Crucial Factors of the Inflammatory Microenvironment (IL-1 beta/TNF-alpha/TIMP-1) Promote Maintenance of the Malignant Hemopoietic Clone of Myelofibrosis By Stimulating the in Vitro Survival/Proliferation/Migration of Circulating CD34+ stem/Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4094-4094
Author(s):  
Dorian Forte ◽  
Daria Sollazzo ◽  
Nicola Polverelli ◽  
Romano Marco ◽  
Lara Rossi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Inflammatory Cytokines ◽  
Stromal Cells ◽  
Colony Formation ◽  
Inflammatory Factors ◽  
Inflammatory Microenvironment ◽  
Cd34 Cells ◽  
Tnf Α ◽  
Pro Inflammatory Cytokines

Abstract Introduction. Myelofibrosis (MF), an acquired clonal disorder of the hematopoietic stem/progenitor cell (HSPC) with a dysregulation in JAK/STAT signalling (mutations in JAK2, MPL and Calreticulin (CALR) genes), is characterized by a state of chronic inflammation. It is argued that the up-regulated production of proinflammatory cytokines by both HSPCs and the surrounding stromal cells generates a microenvironment that selects for the malignant clone. Only recently, it has been hypothesized that the sustained inflammatory microenvironment of MF can alter crucial biological processes, leading to genomic instability and cancer progression. Here we tested the in vitro functional effects of pivotal players of the inflammatory microenvironment (the extracellular ATP nucleotide and selected cytokines, such as Interleukin (IL)-1β, Tumor Necrosis Factor (TNF)-α or the Tissue Inhibitor of Metalloproteinases-1 (TIMP-1)) on the HSPCs from MF patients. Methods: Circulating CD34+/CD34+ CD38- cells from MF patients (JAK2V617F (17 cases) and CALR (9 cases) mutations) or cord blood (CB; 8 samples) were phenotypically and functionally characterized after in vitro incubation with or without ATP (1000 μM), IL-1β (10 ng/mL), TNF-α (10 ng/mL) or TIMP-1 (100 ng/mL) (alone or in combination). Cells were then analyzed for survival/apoptosis (Annexin-V/Propidium Iodide staining), phenotype (evaluation of CD63 (TIMP-1 receptor), CXCR4 and CD38 expression), cell cycle and clonogenic capacity. Migration was assessed first towards a CXCL12 gradient in the presence or absence of the pro-inflammatory factors. In parallel experiments, CD34+ cells from MF patients were co-cultured with normal mesenchymal stromal cells (MSCs) in the presence or absence of the pro-inflammatory cytokines and then evaluated for their ability to migrate towards a CXCL12 gradient. Plasma TIMP-1, TNF-α, IL-1β and CXCL12 were measured by ELISA assay. Results: The plasma levels of TIMP-1, TNF-α, IL-1β, CXCL12 and the number of circulating CD34+, CD34+ CD38-, CD34+ CD63+, CD34+ CD184+ cells were increased in MF patients. According to mutational status, the CD34+ CD63+ cells were higher in the CALR+ patients. The survival of MF CD34+ cells was strongly stimulated by in vitro incubation with TNF-α or IL-1β as compared with the CB-derived CD34+ cells or untreated cells. By multiple cytokine combinations, IL-1β/TIMP-1, IL-1β /ATP or IL-1β /TNF-α treatments significantly promote the survival of MF CD34+ cells as compared with the normal counterparts or the untreated cells. Various combinations with IL-1β were also effective in stimulating survival of CD34+CD38- cells. IL-1β/TIMP-1 and IL-1β/TNF-α/TIMP-1, but not factors alone, significantly increased the CFU-C growth of MF patients as compared with the CB-derived counterparts and the untreated cells. Moreover, comparing CALR+ vs JAK2V617F+ patients, the colony formation of JAK2V617F+ patients was mainly promoted by the IL-1β/TNF-α treatment. Along with clonogenic capacity stimulation, exposure of CD34+ cells from MF patients to IL-1β/TNF-α/TIMP-1 significantly increases the S-phase cells, suggesting that these pro-inflammatory factors stimulated cell-cycle progression in dormant CD34+ MF cells. Migration of CD34+ cells from MF was significantly increased in CXCL12 treated cells. In addition, exposure of MF CD34+ cells to IL-1β/TNF-α, IL-1β/TIMP-1 or IL-1β/TNF-α/TIMP-1 significantly promotes cell migration in comparison with the CB-derived counterparts or SDF-1 alone. MF migrated cells in the presence of IL-1β/TNF-α significantly upregulate CD63 expression. Intriguingly, colony formation of MF migrated CD34+ cells in the presence of IL-1β/TNF-α or IL-1β/TNF-α/TIMP-1 was potently increased. Finally, co-culture systems with normal MSCs in the presence of pro-inflammatory factors revealed that MF CD34+ cells display increased migration ability toward CXCL12 gradient. Conclusions: Altogether our findings suggest that in MF the inflammatory niche plays a key role in the maintenance of the malignant clone. Thus, the interplay between the pro-inflammatory cytokines promote and select the HSPCs with higher proliferative activity, clonogenic potential and migration capability. Targeting these microenvironmental interactions may be a clinically relevant approach. D.F. and D.S. equally contributed Disclosures Martinelli: Pfizer: Consultancy; Ariad: Consultancy; Novartis: Consultancy, Speakers Bureau; MSD: Consultancy; AMGEN: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy.

