Platelet-derived microparticles that are produced during platelet activation bind to traumatized endothelium. Such endothelial injury occurs during percutaneous transluminal coronary angioplasty. Approximately 20% of these patients subsequently develop restenosis, although this is improved by treatment with the anti-platelet glycoprotein IIb/IIIa receptor drug abciximab. As platelet activation occurs during angioplasty, it is likely that platelet-derived microparticles may be produced and hence contribute to restenosis. This study population consisted of 113 angioplasty patients, of whom 38 received abciximab. Paired peripheral arterial blood samples were obtained following heparinization and subsequent to all vessel manipulation. Platelet-derived microparticles were identified using an anti-CD61 (glycoprotein IIIa) fluorescence-conjugated antibody and flow cytometry. Baseline clinical characteristics between patient groups were similar. The level of platelet-derived microparticles increased significantly following angioplasty in the group without abciximab (paired t test, P = 0.019). However, there was no significant change in the level of platelet-derived microparticles following angioplasty in patients who received abciximab, despite requiring more complex angioplasty procedures. In this study, we have demonstrated that the level of platelet-derived microparticles increased during percutaneous transluminal coronary angioplasty, with no such increase with abciximab treatment. The increased platelet-derived microparticles may adhere to traumatized endothelium, contributing to re-occlusion of the arteries, but this remains to be determined.

OBJECTIVE

To explore whether T lymphocytes subgroup, B lymphocytes, platelet antigen CD41a, CD61 or platelet antibodies are involved in the platelet transfusion refractoriness.

METHODS

Forty-seven patients diagnosed as platelet transfusion refractoriness and 32 patients that achieved effective platelet therapy were ennolled in this study. Flow cytometry was performed to detect the proportion of cytotoxic T cell (CD3(+)CD4(-)CD8(+)), helper T cell (CD3(+)CD4(+)CD8(-)) and B lymphocytes (CD19 (+) ), and the expression of platelet glycoproteins, including CD41a and CD61, while platelet antibodies were also measured by solid-phase agglutination.

RESULTS

The significant lower level of helper T cell (36.60% vs 48.53%), higher level of cytotoxic T cell (53.26% vs 44.02%) and lower cytotoxic/helper T cell ratio (0.85 vs 1.31) were observed in platelet refractoriness group when compared with effective platelet therapy group (P<0.05). However, the significant difference was not observed in B lymphocytes between the two group (3.02% vs 2.85%, P>0.05). Platelet glycoproteins CD41a and CD61 and antibodies were both expressed at high levels in platelet refractoriness group (88.10% vs 51.69%, 88.36% vs 51.83%, 85.37% vs 14.82%, respectively, P<0.05).

CONCLUSIONS

Activation of cytotoxic T cells, suppression of helper T cells, higher expression level of platelet glycoproteins CD41a and CD61 as well as the development of anti-platelet antibodies are involved in the immunologic mechanism of platelet refractoriness.

To assess possible alterations of megakaryocytes associated with interferon (IFN) and busulfan (BU) therapy of Ph(1+)-CML, an immunohistochemical and morphometric study was performed on trephine biopsies of the bone marrow taken before and at varying intervals during treatment. For the identification of megakaryopoiesis and its endoreduplicative activity the monoclonal antibodies CD61 (anti-platelet glycoprotein IIIa) and PC10 raised against proliferating cell nuclear antigen (PCNA) were used. We compared 60 specimens from 20 patients following IFN alpha-2b administration (in combination with IFN gamma in seven patients) with 57 specimens from 22 patients after monotherapy with BU. A close correlation with clinical follow-up studies revealed that in the IFN-treated group the prevalence of atypical micro-megakaryocytes, usually characterizing CML, was conspicuously reduced in repeatedly taken bone marrow samples. Initially, even an increase in size which was levelled to normal values during maintenance therapy was observed. These features were most prominently expressed in the 13 patients with a complete hematologic and/or partial cytogenetic response. Associated with this phenomenon was a significant enhancement of the PCNA-labelling index which indicated a stimulation of endoreduplicative (endomitotic) activity necessary for achieving normal size and ploidy. In the second group of patients treated by BU these changes were absent. For this reason, our findings are in keeping with the assumption that during IFN treatment, there is at least partial recovery and expansion of a putative normal (Ph1-) megakaryopoiesis. In conclusion, megakaryocyte morphology, i.e. normalization in size, is thought to be a useful indicator to evaluate the response to IFN in CML patients.

