scholarly journals Defective Autophagy in MPN Were Predominantly Detected in Megakaryocytes in Philadelphia Negative (Ph -) Myeloproliferative Neoplasm

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5386-5386
Author(s):  
Jen-Chin Wang ◽  
Guanfang Shi ◽  
Karan Josan ◽  
Preethi Ramachandran ◽  
Vladimir Gotlieb ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Western Blot ◽  
Cell Signaling ◽  
Mononuclear Cells ◽  
Myeloproliferative Neoplasm ◽  
Beclin 1 ◽  
Positive Staining ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear ◽  
Cytoplasmic Staining

We previously reported that autophagy was defective in classical Philadelphia negative (Ph -) Myeloproliferative Neoplasm (MPN) by demonstrating increased p62, decreased Beclin-1 by RT-PCR and Western Blot (WB) methods and decreased LC3-II by WB (ASH annual meeting poster 2018) in peripheral blood mononuclear cells. Now, we further performed immunohistochemical staining of these autophagy markers on the bone marrow biopsy specimens. Methods: Formalin-fixed and paraffin-embedded bone marrow samples were stained with primary antibodies against Beclin-1 (mouse monoclonal, Millipore), LC3B (rabbit monoclonal, Cell Signaling Technology), and p62 (mouse monoclonal, Cell Signaling Technology). The tissue sections were analyzed using VENTANA BenchMark Ultra System (Ventana Medical Systems, Inc.) according to the manufacturer's protocol. Patients who were studied included 12 essential thrombocythemia (ET), 10 polycythemia (PV), and 5 myelofibrosis (MF) (including 1 post-ET MF and 1 post-PV MF). Scoring was given based on the degree of cytoplasmic staining. Score 1 included weak cytoplasmic stain, score 2 included moderate cytoplasmic stain and score 3 was given for strong cytoplasmic staining . Results: 1) As shown in Fig1, the predominant cells which stained positive including p62, Beclin-1 , or LC3 B were mostly found on the megakaryoctes, although very few of other cell types were found to have positive staining as well 2) As shown in Table 1, the immunostaining results were in general correlated to the RT-PCR , or western blot findings that a defective autophagy was demonstrated in MPN patients with combined finding of a stronger staining in p62,and weak staining in Beclin-1 and LC3B. Conclusion: We have demonstrated defective autophagy in these Ph (-) MPN, by immunostaining of the bone marrow specimens, and demonstrating positivity for defective autophagy predominantly in megakaryocytes. Since megakaryocytes are the most important cells involved in the pathogenesis of MPN, we propose that defective autophagy in megakaryocytes play an important role in the pathogenesis of these diseases. Disclosures Wang: Incyt: Research Funding.

scholarly journals POS0430 EXPRESSION AND CLINICAL SIGNIFICANCE OF AUTOPHAGY-RELATED GENES IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF SYSTEMIC SCLEROSIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 443.2-443
Author(s):  
Y. F. Qing ◽  
J. Zheng ◽  
S. B. Wang ◽  
F. Dai ◽  
Y. Jiang ◽  
...  
Keyword(s):  
Systemic Sclerosis ◽  
Clinical Significance ◽  
Normal Control ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Beclin 1 ◽  
Peripheral Blood Mononuclear ◽  
Mesenchymal Transition ◽  
Blood Mononuclear Cells

