scholarly journals High-Dose Dexamethasone Corrects the Elevated Level of IL-16 in Immune Thrombocytopenia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2431-2431
Author(s):  
Shuwen Wang ◽  
Qi Feng ◽  
Yu Hou ◽  
Anli Liu ◽  
Mingqiang Hua ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Caspase 3 ◽  
Mononuclear Cells ◽  
Single Agent ◽  
High Dose ◽  
Mrna Levels ◽  
Healthy Controls ◽  
High Dose Dexamethasone

Abstract Introduction: Adult primary immune thrombocytopenia (ITP) is an autoimmune-mediated haemorrhagic disorder characterized by excessive platelet destruction and decreased platelet production. ITP patients have a Th1 dominant profile, which was involved in the pathogenesis of ITP. Interleukin-16 (IL-16) can directly affect cellular or humoral immunity by mediating the cellular cross-talk among T cells, B cells and dendritic cells. Several studies have focused on IL-16 as an immunomodulatory cytokine that takes part in Th1 polarization in autoimmune diseases, but the roles of IL-16 in ITP remain unknown. Treatment with high-dose dexamethasone (HD-DXM) as a single-agent for four days has been widely recognized as the first-line therapy for ITP patients in need of clinical management. The aim of this study is to investigate the roles of IL-16 and effect of HD-DXM on IL-16 in ITP. Methods: We investigated the differences in IL-16 expression in the bone marrow and peripheral blood between ITP patients and healthy controls by ELISA. The mRNA expression of pro-IL-16, caspase-3 and T-bet in active ITP patients and healthy controls were quantified by real-time PCR. Furthermore, we detected changes in IL-16 concentration and gene expression before and after the 4-day HD-DXM therapy. Bone marrow samples were obtained from 23 patients and 10 healthy bone marrow donors, and peripheral blood samples were obtained from 64 patients (among them, 21 received single-agent HD-DXM therapy, the peripheral blood samples were obtained before HD-DXM therapy and 28 days after HD-DXM administration) and 38 healthy blood donors. Adult primary ITP patients with active disease were enrolled in this study between May 2015 and January 2018 at the Department of Haematology, Qilu Hospital, Shandong University, Jinan, China. Results: The concentration of IL-16 in the bone marrow supernatants and plasma of active ITP patients was significantly higher compared with that of the healthy controls (P < 0.05) (Fig. 1A, B). Gene expression of pro-IL-16 and caspase-3 in bone marrow mononuclear cells (BMMCs) in ITP patients elevated compared with healthy controls (P < 0.05) (Fig. 2A). Besides, gene expression of pro-IL-16, caspase-3 and T-bet in peripheral blood mononuclear cells (PBMCs) in ITP patients elevated (P < 0.05) (Fig. 2B). Among the 21 patients which received single-agent HD-DXM therapy, 18 responded effectively to the HD-DXM therapy according to the standard definition. The post-treatment plasma (n=21) IL-16 level decreased significantly compared with that of pre-treatment level (P < 0.05), and the level of IL-16 in the plasma of ITP patients before and after HD-DXM treatment was significantly higher than that of healthy controls (P < 0.05) (Fig. 1C). There was no correlation between IL-16 levels in the bone marrow supernatant or plasma and platelet count (data not shown). In addition, pro-IL-16, caspase-3, T-bet mRNA expression was significantly decreased after treatment with HD-DMX (P < 0.05) (Fig. 2C), although the corresponding values in patients were not statistically higher than those of healthy controls. The correlation between the plasma IL-16 concentration and pro-IL-16, caspase-3 and T-bet mRNA levels was also analysed in ITP patients (n = 21). The results demonstrated that the mRNA levels of the three detected factors were positively correlated with plasma IL-16 concentration (r2 = 0.5846, P < 0.05, r2 = 0.3980, P < 0.05 and r2 = 0.3145, P <0.05 for pro-IL-16, caspase-3 and T-bet, respectively) (Fig. 2D-F). Conclusions: ITP is an autoimmune disorder; complex interactions among antigen-presenting cells, T cells and B cells are pivotal to its pathogenesis. Our data suggest that IL-16 plays an important role in the pathogenesis of ITP by polarization of Th1. Furthermore, by modulating the abnormal IL-16 level associated with the Th1 imbalance via treatment with pulsed HD-DXM provided us with new insights into the immune regulatory mechanisms for the treatment of ITP. In our future study, we will use an in vitro study system to test and verify if the anti-IL-16 antibody can be used to treat adult ITP. Disclosures No relevant conflicts of interest to declare.