An elevated concentration of fibrinogen in blood is a significant risk factor during many pathological diseases, as it leads to an increase in red blood cells (RBC) aggregation, resulting in hemorheological disorders. Despite the biomedical importance, the mechanisms of fibrinogen-induced RBC aggregation are still debatable. One of the discussed models is the non-specific adsorption of fibrinogen macromolecules onto the RBC membrane, leading to the cells bridging in aggregates. However, recent works point to the specific character of the interaction between fibrinogen and the RBC membrane. Fibrinogen is the major physiological ligand of glycoproteins receptors IIbIIIa (GPIIbIIIa or αIIββ3 or CD41/CD61). Inhibitors of GPIIbIIIa are widely used in clinics for the treatment of various cardiovascular diseases as antiplatelets agents preventing the platelets' aggregation. However, the effects of GPIIbIIIa inhibition on RBC aggregation are not sufficiently well studied. The objective of the present work was the complex multimodal in vitro study of the interaction between fibrinogen and the RBC membrane, revealing the role of GPIIbIIIa in the specificity of binding of fibrinogen by the RBC membrane and its involvement in the cells' aggregation process. We demonstrate that GPIIbIIIa inhibition leads to a significant decrease in the adsorption of fibrinogen macromolecules onto the membrane, resulting in the reduction of RBC aggregation. We show that the mechanisms underlying these effects are governed by a decrease in the bridging components of RBC aggregation forces.

Keywords: RBC membrane; fibrinogen macromolecules; flow cytometry; glycoproteins IIbIIIa inhibition; microfluidics; optical (laser) tweezers.

Cardiovascular diseases remain the leading cause of death worldwide. Patency rates of clinically-utilized small diameter synthetic vascular grafts such as Dacron® and expanded polytetrafluoroethylene (ePTFE) to treat cardiovascular disease are inadequate due to lack of endothelialization. Sodium trimetaphosphate (STMP) crosslinked PVA could be potentially employed as blood-compatible small diameter vascular graft for the treatment of cardiovascular disease. However, PVA severely lacks cell adhesion properties, and the efforts to endothelialize STMP-PVA have been insufficient to produce a functioning endothelium. To this end, we developed a one-pot method to conjugate cell-adhesive protein via hydroxyl-to-amine coupling using carbonyldiimidazole by targeting residual hydroxyl groups on crosslinked STMP-PVA hydrogel. Primary human umbilical vascular endothelial cells (HUVECs) demonstrated significantly improved cells adhesion, viability and spreading on modified PVA. Cells formed a confluent endothelial monolayer, and expressed vinculin focal adhesions, cell-cell junction protein zonula occludens 1 (ZO1), and vascular endothelial cadherin (VE-Cadherin). Extensive characterization of the blood-compatibility was performed on modified PVA hydrogel by examining platelet activation, platelet microparticle formation, platelet CD61 and CD62P expression, and thrombin generation, which showed that the modified PVA was blood-compatible. Additionally, grafts were tested under whole, flowing blood without any anticoagulants in a non-human primate, arteriovenous shunt model. No differences were seen in platelet or fibrin accumulation between the modified-PVA, unmodified PVA or clinical, ePTFE controls. This study presents a significant step in the modification of PVA for the development of next generation in situ endothelialized synthetic vascular grafts.

Keywords: anticoagulation; blood clotting; endothelial adhesion; hemocompatibility; surface modification; thrombogenesis.

The objective of this study was to evaluate the effect of clopidogrel response in patients with untreated type 2 diabetes mellitus as compared with normal individuals. One hundred and seven subjects i.e. 32 normal and 75 patients with untreated type 2 diabetes mellitus were enrolled in this study. In the first step, normal subjects as well as diabetic patients were selected and tested for various laboratory parameters and platelets flow cytometry. In the second step, an antiplatelet drug (clopidogrel) was administered for 10 days to each individual enrolled in the study. After 10 days blood samples were collected for platelets flow cytometry. CD41 and CD61 did not show any change after the administration of clopidogrel in resting and activated platelets. CD63 and CD62p positivity was increased in normal and in diabetic patients' platelets after activation with ADP before clopidogrel. It was decreased in normal resting and ADP stimulated platelets after clopidogrel treatment. CD63 and CD62p positivity in resting and ADP stimulated patients' platelets was also decreased after clopidogrel treatment. The change was, however, not as marked as in normal subjects.

CD71 is an immunohistochemical marker used in diagnosing acute myeloid leukemia (AML) M6-Er in humans; however, to our knowledge, it has not been reportedly used for immunohistochemistry in veterinary medicine. We evaluated the pathologic features of AML M6-Er in a retrovirus-negative cat and used CD71 to support the diagnosis. A 4-y-old spayed female Scottish Fold cat was presented with lethargy, anorexia, and fever. Whole-blood PCR assay results for pro feline leukemia virus/pro feline immunodeficiency virus and feline vector-borne diseases were negative. Early erythroid precursors were observed in the peripheral blood smear. Fine-needle aspiration of the enlarged spleen and splenic lymph node showed many early erythroid precursors. Bone marrow aspirate smears revealed erythroid hyperplasia with 68.4% erythroid lineage and 3.6% rubriblasts. Dysplastic cells infiltrated other organs. The patient was diagnosed with myelodysplastic syndrome, progressing to the early phase of AML M6-Er. The patient died on day 121 despite multidrug treatments. Postmortem examination revealed neoplastic erythroblasts infiltrating the bone marrow and other organs. Neoplastic cells were immunopositive for CD71 but immunonegative for CD3, CD20, granzyme B, von Willebrand factor, CD61, myeloperoxidase, and Iba-1. Although further studies are necessary for the application of CD71, our results supported the morphologic diagnosis of AML M6-Er.

