IACUC Research Articles

Ficolin A Derived from Local Macrophages and Neutrophils Protects Against LPS-Induced Acute Lung Injury by Activating Complement

Background: Ficolins are important and widely distributed secreted pattern recognition molecules that can induce lectin complement pathway activation and initiate the innate immune response. Although ficolins can bind lipopolysaccharide (LPS) in vitro, the sources, dynamic changes and roles of ficolins in LPS-induced local pulmonary inflammation and injury remain poorly understood. Methods: C57BL/6 and Ficolins-KO mice were intranasally administrated 10 μg LPS to induce acute lung inflammation and injury. Flow cytometry and immunofluorescence were performed to reveal the expression of ficolins. Lung injury was analyzed by hematoxylin & eosin staining. Complement activation was confirmed by immunohistochemistry and sandwich ELISA. Findings: In this study, we found that ficolin A and ficolin B are expressed in leukocytes from the bone marrow, peripheral blood, lung and spleen. Further analyses showed that macrophages and neutrophils are the main sources of ficolin A and ficolin B, and T and B cells also express a small amount of ficolin B. The intranasal administration of LPS induced local pulmonary inflammation with the increased recruitment of macrophages and neutrophils. LPS stimulation induced increased expression of ficolin A and ficolin B in neutrophils at the acute stage, and in macrophages at the late stage. The severity of the lung injury and local inflammation of Fcna−/− mice was increased via the induction of extracellular complement activation. The recovery of LPS-induced local lung inflammation and injury was delayed in Fcnb−/− mice. Interpretation: The local macrophage- and neutrophil-derived ficolin A protects against LPS-induced acute lung injury by mediating extracellular complement activation. These findings suggested that ficolins might be novel therapeutic targets for gram-negative bacteria infection induced pneumonia. Funding Statement: This work was supported by National Science Grant for Distinguished Young Scholars (81425001/H0104), National Natural Science Foundation of China, China (81373114), Beijing Municipal Natural Science Foundation, China (7182013), CAMS Innovation Fund for Medical Sciences, China (CIFMS 2018-I2M-1-003, CIFMS 2016-2M-1-014, 2016-12M-006), the Chinese National Major S & T Project (2017ZX10304402-001). Declaration of Interests: All authors declare no conflicts of interest associated with this manuscript. Ethics Approval Statement: All experiments were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) of Capital Medical University.

A Natural Product Exhibits Potent Anti-Botulism Efficacy by Accelerating Degradation of Intracellular Toxins

Background: Botulinum neurotoxins (BoNTs) are among the deadliest toxins known to humans. To date, there are no approved drugs to treat botulism after intoxication of the motor neuron. Intensive efforts have been made to develop small-molecule inhibitors, however, few possess noteworthy in vivo efficacy. Methods: The primary rat cerebellar granule neuron (CGN)-based screening was performed to discover novel effective inhibitors against BoNT/A in neural cells. The anti-botulism mechanism of embelin was preliminary studied in the impact of embelin on the main steps of BoNTs intoxication and intracellular duration. The anti-botulism efficacy of natural product embelin on botulinum toxin type A and type B was assessed in BALB/c mouse. Findings: We discovered a promising anti-BoNTs natural poduct embelin, which could protect 80% and 60% of the mice against lethal dose of BoNT/A or BoNT/B at 1 h after toxin exposure, respectively. Moreover, at 8 h after toxin exposure, embelin still protect 30% and 35% of the mice against lethal dose of BoNT/A or BoNT/B. In addition, mechanism analysis proved that embelin could significantly speed up the clearance of BoNTs in neurons. Interpretation: We discovered a promising anti-BoNTs natural product embelin, which could protect mice against lethal dose of BoNTs. The novel botulism antidote will be useful for reducing the morbidity of BoNTs intoxication and have potential biodefense implications. Funding Statement: We acknowledge this work was supported by National Key R&D Program of China (2017YFC1200905), National Key Basic Research Program (973) of China (2015CB554202), and the Beijing Nova Program (Z181100006218111) for funding. Declaration of Interests: The authors declare no competing financial interest. Ethics Approval Statement: The Beijing Institute of Microbiology and Epidemiology Animal Care and Use Committee approved our study protocol. The animal care and use protocol adhered to the regulations of the Institutional Animal Care and Use Committee (IACUC).

