[Efficacy and Safety of Venetoclax-Based Induction Therapy in Acute Myeloid Leukemia].

AbstractObjective: To investigate the efficacy and safety of venetoclax-based induction chemotherapy in newly diagnosed (ND) patients ineligible for intensive therapy and patients with relapsed/refractory (R/R) acute myeloid leukemia (AML). METHODS: The clinical data of 51 newly diagnosed patients ineligible for intensive therapy and patients with R/R AML treated in the Department of Hematology of Xijing Hospital from February 1, 2021 to April 30, 2022 were retrospectively analyzed. The incidence of complete remission (CR)/CR with incomplete hematological recovery (CRi), objective remission rate (ORR), minimal residual disease (MRD) status, advense events (AE), overall survival (OS) and progression-free survival (PFS) were analyzed. RESULTS: Among 51 patients, 32 patients were newly diagnosed patients unfit for intensive therapy, with a median age of 60 (29-88) years, and 19 patients were R/R patients, with a median age of 49 (22-92) years. The median cycles of VEN-based treatment in the two groups were both 2. The CR/CRi rates in the ND-AML and R/R-AML group after one course of induction treatment were 65.6% and 36.9%, respectively, and the ORR were 81.3% and 42.1%, respectively. The cumulative CR/CRi rates after 1-3 courses of VEN-based treatment were 71.9% and 47.4%, respectively. The MRD negativity rates of patients achieving CR/CRi were 69.6% and 33.3%, respectively. In the ND-AML and R/R-AML group, the median PFS were 8(5-11) and 3(1-5) months, and the median OS were 13 (6-20) and 5 (3-7) months, respectively. The median OS of patients achieving CR/CRi in both groups was significantly better than that of patients not achieving CR/CRi (13 months vs 4 months; OS not reached vs 4 months). During the first induction cycle, the incidence of grade 3 or higher granulocytopenia, anemia and thrombocytopenia was 96%, 90.2% and 84.3%, respectively. 30 patients (58.8%) had granulocytopenia with fever. The most common non-hematological AE was infection (12/51, 23.5%), followed by gastrointestinal symptoms (6/51, 11.8%). CONCLUSION: The VEN-based strategy has good treatment response and tolerance in newly diagnosed patients unfit for intensive therapy and R/R AML. The most common AEs are hematological toxicities and infection.

[Clinical Characteristics and Prognosis of Systemic Anaplastic Large Cell Lymphoma].

OBJECTIVE: To evaluate the clinical characteristics, treatment and prognosis of systemic anaplastic large cell lymphoma(sALCL). METHODS: The clinical data of 90 cases with sALCL treated in the Department of Hematology of the Affiliated Xijing Hospital of Air Force Medical University from November 2018 to October 2021 were retrospectively analyzed. The clinical features, treatment and prognosis were summarized and the prognostic factors were investigated. RESULTS: There were 58 males and 32 females, with a median age of 32 (12-73) years old. 69 (76.7%) patients had Ann Arbor stage Ⅲ-Ⅳ disease and half of the patients had extranodal infiltration. The median age was 27(12-72) years of the 60 ALK+ patients while 53(15-73) years of the 30 ALK- patients, and it was significantly different in the age of onset between the two group(P<0.01). 88 patients received first line chemotherapy, and 50(568%) cases achieved complete remission(CR). IPI score≥3 was an independent risk factor for CR. The median progressive free survival(PFS) and overall survival(OS) of the patients were not reached. Multivariate analysis showed that no achievement of CR after first-line therapy was a significant prognostic factor influencing PFS and OS. CONCLUSION: sALCL mainly occurs in males and most patients were in advanced stage. Half of the patients had extranodal involvement. The CR rate after first-line chemotherapy was 568%, and IPI score≥3 was a significant prognostic factor for CR. No achievement of CR after first-line therapy is poorly prognostic for PFS and OS.

[Clinicopathological Characteristics and Prognosis Analysis of the Patients with Plasmablastic Lymphoma].

