scholarly journals Extracellular vesicles from human cardiovascular progenitors trigger a reparative immune response in infarcted hearts

2020 ◽  
Author(s):  
Bruna Lima Correa ◽  
Nadia El Harane ◽  
Ingrid Gomez ◽  
Hocine Rachid Hocine ◽  
José Vilar ◽  
...  
Keyword(s):  
Immune Response ◽  
Extracellular Vesicles ◽  
Inflammatory Cytokines ◽  
Nk Cell ◽  
Inflammatory Monocytes ◽  
Ifn Γ ◽  
Anti Inflammatory ◽  
Pro Inflammatory Cytokines

Abstract Aims The cardioprotective effects of human induced pluripotent stem cell-derived cardiovascular progenitor cells (CPC) are largely mediated by the paracrine release of extracellular vesicles (EV). We aimed to assess the immunological behaviour of EV-CPC, which is a prerequisite for their clinical translation. Methods and results Flow cytometry demonstrated that EV-CPC expressed very low levels of immune relevant molecules including HLA Class I, CD80, CD274 (PD-L1), and CD275 (ICOS-L); and moderate levels of ligands of the natural killer (NK) cell activating receptor, NKG2D. In mixed lymphocyte reactions, EV-CPC neither induced nor modulated adaptive allogeneic T cell immune responses. They also failed to induce NK cell degranulation, even at high concentrations. These in vitro effects were confirmed in vivo as repeated injections of EV-CPC did not stimulate production of immunoglobulins or affect the interferon (IFN)-γ responses from primed splenocytes. In a mouse model of chronic heart failure, intra-myocardial injections of EV-CPC, 3 weeks after myocardial infarction, decreased both the number of cardiac pro-inflammatory Ly6Chigh monocytes and circulating levels of pro-inflammatory cytokines (IL-1α, TNF-α, and IFN-γ). In a model of acute infarction, direct cardiac injection of EV-CPC 2 days after infarction reduced pro-inflammatory macrophages, Ly6Chigh monocytes, and neutrophils in heart tissue as compared to controls. EV-CPC also reduced levels of pro-inflammatory cytokines IL-1α, IL-2, and IL-6, and increased levels of the anti-inflammatory cytokine IL-10. These effects on human macrophages and monocytes were reproduced in vitro; EV-CPC reduced the number of pro-inflammatory monocytes and M1 macrophages, while increasing the number of anti-inflammatory M2 macrophages. Conclusions EV-CPC do not trigger an immune response either in in vitro human allogeneic models or in immunocompetent animal models. The capacity for orienting the response of monocyte/macrophages towards resolution of inflammation strengthens the clinical attractiveness of EV-CPC as an acellular therapy for cardiac repair.

scholarly journals Sarcoma IL-12 overexpression facilitates NK cell immunomodulation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mary Jo Rademacher ◽  
Anahi Cruz ◽  
Mary Faber ◽  
Robyn A. A. Oldham ◽  
Dandan Wang ◽  
...  
Keyword(s):  
Immune Response ◽  
Cell Lines ◽  
Nk Cell ◽  
Autologous Tumor ◽  
Murine Models ◽  
Lentiviral Transduction ◽  
Ifn Γ ◽  
Human Sarcoma

AbstractInterleukin-12 (IL-12) is an inflammatory cytokine that has demonstrated efficacy for cancer immunotherapy, but systemic administration has detrimental toxicities. Lentiviral transduction eliciting IL-12-producing human sarcoma for autologous reintroduction provides localized delivery for both innate and adaptive immune response augmentation. Sarcoma cell lines and primary human sarcoma samples were transduced with recombinant lentivirus engineering expression of human IL-12 (hu-IL-12). IL-12 expressing sarcomas were assessed in vitro and in vivo following implantation into humanized NSG and transgenic human IL-15 expressing (NSG.Tg(Hu-IL-15)) murine models. Lentiviral transduction (LV/hu-IL-12) of human osteosarcoma, Ewing sarcoma and rhabdomyosarcoma cell lines, as well as low-passage primary human sarcomas, engendered high-level expression of hu-IL-12. Hu-IL-12 demonstrated functional viability, eliciting specific NK cell-mediated interferon-γ (IFN-γ) release and cytotoxic growth restriction of spheroids in vitro. In orthotopic xenograft murine models, the LV/hu-IL-12 transduced human sarcoma produced detectable IL-12 and elicited an IFN-γ inflammatory immune response specific to mature human NK reconstitution in the NSG.Tg(Hu-IL-15) model while restricting tumor growth. We conclude that LV/hu-IL-12 transduction of sarcoma elicits a specific immune reaction and the humanized NSG.Tg(Hu-IL-15) xenograft, with mature human NK cells, can define in vivo anti-tumor effects and systemic toxicities. IL-12 immunomodulation through autologous tumor transduction and reintroduction merits exploration for sarcoma treatment.

