A preliminary study on SLAMF6 expression in patients with severe aplastic anemia / 中华血液学杂志

Objective@#To explore the expression of SLAMF6 on CD8+ T cells in patients with severe aplastic anemia (SAA) and its correlation with disease immune status.@*Methods@#By flow cytometry (FCM), SLAMF6 expression level in peripheral blood CD8+ T cells was detected in 21 patients with SAA and 15 normal controls respectively from February 2017 to April 2018. The correlation between SLAMF6 expression level and hematopoietic functions, including HGB, PLT, the neutrophil granulocyte and reticulocyte absolute value in peripheral blood, hyperplasia degree (percentage of granulocytes, erythrocytes, lymphocytes and megakaryocytes in bone marrow) and perforin, granzyme B, IFN-γ expression level in CD8+ T cells were evaluated. To further confirm the effect of SLAMF6 on CD8+ T cells, anti-SLAMF6 Ab was used to block SLAMF6 pathway (IgG as control), and FCM was used to detect the perforin, granzyme B, and IFN-γ production of CD8+ T cells.@*Results@#The expression of SLAMF6 on CD8+ T cells in untreated SAA patients[(56.29±12.97)%]was significantly lower than that of normal controls[(80.96±7.36)%](t=-7.672, P<0.001). The expression of SLAMF6 on CD8+ T cells in SAA patients were positively correlated with the HGB, PLT, the neutrophil granulocyte and reticulocyte absolute value in peripheral blood, percentage of granulocytes, erythrocytes in bone marrow (all P<0.05), but they were negatively correlated with the percentage of lymphocytes in bone marrow, and the expression of perforin, granzyme B, and IFN-γ of CD8+ T cells (all P<0.05). After blocking SLAMF6 pathway by anti-SLAMF6 Ab, the expression levels of perforin, granzyme B and IFN-γ in SAA patients were significantly higher than those in the untreated group, and the differences were statistically significant (all P<0.05).@*Conclusions@#SLAMF6 is significantly down-regulated on CD8+ T cells in SAA patients, which may act as a negative immunoregulatory molecule participating in the mechanism of SAA by affecting the functional molecules secretion on CD8+ T cells.

Study on C5b-9 deposited on the membrane of platelets and its dysfunction in patients with paroxysmal nocturnal hemoglobinuria / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the expression levels of terminal complement complex (C5b-9) and CD62p on platelets and the soluble C5b-9 (sC5b-9) level in serum in patients with PNH or PNH-aplastic anemia (AA).</p><p><b>METHODS</b>Serum levels of sC5b-9, complement C3 and C4 were detected by using ELISA in 25 patients with PNH/PNH-AA. The quantities of C5b-9 and CD62p on the membrane of platelets were detected by flow cytometry.</p><p><b>RESULTS</b>①In PNH/PNH-AA group, the serum sC5b-9 level [390.27(265.73-676.87) μg/L] was lower than that in control group [540.39(344.20-1 576.78) μg/L] (P<0.01). ②The platelet PNH clone (CD59⁻CD61⁺/CD61⁺) size [50.58(23.29-81.60)%] was bigger in the PNH/PNH-AA group than that [23.57(15.58-29.02)%] in control group (P<0.01). The percentages of C5b-9 deposition (C5b-9⁺CD61⁺/CD61⁺) were higher on the PNH clone platelets (CD59⁻CD61⁺) in the PNH/PNH-AA group [(17.53 ± 6.27)%] than those on the normal platelets (CD59⁺CD61⁺) in PNH patients 11.33±5.03）%］ and control [(10.88±3.58)%] group (P<0.01). ③ The expression of CD62p (CD62p⁺CD61⁺/CD61⁺) on PNH clone platelets in PNH patients [(61.98 ± 11.71)%] was higher than that on the normal platelets in PNH patients [(43.76±11.30)%] and control group [(38.23±18.07)%] (P<0.01). In addition, the expression of CD62p on normal platelets was higher in PNH patients than control (P<0.05). ④The deposition of C5b-9 positively correlated with the expression of CD62p on the platelets (r=0.559, P=0.002).</p><p><b>CONCLUSION</b>Deficiency of CD59 antigen on platelets in PNH patients may lead to the deposition of C5b-9 on its membrane and its dysfunction, which may contribute to thrombosis events in PNH.</p>

