Qualitative study on cognition and attitude of palliative care of nephrology nurse in patients with end-stage renal disease / 中国实用护理杂志

Objective:To investigate the cognition and attitude of nephrology nurse to palliative care for patients with end-stage renal disease, and to provide reference for the promotion and application of palliative care in patients with end-stage renal disease.Methods:Using the phenomenological research method, the purpose sampling method and the principle of data saturation, the semi-structured interviews were conducted among 12 nurses from the Department of Nephrology in Xiang Yang NO.1 People ′s Hospital, Hubei University of Medicine in November 2021. The data were analyzed, summarized and extracted by Colaizzi 7-step analysis method. Results:The cognition and attitude of nephrology nurses to palliative care of patients with end-stage renal disease were summarized into three themes. They were: different cognition of palliative care; attitudes towards palliative care for end-stage renal disease patients (positive feelings, negative feelings); difficulties in the implementation of palliative care (difficult decision-making, unknown timing and effect, lack of social policy support, traditional thought constraints).Conclusions:Interviewees lacked cognition to palliative care and have different attitudes. In order to promote the application of palliative care in patients with end-stage renal disease, relevant publicity and education should be strengthened, the palliative care system of end-stage renal disease should be improved, and social support for palliative care should be enhanced.

Analysis of factors associated early diagnosis in coronavirus disease 2019 (COVID-19)

BackgroundThe pandemic of coronavirus disease 2019 (COVID-19) has become the first concern in international affairs as the novel coronavirus (SARS-CoV-2) is spreading all over the world at a terrific speed. The accuracy of early diagnosis is critical in the control of the spread of the virus. Although the real-time RT-PCR detection of the virus nucleic acid is the current golden diagnostic standard, it has high false negative rate when only apply single test. ObjectiveSummarize the baseline characteristics and laboratory examination results of hospitalized COVID-19 patients. Analyze the factors that could interfere with the early diagnosis quantitatively to support the timely confirmation of the disease. MethodsAll suspected patients with COVID-19 were included in our study until Feb 9th, 2020. The last day of follow-up was Mar 20th, 2020. Throat swab real-time RT-PCR test was used to confirm SARS-CoV-2 infection. The difference between the epidemiological profile and first laboratory examination results of COVID-19 patients and non-COVID-19 patients were compared and analyzed by multiple logistic regression. Receiver operating characteristic (ROC) curve and area under curve (AUC) were used to assess the potential diagnostic value in factors, which had statistical differences in regression analysis. ResultsIn total, 315 hospitalized patients were included. Among them, 108 were confirmed as COVID-19 patients and 207 were non-COVID-19 patients. Two groups of patients have significance in comparing age, contact history, leukocyte count, lymphocyte count, C-reactive protein, erythrocyte sedimentation rate (p<0.10). Multiple logistic regression analysis showed age, contact history and decreasing lymphocyte count could be used as individual factor that has diagnostic value (p<0.05). The AUC of first RT-PCR test was 0.84 (95% CI 0.73-0.89), AUC of cumulative two times of RT-PCR tests was 0.92 (95% CI 0.88-0.96) and 0.96 (95% CI 0.93-0.99) for cumulative three times of RT-PCR tests. Ninety-six patients showed typical pneumonia radiological features in first CT scan, AUC was 0.74 (95% CI 0.60-0.73). The AUC of patients age, contact history with confirmed people and the decreased lymphocytes were 0.66 (95% CI 0.60-0.73), 0.67 (95% CI 0.61-0.73), 0.62 (95% CI 0.56-0.69), respectively. Taking chest CT scan diagnosis together with patients age and decreasing lymphocytes, AUC would be 0.86 (95% CI 0.82-0.90). The age threshold to predict COVID-19 was 41.5 years, with a diagnostic sensitivity of 0.70 (95% CI 0.61-0.79) and a specificity of 0.59 (95% CI 0.52-0.66). Positive and negative likelihood ratios were 1.71 and 0.50, respectively. Threshold of lymphocyte count to diagnose COVID-19 was 1.53x109/L, with a diagnostic sensitivity of 0.82 (95% CI 0.73-0.88) and a specificity of 0.50 (95% CI 0.43-0.57). Positive and negative likelihood ratios were 1.64 and 0.37, respectively. ConclusionSingle RT-PCR test has relatively high false negative rate. When first RT-PCR test show negative result in suspected patients, the chest CT scan, contact history, age and lymphocyte count should be used combinedly to assess the possibility of SARS-CoV-2 infection.