Background:Growing evidences have demonstrated that autophagy is a powerful regulators in the pathogenesis of fibrosis and autoimmune diseases. Autophagy abnormalities in SSc involve abnormal autophagy-related protein and autophagy-related gene polymorphism[1-2], however there is a few reports on the expression and clinical significance of autophagy-related genes.Objectives:To investigate the expression and clinical significance of autophagy-related genes LC-3 mRNA, Becline-1 mRNA, Agt-3 mRNA, Agt-5 mRNA, Agt-12 mRNA and Agt-16L1 mRNA in peripheral blood mononuclear cells (PBMC) of systemic sclerosis (SSc).Methods:51 cases of SSc and 60 cases of normal control were received from the Affiliated Hospital of North Sichuan Medical College, and autophagy-related genes were detected by RT-PCR. SPSS19.0 statistical software was used to compare the expression of autophagy-related genes between groups and analyze the relationship between autophagy-related genes and clinical data, P<0.05 was considered statistically significantResults:LC-3, Becline-1, and Agt-3 were highly expressed in SSc compared with normal control [LC-3: 0.78(0.60) ×10-3 vs. 0.52(0.54) ×10-3; Beclin-1: 6.68(3.56)×10-3 vs. 5.22(3.54)×10-3; Agt-3: 17.58(12.33)×10-3 vs. 11.00(4.56)×10-3, P<0.05], however Agt-5, Agt-12 and Agt-16L1 of autophagy-related genes were not statistically significant [AGT-5: 6.67(3.58) ×10-3 vs. 6.67(2.64) ×10-3; AGT-12: 8.64(5.56)×10-3 vs. 8.57(4.66)×10-3; Agt-16L1: 2.69(2.19)×10-3 vs. 2.52(2.26)×10-3] (Figure 1). Beclin-1 and Agt-5 high expressed in SSc with the positive of anti-SSA/Ro antibody. LC-3 was positively correlated with Age(r=0.662) and ESR(r=0.355) (all P<0.05).Conclusion:Autophagy-related genes were increased in PBMC of SSc, and were correlated with Age, ESR and autoantibody, suggested that autophagy is a key feature in the pathogenesis of systemic sclerosis.Figure 1.The relative expression of autophagy-related genesReferences:[1]LIU C, ZHOU X, LU J, et al. Autophagy mediates 2-methoxyestradiol-inhibited scleroderma collagen synthesis and endothelial-to-mesenchymal transition induced by hypoxia[J]. Rheumatology, 2019;58(11):1966–1975.[2]Mayes M D, Bossini-Castillo L, Gorlova O, et al. Immunochip Analysis Identifies Multiple Susceptibility Loci for Systemic Sclerosis[J]. The American Journal of Human Genetics, 2014,94(1):47-61.DOI:10.1016/j.ajhg.2013.12.002.Disclosure of Interests:None declared

scholarly journals Virological and Parasitological Characterization of Mini-LEWE Minipigs Using Improved Screening Methods and an Overview of Data on Various Minipig Breeds

2021 ◽  
Vol 9 (12) ◽  
pp. 2617
Author(s):  
Sabrina Halecker ◽  
Julia Metzger ◽  
Christina Strube ◽  
Ludwig Krabben ◽  
Benedikt Kaufer ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Massachusetts General Hospital ◽  
Hepatitis E ◽  
Human Cells ◽  
Gastrointestinal Parasites ◽  
Endogenous Retroviruses ◽  
Detection Methods ◽  
Screening Methods ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear

Minipigs play an important role in biomedical research and have also been used as donor animals in xenotransplantation. To serve as a donor in xenotransplantation, the animals must be free of potential zoonotic viruses, bacteria and parasites. Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pigs and cannot be eliminated as most of the other pig viruses can. PERV-A and PERV-B infect human cells in cell culture and are integrated in all pigs, whereas PERV-C infects only pig cells and it is found in many, but not all pigs. Minipigs are known for a high prevalence of recombinant PERV-A/C viruses able to infect human cells (Denner and Schuurman, Viruses, 2021;13:1869). Here, Mini-LEWE minipigs are screened for the first time for pig viruses including PERV. Peripheral blood mononuclear cells (PBMCs) from 10 animals were screened using PCR-based methods (PCR, RT-PCR, and real-time PCR). In comparison with our previous screening assays, numerous improvements were introduced, e.g., the usage of gene blocks as a PCR standard and foreign RNA to control reverse transcription in RT-PCR. Using these improved detection methods, Mini-LEWE pigs were found to be negative for porcine cytomegalovirus (PCMV), porcine lymphotropic herpesviruses (PLHV-1, -2 and -3), porcine circoviruses (PCV1, 2, 3 and 4), porcine parvovirus (PPV) and hepatitis E virus (HEV). All animals carried PERV-A, PERV-B and PERV-C in their genome. PERV-A/C was not found. In contrast to all other minipig breeds (Göttingen minipigs, Aachen minipigs, Yucatan micropig, Massachusetts General Hospital miniature pigs), Mini-LEWE minipigs have less viruses and no PERV-A/C. Parasitological screening showed that none of the Mini-LEWE minipigs harbored ecto- and gastrointestinal parasites, but at least one animal tested positive for anti-Toxoplasma gondii antibodies.

scholarly journals Reprogramming of bone marrow myeloid progenitor cells in patients with severe coronary artery disease