scholarly journals Apoptotic Cells for the Prevention of Cytokine Release Syndrome (CRS) in CAR T-Cell Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1626-1626
Author(s):  
Dror Mevorach ◽  
Veronique Amor ◽  
Yehudith Shabat
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Inflammatory Cytokines ◽  
Apoptotic Cells ◽  
Car T ◽  
Ifn Γ ◽  
Pro Inflammatory Cytokines

Abstract Background: Chimeric antigen receptor (CAR)-modified T cells with specificity against CD19 have demonstrated dramatic promise against highly refractory hematologic malignancies. Clinical responses with complete remission rates as high as 90% have been reported in children and adults with relapsed/refractory acute lymphoblastic leukemia (ALL). However, very significant toxicity has been observed and as many as 30% in average developing severe forms of CRS and possibly related neurotoxicity. CRS is occurring due to large secretion of pro-inflammatory cytokines, mainly from macrophages/monocytes, and resembles macrophage-activating syndrome and hemophagocytosis in response to CAR T-secreting IFN-g and possibly additional cytokines. To better understand the mechanisms leading to CRS and to treat or prevent it, we have developed in vitro and in vivo models of CRS with and without CAR-modified T cells. Early apoptotic cells that have been successfully tested for the prevention of acute GVHD, including in 7 ALL patients, were tested in these models for their effect on cytokines and CAR T cell cytotoxicity. Methods: CD19-expressing HeLa cells were used alone or with co-incubation with human macrophages for in vitro experiments and intraperitoneal experiments. Raji was used in vivo for leukemia induction. LPS and IFN-γ were used to trigger additional cytokine release. CD19-specific CAR-modified cells were used (ProMab) for anti-tumor effect against CD19-bearing cells. Cytotoxicity assay was examined in vivo using 7-AAD with flow cytometry and in vitro by survival curves and analysis of tumor load in bone marrow and liver. CRS occurred spontaneously or in response to LPS and IFN-γ. Mouse IL-10, IL-1β, IL-2, IP-10, IL-4, IL-5, IL-6, IFNα, IL-9, IL-13, IFN-γ, IL-12p70, GM-CSF, TNF-α, MIP-1α, MIP-1β, IL-17A, IL-15/IL-15R, and IL-7, as well as 32 human cytokines were evaluated by Luminex technology using the MAPIX system analyzer (Mereck Millipore) and MILLIPLEX Analyst software (Merek Millipore). Mouse IL-6Rα, MIG (CXCL9), and TGF-β1 were evaluated by Quantikine ELISA (R&D systems). Bone marrow and liver were evaluated using flow cytometry and immunohistochemistry. The IFN-γ effect was evaluated by STAT1 phosphorylation and biological products. Human macrophages and dendritic cells were generated from monocytes. Early apoptotic cells were produced as shown in GVHD clinical trial; at least 50% of cells were annexin V-positive and less than 5% were PI-positive. Results: Apoptotic cells had no negative effect in vitro or in vivo on CAR-modified T cells with specificity against CD19. There were comparable E/T ratios for CAR T in the presence or absence of apoptotic cells in vitro, and comparable survival curves in vivo. On the other hand, significant downregulation (p<0.01) of pro-inflammatory cytokines, including IL-6, IP-10, TNF-a, MIP-1α, MIP-1β, was documented. IFN-γ was not downregulated, but its effect on macrophages and dendritic cells was inhibited at the level of phosphorylated STAT1 and IFN-γ-induced expression of CXCL10 and CXCL9 was reduced. Conclusion: CRS evolves from several factors, including tumor biology, interaction with monocytes/macrophages/dendritic cells, and as a response to the CAR T cell effect and expansion. Apoptotic cells decrease pro-inflammatory cytokines that originate from innate immunity and inhibit the IFN-γ effect on monocyte/macrophages/ dendritic cells without harming IFN-γ levels or CAR-T cytotoxicity. Disclosures Mevorach: Enlivex: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Amor:Enlivex: Employment. Shabat:Enlivex: Employment.