The clinical significance of CD4+CD69+T lymphocytes in patients with autoimmune hemolytic anemia/Evans syndrome / 中华内科杂志

Objective To investigate the significant of peripheral CD4+CD69+T lymphocytes in patients with autoimmune hemolytic anemia (AIHA)/Evans syndrome (ES).Methods In this study peripheral blood samples from 32 patients with AIHA/ES (15 hemolytic episode patients,17 remission patients) and 13 healthy controls were collected.Patients with AIHA/ES were recruited in Tianjin Medical University General Hospital from October 2015 to May 2016.The percentages of CD69+ T lymphocytes were analyzed by flow cytometry.The expression of CD69 mRNA in CD4+T lymphocytes which was sorted from peripheral blood by magnetic activated cell sorting (MACS) was detected using real-time PCR.Soluable CD69 was measured by ELISA.Results In hemolytic episode patients,the ratio of CD3+CD69+/CD3+T lymphocytes [(3.08 ± 1.48)％] was significantly higher than that in healthy controls [(1.28 ± 0.83)％,P＜0.01] and in remission group[(1.96± 1.33)％,P＜0.05].The absolute count of CD3+CD69+T lymphocytes in hemolytic episode group [(2.94± 1.81)× 107/L] was higher than that in healthy controls [(1.48± 1.42)× 107/L,P＜0.05].The ratio of CD3+CD4+CD69+/CD3+CD4+T cells in hemolytic episode group [(2.16± 1.56)％] was significantly higher than that in remission group [(1.16±0.62)％,P＜0.05] and healthy controls[(0.94±0.78)％,P＜0.05].The quantity of CD3+CD4+CD69+T lymphocytes in hemolytic episode group[(1.04±0.98)× 107/L] was higher than in healthy controls [(0.44± 0.38) × 107 / L,P＜0.05].The ratio of CD3+CD8+CD69+/CD3+ CD8+T lymphocyte in hemolytic episode group [(4.87±2.56)％] was significantly higher than that in healthy controls[(1.83± 1.27)％,P＜0.01].The quantity of CD3+CDs+CD69+T lymphocytes in three groups did not show significant difference.The ratio of CD3+CD4+CD69+/CD3+CD4+T lymphocytes in hemolytic episode group was negatively correlated with hemoglobin (Hb) (P＜0.01),positively correlated with the percentage of reticulocytes (Ret％)(P=0.01)total bilirubin(TBil),indirect bilirubin(IBil) (P＜0.01) and not correlated with absolute reticulocytes count,lactic dehydrogenase (LDH),complement 3(C3),complement 4 (C4).The ratio of CD3+CD4+CD69+/CD3+CD4+T lymphocytes in remission group was negatively correlated with Hb (P＜0.05).In hemolytic episode patients CD69 mRNA (32.26±35.11) was significantly higher than that in remission group(6.05±5.87)(P＜0.05)and healthy controls (1.76± 1.85)(P＜0.01).CD69 mRNA in remission group was significantly higher than healthy controls (P＜0.05).Serum CD69 in hemolytic episode patients [(494.21 ± 16.06) ng/L] was significantly higher than that in healthy controls [(441.39± 104.6) ng/L,P＜0.05].Conclusion Our findings suggest that the proportion of CD4+CD69+ T lymphocytes increase in AIHA/ES patients,which is correlated with the severity of disease.

Characteristic and function of peripheral blood mononuclear cells-induced macrophages in patients with myelodysplastic syndrome / 中华血液学杂志

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 / 中华血液学杂志

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.

