scholarly journals Expression of Paraoxonase-2 in Different Stages of Cutaneous T Cell Lymphoma: Is It a Potential Biomarker for Aggressiveness?

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4496-4496
Author(s):  
Erika Morsia ◽  
Serena Rupoli ◽  
Elisa Molinelli ◽  
Davide Sartini ◽  
Anna Maria Offidani ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Enzyme Level ◽  
Stage I ◽  
Positive Staining ◽  
Interesting Phenomenon ◽  
Expression Levels ◽  
Potential Biomarker ◽  
Study Results

Abstract Introduction: Paraoxonase 2 (PON2) is an intracellular membrane protein that belong to the PON gene family. This enzyme exerting a crucial role against production of reactive oxygen species within mithochondrial respiratory chain. Some reports provided clinical evidence for a link between PON2 and different types of malignancies and recently a growing attention has been focused on exploring the role of PON2 in cancer. Moreover, in contrast to solid tumors, a low expression levels of PON2 protein were detected in hematological malignancies like acute myeloid leukemia and B-cell lymphomas. The aim of this study was to investigate the role of PON2 in the cutaneous T cells Lymphoma (CTCL). Methods: We performed an immunohistochemistry analysis of PON2 protein expression at various stages CTCL. We analyzed PON2 protein using skin biopsies from stage I (n= 3) and stage II (n= 6) Mycosis Fungoides (MF) and stage III/IV (n= 5) erythrodermic MF/Sézary Syndrome (SS) and control (n=6) tissues. Values were expressed as percentage of PON2-positive staining cells, whereas the intensity of PON2 positivity was semi-quantitatively scored from negative to -/+/+++. Subsequent statistical analysis was carried out to explore the existence of correlations between intra-tumor enzyme level and clinical-pathological features at diagnosis. Results: In our preliminary study, results showed PON2 down-expression in SS compared to controls. Among CTCL, we found significant differences in enzyme levels between MF and erythrodermic MF/SS; in fact, PON2 expression was higher in patients with not erythrodermic MF compared to patients with erythrodermic MF/SS. (Table I) Moreover, we found no significant differences in enzyme levels between MF patients and controls. Conclusion: Our study is the first to demonstrate downregulation of PON2 intra-tumoral in SS patients while protein levels were higher in stage I and II MF patients. These findings seem to suggest that PON2 expression levels could be negatively related with tumor aggressiveness in CTCL: an interesting phenomenon where PON2 is upregulated in the early stages and downregulated in the late stages of CTCL. Further, in vitro studies are needed to clarify the mechanism related to PON2 expression. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Predominant Complement Mediated Lysis of B-CLL Cells by Therapeutic MAbs Rituximab and Campath-1H in Whole Blood Assays.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3445-3445 ◽  
Author(s):  
Josee Golay ◽  
Luca Bologna ◽  
Elisa Gotti ◽  
Alessandro Rambaldi ◽  
Renato Bassan ◽  
...  
Keyword(s):  
Target Cell ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Leukemic Cells ◽  
Human Igg ◽  
Blood Samples ◽  
Expression Levels ◽  
Cd20 Expression

