[Serological Characteristics of Patients with Autoimmune Hemoly-tic Anemia and Efficacy and Safety of Incompatible Transfusion of Red Blood Cells].

OBJECTIVE: To explore the serological characteristics of patients with autoimmune hemolytic anemia（AIHA） and analyze its clinical efficacy and safety of incompatible red blood cell transfusion. METHODS: Sixty AIHA patients admitted in our hospital from January 2014 to January 2018 were selected. The blood type serological characteristics of 60 patients were analyzed retrospectively. According to the type of autoantibody and the composition of different red blood cells, the efficacy and safety of erythrocyte infusions were evaluated respectively. RESULTS: The screen results of irregular antibody in 60 AIHA patients were positive, and the direct anti-human globulin test also was positive, including 8 cases of cold autoantibodies (13.33%), 49 cases of IgG warm autoantibodies (81.67%), and 3 cases of warm cold double autoantibodies (5%). The irregular anti-body identification test confirmed the existence of homoantiboby in 17 cases (28.33%). Out of 60 cases 34 received incompatible red blood cell (RBC) infusion for 108 time including ABO homotype non washing RBC (81 tirnes) and O type washing RBC (27 times). The infusion results showed that the total [JP2]effective rate was 57.41(62/108), total partial effective rate was 14.81% (16/108) and total ineffective rate was 27.78% (30/108).The infusion of ABO homotype non-washing RBC for 81 time showed that the effective rate was 58.02%[JP] (47/81) , partial effective rate was 12.35 (10/81) and ineffective rate was 29.67% (24/81); the infusion of O type washing RBC for 27 times showed that the effective rate was 55.56% (15/27), partial effective rate was 22.22% (6/27) and ineffective rate was 22.22% (6/27), there was no significant difference in effective rate between 2 kinds of infusion (P>0.05). The comparison of different antibody type infusion showed that in the infusion of IgM cold autoantiboay for 12 times, the effective rate was 41.67% (5/12), partial effective rate was 33.33% (4/12) and ineffective rate was 25% (3/12); in the infusion of IgG warm antoantibody for 93 times. The effective rate was 58.06% (54/93),partial effective rate was 12.90% (12/93) and ineffective rale was 29.04% (27/93), there was also no significant difference in effective rate between 2 kinds of infusion(P>0.05). However, in infusion of cold/warm double autoantibody for 3 times, the effective rate was 100% (3/3), moreover, the hemotytic reaction of infusion was not observed during the treatment . CONCLUSION: The infusion of ABO homotype non-washing RBC and O type washing RBC both possess the high safely and efficacy for treatment of patients with AIHA, but the use of ABO homotype non-washing RBC can effectively avoid the excessive use of O type washing RBC.

Elaphroporia ailaoshanensis gen. et sp. nov. in Polyporales (Basidiomycota).

A new poroid wood-inhabiting fungal genus, Elaphroporia, typified by E. ailaoshanensissp. nov., is proposed based on a combination of morphological features and molecular evidence. The genus is characterised by an annual growth habit, resupinate basidiocarps, becoming rigid and light-weight up on drying, a monomitic hyphal system with thick-walled generative hyphae bearing both clamp connections and simple septa, slightly amyloid, CB+ and ellipsoid, hyaline, thin-walled, smooth and IKI-, CB- basidiospores. Sequences of ITS and LSU nrRNA gene regions of the studied samples were generated, and phylogenetic analyses were performed with maximum likelihood, maximum parsimony and bayesian inference methods. The phylogenetic analysis based on molecular data of ITS+nLSU sequences showed that Elaphroporia belonged to the residual polyporoid clade and was closely related to Junghuhnia crustacea. Further investigation was obtained for more representative taxa in the Meruliaceae based on ITS+nLSU sequences, in which the result demonstrated that the genus Elaphroporia formed a monophyletic lineage with a strong support (100 % BS, 100 % BP, 1.00 BPP) and then grouped with Flaviporus and Steccherinum.

PIM-1 kinase inhibitor SMI-4a exerts antitumor effects in chronic myeloid leukemia cells by enhancing the activity of glycogen synthase kinase 3ß.