scholarly journals Nobiletin exerts anti-diabetic and anti-inflammatory effects in an in vitro human model and in vivo murine model of gestational diabetes

2020 ◽  
Vol 134 (6) ◽  
pp. 571-592 ◽  
Author(s):  
Caitlyn Nguyen-Ngo ◽  
Carlos Salomon ◽  
Stephanie Quak ◽  
Andrew Lai ◽  
Jane C Willcox ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Glucose Metabolism ◽  
Gestational Diabetes ◽  
Mrna Expression ◽  
Inflammatory Cytokines ◽  
Cytokines And Chemokines ◽  
Anti Inflammatory ◽  
Pro Inflammatory Cytokines

Abstract Gestational diabetes mellitus (GDM) is a global health issue, whereby pregnant women are afflicted with carbohydrate intolerance with first onset during pregnancy. GDM is characterized by maternal peripheral insulin resistance, thought to be driven by low-grade maternal inflammation. Nobiletin, a polymethoxylated flavonoid, possesses potent glucose-sensitizing and anti-inflammatory properties; however, its effects in GDM have not been assessed. The present study aimed to determine the effects of nobiletin on glucose metabolism and inflammation associated with GDM in both in vitro human tissues and an in vivo animal model of GDM. In vitro, treatment with nobiletin significantly improved TNF-impaired glucose uptake in human skeletal muscle, and suppressed mRNA expression and protein secretion of pro-inflammatory cytokines and chemokines in human placenta and visceral adipose tissue (VAT). Mechanistically, nobiletin significantly inhibited Akt and Erk activation in placenta, and NF-κB activation in VAT. In vivo, GDM mice treated with 50 mg/kg nobiletin daily via oral gavage from gestational day (gd) 1-17 or via i.p. injections from gd 10-17 significantly improved glucose tolerance. Pregnant GDM mice treated with nobiletin from either gd 1-17 or gd 10-17 exhibited significantly suppressed mRNA expression of pro-inflammatory cytokines and chemokines in placenta, VAT and subcutaneous adipose tissue (SAT). Using a quantitative mass spectrometry approach, we identified differentially abundant proteins associated with the effect of nobiletin in vivo. Together, these studies demonstrate that nobiletin improves glucose metabolism and reduces inflammation associated with GDM and may be a novel therapeutic for the prevention of GDM.

scholarly journals Anti-inflammatory homoeopathic drug dilutions restrain lipopolysaccharide-induced release of pro-inflammatory cytokines: In vitro and in vivo evidence

2017 ◽  
Vol 11 (3) ◽  
pp. 158 ◽  
Author(s):  
ChanakyaNath Kundu ◽  
ChandragoudaR Patil ◽  
UmeshB Mahajan ◽  
AjitK Walke ◽  
MahendraV Kardile ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Anti Inflammatory ◽  
Pro Inflammatory Cytokines

scholarly journals CD1d Mediates T-Cell-Dependent Resistance to Secondary Infection with Encephalomyocarditis Virus (EMCV) In Vitro and Immune Response to EMCV Infection In Vivo

2006 ◽  
Vol 80 (14) ◽  
pp. 7146-7158 ◽  
Author(s):  
Petr O. Ilyinskii ◽  
Ruojie Wang ◽  
Steven P. Balk ◽  
Mark A. Exley
Keyword(s):  
Immune Response ◽  
T Cell ◽  
Cell Activation ◽  
Nk Cell ◽  
Nkt Cells ◽  
Encephalomyocarditis Virus ◽  
Wild Type ◽  
Ifn Γ