Abstract Abstract 3445 Poster Board III-333 The mechanism of action of unconjugated MAbs such as Rituximab and Campath-1H in vivo is still a matter of debate. Most in vitro assays with antibodies rely upon purified effector cells or proteins taken outside their natural context, and on target cell lines rather than patients cells. In order to analyse the activity of therapeutic MAbs on circulating leukemic cells in more physiological conditions and in a system the least manipulated as possible, we have set up a whole blood assays using Rituximab and Campath-1H. Peripheral blood samples were drawn from B-CLL patients or normal donors in sodium citrate and antibodies were directly added at different concentrations. We first demonstrated that neither apoptosis, induced by cross-linked anti-CD20 antibody, nor complement mediated cytotoxicity (CDC) induced by Campath-1H or Rituximab were significantly inhibited by citrate used at the standard concentration (0.1 M). We then performed a number of experiments using whole blood samples in citrate, into which increasing concentrations of Rituximab or Campath-1H were added. Lysis was analysed by FACS analysis after different incubation times at 37°C. We observed that Campath-1H very rapidly and efficiently lysed normal B cells or B-CLL targets in vitro in whole blood: maximal lysis was reached within 4 hours and was observed already with 1 and 10 μg/ml antibody (61 %), even though it was still more effective at 25 or 50 μg/ml (up to 90 % lysis). 25 μg/ml is known to be reached in the circulation after 30mg infusions of the antibody 3 times a week. Lysis by Campath-1H was fully complement dependent since it was inhibited by 90% in presence of excess blocking anti-C5 antibody Eculizumab (200 μg/ml). Eculizumab alone in contrast had no effect on cell viability. We then analysed the efficacy of increasing concentrations of Rituximab in the same assay conditions. We observed in general a much reduced lysis with Rituximab compared to Campath-1H, even using antibody up to 200 μg/ml, a concentration that is reached in the circulation after standard 375 mg/m2 administration of the antibody once a week. Lysis showed also slower kinetics, with limited lysis at 4 hours (mean 6.4%) and maximal lysis with Rituximab reached only after 24 hours incubation (mean 18.8%). Also in this case, target cell death was inhibited by at least 90% in presence of Eculizumab, suggesting a major role of complement. Lysis by Rituximab correlated directly with CD20 expression levels (R=0.8) in 13 B-CLL samples analysed, as expected for a mechanism complement dependent. Indeed a mean 29.3% and 73.2% killing could be observed in the two CD20 bright B-CLL, at 4 and 24 hours respectively, whereas a mean of 3.1% and 10.9% lysis was observed in the 11 low-intermediate CD20 samples analysed at the same time points. These data in whole blood confirm our previously published results on the role of CD20 expression levels in CDC of isolated B-CLL cells (Golay et al., Blood 98, 3383-3389, 2001). In contrast to CDC and apoptosis, ADCC was strongly inhibited by citrate as well as several anti-coagulants tested and therefore could not be analysed in this type of assay. Nonetheless in B-CLL samples, NK cells were below detection limit (<0.1%) in most cases analysed, suggesting that ADCC in the circulation is not a major mechanism of lysis in this disease subtype. Finally we determined the effect of citrate on phagocytosis mediated by Rituximab and in vitro differentiated human macrophages. Phagocytosis could be observed in presence of 0.1M citrate (31%, compared to 44% in absence of citrate). Phagocytosis of B-CLL in whole blood was therefore analysed by layering samples directly onto the macrophages. We observed that phagocytosis of B-CLL targets in whole blood was very low (less than 1% over background) compared to a mean of 47% for purified B-CLL targets phagocytosed in normal culture medium. Phagocytosis in whole blood was low presumably due to the presence of high concentration of human IgG in whole blood since as low as 50 μg/ml human IgG is known to inhibit phagocytosis by 90%. We conclude that the major activity of Campath-1H and Rituximab in the circulation is through complement. Apoptosis, ADCC and phagocytosis appear to play a marginal role in this context but may become more important in tissues. The method presented could be used to rapidly screen novel antibodies for their efficacy through either as apoptosis or CDC directly on unmanipulated patients material. Disclosures No relevant conflicts of interest to declare.

scholarly journals MicroRNA-30-3p Suppresses Inflammatory Factor-Induced Endothelial Cell Injury by Targeting TCF21

2019 ◽  
Vol 2019 ◽  
pp. 1-14 ◽  
Author(s):  
Zhenyu Zhou ◽  
Yu Chen ◽  
Dongying Zhang ◽  
Shiyong Wu ◽  
Tao Liu ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Functional Role ◽  
Target Gene ◽  
Luciferase Reporter ◽  
Inflammatory Factor ◽  
Expression Levels ◽  
Potential Biomarker ◽  
Progression Of Atherosclerosis

