Donor-derived CD19 CAR-T Cells versus Chemotherapy Plus Donor Lymphocyte Infusion for Treatment of Recurrent CD19-positive B-ALL After Allogeneic Hematopoietic Stem Cell Transplantation.

OBJECTIVE: This study aimed to compare the efficacy of anti-CD19 chimeric antigen receptor T cells (CAR-T cells) versus chemotherapy plus donor lymphocyte infusion (chemo-DLI) for treating relapsed CD19-positive B-cell acute lymphoblastic leukemia (B-ALL) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS: Clinical data of 43 patients with B-ALL who relapsed after allo-HSCT were retrospectively analyzed. Twenty-two patients were treated with CAR-T cells (CAR-T group), and 21 with chemotherapy plus DLI (chemo-DLI group). The complete remission (CR) and minimal residual disease (MRD)-negative CR rates, leukemia-free survival (LFS) rate, overall survival (OS) rate, and incidence of acute graft-versus-host disease (aGVHD), cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were compared between the two groups. RESULTS: The CR and MRD-negative CR rates in the CAR-T group (77.3% and 61.5%) were significantly higher than those in the chemo-DLI group (38.1% and 23.8%) (P=0.008 and P=0.003). The 1- and 2-year LFS rates in the CAR-T group were superior to those in the chemo-DLI group: 54.5% and 50.0% vs. 9.5% and 4.8% (P=0.0001 and P=0.00004). The 1- and 2-year OS rates in the CAR-T versus chemo-DLI group were 59.1% and 54.5% vs. 19% and 9.5% (P=0.011 and P=0.003). Six patients (28.6%) with grade 2-4 aGVHD were identified in the chemo-DLI group. Two patients (9.1%) in the CAR-T group developed grade 1-2 aGVHD. Nineteen patients (86.4%) developed CRS in the CAR-T group, comprising grade 1-2 CRS in 13 patients (59.1%) and grade 3 CRS in 6 patients (27.3%). Two patients (9.1%) developed grade 1-2 ICANS. CONCLUSION: Donor-derived anti-CD19 CAR-T-cell therapy may be better, safer, and more effective than chemo-DLI for B-ALL patients who relapse after allo-HSCT.

miR424-5p functions as an anti-oncogene in cervical cancer cell growth by targeting KDM5B via the Notch signaling pathway.

AIMS: Aberrant expression of miRNAs exert the critical roles in carcinogenesis, including cervical cancer. Recent study corroborated the down-regulation of miR424-5p in uterine cervix adenocarcinoma. This research aimed to investigate the function and underlying mechanisms of miR424-5p in cervical cancer cell growth. MAIN METHODS: Tissues samples were collected from patients with cervical cancer and healthy control. The expression levels of miR424-5p were determined by qRT-PCR. After transfection with miR424-5p mimics or inhibitor, cervical cancer cell proliferation and apoptosis were evaluated by WST-1 and flow cytometry assay, respectively. The underlying mechanism involved in aforementioned processes was also explored. KEY FINDINGS: Expression of miR424-5p was notably decreased in cervical cancer tissues and cells. Overexpression of miR424-5p restrained cell proliferation and promoted cell apoptosis, but with little function in miR424-5p inhibitor-treated groups. Furthermore, KDM5B was identified as a direct target of miR424-5p as the evidence that miR-424-5p inhibited KDM5B expression and luciferase activity of KDM5B 3'-UTR. Here, KDM5B elevation majorly reversed miR424-5p-triggered inhibition in cell proliferation and increase in cell apoptosis. Moreover, silencing KDM5B expression also restrained cell growth. Additionally, miR424-5p overexpression inhibited the expression of Notch1 and Notch2, which was obviously rescued after KDM5B up-regulation. Simultaneously, blocking KDM5B also attenuated the activation of Notch pathway. Importantly, treatment with Notch agonist Jagged1 antagonized miR424-5p-mediated suppression on cell growth. SIGNIFICANCE: This research suggests that miR424-5p may act as a novel anti-oncogene in cervical cancer by blocking cell growth through targeting KDM5B-Notch pathway. Accordingly, our study will support a promising therapeutic strategy against cervical carcinoma.

