The role of TIM3+ NK and TIM3- NK cells in the immune pathogenesis of severe aplastic anemia.

Background: Natural killer (NK) cells play important immunoregulatory roles in the immune pathogenesis of severe aplastic anemia (SAA). Our previous research showed that SAA caused a decrease in T cell immunoglobulin mucin-3 (TIM3) expression on NK cells. Here we investigated the expression of surface receptors, and the cytotoxicity of peripheral TIM3+ NK and TIM3- NK cells in patients with SAA. Methods: The expressions of surface receptors and cytoplasmic protein of TIM3+ NK and TIM3- NK cells from peripheral blood were detected by FCM. The functions of mDCs, and apoptosis rate of K562 cells after co-culture with TIM3+ NK and TIM3- NK cells were maesured by FCM. Westren-blot was used to detect the changes of TIM3+ NK and TIM3- NK signaling pathway proteins (AKT, P-AKT) and compare the functional activity of the two groups. Results: Activating receptors NKG2D and Granzyme B were higher, while inhibiting receptors NKG2A, CD158a and CD158b were lower on TIM3- NK cells compared with TIM3+ NK cells in patients with SAA. In SAA, the expression of CD80 and CD86 on mDCs (Myeloid dendritic cells) was significantly decreased after incubation with TIM3- NK cells. The apoptosis rate (AR) of K562 cells was significantly increased after being incubated with TIM3- NK cells in SAA. The level of signal pathway protein AKT of TIM3- NK cells in SAA was similar to that of TIM3+ NK cells, and the levels of P-AKT and P-AKT/AKT ratio of TIM3- NK cells were significantly higher than those of TIM3+ NK cells. Conclusions: Therefore, TIM3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. Low expression of TIM3 contributes to the enhancement of NK cell activity which in turn inhibits the immune activation state of SAA and improves the disease state. Our research may aid the development of new therapeutic strategies based on TIM3-NK cells infusion for the treatment of SAA.

Molecular mechanism of ATF6 in unfolded protein response and its role in disease.

Activating transcription factor 6 (ATF6), an important signaling molecule in unfolded protein response (UPR), plays a role in the pathogenesis of several diseases, including diseases such as congenital retinal disease, liver fibrosis and ankylosing spondylitis. After endoplasmic reticulum stress (ERS), ATF6 is activated after separation from binding immunoglobulin protein (GRP78/BiP) in the endoplasmic reticulum (ER) and transported to the Golgi apparatus to be hydrolyzed by site 1 and site 2 proteases into ATF6 fragments, which localize to the nucleus and regulate the transcription and expression of ERS-related genes. In these diseases, ERS leads to the activation of UPR, which ultimately lead to the occurrence and development of diseases by regulating the physiological state of cells through the ATF6 signaling pathway. Here, we discuss the evidence for the pathogenic importance of ATF6 signaling in different diseases and discuss preclinical results.

Luspatercept for the treatment of congenital sideroblastic anemia: Two case reports.

Congenital sideroblastic anemia (CSA) is a group of disorders caused by different genetic mutations that result in low iron utilization and ineffective erythropoiesis. Current treatments are limited, and some patients do not respond to vitamin B6 therapy. Luspatercept is a novel erythropoietic maturation agent approved for adult ß-thalassemia and Myelodysplastic syndromes with ring sideroblasts (MDS-RS) associated with ineffective erythropoiesis. Here we report 2 patients with CSA due to mutations in ALAS2 and SLC25A38 genes who became unresponsive after a period of treatment with vitamin B6 and iron chelators but achieved transfusion independence and a markedly reduced spleen after combination with luspatercept.

The effectiveness of a novel treatment of TIM-3(-) NK cells infusion in murine models of immune-mediated bone marrow failure.