Beta-nerve growth factor gene therapy alleviates pyridoxine-induced neuropathic damage by increasing doublecortin and tyrosine kinase A in the dorsal root ganglion.

Beta-nerve growth factor (β-NGF) is known to be a major leading cause of neuronal plasticity. To identify the possible action mechanisms of β-NGF gene therapy for sciatic nerve recovery, experimental dogs were randomly divided into control, pyridoxine, and pyridoxine + β-NGF groups. We observed chronological changes of morphology in the dorsal root ganglia in response to pyridoxine toxicity based on cresyl violet staining. The number of large neurons positive for cresyl violet was dramatically decreased after pyridoxine intoxication for 7 days in the dorsal root ganglia and the neuron number was gradually increased after pyridoxine withdrawal. In addition, we also investigated the effects of β-NGF gene therapy on neuronal plasticity in pyridoxine-induced neuropathic dogs. To accomplish this, tyrosine kinase receptor A (TrkA), βIII-tubulin and doublecortin (DCX) immunohistochemical staining was performed at 3 days after the last pyridoxine treatment. TrkA-immunoreactive neurons were dramatically decreased in the pyridoxine group compared to the control group, but strong TrkA immunoreactivity was observed in the small-sized dorsal root ganglia in this group. TrkA immunoreactivity in the dorsal root ganglia was similar between β-NGF and control groups. The numbers of βIII-tubulin- and DCX-immunoreactive cells decreased significantly in the pyridoxine group compared to the control group. However, the reduction of βIII-tubulin- and DCX-immunoreactive cells in the dorsal root ganglia in the β-NGF group was significantly ameliorated than that in the pyridoxine group. These results indicate that β-NGF gene therapy is a powerful treatment of pyridoxine-induced neuropathic damage by increasing the TrkA and DCX levels in the dorsal root ganglia. The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University, South Korea (approval No. SNU-060623-1, SNU-091009-1) on June 23, 2006 and October 9, 2009, respectively.

Everything You Need to Know About Satisfying IACUC Protocol Requirements.

There have been recent efforts to reduce the administrative burden imposed on investigators. Although a complete and thorough review of proposed animal studies is an essential function of the Institutional Animal Care and Use Committee (IACUC), efforts to streamline and clarify this process may help investigators spend less time writing animal use protocols and responding to committee comments. The IACUC relies on well-written protocols for an efficient review process. A well-designed protocol form is also critical in guiding investigators through the process. However, it is ultimately the investigators' responsibility to ensure that the information they provide answers all the IACUC's questions with enough detail and quality for a fast and effective review. This article, aimed primarily for researchers but also IACUC administrators, provides an overview of the IACUC protocol review and approval process, the criteria that the IACUC uses for evaluations, and the type of information that should be included in the various sections of the protocol form. Some specific examples are also provided.

Fatty-Acid Receptor CD36 Functions As a Hydrogen Sulfide-Targeting Receptor with Its Cys333-Cys272 Disulfide Bond Serving As a Specific Molecular Switch to Accelerate Gastric Cancer Metastasis

Background: There is a large body of evidence suggesting that hydrogen sulfide (H2S) plays an important role in cancer metastasis; however, the molecular mechanisms of H2S-mediated acceleration of cancer metastasis remain unknown. Methods: We used RNA-sequencing to identify potential differences in gene expression in vitro. Using mass spectrometry, we identified a disulfide complex within CD36 that was labile to H2S-mediated modification. Targeting CD36 in vivo blocked H2S-promoted gastric cancer (GC) metastasis and improved animal survival. Tumor patients' samples were used for clinical translational significance evaluation. Findings: H2S triggered lipid metabolism reprogramming by significantly up-regulating the expression of the fatty-acid receptor CD36 and directly activating CD36 in GC cells. Mechanistically, a disulfide bond located between cysteine (Cys)333 and Cys272 within the CD36 protein structure that was labile to H2S-mediated modification. The long chain-fatty acid (LC-FA) binding pocket was capped by a turn in the CD36 protein, located between helical and sheet structures that were stabilized by the Cys333-Cys272. This limited the secondary binding between LC-FAs and lysine (Lys)334. Breaking the Cys333-Cys272 disulfide bond restored the second LC-FA binding conformation of CD36. Interpretation: CD36 gene transcription and CD36 subcellular recycling formed a positive feedback loop, and this disulfide bond within CD36 acted as a special molecular for this positive feedback. H2S opened the switch and induced LC-FA-mediated signaling, thus accelerating the metastasis of GC. Genetic and pharmacological blockade of CD36 function could be harnessed as an anti-metastasis therapeutic strategy. Therefore, we propose an effective strategy for anti-metastasis by blocking CD36. Funding: This work was supported by the Strategic Priority Research Program of the Science and Technology Commission of Shanghai (1312JC1408402 for Zhirong Wang). Declaration of Interest: All authors have no potential conflicts of interest. Ethical Approval: This animal study was conducted in accordance with the rules and regulations of the Institutional Animal Care and Use Committee (IACUC) at the Department of Laboratory Animal Science, Fudan University (Shanghai, P.R. China). Informed consent was approved by the Ethics Committees of Tongji Hospital and all subjects gave written informed consent.