Comparison of temporary abdominal aortic occlusion with internal iliac artery occlusion for patients with placenta accreta - a non-randomised prospective study.

BACKGROUND: To compare the efficacy of temporary abdominal aortic occlusion with internal iliac artery occlusion for the management of placenta accreta. PATIENTS AND METHODS: 105 patients with placenta accreta were selected for treatment with temporary abdominal aortic occlusion (n = 57, group A) or bilateral iliac artery occlusion (n = 48, group B). Temporary abdominal aortic and internal iliac artery balloon occlusions were performed during caesarean sections. Data regarding the clinical success, blood loss, blood transfusion, balloon insertion time, fluoroscopy time, balloon occlusion time, foetal radiation dose, and complications were collected. RESULTS: Temporary abdominal aortic occlusion and bilateral internal iliac artery occlusion were technically successful in all patients. The amount of blood loss (P < 0.001), amount of blood transfusion (P < 0.001), balloon insertion time (P < 0.001), foetal radiation dose (P < 0.001) and fluoroscopy time (P < 0.01) in group A were significantly lower than those of patients in group B. No marked differences were found between these 2 groups with respect to age, mean postoperative hospital stay, balloon occlusion time, and Apgar score (p > 0.05). CONCLUSIONS: Temporary abdominal aortic balloon occlusion resulted in better clinical outcomes with less blood loss, blood transfusion, balloon insertion time, fluoroscopy time and foetal radiation dose than those in bilateral internal iliac balloon occlusion.
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Pseudomonas aeruginosa injection enhanced antitumor cytotoxicity of cytokine-induced killer cells derived from cord blood.

Cord blood (CB) is becoming an extensive source of cytokine-induced killer cells. It had been used in several clinical settings and proven to be efficacious and safe. Therefore, we investigated the possibility of combining CIK cells derived from cord blood (CB-CIK) and Pseudomonas aeruginosa injection (PA-MSHA) in order to enhance the cytotoxicity of CB-CIK cells against tumors. Compared with the CB-CIK cells, the PA-MSHA-treated CB-CIK cells demonstrated with increased proliferation rates, higher expression of activated cell surface marker CD28 and lower expression of inhibited cell surface markers PD-1 and CTLA-4. Furthermore, PA-MSHA-treated CB-CIK cells exhibited more effectively for secreting pro-inflammatory cytokine such as IFN-γ and expressing high levels of TLR2, TLR4 and TLR6. The expression of CD107a was higher in the CD3(+)CD56(+) subset of PA-MSHA-treated CB-CIK cells. Our results indicate that the PA-MSHA-treated CB-CIK cells exhibited a more potent in cytotoxic activity against tumor cells. Thus, PA-MSHA enhanced the antitumor ability of CB-CIK cells.

The TLR3, PI3K, survivin, FasL, and Fas genes as major risk factors of occurrence and development of cervical cancer disease.

To investigate the role of TLR3/PI3K signals in the occurrence and development of cervical cancer disease, TLR3-siRNA was used to block key signaling pathways involved in cervical cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Results show that tumor U14 cell growth, migration and invasion in TLR3-siRNA treatment group were significantly decreased. Through LY294002 suppressing targeted gene, the LY294002 treatment specifically and significantly knocked down the expressions of tumor TLR3 and PI3K proteins in cervical cancer mice. Furthermore, expressions of tumor Survivin and FasL proteins were markedly suppressed, whereas expressions of Fas protein were upregulated in LY294002 treatment group mice. LY294002 treatment suppressed tumor growth and increased the thymus and spleen indeces and survival days of cervical cancer mice. This study demonstrates that TLR3-siRNA and LY294002 treatments can markedly suppress cervical cancer cell invasion and tumor growth and increase survival life by silencing targeted genes.

[Effect of DNA methyltransferase 1 gene silencing on proliferation and apoptosis of HeLa cells].

