The purpose of this study was to investigate the lethal effect of cytotoxic lymphocytes against U266 cells induced by DCs pulsed with multiple myeloma (MM) U266 lysate and transfected with GM-CSF recombinant adenovirus. The cytotoxic lymphocytes against U266 cells were induced by culturing with DCs, which pulsed with MM U266 antigens and transfected with GM-CSF recombinant adenovirus. The effect of cytotoxic lymphocytes against U266 cells were measured by LDH release detection. Experiments were divided into 3 groups: N-DC group as control in which DCs were normal; U-DC group in which DCs were pulsed by U266 soluble antigen, and G-U-DC group in which DCs were stimulated by U266 soluble antigen and GM-CSF transfected with Ad-CMV. The results showed that there was significant difference on killing rate against U266 cells between 3 groups (F = 10.939, p < 0.05). The killing rate of G-U-DC group was the highest (p < 0.001), and killing rate of U-DC group was higher than that of N-DC group (p < 0.001). It is concluded that the cytotoxic lymphocytes against U266 cells can be induced by DCs pulsed with U266 lysate, and the lethal effect of CTLs can be enhanced when DCs transfected by recombinant adenovirus with exogenous gene GM-CSF.
Adenoviridae ; genetics ; Antigens, Neoplasm ; genetics ; immunology ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; Humans ; Multiple Myeloma ; Recombinant Proteins ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection

BACKGROUNDSurvivin is a rather specific gene in tumor tissue. We transfected dendritic cells (DCs) with recombinant adenovirus (Ad) containing survivin gene and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene and tested the inducing effect of the transfected DCs on cytotoxic T lymphocytes (CTL) to kill leukemic cells.

METHODSAfter derived from the peripheral, DCs was assayed by mixed leukocyte reaction (MLR) tests. Lactate dehydrogenase (LDH) release test was used to evaluate cytotoxicity of CTL.

RESULTSExpression of survivin in transfected DCs was confirmed by Western blotting analysis. GM-CSF expression was confirmed by enzyme-linked immunosorbent assay (ELISA). In MLR assay, DCs coinfected with Ad-survivin and Ad-GM-CSF induced higher allogeneic lymphocyte reaction than control DCs at ratios of 1:5, 1:10, 1:50 and 1:100. DCs coinfected with Ad-survivin and Ad-GM-CSF had much higher activity of CTL to HL-60 cells than DCs infected with Ad-survivin only, Ad-GM-CSF only, or control DCs. Levels of interleukin-12 (IL-12) and interferon gamma (IFN-gamma) in lymphocyte supernatants containing DCs coinfected with Ad-survivin and Ad-GM-CSF were significantly higher than those in the control group.

CONCLUSIONDCs coinfected with Ad-survivin and Ad-GM-CSF induce much higher anti-leukemic response in vitro than those infected with either factor. Therefore, adenovirus vectors containing survivin and GM-CSF genes may be promising vaccine candidates for leukemia therapy.

Adenoviridae ; genetics ; Cytotoxicity, Immunologic ; Dendritic Cells ; physiology ; ultrastructure ; Genetic Therapy ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; HL-60 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Interferon-gamma ; biosynthesis ; Interleukin-12 ; biosynthesis ; Leukemia ; therapy ; Lymphocyte Activation ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection

This study was purposed to investigate the immunological effects of modified dendritic cells (DCs) in inducing cytotoxic T cells (CTLs) effect against lymphoma cells. The DCs were derived from human peripheral blood and transfected with recombined adenovirus vector carrying survivin gene, Western blot was used to detect the expression of survivin, the lactate dehydrogenase (LDH) release test was used to determine the cytotoxicity of CTLs, the mixed lymphocyte reaction (MLR) was used to measure the ability to proleferate allo-lymphocyte by DCs, ELISA was used to assay IL-12 level in supernatant. The results showed that the expression of survivin in transfected dentritic cells was confirmed by Western blot analysis. In MLR assay, DCs transfected with Ad-survivin could induce higher allogeneic lymphocytes reaction at the ratios of 1:5, 1:10, 1:50 and 1:100. DCs transfected with Ad-survivin had much higher activity of CTL to CA46 cells than control DCs. The levels of IL-12 of supernatants containing DCs transfected with Ad-survivin were significantly higher than that in the control group. It is concluded that DCs transfected with Ad-survivin can induce CTL response in vitro against lymphoma cells.
Adenoviridae ; genetics ; metabolism ; Apoptosis ; immunology ; Dendritic Cells ; immunology ; Genetic Vectors ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Interleukin-12 ; metabolism ; Lymphoma ; immunology ; Microtubule-Associated Proteins ; genetics ; immunology ; Recombination, Genetic ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Tumor Cells, Cultured

