Effects of human immunodeficiency virus on the erythrocyte and megakaryocyte lineages

Anaemia and thrombocytopenia are haematological disorders that can be detected in many human immunodeficiency virus(HIV)-positive patients during the development of HIV infection. The progressive decline of erythrocytes and platelets plays an important role both in HIV disease progression and in the clinical and therapeutic management of HIV-positive patients. HIV-dependent impairment of the megakaryocyte and erythrocyte lineages is multifactorial and particularly affects survival, proliferation and differentiation of bone marrow(BM) CD34+ haematopoietic progenitor cells, the activity of BM stromal cells and the regulation of cytokine networks. In this review, we analyse the ma-jor HIV-related mechanisms that are involved in the genesis and development of the anaemia and thrombocytopenia observed in HIV positive patients.

Megakaryocyte aplastic thrombocytopeniaafter CAR T-cell therapy in a patient withmultiple myeloma:A case report

Chimeric antigen receptor(CAR)T-cell therapy is an effective new treatment strategy for hematologic malignancies.The success of CAR T-cell therapy in treating leukemia and lymphoma has promoted its development for multiple myeloma(MM),and the initial results of CAR T cell therapy have been encouraging.CAR T-cell therapy target antigens that have been clinically evaluated in MM;these antigens include CD19,B cell maturation antigen(BCMA),CD38,and CD138.A barrier to the widespread use of CAR T-cell therapy is its toxicity,primarily cytokine release syndrome(CRS),and neurologic toxicity.This study reports a patient with refractory MM who also developed megakaryocyte aplastic thrombocytopenia after receiving CAR T-cell therapy;such a case or the unusual side effects involving medications are yet unreported.There are risks in using cyclosporine and other immunosuppressants that may lead to MM recurrence as the use of such substances is contradictory to previous treatments;therefore,we temporarily administered platelet infusion as supportive care.Thus far,the condition of the patient has been steady and the patient regularly takes blood test in the hospital.

Direct Infection of Colony Forming Unit-Megakaryocyte by Human Cytomegalovirus Contributes the Pathogenesis of Idiopathic Thrombocytopenic Purpura

Human cytomegalovirus （HCMV） late mRNA expression in megakaryoblast and in turn the pathogenesis of idiopathic thrombocytopenic purpura （ITP） patients with HCMV infection, and effectiveness of ganciclovir were investigated. Colony forming unit-megakaryocytes （CFU-MK） of 46 ITP patients with HCMV infection were incubated from patients＇ bone marrow mononuclear cells （MNC）. Reverse transcriptase-polymerase chain reaction （RT-PCR） was subsequently used for CFU-MK for HCMV-late mRNA detection, Ganciclovir therapy was given to both HCMV-late mRNA positive and negative groups for comparison of therapeutic effectiveness, The results in 19 of 46 CFU-MK culture cells specimens with positive HCMV-DNA by PCR or positive CMV-IgM by enzyme linked immunosorbent assay （ELISA） in the correspondent serum of peripheral blood were positive for HCMV-late mRNA, Sixteen out of 19 patients with positive HCMV-late mRNA CFU-MK had a positive response to ganciclovir. Amongst 27 patients with negative HCMV-late mRNA CFU-MK, only 4 positive responders to ganciclovir therapy were observed. Curative effectiveness of ganciclovir in HCMV-late mRNA positive group was significantly higher than that in HCMV-late mRNA negative group （P〈0.01）, It was suggested that HCMV could directly infect CFU-MK, which might be one of the mechanisms responsible for HCMV related ITE The ganci- clovir is an effective therapy in resulting in the increases in thrombocyte in the ITP patients whose HCMV- late mRNA was positive in their CFU-MK.

MXRA7 is involved in megakaryocyte differentiation and platelet production

Matrix remodeling is a critical process in hematopoiesis.The biology of MXRA7,as a matrix remodeling associated gene,has still not been reported in hematopoietic process.Public databases showed that MXRA7 expressed in hematopoietic stem cells,suggesting that it may be involved in hematopoiesis.We found that the amounts of megakaryocytes were lower in bone marrow and spleen from Mxra7−/−mice compared with that from wild-type mice.Knock-out of MXRA7 also reduced the amount of platelet in peripheral blood and affected the function of platelets.Knock-out of MXRA7 inhibited hematopoietic stem/progenitor cells differentiate to megakaryocytes possibly through down-regulating the expression of GATA-1 and FOG-1.Moreover,knockdown of MXRA7 in MEG-01 cells could inhibit the cell proliferation and cell apoptosis.Knockdown of MXRA7 inhibited the differentiation of MEG-01 cells and proplatelet formation through suppressing the ERK/MAPK signaling pathway and the expression ofβ-tubulin.In conclusion,the current study demonstrated the potential significance of MXRA7 in megakaryocyte differentiation and platelet production.The novel findings proposed a new target for the treatment of platelet-related diseases,and much more investigations are guaranteed to dissect the mechanisms of MXRA7 in megakaryocyte differentiation and platelet production.