OBJECTIVE: To evaluate the clinicopathological characteristics, treatment and prognosis of the patients with plasmablastic lymphoma(PBL). METHODS: The clinical and pathological data of 21 patients with PBL diagnosed and treated in our center between January 2009 and September 2017 were retrospectively analyzed. The clinical and pathological features, treatment and therapentic outcome were summarized and the high risk factors affecting the prognosis of patients were investigated. RESULTS: The 21 PBL patients included 12 males and 9 females, and their median age was 52 years old. The human immunodeficiency virus (HIV) was negative in all patients. The primary involved sites of 16 patients were extranodal, and the patients staged in III-IV accounted for 81%; 18 patients receved first-line chemotherapy with standard CHOP(E) (cyclophosphamide +epirubicin +vincristine +prednisone±etoposide). After treatment, only 1 patient achieved complete response (CR), and 8 patients achieved partial response (PR). The median overall survival time was 6.3 months. Multivariate analysis showed the America Eastern Cooperative Oncology Group (ECOG) physical score and bone marrow infiltration were significant prognostic factors (P<0.01). CONCLUSION: Plasmablastic lymphoma frequently occurrs in the middle-old aged persons with all HIV negative. Primary extranodal lesions are frequent. Most patients were in advanced stage with poor treatment response. ECOG score≥2 and bone marrow infiltration are independent prognostic factors related with worse prognosis.

A phase 2 study of methotrexate, etoposide, dexamethasone, and pegaspargase chemotherapy for newly diagnosed, relapsed, or refractory extranodal natural killer/T-cell lymphoma, nasal type: a multicenter trial in Northwest China.

The nasal type of extranodal natural killer/T-cell lymphoma is a rare aggressive lymphoma with poor prognosis. To discover a successful treatment, we investigated the efficacy and safety of chemotherapy with methotrexate, etoposide, dexamethasone, and polyethylene glycol-asparaginase (MESA). Three cycles of MESA were administered to 46 patients with new or relapsed/refractory natural killer/T-cell lymphoma. Complete response after 3 treatment cycles was 43.5%, the overall response rate was 87%, and 2-year overall survival was 83.4%. Complete response was significantly better for newly diagnosed patients than for patients with relapsed/refractory disease. Patients with newly diagnosed disease had a significantly better overall response rate after 1, but not after 2 or 3 treatment cycles. Overall survival and progression-free survival did not differ over 2 years. Grade 1/2 toxicities were frequent, but MESA was associated with fewer grade 3/4 events or treatment-related deaths. These results will require confirmation in larger prospective trials.

Natural Killer/T Cell Lymphoma, Nasal Type: A Retrospective Clinical Analysis in North-Western China.

BACKGROUND: Extranodal natural killer (NK)/T cell lymphoma (ENKTL) is an aggressive non-Hodgkin's lymphoma with high mortality and poor prognosis despite radiotherapy and chemotherapy. The current analysis aimed to assess the pathological features, clinical features, and prognostic indicators of ENKTL. MATERIAL AND METHODS: 120 ENKTL patients were analyzed for pathologic diagnosis and clinical disease manifestations from April 2007 to October 2012. Complete remission, 2-year overall survival, and progression-free survival were analyzed. RESULTS: Compared with the nasal group, a greater percentage of patients in the non-nasal group intended to receive autologous stem cell transplantation had Epstein-Barr virus (EBV) DNA, Ann Arbor stage IV, Ki-67 expression ≥ 60%, and abnormal ferroprotein and ß-microglobulin levels. The rate of complete remission in the non-nasal group was higher than that in the nasal group. The overall survival rate was 74.9% at 24 months. Patients receiving chemotherapy and radiotherapy were more likely to have disease progression compared with patients who received chemotherapy or radiotherapy alone. CONCLUSIONS: Further understanding the pathological and clinical features of ENKTL will be critical for moving forward. Ki-67, ß-microglobulin, EBV DNA, and primary site prognostic indicators may be useful to stratify patients into different risk groups, to gain insight into patient-specific treatments, and to potentially improve survival.

[Influence of CD117 Expression on Response of Multiple Myeloma Patients to Chemotherapy].

