Fli-1 and EKLF Gene Expression in Patients with MDS 5q- Syndrome.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2788-2788 ◽  
Author(s):  
Radana Neuwirtova ◽  
Ota Fuchs ◽  
Dana Provaznikova ◽  
Jaroslav Cermak ◽  
Magda Siskova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Transcription Factors ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Direct Sequencing ◽  
Macrocytic Anemia ◽  
Gene Expression Measurement ◽  
Healthy Controls ◽  
Average Value

Abstract Abstract 2788 Poster Board II-764 Introduction. Patients with MDS-5q- syndrome have macrocytic anemia often with hypoplastic erythropoiesis and on the contrary thrombocythemia with effective though dysplastic megakaryopoiesis. Megakaryocytes and erythroid cells are thought to share a common progenitor MEP (T.P.McDonald et al., Exp. Hematology 1993). There are two key transcription factors which together with other transcription factors and relevant cytokines and receptors determine the hemopoietic differentiation of the common stem cell: erythroid Krüppel-like factor (EKLF) for erythroid lineage and Friend leukemia virus integration 1 (FLi-1) for megakaryopoiesis (Pilar Frontelo et al., Blood 2007; G.A.Blobel, Blood 2007; F.Bouilloux et al., Blood 2008). There is functional cross antagonism between FLi-1 and EKLF (J.Starck et al., Mol. Cel. Biology 2003). FLi-1 is active only if dephosphorylated (H.Huang et al., ASH Abstracts 2008). The question is whether both factors play any role in 5q- syndrome. Methods. FLi-1 and EKLF gene expressions were determined in mononuclear cells isolated from the whole blood or bone marrow using Ficoll-Paque PLUS. Expression of both factors was measured by quantitative real-time PCR. RT-PCR products were verified by electrophoresis and direct sequencing. The assays were performed for sample in duplicate. Glyceraldehyd-3-phosphate dehydrogenase (GAPDH), FLi-1 and EKLF were amplified in 25 μl reaction mixture containing 12.5 μl SYBR Green JumpStart Taq Ready Mix, 2.5 μl 2 μM FLi-1 or EKLF forward and reverse primers, 0,25 μl internal reference dye and 1 μl cDNA. Relative levels of FLi-1 and EKLF mRNAs were calculated to the level of housekeeping GAPDH mRNA. Results. FLi-1 and EKLF were measured in blood mononuclear cells of 8 patients fulfilling all criterias of 5q- syndrome. FLi1mRNA/GAPDHmRNA was higher in all samples, average value was 0.0930 (0.0242-0.4274) compared to control value 0.0194. FLi1mRNA/GAPDHmRNA in bone marrow mononuclear cells of 7 patients with 5q- syndrome was higher in all samples but one. The average value was 0.0827 (0.0070-0.2554) compared to healthy controls 0.0044. EKLF gives very low values in the majority of patients′ blood and bone marrow samples as well as in healthy controls. The evaluation is therefore less reliable then FLi-1 assessment. EKLFmRNA/GAPDHmRNA in blood was 0.0004 (0.0-0.0023) compared to the control 0.0222. The results of EKLF in 5 bone marrow samples are inconsistent. Three are lower than the control (0.0068), 1 of remaining 2 samples is extremely high (0.3491). It is interesting that this patient is the only one who responded to erythropoietin and is transfusion independent. Summary. Our preliminary results with FLi-1 and EKLF gene expression measurement are in agreement with expected findings: increased FLi-1 expression corresponds to thrombocytemia in 5q- syndrome patients and expression of EKLF, lower than in controls would correspond to anemia in these patients. However, EKLF values are less reliable because of very low values in patients as well as in controls and because of inconsistent results in bone marrow samples. We prepare to follow both factors in 5q- patients after the treatment with lenalidomide. Lenalidomide improves anemia in 5q- syndrome patients and temporarily causes decrease of thrombocytes (A.List et al., N.Engl.J.Med. 2005, 2006). Inhibition of phosphatases by lenalidomide (S.Wei et al., Proc.Natl.Acad.Sci.USA 2009) can stop FLi-1 dephosphorylation which leads to FLi-1 inactivation. Hypotetically inactive FLi-1 would enable EKLF to induce MEP into erythroid lineage. Supported by MSM 0021620808 Disclosures: No relevant conflicts of interest to declare.