High Cereblon Expression In Lower Risk Myelodysplastic Syndromes With 5q Deletion Is Associated With The Efficacy Of Lenalidomide

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1529-1529
Author(s):  
Anna T Jonasova ◽  
Jaroslav Polak ◽  
Martin Vostry ◽  
Arnost Kostecka ◽  
Radka Bokorova ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Myelodysplastic Syndromes ◽  
Peripheral Blood ◽  
Lower Risk ◽  
Mononuclear Cells ◽  
Low Risk ◽  
Mrna Levels ◽  
Healthy Controls ◽  
5Q Deletion

Abstract Introduction The binding of immunomodulatory drugs (IMiDs), including lenalidomide, to CRBN is associated with cytotoxicity of IMiDs used in the treatment of multiple myeloma, myelodysplastic syndromes (MDS) and lymphomas. Cereblon (CRBN) is named for its putative role in cerebral development, especially in memory and learning. CRBN forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), cullin-4 (CUL4) and regulator of cullin 1 (ROC1). This complex regulates DNA repair, DNA replication and transcription. CRBN is a primary target of thalidomide teratogenicity (Ito et al., Science 2010; 327:1345-1350). Down-regulation of the CRBN expression is associated with the development of marked IMiDs resistance in human multiple myeloma cells (Zhu et al., Blood 2011; 118: 4771-4779; Lopez Girona et al., Leukemia 2012; 26: 2326-2335; Heintel et al., Br J Haematol 2013; 161: 695-700; Broyl et al., Blood 2013; 121: 624-627; Lodé et al., Br J Haematol 2013; Jul 17. doi: 10.1111/bjh.12478). CRBN expression is thus required for the antimyeloma activity of IMiDs. Aims To gain insight into the role of CRBN in lower risk myelodysplastic syndromes with or without 5q deletion and into the mechanisms of lenalidomide action, we study CRBN expression in these two groups of lower risk MDS patients and in healthy controls. We further study the expression of DDB1 and IRF4 (interferon regulatory factor 4, one of target genes of CRBN). Methods Mononuclear cells were isolated both from bone marrow [23 low risk MDS patients with 5q- deletion, 37 low-risk MDS patients with normal chromosome 5 (non5q-) and 24 healthy controls] and from peripheral blood [38 patients with 5q- , 52 non 5q- patients and 25 healthy controls] by Ficoll-Paque PLUS gradient separation, and washed with phosphate-buffered saline. The rest of the red cells were lysed. Total RNA was isolated using RNA isolation solvent and complementary DNA was synthesized from total RNA using SuperScript II reverse transcriptase. Relative levels of the CRBN, DDB1 and IRF4 mRNAs were determined by TaqMan-based quantitative real-time PCR and by calculation to the level of housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The experiments were performed in duplicate. Informed consent was obtained from all patients and healthy controls. Evaluation of 2-ddCt indicates the fold change in gene expression relative to the control. Results The median of CRBN mRNA levels in total RNA isolated from peripheral blood mononuclear cells was the highest (3.6) in lower risk MDS with 5q deletion, lower (1.5) in non5q- MDS and lowest (1.2) in healthy controls. Similar results were obtained in bone marrow mononuclear cells (medians 3.3 in 5q- syndrome, 2.2 in non5q- MDS and 1.3 in healthy controls). The differences between the 3 groups were statistically significant (p˂0.05, Mann-Whitney test) with the exception of non5q- MDS in comparison with healthy controls in mononuclear cells of peripheral blood. Very similar results were obtained in the analysis of DDB1 and IRF4 mRNAs levels, which correlated with CRBN levels. We analyzed CRBN mRNA levels before and in the course of the lenalidomide treatment of 5q- low risk MDS (6 patients). We obtained high CRBN mRNA levels before and during the treatment by lenalidomide in all lenalidomide responders (4 patients). We found a sharp decrease of CRBN mRNA in mononuclear cells of bone marrow in two patients who stopped responding to lenalidomide therapy and subsequently progressed to higher risk MDS. A/G polymorphism located at -29 nt of the 5x UTR of CRBN does not act as a biomarker of response to treatment with lenalidomide in 5q- syndrome. Conclusions 5q- low risk MDS patients have the highest levels of CRBN mRNA in comparison to lower risk myelodysplastic syndromes with normal chromosome 5 and to healthy controls. DDB1 and IRF4 mRNAs levels correlate with CRBN levels. High levels of CRBN mRNA were detected in all lenalidomide responders during the course of the therapy. Significant decrease of CRBN levels during treatment by lenalidomide is associated with loss of response to treatment and disease progression. Similarly to the treatment of multiple myeloma, these results suggest that high levels of CRBN mRNA are necessary for the efficacy of lenalidomide in low risk 5q- patients. Supported by the MH CR grant NT/13836-4/2012, PRVOUK-27/LF1/2 and by the MH CR project for development of research institute (UHKT). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Atypical hodgkin and reed-sternberg cells in peripheral blood of a patient with advanced stage of Hodgkin's disease - a case report