OBJECTIVE: To investigate the influence of CD117 expression on response of multiple myeloma patients to chemo-therapy. METHODS: A total of 65 cases of newly diagnosed multiple myeloma in our hospital from 2011 to 2013 were enrolled in this study. Cytogenetic abnormalities and immunophenotype were detected by using fluorescence in situ hybridization and flow cytometry before chemotherapy. The therapeutic efficacy of patients was evaluated after 4 cycles of PAD or TAD regimen. RESULTS: The positive rates of 1q21 amplification, RB1: 13q14 deletion, D13S319: 13q14.3 deletion, IgH: 14q32 rearrangement and p53: 17p13 deletion were 32.2%, 40%, 40%, 20% and 3.1% respectively; the positive rates of CD38, CD138, CD56, CD117, CD20 were respectively 100%, 100%, 60%, 20%, 10.8%; the positive rates of CD19 and CD10 were 4.6% and 4.6% respectively; the positive CD22, CD7, CD5, CD103 did not found in any patients. The therapeutic efficacy of CD117⁻ patients was better than that of CD117⁺ patients (P < 0.05), there was no correlation of the remaining indicators with efficacy; the proportion of CD117⁺ patients with ß2-microglobulin ≥ 5.5 mg/L was significantly higher than that of CD117⁻ patients (P < 0.05); the rest of baseline data had no significant difference (P > 0.05). CONCLUSION: CD117 can be used as an indicator for evaluating efficacy of patients with newly diagnosed multiple myeloma.

[Valproic Acid Represses Autophagy and Enhances the Anti-myeloma Activity of DNA-damaging Drugs].

OBJECTIVE: To explore the effect of valproic acid(VPA) on anti-myeloma activity of Doxorubicin(DOX) or Melphalan(MEL) and its related mechanism. METHODS: Human multiple myeloma(MM) cells were treated with VPA of non-toxic dose in absence and presence of DOX or MEL at different concentrations (ie. IC10, IC20, IC40). The cell proliferation was detected by MTT method. Western blot was used to detect the expression levels of autophagy-related proteins (LC3, ATG5, ATG7) and acetylated histone H4K16ac. RESULTS: Cell proliferation inhibition markedly increased in VPA plus DOX or MEL as compared with DOX or MEL alone (P<0.05). Both LC3 and H4K16ac expression levels in co-treatment were between VPA and DOX or MEL treated alone. Importantly, VPA of non-toxic dose not only augmented the anti-myeloma activity of DOX or MEL, but also down-regulated the autophagy-related protein expression and increases H4K16ac protein levels. CONCLUSION: H4K16ac can inhibit the transcription of autophagy-related genes, The VPA enhance the anti-myeloma activity of DNA-damaging drugs, at least in part, via H4K16ac-mediated suppression of cytoprotective autophagy.

Increased 14-3-3ζ expression in the multidrug-resistant leukemia cell line HL-60/VCR as compared to the parental line mediates cell growth and apoptosis in part through modification of gene expression.

BACKGROUND: Acute myeloid leukemia (AML) recurrence is largely a result of multidrug resistance (MDR). We aimed to examine the role of 14-3-3ζ in AML chemosensitivity using HL-60 and vincristine-resistant HL-60/VCR cells. METHODS: The effects of 14-3-3ζ siRNA on the growth and cell cycle progression of HL-60 and HL-60/VCR cells were determined. The effect of 14-3-3ζ siRNA on topotecan (TPT)-induced apoptosis was evaluated by several assays. RESULTS: Compared to HL-60 cells, HL-60/VCR cells had increased 14-3-3ζ mRNA and protein expression. Increased mdr-1 mRNA as well as mdr-1, Bcl-2 and Mcl-1 protein expression were observed in HL-60/VCR cells. In both HL-60 and HL-60/VCR cells, 14-3-3ζ was observed in the cytoplasm and nuclear compartments. 14-3-3ζ siRNA significantly reduced HL-60 and HL-60/VCR cell growth after 48 h and increased the proportion of cells in the G0/G1 phase. Moreover, 14-3-3ζ siRNA significantly increased the sensitivity of both HL-60 and HL-60/VCR cells to TPT, possibly through the inhibition of Bcl-2, Mcl-1 and mdr-1 protein expression. CONCLUSIONS: Silencing of 14-3-3ζ increased the sensitivity of both sensitive and resistant HL-60 cells to TPT-induced apoptosis, possibly through altering the expression of apoptosis-associated proteins, suggesting that it may be a potential target for MDR AML.