The development of targeted tyrosine kinase inhibitors (TKIs) has succeeded in altering the course of chronic myeloid leukemia (CML). However, a number of patients have failed to respond or experienced disease relapse following TKI treatment. Proviral integration site for moloney murine leukemia virus­1 (PIM­1) is a serine/threonine kinase that participates in regulating apoptosis, cell cycle, signal transduction and transcriptional pathways, which are associated with tumor progression, and poor prognosis. SMI­4a is a selective PIM­1 kinase inhibitor that inhibits PIM­1 kinase activity in vivo and in vitro. The present study aimed to explore the mechanism underlying the antitumor effect of SMI­4a in K562 and imatinib­resistant K562 (K562/G) cell lines. It was demonstrated that SMI­4a inhibited the proliferation of K562 and K562/G cells using a WST­8 assay. The Annexin V­propidium iodide assay demonstrated that SMI­4a induced apoptosis of K562 and K562/G cells in a dose­, and time­dependent manner. Furthermore, Hoechst 33342 staining was used to verify the apoptosis rate. The clone formation assay revealed that SMI­4a significantly inhibited the colony formation capacity of K562 and K562/G cells. Western blot analysis demonstrated that SMI­4a decreased phosphorylated (p)­Ser9­glycogen synthase kinase (GSK) 3ß/pGSK3ß and inhibited the translocation of ß­catenin. In addition, the downstream gene expression of apoptosis regulator Bax and poly(ADP­ribose) polymerase­1 was upregulated, and apoptosis regulator Bcl­2 and Myc proto­oncogene protein expression levels were downregulated. Immunofluorescence results demonstrated changes in the expression level of ß­catenin in the plasma and nucleus. The results of the present study suggest that SMI­4a is an effective drug to use in combination with current chemotherapeutics for the treatment of imatinib-resistant CML.

Celecoxib exerts antitumor effects in HL-60 acute leukemia cells and inhibits autophagy by affecting lysosome function.

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, has been demonstrated to exert antitumor activity in a variety of cancer cells. The underlying mechanism involves inhibition of cell cycle progression and induction of apoptosis. Besides, celecoxib has also been found to induce autophagy in some solid tumor cells. The aim of this study was to investigate the effect of celecoxib on cell proliferation in HL-60 human acute leukemia cells and to explore the potential mechanism. HL-60 cells were exposed to various concentrations of celecoxib and cell viability was evaluated by the MTT assay. Apoptosis was analyzed with flow cytometry and the amount of autophagosome was evaluated by LysoTracker probe labelling. The expression of apoptosis- and autophagy-related proteins was assayed by Western blot and LysoSensor probe labelling was used to detect the effect of celecoxib on the lysosomal functions. The results of this study indicated that celecoxib inhibited cell proliferation in a time- and dose-dependent fashion. The flow cytometry analysis showed that celecoxib induced apoptosis at low concentrations and mainly cell necrosis at high concentrations. The Western blot test confirmed the induction of apoptosis by the upregulation of apoptosis-related proteins cleaved caspase-3 and cleaved PARP. Furthermore, this study demonstrated that celecoxib prevented the autophagic flux by inhibiting lysosome function; the fluorescence intensity of the LysoTracker probe and the level of autophagy-related proteins LC3-II and p62 were increased, but the fluorescence intensity of the LysoSensor probe was weakened. These findings show that celecoxib is an autophagy suppresser and has antitumor effects in HL-60 cells by inducing cell apoptosis and necrosis.

Zoledronic acid overcomes adriamycin resistance in acute myeloid leukemia cells by promoting apoptosis.

Zoledronic acid (ZOL), a nitrogen­containing bisphosphonate, is widely used in metastatic bone disease. Previous studies indicate that ZOL has marked anti­leukemia activity, however, the underlying mechanism of action remains to be elucidated. The present study aimed to explore the mechanism of the anti­leukemia effect of ZOL in leukemia cells. It was observed that ZOL inhibited the proliferation of HL­60 and adriamycin­resistant HL­60 (HL­60/A) cells using a WST­8 assay. An Annexin V­propidium iodide indicated that ZOL induced apoptosis of the two cell types in a dose­ and time­dependent manner. Hoechst 33342 staining was also used to verify the levels of apoptosis. The colony formation assay demonstrated that ZOL significantly inhibited colony formation capacity in acute myeloid leukemia (AML) cells. This was achieved by the induction of S­phase cell cycle arrest, downregulation of B­cell lymphoma 2 (Bcl­2) and upregulation of Bcl­2 associated X protein and cleaved poly (ADP­ribose) polymerase. The results indicate that ZOL inhibited cell proliferation by inducing apoptosis via the mitochondrial apoptotic pathway and this anti­leukemic activity appeared notably enhanced in HL­60/A cells. As ZOL is already available for clinical use, these results indicate that it may be an effective addition to the chemotherapeutic strategies for AML.

