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Objective:To construct pancreatic cancer cell lines expressing luciferase and mesothelin (MSLN), and evaluate the feasibility of using them as target cells in analyzing the cytotoxicity activity of immune cells.Methods:Lentiviral vectors expressing luciferase and MSLN genes were constructed, and pancreatic cancer cell lines were infected after lentivirus packaging. Single-cell clones were obtained by limited dilution following antibiotic screening, and the stable expression of the target genes were verified. These cells were used as target cells to detect the cytotoxicity of immune cells by real-time cell analysis (RTCA) and luciferase activity. Besides, these luciferase-expressing cells were transplanted into B-NDG mice to establish the animal models of pancreatic cancer, and in vivo optical imaging technology was used to detect the expression of luciferase and monitor the tumor growth in mice. The cytotoxicity of chimeric antigen receptor T (CAR-T) cells was verified in these animal models. Results:Three pancreatic cancer cell lines, panc-1-luc, panc-1-luc-MSLN and capan-2-luc, that could stably express luciferase and MSLN genes were successfully constructed. The expression of the reporter gene in these cells were high, and positively correlated with the number of cells. There were 95.6% of panc-1-luc-MSLN cells expressing MSLN. MSLN-CAR-T cells had specific killing effect on MSLN-positive panc-1-luc-MSLN cells and capan-2-luc cells, with the minimum killing rates of (70.00±18.19)% and (57.00±5.29)%, respectively. But they had no cytotoxicity to MSLN-negative panc-1-luc cells. RTCA results showed that MSLN-CAR-T cells were able to lyse all three pancreatic cancer cell lines, and the minimum killing rates were (56.33±7.64)%, (93.00±2.65)% and (26.33±28.15)%, respectively. The killing of target cells by NK-92MI cells was not depended on MSLN expression. The cytotoxicity in the mice models of pancreatic cancer was consistent with the results in vitro. The in vivo and in vitro test results suggested that the expression of luciferase by target cells could reflect the cytotoxicity of immune cells. Conclusions:This study establishes three pancreatic cancer cell lines stably expressing luciferase, which can be used to evaluate the cytotoxicity of immunotherapy products targeting tumor cells in vitro and in vivo.
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Objective:To evaluate the feasibility of 8E5 cells and CD19-CAR-Jurkat cells used as reference cells in the detection of lentiviral vector integration sites with different methods.Methods:Single clones of 8E5 cells and CD19-CAR-Jurkat cells were selected using limiting dilution method. Digital PCR was established to detect the copy number of HIV-1 in 8E5 cells and the copy number of CAR in CD19-CAR-Jurkat cells. High-throughput sequencing techniques (whole-genome resequencing, modified genome sequencing and probe hybridization capture) were used to detect integration sites in 8E5 cells and CD19-CAR-Jurkat cells, and optical genome mapping (OGM) technology was used for further confirmation.Results:Three clones of 8E5-D8 cells and six clones of CD19-CAR-Jurkat 2-6 cells were selected using the limiting dilution method. 8E5-D8 and CD19-CAR-Jurkat 2-6 were chosen as candidate cells based on their gene copy numbers detected by digital PCR and flow cytometry. These cells were then expanded and cryopreserved. Digital PCR showed that 8E5-D8 cells contained approximately 1 copy per cell, while CD19-CAR-Jurkat 2-6 cells contained approximately 13 copies per cell. High-throughput sequencing revealed one integration site in 8E5 cells and 13 integration sites in CD19-CAR-Jurkat cells, which matched the copy number detection results. All these integration sites were further confirmed at the submicroscopic level of chromosomes using OGM.Conclusions:Based on the insertion copy numbers and integration sites, 8E5-D8 cells and CD19-CAR-Jurkat 2-6 cells could be used as reference cells in further development of methods for detecting integration sites in CAR-T cell lentiviral vectors.