Abnormal WT1 gene expression in paroxysmal nocturnal hemoglobinuria / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the pathogenesis of abnormal WT1 expression in paroxysmal nocturnal hemoglobinuria (PNH).</p><p><b>METHODS</b>The expression of WT1 mRNA in CD59⁻ and CD59⁺ bone marrow mononuclear cells (BMMNC) were measured by semi-quantitative reverse transcription PCR. After WT1 gene silence by RNA interference (RNAi) technology, biological characteristics of BMMNC were investigated by flow cytometry.</p><p><b>RESULTS</b>The relative expression of WT1 mRNA in PNH CD59⁻ BMMNC (1.06 ± 0.12) was significantly higher than that in PNH CD59⁺ BMMNC (0.90 ± 0.12) and normal BMMNC (0.86 ± 0.05, P<0.05), but there was no significant difference between PNH CD59⁺ BMMNC and normal BMMNC (P>0.05). WT1 mRNA expression in PNH was positively correlated with the proportion of CD59⁻ cells (r²=0.490, P=0.016), but had no relationship with the proportion of CD59⁺ cells. After WT1 gene silence by siRNA in PNH CD59⁻ BMMNC, WT1 mRNA expression was decreased. The proportions of G0/G1 phase in PNH CD59⁻ cell blank control group and siRNA-scr transfected group were (92.73 ± 3.71)% and (93.06 ± 4.14)%, and the proportions of S phase were (6.99 ± 3.61)% and (6.73 ± 4.08)%, respectively. The proportions of G0/G1 and S phase in siRNA-WT1 transfected group was (94.46 ± 3.71)% and (5.40 ± 3.55)%, respectively. There were significant differences in the proportions of G0/G1 phase and S phase among the controls, siRNA-WT1 transfected group and siRNA-scr transfected group (P<0.05). The rate of apoptosis in siRNA-WT1 transfected group [(35.91 ± 22.36)%] was significantly higher than those in controls [(26.12 ± 17.10)%] and siRNA-scr transfected group [(27.39 ± 18.99)%] (P<0.05).</p><p><b>CONCLUSION</b>siRNA-WT1 could effectively suppress the WT1 gene expression of CD59⁻ clone in PNH patients, inhibit its proliferation, and promote its apoptosis. WT1 gene expression might contribute to PNH clone proliferation.</p>

Telomere length of peripheral lymphocytes in patients with immuno-related pancytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the changes of relative telomere length (RTL) of peripheral blood (PB) CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺T lymphocytes, CD19⁺B lymphocytes and bone marrow (BM) CD34⁺ cells and its association with disease severity in untreated patients with immuno-related pancytopenia (IRP).</p><p><b>METHODS</b>The PB CD3⁺ , CD3⁺ CD4⁺ , CD3⁺ CD8⁺ T lymphocytes, CD19⁺ B lymphocytes, and BM CD34⁺ cells were purified by magnetic activated cell sorting (MACS), and RTL were measured with flow-fluorescence in situ hybridization (FLOW-FISH).</p><p><b>RESULTS</b>The RTL of CD3⁺, CD3⁺CD4⁺ , and CD3⁺CD8⁺T lymphocytes in untreated IRP patients were (27.754 ± 16.323)%, (7.526 ± 3.745)% and (25.854 ± 14.789)%, respectivly, which were significantly shorter than those in healthy-controls (54.555 ± 19.782)%, (12.096 ± 2.805)%, and (38.367 ± 4.626)% (P<0.05). The RTL of CD19⁺ lymphocytes in untreated IRP patients was (22.136 ± 16.142)%, which was significantly shorter than that in healthy controls (42.846 ± 16.353)% (P<0.01). There was no significant difference of BM CD34⁺ cells RTL between the untreated IRP patients (22.528 ± 21.601)% and the healthy controls (23.936 ± 19.822)% (P>0.05). There were significantly positive correlations between the RTL of B lymphocytes and the count of white blood cell (r=0.706, P=0.015). There were negative correlations between RTL of B lymphocytes and the clinical symptoms (r=-0.613, P=0.045) and positive correlations with therapeutic effect (r=0.775, P=0.005).</p><p><b>CONCLUSION</b>The shorter RTL of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD19⁺ lymphocytes, and the normal RTL of BM CD34⁺ cells in untreated IRP patients were identified, which might imply that IRP is a type of acquired autoimmune diseases.</p>

Effect of CCL3 on osteoblast in myeloma bone disease / 中华血液学杂志

<p><b>OBJECTIVE</b>To culture osteoblast in vitro and evaluate CCL3 receptor CCR1 expression in patients with multiple myeloma (MM).</p><p><b>METHODS</b>Bone marrow osteoblasts from MM patients were cultured in vitro with dexamethasone, β-sodium glycerophosphate and vitamin C, which were identified by alkaline phosphatase staining, Von Kossa's staining. The CCL3 receptor expression was evaluated by flow cytometry. The morphology and quantity of osteoblast were observed after exposure to CCL3.</p><p><b>RESULTS</b>Bone marrow osteoblasts from MM patients could be cultured in vitro and be identified by positive staining of alkaline phosphatase and Von Kossa's. MM-derived osteoblasts expressed higher levels of CCR1 (74.48 ± 7.31)%, compared with normal controls (48.35 ± 8.81)%. Calcium deposition of osteoblasts after exposure to CCL3 was less than that of controls.</p><p><b>CONCLUSION</b>Bone marrow osteoblasts could be cultured in vitro from MM Patients. CCL3 may contribute to the development of myeloma bone disease.</p>