Targeting the COX1/2-Driven Thromboxane A2 Pathway Suppresses Barrett's Esophagus and Esophageal Adenocarcinoma Development

Background: Barrett's esophagus (BE), a complication of gastroesophageal reflux disease (GERD), predisposes patients to esophageal adenocarcinoma (EAC). Reliable biomarkers for early detection and discovery of potential drug targets are urgently needed for improved BE and EAC patient outcomes. Methods: Patient biopsy samples were evaluated for COX1, COX2, and thromboxane A2 synthase (TBXAS) expression. Circulating prostaglandins biosynthesis was determined using enzyme immunoassay kits. Anchorage-independent cell growth assay, crystal violet staining assay, and xenograft experiments were conducted to assess BE and EAC cell growth. A surgical mouse model of reflux (i.e., esophagoduodenostomy) was established and samples were analyzed using an enzyme immunoassay kit, immunohistochemistry, immunoblotting, or RT-PCR. Esophageal biopsy samples (pre- and post-intervention) were obtained from a randomized clinical trial in which participants were administered esomeprazole (40 mg) twice daily in combination with an aspirin placebo or 81 or 325 mg aspirin for 28 days. Esophageal biopsy specimens before and after the intervention period were analyzed. Findings: COX2 and TBXAS are highly expressed in BE and EAC patients accompanied by a pronounced elevation of circulating TXA2 levels. Aspirin suppressed BE and EAC growth by targeting the TXA2 pathway. Additionally, biopsies from 49 patients (with similar baseline characteristics) showed that aspirin substantially decreased serum TXA2 levels, resulting in reduced inflammation. Interpretation: This study establishes the importance of the COX1/2-driven TXA2 pathway in BE and EAC pathophysiology and lays the groundwork for introducing a TXA2-targeting strategy for EAC prevention and early detection. Funding Statement: This work was supported by the Hormel Foundation and National Institutes of Health grants CA166011, CA187027, and CA196639 (Z. Dong). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: All animal studies were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC).

Maintaining balance: what (dis)qualifies a nonaffiliated IACUC candidate?

Maintaining balance: what (dis)qualifies a nonaffiliated IACUC candidate?

An investigation of inter-shock timing and electrode placement for double-sequential defibrillation

BackgroundDouble-Sequential Defibrillation (DSD) is the near-simultaneous use of two defibrillators to treat refractory VF. We hypothesized that (1) risk of DSD-associated defibrillator damage depends on shock vector and (2) the efficacy of DSD depends on inter-shock time. MethodsPart 1: risk of defibrillator damage was assessed in three anaesthetized pigs by applying two sets of defibrillation electrodes in six different configurations (near-orthogonal or near-parallel vectors). Ten 360J shocks were delivered from one set of pads and peak voltage was measured across the second set. Part 2: the dependence of DSD efficacy on inter-shock time was assessed in ten anaesthetized pigs. Electrodes were applied in lateral-lateral (LL) and anterior-posterior positions. Control (LL Stacked Shocks; one vector, two shocks ∼10 s apart) and DSD therapies (Overlapping, 10 ms, 50 ms, 100 ms, 200 ms, 500 ms, 1000 ms apart) were tested in a block randomized design treating electrically-induced VF (n = ∼89 VF episodes/therapy). Shock energies were selected to achieve 25% shock success for a single LL shock. ResultsPart 1: peak voltage delivered was 1833 ± 48 V (mean ± 95%CI). Peak voltage exposure was, on average, 10-fold higher for parallel than orthogonal vectors (p < 0.0001). Part 2: DSD efficacy compared to Stacked LL shocks was higher for Overlapping, 10 ms, and 100 ms (p < 0.05); lower at 50 ms (p < 0.05); and not different at 200 ms or longer inter-shock times. ConclusionRisk of DSD-associated defibrillator damage can be mitigated by using near-orthogonal shock vectors. DSD efficacy is highly dependent on the inter-shock time and can be better, worse, or no different than stacked shocks from a single vector. Institutional Protocol NumberUniversity of Alabama at Birmingham Institutional Animal Care and Use Committee (IACUC) Protocol Number 06860.