Keywords: CD71; erythroleukemia; feline; immunohistochemistry.

Background: Bronchoalveolar lavage (BAL) in patients with severe coronavirus disease 2019 (COVID-19) may provide additional and complementary findings for the management of these patients admitted to intensive care units (ICUs). This study addresses the cytological features of the infection and highlights the more influential inflammatory components. The correlation between pathological variables and clinical data is also analyzed.

Methods: The authors performed a retrospective analysis of the cytopathological features of BAL in 20 COVID-19 patients and 20 members of a matched cohort from a critical ICU who had acute respiratory distress syndrome caused by other pulmonary conditions.

Results: A comparison of the controls (n = 20) and the COVID-19 patients (n = 20) revealed that the latter had a higher neutrophil count (median, 63.8% of the cell count) with lower percentages of macrophages and lymphocytes. An increase in the expression of CD68-positive, monocytic multinucleated giant cells (MGCs) was reported; megakaryocytes were not detected on CD61 staining. Perls staining showed isolated elements. In situ RNA analysis demonstrated scattered chromogenic signals in type II pneumocytes. An ultrastructural analysis confirmed the presence of intracytoplasmic vacuoles containing rounded structures measuring 140 nm in diameter (putative viral particles). In COVID-19 patients, the clinicopathological correlation revealed a positive correlation between lactate dehydrogenase values and MGCs (r = 0.54).

Conclusions: The analysis of BAL samples might be implemented as a routine practice for the evaluation of COVID-19 patients in ICUs in the appropriate clinical scenario. Additional studies using a larger sample size of patients who developed COVID-19 during the second wave of the epidemic in the autumn of 2020 are needed to further support our findings.

Keywords: bronchoalveolar lavage (BAL); coronavirus disease 2019 (COVID-19); multinucleated giant cell; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

We have demonstrated that anticancer drugs at cytostatic concentrations enhance the expression and function of epidermal growth factor (EGF-R) and transferrin (TRF-R) receptors on human tumor cells. We hypothesized that these effects could represent a protective response of tumor cells to sublethal antiproliferative stimuli which could lead to enhanced growth potential. 72 hours exposure of human melanoma GLL-19 cells to 1,000 nM ara-C induced growth inhibition and increased the number of EGF-R, TRF-R and nerve growth factor receptor (NGF-R) on cell surface. Enhanced expression of beta 3 integrins CD49a, CD49c and CD49e, av integrin CD51, beta 3 integrin CD61, CD58/LFA3 and collagen IV and laminin was also detected in ara-C-treated GLL-19 cells. These changes at the tumor cell surface were paralleled by increased in vitro adhesion, invasive potential and clonogenic growth in soft agar and in vivo tumor formation. A more aggressive tumor cell phenotype is induced in human melanoma cells after transient exposure to cytostatic concentrations of ara-C.

Objective: To analyze the clinical characteristics and treatment effects of children with acute megakaryoblastic leukemia without down syndrome (non-DS-AMKL).

Methods: The clinical data of 19 children with non-DS-AMKL treated in the Pediatric Hematology Ward in Sun Yat-sen Memorial Hospital of Sun Yat-sen University from May 2008 to April 2018 were analyzed retrospectively. The clinical characteristics, laboratory test and treatment methods of the children were concluded. All patients were followed up to evaluate the effect of treatment.

Results: The 19 cases of children included nine male and ten female, the median age of onset was 2 years old. The clinical manifestations showed nonspecific. The median white blood cell of peripheral blood was 15.88×10⁹/L, the median hemoglobin was 67 g/L and median platelet was 16×10⁹/L. An increase of primitive and naive megakaryocytes was found in the bone marrow sample. The immunophenotypes of bone marrow detected by flow cytometry in 19 children were all positive expressed for CD41, CD61. Genetic tests showed that five cases carrying EVI1, including one complicating with MLL/AF10, one patient carrying HOX11, one carrying GATA1, IKZF1 and DDX11 mutations, one patient carrying missense mutation of NRAS, one with missense mutation of KRAS and two cases with high expression of WT1. Karyotype analysis were performed in 11 cases of children, including four with normal karyotype, four with complex karyotypes, one with trisomy 8, one with 7/13 trisomy 21 without Down syndrome manifestations (considered as abnormal somatic karyotype) and one Robertsonian translocation carrier involving chromosomes 14 and 21. Ten children received treatment, including three cases with allogeneic hematopoietic stem cell transplantation (allo-HSCT) after complete remission (CR), two cases with complete donor implantation, and one without implanted but hematopoietic recovered, these three children were followed up for 26, 15 and 12 months respectively and the minimal residual disease (MRD) were all less than 10^-4. Another three cases achieving CR after chemotherapy but relapsed in 5, 10 and 12 months after the onset respectively, and all the three children were died eventually. One children died of pulmonary hemorrhage during chemotherapy and three children died from discontinuation of treatment after non remission. The remaining nine children died because of without chemotherapy treatment.