Research on the negative immune regulation of NK cells in patients with primary immune thrombocytopenia / 中华血液学杂志

Objective@#To investigate the levels of NK cells and their relevant cytokines (IL-10, TGF-β and IFN-γ) in patients with primary immune thrombocytopenia (ITP) .@*Methods@#All samples were obtained from 42 patients (22 newly diagnosed and 20 in remission) and 20 healthy volunteers. The levels of IL-10 and IFN-γ in blood serum were detected by enzyme-linked immunosorbent assay (ELISA) . The percentage of CD3- CD56+ NK cell, CD3- CD56bright CD16- NK cell, CD3- CD56dim CD16+ NK cell in peripheral blood lymphocyte were detected by flow cytometry. The NK cells were isolated by immunomagnetic microbeads. The mRNA expression levels of IL-10, TGF-β, and IFN-γ in NK cells were detected by real-time fluorescent quantitative PCR. Correlation between the above measured results was analyzed.@*Results@#① The blood serum level of IFN-γ in newly diagnosed ITP patients [ (653.0±221.6) ng/L] was higher than that in remission ITP patients [ (484.4±219.5) ng/L] and healthy control [ (390.9±253.5) ng/L] (P=0.022, P=0.001) . The blood serum level of IL-10 in newly diagnosed ITP patients was lower than that in healthy control [ (52.09±26.66) ng/L vs (79.44±38.43) ng/L, P=0.007]. ②The percentage of NK cell in newly diagnosed and remission ITP patients [ (9.53±3.93) %, (9.03±3.78) %] were significantly lower than that in healthy control [ (13.72±7.42) %] (P=0.013, P=0.007) . The ratio of CD3- CD56bright CD16- NK cell/total NK cells in newly diagnosed ITP patients was higher than that in healthy control [ (6.85±4.43) % vs (4.05±2.81) %, P=0.032]. The ratio of CD3-CD56dim CD16- NK cell/total NK cells in newly diagnosed ITP patients was lower than that in healthy control [ (93.14±4.43) % vs (95.94±2.81) %, P=0.032]. ③ There was no significant difference in the mRNA expression level of IFN-γ in NK cells of ITP patients and healthy control (all P>0.05) . The mRNA expression levels of IL-10 and TGF-β in NK cells in newly diagnosed ITP patients were significantly higher than that in healthy control (1.82±1.32 vs 1.02±1.03, P=0.023; 2.80±2.31 vs 1.46±1.37, P=0.028) . The ratio of CD3-CD56bright CD16- NK cell/total NK cells was positively correlated with the mRNA expression levels of IL-10, TGF-β in NK cells (r=0.424, P=0.001; r=0.432, P<0.001) .@*Conclusion@#NK cells may compensate for the deficiency of the number by enhancing the secretion of negative regulation cytokines, acting as "protective" roles in the disease.

Autophagy level of bone marrow mononuclear cells in patients with myelodysplastic syndromes / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the change of autophagy level of bone marrow mononuclear cells（BMMNCs）in patients with myelodysplastic syndromes（MDS）.</p><p><b>METHODS</b>Thirty- eight patients with MDS and 26 megaloblastic anemia patients were enrolled in this study. The autophagic vacuoles were observed by transmission electron microscopy （TEM） and the quantity of autophagic vacuoles was detected by monodansylcadaverine （MDC） staining. The LC3 protein positive cells were counted by immunofluorescence assays. The expression of Beclin 1, LC3A, mTOR mRNA were measured by real time PCR. The expression of Beclin 1 proteins were detected by Western blotting.</p><p><b>RESULTS</b>The autophgic vacuoles of double membrane that surrounds lysosomes appeared in MDS patients. The percentage of MDC positive cells was significantly higher in MDS patients［（9.75±2.63）%］than that of controls［（2.90± 0.89）%, P＜0.05）. The percentage of LC3 protein cells was also increased in MDS patients（6.13±1.03）% vs（1.5±0.58）%, P＜0.05）. The expression of Beclin 1 and LC3A mRNA in low-risk and intermediate-1 MDS were higher compared with controls （3.61 ± 3.02 vs 1.55 ± 1.03 and 6.56 ± 3.97 vs 1.21 ± 0.95 respectively, both P＜0.05）. The expression of mTOR mRNA was down- regulated in low- risk and intermediate-1 MDS compared with controls（0.39±0.37 vs 1.50±1.03, P＜0.05）. There were no significant difference in expression of Beclin 1, LC3 and mTOR mRNA among intermediate-2 and high-risk MDS and controls. Beclin 1 protein expression was higher in low- risk and intermediate- 1 MDS patients（1.257 ± 0.197）than that of controls（0.528±0.086）and inermediate-2 and high-risk MDS patients（0.622±0.118）.</p><p><b>CONCLUSION</b>The autophagy levels were increased in low- risk and intermediate- 1 MDS, while not enhanced in intermediate-2 MDS. Autophagy might be considered as a cell protective mechanism in MDS. The relatively defective autophagy in intermediate- 2 and high- risk MDS might contribute to disease's progression.</p>