Primary Bone Marrow-Derived MSC Subsets Have Distinct Wnt-Signatures Compared to Conventionally Cultured MSC and Differ in Their Capacity to Support Hematopoiesis in Vitro,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3414-3414 ◽  
Author(s):  
Marijke W Maijenburg ◽  
Marion Kleijer ◽  
Kim Vermeul ◽  
Erik P.J. Mul ◽  
Floris P.J. van Alphen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Wnt Signaling ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Wnt Proteins

Abstract Abstract 3414 Mesenchymal stromal cells (MSC) are of promising therapeutic use to suppress immunogenic responses following transplantation, and to support expansion of hematopoietic stem- and progenitors cells (HSPC) from small transplants derived for instance from cord blood. Culture-expanded MSC produce a wide variety and quantity of Wnt-proteins and the crucial role of Wnt-signaling in the hematopoietic stem cell niche is well established. However, studies addressing Wnt-signaling in MSC have (i) only focused on culture-expanded MSC and (ii) did not discriminate between phenotypically distinct subpopulations which are present in bulk cultures of expanded MSC. Recently we identified three new subpopulations of MSC in human bone marrow (BM) based on expression of CD271 and CD146: CD271brightCD146−, CD271brightCD146+, CD271−CD146+. These fractions co-express the “classical” MSC markers CD90 and CD105 and lack expression of CD45 and CD34 (Maijenburg et al, Blood 2010, 116, 2590). We and others demonstrated that the adult BM-derived CD271brightCD146− and CD271brightCD146+ cells contain all colony forming units-fibroblasts (Maijenburg et al, Blood 2010, 116, 2590; Tormin et al, Blood 2010, 116, 2594). To investigate how these primary subsets functionally compare to conventional, culture-expanded MSC, we investigated their Wnt-signature and hematopoietic support capacity. To this end, we sorted CD271brightCD146− and CD271brightCD146+ cells from human adult BM (n=3) and compared their Wnt-signatures obtained by Wnt-PCR array to the profiles from cultured MSC from the same donors. Fifteen genes were consistently differentially expressed in the two sorted uncultured subsets compared to their conventionally cultured counterparts. Expression of CCND1, WISP1 and WNT5B was strongly increased, and WNT5A was only detected in the conventionally cultured MSC. In contrast, WNT3A was exclusively expressed by sorted primary CD271brightCD146− and CD271brightCD146+ cells, that also expressed higher levels of JUN, LEF1 and WIF1. The differences in Wnt (target)-gene expression between CD271brightCD146− and CD271brightCD146+ cells were more subtle. The Wnt-receptors LRP6 and FZD7 were significantly higher expressed in CD271brightCD146+ cells, and a trend towards increased expression in the same subset was observed for CTNNB1, WNT11 and MYC. When the sorted subsets were cultured for 14 days (one passage), the differences in Wnt-related gene expression between the subsets was lost and the expanded sorted cells acquired an almost similar Wnt-signature as the MSC cultured from BM mononuclear cells from the same donors. The cultured subsets lost the expression of Wnt3a and gained the expression of Wnt5a, similar to the unsorted MSC cultured from the same donors in parallel. Despite the loss of a distinct Wnt-signature, co-culture experiments combining the sorted MSC subsets with human HSPC revealed that CD271brightCD146+ cells have a significantly increased capacity to support HSPC in short-term co-cultures (2 weeks) compared to CD271brightCD146− cells (p<0.021, n=3), which was analyzed in hematopoietic colony assays following co-culture. In contrast, a trend towards better long-term hematopoietic support (co-culture for 6 weeks) was observed on CD271brightCD146− cells. In conclusion, we demonstrate for the first time that primary sorted uncultured MSC subsets have a distinct Wnt-signature compared to cultured unsorted MSC and display differences in hematopoietic support. As it was recently shown that CD271brightCD146− and CD271brightCD146+ MSC localize to separate niches in vivo (Tormin et al, Blood 2011), our data indicate that the two MSC subsets are not necessarily distinct cell types and that the different Wnt-signature may be a reflection of these distinct microenvironments. Cell culturing for only one passage dramatically changed the Wnt-signature of the sorted MSC subsets, indicating that Wnt-signaling in in vitro expanded MSC does not resemble the Wnt-signature in their tissue resident counterparts in vivo. Disclosures: No relevant conflicts of interest to declare.

