epigenetic alterations affect the onset of ischemic stroke, brain injury after stroke, and mechanisms of poststroke recovery. In particular, DNA methylation can be dynamically altered by maintaining normal brain function or inducing abnormal brain damage. DNA methylation is regulated by DNA methyltransferase (DNMT), which promotes methylation, DNA demethylase, which removes methyl groups, and methyl-cytosine-phosphate-guanine-binding domain (MBD) protein, which binds methylated DNA and inhibits gene expression. Investigating the effects of modulating DNMT, TET, and MBD protein expression on neuronal cell death and neurorepair in ischemic stroke and elucidating the underlying mechanisms can facilitate the formulation of therapeutic strategies for neuroprotection and promotion of neuronal recovery after stroke. In this review, we summarize the role of DNA methylation in neuroprotection and neuronal recovery after stroke according to the current knowledge regarding the effects of DNA methylation on excitotoxicity, oxidative stress, apoptosis, neuroinflammation, and recovery after ischemic stroke. This review of the literature regarding the role of DNA methylation in neuroprotection and functional recovery after stroke may contribute to the development and application of novel therapeutic strategies for stroke.

Rodent Models of Post-Stroke Dementia.

Post-stroke cognitive impairment is one of the most common complications in stroke survivors. Concomitant vascular risk factors, including aging, diabetes mellitus, hypertension, dyslipidemia, or underlying pathologic conditions, such as chronic cerebral hypoperfusion, white matter hyperintensities, or Alzheimer's disease pathology, can predispose patients to develop post-stroke dementia (PSD). Given the various clinical conditions associated with PSD, a single animal model for PSD is not possible. Animal models of PSD that consider these diverse clinical situations have not been well-studied. In this literature review, diverse rodent models that simulate the various clinical conditions of PSD have been evaluated. Heterogeneous rodent models of PSD are classified into the following categories: surgical technique, special structure, and comorbid condition. The characteristics of individual models and their clinical significance are discussed in detail. Diverse rodent models mimicking the specific pathomechanisms of PSD could provide effective animal platforms for future studies investigating the characteristics and pathophysiology of PSD.

Changes in Mitochondria-Related Gene Expression upon Acupuncture at LR3 in the D-Galactosamine-Induced Liver Damage Rat Model.

Hepatic diseases, such as hepatonecrosis, hepatitis, and hepatocirrhosis, are associated with mitochondrial dysfunction and increased reactive oxygen species generation and inflammation, ultimately leading to liver failure. In this study, we examined if acupuncture at LR3 can affect mitochondria-related gene expression in a liver damage model of experimentally induced acute liver failure (ALF). ALF was induced by the intraperitoneal injection of D-galactosamine (D-GalN) in experimental rats, who then received either sham (ALF), manual acupuncture (MA), electroacupuncture (EA), or silymarin (PC, positive control) treatment. Liver tissues were extracted from experimental and untreated control rats for histopathological analysis and expression profiling of genes involved in mitochondrial function. Of the 168 mitochondria-related genes profiled, two genes belonging to the solute-carrier transporter family (Slc25a15 and Slc25a25) and Ndufb7 were upregulated. Gamma-glutamylcysteine synthetase was more downregulated in MA than ALF. Furthermore, MA reversed D-GalN-induced inflammatory cell infiltration, destruction of hepatic cell plates, and increase in the levels of the proinflammatory cytokine TNF-α. MA at LR3 can reduce the risk of D-GalN-induced ALF by inducing the expression of metabolic and inflammation-related genes and regulating proinflammatory factor production in hepatic mitochondria.

Effects of Elevated Progesterone Levels on the Day of hCG on the Quality of Oocyte and Embryo.

This study is designed to investigate the effects of increased progesterone (P4) levels on the quality of retrieved oocytes and embryos during IVF. This retrospective analysis included 982 all-freezing in vitro fertilization (IVF) cycles (conducted between November 2019 and June 2020 at CHA Fertility Center Bundang, South Korea) in which serum P4 levels were measured on the day of human chorionic gonadotropin (hCG) administration. Our study revealed that the serum P4 levels on the day of hCG administration are strongly associated with the rates of oocyte maturation, displaying a positive correlation in patients with serum P4 < 2.25 ng/mL (p = 0.025). Moreover, patients with serum P4 < 1.25 ng/mL showed relatively low fertilization rates (p = 0.037), and the rates of good embryo retrieval were significantly increased with the serum P4 level < 1.5 ng/mL (p = 0.001). Interestingly, serum P4 level on the day of hCG administration affects the rate of good-quality embryo development, especially at the cleavage stage, and is associated with the status of ovarian responses. Our current study suggests that serum P4 level on the day of hCG administration negatively affects the rates of oocyte maturation, fertilization, and the development of good embryos.

