Clinical Characteristics, Prognostic Factors and Misdiagnosis of Bone Marrow Necrosis Patients / 中国实验血液学杂志

OBJECTIVE@#To analyze the clinical characteristics, diagnosis and prognostic factors of bone marrow necrosis (BMN) patients, aim to avoid misdiagnosis, missed diagnosis or delayed treatment.@*METHODS@#The clinical data of 51 BMN patients treated in the Affiliated Hospital of Xuzhou Medical University from January 2010 to December 2017 were retrospectively analyzed. The types of primary disease, etiology, clinical manifestations, laboratory tests, radiological findings, treatment outcomes and prognostic factors were summrized, and the reasons for misdiagnosis were analyzed.@*RESULTS@#Among 51 BMN patients, the hematologic tumor was detected out in 32 patients; solid tumors caused- BMN was detected out in 14 patients, benign lesions for 5 patients. The time of interval from the appearance of symptoms to the confirmation of BMN was 7 days to 6 months, with a median of 35 days. Misdiagnosis and missed diagnosis occurred in 25.5% of the BMN patients. Anemia was found in all of the 51 BMN patients, fever accounted for 58.8%, systemic bone pain for 52.9%, bleeding for 29.4%, lymphadenectasis for 37.3%, and hepatosplenomegaly for 19.6%. Leukoerythroblastic anemia accounted for 84.3%, bicytopenia for 51.0%, pancytopenia for 25.5%, and monocytopenia for 23.5%. The serologic test revealed no specific results. The first bone marrow aspiration were 38 patients and multi-site puncture were 7 patients. The diagnostic coincidence rate of bone marrow smear was 88.2%. Among 51 BMN patients, 41 patients received bone marrow biopsy, and the diagnostic coincidence rate of bone marrow biopsy was 75.6%. The abnormal signals were found in multiple vertebral bodies by spinal/pelvic MRI scan in 13 BMN patients; PET-CT scan revealed a diffuse pattern of low FDG uptake in the bone marrow in 16 patients, with a local increase in FDG uptake accompanied by bone marrow involvement. For 46 patients with BMN combined with malignancies, among which 35 patients died (76.1%) and the median survival time was 25 days. Among the 32 patients with hematologic tumors, early death occurred in 12 patients, BMN disappeared in 11 out of 20 patients received active chemotherapy for the primary disease, 9 patients died within 1 week to 3 months. Fourteen patients combined with bone marrow metastatic carcinoma died within 2 weeks to 3 months. Focal necrosis disappeared in 4 out of 5 BMN patients secondary to non-malignant diseases after symptomatic supportive treatment and still alived. Multiple logistic regression was performed to analyze factors affecting the prognosis of BMN patients, the result showed that the prognosis of BMN was closely related to the factors of primary disease (benign and malignant). The reasons for misdiagnosis and missed diagnosis were as follows: hidden onset of the primary disease, nonspecific symptoms, insufficient understanding and alertness of the physicians regarding the primary clinical characteristics and hematological abnormalities, and failure to receive multiple sites bone marrow punctures or bone marrow biopsies.@*CONCLUSION@#BMN usually occurs concomitantly to hematologic tumors and bone marrow metastases from solid tumors. Its prognosis is closely related to the nature and severity of the primary disease and its own severity. In the clinic, BMN should be suspected in patients with severe bone pain, fever, hepatosplenomegaly, hemocytopenia, lymphadenectasis and leukoerythroblastic anemia. Bone marrow puncture at multiple positions and bone marrow biopsy can compensate for each other in the diagnosis of BMN. The combined use of the two methods can improve the diagnostic coincidence rate of BMN, and the positive rate of the etiological diagnosis of BMN.

Expression and Significance of PD-1, TIM-3 and VISTA on T Cell of Acute Myeloid Leukemia Patients / 中国实验血液学杂志