The study was aimed to construct the recombinant adenovirus vectors containing human survivin gene, and to investigate their expression in transfected dendritic cells. Full length cDNA encoding survivin was obtained by PCR amplification from plasmid pcDNA3.0-survivin. The PCR product was restricted, and then inserted into pShuttle-CMV. The plasmids of pShuttle-CMV-survivin were linearized with PmeI, and the fragment containing survivin was ligated with pShuttle-CMV and transfected into E. coli BJ5183. After homologous recombination in bacteria, the extracted plasmid from the positive bacteria were linearized with PacI, transfected into HEK293 cells with liposome Lipofectamine 2000. Then, the harvested adenovirus supernatants were transfected into dendritic cells. The results showed that the recombinant adenovirus-survivin was constructed successfully and its titer was about 2.65 x 10(9) pfu/ml. The expression of survivin in transfected dendritic cells was confirmed by Western blot analysis. It is concluded that the recombinant adenovirus vector containing human survivin was constructed successfully, which may provide preliminary laboratory evidence for anti-leukemia immunotherapy.
Adenoviridae ; genetics ; metabolism ; Dendritic Cells ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Viral ; Genetic Vectors ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Neoplasm Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVE: To investigate the effect of steadily down-regulating the expression of calreticulin (CALR) on the invasion of natural killer/T-cell lymphoma SNK6 cells, and explore its possible mechanism. METHODS: The sequences of specific short hairpin RNA (shRNA) targeting on human CALR were designed, and were inserted into pLKO.1-puro lentivirus vector, and the reconbinant lentivirus vector was obtained; the lentivirus particles were backed by three-plasmid system and transfected into SNK6 cells, the SNK6 cells stably down-regulating the CALR expression were sercened by puromytain, the CALR-silencing effect was verified by real-time PCR and Western blot. CCK-8 assay was used to evaluate the cell viability, The transwell invasion assays was used to analyse invasion of SNK6 cells. The mRNA expression of Calreticulin, MMP2, MMP9 and VEGF was determined by real time PCR, the protein expression of Calreticulin and GAPDH was analyzed by Western blot. RESULTS: The recombinant lentiviral vector pLKO.1-puro-shCALR was successfully constructed, packed into the lentivirus, then the SNK6 cells stably down-regulating Calreticulin expression was obtained. When Calreticulin was down-rengulated in SNK6 cells, the proliferation rate was reduced and the invasion ability was decreased; the mRNA levels of VEGF and MMP-2/9 also were reduced. CONCLUSION The stable down-regnlation of CALR expression in SNK6 cells can attenuate the imvasiveness of SNK6 cells, which maybe related with transcriptional decrease of MMP2, MMP9 and VEGF.
Calreticulin ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Genetic Vectors ; Humans ; Lentivirus ; Matrix Metalloproteinase 2 ; Matrix Metalloproteinase 9 ; RNA Interference ; RNA, Small Interfering ; Transfection ; Vascular Endothelial Growth Factor A

OBJECTIVETo explore the effects of mTOR inhibitor rapamycin on proliferation, cell cycle and apoptosis of Burkitt's lymphoma cell line Raji and CA46 cells and its mechanism, so as to provide the experimental evidence for a therapeutic target of Burkitt's lymphoma.

METHODS3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay was performed to assess the inhibitory effect of rapamycin on proliferation of Burkitt's lymphoma cell line Raji and CA46 cells. The cell cycle distribution of Raji and CA46 cells was analyzed by flow cytometry with propidium iodide(PI) single staining. The cell apoptosis of Raji and CA46 cells was analyzed by flow cytometry with FITC Annexin V+PI double staining. The expressions of RPS6, p-RPS6, survivin and caspase-3 proteins were detected by Western blot after treating with rapamycin.

RESULTSRapamycin markedly inhibited the proliferation of both Raji and CA46 cells in a time- and concentration-dependent manners, showing good biological activity, the cell proliferation inhibition rate reached about 20% after treatment with 1 nmol/L rapamycin. After treatment with different concentrations of rapamycin for 24 and 48 hours, the proportion of both cells in G/Gphase in the treated groups was significantly increased in a time- and concentration-dependent manners in comparison with the solvent control group. With regard to the cells in S and G/M phase, the decreased population was accompanied by the increase of G/Gphase cells. After treatment with 100 nmol/L rapamycin for 48 hours, both Raji and CA46 cells demonstrated an apparent apoptosis,especially late apoptosis by flow cytometry with Annexin V+PI staining. After treatment with rapamycin, the expression of p-RPS6 and survivin of Raji and CA46 cells was obviously down-regulated, the expression of caspase-3 was obviously up-regulated in a time- and dose-dependent manners. However, rapamycin did not obviously affect the expression of RPS6.

CONCLUSIONThe rapamycin can effectively inhibit cell proliferation, arrest Raji and CA46 cells in G/Gphase, and this effect associates with inhibiting the activation of mTOR/RPS6 signal pathway through down-regulating the expression of phosphorylated RPS6, i.e. mTOR downstream signal pathway. It also can induce apoptosis by down-regulating the expression of anti-apoptotic protein survivin and activating the intrinsic pro-apoptotic protein caspase-3.