scholarly journals P0527AROLE OF PROMOTING INFLAMMATION OF KLF6 IN ACUTE KIDNEY INURY

2020 ◽  
Vol 35 (Supplement_3) ◽  
Author(s):  
Dan Li ◽  
Chenyu Li ◽  
Yan Xu
Keyword(s):  
Inflammatory Cytokines ◽  
Inflammatory Cytokine ◽  
Ischemia Reperfusion ◽  
Kidney Injury ◽  
Tnf Α ◽  
Public Dataset ◽  
Anti Inflammatory ◽  
Anti Inflammatory Cytokine ◽  
Pro Inflammatory Cytokines

Abstract Background and Aims Acute kidney injury (AKI), commonly appeared in cardiac arrest, surgery and kidney transplantation which involved in ischemia-reperfusion (IR) injury of kidney. However, the mechanisms underlying inflammatory response in IR AKI is still unclear. Method Public dataset showed kruppel-like factor 6 (KLF6) was significantly highly expressed (P<0.05) in AKI, implies KLF6 might be associated with AKI. To evaluate the mechanism of KLF6 on IR AKI, 30 rats were randomly divided into sham and IR group, and were sacrificed at 0 h, 3 h, 6 h, 12 h or 24 h after IR. Results The results showed KLF6 expression was peaking at 6 h after IR, and the expression of pro-inflammatory cytokines MCP-1 and TNF-α were increased both in serum and kidney tissues after IR, while anti-inflammatory cytokine IL-10 was decreased after IR. Furthermore, in vitro results showed KLF6 knock-down reduced the pro-inflammatory cytokines expression and increased the anti-inflammatory cytokines expression. Conclusion These results suggest that (1) KLF6 might be a novel biomarker for early diagnosis of AKI and (2) targeting KLF6 expression may offer novel strategies to protect kidneys from IR AKI Figure KLF6, AKI, Control Inflammation

scholarly journals Steroid Sulfates from Ophiuroids (Brittle Stars): Action on Some Factors of Innate and Adaptive Immunity

2016 ◽  
Vol 11 (6) ◽  
pp. 1934578X1601100
Author(s):  
Anna K Gazha ◽  
Lyudmila A. Ivanushko ◽  
Eleonora V. Levina ◽  
Sergey N. Fedorov ◽  
Tatyana S. Zaporozets ◽  
...  
Keyword(s):  
Adaptive Immunity ◽  
Inflammatory Cytokines ◽  
Brittle Stars ◽  
Innate And Adaptive Immunity ◽  
Functional Activities ◽  
In Vivo Experiments ◽  
Tnf Α ◽  
Pro Inflammatory Cytokines

The action of seven polyhydroxylated sterol mono- and disulfates (1-7), isolated from ophiuroids, on innate and adaptive immunity was examined in in vitro and in vivo experiments. At least, three of them (1, 2 and 4) increased the functional activities of neutrophils, including levels of oxygen-dependent metabolism, adhesive and phagocytic properties, and induced the expression of pro-inflammatory cytokines TNF-α and IL-8. Compound 4 was the most active for enhancing the production of antibody forming cells in the mouse spleen.

