Inhibitory effect of activin A on IL-9 production by mouse NK cells through Smad3 signaling

2020 ◽  
Vol 401 (2) ◽  
pp. 297-308
Author(s):  
Chunhui Ma ◽  
Yan Qi ◽  
Haiyan Liu ◽  
Chengdong Wu ◽  
Xueling Cui ◽  
...  
Keyword(s):  
Nk Cells ◽  
Inhibitory Effect ◽  
Activin A ◽  
Enzyme Linked Immunosorbent Assay ◽  
T Helper ◽  
Inhibitory Effects ◽  
Th Cells ◽  
Smad3 Protein ◽  
Ifn Γ ◽  
Interleukin 9

AbstractInterleukin-9 (IL-9) is a cytokine secreted by T-helper (Th)9 cells, and activin A can enhance Th9 cell differentiation. However, whether activin A affects IL-9 production by natural killer (NK) cells remains unclear. Herein, we found that not only Th cells, but also CD3−CD49b+NKp46+ NK cells of Balb/c mice produced IL-9. Although activin A promoted IL-9 expression in CD4+ Th cells, it inhibited IL-9 production by CD49b+NKp46+ NK cells in mice. Furthermore, the enzyme-linked immunosorbent assay (ELISA) results showed that mouse NK cells could secrete mature IL-9 protein, and activin A inhibited IL-9 release by NK cells. Additionally, activin A inhibited interferon (IFN)-γ production in splenic NK cells in mice, but promoted IL-2 production, and did not alter the production of IL-10. Western blotting results showed that levels of activin type IIA receptor (ActRIIA), Smad3 and phosphorylated-Smad3 (p-SMAD3) protein increased in activin A-treated splenic NK cells, compared with that in control NK cells. The inhibitory effects of activin A on IL-9 production by NK cells were attenuated in the presence of activin antagonist follistatin (FST) or Smad3 knockdown to NK cells. These data suggest that although activin A up-regulates IL-9 expression in Th cells, it inhibits IL-9 production in NK cells through Smad3 signaling.

scholarly journals A Signal Transducer and Activator of  Transcription (Stat)4-independent Pathway for the Development of  T Helper Type 1 Cells

1998 ◽  
Vol 188 (6) ◽  
pp. 1191-1196 ◽  
Author(s):  
Mark H. Kaplan ◽  
Andrea L. Wurster ◽  
Michael J. Grusby
Keyword(s):  
Delayed Type Hypersensitivity ◽  
Th2 Cells ◽  
Signal Transducer ◽  
T Helper ◽  
Th1 Cell ◽  
Th Cells ◽  
Th Cell ◽  
Ifn Γ

The differentiation of T helper (Th) cells is regulated by members of the signal transducer and activator of transcription (STAT) family of signaling molecules. We have generated mice lacking both Stat4 and Stat6 to examine the ability of Th cells to develop in the absence of these two transcription factors. Stat4, Stat6−/− lymphocytes fail to differentiate into interleukin (IL)-4–secreting Th2 cells. However, in contrast to Stat4−/− lymphocytes, T cells from Stat4, Stat6−/− mice produce significant amounts of interferon (IFN)-γ when activated in vitro. Although Stat4, Stat6−/− lymphocytes produce less IFN-γ than IL-12–stimulated control lymphocytes, equivalent numbers of IFN-γ–secreting cells can be generated from cultures of Stat4, Stat6−/− lymphocytes activated under neutral conditions and control lymphocytes activated under Th1 cell–promoting conditions. Moreover, Stat4, Stat6−/− mice are able to mount an in vivo Th1 cell–mediated delayed-type hypersensitivity response. These results support a model of Th cell differentiation in which the generation of Th2 cells requires Stat6, whereas a Stat4-independent pathway exists for the development of Th1 cells.