OBJECTIVE@#To study the expression of multiple negative costimulatory molecules on peripheral blood T cells in patients with acute myeloid leukemia (AML) and its affection on prognosis.@*METHODS@#The peripheral blood samples from patients with newly diagnosed AML, complete remission (CR), and no-remission (NR) were collected, the expression levels PD-1、VISTA and TIM-3 in CD4 and CD8 T cells were detected by flow cytometry , and the clinical data of patients were analyzed.@*RESULTS@#The expression levels of PD-1、VISTA and TIM-3 of CD4 and CD8 T cells in the newly diagnosed AML patients were significantly higher than those in control group (P＜0.05). The expression levels of PD-1、TIM-3 and VISTA of CD4 and CD8 T cells in the CR group were significantly lower than those in newly diagnosed and the NR group (P＜0.05). The TIM-3 expression level positively correlated with VISTA expression level of CD4 and CD8 T cells in newly diagnosed AML patients (r=0.85 and 0.73). The VISTA and PD-1 expression level of CD4 T cells in newly diagnosed AML, NR after first induction chemotherapy and high risk patients significantly increased (P＜0.05), the TIM-3 expression level of CD8 T cells in high risk group significantly increased (P＜0.05), and the VISTA expression level of CD8 T cells in CBFβ-MYH11 mutation-positive group significantly decreased (P＜0.05).@*CONCLUSION@#The expression of PD-1、TIM-3 and VISTA in AML peripheral blood T cells may be involved in the immune escape of AML and can be the targets of treatment for acute myeloid leukemia patients.

Advances in the Diagnosis and Therapy of Double-hit Lymphoma--Review / 中国实验血液学杂志

Abstract　　Double-hit lymphoma (DHL) is a high-grade B-cell lymphoma with MYC and BCL-2/BCL-6 rearrangement, which is a invasive disease with a poor prognosis. FISH is the most important diagnostic method. There is no standard protocol for this disease yet. New therapeutic approaches including targeted therapy，checkpoint inhibitors and chimeric antigen receptor T-cell therapy are changing the paradigm of treatment for DHL. This review summarizes new developments in diagnosis and treatment of double-hit lymphoma.

Study on the immune functions of dendritic cells regulated by histone deacetylase inhibitor Belinostat / 中华血液学杂志

Objective: To explore effects of histone deacetylase inhibitor Belinostat on the immunologic function of dendritic cells (DC) and its possible mechanism. Methods: Cultured mouse bone marrow-derived DC from C57BL/6 mouse in vitro. The experiments were divided into 0, 50, 100 nmol/L Belinostat + immature DC (imDC) group, and 0, 50, 100 nmol/L Belinostat mature DC (mDC). The changes of the ultrastructure of DC were observed by transmission electron microscope (TEM). Immunophenotype and CCR7 expression rate were detected by FCM, and the migration rate was observed by chemotaxis assay. The proliferation of lymphocytes stimulated by different DC was detected by mixed lymphocyte culture reaction. The cytokines in the culture supernatant, including TNF-α, IL-12 and IL-10, were examined by ELISA. RQ-PCR was used to examine the relative expression of mRNA in RelB. Results: Successful cultured and identified the qualified imDC and mDC. Belinostat decreased the expression of CCR7 on imDC [(25.82±7.25)% vs (50.44±5.61)% and (18.71±2.00)% vs (50.44±5.61)%], meanwhile increased the rate on mDC [(71.14±1.96)% vs (64.90±1.47)%]. Chemotaxis assay showed that the migration rate of Belinostat+imDC and Belinostat+mDC group were both decreased, but the difference in imDC was not significant. T lymphocyte proliferation rate stimulated by 100 nmol/L Belinostat+imDC group was lower than imDC group in condition irritation cell∶reaction cell=1∶2 [(227.09±13.49)% vs (309.49±53.69)%]. Belinostat significantly suppressed the secretion of cytokines TNF-α, IL-12 and IL-10 (all P<0.01). The relative expression of mRNA in RelB was slightly decreased in Belinostat+imDC and Belinostat+mDC group (all P<0.05). Conclusion: Belinostat could effectly suppress DC maturation and regulate immune tolerance of DC, which may be due to the down-regulation of mRNA level of RelB in DC.