The PI3K Specific Inhibitor BKM120 Results Effective and Synergizes With Ruxolitinib In Preclinical Models Of Myeloproliferative Neoplasms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1599-1599 ◽  
Author(s):  
Niccolò Bartalucci ◽  
Costanza Bogani ◽  
Serena Martinelli ◽  
Carmela Mannarelli ◽  
Jean-Luc Villeval ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Colony Formation ◽  
Myeloproliferative Neoplasms ◽  
Janus Kinase ◽  
Preclinical Models ◽  
Mtor Inhibition ◽  
Jak2 Inhibitor ◽  
Cd34 Cells

Abstract Background and Aims A gain-of-function mutation in Janus kinase 2 (JAK2V617F) is at the basis of the majority of chronic myeloproliferative neoplasms (MPN). The dual JAK1/JAK2 inhibitor ruxolitinib (ruxo) determined rapid and sustained responses in splenomegaly and symptomatic improvement in patients with myelofibrosis (MF), supporting the central role of dysregulated JAK2 signaling. Enhanced activation of other downstream pathways including the PI3K/mTOR pathway has been documented as well. We previously reported (Bogani et al, PlosOne 2013;8:54828) that targeting mTOR by the allosteric inhibitor RAD001 resulted in inhibition of JAK2VF mutated cells and produced clinical benefits in a phase I/II trial (Guglielmelli et al, Blood 2011;118:2069). In this study we evaluated the effects of BKM120, a specific PI3K inhibitor, alone and in combination with ruxolitinib, in in-vitro and in-vivo MPN models. Methods To evaluate cell proliferation, colony formation, apoptosis, cell cycle and protein phosphorylation status we used mouse BaF3 and BaF3-EPOR cells expressing wild type (WT) or VF mutated JAK2, the human VF-mutated HEL and SET2 cell lines, and primary MPN CD34+ cells from patients with MF or polycythemia vera (PV). Effect of drug combination was analyzed according to Chou and Talalay calculating the combination index (CI); a CI <1 indicates synergistic activity. For in vivo studies we used two mouse models: (1) SCID mice receiving iv BaF3-EPOR VF-luciferase (luc) cells (gift of T. Radimerski) were randomized on day 6 to different treatment groups based on baseline luminescence. (2) C57Bl6/J JAK2 VF Knock-in mice were generated by insertion of the reversed JAK2V617F exon 13 sequence; mating with Vav-Cre transgenic mice activates the VF allele producing a MPN phenotype in progenies with VF heterozygous expression (Hasan et al, Blood 2013;Epub). Mice were treated for 15 days, then blood, spleen and bone marrow cells were analyzed. Results We found that BKM120 preferential inhibited BAF3 VF and BaF3-EpoR VF cells (IC50: 364±200nM and 1100±207nM, respectively) compared to their respective WT counterpart (5300±800nM and 3122±1000nM: p<.05). HEL and SET2 cells resulted also sensitive to BKM120 (2000±500nM and 1000±300nM). Interestingly we found that BKM120 significantly increased G2/M phase and decreased S phase of cell cycle (p<.01) and induced apoptosis (IC50, SET2=10µM, BaF3-EPOR VF=1.8 µM). Western blot analysis showed marked reduction of phospho-mTOR and its target phospho-4EBP1 as well as downregulation of phospho-STAT5 at 6 and 24h of treatment. BKM120 impaired colony formation from MF and PV CD34+ cells at doses 2 to 8-fold lower than healthy controls (p<.01). BKM120 strongly inhibited EEC colony growth from PV pts (IC50, 9±4nM). Co-treatment of BKM120+ruxo resulted in synergistic inhibition of proliferation of SET2 (median CI=0.45) and BaF3-EPOR VF (median CI=0.8) cells. Triple combinations including BKM120/ruxo plus either RAD001 (Torc1 inhibitor) or PP242 (Torc1/2 inhibitor) resulted highly synergistic (median CI=0.27 and 0.52) to indicate the importance of complete mTOR inhibition. BKM120 at 45mpk and 60mpk increased mean lifespan of BaF3 VF luc mouse model from 21d in control mice to 27.2d and 28d in BMK120 treated mice. In KI mice, co-treatment with 60mpk BKM120 + 60mpk ruxo resulted in improvement of splenomegaly (median spleen weight: 1.4, 0.82, 0.8 and 0.6 g respectively for controls, 60mpk BKM120, 60mpk ruxo and 60mpk BKM120+60mpk ruxo) and reduction of leukocytosis and reticulocyte count. The level of phosho-STAT5 and -4EBP1 in the spleen was significantly reduced in mice receiving BKM120+ruxo as compared to single drug treatment. We finally analyzed the effects of BKM120+/-ruxo on the in-vitro clonogenic growth of BM cells from VF and WT KI mice mixed in a 1:1 ratio. The proportion of VF-positive colonies resulted reduced in a dose dependent manner by 19%, 33% and 44% (p<.03) compared to controls with 50nM, 100nM and 300nM BKM120 respectively. A 25% and 39% of VF-positive colonies reduction was achieved with 50nM and 100nM ruxolitinib. The combined treatment with 100nM BKM120 + 50nM ruxo resulted in a 50% decrease of the number of mutated colonies (p<.02). Conclusions Inhibition of PI3K by BKM120 alone and combined with JAK2 inhibitor ruxolitinib resulted in enhanced activity in preclinical models of MPN, providing a rationale for the ongoing combination clinical trial. Disclosures: Vannucchi: Novartis: Membership on an entity’s Board of Directors or advisory committees.