2003 ◽  
Vol 131 (9-10) ◽  
pp. 400-402 ◽  
Author(s):  
Rajko Milosevic ◽  
Milica Colovic ◽  
Vesna Cemerikic-Martinovic ◽  
Natasa Colovic ◽  
Marina Bogunovic
Keyword(s):  
Bone Marrow ◽  
Lymph Nodes ◽  
Peripheral Blood ◽  
Hodgkin's Disease ◽  
Mononuclear Cells ◽  
Hodgkin’S Disease ◽  
Lymphoid Cells ◽  
High Dose ◽  
Advanced Stage ◽  
Neck Lymph Nodes

The occurrence of abnormal Hodgkin's and Reed-Sternberg cells in the peripheral blood in a patient suffering from Hodgkin's disease has been noticed exceptionally rare in a previous period, and especially rare in last ten years primarily due to successfull treatment of this disease. The presence of atypical mononuclear cells in peripheral blood which cytomorphologically resembled Reed-Sternberg cells was registered in 8 patients till 1966. During the last decade, the presence of atypical mononuclear cells in the peripheral blood was used for their isolation cultivation, and detailed immunophenotypic and genetic analysis. The analysis of mononuclear cells in rare patients with Hodgkin's disease was established that they belong to the B-lymphoid cells with expression of CD30 and CD15 antigens. The examination of presence of Hodgkin's cells in the peripheral blood of patients with Hodgkin's disease is important for patients with advanced stage of the disease in which autologous stem cell transplantation and high dose chmeotherapy is planned. The authors present a 33-year-old patient, who noticed enlarged neck lymph nodes in September 2000, high temperature and loss in weight. On physical examination enlarged neck lymph nodes 5x8 cm and hepatosplenomegaly were found. There was anemia and thrombo-cytopenia, and normal WBC count with 24% of lymphoid elements in differential formula. On histologic examination of lymph nodes Hodgkin?s disease, type nodular sclerosis with mixed cellularity was found. Histology of bone marrow showed nodal lymphomatous infiltration. Immunohistochemistry with monoclonal antibodies of concentrate of peripheral blood cells showed expression of CD30+ and CD15+, immunophenotypically and morphologically matching Reed-Sternberg cells. Cytogentic analysis of mononuclear cells of the bone marrow showed normal karyotype. The patient was in clinical stage IV/V of the disease and chemotherapy with 9 cycles of ABVD+Mp protocol was applied. He is still in remission.