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>

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>

Culture and regulation of osteoblasts in multiple myeloma patients / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the biological characteristics of osteoblasts cultured in vitro from bone marrow (BM) of multiple myeloma (MM) patients and to explore their generation and osteogenic potential. Effects of some factors such as bortezomib and MM patient serum on the osteoblasts were observed.</p><p><b>METHODS</b>Twenty MM patients and 10 healthy donors as controls were enrolled in this study. Osteoblasts from MM patients'BM were cultured in vitro. The generation and osteogenic potential of osteoblasts from MM patients and normal subjects were compared. The changes of their osteogenic potential and biological characteristics were observed. The antigens (CD34,CD138,CD45) on osteoblasts were catalyzed by flow cytometry. The levels of IL-7 were measured by ELISA. The BMP2 mRNAs were measured by RT-PCR.</p><p><b>RESULTS</b>Osteoblasts from MM patients'BM could be cultured in vitro. The quantity of osteoblasts from MM patients (6.3±1.5) was less than normal subjects (8.2±2.6) (P<0.05). The osteoblasts cultured with MM patient serum (7.4±1.1) were less than those without patient serum (8.2±2.6) (P<0.05). Bortezomib increased those from MM patients after 6 days culture (8.9±2.1 vs 6.3±1.5) (P<0.05). Von Kossa staining showed that there were more calcium depositions in MM osteoblasts with bortezomib (8.8±1.9) than those without bortezomib (6.1±1.6) (P<0.05), but less than those from normal controls (15.8±2.2) (P<0.05). CD138, CD45, CD34 were not detected on the cultured cells. The level of IL-7 in MM patients'serum (2.07±0.71) was higher than that in normal controls (1.62±0.15) (P<0.05). The expression of BMP2 mRNA was seen in the normal osteoblasts and MM patients'osteoblasts cultured with bortezomib, but not be seen in those without bortezomib.</p><p><b>CONCLUSION</b>Osteoblasts could be cultured in vitro from MM patients' BM. The proliferation and osteogenic potential of osteoblasts from MM patients were decreased. Bortezomib was a positive regulatory of osteoblasts and MM patient serum was a negative one. They both could affect the proliferation and osteogenic potential of osteoblasts.</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.

TET2 and DLK1 gene expression and their clinical significance in bone marrow CD3+ T cells of patients with myelodysplastic syndrome / 中华内科杂志

Objective To investigate the expression of TET2 and DLK1 mRNA in bone marrow CD3+ T cells of patients with myelodysplastic syndrome (MDS) and their clinical significance and to explore the potential mechanism of abnormal cell-mediated immunity.Methods CD3+ T cells were sorted by magnetic activated cell-sorting system.The expressions of TET2 and DLK1 mRNA in bone marrow CD3+ T cells from 26 MDS patients and 16 healthy controls were detected by fluorescence quantitative PCR.Results The expression of TET2 mRNA in CD3+ T cells was down-regulated in the MDS patients by (0.16 ±0.15) fold compared with the controls ( P ＜ 0.05 ).The expression of TET2 mRNA in CD3+ T cells of MDS patients was positively correlated with serum complement C3 ( r =0.404,P ＜ 0.05 ).The expression of DLK1 mRNA in CD3+ T cells was up-regulated in the MDS patients by (1.61 ±0.88) folds compared with the controls (P＜0.05).Grouped by the chromosomes,the patients with chromosome abnormalities presented significantly higher DLK1 mRNA level than those with normal chromosomes [ ( 1.45 ± 0.44 ) folds,P ＜0.05 ].The expression of DLK1 mRNA in CD3+ T cells of MDS patients was positively correlated with the proportion of bone marrow blasts ( r =0.343,P ＜ 0.05 ).Conclusions The mRNA expression of TET2 in CD3+ T cells of MDS patients was decreased while the mRNA expression of DLK1 was increased,which might decline the immune surveillance function.The findings would be useful for exploring the mechanism of immune tolerance.

