A Study of Immune Functionality of Newly Diagnosed Severe Aplastic Anemia Patients with Virus Infection.

BACKGROUND: Previous research showed that virus infection is correlated with the occurrence, development, and prognosis of AA. This study was designed to explore the influence of virus infection on the immune functionality and immunosuppressive therapy (IST) efficiency of newly diagnosed SAA patients. METHODS: Fifty-six newly diagnosed SAA patients combined with virus infection treated in the Hematology Department of Tianjin Medical University General Hospital from October 2004 to July 2014 were studied. Various immune parameters were tested and compared for SAA patients with and without virus infection. RESULTS: When compared with SAA patients without corresponding virus infection, SAA patients with CMV-IgM, PVB19-IgM, and EBV infection had increased CD8+ T cell percentage, decreased CD4+/CD8+ T cell ratios, and increased CD8+HLA-DR+/CD8+ percentage. The absolute value of CD8+ T cell of CMV-IgM group had increased as well. The CMV-IgM and PVB19-IgM groups showed decreased CD4+ T cell percentage, and decreased CD4+HLA-DR+/CD8+HLA-DR+ ratio. The PVB19-IgM group exhibited decreased CD4+HLA-DR+/CD4+ percentage, increased Th1 percentage and increased pDC percentage. Patients with EB virus infection showed lower NK cell percentage. Three years after IST, the treatment is significantly less effective for the SAA patients combined with virus infection than those without. CONCLUSIONS: CMV, PVB19, and EBV infection worsen the immune functionality abnormality of newly diagnosed SAA patients and reduce the IST efficiency.

Clinical features and treatment outcome of elderly multiple myeloma patients with impaired renal function.

BACKGROUND: Renal impairment (RI) is a most common complication of multiple myeloma (MM), which is associated with an increased risk of early death and worse survival. METHODS: We retrospectively analyzed clinical features and outcomes of 77 MM patients over 70 years old and compared the differences between with and without RI groups. RESULTS: The percentage of elder MM patients with RI was 61%. Hemoglobin level was a protective factor (OR = 0.954, P = 0.033), while creatinine and hypertension were hazards (OR = 1.288, P < 0.001 and OR = 30.12, P = 0.008). And the percentages of patients with mild-to-moderate RI and moderate-to-severe RI were 40.4% and 59.6%. Complete remission (CR) rate was higher in patients treated with bortezomib (33.3%) than those with non-bortezomib treatment (3.33%) (P = 0.007). Meanwhile, CRrenal was higher in patients with bortezomib (58.3%) than non-bortezomib treatment (22.2%) (P = 0.025). The median OS of the patients with RI treated with bortezomib was longer than those with non-bortezomib regimens (15.0 vs 6.0 months, P = 0.001). The same result was observed in the patients with moderate-to-severe RI (13.0 vs 6.0 months, P = 0.007). The median OS of the patients with RI receiving the bortezomib regimens (15 months) was longer than those with non-bortezomib regimens (6.0 months) (P = 0.001). CONCLUSION: Hemoglobin is a protective factor in elder patients with RI, while creatinine and hypertension were hazards. The median OS of elderly patients with RI was worse, and bortezomib can improve the CR rate in these patients.

A cohort study of immune and hematopoietic functionality changes in severe aplastic anemia patients treated with immunosuppressive therapy.