OBJECTIVE: To observe the effect of DNA methyltransferase 1 (DNMT1) gene silencing by RNA interfering technology on the proliferation and apoptosis of HeLa cells. METHODS: Recombinant plasmid pshRNA-DNMT1-A, B and C were respectively transfected into HeLa cells by lipofectamine 2000, while cells transfected plasmid vector pSilencer3. 1-H1 and cells untreated as control groups. RT-PCR was adopted to select the recombinant plasmid which showed the most optimal inhibition effect. RT-PCR and western blotting was used to detected the mRNA and protein expression of DNMT1 in HeLa cells transfected for 24, 48 and 72 hours. Cell counting kit-8 (CCK-8) assay was used to investigate the proliferation of the HeLa cells after transfection, while apoptosis was detected by flowcytometry (FCM) method. RESULTS: Three DNMT1-targeted short hairpin RNA (shRNA) A,B and C were successfully inserted into the plasmid vector pshRNA, and the coding sequences of the obtained shRNA were consistent with the designed fragments. The results indicated that both recombinant plasmid pshRNA-DNMT1-A and B could effectively knock down the expression of DNMT1 gene in human cervical cancer cells, of which pshRNA-DNMT1-B was the better choice. While no effect of pshRNA-DNMT1-C was seen. RT-PCR results showed that the relative mRNA expression of DNMT1 gene in HeLa cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were 0.406 +/- 0.057, 0.191 +/- 0.036 and 0. 104 +/- 0.015, which were significantly lower than that in HeLa cells transfected by empty vector and non-transfected cells (0.520 +/- 0.020, 0.537 +/- 0.041, respectively, P < 0. 05). The western blotting analysis manifested that the relative expression of DNMT1 protein of HeLa cells transfected by pshRNA-DNMT1 for 24, 48 and 72 hours were 0.197 +/- 0.024, 0.075 +/- 0.015, 0.040 +/- 0. 013, which were significantly lower than that in transfected cells by empty vector and non-transfected cells (0.273 +/- 0.010, 0.283 +/- 0.016, respectively, P < 0.05). The CCK-8 results showed that the cell survival rates of HeLa cells transfected by pshRNA-DNMT1 for 24, 48, 72, 96 and 120 hours were 70.8%, 64.8%, 51.6%, 45.3% and 38.0%, there were statistically different compared with cells transfected by empty vector and non-transfected cells at different time-points (P < 0.01). The results of FCM indicated that the apoptosis rate of HeLa cells transfected with pshRNA-DNMT1 for 24, 48 and 72 hours were (17.7 +/- 1.3)%, (35.3 +/- 1.3)%, (47.6 +/- 1.6)%, which were significantly higher than empty vector transfected cells and non-transfected cells [(4.9 +/- 0.5)%, (5.1 +/- 0.7)%, respectively, P < 0.05]. CONCLUSIONS: DNMT1 can be successfully silenced by RNA interfering in cervical HeLa cells. Downregulation of DNMT1 can inhibit cervical cancer cells proliferation and induce cell apoptosis.

[Promoter methylation of DAPK gene in cervical carcinoma].

BACKGROUND & OBJECTIVE: Aberrant DNA methylation plays important roles during multistage carcinogenesis in various human organs. This study was to explore the relationship between the promoter methylation and inactivation of DAPK gene in cervical cancer. METHODS: The promoter methylation of DAPK was investigated with methylation-specific polymerase chain reaction (MSP) in 52 specimens of cervical cancer, 60 specimens of cervical intraepithelial neoplasia (CIN) and 20 specimens of normal cervical squamous epithelial tissues. Its correlation to clinicopathologic features of cervical cancer was analyzed. The protein expression of DAPK was detected by immunohistochemistry. RESULTS: The methylation rate of DAPK gene promoter was significantly higher in cervical cancer tissues than in CIN (65.4% vs. 18.3%, P<0.05); while no methylation of DAPK gene was found in normal cervical tissues. The methylation rate of DAPK gene was significantly higher in cervical squamous cell carcinomas than in adenocarcinomas (80.0% vs. 16.7%, P<0.001). Promoter methylation of DAPK was negatively correlated to its protein expression (r=-0.849, P<0.001). CONCLUSION: The promoter methylation may lead to inactivation of DAPK gene, and may be related with tumorigenesis of cervical cancer.

[Expression and significance of P16INK4A and PTEN in high-risk human papillomavirus-related cervical cancer].