Oxidative DNA base modifications in peripheral blood mononuclear cells of patients treated with high-dose infusional doxorubicin

Blood ◽  
2001 ◽  
Vol 97 (9) ◽  
pp. 2839-2845 ◽  
Author(s):  
James H. Doroshow ◽  
Timothy W. Synold ◽  
George Somlo ◽  
Steven A. Akman ◽  
Ewa Gajewski
Keyword(s):  
Peripheral Blood Mononuclear Cell ◽  
Mononuclear Cell ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Single Agent ◽  
Gas Chromatography Mass Spectrometry ◽  
High Dose ◽  
Selected Ion Monitoring ◽  
Peripheral Blood Mononuclear ◽  
Dna Base

Abstract In prior studies, it was demonstrated that the redox metabolism of doxorubicin leads to the formation of promutagenic oxidized DNA bases in human chromatin, suggesting a potential mechanism for doxorubicin-related second malignancies. To determine whether a similar type of DNA damage is produced in the clinic, peripheral blood mononuclear cell DNA from 15 women treated with infusional doxorubicin (165 mg/m2) as a single agent was examined for 14 modified bases by gas chromatography/mass spectrometry with selected ion monitoring. Prior to the 96-hour doxorubicin infusion, 13 different oxidized bases were present in all DNA samples examined. Chemotherapy, producing a steady-state level of 0.1 μM doxorubicin, increased DNA base oxidation up to 4-fold compared to baseline values for 9 of the 13 bases studied. Maximal base oxidation was observed 72 to 96 hours after doxorubicin treatment was begun; the greatest significant increases were found for Thy Gly (4.2-fold), 5-OH-Hyd (2.5-fold), FapyAde (2.4-fold), and 5-OH-MeUra (2.4-fold). The level of the promutagenic base FapyGua increased 1.6-fold (P &lt; .02), whereas no change in 8-OH-Gua levels was observed in peripheral blood mononuclear cell DNA during the doxorubicin infusion. These results suggest that DNA base damage similar to that produced by ionizing radiation occurs under clinical conditions in hematopoietic cells after doxorubicin exposure. If doxorubicin-induced DNA base oxidation occurs in primitive hematopoietic precursors, these lesions could contribute to the mutagenic or toxic effects of the anthracyclines on the bone marrow.

scholarly journals Expression of the antiviral protein Mx in peripheral blood mononuclear cells of pregnant and bred, non-pregnant ewes

2001 ◽  
Vol 170 (2) ◽  
pp. R7-11 ◽  
Author(s):  
SJ Yankey ◽  
BA Hicks ◽  
KG Carnahan ◽  
AM Assiri ◽  
SJ Sinor ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune System ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Fold Increase ◽  
Mrna Levels ◽  
Antiviral Protein ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Interferon-tau (IFN tau) acts locally on the endometrium to suppress estrogen and oxytocin receptor expression and block luteolysis in ruminants. Systemic administration of conceptus homogenates or recombinant ovine IFN tau does not block luteolysis or enhance pregnancy rates in sheep or cattle, respectively. However, IFN tau up-regulates expression of the antiviral protein Mx throughout the entire uterine wall during early pregnancy. These studies determined if conceptus-derived IFN tau also up-regulates Mx expression in components of the circulating immune system that migrate through the endometrial wall. In experiment one, peripheral blood mononuclear cells (PBMC) were isolated from ewes at D26 post-artificial insemination (AI) and Mx mRNA levels examined by Northern and slot-blot hybridization. Pregnancy resulted in a two-fold increase in Mx mRNA levels compared to bred, non-pregnant ewes at D26. In experiment two, PBMC were isolated from ewes at AI, and every three days from D9 to D30. Results showed a four-fold increase in Mx mRNA levels in PBMC from pregnant versus bred, non-pregnant ewes at D15. Increased Mx mRNA, which remained elevated through D30, was accompanied by increased levels of Mx protein. These results show that pregnancy recognition signaling rapidly induces Mx gene expression in PBMC, and are the first to suggest that IFN tau activates gene expression in components of the circulating immune system.

scholarly journals Gene expression of chemokines CX3CL1 and CXCL16 and their receptors, CX3CR1 and CXCR6, in peripheral blood mononuclear cells of patients with relapsing-remitting multiple sclerosis - a pilot study