To investigate if variations in immune and hematopoietic parameters correlated with immunosuppressive therapy (IST) in severe aplastic anemia (SAA) patients.A total of 115 SAA patients who received IST were included. Their immune and hematopoietic functionality changes had been evaluated at 0, 0.5, 1, 2, and 3-year(s) IST.For SAA patients with complete remission (CR), the CD4/CD8T cell ratio continued to increase after a year of IST. The T helper (Th)1/Th2 ratio continued to decrease after 6 months of IST, as did the activated CD8 T cell percentage. The myeloid dendritic cell (mDC)/plasmacytoid dendritic cell (pDC) ratio after 3 years of IST was significantly lower compared to that of untreated patients. The mDC/pDC and Th1/Th2 ratios exhibited positive correlation. The activated CD8 T cell percentage and the number of peripheral blood neutrophils showed inverse correlation. For SAA patients with partial remission (PR), the CD4T cell percentage increased at 1-year post-IST, but the later changes were not statistically significant. The other immune indexes of patients in partial remission group and nonremission (NR) group showed no obvious recovery. For all SAA patients, the percentage of T regulatory cells in CD4 lymphocyte was higher in post-IST group compared to the pretreatment group.For SAA patients responded well to IST, increase in peripheral neutrophils and improvement in bone marrow myeloid cells were first observed followed reduction in the activated CD8 T cell percentage, Th1/Th2 ratio, CD4/CD8T ratio, along with mDC/pDC ratio, all of which negatively correlated with the hematopoietic parameters. This demonstrates that IST prompts improvements of hematopoietic functionalities of the SAA patients by regulating their immune functionalities.

De-escalation empirical antibiotic therapy improved survival for patients with severe aplastic anemia treated with antithymocyte globulin.

We aimed to investigate the efficacy and safety of de-escalation empirical therapy for controlling infection in patients with severe aplastic anaemia (SAA) treated with antithymocyte globulin (ATG). Eighty-seven ATG-treated SAA patients who had microbiological culture-confirmed infections from 2006 to 2015 in our center were retrospectively analyzed. The efficacy of de-escalation and non-de-escalation therapy was compared. Among all 87 patients, 63 patients were treated with de-escalation therapy and 24 patients with non-de-escalation therapy. More patients showed response to anti-infection treatment in de-escalation group than in non-de-escalation group both on day 7 (60.32% vs. 25.00%, P = 0.003) and on day 30 (79.37% vs. 58.33%, P = 0.047) since the initial antimicrobial therapy. On day 30, more patients had increased absolute neutrophil count in de-escalation group compared with non-de-escalation group (76.19% vs. 45.83%, P = 0.007), and de-escalation group had lower morality rate (17.46% vs. 37.50%, P = 0.047) and better survival outcome (P = 0.023) on day 90. Twenty-three patients in de-escalation group and 5 patients in non-escalation group received granulocyte transfusions. Granulocyte transfusions helped to control infections in both de-escalation group (P = 0.027) and non-de-escalation group (P = 0.042) on day 7, but did not improve survival on day 90. We concluded that de-escalation antibiotics improved survival in SAA patients after ATG treatment. Early administration of broad-spectrum antibiotics pending microbiological cultures combined with a commitment to change to narrow-spectrum antibiotics should be recommended for controlling infections in SAA patients treated with ATG. Granulocyte transfusions might be an adjunctive therapy in controlling infections.

CYR61/CCN1 stimulates proliferation and differentiation of osteoblasts in vitro and contributes to bone remodeling in vivo in myeloma bone disease.

Cysteine-rich 61 (CYR61/CCN1), a secreted protein in bone marrow (BM) microenvironment, has diverse effects on many cellular activities such as growth and differentiation. However, the effect of CCN1 on osteoblasts (OBs) in myeloma bone disease remains unclear. In our study, the level of CCN1 in multiple myeloma (MM) patients was detected by ELISA and RT-PCR. The proliferation and differentiation of OBs from MM patients were observed after stimulated by CCN1 in vitro. The myeloma cells transduced with CYR61 gene (RPMI­8226/CYR61) were injected in a mouse model to evaluate the efficacy of CCN1 in vivo and compare with zoledronic acid. The results showed that CYR61/CCN1 levels in BM supernatant and OBs both elevated significantly in all newly diagnosed MM patients, especially in patients without bone disease (P=0.001 and P<0.001). After 30 ng/l CCN1 stimulation for 24 h, the quantity and mineralization of OBs increased significantly in vitro (P=0.046 and 0.048). The transcription factors of Wnt pathway, runt-related transcription factor 2 (Runx2) and ß-catenin were upregulated in OBs after CCN1 stimulation (P=0.012 and 0.011). After injection of RPMI­8226 cells, bone lesions were observed obviously by microCT and histochemistry at 7 weeks. Radiographic analysis of the bones showed decreased resorption in CCN1 overexpression group and zoledronic acid group, while severe resorption in negative control. Furthermore, trabecular bone volume in CCN1 overexpression group (1.7539±0.16949) was significantly higher than zoledronic acid group (1.2839±0.077) (P=0.012). In conclusion, CCN1 can stimulate the proliferation and differentiation of OBs in vitro and contribute to bone remodeling in vivo in MBD.