BACKGROUND: T-cell immunoglobulin and mucin-containing domain (TIM)-3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. In this study, we aimed to explore a novel treatment method of TIM-3(+) NK or TIM-3(-) NK cell infusion in combination with immunosuppressive therapy for bone marrow failure (BMF)/aplastic anemia (AA) mice. METHODS: BMF/AA mouse model was constructed. The TIM-3 expression and functional molecules on TIM-3(+) and TIM-3(-) NK cells of the BMF group, total body irradiation (TBI) group, and normal control (NC) group mice were detected by flow cytometry. After treatment, the general condition, whole blood cell and bone marrow cell (BMC) count, and immune condition of mice from each group were compared. RESULTS: TIM-3 expression in the peripheral blood NK cells of BMF mice was significantly lower than that of the TBI and NC group mice. TIM-3(-) NK cells expressed more NKG2D receptors than TIM-3(+) NK cells. The levels of P-Akt and PI3K in TIM-3(-) NK cells were higher than those in TIM-3(+) NK cells. On the 17th day after BMF induction, the weight, peripheral whole blood cell count, and BMC count of BMF mice decreased significantly compared with that of the NC group mice. The therapeutic effect in the TIM-3(-) NK cell treatment group was better than that in the TIM-3(+) NK cell treatment and CsA treatment groups. Concurrently, the ratio of CD4+ T and CD8+ T cells of BMF mice was significantly lower than that of the NC group mice. The therapeutic effect in CsA + TIM-3(-) NK group was more significant than that of the CsA treatment and the CsA + TIM-3(+) NK groups. CONCLUSIONS: In this study, we found that the general condition, peripheral whole blood cell and BMC count, and immune status of BMF mice improved significantly after CsA + TIM-3(-) NK cell treatment. These results may provide further insights into the immune pathogenesis of SAA and novel therapeutic ideas for improving SAA treatment.

Long noncoding RNA FAM157C contributes to clonal proliferation in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disease of hematopoietic stem cells (HSCs). Long noncoding RNAs (lncRNAs) perform a wide range of biological functions, including the regulation of gene expression, cell differentiation, and proliferation, but their role in PNH remains unclear.CD59- and CD59+ granulocytes and monocytes from 35 PNH patients were sorted. High-throughput sequencing was analyzed in 5 PNH patients, and differentially expressed lncRNAs and mRNAs were identified. The mRNAs with fragments per kilobase of exon model per million mapped fragments (FPKM) > 10 in at least 3 patients were selected, and experiments were performed to identify their upstream regulatory lncRNAs. The expression of selected mRNAs and lncRNAs was verified by qRT‒PCR, and the correlation of these expression patterns with clinical data from other 30 PNH patients was analyzed. Then, the functions of the lncRNAs were studied in the PIGA-KO-THP-1 cell line.Transcription analysis revealed 742 upregulated and 1376 downregulated lncRNAs and 3276 upregulated and 213 downregulated mRNAs. After deep screening, 8 highly expressed mRNAs that were related to the NF-κB pathway were analyzed to determine coexpression patterns. LINC01002, FAM157C, CTD-2530H12.2, XLOC-064331 and XLOC-106677 were correlated with the 8 mRNAs. After measuring the expression of these molecules in 30 PNH patients by qRT‒PCR, lncRNA FAM157C was verified to be upregulated in the PNH clone, and its expression levels were positively correlated with the LDH levels and CD59- granulated and monocyte cell ratios. After knockdown of the FAM157C gene in the PIGA-KO-THP-1 cell line, we found that the cells were arrested in the G0/G1 phase and S phase, the apoptosis rate increased, and the cell proliferation decreased.LncRNA FAM157C was proven to promote PNH clone proliferation, and this is the first study to explore the role of lncRNAs in PNH.

[Expression and Significance of PD-1 and ICOS in Patients with Primary Immune Thrombocytopenia].

