Targeting BCMA in multiple myeloma using chimeric antigen receptor-engineered T cells / 中华血液学杂志

Objective@#To construct the BCMA-CAR using the B-cell maturation antigen (BCMA) specific ligand APRIL as antigen binding region and to validate the effect of BCMA-CAR modified T cells (BCMA-CAR-T) on myeloma cells.@*Methods@#The BCMA-CAR was constructed using the BCMA specific ligand APRIL as antigen binding domain and 4-1BB as the costimulatory domain. The specific cytotoxicity against BCMA+ myeloma cell lines and primary multiple myeloma (MM) cells in vitro were evaluated. In addition, BCMA+ myeloma xenograft mouse model was established to assess the anti-tumor effect of BCMA-CAR-T cell therapy in vivo.@*Results@#BCMA-CAR-T cells could specifically kill BCMA+ myeloma cell lines (For BCMA-CAR-T cells, BCMA+ cells are almost undetectable in the E∶T ratio of 1∶4) and MM patients’ bone marrow mononuclear cells (the proportion of residual cells in BCMA-CAR-T and vector-T groups was 16.0% vs 66.85%, P=0.003) with significant degranulation (CAR-T and vector-T cells cocultured with MM1.S, H929 and U266 had degranulation levels of 33.30% vs 5.62%, 16.97% vs 2.95% and 25.87% vs 2.97%, respectively, P<0.001) and cytokines release (P<0.01) in vitro. In a human BCMA+ myeloma xenograft mouse model, BCMA-CAR-T cells could significantly prolong the survival of mice (The median survival time of mice treated with BCMA-CAR-T and vector-T cells was 87.5 days and 67.5 days, respectively, P<0.001) .@*Conclusion@#The ligand-based BCMA-CAR-T cells could be a promising strategy for BCMA+ multiple myeloma treatment.

Spectrum of somatic mutations and their prognostic significance in adult patients with B cell acute lymphoblastic leukemia / 中华血液学杂志

Objective@#To investigate the spectrum of gene mutations in adult patients with B-acute lymphoblastic leukemia (B-ALL), and to analyze the influences of different gene mutations on prognosis.@*Methods@#DNA samples from 113 adult B-ALL patients who administered from June 2009 to September 2015 were collected. Target-specific next generation sequencing (NGS) approach was used to analyze the mutations of 112 genes (focused on the specific mutational hotspots) and all putative mutations were compared against multiple databases to calculate the frequency spectrum. The impact of gene mutation on the patients’ overall survival (OS) and recurrence free survival (RFS) was analyzed by the putative mutations through Kaplan-Meier, and Cox regression methods.@*Results@#Of the 113 patients, 103 (92.0%) harbored at least one mutation and 29 (25.6%) harbored more than 3 genes mutation. The five most frequently mutated genes in B-ALL are SF1, FAT1, MPL, PTPN11 and NRAS. Gene mutations are different between Ph+ B-ALL and Ph- B-ALL patients. Ph- B-ALL patients with JAK-STAT signal pathway related gene mutation, such as JAK1/JAK2 mutation showed a poor prognosis compared to the patients without mutation (OS: P=0.011, 0.001; RFS: P=0.014,<0.001). Patients with PTPN11 mutation showed better survival than those without mutation, but the difference was not statistically significant (P value > 0.05). Besides, in Ph+ B-ALL patients whose epigenetic modifications related signaling pathway genes were affected, they had a worse prognosis (OS: P=0.038; RFS: P=0.047).@*Conclusion@#Gene mutations are common in adult ALL patients, a variety of signaling pathways are involved. The frequency and spectrum are varied in different types of B-ALL. JAK family gene mutation usually indicates poor prognosis. The co-occurrence of somatic mutations in adult B-ALL patients indicate the genetic complex and instability of adult B-ALL patients.

Screening of adult Ph-like acute lymphoblastic leukemia by multiplex real-time quantitative PCR / 中华血液学杂志

Objective@#To investigate the feasibility of multiplex real-time RT-PCR with fluorescent probes in early screening of Ph-like acute lymphoblastic leukemia (ALL) and analyze the clinical feature and prognos.@*Method@#A total of 118 adult B-ALL patients diagnosed between October 2010 and March 2016 were enrolled in this study. Multiplex RT-PCR was used to detect the Ph-like ALL related fusion gene and CRLF2 expression in 58 BCR-ABL and MLL rearrangement negative patients. The clinical features, treatment response and prognosis were analyzed in Ph-like fusion gene positive and/or CRLF2 over-expression patients.@*Result@#Among 58 patients, 9 patients (9/58, 15.5%) showed Ph-like ALL related fusion genes positive and 10 patients (10/58, 17.2%) showed CRLF2 over-expression. There were statistical differences in age, WBC count, immunophenotypes, cytogenetics and risk stratification among Ph-like fusion gene positive or CRLF2 over-expression patients, Ph+ patients, MLL+ patients and B-other patients. The 2-year overall survival rates were 65%, 47%, 64% and 74% respectively among these four groups (P=0.043) . The 2-year relapse free survival rates were 51%, 39%, 62% and 70% respectively among these four groups (P=0.010) .@*Conclusion@#Routine screening of Ph-like ALL by multiplex RTPCR is feasible.