[Efficacy comparison between standard and reduced doses of bortezomib combined with adriamycin and dexamethasone in the treatment of patients with multiple myeloma].

OBJECTIVE: To compare the efficacy and safety of standard or reduced doses of bortezomib combined with adriamycin and dexamethasone (PAD) in patients with multiple myeloma (MM). METHODS: Eighty-two newly diagnosed or refractory/relapsed patients received bortezomib [either 1.2-1.3 mg/m(2) (standard dose) or 1.0-1.1 mg/m(2) (reduced dose) on day 1, 4, 8 and 11], and adriamycin (10 mg/m(2)) plus dexamethasone (40 mg/m(2)) on day 1-4 at 3-week intervals for 1 to 6 courses. The International Myeloma Working Group Criteria were used to evaluate the response. Adverse events were graded according to the National Cancer Institute Common Toxicity Criteria (Version 3.0). RESULTS: Two courses of standard dose of PAD resulted in a similar response rate of partial and very good partial complete remissions (PR) compared with reduced dose (80.0% vs 80.8%, P=0.728). Grade III- Ⅳ neutropenia and thrombocytopenia were higher with standard dose than that with reduced doses of PAD (21.1% vs11.1%, P=0.270; 10.5% vs 6.3%, P=0.619, respectively). Grade III-Ⅳ bortezomib-induced peripheral neuropathy, herpes zoster, fatigue or abdominal distention were significantly higher with standard dose than that with reduced dose of PAD (15.8% vs 1.6%, P=0.037; 26.3% vs 6.3%, P=0.028; 36.8% vs 14.3%, P=0.046; 15.8% vs 1.6%, P=0.037, respectively). CONCLUSION: Reduced dose of PAD appears to result in a similar overall response rate, but a better tolerance and safety compared with standard dose.

[Effects of triptolide on bortezomib-induced apoptosis in multiple myeloma cells].

This study was purposed to investigate the effect of triptolide on bortezomib-induced apoptosis in multiple myeloma cell line NCI-H929(H929). MTT assay was applied to detect the inhibitory effects of triptolide and bortezomib alone or combined at different concentrations on H929 cells, the cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI staining. The results showed that both triptolide (10 - 100 ng/ml) and bortezomib (10 - 100 nmol/L) alone or combination inhibited the proliferation of MM cell line H929 in a concentration-dependent manner. The apoptotic rate of H929 cells in group of triptolide combined with bortezomib was much higher than that in groups of single drug or control; moreover, the apoptotic rate of H929 cells treated by non-inhibitory concentration of triptolide (10 ng/ml) combined with bortezomib (40 nmol/L) for 24 h was significantly higher than that by bortezomib alone (P < 0.05). It is concluded that triptolide can significantly enhance the pro-apoptotic activity of bortezomib in MM cells.

Reduced tumor lysis syndrome with low dose chemotherapy for hyperleukocytic acute leukemia prior to induction therapy.

Patients with hyperleukocytic acute leukemia (HAL) can succumb to leukostasis. In an attempt to reduce its incidence, 45 patients with newly diagnosed HAL and hyperleukocytosis were administered half the conventional dose of etoposide and cytosine arabinoside (EA: 50 mg/m2 daily each) until WBC counts were significantly reduced and standard induction therapy was initiated. We retrospectively reviewed their outcomes and analyzed potential factors with a logistic regression model. The incidence of early mortality (<30 days) was 4.4% (2/45). Patients who achieved complete remission with induction chemotherapy had significantly lower median WBC counts (26x10(9) L-1) after low dose EA treatment than the no response patients (median WBC: 65x10(9) L-1 (P<0.05). Low dose EA treatment of HAL patients reduced WBC for both lymphoid and myeloid leukemic cells and can be considered for preemptive administration to HAL patients prior to the differential diagnosis of the acute leukemia. This approach warrants further studies as a cytoreduction therapy for HAL.

[Effects of xbp-1 gene silencing on bortezomib-induced apoptosis in human multiple myeloma cells].