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Objective:To establish VSV-G qPCR assay for detection of replication competent lentivirus(RCL) and verify its application.Methods:A real-time fluorescent quantitative PCR for VSV-G envelope gene was developed. Several parameters including specificity, linear, amplification efficiency, precision, trueness, dynamic range, limit of detection, limit of quantification and robustness were verified. Preliminary application on CAR-T cells, end of production cells and the harvest of lentivirus vector was performed by using the method developed.Results:The real-time fluorescent quantitative PCR assay for VSV-G was specific for the detection VSV-G without specific amplification on 293T, PBMC and C8166 cells. The linear range of the assay was 1×10 2 copies/test-1×10 9 copies/test with a R2 value more than 0.998 and amplification efficiency between 93% and 98%. The precision (relative standard deviation) of the assay was less than 12% and the trueness (the rate of recovery) of the assay was between 85% and 106%. The limit of detection (LOD) and limit of quantification (LOQ) of the assay was 5 copies/test and 40 copies/test. In addition, the robustness of the assay was also well. All the results of validation illustrated that the assay could meet the detection requirements. All of the 54 samples including CAR-T cells, lentivirus vector and end of product cells after amplification and passage on C8166 cells were negative of RCL by using the established assay. Conclusions:The real-time fluorescent quantitative PCR for VSV-G were established successfully. All of the validation results illustrated that the assay could meet the detection requirements. The application of the assay was conducive to further enhance the safety of the lentivirus vector related products.
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@#The high-purity impurity C was isolated and purified from a new anti-allergic drug, rupatifen, by nucleophilic substitution reaction with bromobutaric acid, and its structure was confirmed by IR, UV, MS, 1H NMR, 13C NMR, DEPT135°,HSQC, HMBC, and 1H-1HCOSY. The preparation process of impurity C in this study was simple and easy to obtain under mild conditions, with the purity of 99.0% and the yield of 25%-30%;and the sample met the target compound by structural confirmation. The preparation process and structure confirmation provided sufficient impurity C reference substance for impurity research of raw materials and preparations of rupatifen fumarate, which laid a solid foundation for quality research of new drugs.
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Objective To optimize and compare four in vitro cytotoxicity assays in order to find a relatively rapid assay that can replace the traditional 51Cr release assay to evaluate the cytotoxicity of immune cells prepared for immunotherapy.Methods Four assays including BATDA, CAM (calcein acetoxymethyl ester), CytoTox-Glo and PKH were optimized and used to measure the in vitro cytotoxicity of NK-92 cells to K562 cell line.Intra-and inter-assay reproducibility of these assays and their correlation with 51Cr release assay were analyzed.Results After optimization, all of the four cytotoxicity assays showed good correlation with effector to target (E/T) ratio in a certain range.Compared with the other three assays, CytoTox-Glo assay showed obvious hook effect at a high E/T ratio of 40∶1.BATDA assay could detect the significant cytotoxicity of NK-92 cells to K562 cells after incubating them for one hour and that was the shortest time taken by the four assays to detect in vitro cytotoxicity.Both CAM and PKH assays took about four hours and CytoTox-Glo assay took six to eight hours to detect the significant cytotoxic potency.All of the four assays, especially BATDA and CAM assays, showed good intra-and inter-assay reproducibility.Among these assays, BATDA assay showed the best correlation with the traditional 51Cr release assay.BATDA assay, as compared with the other three assays, could be used to detect the cytotoxicity of Caov3 cells, an adherent cell line, and showed good results in evaluating the cytotoxic potency of autologous primary NK cells and CD19-CAR T cells.Conclusion Compared with the other three assays, BATDA assay shows the best linear correlation with 51Cr release assay and has the advantages of time-saving and good reproducibility.Besides, it is a better assay for detecting the cytotoxicity of adherent cells.BATDA assay is a promising substitute for 51Cr release assay in evaluating the in vitro cytotoxic potency of NK cells and other immune cells.