Catgut Implantation at Acupoints has an Immunosuppressive Effect on Autoimmune Uveitis by Regulating Th1/Th17 Lymphocytes

2021 ◽  
Author(s):  
Feifei Chen ◽  
Jianying Zhao ◽  
Shuyang Zhong ◽  
Fengming Zheng ◽  
Xiaobo Hao
Keyword(s):  
Flow Cytometry ◽  
Rat Model ◽  
Inhibitory Effect ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Autoimmune Uveitis ◽  
Post Immunization ◽  
Th17 Lymphocytes ◽  
Catgut Implantation At Acupoints ◽  
Ifn Γ

Catgut implantation at acupoints (CIA) has a long history as a medical treatment for a wide variety of diseases, including autoimmune diseases. However, the effect and mechanism of this therapy in autoimmune uveitis is still largely unknown. The aim of this study was to explore the immunity-inhibitory effect of CIA in an experimental autoimmune uveitis (EAU) rat model. EAU was established in Lewis rats by the injection of IRBP1177–1191 peptide. The rats were randomly divided into control and CIA groups. Phenotypic and histological assessments were performed days 9, 13, 18, 23 post-immunization. The percentage of Th1 and Th17 lymphocytes isolated from lymph nodes were determined by flow cytometry. The expression of IL-17 and IFN-γ was detected by real-time quantitative PCR and enzyme-linked immunosorbent assay (ELISA). In the CIA group, delayed mild inflammation was observed. Pathological investigation found alleviated infiltration of lymphocytes and ocular damage. Flow cytometry showed significantly decreased Th17 lymphocytes at day 9, 13, and 18 post-immunization (P<0.05) and no significant changes at day 23 post-immunization (P=0.868) after CIA. The Th1 lymphocytes were significantly decreased at day 13 and 18 post-immunization (P<0.05) and comparable at day 9 (P=0.111) and 23 (P=0.551) post-immunization in the CIA group. IL-17 and IFN-γ mRNA levels were notably decreased at day 9, 13 and 18 post-immunization (P<0.05) and showed a downward trend at day 23 post-immunization, although with no significance (P=0.080 and P=0.137, respectively) after CIA. Serum IL-17 and IFN-γ levels in the CIA group were significantly decreased at day 9, 13 and 18 post-immunization (P<0.05) and were comparable at day 23 post-immunization (P=0.078 and P=0.979, respectively). Ocular inflammation was markedly inhibited after catgut implantation at Pishu (BL20) and Shenshu (BL23) acupoints in an EAU rat model. Moreover, CIA reduced Th1 and Th17 lymphocytes and the expression IFN-γ and IL-17.

scholarly journals CD16+ monocytes control T-cell subset development in immune thrombocytopenia

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3326-3335 ◽  
Author(s):  
Hui Zhong ◽  
Weili Bao ◽  
Xiaojuan Li ◽  
Allison Miller ◽  
Caroline Seery ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Thrombocytopenia ◽  
Critical Role ◽  
Cell Subset ◽  
Inhibitory Effect ◽  
Cell Polarization ◽  
T Cell Subsets ◽  
Monocyte Subset ◽  
Th Cells ◽  
Ifn Γ

Abstract Immune thrombocytopenia (ITP) results from decreased platelet production and accelerated platelet destruction. Impaired CD4+ regulatory T-cell (Treg) compartment and skewed Th1 and possibly Th17 responses have been described in ITP patients. The trigger for aberrant T-cell polarization remains unknown. Because monocytes have a critical role in development and polarization of T-cell subsets, we explored the contribution of monocyte subsets in control of Treg and Th development in patients with ITP. Unlike circulating classic CD14hiCD16− subpopulation, the CD16+ monocyte subset was expanded in ITP patients with low platelet counts on thrombopoietic agents and positively correlated with T-cell CD4+IFN-γ+ levels, but negatively with circulating CD4+CD25hiFoxp3+ and IL-17+ Th cells. Using a coculture model, we found that CD16+ ITP monocytes promoted the expansion of IFN-γ+CD4+ cells and concomitantly inhibited the proliferation of Tregs and IL-17+ Th cells. Th-1–polarizing cytokine IL-12, secreted after direct contact of patient T-cell and CD16+ monocytes, was responsible for the inhibitory effect on Treg and IL-17+CD4+ cell proliferation. Our findings are consistent with ITP CD16+ monocytes promoting Th1 development, which in turn negatively regulates IL-17 and Treg induction. This underscores the critical role of CD16+ monocytes in the generation of potentially pathogenic Th responses in ITP.