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Marlies P Noz ◽  
Siroon Bekkering ◽  
Laszlo Groh ◽  
Tim MJ Nielen ◽  
Evert JP Lamfers ◽  
...  
Keyword(s):  
Coronary Artery Disease ◽  
Bone Marrow ◽  
Coronary Artery ◽  
Immune Cells ◽  
Mononuclear Cells ◽  
Immune Cell ◽  
Ex Vivo ◽  
Peripheral Blood Mononuclear ◽  
Artery Disease ◽  
Myeloid Progenitors

Atherosclerosis is the major cause of cardiovascular disease (CVD). Monocyte-derived macrophages are the most abundant immune cells in atherosclerotic plaques. In patients with atherosclerotic CVD, leukocytes have a hyperinflammatory phenotype. We hypothesize that immune cell reprogramming in these patients occurs at the level of myeloid progenitors. We included 13 patients with coronary artery disease due to severe atherosclerosis and 13 subjects without atherosclerosis in an exploratory study. Cytokine production capacity after ex vivo stimulation of peripheral blood mononuclear cells (MNCs) and bone marrow MNCs was higher in patients with atherosclerosis. In BM-MNCs this was associated with increased glycolysis and oxidative phosphorylation. The BM composition was skewed towards myelopoiesis and transcriptome analysis of HSC/GMP cell populations revealed enrichment of neutrophil- and monocyte-related pathways. These results show that in patients with atherosclerosis, activation of innate immune cells occurs at the level of myeloid progenitors, which adds exciting opportunities for novel treatment strategies.

Abstract 29: Efficient Differentiation and Polarization of Macrophages Differentiated From Human Peripheral Blood Mononuclear Cells-derived iPS Cells

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Hanrui Zhang ◽  
Rhia Shah ◽  
Christine Hinkle ◽  
Wenli Yang ◽  
Daniel VanDorn ◽  
...  
Keyword(s):  
Metabolic Disease ◽  
Mononuclear Cells ◽  
Human Peripheral Blood ◽  
Embryoid Bodies ◽  
Alternative Activation ◽  
Boyden Chamber ◽  
Positive Staining ◽  
Mesoderm Induction ◽  
Erythroid Progenitors ◽  
Peripheral Blood Mononuclear

Macrophages fulfill homeostatic functions beyond defense. Monocytes/macrophages derived from human induced pluripotent stem cells (hiPSCs) are useful tools to study vascular diseases and potential sources of cell-based therapies. The objective of this study is to generate PBMC-hiPSC derived monocytes, macrophages, as well as M1 and M2 type differentiated macrophages and to examine multiple macrophage characteristics of relevance to cardio-metabolic disease. We briefly cultured human adult PBMCs from healthy volunteers to enrich for the erythroid progenitors. These progenitors were used to generate hiPSCs using lentivirus-mediated transduction of Oct3/4, Sox2, Klf4 and c-Myc. A stepwise protocol was used for the differentiation of PBMC-hiPSCs to monocytes/macrophages. The protocol involves primitive streak and mesoderm induction (stage 1), hematopoietic specification (stage 2), hematopoietic cell maturation and myeloid expansion (stage 3), monocytes collection and differentiation to macrophages (stage 4). Differentiation cultures were set up in six-well plate format, from 20 embryoid bodies per well, and produced up to 30 million macrophages. hiPSC-derived macrophages (hiPSC-MΦ) exhibited spindle morphology and incorporated acetylated low-density lipoprotein. Immunocytochemical analysis of hiPSC-MΦ revealed positive staining for CD68, CD11b, and CCL2. FACS analysis indicates >90% expression of macrophage lineage markers, including CD45, CD14, CD16, CD115, CD68, CX3CR1, CD11b, and CD18. Migration of hiPSC-MΦ in response to M-CSF was confirmed by Boyden Chamber assay. hiPSC-MΦ were polarized to M1 (classical activation stimulated by TLR ligands LPS and IFN-gamma) and M2 (alternative activation stimulated by IL-4) types. M2 macrophages were characterized by more efficient phagocytic activity of zymosan particles and greater migration capacities compared with M1 macrophages. The results demonstrated the feasibility of differentiation/polarization of macrophages from PBMC-hiPSCs. This study advanced our understanding of the suitability of subject-specific hiPSC for deriving myeloid/monocyte/macrophages for study of monocyte-macrophage functions of direct relevance to cardio-metabolic disease.