RUNX3 plays an important role in As2O3­induced apoptosis and allows cells to overcome MSC­mediated drug resistance.

The interaction between bone marrow stromal cells and leukemia cells is critical for the persistence and progression of leukemia, and this interaction may account for residual disease. However, the link between leukemia cells and their environment is still poorly understood. In our study, runt­related transcription factor 3 (RUNX3) was identified as a novel target gene affected by As2O3 and involved in mesenchymal stem cell (MSC)­mediated protection of leukemia cells from As2O3­induced apoptosis. We observed induction of RUNX3 expression and the translocation of RUNX3 into the nucleus after As2O3 treatment in leukemia cells. In K562 chronic myeloid leukemia cells, downregulation of endogenous RUNX3 compromised As2O3­induced growth inhibition, cell cycle arrest, and apoptosis. In the presence of MSC, As2O3­induced expression of RUNX3 was reduced significantly and this reduction was modulated by CXCL12/CXCR4 signaling. Furthermore, overexpression of RUNX3 restored, at least in part, the sensitivity of leukemic cells to As2O3. We conclude that RUNX3 plays an important role in As2O3­induced cellular responses and allows cells to overcome MSC­mediated drug resistance. Therefore, RUNX3 is a promising target for therapeutic approaches to overcome MSC­mediated drug resistance.

RUNX3 is involved in caspase-3-dependent apoptosis induced by a combination of 5-aza-CdR and TSA in leukaemia cell lines.

PURPOSE: Epigenetic therapy has had a significant impact on the management of haematologic malignancies. The aim of this study was to assess whether 5-aza-CdR and TSA inhibit the growth of leukaemia cells and induce caspase-3-dependent apoptosis by upregulating RUNX3 expression. METHODS: K562 and Reh cells were treated with 5-aza-CdR, TSA or both compounds. RT-PCR and Western blot analyses were used to examine the expression of RUNX3 at the mRNA and protein levels, respectively. Immunofluorescence microscopy was used to detect the cellular location of RUNX3. Additionally, after K562 cells were transfected with RUNX3, apoptosis and proliferation were studied using Annexin V staining and MTT assays. RESULTS: The expression of RUNX3 in leukaemia cell lines was markedly less than that in the controls. Demethylating drug 5-aza-CdR could induce RUNX3 expression, but the combination of TSA and 5-aza-CdR had a greater effect than did treatment with a single compound. The combination of 5-aza-CdR and TSA induced the translocation of RUNX3 from the cytoplasm into the nucleus. TSA enhanced apoptosis induced by 5-aza-CdR, and Annexin V and Hoechst 33258 staining showed that the combination induced apoptosis but not necrosis. Furthermore, apoptosis was dependent on the caspase-3 pathway. RUNX3 overexpression in K562 cells led to growth inhibition and apoptosis and potentiated the effects of 5-aza-CdR induction. CONCLUSION: RUNX3 plays an important role in leukaemia cellular functions, and the induction of RUNX3-mediated effects may contribute to the therapeutic value of combination TSA and 5-aza-CdR treatment.

[Significance of DNA methylation status of runx3 gene promoter region in acute leukemia].

This study was aimed to investigate the effects of methylation of runx3 gene promoter on pathogenesis of acute leukemia (AL) and its clinical significance. The methylation of runx3 gene promoter in cells of bone marrow or peripheral blood from 40 cases of AL and 10 healthy persons as well as in CHRF, U937 and K562 cell lines were assuaged by methylation specific polymerase chain reaction (MS-PCR), the expression of runx3 gene were detected with reverse transcription polymerase chain reaction (RT-PCR). The results indicated that no methylation was detected in all of cell lines and healthy persons while expression of runx3 gene could be deteted, methylation of runx3 gene promoter was found in 35% (14/40) AL patients and its percentage was significant higher than that healthy persons (0%), the difference in methylation for runx3 between two kinds of samples was statistically significant (p<0.05), while methylation rate in AML was 30.43% (7/23), ALL was 41.18% (7/17), there was no significant difference between them (p>0.25). All of methylated samples had no expression of runx3 gene. Patients without methylation of runx3 gene had a lower percentage of blasts in bone marrow and a higher complete remission rate of first chemotherapy than those with methylation of runx3 gene. It is concluded that methylation of runx3 gene promoter probably plays a role in the pathogenesis of AL and may have clinical significance in predicting prognosis of AL.