Research of Gene Expression in Patients with Myelodysplastic Syndromes.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4433-4433
Author(s):  
Bao-An Chen ◽  
Bo Zhang ◽  
Chong Gao ◽  
Guo-Hua Xia ◽  
Ze-ye Shao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Clinical Significance ◽  
Myelodysplastic Syndromes ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Mononuclear Cells ◽  
Rt Pcr ◽  
Normal People ◽  
Significant Difference

Abstract Abstract 4433 Object This study was aimed to investigate the expression of c-FLIPL, c-FLIPS and DLK1 mRNA in Myelodysplastic Syndromes (MDS) patients, as compared with normal people and AML patients, and to find its clinical significance. Methods The expression of c-FLIPL, c-FLIPS and DLK1 mRNA in bone marrow mononuclear cells (BMNNC) of 16 patients with MDS, 8 patients with AML and 3 controls were detected by RT-PCR. Results The expression of DLK1 mRNA was up-regulated in MDS, including RA and RAEB, as compared with controls(P<0.05). There was no significant difference in expression of DLK1 between RA and RAEB(P>0.05). The expression of DLK1 was significant higher in AML patients, compared with controls(P<0.05). There was no significant difference between MDS and AML patients(P>0.05). The expression of c-FLIPL mRNA was higher than that in controls, both in RA and RAEB(P<0.05). There was no significant difference in expression of c-FLIPL between RA and RAEB(P>0.05). In eight AML patients, c-FLIPL gene's expression was up-regulated, as compared with controls(P<0.05). Between AML and MDS patients, there was no significant difference(P>0.05); The expression of c-FLIPS mRNA had no significant difference between MDS patients and controls(P>0.05), but its expression in RAEB was significant higher as compared with RA patients and controls(P<0.05). And in AML patients, the expression of c-FLIPS was higher than that in controls(P<0.05), but there was no significant difference between AML and MDS patients(P>0.05). Conclusion It is concluded that the expressions of DLK1, c-FLIPL and c-FLIPS mRNA in MDS/AML patients are abnormal as compared with normal people, although there are no significant difference have been found between AML and MDS. These genes may play critical roles in escaping malignant clone of MDS from apoptosis and acquiring the ability to divide unlimitedly, they can become important indexes for evaluating of development in MDS. Disclosures: No relevant conflicts of interest to declare.

Bone Marrow Cells From Low-Risk Myelodysplastic Syndrome Patients Show Features of Enhanced Autophagy

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4961-4961
Author(s):  
Yuan-yuan Wang ◽  
Zi-Xing Chen ◽  
Jian-nong Cen ◽  
Hong-jie Shen ◽  
Xiao-fei Qi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Low Risk ◽  
Beclin 1 ◽  
Healthy Controls ◽  
Aberrant Expression ◽  
Marrow Cells

Abstract Abstract 4961 Myelodysplastic syndrome (MDS) is a preneoplastic condition that frequently develops into overt acute myeloid leukemia (AML). Beclin 1, a gene plays an important role in autophagy, has been recognized as a tumour suppressor. The aberrant expression of Beclin 1 has been correlated to the development and progression of cancer. However, The function and expression of Beclin 1 in MDS is largely unexplored. The present study aimed to investigate Beclin 1 expression and its correlation with IPSS in bone marrow cells from Patients with Low-Risk MDS. We have analyzed the expression of Beclin 1 in BM mononuclear cells (BMMNCs) from patients of Low-Risk MDS (31 cases), 22 AML(22 cases) and healthy control (9 cases) by quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR), immunofluorescence and western blot analysis. The expression level of Beclin 1 mRNA is significantly higher in BMMNCs in patients with Low-Risk MDS(mean± SD=6.26±4.87) when compared to healthy controls (mean± SD= 1.52±1.37), and the IPSS score was negatively correlated with the percentage of Beclin 1 positive cells. A markedly decreased expression of Beclin 1 was observed in AML (mean± SD=1.12±1.04) compared with healthy controls. The amount of Beclin 1 protein determined by either western blotting or immunofluorescence was markedly increased in MDS compared with that in controls (P<0.05). These results indicated that the cells from Patients with Low-Risk Myelodysplasia show features of enhanced autophagy, suggesting that the biological phenotype of autophagy, in addition to apoptosis and senescence, may also participate in the development of MDS and its progression to AML. Disclosures: No relevant conflicts of interest to declare.