scholarly journals IKK-2 inhibitor TPCA-1 represses nasal epithelial inflammation in vitro

2011 ◽  
Vol 49 (2) ◽  
pp. 168-173
Author(s):  
F. Sachse ◽  
K. Becker ◽  
T.J. Basel ◽  
D. Weiss ◽  
C. Rudack
Keyword(s):  
Inflammatory Cytokines ◽  
Inflammatory Disease ◽  
Inflammatory Process ◽  
Nasal Polyposis ◽  
Paraffin Sections ◽  
Tnf Α ◽  
Epithelial Inflammation ◽  
Pro Inflammatory Cytokines

BACKGROUND: Nasal polyposis (NP) is considered a subgroup within chronic rhinosinusitis. NP can be further subdivided into aspirin sensitive- and aspirin tolerant types (ASNP/ ATNP). Although the true etiology of NP has not been identified so far, it is agreed that NP represents an inflammatory disease of the nasal mucosa. Alterations of cellular kinase activities including that of IKK-2 might play a role in this inflammatory process. METHODS: Paraffin sections of ASNP, ATNP and controls were immunostained with Phospho-IkB-α antibody that detects the direct IKK-2 product (IkB-α. Intensity of epithelial staining was analysed semi-quantitatively by two independent observers. In cultured nasal polyp epithelial cells (NPECs) epithelial derived cytokines IL-8 and GRO α were induced by TNF-α or Staphylococcal supernatants and subsequently repressed by IKK-2 inhibitor TPCA-1. RESULTS: Significant Phospho-IkB-α staining was observed in the nasal epithelium of ASNP compared to ATNP and controls suggesting strong IKK-2 activation in patients with ASNP in vivo. In vitro, pro-inflammatory cytokines IL-8 and GRO-α in NPECs were significantly repressed by TPCA-1. CONCLUSION: IKK-2 activity is increased in the subgroup of ASNP. IL-8 and GRO-α responses were repressed by IKK-2 inhibitor TPCA-1 in vitro. IKK-2 inhibitors might represent a potential target for anti-inflammatory intervention in ASNP.

scholarly journals Metformin ameliorates scleroderma via inhibiting Th17 cells and reducing mTOR-STAT3 signaling in skin fibroblasts

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Jeonghyeon Moon ◽  
Seon-yeong Lee ◽  
Jeong Won Choi ◽  
A Ram Lee ◽  
Jin Hee Yoo ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Th17 Cells ◽  
Skin Fibroblasts ◽  
Inflammatory Factors ◽  
Metformin Treatment ◽  
Protective Effects ◽  
Stat3 Signaling ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Pro Inflammatory Cytokines