Inhibitory effect of myocilin on fibronectin expression and adhesion in trabecular meshwork cells of glaucoma / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To investigate the effect of myocilin on the expression of fibronectin (FN) and the adhesion function in trabecular meshwork cells of primary open angle glaucoma (POAG) in vitro. ·METHODS: The concentration of recombinant myocilin protein was 0 (control group), 0. 5, 1, 1. 5, 2 and 2.5μ g/mL, respectively. The expression of FN in the cells and the cell culture medium were detected by Western blot and ELISA. The effect of myocilin protein on the adhesion of cultured POAG trabecular meshwork cells was detected by CCK-8 method. ·RESULTS: Western blot showed that the ratio of FN/β-actin was 34.8±0.6,33.4±1.0,28.9±0.8,21.6±0.9,15.9± 1.1 and 11. 9 ± 0. 8; that of 0. 5μ g/mL group was not significantly different compared with that of control group (P=0.092);that of 1.0,1.5,2 and 2.5μ g/mL group were significantly different compared with that of the control group (F=346.131, P<0.05). The concentration of FN in the cell culture medium was 0. 4654 ± 0. 0039, 0. 4596 ± 0.0032, 0.4216± 0.0037, 0.4214 ± 0.0039, 0.4043 ± 0.0039, 0.3806±0.0071μ g/mL by ELISA;difference between that of 0.5μ g/mL group and that of control group was not statistically significant (P=0.120);the difference between of 1,1.5,2,2.5μ g/mL group and the control group were statistically significant (F=176.054, P<0.05). The mean values of the absorbance values of each group were 1.9814 ± 0.1624, 1.8848 ± 0.0267, 1.4895 ± 0.0916, 1.4120 ± 0.1087,1.3392 ± 0.1391, 1.0310 ± 0.0639 through CCK-8 method; that of 0. 5μ g/mL group was not significantly different compared with that of control group(P=0.300);the difference between of 1,1.5,2,2.5μ g/mL group and the control group were statistically significant (F =177.818,P<0.05). · CONCLUSION: Myocilin protein can inhibit the fibronectin expression in POAG trabecular meshwork cells, showing a dose dependent manner. Myocilin protein can reduce the adhesion of POAG trabecular meshwork cells.

Establishment of Mouse Model with Humanized Chronic Myeloid Leukemia / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To establish a BALB/c nude mouse model with the huamanized chronic myeloid leukemia (CML) for the study of human CML.</p><p><b>METHODS</b>The BALB/c nude mice aged 4 weeks pretreated by splenectomy, the cyclophosphamide intraperitoneal injection and sublethal irradiation (SLI) were transplanted intravenously with bone marrow mononuclear cells from CML patients. The SLI-pretreated nude mice were divided into 2 groups: group A, in which the nude mice were injected with 0.3 ml PBS; group B, in which the nude mice were infused intravenously with 4.5×10mononuclear cells from CML patients. Then the changes of body weight and appetite were observed, the hemogram and cell morphology were determined, the expressions of human CD13 and CD45 were detected by flow cytometry, the pathologic analysis of bone, liver and intestine were performed by biopsy, and the BCR/ABL fusion gene was detected by RT-PCR.</p><p><b>RESULTS</b>The mice in group B displayed weakness, auantic, less foodintake and instabiligy of gait as time want on. The average survival time was 46.2±4.2 d (45-57 d). On the third week, the CD13CD45cells accounted for 0.56±0.05% and 2.56±0.36% respectively in group A and B. While on the sixth week, the CD13CD45cells accounted for 0.44±0.07% and 4.97±0.43% in A and B groups respectively, these results showed that cell count in B group was significantly higher than that in A group(P<0.05). Pathological examination showed that the leukemic cells were found in bone marrow of group B. The BCR/ABL fusion gene could be detected in bone marrow.</p><p><b>CONCLUSION</b>BALB/c nude mouse model with huamanized chronic myeloid leukemia(CML) model has been established by pretreating mice with SLI. The survival time of mice in this model has been long, and the cost to establish the model is low.</p>