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.

Rearrangements of IER3 Represent a Novel and Recurrent Molecular Abnormality in Myelodysplastic Syndromes (MDS)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2679-2679
Author(s):  
David P. Steensma ◽  
Jessemy D Neiger ◽  
Julie C Porcher ◽  
J. Jonathan Keats ◽  
Peter Leif Bergsagel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Direct Sequencing ◽  
Marrow Cell ◽  
Regulatory Elements ◽  
Comparative Genomic ◽  
Healthy Controls ◽  
Monosomy 7 ◽  
Molecular Abnormalities ◽  
Molecular Abnormality

Abstract Background: For most patients with MDS, the disease-associated molecular abnormalities are unknown, which contributes to diagnostic uncertainty and has limited development of effective therapies. Chromosomal deletions such as del(5q) or monosomy 7 are more common than translocations in MDS, but balanced translocations are more likely to be pathobiologically informative. Here we report cloning of an MDS-associated translocation that revealed a novel recurrent molecular abnormality. Methods: We first studied a 71-year old man who presented with MDS and t(6;9)(p21.3;q34) as an isolated chromosomal abnormality in 18 of 20 metaphases. FISH assays for DEK/CAN and ABL rearrangements were unrevealing. We generated somatic hybrid murine/human cell lines from a buffy coat; microdissected and amplified derivative chromosomes; and hybridized to 6p/9q custom comparative genomic hybridization (CGH) arrays. The CGH results considerably narrowed the 6p and 9q breakpoints, which were then amplified by long-range PCR and sequenced to define the rearrangement. Results: The IER3 gene (Immediate Early Response 3; also known as IEX-1) at 6p21.3 was found to be separated from its upstream regulatory elements in this rearrangement and translocated to a transcript-poor region of 9q. RT-PCR confirmed marked downregulation of IER3 expression in the patient compared to healthy controls. IER3 is a plausible candidate for involvement in MDS because of its known role in regulating death receptor-induced apoptosis, interaction with NF-κb pathways, and its importance in response to genotoxic stresses such as ionizing irradiation. We then identified archival bone marrow cell pellets from 204 additional patients with various clonal hematological disorders and chromosomal rearrangements involving 6p21 or 6p22 (i.e., translocations, inversions, deletions, or additions), and designed a splitsignal FISH probe set to assay IER3. FISH results were abnormal in 8 additional patients (9/205 pts total abnormal, 4.4%): 3 split signals and 6 amplifications (one patient had both abnormalities); all 8 patients had MDS and 6p rearrangements as part of a more complex karyotype. FISH studies in MDS patients without 6p rearrangements are ongoing. RTPCR in MDS patients without 6p rearrangements demonstrated down-regulation of IER3 by &gt;2-fold in 38% of patients (67% lower-risk) and up-regulation in 12% of patients (50% higher-risk) when compared to the mean for healthy controls. Direct sequencing of the IER3 coding region and the promoter region in 30 patients without 6p rearrangements revealed no point mutations, but the prevalence of non-synonymous single nucleotide polymorphism rs3094124 (p.A127P) was higher than expected in patients (variant in 28.3 % of MDS alleles vs. 8.6% of 116 HAP-CEU controls, p=0.0015). IER3−/− mice are known to have hypertension, cardiac hypertrophy, and epithelial proliferation, but are not anemic (Sommer SL et al J Appl Physiol 2006). We performed methylcellulose hematopoietic colony growth assays using bone marrow mononuclear cells from IER3−/− mice and wildtype controls, and noted no differences in BFU-E, CFU-E, and CFU-GM growth under unstressed conditions. Studies of IER3 expression induction under stressed conditions in primary MDS cells are ongoing. Conclusion: IER3 rearrangements represent a novel clonally-restricted recurrent genetic abnormality in MDS. Dysregulation of IER3 expression is common in MDS, even in patients without 6p rearrangements. This is the first time IER3 has been linked to human disease. Clarification of the role of IER3 in MDS is likely to yield new insights into MDS pathobiology.