Comparison of Reduced-Intensity Idarubicin and Daunorubicin Plus Cytarabine as Induction Chemotherapy for Elderly Patients with Newly Diagnosed Acute Myeloid Leukemia.

BACKGROUND AND OBJECTIVES: The therapy in elderly patients with acute myeloid leukemia (AML) is a big challenge because of poor risk factors and inferior tolerance to intensive chemotherapy. This study aims to compare the efficacy between reduced-intensity idarubicin plus cytarabine and daunorubicin plus cytarabine (IA regimen and DA regimen, respectively) in elderly patients with newly diagnosed AML. METHODS: We retrospectively investigated 74 patients with newly diagnosed non-M3 AML aged >60 years, where 33 patients received IA regimen, 30 patients received DA regimen, while 11 patients received supportive treatment. We observed the complete remission (CR) rates, overall survival (OS) and side effects in different arms. RESULTS: The CR rate in IA arm (70.4 %, 19/27) was significantly higher than that in DA arm (40 %, 10/25) in de novo AML (p = 0.028), and further significantly higher when white blood cell (WBC) count >10 × 109/L (p = 0.042) and ECOG (Eastern Cooperative Oncology Group) score <2 (p = 0.021). The overall survival of the entire population was poor with a median survival of 10 months, 1- and 2-year survival rates were 40.5 % (30/74) and 9.5 % (7/74). The median survival of the patients with chemotherapy was 12 months, which was significantly longer than patients treated supportively (4 months) (p < 0.001). There were no differences of median survival and duration of CR between two arms. Early mortality decreased in the past 5 years in both groups. Meanwhile, low-dose idarubicin was well tolerated in elderly patients. CONCLUSIONS: Reduced-intensity chemotherapy offered an improvement in survival, and the reduced-intensity IA regimen could improve CR rate in elderly patients with de novo AML.

A monocentric retrospective study comparing pulse cyclophosphamide therapy versus low dose rituximab in the treatment of refractory autoimmune hemolytic anemia in adults.

This retrospective study aims at confirming the efficacy and safety of low dose rituximab and pulse cyclophosphamide in the treatment of refractory AIHA in adults and making comparison of the two. Forty-nine adult patients with refractory AIHA have been enrolled. Results showed low dose rituximab combined with steroid therapy (group B) got more CR (78.9 %, 15/19) compared to that in intermittent intravenous cyclophosphamide combined with steroid therapy (group A) (42.1 %, 8/19) (P = 0.04) at 6 months after treatment. The hemoglobin level in group B was higher than group A at the time point of 1 month (P = 0.02) after treatments. The RFS in group A was 87.9 % at 6 months and 82.7 % at 12 months, which were no significant difference with group B (91.1 % at 6 months and 86.0 % at 12 months) (P = 0.81). Both the two therapies were well tolerated with pulmonary infections as the most common side effects. In conclusion, low dose rituximab combined with steroid therapy presents to be a better choice in the treatment of refractory AIHA in adults comparing with pulse cyclophosphamide therapy.

[GDF11 level in patients with myelodysplastic syndrome and its clinical significance].