Recapitulated Crosstalk between Cerebral Metastatic Lung Cancer Cells and Brain Perivascular Tumor Microenvironment in a Microfluidic Co-Culture Chip.

Non-small cell lung carcinoma (NSCLC), which affects the brain, is fatal and resistant to anti-cancer therapies. Despite innate, distinct characteristics of the brain from other organs, the underlying delicate crosstalk between brain metastatic NSCLC (BM-NSCLC) cells and brain tumor microenvironment (bTME) associated with tumor evolution remains elusive. Here, a novel 3D microfluidic tri-culture platform is proposed for recapitulating positive feedback from BM-NSCLC and astrocytes and brain-specific endothelial cells, two major players in bTME. Advanced imaging and quantitative functional assessment of the 3D tri-culture model enable real-time live imaging of cell viability and separate analyses of genomic/molecular/secretome from each subset. Susceptibility of multiple patient-derived BM-NSCLCs to representative targeted agents is altered and secretion of serpin E1, interleukin-8, and secreted phosphoprotein 1, which are associated with tumor aggressiveness and poor clinical outcome, is increased in tri-culture. Notably, multiple signaling pathways involved in inflammatory responses, nuclear factor kappa-light-chain-enhancer of activated B cells, and cancer metastasis are activated in BM-NSCLC through interaction with two bTME cell types. This novel platform offers a tool to elucidate potential molecular targets and for effective anti-cancer therapy targeting the crosstalk between metastatic cancer cells and adjacent components of bTME.

Correlation of Hemispatial Neglect with White Matter Tract Integrity: A DTI Study.

We investigated the diffusion tensor image (DTI) parameters of superior longitudinal fasciculus (SLF) and inferior fronto-occipital fasciculus (IFOF), and their relationships with hemispatial neglect. Thirteen patients with first-ever ischemic stroke who had the right hemispheric lesion were included. Neglect was assessed using the Albert test and figure discrimination test of Motor-free Visual Perception Test 3 (MVPT-3). The SLF and IFOF were separated by diffusion tensor tractography (DTT) and tract volume (TV) was calculated. We measured the fractional anisotropy (FA) and apparent diffusion coefficient (ADC) values in the total area, seed region of interest (ROI), and target ROI, respectively. Among thirteen patients, seven demonstrated signs of hemispatial neglect on neglect test. Tractography reconstruction showed significantly low TV of the right IFOF in patients with hemispatial neglect. FA values of the right SLF and the right IFOF were significantly lower in neglect patients. ADC values were not significantly different in two groups. This study suggests that damage of SLF and IFOF is associated with hemispatial neglect in right hemispheric stroke patients. DTI may be useful for predicting the severities of hemispatial neglect using values such as TV and FA of each tract.

Gamma irradiation exposure for collapsed cell junctions and reduced angiogenesis of 3-D in vitro blood vessels.

During radiotherapy, microenvironments neighboring the tumor are also exposed to gamma irradiation; this results in unexpected side effects. Blood vessels can serve as microenvironments for tumors and they play an important role in providing nutrients to tumors. This is mostly related to tumor progression, metastasis, and relapse after therapy. Many studies have been performed to obtain a better understanding of tumor vasculature after radiotherapy with in vitro models. However, compared to 3-D models, 2-D in vitro endothelial monolayers cannot physiologically reflect in vivo blood vessels. We previously remodeled the extracellular matrix (ECM) hydrogel that enhanced the tight barrier formation of 3-D blood vessels and the vascular endothelial growth factor (VEGF) gradient induced angiogenesis in a microfluidic device. In this study, the blood vessel model is further introduced to understand how gamma irradiation affects the endothelial monolayer. After the gamma irradiation exposure, we observed a collapsed endothelial barrier and a reduced angiogenic potential. Changes in the cell behaviors of the tip and stalk cells were also detected in the angiogenesis model after irradiation, which is difficult to observe in 2-D monolayer models. Therefore, the 3-D in vitro blood vessel model can be used to understand radiation-induced endothelial injuries.

Drug screening by uniform patient derived colorectal cancer hydro-organoids.