AbstractScleroderma is an autoimmune disease that causes dermal fibrosis. It occurs when collagen accumulates in tissue as a result of persistent inflammation. Th17 cells and pro-inflammatory cytokines such as IL-1β, IL-6, IL-17, and TNF-α play important roles in the pathogenesis of scleroderma. Because metformin, a medication used to treat diabetes, has effective immunoregulatory functions, we investigated its therapeutic function in scleroderma. Mice in a model of bleomycin-induced scleroderma were treated with metformin for 2 weeks. Histological assessment demonstrated protective effects of metformin against scleroderma. Metformin decreased the expression of pro-inflammatory factors in dermal tissue and lymphocytes. It also decreased mRNA expression of pro-inflammatory cytokines (IL-1β, IL-6, IL-17, and TNF-α) and fibrosis-inducing molecules both in vivo and in vitro. These results suggest that metformin treatment has anti-inflammatory effects on lymphocytes via the inhibition of IL-17 and cytokines related to Th17 differentiation, such as IL-1β, IL-6, and TNF-α. To investigate how metformin modulates the inflammatory process in skin fibroblasts, we measured mTOR-STAT3 signaling in skin fibroblasts and found that phosphorylated mTOR and phosphorylated STAT3 protein expression were decreased by metformin treatment. These results suggest that metformin has potential to treat scleroderma by inhibiting pro-inflammatory cytokines and anti-inflammatory activity mediated by mTOR-STAT3 signaling.

scholarly journals MiR-200a-3p Accelerated Hypoxia/Reoxygenation Injury in HCM Cells by Enhancing IGF2R via Wnt/β-catenin Signalling Pathway

2021 ◽  
Vol 21 (02) ◽  
Author(s):  
Yaolei Ge
Keyword(s):  
Cell Viability ◽  
Inflammatory Cytokines ◽  
Caspase 3 ◽  
Signalling Pathway ◽  
Dependent Manner ◽  
Tnf Α ◽  
Reoxygenation Injury ◽  
Pro Inflammatory Cytokines ◽  
Hypoxia Reoxygenation

ABSTRACT The present study examined functions of miR-200a-3p accelerated progressions of HCM cells via IGF2R and Wnt/β-catenin signalling pathway after hypoxia/reoxygenation treatment in vitro. CCK-8 showed that cell viability of HCM was inhibited while apoptosis rates detected by flow cytometry were promoted in a time dependent manner after H/R (12 hours and 24 hours). Beyond that, Bcl-2 and c-IAP1 were decreased but Bax and caspase-3 were upregulated by H/R treatment. IL-1β, IL-6, TNF-α and NLRP3 were also increased after treatment. RT-qPCR showed increased expressions of miR-200a-3p by H/R treatment while its inhibitor elevated cell viability but depressed apoptosis rate and pro-inflammatory cytokines’ expressions. IGF2R was upregulated after H/R treatment and its downregulation magnified effects of suppressed miR-200a-3p. HIF-1α/Wnt/β -catenin signalling pathway was activated by miR-200a-3p and IGF2R while IWP-2 treatment abolished the activation of Wnt3a andβ -catenin, causing decreased apoptosis and pro-inflammatory cytokines’ expressions but accelerated the cell viability.

scholarly journals Maternal Serum Levels of Interferon-gamma, Tumor Necrotic Factor-alpha and Progesterone of Infertile Women on In vitro Fertilization before and after Treatment

2020 ◽  
pp. 38-44
Author(s):  
N. Osakue ◽  
C. C. Onyenekwe ◽  
F. A. Ehiaghe ◽  
J. E. Ahaneku ◽  
J. I. Ikechebelu ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Inflammatory Cytokines ◽  
First Trimester ◽  
Vitro Fertilization ◽  
Tnf Α ◽  
Before And After ◽  
First Trimester Of Pregnancy ◽  
Ifn Γ ◽  
Pro Inflammatory Cytokines

Background: In vitro fertilization (IVF) is an assisted reproductive technology (ART) that is widely used globally in the treatment of infertility. Infertility can occur due to male factors, female factors or both. Aim: This is the first Nigerian study that sets out to observe the levels and relationship between circulating pro-inflammatory cytokines (IFN-γ, TNF-α) and progesterone (PG) in Nigerian women undergoing in vitro fertilization pre and post treatment and their possible effect on pregnancy outcome. Materials and Methods: This observational study randomly selected sixty-two (62) infertile females below 45 year of age who enrolled in the IVF treatment at Lily Hospitals, Warri and Shepherd Specialist Hospital, Warri, Southern Nigeria. Only data of the thirteen (13) infertile females who became pregnant after the IVF treatment where followed up and presented in this study. Five (5) ml of whole blood were collected into plain tubes on day 3 of the menstrual cycle of all the participants from the ante-cubital vein before and after IVF procedure using standard laboratory collection technique. Ovarian stimulation was done using the long gonadotropin-releasing hormone agonist protocol. Oocyte retrieval transfer was done using ultrasound-guided fine-needle aspiration and embryo transfer was done using ultrasound-guided embryo transfer. IFN-γ, TNF-α and PG were estimated using enzyme-linked immunosorbent assay method. Results and Conclusion: Significant increase in the levels of TNF-α and PG at the second trimester and third trimester of pregnancy when compared with the first trimester of pregnancy (p = 0.000). While the level of IFN-γ was significantly increased in the second trimester of pregnancy when compared with the first trimester of pregnancy (p = 0.000). It is evident from the study that both pro-inflammatory cytokines (IFN-γ and TNF-α) act in synergy to maintain the level of progesterone which act as an anti-inflammatory agent to regulate the activities of the pro-inflammatory cytokines for successful oocytes implantation and maturation.