Conclusion: Non-DS-AMKL was rare in children and difficult to be diagnosed. Determination of MICM classification as early as possible was helpful for diagnosis, and genetic testing played an important role for diagnosis and prognosis evaluation. Early hematopoietic stem cell transplantation in patients with CR after chemotherapy might be an effective way to cure AMKL.

题目: 儿童non-DS-AMKL的临床特点及预后分析.

目的: 分析儿童非Down综合征相关急性巨核细胞白血病（non-DS-AMKL）的临床特征及治疗效果.

方法: 对2008年5月至2018年4月中山大学孙逸仙纪念医院儿科血液病区收治的19例non-DS-AMKL患儿的临床资料进行回顾性分析，总结其发病特点、实验室检查结果及治疗方法，并对所有患儿进行随访，统计治疗效果.

结果: 19例non-DS-AMKL患儿，其中男9例，女10例，发病中位年龄为2岁。临床表现无特异性。外周血白细胞中位数15.88×10⁹/L，血红蛋白中位数67 g/L，血小板中位数16×10⁹/L。骨髓象见原始和幼稚巨核细胞增多。19例患儿骨髓流式细胞术检测免疫表型均表达CD41、CD61。基因检测结果中，5例EVI1⁺，其中1例合并MLL/AF10⁺，1例HOX11⁺，1例存在GATA1、IKZF1、DDX11基因变异，2例NRAS错义变异，1例KRAS错义变异，2例WT1高表达。11例行染色体核型分析，其中4例染色体正常，4例为复杂核型，1例为8三体， 1例7/13个细胞核型为21三体但无Down综合征临床表现（考虑为体细胞核型异常），1例为14号和21号染色体罗伯特易位携带者。10例患儿接受治疗，有6例患儿获得完全缓解（CR），其中3例CR后接受异基因造血干细胞移植术，2例完全供体植入，1例未植入但自身造血恢复，分别随访26、15及12个月，微小残留病灶监测（MRD）均＜10^-4；另外3例CR患儿分别于发病5、10和12个月复发，最终均未能再次达到CR而死亡。1例化疗中因肺出血死亡，另外3例中途放弃化疗最终死亡，其余9例患儿均未接受化疗最终死亡.

结论: 儿童non-DS-AMKL较为少见，临床诊断困难，尽早明确MICM分型有助于确诊，基因检测对于明确诊断和评估预后有重要意义。化疗后获得CR的患者，尽早行造血干细胞移植术可能是治愈AMKL的有效手段.

Dengue virus (DENV) infection causes dengue fever in humans, which can lead to thrombocytopenia showing a marked reduction in platelet counts, and dengue hemorrhagic fever. The virus may cause thrombocytopenia either by destroying the platelets or by interfering with their generation via the process of megakaryopoiesis. MEG-01 is the human megakaryoblastic leukemia cell line that can be differentiated in vitro by phorbol-12-myristate-13-acetate (PMA) treatment to produce platelet-like-particles (PLPs). We have studied DENV infection of MEG-01 cells to understand its effect on megakaryopoiesis and the generation of PLPs. We observed that DENV could infect only naive MEG-01 cells, and differentiated cells were refractory to virus infection/replication. However, DENV-infected MEG-01 cells, when induced for differentiation with PMA, supported an enhanced viral replication. Following the virus infection, the MEG-01 cells showed a marked reduction in the surface expression of platelet markers (CD41, CD42a, and CD61), a decreased polyploidy, and significantly reduced PLP counts. DENV infection caused an enhanced Notch signaling in MEG-01 cells where the virus envelope protein was shown to interact with TAL-1, a host protein important for megakaryopoiesis. These observations provide new insight into the role of DENV in modulating the megakaryopoiesis and platelet production process.

To develop a novel fluorine-18 (¹⁸F)-labeled arginine-glycine-aspartic acid (RGD)-coupled ultra-small iron oxide nanoparticle (USPIO) (hereafter, referred to as ¹⁸F-RGD@USPIO) and conduct an in-depth investigation to monitor the anti-angiogenic therapeutic effects by using a novel dual-modality PET/MRI probe.