mRNA expression analysis confirms CD44 splicing impairment in systemic lupus erythematosus patients

Lupus ◽  
2021 ◽  
pp. 096120332110047
Author(s):  
Andrea Latini ◽  
Lucia Novelli ◽  
Fulvia Ceccarelli ◽  
Cristiana Barbati ◽  
Carlo Perricone ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
T Cells ◽  
Mrna Expression ◽  
Lupus Erythematosus ◽  
Mononuclear Cells ◽  
Direct Sequencing ◽  
Healthy Controls ◽  
Systemic Lupus ◽  
Peripheral Tissues ◽  
Variant Isoforms

Background Systemic Lupus Erythematosus (SLE) is a complex chronic autoimmune disease characterized by several immunological alterations. T cells have a peculiar role in SLE pathogenesis, moving from the bloodstream to the peripheral tissues, causing organ damage. This process is possible for their increased adherence and migration capacity mediated by adhesion molecules, such as CD44. Ten different variant isoforms of this molecule have been described, and two of them, CD44v3 and CD44v6 have been found to be increased on SLE T cells compared to healthy controls, being proposed as biomarkers of disease and disease activity. The process of alternative splicing of CD44 transcripts is not fully understood. We investigated the mRNA expression of CD44v3 and CD44v6 and also analyzed possible CD44 splicing regulators (ESRP1 molecule and rs9666607 CD44 polymorphism) in a cohort of SLE patients compared to healthy controls. Methods This study involved 18 SLE patients and 18 healthy controls. Total RNA and DNA were extracted by peripheral blood mononuclear cells. The expression study was conducted by quantitative RT-polymerase chain reaction, using SYBR Green protocol. Genotyping of rs9666607 SNP was performed by direct sequencing. Results CD44v6 mRNA expression was higher in SLE patients compared to healthy controls (p = 0.028). CD44v3/v6 mRNA ratio in healthy controls was strongly unbalanced towards isoform v3 compared to SLE patients (p = 0.002) and decreased progressively from healthy controls to the SLE patients in remission and those with active disease (p = 0.015). The expression levels of CD44v3 and CD44v6 mRNA correlated with the disease duration (p = 0.038, Pearson r = 0.493 and p = 0.038, Pearson r = 0.495, respectively). Splicing regulator ESRP1 expression positively correlated with CD44v6 expression in healthy controls (p = 0.02, Pearson r = 0.532) but not in SLE patients. The variant A allele of rs9666607 of CD44 was associated with higher level of global CD44 mRNA (p = 0.04) but not with the variant isoforms. Conclusions In SLE patients, the increase in CD44v6 protein correlates with a higher transcript level of this isoform, confirming an impairment of CD44 splicing in the disease, whose regulatory mechanisms require further investigation.

The Auto-Antibodies on Bone Marrow Cells in the Patients with Systemic Lupus Erythematosus.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3856-3856
Author(s):  
Rong Fu ◽  
Jizi Deng ◽  
Shang Yuan ◽  
Lu Gong ◽  
Jun Sun ◽  
...  
Keyword(s):  
Systemic Lupus Erythematosus ◽  
Bone Marrow ◽  
Lupus Erythematosus ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
Coombs Test ◽  
Systemic Lupus ◽  
Cell Counts ◽  
Significant Difference ◽  
Positive Rate