Atherosclerosis is one of the leading causes of mortality worldwide. Growing evidence suggested that miRNAs contributed to the progression of atherosclerosis. miR-30-5p was found involved in various diseases. However, the role of miR-30-5p in regulation of atherosclerosis is not known. Here, we aim to investigate the effects of miR-30-5p on regulating the progression of atherosclerosis. The expression levels of miR-30-5p in serum collected from atherosclerosis patients and normal healthy people were analyzed by qRT-PCR. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway bioinformatics were carried out to reveal the possible signaling pathways involved in the mode of action of miR-30-5p. A potential target gene of miRNA-30-5p was searched and examined by a luciferase reporter assay. ELISA, Western blot, proliferation, and flow cytometry assays were performed to assess the biological functional role of miR-30-5p in vitro. Also, an in vitro monocyte-endothelial cell coculture model was used to study the functional role of miR-30-5p in atherosclerosis. We found that miR-30-5p was significantly decreased in serum samples from atherosclerosis patients compared with control subjects. GO and KEGG analysis results showed that miR-30-5p is highly associated with genetic profile of cardiovascular disease. TCF21 was verified as a target gene of miR-30-5p. Overexpression of miR-30-5p in THP-1 not only protected endothelial cell viability but also inhibited endothelial cell apoptosis, and similar results were observed in cells with that of TCF21 knocked down. Moreover, miR-30-5p decreased the expression levels of lactate dehydrogenase (LDH) and tumor necrosis factor-α (TNF-α) and reduced reactive oxygen species (ROS) accumulation. NF-κB and MAPK/p38 pathways played an indispensable role in the protection ability of miR-30-5p against atherosclerosis. Our results reveal that miR-30-5p suppresses the progression of atherosclerosis through targeting TCF21 in vitro. Therefore, the miR-30-5p-TCF21-MAPK/p38 signaling pathway may be a potential biomarker or therapeutic target in atherosclerosis.

scholarly journals Hsa_circ_0005273 facilitates breast cancer tumorigenesis by regulating YAP1-hippo signaling pathway

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Xuehui Wang ◽  
Changle Ji ◽  
Jiashu Hu ◽  
Xiaochong Deng ◽  
Wenfang Zheng ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Hippo Signaling ◽  
Hippo Signaling Pathway ◽  
Potential Biomarker

Abstract Background Circular RNAs (circRNAs), a novel class of endogenous RNAs, have shown to participate in the development of breast cancer (BC). Hsa_circ_0005273 is a circRNA generated from several exons of PTK2. However, the potential functional role of hsa_circ_0005273 in BC remains largely unknown. Here we aim to evaluate the role of hsa_circ_0005273 in BC. Methods The expression level of hsa_circ_0005273 and miR-200a-3p were examined by RT-qPCR in BC tissues and cell lines. The effect of knocking down hsa_circ_0005273 in BC cell lines were evaluated by examinations of cell proliferation, migration and cell cycle. In addition, xenografts experiment in nude mice were performed to evaluate the effect of hsa_circ_0005273 in BC. RNA immunoprecipitation assay, RNA probe pull-down assay, luciferase reporter assay and fluorescence in situ hybridization were conducted to confirm the relationship between hsa_circ_0005273, miR-200a-3p and YAP1. Results Hsa_circ_0005273 is over-expressed in BC tissues and cell lines, whereas miR-200a-3p expression is repressed. Depletion of hsa_circ_0005273 inhibited the progression of BC cells in vitro and in vivo, while overexpression of hsa_circ_0005273 exhibited the opposite effect. Importantly, hsa_circ_0005273 upregulated YAP1 expression and inactivated Hippo pathway via sponging miR-200a-3p to promote BC progression. Conclusions Hsa_circ_0005273 regulates the miR-200a-3p/YAP1 axis and inactivates Hippo signaling pathway to promote BC progression, which may become a potential biomarker and therapeutic target.

scholarly journals Cerebrospinal Fluid Pulsation Stress Promotes the Angiogenesis of Tissue-Engineered Laminae

2020 ◽  
Vol 2020 ◽  
pp. 1-12
Author(s):  
Linli Li ◽  
Yiqun He ◽  
Han Tang ◽  
Wei Mao ◽  
Haofei Ni ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Cerebrospinal Fluid ◽  
Engineering Technology ◽  
Expression Levels ◽  
Fluid Pulsation ◽  
Endothelial Growth Factor ◽  
The Impact