ABSTRACT The innate and adaptive immune responses have evolved distinct strategies for controlling different viral pathogens. Encephalomyocarditis virus (EMCV) is a picornavirus that can cause paralysis, diabetes, and myocarditis within days of infection. The optimal innate immune response against EMCV in vivo requires CD1d. Interaction of antigen-presenting cell CD1d with distinct natural killer T-cell (“NKT”) populations can induce rapid gamma interferon (IFN-γ) production and NK-cell activation. The T-cell response of CD1d-deficient mice (lacking all NKT cells) against acute EMCV infection was further studied in vitro and in vivo. EMCV persisted at higher levels in CD1d-knockout (KO) splenocyte cultures infected in vitro. Furthermore, optimal resistance to repeat cycles of EMCV infection in vitro was also shown to depend on CD1d. However, this was not reflected in the relative levels of NK-cell activation but rather by the responses of both CD4+ and CD8+ T-cell populations. Repeated EMCV infection in vitro induced less IFN-γ and alpha interferon (IFN-α) from CD1d-deficient splenocytes than with the wild type. Furthermore, the level of EMCV replication in wild-type splenocytes was markedly and specifically increased by addition of blocking anti-CD1d antibody. Depletion experiments demonstrated that dendritic cells contributed less than the combination of NK and NKT cells to anti-EMCV responses and that none of these cell types was the main source of IFN-α. Finally, EMCV infection in vivo produced higher levels of viremia in CD1d-KO mice than in wild-type animals, coupled with significantly less lymphocyte activation and IFN-α production. These results point to the existence of a previously unrecognized mechanism of rapid CD1d-dependent stimulation of the antiviral adaptive cellular immune response.

The effect of antidepressants on inflammatory markers in animal models of depression

2011 ◽  
Vol 26 (S2) ◽  
pp. 2091-2091
Author(s):  
A. Harkin ◽  
T.J. Connor
Keyword(s):  
Animal Models ◽  
Inflammatory Cytokines ◽  
Glial Cells ◽  
Inflammatory Cytokine ◽  
Animal Models Of Depression ◽  
In Vitro Exposure ◽  
Anti Inflammatory ◽  
Pro Inflammatory Cytokines

Considering the evidence that pro-inflammatory cytokines play a causal role in depressive illness, the ability of antidepressants to induce anti-inflammatory effects is a subject of considerable interest. In an in vivo context we observe that antidepressants that enhance noradrenaline availability are the most effective anti-inflammatory agents; a fact consistent with the established anti-inflammatory actions of noradrenaline. Specifically, we have observed that noradrenaline reuptake inhibitors (NRIs) inhibit microglial activation and inhibit expression of pro-inflammatory cytokines (IL-1beta and TNF-alpha), iNOS, and inflammatory chemokines (IP-10 and RANTES) in rat brain following a systemic inflammatory challenge. These in vivo anti-inflammatory actions of NRIs are mimicked by in vitro exposure of primary glial cells to noradrenaline, but not by in vitro exposure of glial cells to the drugs themselves. These data suggest that NRIs promote an anti-inflammatory environment in rat brain in vivo by increasing noradrenaline availability at glial cells. We have also observed that even in the absence of any overt inflammation, chronic treatment with the NRI reboxetine promotes an anti-inflammatory phenotype in the CNS characterised by reduced expression of pro-inflammatory cytokine IFN-gamma, and increased expression of the anti-inflammatory cytokine IL-10. Current experiments are focused on the activation of the inflammatory response system in animal models of depression secondary to inflammation. The models are used subsequently to assess the anti-inflammatory effects of antidepressants in vivo.

scholarly journals Role of cholinergic anti-inflammatory pathway in protecting sepsis-induced acute lung injury through regulation of the dendritic cells

2021 ◽  
Author(s):  
Ruiting Li ◽  
Xuemei Hu ◽  
Huibin Chen ◽  
Yin Yuan ◽  
Huiling Guo ◽  
...  
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Acute Lung Injury ◽  
Lung Injury ◽  
Inflammatory Response ◽  
Inflammatory Cytokines ◽  
Mice Model ◽  
Pro Inflammatory Cytokines