This study was purposed to investigate the effect of xbp-1 gene silencing on bortezomib-induced apoptosis in multiple myeloma cell line NCI-H929 (H929). After xbp-1 gene expression was interfered by small hairpin RNA, the cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI staining, and the expression level of XBP-1 protein was detected by Western blot. The results showed that XBP-1 protein level of H929 cells was inhibited effectively by the PLL3.7 lentiviral vector mediated expression xbp-1 shRNA. The apoptosis rate was significantly higher in xbp-1 shRNA-expressing cells than in untreated control group [(10.13±0.61)% vs (2.5±0.2)%, p<0.05]. After treatment with bortezomib, the apoptosis rate of XBP-1 protein functionally deficient H929 cells was significantly higher than those in vector control group [(45.07±1)% vs (19.53±0.8)%, p<0.05]. It is concluded that xbp-1 gene silencing can significantly enhance the pro-apoptotic activity of bortezomib in multiple myeloma cells.

[PI3-kinase mediates thrombin-induced platelet aggregation through mDia1 pathway.].

OBJECTIVE: To investigate the expression of mDia1 (mammalian diaphanous 1)in platelet and the role of mDia1 or phosphatidylinositol 3-kinase (PI3K) in the process of thrombin-induced platelet aggregation. METHODS: The extent of platelet aggregation was measured by a platelet aggregation system and the expression of mDia1 and its relation with F-actin in quiescent, spreading or aggregated platelets by Western blot. RESULTS: There was no significant difference in mDia1 expression level between quiescent and activated platelets. mDia1 moved from a Triton-X100-soluble cytosolic fraction to insoluble cytoskeleton fraction after thrombin induced platelets aggregation. Anti-mDia1 antibody could inhibit this aggregation. PI3K inhibitor Wortmannin or Ly294002 inhibited the thrombin induced platelet aggregation and the above mentioned mDia1 translocation. CONCLUSION: PI3-kinase mediates the thrombin-induced platelet aggregation through mDia1 pathway.

[PI3-kinase mediates activity of RhoA and interaction of RhoA with mDia1 in thrombin-induced platelet aggregation].

The aim of this study was to investigate the role of RhoA/mDia1 pathway in the process of thrombin-induced platelet aggregation and regulatory effect of PI3K inhibitor on this process. The human platelets were isolated from peripheral blood, the activation of RhoA, Rac1 and Cdc42 in the platelet aggregation was detected by GST pull-down assay and immune co-precipitation, the interaction of RhoA, Rac1 and Cdc42 with mDia1 and the formation of complex in the process of platelet aggregation were determined by immune coprecipitation, and the effect of PI3K inhibitor (wortmannin) on above-mentioned process was assayed. The results showed that thrombin elevated the activity of RhoA and the binding capability of RhoA with mDia1 during thrombin-induced platelet aggregation and spreading on Fg coated coverslips. Wortmannin inhibited the rising of RhoA activity and the binding level of RhoA with mDia1 induced by thrombin. Thrombin elevated the activity of Rac1 and Cdc42 during thrombin-induced platelet aggregation, but could not induce binding of Rac1 or Cdc42 with mDia1. Wortmannin could not inhibit the rising of Rac1 and Cdc42 activity induced by thrombin. It is concluded that the PI3-kinase regulates the thrombin-induced actin cytoskeleton reconstitution in platelets by RhoA-mDia1 pathway.

[PAD regimen for relapsed or refractory patients with multiple myeloma].