Identification of Molecular Mechanisms in Parkinson’s Disease Pathogenesis: Significance of Survival and Inflammation Pathways

2020 ◽  
Author(s):  
Zerrin Karaaslan ◽  
Ozlem Timirci Kahraman ◽  
Elif Sanli ◽  
Hayriye Arzu Ergen ◽  
Basar Bilgic ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Functional Annotation ◽  
Molecular Mechanisms ◽  
Mononuclear Cells ◽  
Neuronal Degeneration ◽  
Expression Profiles ◽  
Quantitative Polymerase Chain Reaction ◽  
Rt Pcr ◽  
Peripheral Blood Mononuclear

Abstract Background: Our aim was to identify the differentially expressed genes (DEGs) between Parkinson’s disease (PD) patients and controls by microarray technology and analysis of related molecular pathways by functional annotation. Methods: Thirty PD patients and 30 controls were enrolled. Agilent Human 8X60 K Oligo Microarray was used for gene level expression identification. Gene ontology and pathway enrichment analyses were used for functional annotation of DEGs. Protein-protein interaction analyses were performed with STRING. Expression levels of randomly selected 5 genes among DEGs were quantified by real time quantitative polymerase chain reaction (RT-PCR) for validation. Flow cytometry was done to determine frequency of regulatory T cells (Tregs) in peripheral blood mononuclear cells. Results: A total of 361 DEGs (143 upregulated and 218 downregulated) were identified after GeneSpring analysis. DEGs were involved in 28 biological processes, 12 cellular components and 26 molecular functions. Pathway analyses demonstrated that upregulated genes mainly enriched in p53 (CASP3, TSC2, ATR, MDM4, CCNG1) and PI3K/Akt (IL2RA, IL4R, TSC2, VEGFA, PKN2, PIK3CA, ITGA4, BCL2L11) signaling pathways. TP53 and PIK3CA were identified as most significant hub proteins. Expression profiles obtained by RT-PCR were consistent with microarray findings. PD patients showed increased proportions of CD49d+ Tregs, which correlated with disability scores. Discussion: Survival pathway genes were upregulated putatively to compensate neuronal degeneration. Bioinformatics analysis showed an association between survival and inflammation genes. Increased CD49d+ Treg ratios might signify the attempt of the immune system to suppress ongoing inflammation. Conclusion: Altered functions of Tregs might have an important role in PD pathogenesis and CD49d expression could be a prognostic biomarker of PD.

scholarly journals The Human SHIP Gene Is Differentially Expressed in Cell Lineages of the Bone Marrow and Blood

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 1876-1885 ◽  
Author(s):  
Susan J. Geier ◽  
Paul A. Algate ◽  
Kristen Carlberg ◽  
Dave Flowers ◽  
Cynthia Friedman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mononuclear Cells ◽  
Sh2 Domain ◽  
Protein Species ◽  
Peripheral Blood Mononuclear ◽  
Phosphorylated Proteins ◽  
Cd34 Cells ◽  
Negative Effect ◽  
Mrna And Protein Expression ◽  
Macrophage Colony

Abstract The macrophage colony-stimulating factor receptor and several other hematopoietic growth factor receptors induce the tyrosine phosphorylation of a 145- to 150-kD protein in murine cells. We have previously cloned a cDNA for the murine 150-kD protein, SHIP, and found that it encodes a unique signaling intermediate that binds the SHC PTB domain through at least one tyrosine phosphorylated (NPXY) site in the carboxyl-terminal region. SHIP also contains several potential SH3 domain-binding sites, an SH2 domain for binding other tyrosine phosphorylated proteins, and an enzymatic activity that removes the phosphate from the 5 position of phosphatidylinositol 3,4,5-phosphate or from inositol 1,3,4,5-phosphate. SHIP has a negative effect on cell growth and therefore loss or modification may have profound effects on hematopoietic cell development. In this study, we have cloned a cDNA for human SHIP and examined mRNA and protein expression of SHIP and related species in bone marrow and blood cells. Flow cytometry indicates that at least 74% of immature CD34+ cells express SHIP cross-reacting protein species, whereas within the more mature population of CD33+ cells, only 10% of cells have similar expression. The majority of T cells react positively with the anti-SHIP antibodies, but significantly fewer B cells are positive. Immunoblotting detects up to seven different cross-reacting SHIP species, with peripheral blood mononuclear cells exhibiting primarily a 100-kD protein and a CD34+ acute myeloblastic leukemia expressing mainly 130-kD and 145-kD forms of SHIP. Overall, these results indicate that there is an enormous diversity in the size of SHIP or SHIP-related mRNA and protein species. Furthermore, the expression of these protein species changes according to both the developmental stage and differentiated lineage of the mature blood cell.

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