Abstract Objective:To explore the pathogenesis of cytopenia in the patients with systemic lupus erythematosus (SLE), the auto-antibodies on bone marrow mononuclear cells (BMMBC) in the patients with SLE were determined. Methods:Twenty one patients with SLE and ten healthy controls were enrolled in this study. BMMNC Coombs test was used to determine the aotoantibodies. The correlation between the types of auto-antibodies on BMMNC, the types of serum auto-antibodies and the counts of blood cells in the patients with SLE were also investigated. Results:Positive results of BMMBC-Coombs test were seen in 12 patients with SLE (57.1%), among them, 10 with hemocytopenia (58.82%), and 2 without hemocytopenia (50%). The positive rate of BMMNC Coombs test was higher in the patients with SLE than that in healthy controls, and was higher in SLE patients with hemocytopenia than that in healthy controls. There were no significant difference of BMMNC-Coombs positive rate between the SLE patients without hemocytopenia and healthy controls, and there were also no significant differences between the SLE patients without hemocytopenia and SLE patients with hemocytopenia. In the 12 SLE patients with positive BMMBC-Coombs tests, IgM auto-antibody accounted for 75.0%, and C3 50.0%, IgG 8.33%, IgG+IgM 8.33%, C3+IgM 16.67%, IgG+IgM+C3 16.67%. In the SLE patients without hemocytopenia, IgG+IgM accounted for 8.33%, C3 8.33%, but IgA autoantibody were not seen in any case. There was a significant positive correlation between the auto-antibodies on BMMNC and peripheral anti-SSA, but there was no significant correlation between the results of BMMBC Coombs tests and peripheral blood cell counts. Conclusion:There were auto-antibodies on BMMNC in the patients with SLE. The hemocytopenia in the patients with SLE maybe resulted from the destructions of bone marrow hematopoietic cells by the autoantibodies.

Transcriptional Regulation of Erythropoiesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. SCI-7-SCI-7
Author(s):  
Mitchell J. Weiss
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Transcriptional Activation ◽  
Zinc Finger Protein ◽  
Conflicts Of Interest ◽  
Globin Gene ◽  
Globin Genes ◽  
Erythroid Development ◽  
Globin Gene Expression

Abstract Abstract SCI-7 Efforts to define the mechanisms of globin gene expression and transcriptional control of erythrocyte formation have provided key insights into our understanding of developmental hematopoiesis. Our group has focused on GATA-1, a zinc finger protein that was initially identified through its ability to bind a conserved cis element that regulates globin gene expression. GATA-1 is essential for erythroid development and mutations in the GATA1 gene are associated with human cytopenias and leukemia. Several general principles have emerged through studies to define the mechanisms of GATA-1 action. First, GATA-1 activates not only globin genes, but also virtually every gene that defines the erythroid phenotype. This observation sparked successful gene discovery efforts to identify new components of erythroid development and physiology. Second, GATA-1 also represses transcription through multiple mechanisms. This property may help to explain how GATA-1 regulates hematopoietic lineage commitment and also how GATA1 mutations contribute to cancer, since several directly repressed targets are proto-oncogenes. Third, GATA-1 regulates not only protein coding genes, but also microRNAs, which in turn, modulate erythropoiesis through post-transcriptional mechanisms. Fourth, GATA-1 interacts with other essential erythroid-specific and ubiquitous transcription factors. These protein interactions regulate gene expression by influencing chromatin modifications and controlling three-dimensional proximity between widely spaced DNA elements. Recently, we have combined transcriptome analysis with ChIP-chip and ChIP-seq studies to correlate in vivo occupancy of DNA by GATA-1 and other transcription factors with mRNA expression genome-wide in erythroid cells. These studies better elucidate how GATA-1 recognizes DNA, discriminates between transcriptional activation versus repression and interacts functionally with other nuclear proteins. I will review published and new aspects of our work in these areas. Disclosures No relevant conflicts of interest to declare.