scholarly journals T Helper Cells in the Immunopathogenesis of Systemic Sclerosis – Current Trends

2017 ◽  
Vol 44 (1) ◽  
pp. 57-63
Author(s):  
E. Krasimirova ◽  
D. Kyurkchiev
Keyword(s):  
Systemic Sclerosis ◽  
Peripheral Blood ◽  
Absolute Number ◽  
Inhibitory Effect ◽  
Major Effect ◽  
Autoimmune Response ◽  
T Helper ◽  
Th Cell ◽  
Cutaneous Form ◽  
Ifn Γ

Abstract Systemic sclerosis (SSc) is a chronic progressive autoimmune disease characterized by skin and multiorgan involvement with alterations in both the innate and adaptive immunities. The hallmark of the disease is widespread fibrosis engaging the skin and multiple internal organs, as well as the musculoskeletal system. There is mounting evidence that T cells are key players in the pathogenesis of scleroderma. The current review discusses the role of the different T helper (Th) lymphocyte subsets in the processes of inflammation and fibrosis, characteristics for the pathogenesis of the disease. Cytokines produced by Th cell populations have a major effect on endothelial cells and fibroblasts in the context of favoring/inhibiting the vasculopathy and the fibrosis spread. The Th2 pro-fibrotic cytokines IL-4 and IL-13 have been shown to induce collagen synthesis by fibroblasts, whereas IFN-γ demonstrates an inhibitory effect. Increased Th17 cells are present in the scleroderma skin infiltrates. The combination of IL-17, IFN-γ and TGF-β levels in CD45RO and CD45RA cells from patients with SSc is useful to distinguish between the limited and the diffuse phenotype of the disease. There are accumulating data for functional and numerical alterations in the Tregs in SSc. High levels of TNF-α which might reduce the suppressive ability of Tregs have been described. According to some studies, the number of Tregs in scleroderma skin biopsies has been decreased against the normal absolute number of Tregs in peripheral blood of the same patients, which suggests suppressed immunomodulatory response. Other studies reported increased frequency of Tregs in peripheral blood of patients with systemic sclerosis and established a correlation with disease activity. The main immunological challenge remains the identification of the trigger of the autoimmune response in SSc, the causes for preferential Th2-type cell responses and the immunological differences between the diffuse and the limited cutaneous form of the disease.

scholarly journals Breaking the co-operation between bystander T-cells and natural killer cells prevents the development of immunosuppression after traumatic skeletal muscle injury in mice

2015 ◽  
Vol 128 (11) ◽  
pp. 825-838 ◽  
Author(s):  
Florian Wirsdörfer ◽  
Jörg M. Bangen ◽  
Eva Pastille ◽  
Wiebke Hansen ◽  
Stefanie B. Flohé
Keyword(s):  
Skeletal Muscle ◽  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Muscle Injury ◽  
Adaptive Immune System ◽  
Th Cells ◽  
Skeletal Muscle Injury ◽  
Ifn Γ

Nosocomial infections represent serious complications after traumatic or surgical injuries in intensive care units. The pathogenesis of the underlying immunosuppression is only incompletely understood. In the present study, we investigated whether injury interferes with the function of the adaptive immune system in particular with the differentiation of antigen-specific T helper (Th)-cell responses in vivo. We used a mouse model for traumatic gastrocnemius muscle injury. Ovalbumin (OVA), which served as a foreign model antigen, was injected into the hind footpads for determination of the differentiation of OVA-specific Th-cells in the draining popliteal lymph node (pLN). The release of interferon (IFN)-γ from OVA-specific Th-cells was impaired within 24 h after injury and this impairment persisted for at least 7 days. In contrast, the proliferation of OVA-specific Th-cells remained unaffected. Injury did not modulate the function of antigen-presenting cells (APCs) in the pLN. Adoptive transfer of total T-cells from pLNs of injured mice inhibited IFN-γ production by OVA-specific Th-cells in naive mice. Suppressed Th1 priming did not occur in lymphocyte-deficient mice after injury but was restored by administration of T-cells before injury. Moreover, the suppression of Th1 differentiation required the presence of natural killer (NK) cells that were recruited to the pLN after injury; this recruitment was dependent on lymphocytes, toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88). In summary, upon traumatic skeletal muscle injury T-cells and NK cells together prevent the development of protective Th1 immunity. Breaking this co-operation might be a novel approach to reduce the risk of infectious complications after injury.