OBJECTIVE: To investigate the efficacy and safety of PAD [bortezomib (PS-341), doxorubicin and dexamethasone] regimen for relapsed or refractory multiple myeloma (MM). METHODS: Seventeen patients with relapsed or refractory MM received two to four 21-day cycles of PAD: an intravenous bolus of bortezomib (1.3 mg/m2) on days 1, 4, 8, and 11; doxorubicin 10 mg per day on days 1 to 4, and dexamethasone 40 mg on days 1-4. Response was evaluated according to International Myeloma Working Group Criteria (IMWG 2006), toxicity was graded according to NCI CTCAE (common terminology criteria for adverse events) v 3.0. RESULTS: After 2-4 courses of PAD, 14 patients (82.4%) response, including complete response (CR) in 4 (23.5%), very good partial response (VGPR) in 4 (23.5%), partial response (PR) in 6 (35.3%) and stable disease (SD) in 3 (17.6%). Median time to progression was 9.5 months. The median course to response was 1.6 (1-3). All of 5 patients with extramedullary plasmacytoma achieved at least PR after the first cycle therapy; the plasmacytoma disappeared after 1-2 cycles of PAD. The efficacy was independent of other prognostic factors such as beta2-MG. Adverse events included thrombocytopenia in 9 patients (52.9%), leukopenia in 4 (23.5%), peripheral neuropathy in 4 (23.5%), varicella herpes zoster in 3 (17.6%), fatigue in 6 (35.3%) and diarrhea in 2 (11.7%). All of these adverse reactions could be controlled with routine supportive treatment, only one patient died from respiratory failure during his fifth PAD cycle. CONCLUSIONS: PAD regimen should be considered as an appropriate treatment for relapsed or refractory MM, especially for MM with extramedullary plasmacytoma. Its efficacy is independent of traditional prognostic factors. The side effects are usually manageable.

[Bortezomib-induced BiP expression and apoptosis in multiple myeloma cells].

This study was aimed to explore the effect of bortezomib on the apoptosis and expression of the molecular chaperone BiP in human multiple myeloma cell line NCI-H929 (H929). After treatment of H929 cells with different concentrations of bortezomib for 24 hours, cell apoptosis was assayed by flow cytometry with Annexin V-FITC/PI staining, and the expression levels of BiP mRNA and protein were detected by RT-PCR and Western blotting analysis. The results showed that bortezomib of different concentrations (20, 40 and 80 nmol/L) induced apoptosis of H929 cells in dose-dependent manner, with apoptotic rates (15.73 +/- 0.67)%, (27.83 +/- 1.26)% and (44.17 +/- 2.25)% respectively, which were significantly higher than that in control (1.21 +/- 0.07%) (p < 0.05). Bortezomib-induced up-regulation of BiP mRNA levels was almost on a parallel with BiP protein when compared with control. Under the similar apoptosis-stimulating conditions with apoptotic rates varying from 40% to 50%, expression levels of BiP mRNA and BiP protein induced by the classical endoplasmic reticulum stressor Brefeldin A (500 ng/ml, 24 h) were almost consistent with those by bortezomib (80 nmol/L, 24 h). It is concluded that bortezomib-induced apoptosis in H929 cells correlates closely with endoplasmic reticulum stress.

[Effect of shRNA-mediated silence of H-ras gene on proliferation of human SACC-M cells].

OBJECTIVE: To examine the effects of H-ras gene silence on cell cycle, proliferation and apoptosis of salivary adenoid cystic carcinoma -M (SACC-M) cell lines. METHODS: The plasmid H-ras-shRNA, containing the sequence of shRNA targeting H-ras, and HK-shRNA (without interfering effect) were constructed and transfected into SACC-M cells. The cell line with shRNA plasmid stable expression was isolated by G418. The expression levels of H-ras were detected by RT-PCR and protein immunofluorescent assay; cell cycle and cell apoptosis were analyzed by flow cytometry (FCM). The proliferation of cell was also determined by subcutaneous tumor formation in nude mice. RESULTS: After transfection of H-ras-shRNA plasmid, the mRNA expression of H-ras in SACC-M cells was down-regulated by 61.80% and protein expression of H-ras was inhibited by 62.76%; the cell proliferation was inhibited obviously; the G0G1 phase cells were increased. The cell apoptosis rate of H-ras-shRNA group was significantly higher than that of HK-shRNA group (P <0.05). The volume of subcutaneous tumor in nude mice was significantly smaller in Hras-shRNA group than in control group. CONCLUSIONS: The recombinant plasmid HRAS-shRNA could efficiently down-regulate the expression of H-ras gene and protein, induce apoptosis of SACC-M cells and simultaneously inhibit proliferation of these cells in vitro and in vivo.

[Construction of eukaryotic expression vector of short hairpin RNA targeting human xylosyltransferase-I gene].