OBJECTIVE: To investigate the expression of programmed death receptor-1 (PD-1) and inducible costimulator (ICOS) on the surface of CD8+ T cells in peripheral blood of patients with primary immune thrombocytopenia (ITP), and explore the roles of PD-1 and ICOS in the occurrence and development of ITP. METHODS: A total of 28 ITP patients treated in Tianjin Medical University General Hospital from September to December 2020 were selected, including 13 patients with newly diagnosed ITP, 15 patients with chronic ITP, and 22 healthy volunteers were recruited as control group. Flow cytometry was used to detect the expression levels of PD-1 and ICOS, and evaluate their correlation with clinical indicators. RESULTS: The percentage of CD8 + T cells in ITP patients of chronic group was higher than that of the newly diagnosed group and the control group (P<0.05). The expression level of PD-1 on CD8+ T cells in ITP patients of newly diagnosed group and chronic group were significantly lower than that of the control group (P<0.05), while the expression level of ICOS were significantly higher (P<0.05). In ITP patients, PD-1 was negatively correlated with platelet count (r=-0.4942, P<0.01), but positively with ICOS (r=0.4342). PD-1 and ICOS were both negatively correlated with lymphocyte count (rPD-1=-0.4374; rICOS=-0.4492). CONCLUSION: In ITP patients, the unbalanced expression of PD-1 and ICOS may interfere with the immune homeostasis of the body, which can be used as a therapeutic target for ITP patients.

New Trends in Nontransplant Therapy for Acquired Aplastic Anemia.

Aplastic anemia (AA) is a hematological disease characterized by pancytopenia and hypofunctional bone marrow hematopoiesis. Patients with AA are treated with either immunosuppressive therapy (IST) using anti-thymocyte globulin (ATG) and cyclosporine (CsA) or hematopoietic stem cell transplantation (HSCT), if a matched donor is available. The standard IST regimen for AA patients results in response rates up to 70% and even higher overall survival. However, primary and secondary failures after IST remain frequent, and to date, all attempts aiming to overcome this problem have been unfruitful. The nontransplant therapeutic options for AA have significantly expanded during the last few years. Here, we review the new trends of nontransplant therapy for AA and summarize the current therapeutic effect of AA.

The Effectiveness of Rapamycin Combined with Eltrombopag in Murine Models of Immune-Mediated Bone Marrow Failure.

Severe aplastic anemia (SAA) is a rare disease characterized by severe pancytopenia and bone marrow failure. Most patients with AA respond to immunosuppressive therapy (IST), usually as antithymocyte globulin (ATG) and cyclosporine (CsA), but some relapse on CsA withdrawal or require long-term administration of CsA to maintain blood counts. Recent research has found that rapamycin (Rapa) was an effective therapy in mouse models of immune-mediated bone marrow failure. However, it has not achieved a satisfactory effect in clinical application. At present, many studies have confirmed that eltrombopag (ELT) combined with IST can improve the curative effect of AA patients. Then, whether Rapa combined Elt in the treatment of AA will acquire better efficacy than a single drug application remains unclear. In this study, an immune attack-mediated AA mouse model was constructed by total body irradiation (TBI) and allo-lymphocyte infusion. In our study, we tested the efficacy of Rapa combined with Elt as a new treatment in mouse models of immune-mediated bone marrow failure. It showed that treatment with Rapa in combination Elt in the AA mouse model ameliorated pancytopenia and extended animal survival in a manner comparable to the standard dose of CsA and Rapa alone. However, there was no significant improvement effect on the number and function of NK cells and their subsets, mDCs, and CD4+/CD8+ ratio in AA mice after the therapy of Rapa combined with Elt compared with Rapa alone. Furthermore, the secretion of IL-10 of Tregs in AA mice increased significantly after the therapy of Rapa combined with Elt, but there was no significant difference in the number of Treg cells. We did not observe the difference in the curative effect of the Rapa group and CsA group, but for IL-10/Tregs ratio, the Rapa group was superior to the CsA group. And the IFN-r secretion of CD8+T cells in AA mice decreased significantly after the combination therapy of Rapa and Elt than Rapa alone. Compared with the AA group, the level of plasma IFN-γ, IL-2, and TNF-α decreased significantly (P < 0.05), but IL-10, IL-4, IL-5, and IL-1ß increased significantly in the Rapa group (P < 0.05). As for IL-10, IL-12p70, IL-2, IL-6, KC/GRO, and TNF-α, the therapy of Rapa combined with Elt showed a more significant effect than Rapa alone in AA mice. To some extent, this study had shown a relatively better synergistic effect in murine models of immune-mediated bone marrow failure after the combination therapy of Rapa and Elt, which was a promising clinical utility in SAA treatment.