scholarly journals N-Acetylcysteine (NAC) Inhibits Synthesis of IL-18 in Macrophage by Suppressing NLRP3 Expression to Reduce the Production of IFN-γ from NK Cells

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Xiaopeng Liu ◽  
Xufeng Lu ◽  
Zhixiong Hu
Keyword(s):  
Mouse Model ◽  
Nk Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Chronic Obstructive ◽  
Related Protein ◽  
Obstructive Pulmonary Disease ◽  
Copd Patients ◽  
Tnf Α ◽  
Ifn Γ ◽  
Nlrp3 Expression

Background. N-Acetylcysteine (NAC) had exerted antioxidation and anti-inflammation effects on chronic obstructive pulmonary disease (COPD) patients. However, its effect in regulating interleukin- (IL-) 18 was not fully understood. This study was designed to evaluate the specific mechanism of NAC regulating IL-18. Materials and Methods. A total of 112 COPD patients and 103 health individuals were recruited in the study. Cytokine level in patients’ serum was measured by enzyme-linked immunosorbent assay (ELISA). A COPD mouse model was established by administration of lipopolysaccharide (LPS) and cigarette smoke. The expression of cytokines was measured by ELISA and flow cytometry. Inflammasome-related protein was measured by Western blot. Result. NAC could effectively improve the immune status of COPD patients as well as the COPD mouse model by downregulating proinflammation and inflammation cytokines including IL-1β, interferon- (IFN-) γ, tumor necrosis factor- (TNF-) α, and IL-18. It also had the capability to suppress synthesis of IL-18 in macrophage to inhibit the secretion of IFN-γ from natural killer (NK) cells through influencing the inflammasome-related protein in macrophages. Conclusion. NAC could effectively inhibit the production of IL-18 by suppressing NLRP3 expression in macrophages to reduce the production of IFN-γ in NK cells.

scholarly journals Increased T-helper interferon-γ-secreting cells in obese children

2006 ◽  
Vol 154 (5) ◽  
pp. 691-697 ◽  
Author(s):  
Lucia Pacifico ◽  
Livia Di Renzo ◽  
Caterina Anania ◽  
John F Osborn ◽  
Flora Ippoliti ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Geometric Mean ◽  
Assessment Model ◽  
Th2 Cells ◽  
Potential Candidate ◽  
Model Assessment ◽  
T Helper ◽  
Obese Children ◽  
Th Cells ◽  
Ifn Γ

Objective: Leptin, an adipocyte-secreted hormone, has emerged as a potential candidate for the link between obesity and the proinflammatory state. Specifically, leptin modulates T-helper (Th) cells toward a Th1 phenotype, with the secretion of proinflammatory cytokines. The aim of this study was to evaluate the Th1/Th2 balance in obese children and its relation with hormonal and metabolic features. Study design: In 50 obese children and 20 control children, we measured the CD4-positive Th cells that secrete interferon (IFN)-γ or interleukin (IL)-2 (taken as an index of Th1 cells), and IL-4 (taken as an index of Th2 cells) as well as serum glucose, insulin, insulin resistance (IR) index (as homeostasis model assessment model (HOMA)), lipid profile, aminotransferases, leptin and ghrelin. Obese children also underwent dual energy X-ray absorptiometry scan measurements, and liver ultrasound scanning. Results: Geometric mean percentages of IL-2- and IL-4-CD4 secreting cells in obese children were not significantly different from those found in control children. However, the geometric mean percentage of CD4-positive T cells secreting IFN-γ was significantly higher in the obese than in the control (P < 0.0001, t-test) group. Within the entire group of study children, the percentage of IFN-γ-positive cells was positively associated with leptin (P = 0.002), insulin (P < 0.00 005), and HOMA-IR values (P < 0.00 005). However, when these associations were restricted to the group of obese subjects, insulin and HOMA-IR values, but not leptin, retained statistical significance. Yet, in the obese group, the percentage of IFN-γ-positive cells was associated with nonalcoholic steatohepatitis (NASH) (P = 0.001), but not with body mass index-standard deviation score and total body fat mass. Conclusions: In obese children, a shift to Th1-cytokine profile dominated by the production of IFN-γ is related to insulin resistance as well as to NASH independently of anthropometric features and other metabolic characteristics. The prevalent Th1 pattern of secreted cytokines may be regarded as a mechanism contributing to inflammation in obesity.