Memory B (CD5⁺ CD19⁺ CD27⁺) lymphocyte in patients with immune-related pancytopenia / 中华血液学杂志

<p><b>OBJECTIVE</b>To detect memory B lymphocyte (Bm) in peripheral blood (PB) of immune-related pancytopenia (IRP).</p><p><b>METHODS</b>86 patients with IRP and 11 health volunteers were enrolled in this study. Bm (CD5⁺ CD19⁺ CD27⁺) and bone marrow mononucleated cell antibodies (BMMNC-Ab) were determined via fluorescence-activated cell sorting, and clinical outcomes of these patients were analyzed.</p><p><b>RESULTS</b>(1)43 initial patients achieved obvious remission in all 52 initial cases after conventional immunosuppression therapy. 16 relapsed patients with IRP received Rituximab (RTX) and 14 cases achieved obvious remission, among which 7 cases were refractory to conventional immunosuppression therapy, 5 cases exhibited obvious remission, and 2 cases did not respond. Other 18 relapsed cases received conventional immunosuppression therapy and 13 cases achieved obvious remission. (1)The level of Bm in PB in 52 initial patients with IRP was(1.81 ± 0.97)%, and no significant difference was observed between the initial patients and health volunteers (1.75 ± 0.55)% (P>0.05). The level of Bm in PB in 34 relapsed patients with IRP was obviously higher than that in the initial IRP patients and health volunteers (P<0.05). Significant difference was observed in the level of Bm in PB in 16 relapsed IRP patients between pre-therapy and post-therapy with RTX (P<0.05). No statistical difference was found between the remission and no-response groups in relapsed patients treated with RTX. RTX regimen produced more effective outcome than conventional immunosuppression therapy, which better eliminated Bm than the latter (P<0.05). Initial patients with IRP who relapsed within a two-year follow-up period had a lower level of Bm in PB compared with un-relapsed patients (P<0.05). Majority of BMMNC- Ab antibodies in relapsed patients were IgG (82.4%) and IgM (69.2%) autoantibodies in patients with initial IRP.</p><p><b>CONCLUSION</b>The level of Bm in PB was associated with relapsed patients with IRP. Bm did not respond to conventional immunosuppression therapy,but responded to RTX.</p>

Quantity and apoptosis-related protein levels of CD4+, CD25+, and CD127low regulatory T cells in peripheral blood of multiple myeloma patients / 中国肿瘤临床

To investigate the role of CD4+, CD25+, and CD127low regulatory T cells (Tregs) in multiple myeloma (MM). Methods:Levels of CD4+T cells and Tregs, as well as expression of CTLA-4 and apoptosis-related proteins, such as CD95, bcl-2, and Caspase3 of Tregs in peripheral blood of 30 patients with newly diagnosed cases, 27 patients under of complete remission (CR) from multiple myeloma patients, and 25 healthy adults were analyzed by flow cytometry. Results:The percentage of CD4+T cells in the untreated group was significantly lower than that of the control group (P<0.05). The percentage of Tregs in CD4+T cells in the untreated group was significantly higher than that of the CR group and control group (P<0.05), which in ISSⅢpatients of the untreated group was significantly higher than that in I/II(P<0.05). No significant difference of CD95 expression in Tregs was observed among the three groups. The expression of CTLA-4 in Tregs from the untreated group was significantly higher than that of the CR group (P<0.05) and control group (P<0.01), and so was in CR group than this in controls (P<0.05). The expression of bcl-2 in Tregs in the untreated group was significantly higher than that of the CR group (P<0.05) and control group (P<0.01), and so was in CR group than this in controls(P<0.05). The expression of Caspase3 in Tregs from the untreated group and CR group were all significantly lower than that of the control group (P<0.05). The percentage of Tregs in CD4+T cells in the untreated group was positively correlated with the proportion of bone marrow plasma cells (P<0.05). The percentage of Tregs in CD4+T cells from 15 MM patients who received bortezamib and dexamethasone (VD) chemotherapy was negatively correlated to the ratio of plasma cell reduction after the first VD chemotherapy (r=0.735, P<0.01). Conclusion:The level of Tregs in the peripheral blood of MM patients was positively correlated with tumor burden and progression of disease, but was negatively correlated with curative effect. The increased level of Tregs was associated with their strengthened anti-apoptosis function.