2020 ◽  
Vol 77 (9) ◽  
pp. 967-973
Author(s):  
Ljiljana Stojkovic ◽  
Aleksandra Stankovic ◽  
Ivan Zivotic ◽  
Evica Dincic ◽  
Dragan Alavantic ◽  
...  
Keyword(s):  
Gene Expression ◽  
Multiple Sclerosis ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Fold Change ◽  
Mrna Levels ◽  
Peripheral Blood Mononuclear ◽  
Relapsing Remitting ◽  
Blood Mononuclear Cells

Background/Aim. In vitro and in vivo studies show that CX3CL1 and CXCL16 chemokines and their specific receptors, CX3CR1 and CXCR6, respectively, mediate mechanism of neuroinflammation during the pathogenesis of multiple sclerosis (MS). The aim of this study was to investigate relative messenger ribonucleic acid (mRNA) levels of CX3CL1, CXCL16, CX3CR1 and CXCR6 in peripheral blood mononuclear cells, as potential molecular markers of relapsing-remitting (RR) MS. Methods. The study included 43 unrelated RR MS patients, 20 of them with clinically active disease (relapse) and 23 with clinically stable disease (remission), and 28 unrelated healthy subjects as controls. Real-time polymerase chain reactions (PCR) were performed using TaqMan? gene expression assays. Relative expression (mRNA) level of each target gene in each sample of peripheral blood mononuclear cells was calculated as the mean normalized expression. Results. The levels of CX3CR1 mRNA were significantly higher in clinically active RR MS patients compared to controls [fold change = 1.38, p (Mann-Whitney U test) = 0.009], and significantly lower in clinically stable vs active RR MS patients [fold change = - 1.43, p (t-test) = 0.03]. Stable RR MS patients had significantly higher CXCL16 mRNA levels than controls [fold change = 1.33, p (Mann-Whitney U test) = 0.006]. A trend of increased CXCR6 gene expression was found in active RR MS patients compared to controls [fold change = 1.23, p (Mann-Whitney U test) = 0.08]. In either active or stable RR MS patients there were no significant correlations of the clinical parameters with expression levels of the target genes. Conclusion. The current results show that increased CX3CR1 mRNA levels in peripheral blood mononuclear cells could represent a proinflammatory molecular marker of clinically active RR MS.

Effect of β-D-Mannuronic Acid (M2000) on Oxidative Stress Enzymes’ Gene Using Healthy Donor Peripheral Blood Mononuclear Cells for Evaluating the Anti-Aging Property

2019 ◽  
Vol 16 (3) ◽  
pp. 265-271 ◽  
Author(s):  
Mahsa Taeb ◽  
Abdollah Jafarzadeh ◽  
Seyed Shahabeddin Mortazavi-Jahromi ◽  
Nahid Zainodini ◽  
Mohammad Reza Mirzaei ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Expression Level ◽  
High Dose ◽  
Peripheral Blood Mononuclear ◽  
Expression Levels ◽  
Blood Mononuclear Cells

Objective: This research aimed to study the anti-aging and anti-inflammatory effects of low and high doses of the β-D-mannuronic (M2000) on gene expression of enzymes involved in oxidative stress (including SOD2, GST, GPX1, CAT, iNOS, and MPO) in peripheral blood mononuclear cells (PBMCs) of healthy donors under in vitro conditions. Methods: The PBMCs were separated and the RNAs were then extracted and the cDNAs synthesized, and expression levels of the mentioned genes were detected by qRT-PCR. Results: Our results indicated that the high dose of this drug could significantly reduce the expression level of the SOD2 gene compared to the lipopolysaccharide (LPS) group (p < 0.0001). Moreover, it was found that the high dose of this drug could significantly decrease the expression level of the GST gene compared to the LPS group (p < 0.0001). However, no significant reductions were observed in expression levels of the CAT and GPX1 genes compared to the LPS group. Furthermore, our data revealed that the level of iNOS and MPO gene expression was significantly reduced, in both doses of M2000, respectively, compared to the LPS group (p < 0.0001). Conclusion: This research showed that M2000 as a novel NSAID with immunosuppressive properties could modify oxidative stress through lowering expression levels of the SOD2, GST, iNOS, and MPO genes compared to the healthy expression levels, with a probable reduction of the risk of developing inflammatory diseases related to age and aging.