scholarly journals Gamma Interferon Production by Hepatic NK T Cells during Escherichia coli Infection Is Resistant to the Inhibitory Effects of Oxidative Stress

2003 ◽  
Vol 71 (5) ◽  
pp. 2468-2477 ◽  
Author(s):  
Guochi Zhang ◽  
Robert Dru Nichols ◽  
Masaru Taniguchi ◽  
Toshinori Nakayama ◽  
Michael J. Parmely
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Escherichia Coli ◽  
T Cells ◽  
Nk Cells ◽  
Inhibitory Effects ◽  
Gram Negative ◽  
E Coli ◽  
Nk T Cells ◽  
Ifn Γ

ABSTRACT The reductive-oxidative status of tissues regulates the expression of many inflammatory genes that are induced during gram-negative bacterial infections. The cytokine gamma interferon (IFN-γ) is a potent stimulus for host inflammatory gene expression, and oxidative stress has been shown to inhibit its production in mice challenged with Escherichia coli bacteria. The objective of the present study was to characterize the cells that produced IFN-γ in a mouse bacterial peritonitis model and determine the effects of oxidative stress on their activation. The liver contained large numbers of IFN-γ-expressing lymphocytes following challenge with viable E. coli bacteria. The surface phenotypes of IFN-γ-expressing hepatic lymphocytes were those of natural killer (NK) cells (NK1.1+ CD3−), conventional T cells (NK1.1− CD3+), and NK T cells (NK1.1+ CD3+). Treating mice with diethyl maleate to deplete tissue thiols significantly impaired IFN-γ production by NK cells, conventional T cells, and CD1d-restricted NK T cells in response to E. coli challenge. However, IFN-γ expression by a subset of NK T cells, which did not bind α-galactosylceramide-CD1d tetramers, was resistant to the inhibitory effects of tissue oxidative stress. Stress-resistant IFN-γ-expressing cells were also predominantly CD8+ and bore γδ T-cell antigen receptors. The residual IFN-γ response by NK T cells may explain previous reports of hepatic gene expression following gram-negative bacterial challenge in thiol-depleted mice. The finding also demonstrates that innate immune cells differ significantly in their responses to altered tissue redox status.

IL-4 synergistically enhances both IL-2– and IL-12–induced IFN-γ expression in murine NK cells

Blood ◽  
2003 ◽  
Vol 102 (1) ◽  
pp. 207-214 ◽  
Author(s):  
Jay H. Bream ◽  
Rafael E. Curiel ◽  
Cheng-Rong Yu ◽  
Charles E. Egwuagu ◽  
Michael J. Grusby ◽  
...  
Keyword(s):  
Nk Cells ◽  
Molecular Mechanisms ◽  
Synergistic Effects ◽  
Interferon Γ ◽  
Interleukin 4 ◽  
T Helper ◽  
T Helper 1 ◽  
Ifn Γ ◽  
Cytokine Stimulation ◽  
Positive Effect