Proteomics analysis reveals alterations of NK cells in patients with severe aplastic anemia.

INTRODUCTION: Severe aplastic anemia (SAA) is a disease characterized by severe pancytopenia and hematopoietic failure of bone marrow. Natural killer (NK) cells are a class of large granular lymphocytes that perform killing and immunomodulatory functions. Our previous study demonstrated that NK cells played the "protective" role in SAA, which is weakened. However, the mechanism remains unclear. METHODS: Peripheral blood NK cells from SAA patients and normal controls were sorted and total proteins were extracted. Then, mass spectrometry was performed to screen differentially expressed proteins (DEPs). RESULTS: Significant differences in the expression levels of 93 proteins were observed in NK cells of SAA patients compared with normal controls. Among them, 48 were upregulated proteins, including histone H1.2, histone H1.3, heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1), and interferon regulatory factor 1 (IRF-1), and 45 were downregulated proteins, including actin-related complex (ARP2/3), histone H3, histone H4, phosphoglycerate kinase 1 (PGK1), talin-1. Gene Ontology (GO) function indicated that the DEPs most involved were vesicle-mediated transport, innate immune response, and DNA binding. KEGG analysis showed 3 upregulated and 12 downregulated pathways, in which cell endocytosis and FC-γ receptor-mediated phagocytosis were most closely related to NK cell functions. CONCLUSION: Our study is the first analysis of proteomic profile in NK cells in SAA and found many DEPs involving in dysfunction of NK cells, which provides potential targets for deeper research of inadequate immunomodulation.

Analysis of clinical characteristics of 92 patients with paroxysmal nocturnal hemoglobinuria: A single institution experience in China.

OBJECTIVES: We performed a retrospective analysis to investigate the clinical characteristics and therapeutic strategies of Chinese paroxysmal nocturnal hemoglobinuria (PNH) patients, and assessed the efficacy and safety of glucocorticoid in PNH patients. METHODS: The clinical data of 92 PNH cases in our hospital were analyzed, including clinical manifestation, laboratory examination, treatment efficacy, and survival. RESULTS: The main clinical manifestations of these patients included hemoglobinuria, anemia, fatigue, dyspnea, headache, abdominal pain, and erectile dysfunction. Glucocorticoid is still the first-line treatment for PNH patients to control hemolytic attack, and the short-term remission rate (12 months) is 79.01% (64/81). Meanwhile, the overall survival (OS) of 10 years after diagnosis was estimated as 70.77% (46/65). Moreover, Cox proportional risk model for multivariate analysis showed that the increase in LDH multiple, thrombosis complications, and complicated with bone marrow failure were the independent adverse prognostic factors affecting the survival of PNH patients. CONCLUSION: Paroxysmal nocturnal hemoglobinuria patients in mainland China have various clinical features, while lower incidences of thrombosis and renal damage. Thrombosis and bone marrow failure are two complications with worse prognosis.

Availability of NK cell expansion agent combined with recombinant IL­2 and IL­15 stimulation on the expansion and high­purity of NK cells in patients with immune­related pancytopenia in vitro.