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Objective To establish an assay for the detection of Torque teno sus virus ( TTSuV) strains and to analyze its preliminary application to biologics.Methods Primers and probe were designed according to the conserved sequences.A fluorescent PCR assay for the detection of TTSuV strains was estab-lished.Several parameters including the specificity, linearity, accuracy, sensitivity and anti-interference of the established assay were verified.The fluorescent PCR assay was performed to detect the samples of por-cine blood, cell substrate and rotavirus vaccine.The porcine blood samples positive for TTSuV strains were further genotyped.Results The established fluorescent PCR assay was confirmed to have high specificity as no cross-reactions with parvovirus virus of various species, SV40 and porcine circovirus strains were detec-ted.The linear range of the assay was 1×109-1×103 copies/μl for TTSuV1 genotype and 1×109-1×102 cop-ies/μl for TTSuV2 genotype with a R2 value more than 0.993.The sensitivity of the fluorescent PCR assay was 1×103 copies/μl for TTSuV1 genotype and 1×102 copies/μl for TTSuV2 genotype.The intra-and inter-CVs were both less than 7%in Ct values and less than 25% and 45% respectively in copies.No interfer-ence was found in the detection of TTSuV nucleic acids from cell samples.8 out of 20 porcine blood samples were positive for TTSuV strains, among which one sample was positive for TTSuV1 genotype, four samples were positive for TTSuV2 genotype and the rest were positive for both TTSuV1 and TTSuV2 genotypes.Com-pared with the reference strain, strains genotyped as TTSuV1 and TTSuV2 were respectively shared 98%-99%and 98%homologies in sequences.All of the cell substrate and rotavirus vaccine samples detected by the fluorescent PCR assay were negative for TTSuV strains.Conclusion The fluorescent PCR assay for the detection of TTSuV was established successfully, the application of which would further improve the safety of biologics.
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Objective:To provide the basic data for the further revision of mycoplasma test method described in Chinese Pharma-copoeia and some references for the operation standardization of drug mycoplasma test. Methods:Two incubation conditions,namely aerobic conditions and microaerophilic conditions,were compared with respect to the growth status of mycoplasma in liquid and solid media. Results:The growth of mycoplasma was obvious difference between the two incubation conditions,and the microaerophilic con-ditions were better than the aerobic conditions. Conclusion:The microaerophilic conditions can be used in the incubation of mycoplas-ma test,which should be defined and standardized in the future Chinese Pharmacopoeia.
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Objective:To investigate the growth-promoting properties and applicability of the mycoplasma test culture media pre-scribed in Chinese pharmacopoeia, and provide reference for the standardization of the drug mycoplasma test method. Methods: Re-spectively using four quantitative detection methods including sensitivity, degree of color change, colony counts and colony diameter, and mycoplasma media widely used in the world as the reference media, the growth-promoting properties of 4 batches of mycoplasma broth and 4 batches of arginine mycoplasma broth from four domestic manufacturers were investigated. Results:The results of sensitivity assay and absorbance detection showed that all the media inoculated with below 100 CFU test microorganisms exhibited visible color change. Furthermore, the results of color change degree and colony diameter showed that there were significant differences among the media products from different manufacturers(P<0. 01). Conclusion:Mycoplasma broth and arginine mycoplasma broth both can sup-port the growth of below 100 CFU test microorganisms. Due to the difference in the growth-promoting properties among the media prod-ucts from different manufacturers, the drug mycoplasma test workers should use more sensitive methods to examine the applicability of the media.
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Abstract] Objective To establish an assay for the detection of porcine parvovirus ( PPV) and to verify its application for monitoring cells used for production.Methods A pair of primers and one probe were designed according to the conserved sequence encoding non-structural protein 1 (NS1).Based on the designed primers, a real-time fluorescent quantitative PCR assay for the detection of PPV was developed. Several parameters including the linearity, precision, minimum detection limit and anti-interference of the established assay were evaluated.A stock of PPV strains was prepared by infecting swine testicle ( ST) cells with PPV strains.An assay for the detection of PPV infection was developed by using ST cells as sensitive cells.A combined ST cell infection-PCR test was developed by combining the ST cell infection assay with the real-time fluorescent quantitative PCR assay.The sensitivity of ST cell infection-PCR test was analyzed.The cell samples used for production of biological products were detected by using the established assay.Results The real-time fluorescent quantitative PCR assay was specific for the detection of PPV without cross-reaction to other species of parvovirus virus, SV40 virus and other porcine viruses.The linear range of the assay was 1×109-1×104 copies/μl with a R2 value more than 0.98.The sensitivity of the real-time quantitative PCR assay was 1×104 copies/μl.Both of the intra-and inter-coefficient of variation (CV) were less than 5%in Ct values.The intra-and inter-CV in copies of detection were 5%-15% and 30%-40% respectively.The minimum detection limit of the real-time fluorescent quantitative PCR assay was 1CCID50/ml.The PPV strains were detected in cell samples with no interference.The sensitivity of ST cells infection-PCR test was 0.01CCID50/ml.All of the 22 cell samples were negative for PPV by using the real-time fluorescent quanti-tative PCR assay.Conclusion The real-time fluorescent quantitative PCR and the ST cell infection-PCR test for the detection of PPV in cells were established successfully.The application of the two assays was conducive to further enhance the safety of using cells for production and therapy.