Abstract Interleukin-4 (IL-4) is thought to influence T and natural killer (NK) cells by down-regulating T helper 1 (Th1)–type cytokines like interferon-γ (IFN-γ). While investigating IL-4 regulation of IFN-γ expression, we found that IL-4 synergized with IL-2 or IL-12 to enhance IFN-γ production and mRNA expression in spleen-derived, IL-2–cultured NK cells, as well as negatively sorted fresh DX5+/CD3- NK cells albeit at lower levels. The positive effect of IL-4 on IL-2–induced IFN-γ production was dependent upon signal transducer and activator of transcription 6 (Stat6) because this response was virtually abrogated in Stat6-/- mice. Notably, though, IL-12 plus IL-4 synergy on IFN-γ expression was intact in Stat6-/- mice. In exploring possible molecular mechanisms to account for the synergistic effects of IL-4 on murine NK cells, we found that IL-2 plus IL-4 stimulation resulted in a modest increase in tyrosine phosphorylation of Stat5, while IL-12 plus IL-4 treatment resulted in a more substantial increase in tyrosine-phosphorylated Stat4. Finally, to identify regions of the IFN-γ promoter that may be involved, NK cells from human IFN-γ promoter/luciferase transgenic mice were treated with cytokines. NK cells from proximal (-110 to +64) promoter region mice did not respond to cytokine stimulation; however, the intact -565 to +64 IFN-γ promoter responded synergistically to IL-2 plus IL-4 and to IL-12 plus IL-4 in NK cells. These data demonstrate a role for IL-4 in enhancing IFN-γ expression in murine NK cells that is partially dependent on Stat6 in IL-2 costimulation and completely independent of Stat6 in IL-12 costimulations. (Blood. 2003;102:207-214)

Glucocorticoid-Induced TNFR-Related Protein (GITR) Ligand Mediates Tumor Immunoediting in Chronic Lymphocytic Leukemia and Impairs Direct and Rituximab-Induced NK Cell Anti-Leukemia Reactivity.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4403-4403
Author(s):  
Corina Buechele ◽  
Tina Baessler ◽  
Benjamin J Schmiedel ◽  
Lothar Kanz ◽  
Helmut R Salih
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Nk Cells ◽  
Tumor Immunity ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Inhibitory Effect ◽  
Lymphocytic Leukemia ◽  
Cell Reactivity ◽  
Immune Effector ◽  
Ifn Γ

Abstract Abstract 4403 Members of the TNF/TNF receptor (TNFR) family of proteins govern differentiation, proliferation, activation, and death of both tumor and immune effector cells and thus play an important role in tumor immunoediting, the reciprocal interaction of tumor cells and anti-tumor immunity. Activation of the TNFR family member GITR has recently been shown to stimulate T cell-mediated anti-tumor immunity in mice. However, available data suggest that GITR mediates different effects in mice and men, and may impair anti-tumor immunity of human NK cells. Here we studied the expression and function of GITR ligand (GITRL) in patients with chronic lymphocytic leukemia (CLL) and the consequences of GITR-GITRL interaction for NK cell reactivity against CLL cells. Substantial GITRL expression was detected on primary B-CLL cells in 38 of 48 (79%) investigated patients. Upon interaction with its cognate receptor, GITRL induced the release of immunoregulatory cytokines like TNF by the leukemia cells, which demonstrated that CLL-expressed GITRL is functional and capable to transduce bidirectional signals. Moreover, disruption of GITR-GITRL interaction in cultures of allogenic NK cells with patient CLL cells by addition of blocking antibody caused a significant increase in NK cell granule mobilization, cytotoxicity and IFN-γ production. The inhibitory effect of tumor-expressed GITRL on the reactivity of human NK cells was also confirmed in cocultures of C1R lymphoma cells transfected to express GITRL with mock transfectants serving as control. In addition, blocking GITR-GITRL interaction also considerably augmented both antibody-dependent cellular cytotoxicity (ADCC) and antibody-induced IFN-γ production of NK cells in cultures with allogenic CLL cells upon Rituximab exposure. Of note, GITR blockade also significantly enhanced anti-leukemia reactivity of autologous NK cells among PBMC of B-CLL patients, and this reinforcement of NK cell effector functions was observed both regarding the direct and, more pronounced, Rituximab-induced anti-leukemia reactivity (both n=10, p<0.01, Student's T test). Thus, expression of functional GITRL by CLL cells potently influences tumor immunoediting and impairs anti-tumor immunity by diminishing both direct and Rituximab-dependent anti-leukemia reactivity of NK cells. Modulation of the GITR-GITRL system might therefore serve to enhance the efficacy of therapeutic approaches in CLL which, like Rituximab-induced ADCC or stem cell transplantation, rely on a sufficient NK cell anti-tumor response. Disclosures: No relevant conflicts of interest to declare.

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