Abstract Background The cholinergic anti-inflammatory pathway (CAP) connects the immune response system and the nervous system via the vagus nerve. The key regulatory receptor is the α7-subtype of the nicotinic acetylcholine receptor (α7nAChR), which is localized on the surface of the cells of immune system. CAP has been proved to be effective in suppressing the inflammation responses in acute lung injury (ALI). Dendritic cells (DCs), the important antigen-presenting cells (APCs), also express the α7nAChR. They not only play an important role in immune response priming but also in participating in the pathological process of ALI. Past studies have indicated that reducing the quantity of mature conventional DCs (cDCs) and inhibiting the maturation of pulmonary DCs may prove effective for the treatment of ALI. However, the effects of CAP on maturation, function and quantity of DCs and cDCs in ALI remain unclear. Objective It was hypothesized that the activation of CAP may inhibit the inflammatory response of ALI by regulating maturation, phenotype, and quantity of DCs and cDCs. This can be considered as an important intervention strategy for treating ALI. Methods GTS-21 (GTS-21 dihydrochloride), an α7nAchR agonist was administered in sepsis-induced ALI mice model and LPS-primed bone marrow-derived dendritic cells (BMDCs). The effects of GTS-21 were observed with respect to maturation, phenotype, and quantity of DCs, cDCs, and cDCs2 (type 2 cDCs), and the release of DC-related pro-inflammatory cytokines (such as IL-6, TNF-α, IL-18 IL-1β, IL-12p40, and HMGB1) in vivo and in vitro conditions. Results The results of the present study revealed that, GTS-21 treatment regulated the maturation of DCs and the production of DC-related pro-inflammatory cytokines in vitro and in sepsis-induced ALI mice model, it reduced the quantity of CD11c+MHCII+ cDCs and CD11c+CD11b+ cDCs2 in vivo experiment. Conclusions The activation of CAP contributes to the reduction in the inflammatory response in ALI by regulating maturation, phenotype, and quantity of DCs, cDCs, and cDCs2.

scholarly journals Adipose Stem Cell-Derived Exosomes Exert a Synergic Anti-Inflammatory Effect with Glucocorticoids and Override Their Detrimental Effects on Rotator Cuff Tendons

2021 ◽  
Author(s):  
Xuancheng Zhang ◽  
Ang Li ◽  
Kang Han ◽  
He Zhang ◽  
Xiaoqiao Huangfu ◽  
...  
Keyword(s):  
Rotator Cuff ◽  
Inflammatory Cytokines ◽  
Cellular Proliferation ◽  
In Vivo Studies ◽  
Degradative Enzymes ◽  
Anti Inflammatory ◽  
Pro Inflammatory Cytokines ◽  
Inflammatory Effect

Abstract Background: Glucocorticoids (GCs) injections are commonly used to relieve pain and improve function in patients with multiple shoulder disability, they cause detrimental effects on the rotator cuff tendons. Adipose stem cell-derived exosomes (ASC-Exos) reportedly recover impaired tendon matrix metabolism by maintaining tissue homeostasis. It is unclear whether additional ASC-Exos treatment overrides the detrimental effects of GCs without interfering with their anti-inflammatory effects.Methods: The in vitro studies included inflammatory analysis and cytoprotective analysis. In the inflammatory analysis, rat raw cells were treated with saline, GCs, or GCs + ASC-Exos and evaluated regarding cellular proliferation, migration, and secretion of inflammatory-related cytokines. In the cytoprotective analysis, rat tenocytes were treated with saline, GCs, or GCs + ASC-Exos and evaluated regarding cellular proliferation, migration, senescence, apoptosis, and transcription of tenocytic genes. In the in vivo studies, a subacromial injection of saline, GCs, or GCs + ASC-Exos was performed in a chronic injured-intact rotator cuff rat model. Histological and biomechanical analysis were performed 1 week to evaluate the protective effect of ASC-Exos against GCs-induced detriments on injured-intact in rotator cuffs.Results: In the in vitro inflammatory analysis, GCs treatment significantly decreased the proliferation, migration, and secretion of pro-inflammatory cytokines in raw cells, and increased the secretion of anti-inflammatory cytokines; additional ASC-Exos treatment further significantly decreased the secretion of pro-inflammatory cytokines and increased the secretion of anti-inflammatory cytokines, while restoring GCs-suppressed cellular proliferation and migration. In the in vitro cytoprotective analysis, GCs treatment significantly decreased the proliferation, migration, and transcription of tenocytic matrix molecules of tenocytes, and significantly increased their senescence, apoptosis, and transcription of ROS and tenocytic degradative enzymes; additional ASC-Exos treatment significantly improved the GCs-suppressed cellular proliferation, migration, and transcription of tenocytic matrix molecules, transcription of tenocytic degradative enzyme inhibitors, and significantly decreased the GCs-induced cell senescence, apoptosis, and transcription of ROS and tenocytic degradative enzymes. In the in vivo studies, an additional ASC-Exos injection restored the impaired histological and biomechanical properties owing to GCs administration.Conclusion: ASC-Exos may exert a stronger anti-inflammatory effect in combination with GCs, and override their detrimental effects on the rotator cuff.