OBJECTIVE: To detect the expression level of growth differentiation factor 11 (GDF11) in patients with myelodysplastic syndrome (MDS), and to evaluate the relationship between GDF11 level and erythropoiesis functions. METHODS: A total of 44 MDS patients (18 low-risk group patients and 26 high-risk group patients) in Department of Hematology in Tianjin Medical University General Hospital and 10 normal controls were selected from September 2014 to June 2015. The concentration of GDF11 in peripheral blood was detected using enzyme-linked immunosorbent assay (ELISA). GDF11 mRNA expression in bone marrow mononuclear cells (BMMNC) was detected using RT-PCR method. The percentage of erythroid cells (CD235a) in bone marrow was detected by flow cytometry. The correlation between these indexes and erythropoiesis functions (including red blood cell count (RBC), hemoglobin level (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), reticulocyte percentage (Ret%), and proportion of nucleated eryhrocyte in bone marrow) were evaluated. RESULTS: (1) The GDF11 level in peripheral blood was significantly higher in high-risk group ((128.67±47.62) µg/L) than in low-risk group ((65.96±36.55) µg/L, P<0.01) and in normal controls ((29.76±10.10) µg/L, P<0.01), also significantly higher in low-risk group than in normal controls (P<0.05). The concentration of GDF11 in severe/moderate anemic MDS patients ((80.97±9.94) µg/L) was higher than that in normal controls/ mild anemic MDS patients((66.82±19.52) µg/L), but with no statistically significant (P>0.05). (2) The percentages of CD235a(+) cells in high-risk and low-risk groups were 38.49%±5.42% and 42.64%±7.36%, respectively, showing no statistically significant difference (P>0.05). (3) In high-risk group, the GDF11 level in peripheral blood was negatively correlated with Hb, RBC and Hct in peripheral blood (r=-0.437, -0.430, -0.306, all P<0.05), and positively correlated with nucleated eryhrocyte, Ret%, MCV and CD235a(+) cells in bone marrow (r=0.465, 0.392, 0.505, 0.387, all P<0.05), but not correlated with MCH and MCHC (both P>0.05). In low-risk group, the GDF11 level in peripheral blood was positively correlated with CD235a(+) cells in bone marrow (r=0.429, P<0.05), and not correlated with Hb, RBC, Ret%, MCHC, MCV, MCH, Hct and nucleated eryhrocyte (all P>0.05). (4) The mRNA expression of GDF11 in MDS patients (39.82±14.55) was higher than that in the controls (1.84±0.64, P<0.01). CONCLUSIONS: GDF11 level in peripheral blood is higher in patients with MDS and increases with the disease risk. The more severe the anemia, the higher the GDF11 level. It may be closely correlated with erythropoiesis indicators in MDS.

[Clinical analysis on malignant clonal hematopoiesis in severe aplastic anemia patients with immunosuppressive therapy].

OBJECTIVE: To investigate the clinical characteristics and risk factors of monosomy 7 malignant clonal evolution in patients with severe aplastic anemia (SAA) treated with combined immunosuppressive therapy (IST). METHODS: The clinical data of SAA patients treated with IST who had monosomy 7 malignant clonal evolution from October 2004 to January 2012 were analyzed respectively. RESULTS: Six patients (4.2%) had monosomy 7 clonal evolutions. The median time to monosomy 7 was 36 (12-75) months after IST. All 6 patients were diagnosed myelodysplastic syndromes (MDS). Among them, 3 patients transformed to acute myeloid leukemia following MDS. The time was 24, 45 and 51 months after IST. The median following time was 42 (17-84) months. Four patients died during the following time. The median time from MDS to death was 9 (5-17) months. Among them, three patients died with infection, one died with cerebral hemorrhage. Six patients had the clinical characteristics that they had no response to IST after 6 months, high monocyte percentage in one month after IST combined with recombinant human granulocyte colony stimulating factor (rHu-GCSF) and agranulocytosis in 3 months after IST. CONCLUSION: Poor myeloid response to IST suggests malignant clonal hematopoiesis and poor prognosis in SAA patients.

[CD22 signal abnormalities in the pathogenesis of immune related pancytopenia].

