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Our aim is to evaluate initial efficacy, safety, and durable response of eltrombopag in the treatment of Chinese children with chronic immune thrombocytopenia (cITP). This was a retrospective, single-center cohort study including 30 pediatric patients with cITP administered eltrombopag between 1 July 2017 and 1 January 2019. Patients with at least 12 weeks of eltrombopag treatment and available follow-up data were included. Initial response rate, durable response rate, bleeding events, and adverse events were assessed during the follow-up period. The median duration of eltrombopag administration was 6 months (range 3–8 months). The initial response rate was 73.3%. Patients with megakaryocyte count ≥100/slide or Treg <4.5% were more likely to achieve initial response. The median follow-up period was 10 months (range 6–20 months). A total of 53.2% of pediatric patients had a durable response of up to 20 months. Patients with megakaryocyte count ≥100/slide and Treg<4.5% had more than 60% durable response rates compared with individuals with megakaryocyte count<100/slide and Treg≥4.5%, respectively. No serious bleeding events or serious adverse events occurred during the study period. Eltrombopag not only shows excellent initial response but also has continued efficacy and safety. Patients with megakaryocyte count ≥100/slide and Treg<4.5% achieve increased initial response and more frequent durable response. [ABSTRACT FROM AUTHOR]

Objective: To explore the effects of methylenetetrahydrofolate reductase (MTHFR) C677T gene polymorphism on the adverse reactions of high-dose methotrexate (MTX) in pediatric patients with acute lymphoblastic leukemia (ALL)., Methods: A total of 69 children with ALL admitted to the department of Pediatrics of Sun Yat-sen Memorial Hospital of Sun Yat-sen University from November 2018 to October 2020 were included in this study. The clinical data of the children were collected, leukocytes were isolated from their peripheral blood, and MTHFR genotyping was performed by digital fluorescence molecular hybridization techniques. The adverse reactions and plasma drug concentration of MTX were monitored during the chemotherapy. Moreover, the effect of MTHFR gene polymorphism on MTX adverse reactions and plasma drug concentration were analyzed., Results: Among the middle and high risk children, compared with the wildtype group (CC genotype), patients with MTHFR C677T mutations (CT+TT genotypes) had a higher risk of leukopenia ( OR =2.38), neutropenia ( OR =2.2), anemia ( OR =1.83) and hepatoxicity ( OR =1.98). However, no significant difference was found in the MTX plasma concentration between the MTHFR C677T mutantion group and the wildtype group at different timepoints (24 h, 48 h and 72 h). Multivariate analysis revealed that the plasma concentration of MTX (48 h), clinical risk level of ALL and MTHFR C677T gene polymorphism were independent factors for the adverse reactions of high-dose MTX., Conclusion: The MTHFR C677T mutations may be associated with myelosuppression and hepatotoxicity in children with ALL after high-dose MTX treatment.

Objective: To investigate the clinical characteristics and treatment of pneumocystis carinii pneumonia (PCP) in children with acute lymphoblastic leukemia (ALL), in order to improve the early diagnosis and effective treatment., Methods: Clinical data of five children with ALL developing PCP in the post-chemotherapy granulocyte deficiency phase were analyzed retrospectively. The clinical manifestations, laboratory tests, imaging findings, treatment methods and effect were summarized., Results: The male-to-female ratio of the five children was 1∶4, and the median age was 5.5 (2.9-8) years old. All patients developed PCP during granulocyte deficiency phase after induction remission chemotherapy. The clinical manifestations were generally non-specific, including high fever, tachypnea, dyspnea, non-severe cough, and rare rales in two lungs (wet rales in two patients). Laboratory tests showed elevated C-reactive protein (CRP), serum procalcitonin (PCT), (1,3)-β-D-glucan (BDG), lactate dehydrogenase (LDH) and inflammatory factors including IL-2R, IL-6 and IL-8. Chest CT showed diffuse bilateral infiltrates with patchy hyperdense shadows. Pneumocystis carinii(PC) was detected in bronchoalveolar lavage fluid (BALF) or induced sputum by high-throughput sequencing in all patients. When PCP was suspected, chemotherapy was discontinued immediately, treatment of trimethoprim-sulfame thoxazole (TMP-SMX) combined with caspofungin against PC was started, and adjunctive methylprednisolone was used. Meanwhile, granulocyte-stimulating factor and gammaglobulin were given as the supportive treatment. All patients were transferred to PICU receiving mechanical ventilation due to respiratory distress during treatment. Four children were cured and one died., Conclusion: PCP should be highly suspected in ALL children with high fever, dyspnea, increased LDH and BDG, and diffuse patchy hyperdense shadow or solid changes in lung CT. The pathogen detection of respiratory specimens should be improved as soon as possible. TMP/SMZ is the first-line drug against PCP, and the combination of Caspofungin and TMP/SMZ treatment for NH-PCP may have a better efficacy. Patients with moderate and severe NH-PCP may benefit from glucocorticoid.