Evidence for Reversal of Immunosuppression by Homeopathic Medicine to a Predominant Th1-type Immune Response in BALB/c Mice Infected with Leishmania donovani

Homeopathy ◽  
2021 ◽  
Author(s):  
Jyoti Joshi ◽  
Chetna Bandral ◽  
Raj Kumar Manchanda ◽  
Anil Khurana ◽  
Debadatta Nayak ◽  
...  
Keyword(s):  
Immune Response ◽  
Leishmania Donovani ◽  
Experimental Approach ◽  
Axenic Culture ◽  
Parasite Load ◽  
Ifn Γ ◽  
Immunomodulatory Potential ◽  
Pro Inflammatory Cytokines

Abstract Background Visceral leishmaniasis (VL) is a neglected tropical disease that is fatal if treatment is not given. The available chemotherapeutic options are unsatisfactory, and so complementary therapies like homeopathy might be a promising approach. Methods A nosode from a pure axenic culture of Leishmania donovani was prepared and screened for its anti-leishmanial potential both in an in-vitro and an in-vivo experimental approach. Results Leishmania donovani amastigote promastigote nosode (LdAPN 30C) exhibited significant anti-leishmanial activity against the promastigote forms of Leishmania donovani and was found to be safe. A study conducted on VL-infected mice revealed that LdAPN 30C resolved the disease by modulating the host immune response toward the Th1 type through upregulating the pro-inflammatory cytokines (IFN-γ and IL-17) and inducing nitric oxide (NO) levels in the infected macrophages. The hepatic parasite load was also found to be significantly decreased. The nosode was found to be safe, as no histological alterations in the liver or kidney were observed in the animals treated with the LdAPN 30C. Conclusion This is the first study in which an axenic culture of Leishmania donovani has been used for the preparation of a homeopathic medication. The study highlights the anti-leishmanial and immunomodulatory potential of a homeopathic nosode in experimental VL.

Methylmercury-induced pro-inflammatory cytokines activation and its preventive strategy using anti-inflammation N-acetyl-l-cysteine: a mini-review

2020 ◽  
Vol 35 (3) ◽  
pp. 233-238
Author(s):  
Muflihatul Muniroh
Keyword(s):  
Inflammatory Cytokines ◽  
Health Concern ◽  
Brain Cells ◽  
Preventive Strategy ◽  
Hearing Impairments ◽  
Sensory Disturbances ◽  
Anti Inflammation ◽  
Pro Inflammatory Cytokines

AbstractThe exposure of methylmercury (MeHg) has become a public health concern because of its neurotoxic effect. Various neurological symptoms were detected in Minamata disease patients, who got intoxicated by MeHg, including paresthesia, ataxia, gait disturbance, sensory disturbances, tremors, visual, and hearing impairments, indicating that MeHg could pass the blood-brain barrier (BBB) and cause impairment of neurons and other brain cells. Previous studies have reported some expected mechanisms of MeHg-induced neurotoxicity including the neuroinflammation pathway. It was characterized by the up-regulation of numerous pro-inflammatory cytokines expression. Therefore, the use of anti-inflammatories such as N-acetyl-l-cysteine (NAC) may act as a preventive compound to protect the brain from MeHg harmful effects. This mini-review will explain detailed information on MeHg-induced pro-inflammatory cytokines activation as well as possible preventive strategies using anti-inflammation NAC to protect brain cells, particularly in in vivo and in vitro studies.

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