Background. Angiogenesis is a prerequisite step to achieve the success of bone regeneration by tissue engineering technology. Previous studies have shown the role of cerebrospinal fluid pulsation (CSFP) stress in the reconstruction of tissue-engineered laminae. In this study, we investigated the role of CSFP stress in the angiogenesis of tissue-engineered laminae. Methods. For the in vitro study, a CSFP bioreactor was used to investigate the impact of CSFP stress on the osteogenic mesenchymal stem cells (MSCs). For the in vivo study, forty-eight New Zealand rabbits were randomly divided into the CSFP group and the Non-CSFP group. Tissue-engineered laminae (TEL) was made by hydroxyapatite-collagen I scaffold and osteogenic MSCs and then implanted into the lamina defect in the two groups. The angiogenic and osteogenic abilities of newborn laminae were examined with histological staining, qRT-PCR, and radiological analysis. Results. The in vitro study showed that CSFP stress could promote the vascular endothelial growth factor A (VEGF-A) expression levels of osteogenic MSCs. In the animal study, the expression levels of angiogenic markers in the CSFP group were higher than those in the Non-CSFP group; moreover, in the CSFP group, their expression levels on the dura mater surface, which are closer to the CSFP stress stimulation, were also higher than those on the paraspinal muscle surface. The expression levels of osteogenic markers in the CSFP group were also higher than those in the Non-CSFP group. Conclusion. CSFP stress could promote the angiogenic ability of osteogenic MSCs and thus promote the angiogenesis of tissue-engineered laminae. The pretreatment of osteogenic MSC with a CSFP bioreactor may have important implications for vertebral lamina reconstruction with a tissue engineering technique.

scholarly journals 2114

2017 ◽  
Vol 1 (S1) ◽  
pp. 57-57
Author(s):  
Jennifer Knudtson ◽  
Ya-Guang Liu ◽  
Marlen Tellez Santos ◽  
Rajeshwar Tekmal ◽  
Ratna Vadlamudi ◽  
...  
Keyword(s):  
Positive Staining ◽  
Test Results ◽  
Endometriotic Lesion ◽  
Vitro Assay ◽  
Epithelial Origin ◽  
Immortalized Cells ◽  
Study Population ◽  
Chromosomal Profile

OBJECTIVES/SPECIFIC AIMS: To further elucidate the role of estrogen receptor β (ER-β) in the early endometriotic lesion attachment. METHODS/STUDY POPULATION: EECs were immortalized using a telomerase vector. Immortalized cells and parental cells were characterized by genotyping, and expression of ER-β as well as other epithelial cell markers. ER-β was knocked-down in immortalized EECs using lentivirus-mediated shRNA transduction. ER-β knockdown was confirmed by RT-qPCR and Western analysis. EEC cells with or without ER-β knockdown were used to assess their attachment to PMCs in an established in vitro assay (Lucidi, 2005). Results were analyzed with Student t-test. RESULTS/ANTICIPATED RESULTS: Genotyping using karyotype assay confirmed a normal chromosomal profile. Also positive staining for cytokeratin and lack of any staining with vimentin confirms the epithelial origin of these cells. ER-β knockdown has a significant decrease in attachment compared to control (p=0.02). DISCUSSION/SIGNIFICANCE OF IMPACT: Primary and immortalized cells were 46XX, cytokeratin positive, and vimentin negative confirming their epithelial origin. ER-β knockdown has a significant decrease in attachment compared with control.

scholarly journals Long noncoding RNA 00976 promotes pancreatic cancer progression through OTUD7B by sponging miR-137 involving EGFR/MAPK pathway

2019 ◽  
Vol 38 (1) ◽  
Author(s):  
Shan Lei ◽  
Zhiwei He ◽  
Tengxiang Chen ◽  
Xingjun Guo ◽  
Zhirui Zeng ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Signaling Pathway ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Mapk Signaling Pathway ◽  
Migration And Invasion ◽  
Potential Biomarker