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 15.88×10 9 /L, the median hemoglobin was 67 g/L and median platelet was 16×10 9 /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.

Objective: To analyze the clinical features and prognosis of 28 children with myelodysplastic syndrome (MDS) and to screen the high risk factors affecting the prognosis so as to provide the new ideas for standard of clinical diagnosis and therapy., Methods: The clinical data of 28 children with newly diagnosed MDS treated in our hospital from March 1994 to July 2016 were analyzed retrospectively, the features of disease onset and the results of laboratory examination were summarized, all MDS children were followed up, the prognosis and the high risk factors affecting the prognosis were evaluated., Results: In all 28 MDS children, the ratio of male to female was 1.8∶1, the incidence of MDS was observed in boys, while the low incidence of MDS was found in older children. The clinical manifestations were mainly the decrease of three series blood cells in 16 cases (57.14%), other cases presented simple anemia (7.1%), simple thrombocytopenia (7.1%), neutropenia with anemia (14.29%), and anemia with thrombocytopenia (14.28%).The bone marrow image showed mainly hyperplasia (82.14%), and the pathological hematopoiesis, moreover the manifistation of pathological hematopoiesis was different in forma and degree; the bone marrow biopsy showed the typical abnormal localization of immature precursor(ALIP) accepted for 33.33%; the chromosome karyotype detection showed the detected rate of chronosome abnormality was 41.18%. The median follow-up time was 1.75 years. 5 children with MDS received the hematopoietic stem cell transplantation (HSCT), among them 1 dead and 4 maintained CCR; Out of other 23 patients no-received HSCT, 7 cases given up treatment after confirmed diagnosis, 16 cases received the chemotherapy (2 cases given up treatment after CR, 5 cases transformed into AML, 3 cases relapsed, 3 cases maintained CCR), 11 cases dead, 9 cases failed to be followed up. The 5-years OS rate and EFS rate in all patients were predicted as (38.2±11.3)% and (35.3±11.3)%,respectively, among them, the OS and EFS rates of patients received the HSCT allo superior to those of patients did not received HSCT ［(80.0±17.9）% vs.(22.8±11.5）%］ (P<0.05) and ［(80.0±17.9)% vs (17.5±11.1)%］(P<0.05). Analysis showed that in addition to receiving the HSCT(P<0.05), platelet decrease in peripheral blood(P<0.01), the age, sex, existance of micromegakaryocytes in bone marrow and progressive MDS or no influenced not on the prognosis(P>0.05)., Conclusion: The children MDS is rare and easy to be misdiagnosis, moreover displays more high heterogeneity and poor prognosis, thereby the early diagnosis is crucial, in addition, the system of prognosis evaluation is imperative to be perfected. The HSCT may be the effective method for curative treatment of childhood MDS.

Objective: To study the relationship of Blimp-1 hypoexpression with abnormality of Treg level and pathogenesis of aplastic anemia (AA)., Methods: The mouse model with AA was established by adminis tration of IFN-γ combined with busulfan. The samples were collected at different day establishing AA model, and the spleen Treg number was detected, the Treg cells were sorted and expression level of prdm-1 was detected., Results: The number of Tregs in mice with AA was lower than that in control mice, moreover, the level of Treg decrease positively correlated with the AA severity (r=0.805), the higher the expression level of prdm-1, the higher the ratio of Treg/lymphocytes, showing positive correlation between them (r=0.548)., Conclusion: Blimp-1 expression may promote the proliferation and differentiation of Treg. The hypoexpression of Blimp-1 mediates the pathogenesis of AA and promotes progression of AA through reducing the proliferation of Treg, and decreacing the number of Treg.