Interaction between osteoblast of bone marrow microenvironment with leukemia cells / 白血病·淋巴瘤

Objective To elucidate the interaction between osteoblast of bone marrow microenvironment and leukemia cells,and to investigate the role of osteoblast in the leukemia cells survival and apoptosis and the influence of leukemia cells on the osteoblast.Methods Leukemia cells from AML1-ETO9a-Rac1 mouse leukemia model and osteoblast cells were used.The ratio of GFP+ leukemia cells that co-cultured with or without osteoblast was detected by FACS.In addition,the apoptosis level of leukemia cells was detected by flow cytometry by PI and Annexin Ⅴ labeling.Activation level of PARP was determined by Western-blot.Real-time PCR (RT-PCR) was utilized to detect the mRNA level of TPO,N-cadherin,OPN and Ang1 in osteoblast which was separated from leukemic mice.Results The ratio of GFP+ cells in AE9a-Rac1 leukemia cells co-cultured with osteoblast cell was significantly higher than that of AE9a-Rac1 leukemia cells cultured alone.The apoptotic level of AE9a-Rac 1 leukemia cells cultured alone was significant higher than that of AE9a-Rac 1 leukemia cells in co-culture system.Western blot showed that activated level of PARP in AE9a-Rac1 leukemia cells co-cultured with osteoblast was lower than that cultured alone.RT-PCR result showed that TPO and N-cadherin mRNA levels in primary osteoblast separated from leukemic mice were higher than that from normal mice.Ang1 and OPN mRNA levels of osteoblast from leukemia mice were lower.Conclusion Osteoblast cell can support the survival and inhibit the apoptosis of leukemia cells.Leukemia cells can influence the functions of osteoblast by microenvironment associated cytokines production.

Expression of Rho GTPase family member RhoA and CDC42 protein in patients with acute leukemia / 白血病·淋巴瘤

Objective To detect the proteins levels of RhoA and CDC42 in bone marrow mononucleated cells (BMMC) of patients with primary acute leukemia,and further determine the role of abnormal interactions between hematopoietic progenitor and bone marrow microenvironment on abnormal behaviors of leukemia cells. Methods BMMC samples were separated from 54 primary acute leukemia patients and 22 normal donors and the cell lysis samples were prepared. RhoA and CDC42 proteins were determined by Western blotting. Independent pair T test was conducted to evaluate whether the differences in RhoA and CDC42 expression were statistically significant between leukemia patients and normal donors. Spearman was applied in analyzing the correlation between expression of RhoA and CDC42 proteins and clinical characters of patients. Results RhoA and CDC42 proteins level of primary acute leukemia patients was significantly higher than that of normal samples. Especially, patients with M2,M3 and M5 subtypes exhibited significant higher RhoA proteins levels and M3 subtype exhibited significant higher CDC42 protein levels. Conclusion RhoA and CDC42 protein levels of primary acute leukemia patients are significantly higher than that of normal donors. This result suggests that RhoA and CDC42 associated efficient migration of leukemia cells could be implicated in abnormal interaction of leukemic cell with bone marrow microenvironment.

Determination of ETO interaction domain within nuclear receptor co-repressor / 中华血液学杂志

<p><b>OBJECTIVE</b>To determine the ETO-interaction domain of nuclear receptor co-repressor (N-CoR) for abolishing the biological function of AML1-ETO.</p><p><b>METHODS</b>Ten different regions of N-CoR (N-CoRYs) were generated by means of polymerase chain reaction (PCR), and cloned into yeast expression plasmid pGADGL to construct pGADGL/N-CoRYs. The yeast two-hybrid technique and X-gal staining were used to determine the binding between the 10 different regions of N-CoR and ETO.</p><p><b>RESULTS</b>It was shown that the co-existence of 988-1,126 and 1,551-1,803 amino acid residues of N-CoRY was the ETO-interaction domains required for the binding with ETO.</p><p><b>CONCLUSION</b>Two domains of N-CoR that interact with two zinc fingers of ETO, and keep stable binding between the two proteins were identified.</p>