BACKGROUND & OBJECTIVE: High-risk human papillomavirus (HR-HPV) is the most important etiologic factor for cervical cancer. Recent studies have revealed that abnormal expression of tumor suppressor gene P16INK4A is closely associated with HR-HPV infection during carcinogenesis of cervical epithelium. Tumor suppressor gene PTEN is also involved in cervical tumorigenesis. This study was to investigate the correlations of HR-HPV infection to P16INK4A and PTEN expression and its clinical significance in the carcinogenesis of cervical epithelium. METHODS: The expression of P16INK4A and PTEN in 30 specimens of normal cervical tissues, 11 specimens of cancer in situ (CIS), and 24 specimens of invasive cervical carcinoma (ICC) was detected by SP immunohistochemistry; 13 types of HR-HPV DNA in these cases were detected by Hybrid Capture 2 (HC-2) assay. RESULTS: The positive rates of HR-HPV and P16INK4A were significantly higher in ICC and CIS than in normal tissues (91.7% and 90.9% vs. 30.0%, P<0.001; 87.5% and 81.8% vs. 6.7%, P<0.001). Both HR-HPV DNA and P16INK4A overexpression (moderate or strong expression) were observed simultaneously in 21 specimens of ICC and 9 specimens of CIS; they were simultaneously negative in 20 specimens of normal cervical tissues and 1 specimen of CIS and 2 specimens of ICC. Overexpression of P16INK4A was positively correlated to HR-HPV infection in cervical cancer (rs = 0.690, P<0.001). PTEN was moderately or strongly expressed in 26 specimens of normal cervical tissues. The positive rate of PTEN was significantly lower in ICC and CIS than in normal cervical tissues (37.5% and 36.4% vs. 83.3%, P<0.01). No obvious relationship between PTEN and HR-HPV was found (rs = -0.174, P = 0.167). CONCLUSIONS: P16INK4A is overexpressed in HR-HPV-infected cervical cancer, but its tumor suppressor action might be inhibited. In contrast, the functional down-regulation of PTEN contributes to cervical tumorigenesis through HR-HPV-independent mechanism.

[Vector construction and silencing effect of Edg4 gene targeted small interfering RNA in ovarian cancer cell line].

OBJECTIVE: To construct the recombinant eukaryotic expression vector pRNAT-U6.1-siEdg4 which carries small interfering RNA (siRNA) of Edg4 and observe the silencing effect of Edg4 gene targeted siRNA in ovarian cancer cell line SKOV3. METHODS: The Edg4 gene-targeted hairpin siRNA sequence was designed according to the Edg4 sequence in Genbank, and the two complementary oligo nucleotide strands were synthesized and annealed and inserted into the pRNAT-U6.1 plasmid to build a recombinant Edg4 siRNA eukaryotic expression vector, which was sequenced and identified to contain the correct Edg4 siRNA sequence. The human ovarian carcinoma cell lines SKOV3 were transfected with the vector using lipofectamine method. The efficiency of transfecting cells was observed with fluorescent microscope and the mRNA expression level of Edg4 gene was detected by real time quantitative PCR. The LPA levels in cell supernatants were detected using a biochemical method. And the apoptosis of SKOV3 cells induced by the vector was evaluated by flow cytometry. RESULTS: The recombinant eukaryotic expression vector was confirmed to contain correct Edg4 siRNA sequence by PCR and sequencing. After transfection large amounts of green fluorescence were seen in plasma and nuclei of SKOV3 cells and the positive cell rates were 64%. The expression level of Edg4 mRNA in transfected SKOV3 cell line was significantly decreased (0.05 +/- 0.01vs 0.29 +/- 0.04, P < 0.05). The decrease in LPA level in the cell supernatants was revealed [(3.0 +/- 1.0) vs (7.5 +/- 2.2)micromol/L, P < 0.05]. The apoptosis rate of transfected SKOV3 was increased obviously (53.38% vs 0.51%, P < 0.05). CONCLUSIONS: We have successfully constructed the recombinant eukaryotic expression vector containing Edg4 gene targeted siRNA (pRNAT-U6.1-siEdg4). The vector could effectively transfect SKOV3 cell line, and obviously suppress the Edg4 mRNA expression and induce cell apoptosis in ovarian cancer cell line SKOV3.