Mesenchymal Stromal Cells From Myelodysplastic Syndrome Patients Show Lower DICER1 and DROSHA Expression, as Well as Decreased Mir-155 and Mir-181a MicroRNA Expression, When Compared with Healthy Controls

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 397-397
Author(s):  
Carlos Santamaría ◽  
Olga López-VIllar ◽  
Sandra Muntión ◽  
Belén Blanco ◽  
Soraya Carrancio ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Protein Expression ◽  
Myelodysplastic Syndrome ◽  
Western Blot ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Microrna Expression ◽  
Healthy Controls ◽  
Lower Expression

Abstract Abstract 397 Mesenchymal stromal cells (MSC) are closely related to the regulation of hematopoietic stem cell niche. Recently, Raaijmakers et al (Nature, 2010), published that deletion of Dicer1, a RNase III enzyme involved in microRNA biogenesis, in murine MSC-derived osteoprogenitors triggered peripherical blood cytopenias, myelodysplasia and subsequent AML, showing that molecular alterations in bone marrow microenvironment could result in clonal impaired haematopoiesis. Here, we have investigated whether MSC from myelodysplastic syndrome (MDS) patients show differences in DICER1 and DROSHA, another RNA III endonuclease, in comparison to healthy MSC. In addition, we have analyzed several hematopoietic-related microRNAs in these same samples. Bone marrow MSC from MDS patients (n=35; 10 5q- syndrome, 4 RA, 5 RARS, 10 RCMD, 3 RAEB, 2 MDS-U and 1 hypocellular MDS) and healthy donors (HD, n=20) were isolated and in vitro expanded following standard procedures until the third passage. Additionally, paired mononuclear cells (MNC) from 13 MDS and 8 HD were obtained. Total RNA was isolated using TRIzol reagent (Invitrogen). DICER1 and DROSHA relative gene expressions were assessed by quantitative PCR (Q-PCR) using commercial TaqMan® assay (Applied Biosystems®) with GAPDH as control gene. DICER1 and DROSHA (Abcam) protein expression were evaluated in whole cell lysates by western blot, using calnexin (Stressgen) as control. Several microRNAs with known role in hematopoiesis and immune system regulation were analyzed in 25 MDS and 12 HD by Q-PCR using commercial TaqMan® MicroRNA assay (Applied Biosystems®) with RNU43 as control microRNA. MSC from MDS showed significant lower DICER1 (0.0035±0.0020 vs. 0.0076±0.0092; p=0.044) and DROSHA (0.0070±0.0028 vs. 0.0135±0.0176; p=0.019) gene expression levels than healthy controls. Moreover, MSC from MDS showed lower protein expression of both DICER1 and DROSHA by western blot analysis, confirming Q-PCR findings. By contrast, no difference in either DICER1 (0.0197±0.0151 vs. 0.0173±0.0112; p=0.9) or DROSHA (0.0089±0.0023 vs. 0.0067±0.0037; p=0.09) gene expression were observed between MNC from MDS and HD. As far as microRNA expression, we observed a lower expression of mir-155 (0.63±0.92 vs. 0.94±0.49; p=0.007) and mir-181a (1.30±0.95 vs. 2.02±1.05; p=0.041) in MSC from MDS in comparison to healthy controls. Mir-155 and mir-181a are involved in T-cell and B-cell differentiation, while mir-155 are also related to erythroid and megakarycytic differentiation. We conclude that MSC from MDS patients show lower expression of DICER and DROSHA, two relevant RNA-III endonucleases involved in the microRNA biogenesis, confirming recent findings in murine models. Moreover, the expression of some microRNA is impaired in these cells, raising the possibility that these microenvironmental alterations could be involved in the MDS pathophysiology. Disclosures: No relevant conflicts of interest to declare.

Cytopenias in Large Granular Lymphocyte Leukemia Are Mediated by Pro-Fibrotic Mesenchymal Stem Cells in the Bone Marrow Niche with Functional Restoration by FGFb

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 932-932
Author(s):  
Adam W Mailloux ◽  
Ling Zhang ◽  
Lili Yang ◽  
Cody Wei ◽  
Lubomir Sokol ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Growth Factors ◽  
Collagen Matrix ◽  
Growth Potential ◽  
Healthy Controls ◽  
Collagen Deposition ◽  
Autocrine Growth ◽  
Cd34 Cells ◽  
Control Mscs