The long-term efficacy and safety of rituximab combined with cyclophosphamide in treatment of seven patients with refractory and recurrent autoimmune hemolytic anemia / 中华内科杂志

Objective To assess the efficacy and safety of monoclonal antibody rituximab combined with cyclophosphamide (CTX) in the treatment of refractory and recurrent autoimmune hemolytic anemia.Methods Seven cases with refractory and recurrent autoimmune hemolytic anemia ( including 1 case of Evans syndrome) were recruited during January,2007 to December,2010.Treatment regimens were as follows:rituximab:375 mg/m2,1 time/week,2-6 courses; CTX:1 g,1/10 d,2-7 courses; combined with intravenous immunoglobulin (IVIG) 5 g,1 time/week,given 1 day after rituximab administration.The efficacy and safety of this regimen were assessed during follow-up.Results All the patients showed good responses (7/7).Six patients achieved complete remission (6/7) and one achieved partial remission ( 1/7 ).Average follow-up time for the patients was 27 months.All patients remained in remission during the 12-month follow-up visits.Two patients showed elevated indirect bilirubin and increased reticulocyte counts within 24 months.One patient achieved complete remission after additional rituximab therapy,and another patient remained partial remission after cyclosporine therapy.At the time of 36-month follow-up visit,the patient relapsed and was retreated with 3 courses of rituximab combined with CTX and eventually achieved partial remission.All patients tolerated the treatment well with few mild side effects.Conclusions Rituximab combined with CTX is effective and relatively safe in patients with refractory and recurrent autoimmune hemolytic anemia.Additional treatment to relapse patients about 12-24 months after drug withdrawal continues to be effective.

Circulating myeloid dendritic cells are increased in individuals with severe aplastic anemia.

The objectives of the study were to investigate the number of myeloid dendritic cells (mDC) and plasmacytoid dendritic cells (pDC) present in peripheral blood mononuclear cells (PBMC) from severe aplastic anemia (SAA) patients before and after intensive immunosuppressive therapy (IST) and to assess the expression of co-stimulatory molecules (CD80, CD86, and CD40) expressed by dendritic cells (DC) from SAA patients. The quantities of mDC and pDC and ratios of mDC to pDC in PBMC were measured in 38 SAA patients at active phase, 19 patients at recovery phase, and 17 normal controls. The surface expression of CD80, CD86, and CD40 on DCs and B lymphocytes was analyzed in 16 SAA patients and 15 normal controls. The percentages of mDC and the ratio of mDC:pDC of SAA patients at active phase increased compared to that of healthy controls [0.65% (range 0.10-2.19%) vs. 0.40% (range 0.11-1.54%), 2.64% (range 1.07-4.33%) vs. 1.56% (range 0.89-2.27), respectively (P < 0.05)]. The percentages of mDCs in recovered SAA patients decreased to 0.43% (range 0.06-0.80), and the ratio of mDC:pDC decreased to 1.78% (range 0.49-3.07). The percentages of mDC and pDC in 10 SAA patients were 0.87% (range 0.10-1.85) and 0.35% (range 0.05-0.65) before IST, which decreased to 0.24% (range 0.06-0.52) and 0.14% (range 0.01-0.28) after IST (P < 0.05). The percentages of CD86 expression on DC of SAA patients increased compared to that of healthy controls [29.84% (range 20.28-39.40) vs. 11.97% (range 0.02-24.15), respectively (P < 0.05)]. The number of mDCs increased in SAA patients, which was associated with stage of disease. The increased number of mDCs and the high expression of costimulatory molecules (CD86) on these DCs may contribute to abnormal activation of T lymphocytes in these patients and subsequent immune system-mediated bone marrow failure.