DDAH-2 Alleviates Contrast Medium Iopromide-Induced Acute Kidney Injury Through Nitric Oxide Synthase

Background: Contrast-induced acute kidney injury (CI-AKI) is one of the most common causes of hospital-acquired acute renal failure. However, the pathogenesis of CI-AKI remains unclear. Asymmetric dimethylarginine (ADMA) is an endogenous nitric oxide synthase (NOS) inhibitor that is largely metabolized by dimethylarginine dimethylaminohydroxylase (DDAH) in humans. Two isoforms of DDAH exist, namely, DDAH-1 and DDAH-2. In this study, we examined whether the DDAH-2/ADMA/NOS pathway is involved in the pathogenesis of CI-AKI. Methods and Findings: Exposure to the contrast medium iopromide led to increases of creatinine and blood urea nitrogen levels, accumulation of ADMA, increase of reactive oxygen species (ROS) generation, and an inflammatory response in mice kidney tissue. The injection of adenovirus-harbouring DDAH-2 lowered renal ADMA levels and had a reno-protective effect against contrast-medium injury by decreasing cell apoptosis and fibrosis. By contrast, contrast-medium-induced renal injury was exacerbated in heterozygous DDAH-2 knockout mice. In the in vitro study, overexpression of DDAH-2 increased the levels of nitrite and intracellular cGMP, while the DDAH-2 knockdown induced the opposite effect. These findings were also observed in the in vivo sample. Interpretation: Our findings provide the first evidence that the DDAH-2/ADMA/NOS pathway might be involved in the pathogenesis of CI-AKI and that the protective effect of DDAH-2 probably is due to the modulation of NOS activity, oxidative stress, and the inflammatory process. Funding Statement: This work was supported by grants from the Ministry of Science and Technology of Taiwan [MOST-103-2314-B-075-046, 106-2314-B-075-044, 107-2314-B-075-060] and Veterans General Hospitals and University System of Taiwan Joint Research Program [VGHUST102-G7-5, 104-G7-5; 105-G7-3]. Declaration of Interests: The authors state: None. Ethics Approval Statement: All animal experiments were performed in accordance with the approved guidelines by the Institutional Animal Care and Use Committee (IACUC) of Taipei Veterans General Hospital (#2016-124).

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development.

SummaryThe neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1–900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC.

Institutional protocols for the oral administration (gavage) of chemicals and microscopic microbial communities to mice: Analytical consensus.

Institutional protocols designed for the oral administration of live microbial communities, either complex or microscopic (microcosmic), to mice do not exist. However, this approach is increasingly employed by investigators focusing on the gut microbiome in experimental research. Herein, we propose two analytically Kappa-based consensus protocols to promote reproducibility and standardization in research practices and describe biologically relevant factors in achieving optimal microbial engraftment of communities in germ-free mice.

Humane endpoint in mice by Brazilian researchers in the vaccine sector

ABSTRACT The application of a humane endpoint (HE) for mice in vaccine trials and further challenging tasks with lethal samples is necessary to reduce or prevent pain and suffering in these animals, and is a refinement of the 3R policy enforced for animal testing in both national and international scenarios. In order to investigate the application of HE in Brazil, researchers from the vaccine sector have answered a questionnaire about their research profile, their usage of HE and their knowledge of its defining criteria, monitoring of animals, staff training, and euthanasia methods employed. The main results revealed that researchers failed to recognise the very concept of HE as well as when to apply it. In addition, the Institutional Animal Care and Use Committees (IACUCs) failed upon giving their approval to the trials. HE is an important refinament of animal testing policy, and these results highlight the need for a clear pre-established definition of when and how it should be implemented in order to ensure more effective application. Furthermore, it is important to clarify the ethics involved and the commitment of both the research teams and IACUCs to animal welfare.