Aim: To examine the morphological characteristics and antigen expression patterns of cultured human retinal glia to define novel subtypes., Methods: Morphologic characteristics and marker expression were examined during cultivation using hematoxylin and eosin (HE) and immunostaining for glial fibrillary acidic protein (GFAP) and vimentin., Results: A subtype of human retinal glia distinct from radial glia (Müller cells) was successfully isolated by digesting the retina first in diastase vera (pancreatin) and then in clostridiopeptidase, followed by culture on fibronectin substrate in human endothelial cell medium (supplemented with 10% fetal bovine serum, growth factors, and heparin sodium). Adherence was detected at 72h and cell-cell coupling at 9-10d after seeding. These cells were extensively and strongly immunopositive for GFAP and vimentin, consistent with glial expression patterns in the human retina, but were morphologically and immunohistochemically distinct from previously reported cultured retinal glia, including GFAP-positive and glutamine synthetase (GS)-positive Müller cells., Conclusion: A unique human retinal glial cell type can be isolated using diastase vera and clostridiopeptidase and then maintained in vitro. Further studies are required to characterize the physiological and pathological functions of these cells.

Aim: To construct the recombinant adenovirus expressing small hairpin RNA (shRNA) targeting human leukocyte-derived arginine aminopeptidase (LRAP) gene and silence the expression of LRAP in human retinal microvascular endothelial cells (HRMECs)., Methods: Three pairs of oligonucleotides coding for shRNAs targeting human LRAP gene were designed and synthesized, and were cloned into the shuttle vector pRNAT-H1.1/Adeno after annealing. The three constructed shuttle plasmids, through homologous recombination with adenoviral backbone vector pAdEasy-1, were transformed into E.coli BJ5183-AD- to produce recombinant adenoviral plasmids. And then the recombinants linearized with Pac I were transfected into 293a cells to package adenoviruses. After amplification and titter determination, the recombinant adenoviruses were used to infect the primary HRMECs. Quantitative real-time PCR was taken to determine the relative amount of LRAP mRNA to screen the adenovirus with the highest silencing efficiency. The level of LRAP protein after RNA interference in HRMECs was further determined by Western blotting., Results: PCR, restriction digestion and DNA sequencing confirmed that the purpose shRNA-coding sequences were correctly inserted into the shuttle vectors and adenoviral plasmids, and the recombinant adenoviruses were packaged successfully in 293a cells. The most effective adenovirus with the silencing efficiency up to 79% was selected by quantitative real-time PCR, which significantly lowered the expression of LRAP in HRMECs., Conclusion: The recombinant adenovirus expressing shRNA effectively silencing LRAP gene was constructed successfully, which would facilitate further study of the role that LRAP plays in the development of diabetic retinopathy.

Purpose: Vascular endothelial growth factor (VEGF) is the most potent angiogenic mitogen, and has been associated with angiogenesis. Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains, which can induce VEGF expression. The aims of the present study were to evaluate the heparanase expression and its relationship with VEGF in the retina of oxygen-induced retinopathy (OIR) mice, and to investigate the effect of the heparanase inhibitor phosphomannopentaose sulfate (PI-88) in the OIR retinas., Methods: Seventy-seven newborn C57BL/6 mice were involved in this study. On postnatal day 7 (P7), pups were exposed to a hyperoxia condition (75% oxygen) for 5 days, and on P12, the mice were returned to room air. Control mice were exposed to room air from birth until P17, with normally developing retinal vasculature. PI-88 was administered intraperitoneally to OIR mice at a dose of 35.7 mg/kg/day for 5 consecutive days. The expression level of heparanase and VEGF in the retinas was assayed using immunohistochemistry, Q-RT-PCR, and western blot., Results: The expression levels of heparanase and VEGF were increased in the OIR retinas compared with the control mice. The Q-RT-PCR results showed that the mRNA expression levels of heparanase and VEGF in OIR retina were increased 1.71 fold (p<0.0001) and 4.34 fold (p<0.0001), respectively. The western blot results showed that the protein expression levels of heparanase and VEGF were increased 1.49 fold (p<0.0001) and 1.72 fold (p<0.0001), respectively, in the OIR retinas compared with the normal retinas. The immunohistochemistry analysis revealed that the heparanase and VEGF signals were intense in the retinal vascular endothelia of the OIR mice but faint in those of the normal controls. The increased protein and mRNA expression levels of heparanase and VEGF in the mouse retinas were significantly decreased by PI-88 administration (p<0.0001)., Conclusions: Heparanase expression was upregulated and correlated with an increase in VEGF expression in the OIR mouse retinas, and might be involved in the progress of retinopathy of prematurity. Inhibition of heparanase expression by PI-88 could be used as a novel therapeutic method for retinopathy of prematurity.