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Objective To conduct extensive quality control tests on Madin-Darby Canine Kidney ( MDCK) cells used for the production of influenza vaccine .Methods Tests for characteristics , extraneous agents, endogenous agents and tumorigenicity were performed on MDCK cells according to Chinese Pharma -copeia Book III .Cell lysate and DNA of MDCK cells were tested for oncogenicity in the light of new interna -tional requirements .Results The MDCK cells extracted from canis were adherent cells with an epithelial morphology, whose average number of chromosome was 80±1.No bacteria, fungi and mycoplasma contami-nation were detected . The detection for extraneous and endogenous virus showed that there was no nonspecific virus causing cytopathic effect , hemadsorption , hemagglutination or animal death .Tests for re-verse transcriptase , bovine viruses and canine viruses were all negative .Each nude mouse was injected with 107 viable cells to observe their tumorigenicity .Twelve weeks after cell injection , no node was found at the injection site and in large organs by gross anatomy .There was no significant difference between test group and negative control group .The test for tumorigenicity of viable cells was negative .Cell lysate and cellular DNA collected from equivalent amount of cells were respectively injected into nude mice , and no node forma-tion was found.There was no significant difference between the test cells and negative controls in pathology indicating that the tested MDCK cells were non-oncogenic .Conclusion It showed the possibility of using MDCK cells for the production of influenza vaccine .
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Objective To study the in vivo expression and biodistribution of Ad5-Fluc (Adenovirus carrying firefly luciferase genes) in mice.Methods The recombinant Ad5-Fluc virus was constructed and infected to BALB/c or nude mice through three different routes.The protein expression level,tissue distribution and the characteristics of infection were analyzed by in vivo bioluminescence imaging technology.Results Compared to other two routes,the BALB/c mice infected through muscular route had the longest expression cycle (over 60 days) and the highest expression level,while the virus was transferred into the liver and spleen after infection.The nude mice had a significantly extended expression cycle than BALB/c mice.Moreover,the characteristic of liver tropism was eliminated after Ad5 F35 infection in mice,while maintained similar expression efficiency.Conclusion Due to the highest expression efficiency,the muscular route would be the optimal route for Ad5 vector based vaccination.In addition,Ad5F35 virus could become an ideal alternative vaccine vector for eliminating the liver tropism.
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Objective To detect the protection induced by HPV-58 L2 11-200 AA in animal, and analyze the relationship between antibody or neutralizing antibody titers and the protection generated by the immunizmg agent. Methods The peptide of HPV-58 L2 11-200 AA was expressed in E. coli and the mice were immunized with the peptide after purification and adsorption with aluminum adjuvant. The protection provided by different immunizing doses was detected in the mouse model against the challenge of the pseud-ovirions of human papiilomavirus types 58. The total antibodies and neutralizing antibody titers of serum were tested with ELISA and neutralization assay against HPV-58 pseudovirus, respectively. The total antibodies or neutralizing antibody titers that can protect the mouse from infection were analyzed. Results The mice can be protected from the challenge with HPV pseudovirus when the immunizing dose was 8 μg. The neutralizing antibody can not be detected in the immune serum by neutralization assay against pseudovirus. The total anti-body level has a corresponding relationship with the protection showed in mouse model. The results of total antibodies detected by ELISA showed that when the titer of total antibodies was ≥25 000, luminescent signal can not be detected and the mice can be protected from pseudovirus infection. Conclusion HPV-58 L2 11-200 AA peptide can protect mice from pseudovirus infection. L2 peptide has a promising perspective to be a candidate vaccine and the level of total antibodies in the immune serum can be used as a surrogate for the evaluation of protection against HPV infection.