Glia-Like Cells From Late-Passage Human MSCs Protect Against Ischemic Stroke Through IGFBP-4

Summary: Early-passage human mesenchymal stem cells (hMSCs) are typically used in clinical trials because of safety and efficacy issues. However, obtaining sufficient cells for treatment is difficult and expensive. Many more late-passage hMSCs can be obtained at lower cost, although efficacy is a large hurdle in clinical trials. The use of late-passage MSCs with better efficacy would be a revolutionary solution for reducing cost and facilitating clinical trials. In the present study, glia-like cells (ghMSCs) were induced from hMSCs and used for both in vitro and in vivo models of ischemia, effectively protecting neurons from ischemia and restoring brain tissue damaged by cerebral infarction. These beneficial effects were significantly blocked by insulin-like growth factor-binding protein-4 (IGFBP-4) antibody. The current study demonstrated that late-passage hMSCs can be efficiently induced into ghMSCs with a better neuroprotective effect against ischemic stroke and that IGFBP-4 may be a key neuronal survival factor secreted by ghMSCs. Funding Statement: This research was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI17C2160). Declaration of Interests: One of the corresponding authors, Mi-Sook Chang, has a patent related to this work, which is noted in the Declaration of Interests section of the manuscript. She obtained a Korean patent for glia-like human mesenchymal stem cells in 2012. Ethics Approval Statements: All procedures involving animals were approved by the Institutional Animal Care and Use Committee, Seoul National University (Seoul, Republic of Korea). In addition, all animal procedures were performed in accordance with the Hanyang University guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee (IACUC) of Hanyang University. In accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, all experiments were carried out in a strictly blinded fashion, inclusion and exclusion criteria were predetermined, and attrition due to mortality and other causes was reported

Prediction of Protocol Approval in a Mock Institutional Animal Care and Use Committee: Evaluation of Type of Research Protocol, Species, and Evaluator Characteristics

Research concerning using animal models that may ultimately benefit the health and well-being of human beings continues to receive substantial but not unequivocal support. Rather, considerable ambivalence driven by attitudes, personality characteristics, and misperceptions about animal research continue to exist. In the present study, individuals comprising various sectors of the academic community (undergraduate and graduate students, faculty, and staff) as well as recruits from the general population were presented with a mock research proposal that varied by goals and species. As part of a fictitious Institutional Animal Care and Use Committee (IACUC), the participants were asked to review the protocol and render a decision to either approve or reject the research proposal. In addition, the participants were queried about the perceived importance of the project, the suffering of the animals, and the amount of perceived scientific detachment from the animals. Lastly, the participants answered a series of items from a research-derived Perceptions about the Use of Animals Scale and the Emotional Intelligence Scale (EIS). Consistent with other research, female respondents were much less accepting of the research protocols than males. Protocol approval rates varied by the research objectives and the species, with research projects seen as having lower biomedical value such as psychology student training and the use of certain species (chimps & cats), generally receiving less support. The ethical issues associated with the use of animals in experiments is briefly considered as well as the need for additional messaging on role of animal research, particularly in the behavioral sciences.

Exosomal MALAT1 Derived From Hepatic Cells Is Involved in the Activation of Hepatic Stellate Cells via miRNA-26b in Fibrosis Induced by Arsenite