Abstract Background Accumulation evidence indicates the vital role of long non-coding RNAs (lncRNAs) in tumorigenesis and the progression of malignant tumors, including pancreatic cancer (PC). However, the role and the molecular mechanism of long non-coding RNA 00976 is unclear in pancreatic cancer. Methods In situ hybridization (ISH) and qRT-PCR was performed to investigate the association between linc00976 expression and the clinicopathological characteristics and prognosis of patients with PC. Subsequently, linc00976 over-expression vector and shRNAs were transfected into PC cells to up-regulate or down-regulate linc00976 expression. Loss- and gain-of function assays were performed to investigate the role of linc00976 in proliferation and metastasis in vitro and vivo. ITRAQ, bioinformatic analysis and rescue assay were used to illustrate the ceRNA mechanism network of linc00976/miR-137/OTUD7B and its downstream EGFR/MAPK signaling pathway. Results linc00976 expression was overexpressed in PC tissues and cell lines and was positively associated with poorer survival in patients with PC. Function studies revealed that linc00976 knockdown significantly suppressed cell proliferation, migration and invasion in vivo and in vitro, whereas its overexpression reversed these effects. Based on Itraq results and online database prediction, Ovarian tumor proteases OTUD7B was found as a downstream gene of linc00976, which deubiquitinated EGFR mediates MAPK signaling activation. Furthermore, Bioinformatics analysis and luciferase assays and rescue experiments revealed that linc00976/miR137/OTUD7B established the ceRNA network modulating PC cell proliferation and tumor growth. Conclusion The present study demonstrates that linc00976 enhances the proliferation and invasion ability of PC cells by upregulating OTUD7B expression, which was a target of miR-137. Ultimately, OTUD7B mediates EGFR and MAPK signaling pathway, suggesting that linc00976/miR-137/OTUD7B/EGFR axis may act as a potential biomarker and therapeutic target for PC.

Characterization of Receptors Involved in Heparin Antibody Complex Mediated Induction of Tissue Factor Expression in Monocytes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 224-224 ◽  
Author(s):  
Sam Glover ◽  
Nigel S. Key ◽  
Gowthami M Arepally ◽  
Nigel Mackman ◽  
Raj S. Kasthuri
Keyword(s):  
Tissue Factor ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Drug Induced ◽  
Peripheral Blood Mononuclear ◽  
Platelet Factor ◽  
Antibody Complex