Natural killer (NK) cells are a group of large granular lymphocytes that play an important regulatory role in innate immunity and adaptive immunity. Immune­related pancytopenia (IRP) is a type of pancytopenia resulting from bone marrow hematopoietic cells that were destroyed or suppressed by auto­antibodies. The specific mechanism of IRP is not clear. In the present study, it was identified that the percentage of NK cells in peripheral blood lymphocytes was decreased in patients with IRP. Subsequently, high purity NK cells were extracted from 6 untreated patients with IRP using the immunomagnetic beads sorting, magnetic­activated cell­sorting method, which were then cultured then in RPMI­1640 medium containing 20% FBS. NK cell expansion agents, with or without recombinant interleukin (IL)­15, were used to amplify high­purity NK cells on the basic of recombinant IL­2. Expression of the activated receptors NKG2­D type II integral membrane protein (NKG2D) and natural killer cell p46­related protein (NKp46), and the inhibitory receptors CD158a and NKG2­A/NKG2­B type II integral membrane protein (NKG2A), in CD56+ NK cells were detected by flow cytometry before and after cell culture. It was observed that treatment with an NK cell expansion agent combined with the stimulation of recombinant IL­2 and recombinant IL­15 could increase the number whilst maintaining the purity of NK cells. There were no significant changes in the expression of NKG2D, NKp46, NKG2A and CD158a in patients with IRP before and after NK cell culture. This new amplification method lays a foundation for clinical NK cell immunotherapy and anti­tumor applications.

Abnormalities of quantities and functions of CD56bright natural killer cells in non-severe aplastic Anemia.

OBJECTIVES: The mechanism of non-severe aplastic anemia (NSAA) is not clear. It may be different from severe aplastic anemia (SAA). CD56bright NK cells (regulatory NK cells) is a subgroup of NK cells that produce immunoregulatory cytokines and express high-affinity IL-2 receptor. To investigate CD56bright NK cells quantities and function in patients with NSAA and to explore how CD56bright NK cells participate in the progress of this disease. METHODS: In this study, we analyzed the quantitative and functional changes of CD56bright NK cells in peripheral blood of patients with NSAA by using Flow Cytometry (FCM) before and after immunosuppressive therapy (IST). The expressions of activating receptor (NKG2D, NKp46, NKp44), inhibitory receptor (NKG2A, CD158a, CD158b) and perforin and granzyme B were detected by FCM. IL-2 and IL-18 levels in serum were detected by ELISA. The correlation between these parameters and clinical indicators of patients were evaluated. RESULTS: We found that the percentage of CD56bright NK cells in newly diagnosed NSAA patients was higher than that in normal controls (p = .011, p < .05). The median expression of NKG2D in patients with NSAA was higher compared to that in normal controls (p = .021, p < .05), and the expression of CD158a was lower (p = .047, p < .05). The concentrations of IL-2 and IL-18 in the serum of patients with NSAA were higher than those in normal group. CONCLUSION: These findings suggest that increased and activated CD56bright NK cells might play a protective role in the pathogenesis of NSAA.

NK cells suppress CD8+ T cell immunity via NKG2D in severe aplastic anemia.

The roles of natural killer (NK) cells in shaping the immune system had raised wide interests. Here we intended to explore the regulatory functions of NK cells on CD8+ T cells in severe aplastic anemia (SAA) using human participants and lymphocyte infusion-induced bone marrow failure (BMF) mouse model. In SAA patients, NK cells had over-expressions of NKG2D and NKp46, under-expression of NKG2A and enhanced cytotoxicity. NK cells limited autologous CD8+ T cell immunity in an effector/target ratio manner. The suppression was dependent on the existence of NKG2D. We also observed upregulated MICA expression on activated CD8+ T cells, which were susceptible to NK cell mediated lysis in SAA. Animal model concurred with the data from patients. Infusion of NK cells suppressed the proliferation of CD8+ T cells and decreased IFN-γ production. In conclusion, NK cells served NKG2D-dependent immunoregulatory roles by attenuating autologous CD8+ T cell response in SAA.