Effects of Panaxadiol Saponins Component as A New Chinese Patent Medicine on Proliferation,Differentiation and Corresponding Gene Expression Profile of Megakaryocytes

Objective： To investigate the effects of panaxadiol saponins component （PDS-C） isolated from total saponins of panax ginseng on proliferation, differentiation and corresponding gone expression profile of megakaryocytes. Methods： Bone marrow culture of colony forming assay of megakaryocytic progenitor cells （CFU-MK） was observed for the promoting proliferation mediated by PDS-C, and differentiation of megakaryocytic blasts caused by PDS-C was analyzed with flow cytometry in CHRF-288 and Meg-01 cells, as well as proliferation, differentiation-related genes expression profile and protein expression levels were detected by human gone expression microarray and western blot. Results： In response to PDS-C 10, 20 and 50 mg/L, CFU-MK from 10 human bone marrow samples was increased by 28.9± 2.7%, 41.0% ± 3.2% and 40.5% ± 2.6% over untreated control, respectively （P〈0.01, each）. Flow cytometry analysis showed that PDS-C treated CHRF-288 cells and Meg-01 cells significantly increased in CD42b, CD41, TSP and CD36 positive ratio, respectively. PDS-C induced 29 genes up-regulated more than two-fold commonly in both cells detected by human expression microarray representing 4000 known genes. The protein expression levels of ZNF91, c-Fos, BTF3a, GATA-1, RGS2, NDRG2 and RUNX1 were increased with western blot in correspond to microarray results. Conclusion： PDS-C as an effective component for hematopoiesis, play the role to enhance proliferation and differentiation of megakaryocytes, also up-regulated expression of proliferation, differentiation-related genes and proteins in vitro. KEYWORDS panaxadiol saponins, megakaryocyte, gone expression profile, proliferation, differentiation

Ultrastructural alterations of megakaryocytes in thrombocytopenia:A review of 43 cases

Thrombocytopenia is a frequent occurrence in a variety of hematopoietic diseases;however,the details of the mechanism leading to low platelet count remain elusive.Megakaryocytes are a series of progenitor cells responsible for the production of platelets.Alterations in megakaryocytes in the bone marrow are a causative factor resulting in thrombocytopenia in varied diseases.Based on ultrastructural analysis of incidentally encountered megakaryocytes in 43 patients with blood diseases marked by low platelet counts,electron micrographs demonstrated that aberrant megakaryocytes predominated in idiopathic thrombocytopenic purpura,aplastic anemia,and myelodysplastic syndrome;autophagy,apoptosis,and cellular damage in megakaryocytes were a prominent feature in aplastic anemia.On the other hand,poorly differentiated megakaryocytes predominated in acute megakaryoblastic leukemia(AMKL)although damaged megakaryocytes were seen in non-AMKL acute leukemia.This paper documents the ultrastructural alterations of megakaryocytes associated with thrombocytopenia and reveals distinctive features for particular blood diseases.A comment is made on future avenues of research emphasizing membrane fusion proteins.

Abivertinib inhibits megakaryocyte differentiation and platelet biogenesis

Abivertinib,a third-generation tyrosine kinase inhibitor,is originally designed to target epidermal growth factor receptor(EGFR)-activating mutations.Previous studies have shown that abivertinib has promising antitumor activity and a well-tolerated safety profile in patients with non-small-cell lung cancer.However,abivertinib also exhibited high inhibitory activity against Bruton’s tyrosine kinase and Janus kinase 3.Given that these kinases play some roles in the progression of megakaryopoiesis,we speculate that abivertinib can affect megakaryocyte(MK)differentiation and platelet biogenesis.We treated cord blood CD34+hematopoietic stem cells,Meg-01 cells,and C57BL/6 mice with abivertinib and observed megakaryopoiesis to determine the biological effect of abivertinib on MK differentiation and platelet biogenesis.Our in vitro results showed that abivertinib impaired the CFU-MK formation,proliferation of CD34+HSC-derived MK progenitor cells,and differentiation and functions of MKs and inhibited Meg-01-derived MK differentiation.These results suggested that megakaryopoiesis was inhibited by abivertinib.We also demonstrated in vivo that abivertinib decreased the number of MKs in bone marrow and platelet counts in mice,which suggested that thrombopoiesis was also inhibited.Thus,these preclinical data collectively suggested that abivertinib could inhibit MK differentiation and platelet biogenesis and might be an agent for thrombocythemia.