An analysis of efficacy and related factors of itraconazole in the treatment of invasive fungal infection in hematological diseases / 中华内科杂志

Objective To investigate the effects and related factors of itraconazole in the treatment of invasive fungal infection (IFI) in the patients with blood diseases ( BD). Methods A total of 156 BD patients with IFI treated with itraconazole in General Hospital, Tianjin Medical University from 2005 to 2008, were retrospectively analyzed. Results Of these patients, 92 were with underlying malignant BD, and 64 with non-malignant BD; 77 possible IFI, and others proven IFI. A total of 94 (63. 5% ) patients were responded to itraconazole successfully, while 54 (36. 5% ) failed. The underlying malignant BD, post-chemotherapy, neutrophil count less than 0. 5 x 109/L, positive fungus culture, and bacteria infection were related with the response to itraconazole significantly, while patient's age, application of other antibiotics,positive C test, IFI localization, haemoglobin level and platelet counts were not Five patients was changed other anti-IFI therapy because of side effects, including gastrointestinal ill (3 cases with nausea or vomiting) and tachycardia (2 cases). Conclusions Itraconazole was effective and safe in the treatment of IFI in the patients with BD. Underlying malignant BD, agranulocytosis, bacteria infection, and delayed anti-IFI therapy might reduce itraconazole therapeutic effects.

Expression of bone marrow macrophages antigen activation and its clinical significance in pancytopenia patients with positive bone marrow mononuclear cells-Coombs test / 中华内科杂志

Objective To explore the expression of antigen activated of macrophages ( MΦ) of bone marrow and its clinical significance in pancytopenia patients with positive bone marrow mononuclear cells (BMMNC)-Coombs test ( immunorelated pancytopenia, IRP) . Methods Sixty-one IRP patients, 10 severe aplastic anemia (SAA) patients and 13 healthy controls were enrolled in this study. The categories of auto-antibodies(IgG, IgM) on BMMNC(CD_(34)~+/CD_(15)~+/GlycoA~+ hematocytes), the quantity (CD_(68)~+/CD_(45)~+)% and expression of antigen activated ( CD_(69) ) of MΦ ( CD_(68)~+ CD_(69)~+/CD_(68)~+ ) % in bone marrow of all cases and controls were measured by fluorescence activated cell sorting( FACS). Results The quantity and expression ratio of activated antigen of bone marrow ( BM ) MΦ in IRP patients [ ( 0. 57 ± 0. 30 ) % and ( 40. 30 ± 18.49)%] were respectively significantly higher than those in SAA [ (0.46 ± 0. 08)% and ( 32. 44 ± 19.37)%] and healthy controls [ (0. 44 ± 0. 69)% and (29.71 ± 11. 67 )% ] ( both P < 0. 05 ). The quantity presented high-positive correlation with the expression ratio of activated antigen of BM MΦ ( r = 0.89, P<0. 01). Patients with IRP were classified into two subgroups according to the quantity of MΦ: Group A (MΦ≥0. 5% , 34 cases) and Group B ( MΦ <0. 5% , 27 cases). Thirty-two cases (94. 12%) were with auto antibody ( IgG) in Group A, while only 2 (7. 41% ) with auto antibody ( IgG) in Group B. There was significant difference in expression ratio of activated antigen of BM MΦ between Group A (49. 19 ± 16. 63) % and Group B (29. 11 ± 14. 30) % ( P < 0. 05 ) , but no difference was found between Group B and the control group (P >0. 05). Total curative rates at 3 and 6 month (47. 06% and 79. 41% ) of Group A were better than those of Group B (22.22% and 51.85%). Thirty-four IRP patients with autoantibody ( IgG) ( + ) were divided into two subgroups according to the quantity of MΦ: high level group ( >0. 75% , 9 cases) and low level group( <0. 75% , 25 cases) , 24 cases (96% ) in MΦ low level group were found auto-antibody (IgG) on one hemotopoietic cell lineage, 1 on two lineages, while 8 (88. 89% ) in MΦ high level group were detected auto-antibody (IgG) on two cell lineages, and 1 on three cell lineages. Expression ratio of activated antigen (56. 12 ± 15. 11) % was much higher in MΦ high level group than that in MΦ low level group (44. 58 ± 18. 16)% (P < 0. 05 ). The count of red blood cell concentration of hemoglobin and platelet in peripheral blood in MΦ high level group were respectively lower than those in MΦ low level group, while the percentage of Ret, the level of total bilirubin and indirect bilirubin, the ratio of erythroid of sternal bone marrow in MΦ high level group were higher than those in MΦ low level group. Conclusion The expression of activated antigen of BM MΦ was enhanced in IRP especially with autoantibody (IgG) , which might be involved in damage process of hemotopoietic cell.