Effects of immature dendritic cells to express CCR7 on graft-versus-host disease in allogeneic bone marrow transplant mouse model / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of immature dendritic cells (imDC) expressing chemokine receptor-7 (CCR7) on acute graft-versus-host disease (aGVHD) in allogeneic bone marrow transposed (allo-BMT) mouse model.</p><p><b>METHODS</b>We constructed the lentiviral vectors carrying mouse CCR7 gene and infect imDC effectively in vitro. GVHD model was established with C57BL/6(H-2b) donor mice and BALB/c (H-2d) recipient mice. After irradiation, recipients were injected with donor bone marrow and spleen cells along with CCR7-modified dendritic cells. Mice were randomized into irradiation, transplant control, pXZ9-imDC (empty vector control) and CCR7-imDC groups. Survival, GVHD score, histopathological analysis and plasma levels of inflammatory cytokines were observed.</p><p><b>RESULTS</b>The mean survival in irradiation, transplantation, pXZ9-imDC and CCR7-imDC groups were (8.20±1.48)d, (12.20±2.78)d, (20.70±6.01)d and (27.5±7.55)d respectively. The survival in CCR7- imDC group was significantly improved compared with other groups (P<0.05). GVHD scores in transplantation, pXZ9-imDC and CCR7-imDC groups were (6.90±1.66), (5.60±0.97) and (4.10±1.79) respectively. CCR7-imDC group had significantly lower GVHD score and minor tissue damages shown by histopathological analysis than the other groups. Plasma IFN-γ level increased and reached the peak at +10 day in transplant group, while it gradually decreased in pXZ9-imDC and CCR7-imDC groups, and then reached the nadir at +20 day post-allo-BMT, with the lowest level in CCR7-imDC group (P<0.01). Plasma IL-4 decreased in transplant group, while it gradually increased in pXZ9-imDC and CCR7-imDC groups and reached the highest level at + 10 day in CCR7- imDC group (P<0.01). The 95%-100% of H-2b positive cells in recipient mice on + 30 day post-allo-BMT demonstrated the complete donor- type implantation.</p><p><b>CONCLUSION</b>Genetically modified immature DC by CCR7 gene could alleviate damages by GVHD and prolong survival of recipient mice after allo-BMT.</p>

Effects of immature dendritic cells genetically modified to express sTNFR I on graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL) in allogeneic bone marrow transplantation mice / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effect of immature dendritic cells (inDC) genetically modified to express sTNFR I on acute graft-versus-host disease (aGVHD) and the graft-versus-leukemia (GVL) effect ofter allogeneic bone marrow transplantation (allo-BMT) in leukemic mice and its mechanism.</p><p><b>METHODS</b>An EL4 leukemia allo-BMT model was established with the BALB/c (H-2d) donor mice (DM)and C57BL/6 (H-2b) recipient mice (RM). The RM received DM bone marrow (BM) cells at a 1:1 ratio with spleen cells intravenously via tail vein at 4 h after TBI. Fifty DM were separated randomly into five groups: (1) Group A: total body irradiation (TBI) group, (2) Group B: lymphoma cell leukemia group, (3) Group C: allo-BMT group, (4) Group D: pXZ9-DC group, (5) Group E: sTNFR I-DC group. Acute GVHD scores, incidence of leukemic cell infiltration, histopathological analysis, survival rate, and survival rate of the recipients were estimated after allo-BMT. Enzyme-linked immunosorbent assay (ELISA) method was used to detect cytokines (INF-gamma and IL-4 ) production. Flow cytometry (FCM) analysis was used to detect allogeneic chimerism.</p><p><b>RESULTS</b>(1) The mice in group A and group B all died of the BM failure and lymphoma cell leukemia, respectively. The mice in group C developed typical clinical signs of a GVHD after BMT with an average survival time(AST) of (11.50 +/- 3.50) d. The signs of aGVHD were less evident in the group D and E, and their AST (21.70 +/- 5.80 and 25.80 +/- 5.20 days, respectively) were all longer than that in group C (P < 0.05). AST of group E was the longest (P < 0.05). The mice in group B all died of leukemia within 18 days after engraftment of EL4 cells. There was was no significant difference in groups C, D and E in the incidence of leukemia (P > 0.05). (2) Serum IFN-gamma level reached peak value. At + 12 d, then decreased gradually in group C, D, and E, and then reached the nadir at +18 d post-BMT, with the lowest in group E (P < 0.05), and the level was significantly lower in group D than in group C (P < 0.05). After BMT, serum IL-4 level slightly decreased in group C, but gradually elevated in group D and E and reached their peak at +12 d, and even more significantly increased in group E (P < 0.05). There was no statistical significance in the pair wise comparison among three group (P < 0.05). (3) The average proportion of H-2d positive cells in RM was 95%-100% on day 30 post-BMT, with complete donor-type implantation.</p><p><b>CONCLUSION</b>Immature DC can induce immuno tolerance. Immature DC genetically modified to express sTNFR I has been shown to prevent acute GVHD in lethally irradiated mice reconstituted with allogeneic bone marrow grafts while maintaining the GVL response.</p>

Prophylaxis of graft-versus-host disease in mice by chemical modification of graft and OX40-OX40L costimulatory pathway / 中华血液学杂志