OBJECTIVE: To investigate the expression of CD22 and its downstream signal molecule spleen tyrosine kinase (SYK) and their phosphorylation of B lymphocytes in patients with immune related pancytopenia(IRP), and to explore the role of CD22 in pathogenesis of IRP. METHODS: The expression of CD22, SYK and their phosphorylation, along with the expression of IgG and IgM, which obtained from B lymphocytes in peripheral blood of 46 patients with IRP(22 new diagnosed and 24 remitted patients returned to normal after treatment), 22 healthy controls and 12 chronic lymphocytic leukemia(CLL) patients from February to December 2014 were analyzed by flow cytometry. And the mRNA expression of CD22 in peripheral blood mononuclear cell was determined by real-time quantitative PCR. RESULTS: The ratios of CD22+ cells and phosphorylated CD22(pCD22)+ cells of B lymphocytes in new diagnosed group (60. 03% ± 20. 94% 71. 32% ± 11. 16%) were significantly higher than those in remission group (46. 92% ± 20. 04%, 55. 82% ± 14. 42%), normal control group (46. 86% ± 17. 78%, 53. 28% ± 14. 76%) and CLL group (39. 74% ± 18. 96%, 59. 07% ± 17.09%) (all P <0.05). The ratios of phosphorylated SYK( pSYK) + cells in the four groups had the same trend (all P <0. 05). The ratio of pCD22+ cells/pSYK+ cells in new diagnosed group was significantly lower than that in normal control group and CLL group (27. 39 (5. 06 - 102. 70) vs 55. 95 (15. 25 - 298. 53), 56. 92(5. 60 - 228. 96), both P <0. 05), and pCD22+ cells positively correlated to pSYK+ cells ( r = 0. 341, P < 0. 05). The expression of IgG in new diagnosed group and remission group was significantly higher than that in normal control group, and the expression of IgM in new diagnosed group was significantly higher than that in normal control group and CLL group (all P <0. 05). The expression levels of CD22 mRNA in new diagnosed group was significantly higher than that in remission group, normal control group and CLL group (all P <0. 05). CONCLUSIONS: The BCR signal pathway of B lymphocyte in IRP patients is enhanced, and the quantity and function of CD22 are increased, while which are still insufficient to inhibit B cell proliferation, and these may have some relationships with the pathogenesis of IRP. [Key words] Pancytopenia; Antigens, CD22; Immune related pancytopenia; Spleen tyrosine kinase; Phosphorylation

[Study on C5b-9 deposited on the membrane of platelets and its dysfunction in patients with paroxysmal nocturnal hemoglobinuria].

OBJECTIVE: 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). METHODS: 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. RESULTS: 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）%ï¼½ 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). CONCLUSION: 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.

[Clinical analysis of 70 cases of polycythemia vera].

OBJECTIVE: To analyze the clinical characteristics and laboratory data, treatment and prognosis of polycythemia vera (PV) and provide evidence for screening of high-risk population and looking for measures to reduce complecations. METHODS: A retrospective study was performed among 70 patients with PV from May 2005 to May 2014, 43 males and 27 females, aged (56.6±13.1) to collect the data about characteristics, laboratory data, myelogram chromosome, karyotypes, BCR/ABL and JAK2 V617F genes, as well as lactate dehydrogenase (LDH) and so on. RESULTS: At diagnosis, there were 42 cases (60.00%) had symptoms, 25 cases (35.71%) had thrombosis and embolism. Hemorrhage occurred in 3 cases (4.29%), splenomegaly in 48 cases (68.57%), hepatomegaly in 7 cases (10.00%). The average hemoglobin at diagnosis was 195.17 g/L, the white blood cells count was 10.12×10(9)/L, the platelet count was 295×10(9)//L. The chromosome karyotypes were all normal. The positive percentage of JAK2 V617F mutation was 87.69% (57/65). The disease outcomes were myelofibrosis for 3 paitents, death from ineffective treatment after transforming to myelofibrosis and then biphenotype acute leukemia for 1 patient, and death from cardiorespiratory failure for 2 patients. The level of erythropoietin in JAK2 V617F mutated group were significantly lower than those in wild-type JAK2 V617F group (P<0.05). The level of hemoglobin and platelet counts in JAK2 V617F mutated group were significantly higher than those in wild-type JAK2 V617F group (both P<0.05). CONCLUSION: PV is one of meyloproliferation neoplasm, characterized by abnormally increasing blood cells, thrombosis and transforming to other myeloproliferative neoplasms.