Objective: To culture human retinal capillary endothelium cells (HRCECs) in vitro and explore the effect of rAAV2-PEDF on proliferation of HRCEs., Methods: Retinas were digested by 2.5% trypsin and 0.1% collagenase I in order. The isolated cells were cultured on fibronectin-coated dishes in media of human endothelial-sFM basal growth medium (HE-SFM BGM) with 10% fetal bovine serum, insulin-transferrin-selenium (ITS) and endothelial cell growth factor (ECGF). The cultured cells were identified by anti-factor VIII related antigen though immunohistochemistry stain. The effect of hypoxia induced by CoCl2 on proliferation of HRCECs was assessed by MTT assay. After rAAV2-PEDF were transfected into HRCECs, the EGPF positive cells were observed by laser confocal scanning microscopy, the protein expression of PEDF were detected by Western blot, and the proliferation of HRCECs were checked by MTT assay. Flow cytometry was used to analyze the apoptosis of HRCECs., Results: Cultured HRCECs attached in the bottom of dishes in 48 h - 72 h and grew to confluence in 2 weeks after seeding. HRCECs were with a positive brown staining for factor VIII. EGPF positive cells were seen under laser confocal scanning microscopy after 48 h of rAAV2-EGFP transfection. The expression level of PEDF protein was higher in experimental group than in control group. The results of MTT assay showed the numeric value OA was 0.085 ± 0.021 in normal group and 0.166 ± 0.024 in hypoxia group (t = 3.938, P < 0.05). In normal oxygen condition, the numeric value OA was 0.171 ± 0.011 in normal control group, 0.178 ± 0.016 in rAAV2-EGFP treated group, and 0.169 ± 0.017 in rAAV2-PEDF treated group (F = 0.01, P > 0.05). In hypoxia condition, the numeric value OA was 0.166 ± 0.013 in CoCl(2) treated group, 0.155 ± 0.012 in CoCl(2) + rAAV2-EGFP treated group, and 0.116 ± 0.015 in CoCl(2) + rAAV2-PEDF treated group. In normal oxygen condition, the ratio of apoptosis was 2.3% in normal control group, and 3.3% in rAAV2-EGFP treated group, and 1.7% in rAAV2-PEDF treated group. In hypoxia condition, the ratio of apoptosis was 3.6% in CoCl(2) treated group, 6.7% in CoCl(2) + rAAV2-EGFP treated group, and 36.4% in CoCl(2) + rAAV2-PEDF treated group., Conclusions: PEDF gene can stably express in HRCECs after rAAV2-PEDF transfection and can obviously inhibit proliferation of HRCECs in hypoxia.

Purpose: We performed human, animal, and in vitro studies to examine the potential role of nuclear transport factor 2 (NTF2) in conferring resistance to diabetic retinopathy (DR)., Methods: Blood NTF2 levels were assessed in two groups of patients with type 2 diabetes mellitus. Group P patients had a history of proliferative DR (PDR), while group N patients did not. The retinal vasculature was examined in diabetic rats three months after they received an intravitreal injection of a recombinant adeno-associated virus (rAAV) vector overexpressing NTF2 (rAAV2-NTF2). Control rats were treated with rAAV2 only. Rat retinal capillary endothelial cells (RRCECs) were infected with rAAV2-NTF2, or with a vector expressing siRNA targeted against NTF2, to assess the effects of overexpression and inhibition of NTF2 on vascular endothelial growth factor (VEGF) expression (mRNA and protein)., Results: There was a strong trend for patients with DR to have lower blood NTF2 levels compared to those who did not have DR (0.10+/-0.01 versus 0.20+/-0.08, p=0.079). There was significantly less retinal blood vessel leakage in diabetic rats infected with rAAV2-NTF2 compared to controls (16.5+/-2.9 versus 24.7+/-7.3, p=0.039). These rats exhibited normal retinal vasculature and blood-retinal barrier function. VEGF expression was inhibited by NTF2 overexpression and stimulated by NTF2 inhibition, (protein [0.41+/-0.05 versus 0.23+/-0.06] and mRNA [0.37+/-0.04 versus 0.23+/-0.06] p<0.01 for all)., Conclusions: These finding suggest that NTF2 is a potential mediator of retinal vasculature integrity. NTF2 may act by altering VEGF expression, thereby influencing the development of DR in patients with diabetes mellitus.