OBJECTIVE: To investigate the current status of antibiotic use for very and extremely low birth weight (VLBW/ELBW) infants in neonatal intensive care units (NICUs) of Hunan Province. METHODS: The use of antibiotics was investigated in multiple level 3 NICUs of Hunan Province for VLBW and ELBW infants born between January, 2017 and December, 2017. RESULTS: The clinical data of 1 442 VLBW/ELBW infants were collected from 24 NICUs in 2017. The median antibiotic use duration was 17 days (range: 0-86 days), accounting for 53.0% of the total length of hospital stay. The highest duration of antibiotic use was up to 91.4% of the total length of hospital stay, with the lowest at 14.6%. In 16 out of 24 NICUs, the antibiotic use duration was accounted for more than 50.0% of the hospitalization days. There were 113 cases with positive bacterial culture grown in blood or cerebrospinal fluid, making the positive rate of overall bacterial culture as 7.84%. The positive rate of bacterial culture in different NICUs was significantly different from 0% to 14.9%. The common isolated bacterial pathogens Klebsiella pneumoniae was 29 cases (25.7%); Escherichia coli 12 cases (10.6%); Staphylococcus aureus 3 cases (2.7%). The most commonly used antibiotics were third-generation of cephalosporins, accounting for 41.00% of the total antibiotics, followed by penicillins, accounting for 32.10%, and followed by carbapenems, accounting for 13.15%. The proportion of antibiotic use time was negatively correlated with birth weight Z-score and the change in weight Z-score between birth and hospital discharge (r=-0.095, -0.151 respectively, P<0.01), positively correlated with death/withdrawal of care (r=0.196, P<0.01). CONCLUSIONS Antibiotics used for VLBW/ELBW infants in NICUs of Hunan Province are obviously prolonged in many NICUs. The proportion of routine use of third-generation of cephalosporins and carbapenems antibiotics is high among the NICUs.
Anti-Bacterial Agents ; Birth Weight ; Humans ; Infant ; Infant, Extremely Low Birth Weight ; Infant, Newborn ; Intensive Care Units, Neonatal ; Surveys and Questionnaires

Objectives: This study aimed to present the Asia-Pacific consensus on long-term and sequential therapy for osteoporosis, offering evidence-based recommendations for the effective management of this chronic condition.The primary focus is on achieving optimal fracture prevention through a comprehensive, individualized approach. Methods: A panel of experts convened to develop consensus statements by synthesizing the current literature and leveraging clinical expertise. The review encompassed long-term anti-osteoporosis medication goals, first-line treatments for individuals at very high fracture risk, and the strategic integration of anabolic and anti resorptive agents in sequential therapy approaches. Results: The panelists reached a consensus on 12 statements. Key recommendations included advocating for anabolic agents as the first-line treatment for individuals at very high fracture risk and transitioning to anti resorptive agents following the completion of anabolic therapy. Anabolic therapy remains an option for in dividuals experiencing new fractures or persistent high fracture risk despite antiresorptive treatment. In cases of inadequate response, the consensus recommended considering a switch to more potent medications. The consensus also addressed the management of medication-related complications, proposing alternatives instead of discontinuation of treatment. Conclusions This consensus provides a comprehensive, cost-effective strategy for fracture prevention with an emphasis on shared decision-making and the incorporation of country-specific case management systems, such as fracture liaison services. It serves as a valuable guide for healthcare professionals in the Asia-Pacific region, contributing to the ongoing evolution of osteoporosis management.

Objective: Several COVID-19 patients have overlapping comorbidities. The independent role of each component contributing to the risk of COVID-19 is unknown, and how some non-cardiometabolic comorbidities affect the risk of COVID-19 remains unclear. Methods: A retrospective follow-up design was adopted. A total of 1,160 laboratory-confirmed patients were enrolled from nine provinces in China. Data on comorbidities were obtained from the patients' medical records. Multivariable logistic regression models were used to estimate the odds ratio ( Results: Overall, 158 (13.6%) patients were diagnosed with severe illness and 32 (2.7%) had unfavorable outcomes. Hypertension (2.87, 1.30-6.32), type 2 diabetes (T2DM) (3.57, 2.32-5.49), cardiovascular disease (CVD) (3.78, 1.81-7.89), fatty liver disease (7.53, 1.96-28.96), hyperlipidemia (2.15, 1.26-3.67), other lung diseases (6.00, 3.01-11.96), and electrolyte imbalance (10.40, 3.00-26.10) were independently linked to increased odds of being severely ill. T2DM (6.07, 2.89-12.75), CVD (8.47, 6.03-11.89), and electrolyte imbalance (19.44, 11.47-32.96) were also strong predictors of unfavorable outcomes. Women with comorbidities were more likely to have severe disease on admission (5.46, 3.25-9.19), while men with comorbidities were more likely to have unfavorable treatment outcomes (6.58, 1.46-29.64) within two weeks. Conclusion Besides hypertension, diabetes, and CVD, fatty liver disease, hyperlipidemia, other lung diseases, and electrolyte imbalance were independent risk factors for COVID-19 severity and poor treatment outcome. Women with comorbidities were more likely to have severe disease, while men with comorbidities were more likely to have unfavorable treatment outcomes.
Adult ; Aged ; COVID-19/virology* ; China/epidemiology* ; Comorbidity ; Female ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Severity of Illness Index ; Treatment Outcome