Expression of C1q in the serum of patients with non­severe aplastic anemia, and its association with disease severity.

A type of aplastic anemia (AA), non-severe aplastic anemia (NSAA) is defined as AA that does not meet the diagnostic criteria of severe aplastic anemia (SAA). Complement component 1q (C1q) has an important role in the pathogenesis of various autoimmune diseases; however, the role of C1q in the immune pathogenesis of NSAA is not clear. The current study aimed to determine whether C1q has an important role in the pathogenesis of NSAA. Isobaric tags for relative and absolute quantitation (iTRAQ) was used to compare the protein expression in bone marrow mononuclear cells from patients with NSAA and healthy volunteers. Pathway enrichment analysis was performed to determine the biological functions involved in NSAA. The differential expression of C1q was marked compared with other proteins. Subsequently, the concentration of C1q in serum samples was determined using ELISA and the correlation of C1q levels and NSAA severity was evaluated. The serum concentrations of C1q were significantly lower in untreated patients with newly diagnosed NSAA compared with NSAA cases in remission and normal controls. Furthermore, there was no significant difference in C1q concentration between newly diagnosed patients with NSAA and patients with autoimmune hemolytic anemia or immune thrombocytopenia. The serum concentration of C1q in newly diagnosed NSAA was significantly lower in patients with SAA (P<0.0001); whereas, there was no significant difference between the patients with SAA, patients with NSAA remission and normal controls (P>0.05). Additionally, the serum C1q concentration was significantly correlated with granulocyte counts, the level of hemoglobin, platelet counts, reticulocyte percentage and remission in patients with NSAA. The serum C1q concentration was also positively correlated with the myeloid/plasmacytoid dendritic cell ratio, and negatively correlated with the CD4(+)/CD8(+) ratio. These findings suggested that C1q may be a reliable serological marker for monitoring and evaluating disease severity in patients with NSAA. C1q may have an important role in the immune pathogenesis of NSAA.

The Risk of Clonal Evolution of Granulocyte Colony-Stimulating Factor for Acquired Aplastic Anemia: A Systematic Review and Meta-Analysis.

OBJECTIVES: This meta-analysis aimed to evaluate the risk of clonal evolution of granulocyte colony-stimulating factor (G-CSF) in acquired aplastic anemia (AA), and whether the use of G-CSF increases the occurrence of secondary malignant neoplasms, mainly myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) or paroxysmal nocturnal hemoglobinuria (PNH). METHODS: Data were gathered from randomized controlled trials (RCTs) to evaluate the effect of G-CSF versus no G-CSF at the risk of developing the clonal complications of acquired AA. Electronic searches in PubMed, Embase, and the Cochrane Library were performed to identify studies up to 1 January 2017. Only RCTs performed on patients who were randomly assigned to receive G-CSF or not to receive G-CSF were included. RESULTS: Four relevant trials that met the inclusion criteria were identified. In a pooled analysis, the G-CSF groups of AA patients were not associated with a statistically significant higher occurrence of secondary malignant neoplasm, mainly MDS and AML (relative risk [RR] 0.86; 95% confidence interval [CI] 0.34-2.19; 4 trials). No significant heterogeneity was found (p = 0.67, I2 = 0%). There was no statistically significant higher occurrence of PNH in the G-CSF groups with AA (RR 1.17; 95% CI 0.51-2.71; 4 trials) and no significant heterogeneity was found (p = 0.42, I2 = 0%). CONCLUSIONS: G-CSF for patients with AA is not associated with a higher occurrence of secondary malignant neoplasm, mainly MDS/AML, or PNH.

Demonstration of IgG Subclass (IgG1 and IgG3) in Immuno-Related Hemocytopenia.