Abstract Abstract 224 Introduction: Heparin-induced thrombocytopenia (HIT) is a major cause of drug-induced thrombocytopenia and occurs in 1–5% of individuals exposed to heparin. Paradoxically, 30–50% of individuals with HIT develop thrombosis. The mechanism of thrombosis in HIT is poorly understood. We recently reported that HIT antibody complexes induce tissue factor (TF) expression in monocytes and result in the release of TF-positive microparticles (MPs). The mechanism by which HIT antibody complexes induce monocyte TF has not been established. The objective of this study is to characterize the receptors involved in HIT antibody complex mediated induction of TF expression in monocytes. As HIT antibody complex mediated activation of platelets is dependent on the FcgRIIA receptor, we evaluated the role of the FcgRII receptor in the induction of monocyte TF by HIT antibody complexes. We also evaluated the role of toll like receptor-4 (TLR4) and the platelet factor 4 (PF4) chemokine receptor CXCR3 in this process. Methods: The combination of heparin, PF4 and the murine monoclonal PF4/heparin-specific antibody KKO has been shown to cause activation of platelets and monocytes, and mimic HIT in vitro. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were pre-incubated for 30 min at 37°C with an inhibitory antibody to the FcgRII receptor (IV.3); anti-CXCR2, 3, or 4 antibodies; anti-TLR4 antibody; or mouse-IgG (mIgG) control. Following pre-incubation with antibodies for 30 minutes, heparin (1U/mL), PF4 (10μg/mL), and KKO (100μg/mL) – together referred to as the HIT antibody complex – were added. Heat-aggregated mIgG and LPS were used as positive controls for the FcgRII and TLR4 receptors, respectively. Following a 6-hour incubation, PBMCs were pelleted by centrifugation and MPs were isolated from the supernatant. The procoagulant activity (PCA) of PBMCs and MPs was measured using clotting assays performed in the presence of the anti-TF antibody HTF-1 or control antibody. TF dependent PCA was calculated by reference to a standard curve generated using relipidated recombinant TF. Results: Incubation of PBMCs with heat aggregated mIgG for 6 hours resulted in significant induction of cellular TF (345 +/− 36 pg/106 cells) which was blocked by 30 min pre-incubation with the antibody IV.3 (146 +/− 17 pg/106 cells, N=3, p<.003). However, pre-incubation with IV.3 had no significant effect on TF induction (140 +/− 5 pg/106 cells) associated with the HIT antibody complex when compared to control mIgG (110 +/− 18 pg/106 cells, N=3, p<0.11). PBMCs incubated with HIT antibody complexes in the presence of a TLR-4 antibody showed less TF activity (52 +/− 4 pg/106 cells) compared to control mIgG (80 +/− 10 pg/106 cells N=3, p<0.025). A similar, partial inhibition of TF activity was also observed in PBMCs incubated with LPS in the presence of an anti-TLR4 antibody (121 +/− 3 pg/106) compared with a control antibody (89 +/− 2 pg/106, N=3, p<.0013). Experiments with a more effective inhibitor of TLR4 are in progress. PBMCs incubated with the HIT antibody complexes in the presence of an anti-CXCR3 antibody showed less TF activity (36 +/− 7 pg/mL) compared to control mIgG (118 +/− 15 pg/106 cells, N=3, p<0.004). Antibodies against CXCR2 and CXCR4 did not have any significant effect on TF induction. Measurement of MP TF activity mirrored the results described above. Using flow cytometry and an anti-CXCR3 antibody labeled with FITC, we found that 5% (± 0.5%) of monocytes expressed CXCR3 (N=3), which is consistent with the reported literature. Conclusions: These data suggest that induction of TF in monocytes by HIT antibody complexes is not mediated by the FcgRII receptor. This is contrary to the mechanism of platelet activation by these antibody complexes, which is an FcgRIIa dependent process. We found that TLR4 plays a role in HIT antibody complex mediated induction of TF in monocytes and blocking TLR4 led to a 30% decrease in TF activity. On the other hand, CXCR3 appeared to play a more significant role with blockade of CXCR3 leading to a 70% decrease in TF activity. Further characterization of the role of these receptors in HIT antibody complex mediated induction of TF expression in monocytes is required. We speculate that the extent of CXCR3 and TLR4 expression in monocytes may influence the susceptibility to developing thrombotic complications in HIT. Disclosures: No relevant conflicts of interest to declare.

A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2332-2332
Author(s):  
Vitalyi Senyuk ◽  
Yunyuan Zhang ◽  
Yang Liu ◽  
Ming Ming ◽  
Jianjun Chen ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Ectopic Expression ◽  
Myeloid Differentiation ◽  
Dna Hypermethylation ◽  
Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

Plasmin Cleaves Complement iC3b, Preventing CR3/4 Mediated Phagocytosis By Macrophages

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1105-1105
Author(s):  
Erica A. Peterson ◽  
Jonathan H Foley ◽  
Michael J Krisinger ◽  
Edward Conway
Keyword(s):  
Flow Cytometry ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Tissue Injury ◽  
Complement Receptors ◽  
Red Cell Membrane ◽  
Western Blots ◽  
Complement Pathways