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Objective To study the profiling and authentication of human cell lines in cell bank of our department using short tandem repeat (STR) loci and to analyze the situation of cell contamination and misjudgment. Methods Sixty-one human cells including cells collected and preserved by our cell bank and cells from the other departments were detected by the 16 STR loci-method. To analyze the cross contamina-tion between human cell lines, the results were aligned with profiles published by international cell banks. Isoenzyme detection was employed to authenticate the cell species when the STR signal can not be detected. Results Among the 61 cells, specific profiles were produced by 41 ceils and there was no cross contamina-tion. Thirty-six cells had the completely same STR profiles in 9 STR loci with the same cells preserved by ATCC or JCRB, while 5 cells have different profiling just in the vWA loci. The cells referred above can be recognized as correct cells; Eleven cells (18.0%) were the false cells. Among them, cancer cells of tongue named Tca8113 and cancer cell of liver named HHCC(changed to FHCC98 now) had the same profile with HeLa and HeLa S3 respectively; Two ceils both named HUT-102 have the completely different profiles with ATCC; The signal of 4 cells was not be detected, and all of them were determined as hamster cell lines by u-sing isoenzyme detection. Also 2 cells were identified to be mixed cells. Conclusion The phenomenon of cell misjudgment and cross contamination between cells is serious. Authentication of cell lines correctly, es-pecially for the re-authenticatian of domestic self-established cells, is very important for the guarantee of the reliability and reproducibility for scientific researches.
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Objective To evaluate five detection methods for the laboratory diagnosis of Clostridium difficile infection in the hospitals of USA, and explore a sensitive, specific, accuracy and rapid regimen for the early diagnosis of Clostridium difficile infection. Methods A total of 174 stool specimens submitted to the clinical microbiology laboratory for Clostridium difficile testing were separately tested by five methods including toxigenic culture (TGC), Premier Toxin A&B EIA( A/B-EIA), C. Diff Quick Chek Complete( DEIA), BD G eneOhm Cdiff assay(BD-PCR) and Laboratory-developed PCR(LD-PCR). The gold standard of TGC was used as a reference criterion, and the sensitivity, specificity, positive predictive value ( PPV )and negative predictive value (NPV) of A/B-EIA, D-EIA, BD-PCR and LD-PCR assays were determined. Results Among the 174 specimens studied, 24 were defined as true positives for Clostridium difficile infection by TGC assay, giving a positive rate of 13.8% (24/174). In comparison to the standard,the sensitivity, specificity, PPV and NPV were 62.5%, 99.3%, 93.8% and 94.3% for A/B-EIA;66.7%, 98.7%, 88.9% and 94.9% for D-EIA; 83.3%, 98.7%, 90.9% and 97.4% for BD-PCR;79.2%, 93.3%, 65.5% and 96.6% for LD-PCR. Among all tested specimens, 34 were positive by atleast one of five methods, and of which 15 were concordant by all five methods. The D-EIA results were divided into three groups depending on results of GDH and (or) toxins A/B: 18 were positive for both GDH and toxins A/B, 23 were positive for only GDH, and 133 were negative for both GDH and toxins A/B. Of 18 positive specimens by D-EIA assay, all were concordant with results of BD-PCR assay and 16 were agreement with results of TGC assay. Twenty-two of 24 positive specimens by TGC assay were included in 41 specimens that were positive for GDH. Among eight false negative specimens by D-EIA assay, four were differentiated as positive results by BD-PCR. According to the present study, the sensitivity, specificity,PPV and NPV of a two-step detection algorithm in combination with D-EIA and BD-PCR assays were 83.3%, 98.7%, 90.9% and 97.4%, respectively. Conclusions From the point of technological evaluation, BD-PCR is preferable. A two-step detection algorithm combining D-EIA with BD-PCR is proposed for the laboratory diagnosis of Clostridium difficile infection. This algorithm has demonstrated an excellent sensitivity and specificity, as well as decreased test turnaround time and test cost.