The Effect of Human Cytomegalovirus on the Formation of CFU-MK In Vitro

To investigate the mechanism and the suppressive effect of human cytomegalovirus (HCMV) on colony forming unit-megakaryocyte (CFU-MK), semi-solid culture system was used to observe the effect of HCMV AD169 strain on CFU-MK's growth of 18 cord blood samples. HCMV DNA and immediate early (IE) protein mRNA in CFU-MK was detected by PCR and reverse transcription-polymerase chain reaction (RT-PCR). Our results showed that HCMV AD169 significantly suppressed the formation of CFU-MK in vitro. Compared with the mock group, the CFU-MK colonies decreased by 21. 6 %, 33. 8 % and 46. 3 %, respectively, in all the 3 infected groups (P<0. 05), suggesting the suppression and the titer of the virus was dose-dependent. Both HCMV DNA and the expression of HCMV IE protein mRNA were positively detected in the colony cells of viral infected group,. It is concluded that HCMV AD169 strain could inhibit the differentiation and proliferation of CFU-MK by directly infecting their progenitors. There was early transcription of HCMV IE protein in CFU-MK infected by virus.

OBSERVATION OF MEGAKARYOCYTOPOIESIS IN HUMAN FETUS BY IMMUNOCYTOCHEMICAL STAINING WITH ANTI-PLATELET GLYCOPROTEIN ⅡB /ⅢA MONOCLONAL ANTIBODY

Fetal liver tissues obtained from 28 human fetuses with gestation age from 3 to 6 months and fetal bone marrow from 35 human fetuses from 3 to 7 months were observed by immunochemical staining with anti-platelet GPⅡ b / Ⅲa monoclonal antibody and ABC technique. In the fetal liver, megakaryocytes were wholly located among growing fetal liver cells and near foci of hemopoiesis. Some megakaryocytes in the fetal liver were small7890- lymphoid-like megakaryocytes. The size of megakaryocytes both in the fetal liver (14.79 ± 4.52μm) and in the fetal bone marrow (16.08±7.39 μm) was small, which did not vary significantly over the gestation age ranging from 3 to 6 or 7 months. However, the maturation stage of megakaryocytes in the fetal liver shifted to more mature stage with the advancement of gestation, although the maturation stage of megakaryocytes in the fetal bone marrow did not change with the advancement of gestation from 4 to 7 months, the megakaryocyte in the fetal bone marrow was less mature

Analysis of gene expression in HIMeg cells before and after induced differentiation:Application of mRNA differential display

Objective: In human megakaryocytic leukemia HIMeg cells, differentially expressed genes regulated byinduced differentiation will be cloned, and from which the key genes in the process of induced differentiation will beidentified. Metbods: The human megakaryocytic leukemia cell line HIMeg was treated with 13-cis retinoic acid for4 d, then total cellular RNA was extracted and reversely transcribed to first strand cDNA. The first strand cDNAwas subjected to PCR based mRNA differential display (mRNA DD), cDNA cloning and sequence analysis- Results: From 5 cDNA fragment candidates obtained, RNA dot blot analysis demonstrated non-regulation in 3, undetectable signals in 1, and altered gene expression in one cDNA fragment which is designated MDI-1 (mRNAdownregulated after induced differentiation ). The nucleotide sequence data reported here will appear in the GenBank under the accession number AF026526. Conclusion: A cDNA fragment was cloned, whose gene expressionwas inhibited after 13-cis retinoic acid-induced differentiation. Further studies are required to determine its fulllength sequence, gene structure and biological function(s).

European vs 2015-World Health Organization clinical molecular and pathological classification of myeloproliferative neoplasms

The BCR/ABL fusion gene or the Ph^1-chromosome in the t(9;22)(q34;q11)exerts a high tyrokinase acticity,which is the cause of chronic myeloid leukemia(CML).The1990 Hannover Bone Marrow Classification separated CML from the myeloproliferative disorders essential thrombocythemia(ET),polycythemia vera(PV)and chronic megakaryocytic granulocytic myeloproliferation(CMGM).The 2006-2008 European Clinical Molecular and Pathological(ECMP)criteria discovered 3variants of thrombocythemia:ET with features of PV(prodromal PV),"true"ET and ET associated with CMGM.The 2008 World Health Organization(WHO)-ECMP and 2014 WHO-CMP classifications defined three phenotypes of JAK2^(V617F)mutated ET:normocellular ET(WHO-ET),hypercelluar ET due to increased erythropoiesis(prodromal PV)and ET with hypercellular megakaryocytic-granulocytic myeloproliferation.The JAK2^(V617F)mutation load in heterozygous WHO-ET is low and associated with normal life expectance.The hetero/homozygous JAK2^(V617F)mutation load in PV and myelofibrosis is related to myeloproliferative neoplasm(MPN)disease burden in terms of symptomaticsplenomegaly,constitutional symptoms,bone marrow hypercellularity and myelofibrosis.JAK2 exon 12mutated MPN presents as idiopathic eryhrocythemia and early stage PV.According to 2014 WHO-CMP criteria JAK2 wild type MPL^(515)mutated ET is the second distinct thrombocythemia featured by clustered giant megakaryocytes with hyperlobulated stag-horn-like nuclei,in a normocellular bone marrow consistent with the diagnosis of"true"ET.JAK2/MPL wild type,calreticulin mutated hypercellular ET appears to be the third distinct thrombocythemia characterized by clustered larged immature dysmorphic megakaryocytes and bulky(bulbous)hyperchromatic nuclei consistent with CMGM or primary megakaryocytic granulocytic myeloproliferation.