BACKGROUND: Immuno-related hemocytopenia (IRH) is defined as idiopathic cytopenia of undetermined significance (ICUS) patients with autoantibodies. In our previous studies, we found that IgG1 levels were increased in IRH patients and might cause the destruction of hematopoietic cells. METHODS: In this study, we analyzed IgG subclasses in 30 IRH patients (male:female = 13:17, median age 32 years, range 18 - 56), 15 IRH remission patients (IRH-R) (male:female = 6:9, median age 34, range 20 - 52) and 20 normal controls (male:female = 8:12, median age 27, range 24 - 36) by Cytometric Bead Array, Flow Cytometry and Immunohistochemical staining. RESULTS: Levels of IgG1/IgG3 in the bone marrow supernatant of IRH patents, as well as the proportion of CD5+ B lymphocytes and Th2 cells (CD3+CD8-IL-4+) were higher than those of IRH-R patients and normal controls, and IgG1 levels had a positive correlation with the proportion of Th2 cells. In IRH patients, IgG1 and IgG3 were positive on nucleated erythrocytes and granulocytes, which were negative in IRH-R patients and healthy controls and had inverse correlations with hematopoietic function. Using immunohistochemical staining, IgG1 were also detected on bone marrow biopsies of IRH patients. CONCLUSIONS: The results indicated that IgG1 and IgG3 autoantibodies in IRH patients might play a key role in the IRH pathogenesis and in the abnormal immune function of IRH patients.

Abnormal populations and functions of natural killer cells in patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized by ineffective hematopoiesis that lead to leukemia. Disorders of the immune system serve important functions in the pathophysiology and progression of this disease. Different levels or mechanisms of natural killer (NK) cells in patients with MDS have been measured in previous studies, making it challenging to understand the pathogenesis of NK cytotoxicity. The present study investigated the frequency of NK cell-mediated antibody-dependent cellular cytotoxicity and explored the function of NK cells by their activating receptors, inhibition signals, degranulation and cytotoxicity factors. In the present study, levels of cluster of differentiation (CD)3-CD56+ NK cells, CD16+-expressing NK cells and subset CD56dim NK cells were decreased in the peripheral blood of patients with MDS. Altered expression of NK protein 44, NK group 2 member D, killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1) and KIR2DL3 on NK cell effector signaling pathways may trigger tumor cell lysis in patients with MDS. The weak cellular adhesion and decreased cytotoxicity of NK cells may lead to ineffective antitumor activity in MDS. These observations suggested that NK cells may serve as immunological determinants in MDS and may permit the development of NK cell-based immunotherapy for the treatment of patients with MDS.

Proteinase 3 expression on the neutrophils of patients with paroxysmal nocturnal hemoglobinuria.

Proteinase 3 (PR3) is released from neutrophils and regulates platelet activity, which is associated with cluster of differentiation (CD)177 antigen (NB1), a glycosylphosphatidylinositol-linked protein. In the present study, the effect of PR3 on thrombosis in paroxysmal nocturnal hemoglobinuria (PNH) and PNH-aplastic anemia (AA) syndrome was explored. The expression of PR3 and NB1 on CD59- neutrophils was detected by flow cytometry, immunofluorescence (IF), reverse transcription-quantitative polymerase chain reaction analysis and western blotting. Serum levels of PR3, proteinase-activated receptor 1 (PAR1) and D-Dimer were measured using ELISAs. The expression of PR3 and NB1 on the plasma membrane of CD59- neutrophils in patients with PNH/PNH-AA was significantly lower compared with their expression on CD59+ neutrophils in patients and controls (P=0.001). However, no correlation between PR3 and NB1 expression was identified. IF staining further demonstrated partially positive PR3 expression on CD59- neutrophils. The serum level of PR3 in patients was identified to be significantly decreased compared with healthy controls (P<0.0001), and significantly negatively correlated with PAR1 (r=-0.456; P=0.043) and D-Dimer (r=-0.503; P=0.028) levels. The mRNA and protein levels of PR3 on PNH clones did not change significantly compared with the control group. In conclusion, PR3 expression on the plasma membrane of neutrophils and in the serum of patients with PNH/PNH-AA decreased, which may result in increased PAR1 expression and increased clotting. The present study provides the basis for further study on platelets in PNH.