In the liver microenvironment, interactions among diverse types of hepatic cells are involved in liver fibrosis. In fibrotic tissues, exosomes act as transporters in intercellular communication. Long non-coding RNAs (lncRNAs) are involved in the activation of hepatic stellate cells (HSCs), which are participants in liver fibrosis. However, the functions of exosomal lncRNAs in liver fibrosis induced by arsenite are undefined. The purposes of the present study were (a) to determine if lncRNAs secreted from human hepatic (L-02) cells exposed to arsenite are shuttled to hepatic stellate LX-2 cells and (b) to establish their effects on LX-2 cells. In mice, MALAT1 was overexpressed in the progression of liver fibrosis induced by arsenite as well as in L-02 cells exposed to arsenite. Co-cultures with arsenite-treated L-02 cells induced the activation of LX-2 cells and overexpression of MALAT1. Arsenite-treated L-02 cells transported MALAT1 into LX-2 cells. Downregulation of MALAT1, which reduced the MALAT1 levels in exosomes derived from arsenite-treated L-02 cells, inhibited the activation of LX-2 cells. Additionally, exosomal MALAT1 derived from arsenite-treated L-02 cells promoted the activation of LX-2 cells via microRNA-26b regulation of COL1A2. Furthermore, circulating exosomal MALAT1 was up-regulated in people exposed to arsenite. In sum, exosomes derived from arsenite-treated hepatic cells transferred MALAT1 to HSCs, which induced their activation. These findings support the concept that, during liver fibrosis induced by arsenite, exosomal lncRNAs are involved in cell-cell communication. Funding Statement: This work was supported by the Natural Science Foundations of China (81730089, 81430077); the National Key Research and Development Program of China (2016YFE0204900); the open project of Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment and Jiangsu Collaborative Innovation Center For Cancer Personalized Medicine, Nanjing Medical University, China (JX21817902/009); the open project of the Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education, Guizhou Medical University, China (GMU-2018-HJZ01); and the Priority Academic Program Development of Jiangsu Higher Education Institutions (2018). Declaration of Interests: The authors declare they have no competing financial interests. Ethics Approval Statement: Animals were treated humanely and with regard for alleviation of suffering according to a protocol approved by the Institutional Animal Care and Use committee (IACUC) of Nanjing Medical University.

IL-7Rα &lt;sup&gt;low&lt;/sup&gt; CD8 &lt;sup&gt;+&lt;/sup&gt; T Cells from Healthy Individuals Are Anergic with Defective Glycolysis

Effector memory (EM) CD8+ T cells expressing lower levels of interleukin-7 receptor alpha (IL-7Rαlow) from healthy individuals are partly compromised in vitro, but the identity of these cells has remained unclear. Here, we demonstrate that IL-7Rαlow EM CD8+ T cells are naturally occurring anergic cells in vivo, impaired in proliferation and IL-2 production, but competent in IFN-γ and TNF-α production, a state that can be restored by IL-2 stimulation. IL-7Rαlow EM CD8+ T cells show decreased expression of GATA3 and c-MYC and are defective in metabolic reprogramming towards glycolysis, a process required for the proliferation of T cells. In particular, mouse IL-7Rαlow memory CD8+ T cells show anergic phenotypes similar to those in humans, but induce severe graft-versus-host disease upon allogeneic T-cell transplantation, suggesting that these cells can be restored to normality or increased activity by inflammatory conditions and may serve as a reservoir for functional immunity. Funding Statement: This work was supported in part by the Creative-Pioneering Researchers Program through Seoul National University (to H.-R. Kim); the Korea Healthcare Technology Research and Development Project, Ministry for Health, Welfare, and Family Affairs (HI17C0534 to H.-R. Kim); the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MOE) (2017R1A4A1015745 to H.-R. Kim); and the SK Telecom Research Fund (3420130260 to H.-R. Kim). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: This work was approved by the Institutional Review Boards of Seoul National University Hospital (# 0905-014-280) and Samsung Medical Center (# 2015-09-053). Written informed consent was obtained from all subjects, in accordance with the Declaration of Helsinki. All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (IACUC No. SNU-171024-1-3 and SNU-171206-1-1). All experiments were performed in accordance with the IACUC guidelines of Seoul National University.