Abstract Abstract 932 Large Granular Lymphocyte Leukemia (LGLL) is a chronic lymphoproliferative syndrome of clonal mature T or NK cells frequently associated with peripheral cytopenias including neutropenia and anemia. The ability of LGLL cells to lyse pro-erythrocytes, the association with secondary autoimmune disorders, and the chronically activated state of LGLL clones suggest that the disease is secondary to a systemic reactive process. Yet, the mechanism causing severe and chronic cytopenias remains unresolved. Because LGLL cells heavily reside in the bone marrow and the bone marrow microenvironment plays such a large role in supporting hematopoiesis, we hypothesized that constituents of the bone marrow microenvironment may be dysfunctional in LGLL patients, and contribute toward the pathogenesis of LGLL and the devolvement of cytopenias. To address this hypothesis, bone marrow core biopsies, aspirates, and peripheral smears taken from 24 patients diagnosed with LGLL were analyzed retrospectively by three independent pathology reviews. Reticulin and trichrome stains revealed clinically relevant myelofibrosis in 21 patients at the time of diagnosis. Of these, 15 had severe myelofibrosis (MF2-3, semi-quantitative grading). The severity of myelofibrosis correlated with neutropenia, anemia, splenomegaly, secondary autoimmune disorders, and the degree of LGLL bone marrow infiltration. The severity of fibrosis also correlated with novel disease aspects reported here for the first time, including increased pseudo-Pelger-Huet and immature neutrophils in peripheral smears, and increased monocytes in the bone marrow and periphery. Because myelofibrosis is so strongly associated with disease severity, we sought to understand the pathogenesis of medullary fibrosis using primary mesenchymal stem cell (MSC) cultures isolated from bone marrow aspirates of LGLL patients or healthy controls. Patient MSC cultures displayed abnormal morphologies, impaired growth kinetics, and reduced growth potential compared to controls, suggesting that patient MSCs may be prematurely senescent. Microarray analysis showed global gene expression changes as healthy MSCs expand in culture including increased expression of pro-hematopoietic cytokines and autocrine growth factors vital to MSC self-renewal such as basic fibroblast growth factor (FGFb) and leukemia inhibitory factor (LIF). Patient MSC cultures appeared incapable of these gene expression changes, instead maintaining low levels of pro-hematopoietic cytokines, FGFb, and LIF expression, while maintaining heightened collagen expression. Cytokine secretion was confirmed using ELISA, and immunofluorescent staining of collagen on MSC cultures verified abnormally elevated collagen deposition from patient MSCs. In particular, collagen I and collagen III matrices were heavily deposited over patient MSCs reiterating the increased trichrome and reticulin staining seen in patient bone marrow biopsies. In an effort to rescue senescence, exogenous FGFb and LIF were added to patient MSCs. FGFb, but not LIF, completely restored the growth kinetics, growth potential, and morphology of these cells. Moreover, collagen deposition of patient MSCs was comparable to healthy donors after exposure to FGFb. We also investigated the ability of MSC cultures to support the growth of hematopoietic progenitors. Healthy bone marrow mononuclear cells were labeled with Carboxyfluorescein succinimidyl ester (CFSE) and placed in co-culture with patient or control MSCs. Four days later, cellular division of CD34+ cells was analyzed by CFSE dilution. Control MSCs were able to support high levels of CD34+ cell division after expansion in culture. Conversely, patient MSCs supported only minimal growth of CD34+ cells. This suggests that gene expression changes seen in healthy MSC cultures during expansion may be required to support hematopoiesis, and that these changes are blocked in senescent MSCs in LGLL patients. Collectively, this data implicates aberrant MSCs and collagen production in the pathogenesis of LGLL, and suggests that autocrine growth factors such as FGFb, may represent a novel therapeutic option to rescue pro-hematopoietic behavior and reduce excessive buildup of collagen fibers in the bone marrow microenvironment.Figure 1MSCs from Patient Bone Marrow Deposite increase collagen matrix compared to healthy controls.Figure 1. MSCs from Patient Bone Marrow Deposite increase collagen matrix compared to healthy controls. Disclosures: No relevant conflicts of interest to declare.

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