Recombinant human thrombopoietin treatment promotes hematopoiesis recovery in patients with severe aplastic anemia receiving immunosuppressive therapy.

OBJECTIVE: To assess the effectiveness of recombinant human thrombopoietin (rhTPO) in severe aplastic anemia (SAA) patients receiving immunosuppressive therapy (IST). METHODS: Eighty-eight SAA patients receiving IST from January 2007 to December 2012 were included in this retrospective analysis. Of these, 40 subjects received rhTPO treatment (15000 U, subcutaneously, three times a week). rhTPO treatment was discontinued when the platelet count returned to normal range. Hematologic response, bone marrow megakaryocyte recovery, and time to transfusion independence were compared. RESULTS: Hematologic response was achieved in 42.5%, 62.5%, and 67.5% of patients receiving rhTPO and 22.9%, 41.6%, and 47.9% of patients not receiving rhTPO at 3, 6, and 9 months after treatment, respectively (P = 0.0665, P = 0.0579, and P = 0.0847, resp.). Subjects receiving rhTPO presented an elevated number of megakaryocytes at 3, 6, and 9 months when compared with those without treatment (P = 0.025, P = 0.021, and P = 0.011, resp.). The time to platelet and red blood cell transfusion independence was shorter in patients who received rhTPO than in those without rhTPO treatment. Overall survival rate presented no differences between the two groups. CONCLUSION: rhTPO could improve hematologic response and promote bone marrow recovery in SAA patients receiving IST.

[Clinical significance of serum bone metabolic markers in diagnosis and monitoring of myeloma bone disease].

OBJECTIVE: To explore the significance of serum bone metabolic markers in the diagnosis and monitoring of multiple myeloma bone disease(MBD). METHODS: Thirty-six newly diagnosed multiple myeloma (MM) patients who were treated in Department of Hematology, Tianjin Medical University General Hospital from January 2013 to December 2014 were collected. Bone morbidity was graded into two stages according to the radiographic evaluation of the skeleton: stage A (n=12) included patients with no lytic lesions or with osteoporosis alone; stage B (n=24) included patients with osteolytic lesions and/or a pathological fracture. All the patients achieved partial or complete remission after treated with bortezomib + dexamethasone + zoledronic acid regimen. A total of 25 aged- and gender-matched healthy individuals were enrolled in this study as controls. The levels of serum tartrate-resistant acid phosphatase isoform 5b (TRACP-5b), carboxy-terminal cross-linking telopeptide of type I collagen (CTX), osteocalcin (OCN), and procollagen I amino-terminal propeptide (PINP) were investigated by ELISA and electrochemiluminescence immunoassay (ECLIA). The differences of these bone metabolic markers before and after treatment, and at different stages of bone disease were observed. RESULTS: The value of TRACP-5b in the newly diagnosed MM was significantly higher than that in the healthy controls and after treatment(median 4.16 vs 2.63 U/L, P=0.014; 4.16 vs 2.61 U/L, P=0.037). Serum level of CTX in the newly diagnosed MM patients was significantly decreased after treatment (median: 0.26 vs 1.05 µg/L, P=0.003). The ratio of CTX/OCN and CTX/PINP decreased after treatment, but there were no significant differences (both P>0.05). The pretreatment level of serum TRACP-5b in stage B patients was higher than that of the healthy controls (median: 4.20 vs 2.63 U/L, P=0.015). The levels of serum CTX in stage A and stage B patients were both higher than that of the healthy controls (median: 1.16 vs 0.48 µg/L, P=0.002; 0.88 vs 0.48 µg/L, P=0.040). The levels of serum OCN and PINP were higher in stage A patients compared with stage B patients, but there were no significant differences (both P>0.05). The ratio of CTX/OCN and CTX/PINP of stage A and stage B patients all increased compared with those of the healthy controls, but there were no significant differences (all P>0.05). CONCLUSIONS: Bone damage of MM patients is improved after effective treatment, but bone imbalance still exists, indicating that the treatment of MBD is a long process. Abnormal serum levels of TRACP-5b and CTX are found before positive X-ray findings in MBD, suggesting that these biochemical markers could be used as indices for early diagnosis of MBD.

