[NIX-mediated mitochondrial autophagy in pathogenesis of myelodysplastic syndrome anemia].

Objective: To investigate the change of NIX level of bone marrow nucleated red blood cells in anemia patients with myelodysplastic syndromes (MDS), to explore the significance of NIX-mediated mitochondrial autophagy in the pathogenesis of MDS anemia. Methods: A total of 54 patients with MDS diagnosed in the Department of Hematology of General Hospital, Tianjin Medical University from July 2015 to July 2016 were enrolled into the MDS group, 33 cases of immune thrombocytopenia or idiopathic leukopenia as controls.The level of NIX, the number of mitochondria, mitochondrial membrane potential, the level of reactive oxygen species (ROS) in GlycoA(+) nucleated red blood cells were measured by flow cytometry; the level of NIX mRNA was measured by PCR. Results: (1) The expression of NIX in GlycoA(+) nucleated red blood cells in high-risk MDS patients (0.61±0.24) was significantly lower than that in controls (0.79±0.16, P=0.027), and lower than that in low-risk MDS patients (0.81±0.15, P=0.011), while there was no significant difference between the controls and low-risk MDS patients. The expression of NIX mRNA in GlycoA(+) nucleated red blood cells in high-risk MDS group (0.36±0.09) was lower than that in the controls (1.44±0.41, P=0.027) and that in the low-risk group (1.02±0.22, P=0.012); there was no significant difference between the controls and the low-risk group. (2) The number of mitochondria in GlycoA(+) nucleated red blood cells in high-risk MDS patients (937.17±707.85) was significantly higher than that in the controls (513.49±372.33, P=0.019) and that in low-risk MDS patients (461.74±438.02, P=0.008); while there was no significant difference between low-risk MDS patients and the controls. (3) The level of mitochondrial membrane potential in GlycoA(+) nucleated red blood cells in high-risk MDS patients (0.33±0.18) was significantly lower than that in the controls (0.61±0.32, P=0.001) and that in low-risk MDS patients (0.61±0.34, P=0.001); with no significant difference between low-risk MDS patients and the controls. (4)The level of ROS in GlycoA(+) nucleated red blood cells in high-risk MDS patients (438.65±322.83) was significantly higher than that in the controls (242.77±136.87, P=0.006), and higher than that in low-risk MDS patients (197.40±95.07, P=0.001); no significantly different between low-risk MDS patients and the controls. (5) The number of mitochondria in GlycoA(+) nucleated red blood cell was positively correlated with the percentage of ring sideroblast (r=0.457, P=0.028) in the MDS patients.(6) The number of mitochondria in GlycoA(+) nucleated red blood cells was negatively correlated with the concentration of hemoglobin (r=-0.521, P=0.009) in high-risk MDS patients, but not correlated with the concentration of hemoglobin in low-risk MDS patients. Conclusion: NIX level is reduced in nucleated red blood cells of high-risk MDS patients, which leads to impaired mitochondrial autophagy, increased damaged mitochondria and apoptosis of nucleated red blood cells, thus related with anemia.

Fas/FasL in the immune pathogenesis of severe aplastic anemia.

Fas/FasL protein expression of bone marrow hematopoietic cells was investigated in severe aplastic anemia (SAA) patients. Fas expression was evaluated in CD34(+), GlycoA(+), CD33(+), and CD14(+) cells labeled with monoclonal antibodies in newly diagnosed and remission SAA patients along with normal controls. FasL expression was evaluated in CD8(+) cells in the same manner. In CD34(+) cells, Fas expression was significantly higher in the newly diagnosed SAA group (46.59 ± 27.60%) than the remission (6.12 ± 3.35%; P < 0.01) and control (8.89 ± 7.28%; P < 0.01) groups. In CD14(+), CD33(+), and GlycoA(+) cells, Fas levels were significantly lower in the newly diagnosed SAA group (29.29 ± 9.23, 46.88 ± 14.30, and 15.15 ± 9.26%, respectively) than in the remission (47.23 ± 31.56, 67.22 ± 34.68, and 43.56 ± 26.85%, respectively; P < 0.05) and normal control (51.25 ± 38.36, 72.06 ± 39.88, 50.38 ± 39.88%, respectively; P < 0.05) groups. FasL expression of CD8(+) cells was significantly higher in the newly diagnosed SAA group (89.53 ± 45.68%) than the remission (56.39 ± 27.94%; P < 0.01) and control (48.63 ± 27.38%; P <0.01) groups. No significant differences were observed between the remission and control groups. FasL expression in CD8(+) T cells was significantly higher in newly diagnosed patients, and CD34(+), CD33(+), CD14(+), and GlycoA(+) cells all showed Fas antigen expression. The Fas/FasL pathway might play an important role in excessive hematopoietic cell apoptosis in SAA bone marrow. Furthermore, CD34(+) cells are likely the main targets of SAA immune injury.

[Characteristic and function of peripheral blood mononuclear cells-induced macrophages in patients with myelodysplastic syndrome].