Livin Expression in Normal Hematopoietic Cells and in Hematologic Malignancies.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4301-4301
Author(s):  
Ihab Abd-Elrahman ◽  
Vered Bucholtz ◽  
Klilah Hershko ◽  
Gail Amir ◽  
Riki Perlman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Hematopoietic Cells ◽  
Hematologic Malignancies ◽  
Inhibitor Of Apoptosis ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Immunohistochemistry Staining

Abstract Livin is a member of the Inhibitor of Apoptosis Proteins (IAP) family, a novel family of intracellular anti-apoptotic proteins that act by binding and inhibiting caspases. We found that Livin is unique among the IAP members as upon strong apoptotic stimuli, it is specifically cleaved by caspases to produce a large C-terminal subunit. This subunit has a paradoxical pro-apoptotic activity. Thus, Livin is not merely an inhibitor of apoptosis. Rather, it is a regulator of apoptosis that can protect against apoptosis but upon continuous apoptotic signals it helps to assure cell death. We showed that Livin plays a major role in melanoma. The level of the Livin protein is directly correlated to the resistance of melanoma cells to chemotherapy and to the survival rate of melanoma patients. Livin was also shown to be over expressed in other solid tumors such as nasopharyngeal, neuroblastoma, colorectal and lung cancers. In this work we studied Livin expression in normal hematopoietic cells as well as hematologic malignancies. Using immunohistochemistry staining for Livin we evaluated its expression in reactive lymph nodes (LN) and showed that in contrast to Bcl2, Livin was detected in highly proliferating germinal centers. In normal bone marrow Livin was detected in Megakaryocytes and immature myeloid precursors. In peripheral blood mononuclear cells, using quantitative RT-PCR, we found that Livin expression was down regulated in activated monocytes and T cells while in B cells, Livin was upregulated upon activation. We studied bone marrow and LNs from 84 patients (pts) with hematologic malignancies. Positive immunohistochemistry staining was found in the malignant cells of all pts with DLBC NHL (31 pts), follicular lymphoma (12 pts) and multiple myeloma (15 pts). Peripheral blood samples from 28 B-CLL pts were compared with healthy controls’ B cells. High mRNA levels were detected in 43% of the pts, in correlation with older age (p&lt;0.05). On the other hand, Livin was not expressed in Hodgkin’s disease (4 pts and 4 cell lines) and only 6/29 pts with AML had high levels of Livin in RT-PCR without any clinical correlation. Our data demonstrate that Livin is over expressed in activated normal B cells both in peripheral blood and LN as well as in most B cell lymphoproliferative diseases. Further investigation will establish the role of Livin over expression in hematologic malignancies.

The Significance of Megakaryocytic Transcription Factor Fli1 and Erythroid Transcription Factor EKLF in the Ribosomopathies: 5q Minus Syndrome and Diamond-Blackfan Anemia. the Role of Fli1 in p53 Regulation and in 5q Minus Syndrome Megakaryopoiesis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3825-3825 ◽  
Author(s):  
Radana Neuwirtova ◽  
Ota Fuchs ◽  
Dagmar Pospisilova ◽  
Radek Cmejla ◽  
Monika Holicka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Ribosomal Proteins ◽  
Mononuclear Cells ◽  
Gene Promoter ◽  
Low Risk ◽  
Normal Chromosome ◽  
Mrna Levels ◽  
Healthy Controls ◽  
Chromosome 5