Abstract Introduction The plasmin(ogen) and complement systems are activated at sites of tissue injury and are involved in hemostasis, wound healing, inflammation and immune surveillance. Although the mechanisms are poorly understood, dysregulation of these systems underlie the pathogenesis and progression of inflammatory and vascular diseases. We aimed to characterize the relevant molecular interactions between the plasmin(ogen) and complement pathways. The three complement pathways converge with formation of C3-convertases that cleave C3 into C3a and C3b. C3a is liberated as an anaphylatoxin while C3b participates in further formation of the C3 and C5 convertases, thereby amplifying complement activation. To dampen the system, negative regulatory mechanisms exist. C3b is degraded to iC3b by the factor I (FI)/FH complex, which in turn is degraded to C3dg by the FI/complement receptor 1 (CR1) complex. iC3b and C3dg induce cellular responses by binding to complement receptors CR3 / CR4 / CR2, and CR2, respectively. Interactions of iC3b with CR3 or CR4 induce phagocytosis by macrophages, and binding of iC3b or C3dg to CR2 promotes B-cell responses. Recent studies show that plasmin proteolyses C3b and iC3b. We further characterized the plasmin cleavage sites in iC3b and evaluated the functional consequences in vitro. Methods and Results Plasmin cleavage of iC3b was examined over a range of concentrations and times. Plasmin (50 nM) generated a 40 kDa iC3b cleavage fragment (946TLD – PSR1303) which was notable for containing both C3dg (1002HLI – PSR1303) and the C3 thioester domain, necessary for opsonic binding to surfaces. We tested the relevance of this cleavage in phagocytosis assays using immunofluorescence and flow cytometry (Figure 1). C3b bound to the surface of fluorescent (Alexa 488) zymosan particles (C3b-zym), was treated with FI/FH to generate iC3b-zym, and subsequently incubated with FI/CR1 or plasmin to yield C3dg-zym or 946TLD – PSR1303-zym, respectively. Western blots confirmed that plasmin generated 946TLD – PSR1303 from iC3b-zym. The C3 fragment-zymosan species (C3b-zym, iC3b-zym, C3dg-zym and 946TLD – PSR1303-zym) were each incubated with macrophages (PMA-differentiated THP-1 cells) for 90 minutes. Cells were washed, stained and fixed for immunofluorescence, or suspended for flow cytometry. Figure 1, panel A shows macrophages stained with CellMask (red, cell membrane) and DAPI (blue, nucleus). Fluorescent zymosan is seen in green. No phagocytosis was detected with zymosan lacking C3 (zym alone), but there was a small amount with C3b-zym. In contrast, iC3b-zym was highly effective in inducing phagocytosis by most macrophages. This effect of iC3b-zym was abolished with FI/CR1 or plasmin, i.e. little phagocytosis was detected with C3dg-zym or 946TLD – PSR1303-zym. Flow cytometry-based quantitative analyses confirmed the preceding findings (Figure 1, panel B), with a similar pattern of phagocytosis induced by the zymosan-bound fragments. No phagocytosis was detected with zymosan lacking C3. Phagocytosis of C3b-zym and iC3b-zym was 7±2% and 17±1% of cells, respectively. C3dg-zym and 946TLD – PSR1303-zym induced phagocytosis was <5%. We also evaluated the role of the complement receptors in mediating the effect of the C3b/iC3b fragments using CR3/4 and CR1 blocking antibodies. These confirmed that phagocytosis of iC3b-zym and C3b-zym is mediated by CR3/4 and CR1, respectively. Conclusions Plasmin cleaves iC3b to form a redundant complement regulatory pathway with the FI/CR1 complex, but which notably does not require a cellular cofactor. Further studies will delineate the role of this and other plasmin-generated complement fragments in modulating innate immune and inflammatory responses. Disclosures: No relevant conflicts of interest to declare.

The Role Of Deregulated HMGA2 Expression With Promoter Methylation Of p16 In Myeloproliferative Neoplasms

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1606-1606
Author(s):  
Kayo Shirado Harada ◽  
Kazuhiko Ikeda ◽  
Kazuei Ogawa ◽  
Hideyoshi Noji ◽  
Hideo Kimura ◽  
...  
Keyword(s):  
Promoter Methylation ◽  
Blood Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasms ◽  
Expression Levels ◽  
Allele Burden ◽  
Polycomb Group Genes ◽  
Micro Rnas ◽  
Jak2v617f Allele Burden