Effects of N-acetylcysteine treatment in acute respiratory distress syndrome: A meta-analysis.

Acute respiratory distress syndrome (ARDS) is a serious complication of acute lung injury. Severe systemic inflammation is the main cause of multiple organ dysfunction and high mortality. Removal of reactive oxygen species by anti-oxidants has been applied in clinical practice. N-acetylcysteine (NAC) is the most commonly used anti-oxidant. However, the benefit of anti-oxidant therapy was not consistently demonstrated by previous studies. In the present study, a meta-analysis was performed to evaluate the effects of NAC for adult patients with ARDS. The PubMed, Cochrane and EMBASE databases were searched to retrieve all of the available randomized controlled trials (RCTs) published until October 2015. Quality evaluation of included studies was performed according to the modified Jadad scale score. The Cochrane Collaboration Review Manager 5.3 software was used to perform the meta-analysis. Five RCTs comprising 183 patients were found to be eligible for inclusion in the meta-analysis. Pooled analysis showed that NAC did not contribute to reduce short-term mortality [risk ratio (RR)=0.73; 95% confidence interval (CI): 0.50-1.07; P=0.10] or 30-day mortality (RR=0.72; 95% CI: 0.44-1.19; P=0.20) when compared with those in the control group. However, duration of intensive care unit (ICU) stay in the NAC group was shortened [weighted mean difference (WMD), -4.56; 95% CI: (-7.32 to -1.80); P=0.001]. There was no significant difference in the ratio of partial arterial oxygen pressure to the fraction of inspired oxygen between the two groups [WMD, 54.34; 95% CI: (-30.50 to 139.17); P=0.21]. No severe adverse reactions were observed in the patients included. Although the duration of ICU stay was shortened, the clinical benefits of NAC were limited for ARDS based on the present meta-analysis. As the number of included trials and patients was small, additional trials are required to provide sufficient evidence for the efficacy of NAC in ARDS.

The dysfunction of platelets in paroxysmal nocturnal hemoglobinuria.

INTRODUCTION: Thrombosis is a dangerous complication of paroxysmal nocturnal hemoglobinuria (PNH) and has a high mortality rate. However, the mechanism underlying the development of thrombosis in PNH remains unclear. To explore this, platelet function and serum complement activity were investigated in 14 patients with classical PNH, 11 with PNH aplastic anemia (AA) and 30 healthy controls. MATERIAL AND METHODS: Serum concentrations of the terminal complement complex (sC5b-9) were determined by enzyme-linked immunofluorescence assay (ELISA), and the levels of C5b-9, CD61 and CD62p on platelet membranes were determined by flow cytometry. Clinical parameters were assessed, including D-dimer and platelet aggregation induced by adenosine diphosphate (ADP) and arachidonic acid (ARA). RESULTS: Serum sC5b-9 concentrations were significantly lower in the PNH/PNH-AA than in the control group (P<0.01). C5b-9 deposition was significantly higher on CD59- platelets than on CD59+ platelets in PNH/PNH-AA patients and healthy controls (P<0.01 for each). D-dimer concentration was significantly higher in PNH/PNH-AA patients - especially those with lactate dehydrogenase (LDH) concentrations>1000U/L - than in controls (P<0.05). CD61 (P<0.05) expression was lower on CD59+ platelets in PNH than in controls and CD5- platelets in PNH. Expression of CD62p (P<0.01) was lower on CD59- and CD59+ platelets (P<0.01) in PNH cases than in controls. Platelet aggregation stimulated by the agonists ADP and ARA in the PNH/PNH-AA patients was significantly lower than that in controls (P<0.05). CONCLUSIONS: The adhesion and aggregation of platelets, especially of CD59+ platelets, were compensatively decreased in PNH/PNH-AA patients without active thrombosis.