[Autophagy level of bone marrow mononuclear cells in patients with myelodysplastic syndromes].

OBJECTIVE: To investigate the change of autophagy level of bone marrow mononuclear cells（BMMNCs）in patients with myelodysplastic syndromes（MDS）. METHODS: 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. RESULTS: 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）%ï¼½than 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）. CONCLUSION: 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.

Osteoblast inhibition by chemokine cytokine ligand3 in myeloma-induced bone disease.

BACKGROUND: Multiple myeloma is a hematologic malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow. A common manifestation of the disease is myeloma bone disease (MBD), which is caused by increased osteoclastic bone resorption and decreased bone formation. The chemokine cytokine ligand 3 (CCL3) is a pro-inflammatory protein and chemokine that stimulates osteoclasts in MBD. However, little is known about the effect of CCL3 on osteoblasts (OB). METHODS: The OBs are induced from patients with MBD and healthy donors, cultured in vitro, and identified by histochemistry. The effects of CCL3 and CCL3 antibody on the OBs in vitro are observed. The CCL3 receptor (CCR1), osteocalcin (OCN), runt-related transcription factor 2 (Runx2), and osterix (Osx) are detected using flow cytometry, enzyme-linked immunosorbent assay, and real-time PCR. RESULTS: Proliferation and osteogenic potential of the OB in patients with MBD are suppressed. Moreover, the CCR1 expression is significantly higher in patients with MBD than in normal controls. The OCN level, quantity of calcium nodules, and Runx2 and Osx levels decrease after CCL3 stimulation, which indicates that CCL3 inhibits OB function. Furthermore, CCL3 antibody partially restores OB activity through the upregulation of the OCN, Runx2, and Osx. CONCLUSIONS: CCL3 contributes to the OB/OC imbalance by inhibiting OB differentiation and function in MBD.

[Memory B (CD5⁺ CD19⁺ CD27⁺) lymphocyte in patients with immune-related pancytopenia].

OBJECTIVE: To detect memory B lymphocyte (Bm) in peripheral blood (PB) of immune-related pancytopenia (IRP). METHODS: 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. RESULTS: (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. CONCLUSION: 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.

[Abnormal WT1 gene expression in paroxysmal nocturnal hemoglobinuria].

OBJECTIVE: To explore the pathogenesis of abnormal WT1 expression in paroxysmal nocturnal hemoglobinuria (PNH). METHODS: 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. RESULTS: 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). CONCLUSION: 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.

[Telomere length of peripheral lymphocytes in patients with immuno-related pancytopenia].

OBJECTIVE: 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). METHODS: 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). RESULTS: 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). CONCLUSION: 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.

[Effect of CCL3 on osteoblast in myeloma bone disease].

OBJECTIVE: To culture osteoblast in vitro and evaluate CCL3 receptor CCR1 expression in patients with multiple myeloma (MM). METHODS: Bone marrow osteoblasts from MM patients were cultured in vitro with dexamethasone, ß-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. RESULTS: 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. CONCLUSION: Bone marrow osteoblasts could be cultured in vitro from MM Patients. CCL3 may contribute to the development of myeloma bone disease.