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Objective To evaluate the clinical significance of the changes of microalbunminuria(MAU) levels in mechanical ventilated patients with severe pneumonia. Methods According to the ratio between the microalbunminuria and the urine creatinine (MAU/CR) (ACR), setting 25mg/mmol as the threshold, 78 mechanical ventilated patients with severe pneumonia were divided into two groups :ACR increasing group and ACR Non-increasing group,then the clinical significance of changes of MAU levels in 72 hours on prognosis of these patients was observed. Results MAU increased in 64 cases(82. 1%) ,of which 46 cases in ACR increasing group and 18 cases in ACR non-increasing group. There showed statistically significant differences on APACHE Ⅱ score, CPIS score,PCT、the success of getting out of mechanical ventilation and the mortality between two groups, (t = 3.50、2. 19 、x~2 = 3. 95、6. 70、5.38 ,P = 0.01,0.03,0.04,0.01,0.02, all P < 0.05). Conclusion Changes of MAU levels have the clinical significance on prognosis of the mechanical ventilated patients with severe pneumonia.
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Objective To study the prevalence and genotype of human parvovirus B19 virus among blood products and plasma pools in China. Methods B19 DNA derived from 16 lots of factor Ⅷ concentrate produced by 4 manufactures and 10 lots of plasma pools were detected by nested PCR. Phylogenetic comparison of the partial B19 sequences obtained from positive samples were performed by direct sequencing. Results Twelve of sixteen lots of factor Ⅷ concentrate and all of ten lots of plasma pools were contaminated by B19 DNA. By comparing the partial B19 sequences,all the isolated viruses were genotype Ⅰ and their nucleotides were high conserved with homology of 98. 3%-100%. Conclusion B19 genotype Ⅰ DNA has been detected in high prevalence in factor Ⅷ concentrate and plasma pools. The genetic diversities were shown to be very low.
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Objective To study the application of short tandem repeat (STR) profiling in quality control of human cell lines used for biological production. Methods The methods detecting 9 and 16 STR loci to identify human cell lines by PCR-capiilary electrophoresis were established respectively. Human cell lines, which were derived from many corporations and including diploid cell strains used for virus-vaccine production and 293 cell lines used for gene therapy products, were analyzed and compared by these two methods. Results The STR profiling methods used for authentication of human cell lines were established. Most of human diploid cell strains(20/21 ) used for virus-vaccine production from 13 corporations were iden-tiffed as the intended cells and no cross-contamination was found. However, one MRC-5 cells was identified as a false cell line and one MRC-5 had 3 alleles in D13S317 locus. For 12 strains of 293 cell lines, there were significant differences in STR profiling from different manufactures, which was likely be explained that the sources and gene modifications of these 293 cell lines are not well known and their genes are unstable during passage. Conclusion The STR profiling method has the advantages of high sensitivity and specifici-ty, and can be used for authentication of each of human cell lines for biological production.
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OBJECTIVE For the reasonable use of antibacterial drugs, drug resisitance diversity of Pseudomonas aeruginosa was studied in our hospital. METHODS Antibacterial activity to all P. aeruginosa collected in four years from 2004 to 2007 were determined by K-B test, and data were analyzed by WHONET software. RESULTS The resistance rate to ampicllin, cefazolin and cefoteton were 100.0%, but low to ceftrazidime, cefoperazone and cefoperazone/sulbactam(3.2% in 2007). The resistance rate to levofloxacin increased year by year, while that to ciprofloxacin descreased from 15.6% to 9.6%.The resistance rate to gentamicin and amikacin descreased from 20.0% to 16.4%,and from 11.1% to 4.8%, respectively. The resistance rate to imipenem and meropenem accounted for about 10.0%, which was as same as ceftazidine. CONCLUSIONS The resistance of P. aeruginosa in our hospital is stable. Clinician should choose right antibacterial drugs on the basis of the test for antibacterial sensitivity and the pharmacological characteristis to improve curative effect and decrease the resistance of bacteria.