Abstract Myeloproliferative Neoplasms (MPNs) are characterized by clonal proliferative hematopoiesis with increased mature blood cells. The signal-activating mutations such as JAK2V617F increase blood cells, but it remains uncertain how an abnormal hematopoietic cell clone expands in MPNs. We have recently showed that overexpression of the high mobility group AT-hook 2 (HMGA2) causes proliferative hematopoiesis with providing a clonal growth advantage to hematopoietic cells in mice (Ikeda et al, Blood, 2011), suggesting the possibility that HMGA2 contributes to the pathogenesis of MPNs. However, since only a few studies have evaluated expression of HMGA2 mRNA in patients with MPNs, the role of HMGA2 in the pathogenesis of MPNs is yet unclear. MPNs also show mutations in epigenetic modifiers involving DNA methylation such as polycomb group genes (PcG) and aberrant expressions of micro RNAs (miRNA) that negatively regulate expressions of targeted genes. Interestingly, deficiency in either PcG-related BMI1 (Oguro et al, J Exp Med, 2012) or let-7-family miRNA (Mayr et al, Science, 2007) causes deregulation of HMGA2 expression, leading to its oncogenic activity in part by negatively regulating tumor suppressor p16. Thus, in this study, to clarify the role of HMGA2 in MPNs, we investigated expression of HMGA2 mRNA in peripheral granulocytes of 56 patients with MPNs including 23 polycythemia vera (PV), 26 essential thrombocythemia (ET) and 7 primary myelofibrosis (PMF) along with clinical findings, JAK2V617F allele burden, expressions of BMI1 mRNA and let-7-family miRNAs, and promoter methylation of p16. Quantitative RT-PCR (qPCR) showed significantly higher expression of HMGA2 mRNA relative to internal control HPRT1 mRNA in PMF (mean ± SD; 31.7 ± 42.8, p<0.01), but not PV (15.7 ± 53.2) or ET (2.14 ± 7.70), compared with 12 healthy volunteers (HV; 0.431 ± 0.366). In addition, deregulated HMGA2 expression (>1.2), which was determined as relative expression level above mean + 2SD of HMGA2 mRNA in 12 HV, was most frequently detected in patients with PMF [7/7 (100%)] (p<0.01), compared with PV [5/23 (21.7%)] and ET [6/26 (23.1%)]. We also found a significant positive correlation in expression levels of HMGA2 mRNA with serum LDH values (r=0.531, p<0.01) rather than JAK2V617F allele burden (r=0.25, p=0.08). These data suggested that expression of HMGA2 mRNA independently correlated with disease phenotype and status in MPNs. We next explored the cause of deregulated expression of HMGA2 mRNA and found lower expression of let-7a (0.19 ± 0.13 vs. 0.42 ± 0.39, p=0.04) and -7c (0.57 ± 0.60 vs. 1.14 ± 0.94, p=0.06) rather than -7b (p=0.2) by qPCR, in patients with deregulated expression of HMGA2 mRNA compared with other patients. However, HMGA2-involved chromosomal abnormality in 12q13-15 was not detected in any patient, and there was no difference in expression of BMI1 mRNA between patients with deregulated expression of HMGA2 mRNA and other patients. Thus, decreased expression of let-7 miRNAs might contribute to deregulated expression of HMGA2 mRNA in MPNs. Finally, we investigated correlation of deregulated expression of HMGA2 mRNA with promoter methylation of p16. Methylation-specific PCR assay detected promoter methylation of p16 in 17/56 (30.4%) patients with MPNs. Strikingly, patients with deregulated expression of HMGA2 mRNA significantly more often showed promoter methylation of p16 compared with other patients [10/18 (55.6%) vs. 7/38 (18.4%), p<0.01]. Furthermore, patients with promoter methylation of p16 showed higher expression levels of HMGA2 mRNA than patients without the methylation, especially in patients with PMF (2.33 ± 0.90 vs. 70.9 ± 38.3, p=0.01). In conclusion, deregulated expression of HMGA2 in association with decreased expression of let-7 miRNAs may play a crucial role in the pathogenesis of MPNs possibly through p16. Disclosures: No relevant conflicts of interest to declare.

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