As the importance of organoids increases, the need to develop organoid culture systems suitable for basic biological and clinical applications is being emphasized. However, there is still an unmet need to produce functionally complex and scalable uniform organoids. Here, we demonstrate a scalable organoid production platform with 8 well strips and a total of 8 × 9 microwells per strip using organoids derived from colorectal cancer tissue. The new culture platform is a format in which single cells are self-organized into organoids in culture medium supplemented with 2% Matrigel. It is functionally compatible with existing 96 well plates and Matrigel based conventional organoid culture methods. The consistency, uniformity and reproducibility of organoid produced on the new platform have been significantly improved compared to those of conventional plates. Importantly, Hydro-organoids are functionally identical to conventional Matrigel organoids, but show better consistency in drug screening. Our results show the possibility that Hydro-organoids can be used in high-throughput assays and incorporated into drug screening models to predict clinical outcomes.

VISTA is an activating receptor in human monocytes.

As indicated by its name, V-domain Ig suppressor of T cell activation (VISTA) is thought to serve primarily as an inhibitory protein that limits immune responses. VISTA antibodies can dampen the effects of several concomitantly elicited activation signals, including TCR and TLR activation, but it is currently unclear if VISTA agonism could singly affect immune cell biology. In this study, we discovered two novel VISTA antibodies and characterized their effects on human peripheral blood mononuclear cells by scRNA/CITE-seq. Both antibodies appeared to agonize VISTA in an Fc-functional manner to elicit transcriptional and functional changes in monocytes consistent with activation. We also used pentameric VISTA to identify Syndecan-2 and several heparan sulfate proteoglycan synthesis genes as novel regulators of VISTA interactions with monocytic cells, adding further evidence of bidirectional signaling. Together, our study highlights several novel aspects of VISTA biology that have yet to be uncovered in myeloid cells and serves as a foundation for future research.

Humulus japonicus rescues autistic­like behaviours in the BTBR T+ Itpr3tf/J mouse model of autism.

Humulus japonicus (HJ) is a traditional herbal medicine that exhibits anti­inflammatory, antimicrobial and anti­tumor effects that is used for the treatment of hypertension, pulmonary disease and leprosy. Recently, it has also been reported that HJ demonstrates neuroprotective properties in animal models of neurodegenerative diseases. The current study hypothesised that the administration of HJ would exhibit therapeutic effects in autism spectrum disorder (ASD), a neurodevelopmental disorder with lifelong consequences. The BTBR T+ Itpr3tf/J mouse model of ASD was used to investigate the anti­autistic like behavioural effects of HJ. Chronic oral administration of the ethanolic extract of HJ significantly increased social interaction, attenuated repetitive grooming behaviour and improved novel­object recognition in BTBR mice. Anti­inflammatory effects of HJ in the brain were analysed using immunohistochemistry and reverse­transcription quantitative PCR analysis. Microglia activation was markedly decreased in the striatum and hippocampus, and pro­inflammatory cytokines, including C­C Motif Chemokine Ligand 2, interleukin (IL)­1ß and IL­6, were significantly reduced in the hippocampus following HJ treatment. Moreover, HJ treatment normalised the phosphorylation levels of: N­methyl­D­aspartate receptor subtype 2B and calcium/calmodulin­dependent protein kinase type II subunit α in the hippocampus of BTBR mice. The results of the present study demonstrated that the administration of HJ may have beneficial potential for ameliorating behavioural deficits and neuroinflammation in ASD.

Herbal Medicine and Acupuncture Combined Treatment Attenuates Colitis in Rats.

This study aimed to verify the efficacy of a combined treatment of Jakyakgamcho-tang (JGT) and acupuncture (CV12, ST25, CV4) on colitis induced by dextrane sulfate sodium (DSS). Changes in immuno-mediated factors and metabolites were investigated. Colitis symptoms such as body weight loss and elevated disease activity index were alleviated by the combined treatment. Moreover, treatment with JGT and acupuncture restored the disturbed architecture of colon by suppressing inflammatory cytokine levels of IFN-[Formula: see text] ([Formula: see text] < 0.05), IL-5 ([Formula: see text] < 0.05), and IL-13 ([Formula: see text] < 0.0001) compared with the DSS group. Analysis of metabolic profiles of serum revealed that treatment groups were clearly separated from the DSS group, suggesting that JGT and acupuncture treatment altered serum metabolites. Furthermore, treatments caused opposite metabolite patterns for dimethylbenzimidazole, 1,5-anhydro-D-glucitol, proline, phosphate, glycolic acid, aspartic acid, tryptophan, phthalic acid, ornithine, and glutamic acid compared with the DSS group. The combined treatment group induced more effective metabolite patterns than the JGT group, implying that acupuncture treatment can restore metabolic changes caused by DSS induction. These results indicate that the simultaneous treatment of JGT administration and acupuncture procedure provides better management of the immune function and inflammatory expression of colitis than a single treatment. It is assumed that intestinal microbial control can be achieved by acupuncture stimulation as well as by taking herbal medicine.