In vitro induction of allo-T lymphocytes proliferation by myeloid dendritic cells in patients with severe aplastic anemia / 中华内科杂志

Objective To investigate the function of myeloid dendritic cells (mDCs) from severe aplastic anemia ( SAA ) patients in stimulating allogeneic T lymphocytes proliferation in vitro and then explore the immunopathogenesis of SAA. Methods Twenty-five SAA patients ( 15 untreated and 10 recovered after immunosuppressive therapy) and 12 normal controls were enrolled in this study. Their mature mDCs were induced from their bone marrow monocytes with recombined human interleukin-4 ( rhIL-4) , recombined human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombined human tumor necrosis factor (rhTNF) in vitro. Then mDCs were co-cultured with allogeneic lymphocytes (mixture lymphocyte reaction, MLR) at a ratio of 1: 100 or 1: 50. The growth rate of lymphocyte was measured with methyl thiazolyl tetrazolium ( MTT) colorimetry.The concentrations of interleukin( IL) -12 and inlerferon -y (IFNγ) in MLR supernatant were measured with EL1SA. The correlation between the growth rate and the concentration of IL-12 or IFNγ was analyzed. Results When mDCs and lymphocytes were co-cultured at the ratio of 1: 100, the growth rates of lymphocytes stimulated with mDCs from untreated, recovered SAA patients and controls were (219. 8 ±94. 0)% , (159. 1 ±66. 0)% and (160. 1 ±91. 9)% respectively. The concentrations of IL-12 in MLR supernatant were (8. 2 ± 3. 6) ng/L, (6. 5 ± 2. 8) ng/L and (6. 1 ± 2. 6) ng/L and the concentrations of IFNγ were (21. 8 ± 8. 7) ng/L, (25. 5 ± 9. 1) ng/L and (22. 6 ± 7. 8) ng/L respectively. All of them had no statistical differences among the three groups ( P > 0. 05 ). When mDCs and lymphocytes were co-cultured at the ratio of 1: 50, the growth rate of lymphocytes stimulated with mDCs from untreated patients was (322. 1 ± 171. 1)% , which was higher than that of recovered patients [ (180. 9 ±79. 1)% and controls (192. 3 ±91. 9)% ]. The concentrations of IL-12 in MLR supernatant in the three groups were (12.6 ±4.4) ng/L, (9.4 ±3.3) ng/L and (8.5 ±3.7) ng/L, and the concentrations of IFNγ were (32. 3 + 9. 2 ) ng/L, ( 27. 4 ± 6. 5) ng/L and (24. 4 ± 7. 4 ) ng/L Both of the values in untreated cases were higher than those of the recovered cases or controls (P < 0. 05 ) , but there were no statistical difference between the recovered and control groups ( P >0. 05 ). The concentration of IL-12 in MLR supernatant correlated positively with the growth rate of lymphocyte (r=0. 529,P <0. 01) and so did the concentration of IFNγ (r = 0. 381, P < 0. 05). Conclusion The function of mDCs to stimulate T lymphocytes proliferation in SAA was enhanced; it might play an important role in the immunopathogenesis of SAA.

Study of the effect of iron overload on the bone marrow hematopoietic function in immuo-related pancytopenia patients / 中国实用内科杂志

Objective To study the effect of iron overload on the bone marrow hematopoietic function on immuo-related pancytopenia(IRP)patients by hematopoietic progenitor cell(HPC)culture of bone marrow(BM).Methods BM liquid 4～5 mL was taken from 46 IRP patients of General Hospital Tianjin Medical University from July 2009 to February 2010 to detect colony-forming-unit of erythrocyte,blast-forming-unit of erythrocyte and colony-forming-unit of granulocyte-monocyteby HPC culture of BM.And according to the serum ferritin(SF)level,these patients were classified into 2 groups to compare HPC proliferation,blood cell counts,BM proliferation,blood transfusion and treatment effects.Results The mean values of 3 different colonies of IRP patients with high SF level [(43.33?17.74),(1.50?2.2),(11.06?5.83)/105BMMNC] were significantly lower than those of the patients with normal SF level [(77.43?40.64),(9.57?7.99),(21.25?11.41)/105BMMNC](P