Objective: To explore characteristic and function of peripheral blood mononuclear cells (PBMNC) -induced macrophages in patients with myelodysplastic syndrome (MDS) to couple with its progression. Methods: A total of 24 MDS patients (11 low-risk patients and 13 high-risk group patients) referred to Department of Hematology of Tianjin Medical University General Hospital and normal controls were enrolled from September 2014 to December 2015. PBMNC was stimulated with GM-CSF to transform to macrophages. The morphology of macrophages was observed by microscope. The quantity of macrophages, CD206 and SIRPα on surface of macrophages were detected by flow cytometry. The phagocytic function of macrophages was analyzed by fluorescence microscopy and flow cytometry. Results: The morphology of macrophages from MDS patients was abnormal. The percentage of transformed macrophages was (5.17±3.47) % in patients with MDS, which was lower than that in controls significantly[ (66.18±13.43) %, t=3.529, P=0.001]. The expression of CD206 on macrophages from MDS patients was significantly lower than that of controls[ (9.73±2.59) % vs (51.15±10.82) %, t=4.551, P<0.001]. The SIRPα level of macrophages from MDS patients was significantly lower than that of controls [ (0.51±0.09) % vs (0.77±0.06) %, t=2.102, P=0.043]. The phagocytic index and the percentage of phagocytic of macrophages from MDS patients were significantly lower than those of macrophages from normal controls[0.45±0.08 vs 0.92±0.07, t=-6.253, P=0.008; (23.69±3.22) % vs (42.75±2.13) %, t=-6.982, P=0.006 respectively]by flow cytometry. The phagocytic index of MDS patients was significantly lower than that of controls (0.24±0.04 vs 0.48±0.96, t=3.464, P=0.001) by fluorescence microscopy. Conclusion: The quantity, recognization receptors and phagocytosis of PBMNC-induced macrophages decreased in MDS patients.

[Study on autophagy in nucleated red blood cells in patients with myelodysplastic syndromes].

Objective: To investigate the change of autophagy level of bone marrow nucleated red blood cell (RBC) in patients with myelodysplastic syndromes (MDS) . Methods: Fifty-four MDS patients and thirty-three controls were enrolled in this study. The mitophagy were observed by transmission electron microscopy (TEM) . The level of autophagy-associated protein LC3B in GlycoA(+) nucleated RBC was measured by flow cytometry. The expressions of ULK1 and mTOR mRNA in GlycoA(+) nucleated RBC were measured by real-time PCR. The expression of the mitochondrial outer membrane protein TOM20 in GlycoA(+) nucleated RBC was detected by Western blot. Results: Autophagosomes or autolysosomes were scarcely observed by TEM in MDS patients. The expression of LC3B in GlycoA(+) nucleated RBC in high-risk MDS patients (0.22±0.12) was significantly lower than that in normal controls (0.43±0.22, P<0.001) , and lower than that in low-risk MDS patients (0.40±0.16, P=0.001) . The expression of AMPK [0.26 (0.60) ] in GlycoA(+) nucleated RBC in high-risk MDS patients was significantly lower than that in controls [1.00 (2.07) , P<0.017) . The expression of ULK1 mRNA in GlycoA(+) nucleated RBC in high-risk MDS patients [0.27 (3.31) ] was significantly lower than that in controls [1.07 (4.41) , P<0.017]. The level of mTOR mRNA in GlycoA(+) nucleated RBC in high-risk MDS patients [1.82 (3.74) ] was significantly higher than that in controls [1.26 (1.38) , P<0.017]. The level of LC3B in GlycoA(+) nucleated RBC was negatively correlated with the HGB (r=0.529, P=0.009) in high-risk MDS patients. The expression of mitochondrial outer membrane protein TOM20 in high-risk MDS patients was 9.42±4.42. Conclusion: Autophagy is impaired in nucleated RBC of MDS patients.

[Quantities and function of NK cells in patients with positive BMMNC-Coombs test and cytopenia].

OBJECTIVE: To test NK cell quantities and function in patients with positive BMMNC-Coombs test (CBCPC) and cytopenia and to explore how NK cell participate in the progress of this disease. METHODS: The percentage of CD3(-)CD56(+) NK cell in peripheral blood lymphocytes, the expression of activating receptor (NKG2D, NKp46, NKp44), inhibitory receptor (CD158a, CD158b), perforin and granzyme-ß were detected by flow cytometry. All samples were taken from 42 patients (22 newly diagnosed and 20 in remission) and 12 healthy volunteers. The correlation between the above parameters and patients' clinical profile were evaluated. RESULTS: ①The percentage of CD3(-)CD56(+) NK cell in new diagnosed and remission CBCPC patients were significantly lower than that in healthy control [(10.04 ± 5.33)% vs (19.94 ± 7.38)%; (11.62 ± 6.80)% vs (19.94 ± 7.38)%, all P<0.01]. ② The expression of activating receptor NKG2D in new diagnosed CBCPC patients was significantly higher than that in remission group and healthy control [(74.03±18.24)% vs (45.97±29.45)%; (74.03±18.24)% vs (41.89± 15.34)% , P <0.01]. ③The expression of inhibitory receptor CD158a in new diagnosed CBCPC patients was significantly lower than that in remission group and healthy control (median: 3.72% vs 16.10%, P= 0.015; 3.72% vs 11.04%, P=0.025). ④The expression of perforin in new diagnosed and remitted CBCPC patients were significantly higher than that in healthy controls [(75.71±10.14) % vs (57.20±18.85)%, P= 0.018; (77.88±22.82)% vs (57.20±18.85)%, P=0.008]. ⑤The product of NK cell percentage and perforin expression in new diagnosed and remission CBCPC patient were significantly lower than that in healthy control [(7.68±4.54)% vs (12.13±5.19)%, P=0.011; (8.24±5.80)% vs (12.13±5.19)%, P=0.023]. The product of NK cell percentage and granzyme-ß expression in the new diagnosed and remission CBCPC patient were significantly lower than that in healthy control [(7.83±5.26)% vs (14.79±8.37)%, P=0.008; (8.37 ± 6.83)% vs (14.79±8.37)%, P=0.012]. CONCLUSION: Deceased quantities and impaired total NK function might play a role in pathogenesis of CBCPC.