Effects of glucocorticoids on the expression of fractalkine and CX3CR1 in kidneys of BXSB mice / 中国病理生理杂志

AIM:To observe the expression of chemokine fractalkine,and its receptor,CX3CR1,in kidneys of lupus-prone BXSB mice,and their changes after treatment with prednisone. The role of fractalkine and CX3CR1 in the pathogenesis of lupus nephritis was also discussed. METHODS:Twelve 12-week-old male BXSB mice were randomly divided into two groups,the prednisone treatment group (BXSB-prednisone group,n=6) and the experimental control group (BXSB group,n=6). Six male C57BL/6J mice at the same weeks of age served as a normal control group (C57BL/6J group). Both the C57BL/6J and the BXSB group of mice received a daily intragastric administration of 0.5 mL normal saline. The BXSB-prednisone group of mice was given a daily intragastric administration of prednisone (0.18 mg/20 g BW) dissolved in 0.5 mL normal saline. All treatments lasted for 10 weeks. The mRNA and protein expressions of fractalkine and CX3CR1 in kidneys of mice were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting analysis respectively. The changes of laboratory index and the kidney histopathology of mice were also investigated. RESULTS:The mRNA and protein expressions of fractalkine and CX3CR1 in kidneys of BXSB mice were significantly higher than those in C57BL/6J mice. The expressions of fractalkine and CX3CR1 in BXSB-prednisone group of mice were much lower than those in BXSB group of mice,accompanied by the lower serum IgG,IgM and anti-dsDNA antibody levels as well as blood urea nitrogen,serum creatinine and urine protein. The glomerular immune complex deposition and the kidney histopathology were also significantly improved in BXSB-prednisone group of mice. CONCLUSION:These results indicate that fractalkine and CX3CR1 participate in the pathogenesis of lupus nephritis in BXSB mice,and the effect of glucocorticoids treatment may be attributed,in part,to its ability to inhibit the expression of fractalkine in kidney.

HIPK2 gene expression and its clinical significance in acute leukemia / 中国病理生理杂志

AIM: To investigate homeodomain-interacting protein kinase 2(HIPK2) gene expression level and its clinical significance in acute leukemia(AL) patients.METHODS: HIPK2 mRNA was determined by semi-quantitative reverse transcriptase polymerase chain reaction(semi-quantitive RT-PCR) in patients with acute leukemia and healthy donors.The relative transcription level was compared between the study group and the control group.RESULTS: ① The relative expression level of HIPK2 mRNA in AL patients was 0.364?0.286,significantly lower than that in control(1.160?0.272,P0.05).CONCLUSION: AL has significantly lower HIPK2 mRNA expression as compared to normal control,which may be associated with leukemogenesis and/or disease progression of AL.

Expression of fractalkine and CX3CR1 in renal tissues of patients with WHO class IV lupus nephritis / 中国病理生理杂志

AIM: To observe the expression of fractalkine, and its receptor, CX3CR1, in renal tissues of patients with diffuse proliferative lupus glomerulonephritis (WHO class IV), minimal glomerular abnormalities, and normal kidney. Meanwhile, the correlation among the expression of fractalkine, CX3CR1 and CD68-positive macrophages was investigated, and the role of fractalkine and CX3CR1 in the pathogenesis of lupus nephritis was discussed. METHODS: The expressions of fractalkine, CX3CR1 and CD68 were detected immunohistochemically in kidney tissue sections obtained from twenty-one patients with WHO class IV lupus nephritis, eighteen cases with minimal glomerular abnormalities, and eight normal kidneys which were no abnormality under light microscope. RESULTS: (1) Fractalkine was generally indistinguishable in tissue sections from normal kidney and minimal glomerular abnormalities. CX3CR1-positive cells and CD68-positive macrophages were sparsely detected in the glomeruli and in the cortical interstitium. (2) There were considerable CX3CR1-positive cells and macrophages in both the glomeruli and the interstitium in sections from class IV lupus nephritis. The number of CX3CR1-positive cells significantly correlated with the number of macrophages in the glomeruli and in the interstitium respectively (r=0.956, P

Relation between myocardial hypertrophy and the circulating insulin-like growth factor-1 in patients with essential hypertension / 中国病理生理杂志

60 mmHg), indicating that the increase in pulse pressure participates in the course of myocardial hypertrophy. ⑤There was no significant correlation between pulse pressure and serum IGF-1 concentra tion, suggesting that different mechanisms are involved in the development of my ocardial hypertrophy between pulse pressure and IGF-1.