The RGD peptide and ¹⁸F were coupled onto USPIO by click chemistry. In vitro experiments including determination of stability, cytotoxicity, cell binding of the obtained ¹⁸F-RGD@USPIO were carried out, and the targeting kinetics and bio-distribution were tested on an MDA-MB-231 tumor model. A total of 20 (n = 10 per group) MDA-MB-231 xenograft-bearing mice were treated with bevacizumab or placebo (intraperitoneal injections of bevacizumab or a volume-equivalent placebo solution at the dose of 5 mg/kg for consecutive 7 days, respectively), and underwent PET/CT and MRI examinations with ¹⁸F-RGD@USPIO before and after treatment. Imaging findings were validated by histological analysis with regard to β₃-integrin expression (CD61 expression), microvascular density (CD31 expression), and proliferation (Ki-67 expression).

Excellent stability, low toxicity, and good specificity to endothelial of ¹⁸F-RGD@USPIO were confirmed. The best time point for MRI scan was 6 h post-injection. No intergroup differences were observed in tumor volume development between baseline and day 7. However, ¹⁸F-RGD@USPIO binding was significantly reduced after bevacizumab treatment compared with placebo, both on MRI (P < 0.001) and PET/CT (P = 0.002). Significantly lower microvascular density, tumor cell proliferation, and integrin β₃ expression were noted in the bevacizumab therapy group than the placebo group, which were consistent with the imaging results.

PET/MRI with the dual-modality nanoprobe, ¹⁸F-RGD@USPIO, can be implemented as a noninvasive approach to monitor the therapeutic effects of anti-angiogenesis in breast cancer model in vivo.

An 87-year-old woman not known to have either a lymphoma or leukemia developed a left multinodular, fish-flesh superior epibulbar and forniceal mass. A biopsy disclosed a blastic tumor with scattered multinucleated immature megakaryoblasts. Immunophenotyping of bone marrow cells revealed strong positivity for CD7, CD31, CD43, CD45, CD61, and CD117; CD71, myeloperoxidase, and lysozyme were also positive in scattered cells. Forty percent of the neoplastic cells were Ki-67 positive. Cytogenetic studies indicated a trisomy 8 (associated with worse prognosis) and a t(12; 17) translocation. Desmin, smooth muscle actin, pancytokeratin, CAM 5.2, adipophilin, tryptase, S100, SOX10, MART1, and E-cadherin were negative, ruling out a nonhematopoietic tumor. The conjunctival lesion was diagnosed as a myeloid sarcoma with megakaryoblastic differentiation, a rare variant. It probably arose from a myelodysplastic syndrome. This is the first case of its type to develop in the conjunctiva.

BACKGROUND

This study aimed to analyze the influence of the interruption of agitation and removal of leukocytes on platelet concentrates (PCs), and determine the maximum amount of time the agitation could be interrupted without impairing PCs' effectiveness during the storage period.

METHODS

Four ABO-identical random donor platelets agitated for 24 hr were pooled, and divided into 4 units, and 2 units of them were leukoreduced. Then 52 pooled units were categorized into 4 groups, non-leukoreduced continuous agitation (Non-LRCA), non-leukoreduced interrupted agitation (Non-LRIA), leukoreduced continuous agitation (LRCA), and leukoreduced interrupted agitation (LRIA), and preserved for 6 days (total 7 days). Mean platelet volume (MPV), pH, HCO(3)(-), pO(2), pCO(2), CD62P, CD61, glucose, lactate, ammonia and free fatty acid were measured during the period.

RESULTS

Starting from the Day 4, the pH and HCO(3)(-) of Non-LRIA group begun to decrease while the amount of lactate production, glucose consumption, and MPV increased compared to the Non- LRCA group (P<0.01). An increase in pO(2) level was observed in the interrupted agitation groups as the storage period prolonged (P<0.01). The pH levels of all the units in the agitation groups remained higher than 6.4 up to Day 7, while those of the non-leukoreduction group did so only up to Day 2, but those of leukoreduction in the interrupted agitation groups did so up to Day 4.

CONCLUSIONS

The interruption of agitation reduced the platelet's capacity to utilize oxygen, increasing lactate amount and reducing pH level. However, the in vitro parameters of the Non-LRIA and Non-LRCA groups on Day 2 were similar to each other and the pH level remained at 6.4 or higher, making one day of agitation interruption possible after 24 hr of agitation. With leukocytes removed, the effective agitation interruption period may become longer.