Dual-Faced Implication of CD4 &lt;sup&gt;+&lt;/sup&gt;Foxp3 &lt;sup&gt;+&lt;/sup&gt; Regulatory T Cells Expanded by Acute Dengue Infection via TLR2/MyD88 Pathway

Background: Dengue infection causes dengue fever (DF) and fatal dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). CD4+Foxp3+ Tregs are expanded in human patients during dengue infection, and appear to be closely associated with clinical severity. However, the molecular pathways involved in Treg proliferation and the reason for their insufficient control of severe clinical disease in dengue infection are poorly understood. Methods: We investigated Treg expansion and their role in DF and DHF/DSS with elaborated immunological analysis using several genetically engineered mouse strains. Results: Here we found that dengue infection induced the proliferation of fully functional CD4+Foxp3+ Tregs via TLR2/MyD88 pathway. Also, surface TLR2 on Tregs was responsible for their proliferation, and dengue-expanded Tregs subverted in vivo differentiation of effector CD8+ T cells. A more interesting finding was that dengue-infected hosts displayed changed levels of susceptibility to other diseases in a TLR2-dependent manner. This change included enhanced susceptibility to tumors and bacterial infection, but highly enhanced resistance to viral infection. Further, transfer of dengue-proliferated Tregs protected the recipients from DF and DHF/DSS caused by dengue infection and LPS-induced sepsis. In contrast, hosts that developed sepsis were more susceptible to dengue infection, an effect attributed to early TLR2-dependent production of proinflammatory cytokines. Interpretation: These facts may explain the reason why in some patients, dengue-proliferated Tregs is insufficient to control DF and DHF/DSS. Our observations lead to new insights into Treg responses activated by dengue infection in a TLR2-dependent manner, which could differentially act on subsequent exposure to other disease-producing situations. Funding: National Research Foundation of Korea (2018R1A2B2001873, 2019R1A6A1A03033084). Declaration of Interest: The authors declare no competing financial interests. Ethical Approval: All animal experiments described in the present study were conducted at Chonbuk National University according to the guidelines set by the Institutional Animal Care and Use Committee (IACUC) of Chonbuk National University, and were pre-approved by the Ethics Committee for Animal Experiments of Chonbuk National University (approval number: 2013-0028). The animal research protocol used in this study followed the guidelines set by the nationally recognized Korea Association for Laboratory Animal Sciences (KALAS). All experimental protocols requiring biosafety were approved by the Institutional. Biosafety Committee (IBC) of Chonbuk National University.

Long Noncoding RNA OIP5-AS1 Promotes the Progression of Liver Hepatocellular Carcinoma &lt;i&gt;via&lt;/i&gt; Regulating miR-26a-3p/EPHA2 Axis

Numerous studies have suggested that dysregulated long noncoding RNAs (lncRNAs) contributed to development and progression of many cancers. LncRNA OIP5 antisense RNA 1 (OIP5-AS1) has been reported to be increased in several cancers. However, the roles of OIP5-AS1 in liver hepatocellular carcinoma (LIHC) remain to be investigated. In this study, we demonstrated that OIP5-AS1 was up-regulated in LIHC tissue specimens and its overexpression was associated with the poor survival of patients with LIHC. Furthermore, loss-of function experiments indicated that OIP5-AS1 promoted cell proliferation and inhibited cell apoptosis both in vitro and in vivo. Moreover, binding sites between OIP5-AS1 and hsa-miR-26a-3p as well as between hsa-miR-26a-3p and EPHA2 were confirmed by luciferase assays. Finally, rescue assay was performed to prove the effect of OIP5-AS1-hsa-miR-26a-3p-EPHA2 axis on LIHC cell biological behaviors. Based on all above findings, our results suggested OIP5-AS1 promoted LIHC cell proliferation and invasion via regulating of hsa-miR-26a-3p/EPHA2 axial. Funding Statement: This study was supported partly by grants from the National Natural Science Foundation of China (81972214, 81772932, 81472202, 81201535, 81302065, 81671716, 81301993, 81372175 and 81472209), The Fundamental Research Funds for the Central Universities (22120170212 and 22120170117), Shanghai Natural Science Foundation (12ZR1436000 and 16ZR1428900), Shanghai Municipal Commission of Health and Family Planning (201540228 and 201440398), Construction of Clinical Medical Center for Tumor Biological Samples in Nantong (HS2016004) and Jiangsu 333 Program (BRA2017205). Declaration of Interests: No potential conflicts of interest were disclosed. Ethics Approval Statement: This study protocol was in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Shanghai Tenth People's Hospital, Tongji University School of Medicine (Approval No: SHSY-IEC-2019K10). All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) guidelines at Tongji University School of Medicine (SYDW-19-215).