OBJECTIVE: To design and construct the plasmids expressing short hairpin RNA (shRNA) targeting human xylosyltransferase- I (XT- I) which is the initiating enzyme in the biosynthesis of proteoglycans (PC). METHODS: Short chain oligonucleotides were designed according to the sequence of XT-I provided by GenBank. The DNA segments were gained through annealing after chemosynthesis, and were cloned into Pgenesil-1 vector. The recombinant XT- I shRNA expression vectors were identified by digestion and sequencing analysis. At last the constructed XT-I expression vectors were transfected into salivary adenoid cystic carcinoma cell line (ACC-M) by Lipofectomine 2000. The expression of green fluorescent protein (GFP) was detected by inverted fluorescent microscope and the rates of transfection were detected by flow cytometer. Semiquantitative RT-PCR was used to detect the expression of mRNA level of XT- I in transfected ACC-M cells and the protein expression of XT- I was detected by Western blot. RESULTS: The plasmids expressing shRNA targeting XT-I gene are called WJ1, WJ2, WJ3, WJ4, WJ5 and WJ6. Successful constructions were identified by digestion and sequencing. The mean rate of transfection was 50.26%. ACC-M cells transfected with WJ1-WJ6 expressed GFP successfully. And by RT-PCR and Western blot, WJ3 showed the most powerful RNAi gene silencing of inhibitory. The inhibition rate was 72.39% of mRNA level and 70.18% of protein level respectively. CONCLUSION: The XT-I shRNA expression vectors were constructed successfully which lays the foundation for RNAi study on the biosynthesis of PG in salivary gland tumors.

[CMV pp65 gene modified dendritic cells activate autologous T cells].

Cytomegalovirus (CMV) infection is a dangerous complication in patients with chronic graft versus host disease (cGVHD). CMV-specific immunity depends on the activity of T cells. This study was aimed to investigate the effect of CMV pp65 gene modified dendritic cells (DCs) on activation of autologous T cells. Lentivirus system was utilized to introduce the CMV full-length pp65 gene into mouse DCs; CpG-DNA was used to induce mature DCs; flow cytometry and immunofluorescence were used to determine the expression of antigen and IFNgamma in T lymphocytes. The results showed that the DCs were infected with lentivirus at a multiplicity of infection (MOI) of 50 with optimal infectious efficiency of 30%-40%; mature DCs expressing pp65 gene could stimulate autologous naive T cells to express CD69 specifically; mature DCs expressing PP65 could stimulate autologous CD4+ or CD8+ T cells to produce IFNgamma. It is concluded that CMV pp65-modified and CpG-DNA-induced mature DCs can activate CMV-specific T lymphocytes in vitro.

[Preparation and identification of anti-human polyclonal antibody against xylosyltransferase-I].

AIM: To prepare the rabbit polyclonal antibody against human xylosyltransferase-I (XT-I) protein and to identify its specificity. METHODS: The predominant epitope of XT-I gene was predicted by the DNAssist software. The DNA fragment of this epitope region was synthesized by PCR and cloned into the pGEX-4T-2 vector. The recombinant plasmid was transformed into E.coli ER2566 and the fusion protein GST-XT was induced and isolated. The purified fusion protein was used to immunize New Zealand rabbits. The antibody titer was determined by ELISA. Purified polyclonal antibody was obtained through affinity chromatography column and the specificity of the purified antibody was characterized by Western blot. RESULTS: The amino acid 175-205 of XT-I (QKHQPELAKKPPSRQK-ELLKRKLEQQEKGKG) was selected as an antigen epitope. The synthesized DNA fragment of XT-I was successfully inserted into pGEX-4T-2 vector and the protein GST-XT was expressed. The purified fusion protein GST-XT was used as the immunogen to immunize rabbits and the polyclonal antibody against XT-I protein was obtained. The result of ELISA showed that the antibody titer was 1:640 000. Western blot analysis showed that the antibody had a good specificity. CONCLUSION: The rabbit polyclonal antibody against human XT-I protein has been successfully prepared, which lays the foundation for further study on the biosynthesis of PG by neoplastic myoepithelial cells in salivary tumors.