The aim of this study was to evaluate the behavior of erythrocyte and platelet, as immunological markers, as well as evaluate the involvement of these factors in hemolytic and hemorrhagic reactions in hamsters experimentally infected by Leptospira interrogans Serovar Canicola. Our experimental design was composed by two randomized groups: Infected Group (IG) (n = 12) and control group (CG) (n = 6). Ninety-six hours after the inoculation, the presence of immunoglobulins (IgG and IgM) and complement C3 levels, related to erythrocytes and platelets, was assessed. Platelet's microparticles marked by CD61, reticulocytes and reticulated platelets were also quantified. Additionally, fibrinogen, prothrombin time, partially activated thromboplastin time and sera levels of IgG and IgM were assessed. Our results showed that levels of platelet decreased in IG (P < 0.001); as well as, there was presence of IgG and C3 associated with erythrocyte surface in the infected animals (P < 0.01, P < 0.05, respectively). Levels of prothrombin time and Activated Partial Thromboplastin Time were increased, while fibrinogen level was decreased (P < 0.01) in IG. CD61 microparticles were higher (P < 0.05) in IG due to platelet activation. Thus, it was established a positive correlation (P < 0.01) between platelets count and fibrinogen (Figure 3, R = 0.84, P < 0.001). Therefore, the platelet consumption component was preponderant in relation to autoimmune causes. Finally, regarding the erythrocytes, the autoimmune component played an important role, did not causing hemolytic reaction in this acute experimental time.

Our previous clinical study demonstrated that low-dose decitabine showed sustained responses in nearly half of refractory immune thrombocytopenia (ITP) patients. The long-term efficacy of decitabine in ITP is not likely due to its simple role in increasing platelet production. Whether decitabine has the potential to restore immune tolerance in ITP is unknown. In this study, we analyzed the effect of decitabine on T cell subpopulations in ITP in vitro and in vivo. We found that low-dose decitabine promoted the generation and differentiation of regulatory T (Treg) cells, and augmented their immunosuppressive function. Splenocytes from CD61 knockout mice immunized with CD61+ platelets were transferred into severe combined immunodeficient (SCID) mouse recipients to induce a murine model of ITP. Low-dose decitabine alleviated thrombocytopenia and restored the balance between Treg and helper T (Th) cells in active ITP mice. Treg deletion and depletion offset the effect of decitabine in restoring CD4+ T cell subpopulations in ITP mice. For patients who received low-dose decitabine, the quantity and function of Treg cells were substantially improved, whereas Th1 and Th17 cells were suppressed compared with the pretreatment levels. Next-generation RNA sequencing and cytokine analysis showed that low-dose decitabine rebalanced T cell homeostasis, decreased proinflammatory cytokines, and down-regulated phosphorylated STAT3 in ITP patients. STAT3 inhibition analysis suggested that low-dose decitabine might restore Treg cells by inhibiting STAT3 activation. In conclusion, our data indicated that the immunomodulatory effect of decitabine provided one possible mechanistic explanation for the sustained response achieved by low-dose decitabine in ITP.

This study was aimed to investigate the protective effects of dimethylsulfoxide (DMSO) combined with trehalose on the cryopreserved platelets. The platelets were preserved at -80 degrees C. The experiments were divided into 5 groups: blank control group composed of apheresis platelet suspension; trehalose group composed of apheresis platelet suspension and 0.25 mol/L trehalose; DMSO group composed of apheresis platelet suspension and 5% DMSO; 5% combined group composed of apheresis platelet suspension, 5% DMSO and 0.25 mol/L trehalose; 2.5% combined group composed of apheresis platelet suspension, 2.5% DMSO and 0.25 mol/L trehalose. All the groups were thawed at 37 degrees C in a waterbath. The recovery rate of platelets and mean platelet volume (MPV) were assayed by using hemocytometer; the ultrastructural changes were examined by electron microscopy; the expressions of CD41, CD42b, CD61 and CD62p on platelets were detected by flow cytometry. The results indicated that single use of trehalose had no strong effect in increasing the recovery rate of platelets, but the morphology of platelets was close to normal. The DMSO showed significant effect in increasing the recovery rate of platelets and maintaining the intact property of platelets, however, the shape of platelets tended to sealing, and partial platelets still displayed heteromorphic changes. The combination of DMSO and trehalose revealed the protective effect on the external morphology and internal structure of platelets to be close to the normal homeostasis, and ensured an ideal recovery rate of the cryopreserved platelets and higher expression levels of CD41, CD42b, CD61 and CD62p in the same time. It is concluded that the combined use of DMSO and trehalose possesses the synergistic protective effect on the cryopreserved platelets, therefore, the combined use of both as the protective agent is hopeful to further raise the effectiveness of clinical infusion of the cryopreserved platelets.

OBJECTIVE

To explore the relationships between the peripheral blood levels of CD61, CD63, PAC-1 and the incidence of acute rejection and tubular necrosis after renal transplantation, and recovery of the graft function.