Pretreated Oenanthe Javanica extract increases anti-inflammatory cytokines, attenuates gliosis, and protects hippocampal neurons following transient global cerebral ischemia in gerbils.

Recently, we have reported that Oenanthe javanica extract (OJE) displays strong neuroprotective effect against ischemic damage after transient global cerebral ischemia. However, neuroprotective mechanisms of OJE have not been fully identified. Thus, this study investigated the neuroprotection of OJE in the hippocampal CA1 area and its anti-inflammatory activity in gerbils subjected to 5 minutes of transient global cerebral ischemia. We treated the animals by intragastrical injection of OJE (100 and 200 mg/kg) once daily for 1 week prior to transient global cerebral ischemia. Neuroprotection of OJE was observed by immunohistochemistry for neuronal nuclear antigen and histofluorescence staining for Fluoro-Jade B. Immunohistochemistry of glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 was done for astrocytosis and microgliosis, respectively. To investigate the neuroprotective mechanisms of OJE, we performed immunohistochemistry of tumor necrosis factor-alpha and interleukin-2 for pro-inflammatory function and interleukin-4 and interleukin-13 for anti-inflammatory function. When we treated the animals by intragastrical administration of 200 mg/kg of OJE, hippocampal CA1 pyramidal neurons were protected from transient global cerebral ischemia and cerebral ischemia-induced gliosis was inhibited in the ischemic hippocampal CA1 area. We also found that interleukin-4 and -13 immunoreactivities were significantly increased in pyramidal neurons of the ischemic CA1 area after OJE pretreatment, and the increased immunoreactivities were sustained in the CA1 pyramidal neurons after transient global cerebral ischemia. However, OJE pretreatment did not increase interleukin-2 and tumor necrosis factor-alpha immunoreactivities in the CA1 pyramidal neurons. Our findings suggest that pretreatment with OJE can protect neurons and attenuate gliosis from transient global cerebral ischemia via increasing expressions of interleukin-4 and -13. The experimental plan of this study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) in Kangwon National University (approval No. KW-160802-1) on August 10, 2016.

Human IL12p80 and Neural Stem Cells Enhance Sciatic Nerve Regeneration

Background: We previously identified mouse interleukin-12p80 (mIL12p80, a homodimer of IL12p40) in the cell extracts of implanted poly(L-lactic acid) (PLA) nerve conduit combined with neural stem cells (NSCs) and showed that mIL12p80 enhances the functional recovery and nerve regeneration in mice. Here, we showed that human IL12p80 (hIL12p80) improves the sciatic nerve regeneration in mice. Methods: Implantation of hIL12p80 and NSCs into nerve conduit promotes repair of the sciatic nerve injury in mouse model. Findings: NSCs transplanted with hIL12p80 showed the best recovery than the other groups in sciatic functional index (SFI), compound muscle action potential (CMAP) and Rotarod analyses. In addition, the group with Conduit+NSC+hIL12p80 (CNI) outperformed the other groups in SFI and Rotarod tests beginning at the fourth-week post-surgery. Immunohistochemistry showed that the CNI group increases the diameter of newly regenerated nerve 98%. In vitro studies showed that hIL12p80 stimulates differentiation of mouse NSCs to oligodendrocyte lineages through phosphorylation of Stat3 at Y705. Further, implantation using poly(L-lactic-co-glycolic acid) (PLGA) nerve conduits (C 2.0 and C 2.1) showed better recovery in Rotarod test and CMAP than using PLA conduits (C 1.0) in FVB/NJ (FVB) mice. In C57BL/6J (B6) mice, the group with C 2.1+NSC+hIL12p80 (C 2.1NI) not only promotes sciatic functional recovery but also reduces experimental autotomy. Interpretation: These results suggest that hIL12p80 together with NSCs enhance the functional recovery and accelerate the regeneration of damaged nerves in mice. These findings could be used in devising potential strategies for nerve injury treatment. Funding Statement: This research is supported by National Health Research Institutes, Taiwan (CS-107-PP-02), and Ministry of Science and Technology, Taiwan (107-1901-01-19-03; 107-0324-01-19-03). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: In this study, all animal experimental procedures followed ethical guidelines and were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Health Research Institutes (Protocol No. NHRI-IACUC-101067A).