Objective: To study the roles of hairy and enhancer of split related-1 (HESR-1) in maintenance of the mature, quiescent vessel and angiogenesis., Methods: Human umbilical vein endothelial cells (HUVEC) were cultured. The full-length coding sequence of HESR-1 was cloned into PcDNA3.1 + using standard protocols. HESR-1 specific siRNA was synthesized and cloned into the RNAi-Ready pSIREN-RetroQ ZsGreen Vector. The constructed PcDNA3.1 + HESR-1 plasmid were transfected into HUVEC for the overexpression of HESR-1, and HESR-1-RNAi plasmid were transfected into HUVEC to silence the HESR-1 gene, the expression of KDR, ALK-1 and Ang-1 in HUVEC were analyzed by RT-PCR and Western blot., Results: The expression of KDR was down-regulated and ALK-1 and Ang-1 were up-regulated in HUVEC with the overexpression of HESR-1; The expression of KDR was up-regulated and ALK-1 and Ang-1 were down-regulated the HESR-1 in HUVEC by RNAi., Conclusion: HESR-1 may play an important role in maintenance of vessel in quiescent and control angiogenesis by the regulation of the expression of KDR, ALK-1 and Ang-1.

Objective: To assess the effect of bcl-X(L), an anti-apoptotic gene, on glutamate-induced apoptosis in cultured retinal photoreceptors., Methods: Glutamate-induced apoptosis in cultured retinal photoreceptors was established. The experiment was divided into three groups: control, glutamate treatment and rAd-gfp-bcl-X(L) + glutamate transfection group, the protection of bcl-X(L) on retinal photoreceptors from apoptosis was evaluated. 6.5 x 10(12) pfu/L rAd-gfp-bcl-X(L) were transfected into retinal photoreceptors in the rAd-gfp-bcl-X(L) + glutamate group 48 h before Glutamate-induced apoptosis was established. The positive photoreceptors with green fluorescence were identified under fluorescence microscopy. The expression of Bcl-X(2) protein in rAd-gfp-bcl-X(L) transfected and non-transfected neurons were assessed by immunohistochemistry assay. DNA fragment was detected by agarose gel electrophoresis. Nuclei were revealed by hoechst33258 staining., Results: rAd-mediated gene delivery can transfect retinal photoreceptors effectively. Increased expression of Bcl-X(L) protein was demonstrated in the rAd-transfecting neurons. Transfection of bcl-X(L) significantly decreased the number of apoptotic retinal photoreceptors in vitro., Conclusion: Transfection of bcl-X(L) protects cultured retinal photoreceptors from apoptosis induced by glutamate. Transfection the gene of bcl-X(L) may be a potential gene therapy for retinal degenerative diseases.

Objective: To observe the therapeutic effect of rAd-gfp-bcl-X(L) on retinal degeneration (RD) in mice., Methods: rAd-gfp-bcl-X(L) granules were injected into the subretinal space of right eyes of RD mice in Group A (postnatal days 10, p10) and Group B (postnatal days 22, p22) (n = 20). The morphological changes were observed by light microscopy and transmission electron microscopy at 15 - 30 days post-injection. Frozen section was used to observe the expression of GFP. The level of bcl-X(L) mRNA and bcl-X(L) protein were assayed by RT-PCR and immunohistochemistry assay., Results: In frozen section, GFP was observed in the treated eyes under fluorescence microscopy, indicating that rAd-gfp-bcl-X(L) had transfected into the retinal cells successfully and that bcl-X(L) was expressed. In Group A, an increased expression of bcl-X(L) mRNA and Bcl-X(L) protein were observed in the treated eyes. The outer nuclear layer of the treated eyes in Group A was thicker than that of the control eyes at 30 days post-injection. The inner segment of treated eyes in Group A was healthier than that of control eyes, as indicated by transmission electron microscopy. No difference was found in the thickness of retina between treated eyes and control eyes in Group B., Conclusion: The bcl-X(L) can be expressed and have an anti-apoptosis effect when the rAd-gfp-bcl-X(L) particles are injected into the subretinal space of RD mice's eyes at an early stage. But the expression and protective effects were not observed in RD mice at a later stage. The therapy of rAd-gfp-bcl-X(L) for retinal degenerative diseases still needs further study.