<p><b>OBJECTIVE</b>To explore the prophylaxis effect of pretreatment of allograft with methoxypolyethylene glycol-succinimidyl-propionic acid ester (mPEG-SPA) and anti-OX40L monoclonal antibody (McAb) on acute graft-versus-host disease (aGVHD) after allogeneic bone marrow transplantation (allo-BMT) in mice.</p><p><b>METHODS</b>Responder splenocytes from C57BL/6 donor mice (H-2(b)) were co-cultured with stimulator splenocytes from BALB/c recipient mice (H-2(d)) for 7 days in the presence or absence of anti-OX40L McAb followed by mPEG-SPA chemical modification. Donor bone marrow cells plus the mixed culture of T-cells were then transplanted into lethally irradiated BALB/c mice. The BALB/c recipient mice were divided into four groups: group A (allo-BMT control group), group B(mPEG-SPA modification group), group C (anti-OX40L McAb pretreated group) and group D (mPEG-SPA and anti-OX40L McAb dual-treated group). Survival time and survival rate of the recipients were observed after allo-BMT. GVHD was assessed by clinical signs and histological changes of skin, liver and small intestines. Enzyme-linked immunosorbent assay (ELISA) was used to detect cytokines (IL-4, IL-10 and INF-gamma) production. Flow cytometry (FCM) analysis was used to detect allogeneic chimerism.</p><p><b>RESULTS</b>(1) The mice in group A developed typical clinical signs of aGVHD and all mice died within 17 days after BMT with an average survival time (AST) of (12.1 +/- 5.5) days. The signs of aGVHD were less evident in mice of groups B, C and D, and their AST (36.2 +/- 24.9, 32.0 +/- 24.8 and 44.3 +/- 23.2 days, respectively) were all longer than that in group A (P < 0.05). AST of group D being the longest (P < 0.05). The survival rates at day 60 post-BMT in groups B, C and D were 50%, 41.7% and 66.7%, respectively. (2) Serum IFN-gamma level was increased after BMT in group A, and peaked in day 10 to day 15 post-BMT, while the level was decreased in groups B, C and D, reached the nadir on the day 10 post-BMT, with the lowest in group D (P < 0.01). After BMT, IL-4 and IL-10 levels were slightly decreased in group A, their levels were elevated in groups B and C (P < 0.05) and even more significantly increased in group D (P < 0.01). IL-4 and IL-10 levels peaked between day 10 and 15 post-BMT. (3) The average proportion of H-2(b) positive cells in recipient mice was 95% - 100% on day 60 post-BMT, with complete donor-type implantation.</p><p><b>CONCLUSION</b>Combination of mPEG-SPA and anti-OX40L McAb can block T-cell activated antigens and co-stimulatory pathway, regulate the T cells differentiation and induce the immune shift of Th0 cells toward Th2 cells. The immune tolerance induced by this method can significantly relieve aGVHD after allo-BMT.</p>

Specific anti-leukemic cell effect mediated by dendritic cells pulsed with chronic myelogenous leukemia lysate antigen in vitro / 中国实验血液学杂志

To investigate the specific antileukemia effect of dendritic cells (DC) pulsed with chronic myelogenous leukemic lysate antigen (CLA), dendritic cells from patients with chronic myelogenous leukemia (CML) were pulsed by CLA, and then cocultured with cytokine-induced killer (CIK) cells from CML patients (CIK + CLA-DC group). The cytotoxic activity in vitro was measured by using a lactate dehydrogenase release assay, and compared with CIK + DC, CIK and CIK + CLA groups. The results showed that under an effector-target ratio of 25:1, the cytotoxic activity of CIK + CLA-DC, CIK + DC, CIK and CIK + CLA groups against autologous CML cells was (68.8 +/- 14.2)%, (52.5 +/- 9.4)%, (20.6 +/- 7.5)% and (24.2 +/- 8.7)%, respectively. CIK + CLA-DC group displayed a strongest cytotoxic activity. When K562 and Raji cells acted as target cells and CIK as effectors, the cytotoxic activity against autologous CML cells in CIK + CLA-DC group (68.8 +/- 14.2)% was much higher than that against K562 cells (14.6 +/- 6.2)% and Raji cells (12.7 +/- 10.2)%, respectively. In conclusion, coculture of CIK cells with DC led to a significant increase in cytotoxic activity. The cytotoxicity could be further increased by DC pulse with CML cell lysate antigen, and cytotoxicity mediated by CML lysate antigen possess stronger specificity.