scholarly journals The Malignant Hemopoietic Clone of Triple Negative Patients with Myelofibrosis Shows in Vitro Functional Defects but Is Highly Responsive to the Pro-Survival Signals of Circulating Autologous Microvesicles

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4334-4334
Author(s):  
Lucia Catani ◽  
Daria Sollazzo ◽  
Dorian Forte ◽  
Martina Barone ◽  
Cristina Morsiani ◽  
...  
Keyword(s):  
Board Of Directors ◽  
Research Funding ◽  
Triple Negative ◽  
Jak2 V617f ◽  
Advisory Committees ◽  
Inflammatory Microenvironment ◽  
Cd34 Cells ◽  
Tnf Α ◽  
In Vitro Survival

Abstract Background Myelofibrosis (MF) is a clonal hemopoietic stem/progenitor cells (HSPCs) disorder with mutations in 3 driver genes (JAK2, MPL, or CALR) and inflammatory microenvironment. Triple negative (TN) patients (pts) do not carry the 3 driver mutations and display a significantly worse survival-rate which can be partially due to a greater mutation complexity. Circulating microvesicles (MVs; 0.1-1 μm), most of megakaryocytes (MKMVs) and platelets (PMVs) origin, play a role in intercellular signalling and are increased in inflammation and cancer including MF. We hypothesized that in TN pts the HSPCs and/or the inflammatory microenvironment show a more aggressive and malignant phenotype. Aims To identify a biomolecular signature of TN pts by comparatively evaluating the circulating HSPCs behaviour and their functional interplay with the inflammatory microenvironment. Methods EDTA-anticoagulated peripheral blood (PB) was collected from 16 MF pts (WHO-2016 criteria-JAK2(V617F) mutated (n=10) and TN (n=6)) and 20 age/sex-matched healthy donors (HD). Driver mutation status was obtained by PCR/NGS. Plasma MVs were isolated by ultracentrifugation (45.000 rpm for 2 hours), counted by Nanosight technology and phenotypically characterized for PMVs (CD61+CD62P+) and MKMVs (CD61+CD62P-) by flow cytometry (size 0.5-0.9 µm). in vitro survival (24 hours), CXCL12-driven migration (12 hours) and clonogenic ability of immunomagnetically isolated CD34+ cells from pts PB or cord blood (CB; n=10) was performed in the presence/absence of the isolated MVs or IL-1β, TNF-α and IL-6 (alone/ in combination). Incorporation of CFSE-labelled MVs into CD34+ cells was analyzed by confocal microscopy. MicroRNA (miRs) profile analysis of the isolated MVs and Gene expression profile (GEP) analysis of CD34+ cells from 3 JAK2(V617F) mutated, 3 TN pts and 3 HD/CB was performed. Plasma inflammatory cytokines levels were evaluated by ELISA. Results We firstly compared the TN and JAK2(V617F) CD34+ cells. TN CD34+ cells showed decreased in vitro survival and clonogenic ability but comparable migration capacity (Fig. 1 A, B, C). Consistently, comparing the GEP, the expression of selected anti-apoptotic (TSPYL5, GFI-1 and FCMR) and pro-apototic (TNFSF10, TP53INP1) genes was significantly down- and up-regulated, respectively, in TN CD34+ cells. We then investigated whether signals from the microenvironment may affect the in vitro behaviour of the TN CD34+ cells. IL-1β, IL-6 and TNF-α plasma levels were significantly increased (p<0.05) in MF pts. However, at variance with the JAK2(V617F) mutated counterparts, survival and clonogenic ability of TN CD34+ cells were not significantly promoted by IL-1β, IL-6 and TNF-α, alone or in combination. This was due, at least in part, to the reduced expression of IL-6 and TNF-α (type I) receptors on TN CD34+ cells. We then analyzed the phenotype of the isolated circulating MVs showing that the PMVs were significantly increased (p<0.05) in the JAK2(V617F) mutated pts only. Conversely, the MKMVs were significantly decreased in both pts groups (p<0.01) as compared to the HD counterparts (Fig. 1 D, E). When we co-cultured CD34+ cells with the isolated MVs we found that TN MVs significantly increased the survival of TN CD34+ cells but inhibited CB CD34+ cells survival (p<0.05, respectively). Conversely, JAK2(V617F) MVs did not significantly affected the survival of CB/pts CD34+ cells. HD MVs significantly increased the survival of CB CD34+ cells (p<0.05) only (Fig. 1 F, G, H). Of note, MVs from pts were transferred into cytosol after co-cultured with CB CD34+ cells (Fig. 1 I). Consistently, comparing the miRs profile of pts and HD MVs, various pro-apoptotic (+) and anti-apoptotic (-) miRs were upregulated. Specifically, miR-155(-), 24(-) and 222(-) were upregulated in TN MVs only; miR-21(-), 19a(-), 34a-5p(+), 423-5p(+) were upregulated in both JAK2(V617F) and TN MVs. Further, miRs regulating inflammation (miR-146a) and proliferation (miR-1274A, downregulated in TN vs JAK2(V617F) MVs), were overexpressed in pts MVs. Conclusions Our results demonstrate that TN CD34+ cells show in vitro defective function and are unresponsive to the inflammatory microenvironment. Interestingly, only the autologous circulating MVs promoted the malignant clone, suggesting that MVs, as signals from microenvironment, may have a pathogenetic role in TN MF and may be the target of novel therapeutic approaches. Figure 1. Figure 1. Disclosures Fabbri: Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Palandri:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