Abstract Abstract 3825 Introduction: 5q- syndrome and DBA belong to ribosomopathies. 5q- syndrome patients (pts) have haploinsufficient RPS14 gene and approximately 50% DBA pts have mutated some of genes for ribosomal proteins. These two disorders share similar erythroid and megakaryocytic characteristics: macrocytic anemia, decreased erythropoiesis, normal platelet count and they differ in: frequent neutropenia in 5q- syndrome, rare thrombocytemia and absence of hypolobulated megakaryocytes in DBA. We recently studied two transcription factors Fli1(Friend leukemia virus integration 1) and EKLF (Erythroid Krüppel like factor, also named KLF1) involved in MEP (common megakaryocytic and erythroid progenitor) differentiation in MDS 5q-syndrome pts. Now we present the examination of these factors in DBA pts. There exists cross-antagonism between Fli1 and EKLF with probable dominance of EKLF. Fli1 mRNA is target for microRNA-l45 (miR-145) localized in common deleted region of 5q. Haploinsuffciency of miR-145 in 5q- syndrome stabilizes Fli1 mRNA and increases Fli1 (Kumar et al. Blood 2009;114 abstr.947). Fli1 is also increased by interkleukin 6. IL-6 is induced by haploinsufficiency of miR-145 and miR-146a. Fli1 gene promoter is upregulated by Fli1 itself. Transcription factor PU.1 is positive regulator of Fli1 gene expression. In mice Fli1 regulates p53 via MDM2 (mouse double minute 2 or HDM2 in humans), an E3 ubiquitin ligase, which promotes p53 degradation in proteasomes (Truong et al. Oncogene 2005;24:962–69). Patients and Methods: Mononuclear cells were isolated from blood of 31 pts with 5q- syndrome, 26 MDS low risk pts with normal chromosome 5, 16 healthy controls and 10 DBA pts (7 with mutation of RP and 3 without proved mutation). Further, mononuclear cells were obtained from bone marrow of 17 pts with 5q-syndrome, 15 MDS low risk pts with normal chromosome 5, and 8 healthy controls. In total RNA of blood and bone marrow mononucklear cells Fli1, EKLF, p53, HDM2 and PU.1 mRNA levels were determined by quantitative real-time PCR. Related levels of mRNAs were calculated to the level of housekeeping GAPDH mRNA. We examined methylation status of EKLF promoter region in 5q- syndrome pts by bisulfite genomic DNA sequencing. All MDS pts involved in this study were examined by FISH and in all 5q- syndrome pts the deletion of 5q31 was confirmed. Results: In 5q- syndrome pts íncreased levels of Fli1 mRNA and decreased EKLF mRNA were detected both in blood and bone marrow mononuclear cells in comparison with healthy controls. In MDS low risk pts with normal chromosome 5 the increased Fli1 mRNA was found only in peripheral blood. There was no difference between EKLF mRNA levels in pts and in controls. In 8 of 10 DBA pts EKLF mRNA was decreased in blood in comparison to healthy controls, while Fli1 mRNA levels did not differ from controls with one pt exception. Methylation of EKLF gene promoter region is not responsible for the decrease EKLF mRNA in 5q- syndrome. Significantly increased level of p53 mRNA was found only in bone marrow of 5q- pts but not in blood of 5q- or DBA pts. There was no significant difference in HDM2 and PU.1 mRNA levels in blood and bone marrow of both groups of MDS pts and also in blood of DBA pts. Discussion: Significant EKLF decrease in all pts with 5q- syndrome and in 8 of 10 DBA pts corresponds to anemia and decreased erythropoiesis in both groups, but the cause of the EKLF decrease remains unknown. In 5q- syndrome low EKLF might be explained by the cross-antagonism between Fli1 and EKLF. Most significant is high Fli1 in all 5q- pts. This finding is important for the explanation of effective megakaryopoiesis contrary to defective erythropoiesis. The situation is different in DBA pts, where Fli1 mRNA is not increased with one exception (pt with atypical RPL5 mutation). Increased Fli1 might be the clue for the maintenance of effective megakaryopoiesis in 5q- syndrome. In erythroid cells ribosomal proteins inhibit HDM2, p53 is not degraded and increased p53 enhances apoptosis of erythroid cells. High Fli1 in megakaryocytic cells transcriptionally stimulates HDM2. In addition, IL-6 stimulates not only Fli1 but also DNA methylase 1, which methylates TP53 promoter and silences TP53 expression. These processes prevent apoptosis of megakaryocytic cells. For effective megakaryopoiesis in DBA pts we have not explanation so far. Disclosures: No relevant conflicts of interest to declare.

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