METHODS

The peripheral blood levels of CD61, CD63, and PAC-1 of 86 patients with uremia in different stages before and after transplantations were analyzed by flow cytometry. The patients were divided into three groups: (1) twenty-nine patients with normal grafts function, (2) hirty with acute rejection and (3) twenty-seven with acute tubular necrosis. The patients with acute rejection were randomly divided into treatment group with anticoagulants and cntrol group.

RESULTS

The peripheral blood levels of CD61, CD63 and PAC-1 significantly increased (P < 0.05) in the patients with acute rejection, in comparison with those with normal grafts function and those with acute tubular necrosis. The peripheral blood levels of CD61, CD63 and PAC-1 in patients with acute rejection in anticoagulants therapy was lower, recovery time of the grafts function was shorter, one-year survival rates of patients and grafts were higher, as compared with those of controls.

CONCLUSIONS

The patients with acute rejection have significantly high peripheral blood levels of CD61, CD63 and PAC-1 before transplantation, however, these values in patients with acute tubular necrosis are not high, this suggesting that acute rejection might relate to platelet activation, while acute tubular necrosis might not relate to it. After anticoagulants therapy in patients with acute rejection, the grafts function might recover faster and their one-year survival rates and grafts might be higher in those with CD61, CD63 and PAC-1 decreasing remarkably.

Our previous study showed that specifically delivering BikDD, a constitutive active mutant of pro-apoptotic protein Bik, to breast cancer cell xenografts in immunocompromised mice has a potent activity against tumor initiating cells (TICs), and that the combination between tyrosine kinase inhibitors (TKI) and BikDD gene therapy yielded synergistic effect on EGFR and HER2 positive breast cancer cells in immunodeficient nude mice. Those encouraging results have allowed us to propose a clinical trial using the liposome-complexing plasmid DNA expressing BikDD gene which has been approved by the NIH RAC Advisory committee. However, it is imperative to test whether systemic delivery of BikDD-expressing plasmid DNAs with liposomes into immunocompetent mice has therapeutic efficacy and tolerable side effects as what we observed in the nude mice model. In this study, we investigated the effects of BikDD gene-therapy on the primary mammary tumors, especially on tumor initiating cells (TICs), of a genetically engineered immunocompetent mouse harboring normal microenvironment and immune response. The effects on TIC population in tumors were determined by FACS analysis with different sets of murine specific TIC markers, CD49f(high)CD61(high) and CD24(+)Jagged1(-). First we showed in vitro that ectopic expression of BikDD in murine N202 cells derived from MMTV-HER2/Neu transgenic mouse tumors induced apoptosis and decreased the number of TICs. Consistently, systemic delivery of VISA-Claudin4-BikDD by liposome complexes significantly inhibited mammary tumor growth and slowed down residual tumor growth post cessation of therapy in MMTV-HER2/Neu transgenic mice compared to the controls. In addition, the anti-tumor effects of BikDD in vivo were consistent with decreased TIC population assessed by FACS analysis and in vitro tumorsphere formation assay of freshly isolated tumor cells. Importantly, systemic administration of BikDD did not cause significant cytotoxic response in standard toxicity assays or body weight changes. Taken together, our findings validated that selective expression of BikDD in the primary mammary tumors in immunocompetent hosts significantly reduced tumor burden and inhibited the residual tumor growth at off-therapy stage by eliminating TICs. Hence, the VISA-Claudin4-BikDD-mediated gene therapy is worthy of further investigation in breast cancer clinical trials.

Sulfaminoheparosans (alternatively known as bioheparins) represent sulfated derivatives obtained from the K5 capsular polysaccharide of Escherichia coli. Previous studies have shown that these agents are structurally comparable to heparins and capable of exerting anticoagulant and antiprotease effects like heparins. Furthermore they are also able to release tissue factor pathway inhibitor (TFPI). Tissue factor (TF) plays a vital role in the pathogenesis of thrombotic and cardiovascular disorders. Anticoagulants such as heparins and bioheparins inhibit this thrombogenic mediator and thereby downregulate the activation of prothrombin and factor X. This study was carried out to determine the effects of several bioheparin fractions and heparins on TF-mediated platelet activation and their direct effect on platelets using human whole blood flow cytometry. Four different sulfaminoheparosan fractions with mean molecular weights of 20, 9, 7, and 6 kDa were tested for their inhibitory effects on platelet activation at two different concentrations (100 and 10 microg/mL). Unfractionated heparin and a low-molecular-weight heparin, tinzaparin, were also tested under the same experimental conditions for comparative modulatory responses. Fresh whole blood from healthy female and male volunteers (n = 5) was mixed with each of these agents and incubated with TF (diluted thromboplastin C) to activate platelets. Platelets were labeled with the antibodies CD61 FITC (GP IIIa) and CD62 PE (P-selectin). The data were analyzed in terms of percent platelet aggregation and platelet P-selectin expression. At 100 microg/mL, all of these agents strongly and significantly inhibited (approximately 40%) the platelet activation induced by TF in comparison to saline control. The inhibitory effects of each of these agents were slightly weaker (approximately 24% inhibition) at 10 microg/mL. The inhibitory effects of these agents on P-selectin expression correspond to their effects on platelet aggregation. At 100 microg/mL all the agents produced greater than 80% inhibition of P-selectin expression whereas at 10 microg/mL, the inhibition is greater than 70% except for bio-20 kDa, which produced less than 50% of inhibition. No molecular weight dependence was observed with bioheparin fractions in terms of inhibitory effects on platelet aggregation or P-selectin expression. None of the bioheparins and heparins exhibited any direct effects on platelets. These observations suggest that both the bioheparins and heparins are capable of inhibiting TF-mediated activation of platelets. Thus the therapeutic effects of bioheparins in the TF-mediated pathogenesis of platelet activation may be similar to those of heparins.