scholarly journals Small molecule therapeutics for COVID-19: repurposing of inhaled furosemide

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e9533 ◽  
Author(s):  
Zhiyu Wang ◽  
Yanfei Wang ◽  
Prachi Vilekar ◽  
Seung-Pil Yang ◽  
Mayuri Gupta ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Small Molecule ◽  
Inflammatory Activity ◽  
Health Concern ◽  
Docking Simulations ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Novel Coronavirus ◽  
Pro Inflammatory Cytokines

The novel coronavirus SARS-CoV-2 has become a global health concern. The morbidity and mortality of the potentially lethal infection caused by this virus arise from the initial viral infection and the subsequent host inflammatory response. The latter may lead to excessive release of pro-inflammatory cytokines, IL-6 and IL-8, as well as TNF-α ultimately culminating in hypercytokinemia (“cytokine storm”). To address this immuno-inflammatory pathogenesis, multiple clinical trials have been proposed to evaluate anti-inflammatory biologic therapies targeting specific cytokines. However, despite the obvious clinical utility of such biologics, their specific applicability to COVID-19 has multiple drawbacks, including they target only one of the multiple cytokines involved in COVID-19’s immunopathy. Therefore, we set out to identify a small molecule with broad-spectrum anti-inflammatory mechanism of action targeting multiple cytokines of innate immunity. In this study, a library of small molecules endogenous to the human body was assembled, subjected to in silico molecular docking simulations and a focused in vitro screen to identify anti-pro-inflammatory activity via interleukin inhibition. This has enabled us to identify the loop diuretic furosemide as a candidate molecule. To pre-clinically evaluate furosemide as a putative COVID-19 therapeutic, we studied its anti-inflammatory activity on RAW264.7, THP-1 and SIM-A9 cell lines stimulated by lipopolysaccharide (LPS). Upon treatment with furosemide, LPS-induced production of pro-inflammatory cytokines was reduced, indicating that furosemide suppresses the M1 polarization, including IL-6 and TNF-α release. In addition, we found that furosemide promotes the production of anti-inflammatory cytokine products (IL-1RA, arginase), indicating M2 polarization. Accordingly, we conclude that furosemide is a reasonably potent inhibitor of IL-6 and TNF-α that is also safe, inexpensive and well-studied. Our pre-clinical data suggest that it may be a candidate for repurposing as an inhaled therapy against COVID-19.

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