HOXC4 up-regulates NF-κB signaling and promotes the cell proliferation to drive development of human hematopoiesis, especially CD43+ cells

The hematopoietic function of HOXC4 has not been extensively investigated.Our research indicated that induction of HOXC4 in co-culture system from D10 significantly promoted productions of most hematopoietic progenitor cells.CD34−CD43+cells could be clearly classified into CD34−CD43^(low) and CD34−CD43^(high) sub-populations at D14.The former cells had greater myelogenic potential,and their production was not significantly influenced by induction of HOXC4.By contrast,the latter cells had greater potential to differentiate into megakaryocytes and erythroid cells,and thus had properties of erythroid–megakaryocyte common progenitors,which abundance was increased by∼2-fold when HOXC4 was induced from D10.For CD34−CD43^(low),CD34+CD43+,and CD34−CD43^(high) sub-populations,CD43 level served as a natural index for the tendency to undergo hematopoiesis.Induction of HOXC4 from D10 caused more CD43+cells sustain in S-phase with up-regulation of NF-κB signaling,which could be counteracted by inhibition of NF-κB signaling.These observations suggested that promotion of hematopoiesis by HOXC4 is closely related to NF-κB signaling and a change in cell-cycle status,which containing potential of clinical applications.

Loss of Tet2 affects platelet function but not coagulation in mice

Ten-eleven translocation 2(TET2)functions as a methylcytosine dioxygenase that catalyzes the iterative oxidation of 5-methylcytosine to 5-hydroxymethylcytosine,5-formylcytosine and 5-carboxylcytosine.TET2 has been shown to be crucial for the maintenance and differentiation of hematopoietic stem cells,and its deletion and/or mutations results in the expansion of HSPCs,and leads to hematological malignancies.TET2 mutations were found in a variety of hematological disorders such as CMML(60%),MDS(30%),MPN(13%)and AML(20%).Interestingly,it was shown that CMML patients with TET2 mutation exhibited fewer platelets than CMML patients without TET2 mutation.However,the role and function of TET2 in platelet hemostasis and thrombogenesis is not well defined.Here in this study,using a genetically engineered Tet2 deletion mouse model,we found that the absence of Tet2 caused a decrease in the proportion of MEP cells and hyperploid megakaryocytes.Additionally,Tet2-deficient mice displayed impaired platelet activation and aggregation under stimulation of ADP and low concentrations of thrombin,although the modestly compromised platelet function and MEP differentiation in Tet2-deficient mice could be compensated without affecting blood coagulation function.Our study indicate that Tet2 deficiency leads to mild impairment of platelet function and thrombopoiesis in mice.

STUDY ON INDUCTION OF DIFFERENTIATION OF A HUMAN MEGAKARYOBLASTIC LEUKEMIA CELL LINE (HIMeg) BY 1,25-DIHYDROXYVITAMIN D_3

It is reported that 1,25-dihydroxyvitamin D_3(1,25(OH)_2D_3), a physiological factor, has aninductive effect on the differentiation of a novel human megakaryoblastic leukemia cell line(HIMeg) in vitro. At the concentrations ranging from 10^(-9) to 10^(-6) mol/L, 1,25(OH)_2D_3 showedinhibition of proliferation on HIMeg cells which was demonstrated by count of survivalcells and cloning efficiency. Meanwhile, using light/electron microscopy, stain of cytochem-istry (including immunoenzymatic technique) and flow cytometry, we found that HIMeg cellscould be further induced into more mature cells in megakaryocytic lineage confirmed by aseries of evidence, including the changes of cell morphology/structure and cytochemistry,increased expression of differentiation antigens on the cell surface, and polyploidization.So, it is possible for 1,25(OH)_2D_3 to promote the differentiation of the cells in megakaryo-cytic lineage in vivo and to be used to treat acute megakaryoblastic leukemia and other di-seases with malignant megakaryocytosis.