Mucosal microvascular thrombi in rectal biopsies were observed in some ulcerative colitis (UC). Heparin may be effective in steroid resistant UC in some studies, however, the new results of meta-analysis demonstrated a non-significant effect of heparin in controlled clinical trials, differing markedly from observational studies. The objective of this study was to identify colonic microvascular thrombi in larger cases with UC, and analyse its possible risk factors: age, gender, histologic score, extent of lesions and operation or biopsy specimens, and assess the significance of microvascular thrombosis in patients with UC. The microvascular thrombi were identified by immunohistochemical staining with anti-CD61 monoclonal antibody and Martius scarlet blue (MSB) staining in 40 colonic tissue samples of UC (31 biopsy specimens and nine operated cases) and 12 cases of normal colon tissue from operated colonic carcinoma. Logistic regression analysis was used to assess the relationship of age, gender, degree of histology, origin of the specimens, extent of lesions and microvascular thrombi examined. Microvascular thrombi were positive in 14 of 40 UC cases, and none in the controls. The presence of microvascular thrombi was related to operation specimens with odds ratio 11.667, P=0.0179, it might be also related to histologic score (OR=1.350) and extent of lesions (OR=1.619). These results suggest that microvascular thrombosis may be one of the important pathogenesis in some UC, and that the effect of anticoagulant treatment still needs to be assessed.

Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

Myeloid sarcoma (MS) is a localized extra-medullary tumor mass of immature myeloid cells, arising de novo or related to acute myeloid leukemia, of which it can be a forerunner, a coinciding or late event. Less commonly, MS represents an acute blastic transformation of myelodysplastic syndromes or myeloproliferative neoplasms. This rare condition commonly consists of a proliferation of more or less immature cells with a myeloid immunophenotype, very exceptional cases showing a megakaryoblastic or erythroid differentiation. The most common localization of MS is the skin, lymph node, soft tissues and bones, but CNS involvement is exceedingly rare, with no cases reported in the sellar region. We report a 54-year-old man, affected by myeloproliferative neoplasm, JAK2 V617F-positive of 13 years duration, who acutely presented with a third cranial nerve palsy; neuroradiology documented a space-occupying lesion at the level of the sellar, upper clival and right parasellar regions, that was sub-totally removed with a trans-sphenoidal approach. The histological examination documented a proliferation of large, blastic cells, frequently multinucleated; a diagnosis of MS with megakaryoblastic differentiation, arising in a background of chronic idiopathic myelofibrosis, was suggested by immunohistochemistry, owing to CD42b, CD45, CD61 and LAT (linker for activation of T cells) positivity. In addition, homozygous JAK2 V617F mutation was detected from the myeloid sarcoma specimen. A few weeks after surgery, an acute blastic leukemic transformation occurred and, despite chemotherapy, the patient died 2 months after surgery. To the best of our knowledge, this is the first MS case with megakaryoblastic differentiation arising within the CNS.

Acute megakaryoblastic leukemia is a rare subtype of acute myeloid leukemia with a characteristic morphologic and immunophenotypic profile. It has to be distinguished from other subtypes of acute myeloid leukemia as well as acute myeloid leukemia with t (1; 22) (p13;q13) and acute megakaryoblastic leukemia in Down Syndrome because of its poor prognosis. We studied ten cases diagnosed over a period of 2 years (from July 2011 to June 2013). All the ten cases were in the pediatric age group ranging from 4 months to 2 years. On morphology, pointers to the diagnosis were clustering of blasts, presence of cytoplasmic blebs and platelet budding. An additional interesting morphological feature observed in our study was nuclear blebs which were seen in nine cases. Diagnosis was confirmed in all cases by positive immunostaining for CD61. Two of the cases had an extremely rare clinical presentation as granulocytic sarcoma. Although rare, acute megakaryoblastic leukemia should be kept in mind especially in leukemia in infants.