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.

BACKGROUND:The elderly hip fracture patients with chronic obstructive pulmonary disease (COPD) have a higher postoperative mortality than those only with hip fractures. In recent years, it has become an issue of concerns. Because the mechanism is unknown, however, there are no effective clinical interventions for these patients. OBJECTIVE:To observe the effect of bone marrow stromal stem cel s (BMSCs) on the level of pulmonary inflammation in aged rats with chronic obstructive pulmonary disease (COPD) after hip fractures. METHODS:Thirty male Sprague-Dawley rats, 12 months old, were exposed to smoking for 4 months and randomized into three groups, smoking, smoking+hip fracture+normal saline, smoking+hip fracture+BMSCs groups. Animal models of hip fracture were made in the latter two groups. Twenty-four hours after hip fracture, the lower lobe and peripheral blood samples were taken from al rats in the three groups, to evaluate the pathological changes of lung tissue and detect levels of inflammatory factors in the lung tissue and peripheral blood. RESULTS AND CONCLUSION:After closed hip fracture, aged COPD rats exhibited inflammatory cel infiltration, mucus secretion, airway stenosis or occlusion;the levels of interleukin-6, tumor necrosis factor-αand interleukin-10 in the lung tissue and peripheral blood were increased. After intravenous injection of BMSCs, the pathological changes of the lung tissue were reduced, and the levels of pro-inflammatory factors, tumor necrosis factor-αand interleukin-6, decreased, but the level of anti-inflammatory factor interleukin-10 further increased, which were significantly different from those in the normal saline group (P<0.05). These findings indicate that BMSCs can relieve acute lung injury in aged COPD rats with hip fractures.

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.

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.

OBJECTIVETo evaluate the effect of light irradiation intensity on bond durability of dual-cured resin luting agents to silanized ceramics.

METHODSLinkmax HV (LMHV), Nexus 2 (NX2), Variolink II HV (VL II HV) as dual-cured resin luting agents were bonded to silanized GN-I glass ceramics, and irradiated by 800, 310 and 80 mW x cm(-2) light intensity to form micro-shear test specimens. After 1, 90 d water storage, micro-shear bond strength of silanized resin/ceramic luting agent were measured. Data of each resin luting agent were analyzed by one-way ANOVA.

RESULTS90 d water storage decreased significantly the bond strength of all test groups, and the weak of irradiation intensity did not deteriorate this reduction of bond strength of luting resin/cermaic, oppositely in which LMHV irradiated by 310 mW x cm(-2) light intensity and NX2 irradiated by 80 mW x cm(-2) showed the higher bond strength than that irradiated by 800 mW x cm(-2).

CONCLUSIONThe weak of irradiation intensity does not affect the bond durability of dual-cured resin luting agents to silanized ceramics.

Ceramics ; Composite Resins ; Dental Cements ; Humans ; Materials Testing ; Resin Cements

OBJECTIVETo evaluate the effect of different silane couplers on bond strength and durability of two machinable glass ceramics to resin cement.

METHODSTwo machinable glass ceramics (A and B) were silanized by three silane couplers (A, B, C), and were bonded with a resin cement (G-CEM) to form micro-shear test specimens of six groups. The specimens of each group were subdivided into two subgroups, and their micro-shear bond strength was measured before and after 10000 thermal cycles. Bond strength data were analyzed by two-way ANOVA.

RESULTSBefore thermal cycles, the bond strength of ceramic A treated by silane coupler A was lower than that of ceramic B (P = 0.002). The bond strength of ceramic A treated by silane coupler C was significantly higher than that treated by silane coupler A and B (P = 0.014, P = 0.019). 10 000 thermal cycles obviously decreased the bond strength of all groups except the group of ceramic A treated by silane coupler B, and no significant difference was found between three silane coupler with either of two ceramic. However the bond strength of ceramic B treated by silane coupler B and C was significantly higher than that of ceramic A (P = 0.003, P = 0.027).

CONCLUSIONAs well as the types of silane coupler, the type of ceramic could affect their bond strength and durability to resin cement.

Ceramics ; Materials Testing ; Resin Cements ; Silanes

OBJECTIVETo analyze the effect of different silane coupling agents on the resin bond durability of glass-infiltrated alumina ceramic. Methods A glass-infiltrated alumina ceramic was silanized or not by three silane coupling agents. The treated ceramic surfaces were bonded with two resin cements. Their micro-bond strength were measured after 0, 30,000 thermal cycles.

RESULTSBefore thermal cycling, resin cement A had lowest bond strength to ceramic, and ceramic treated by silane coupling agent A with two cements had lower bond strength than those treated by silane coupling agent B and C. After thermal cycling, cement A had no bond strength with no treated ceramic, only ceramic treated by silane coupling agent A with two cements had more than 5 MPa bond strength.

CONCLUSIONThe glass-infiltrated alumina cermaic treated by the silane coupling agent activated by 10-methacryloyloxydecyl-dihydrogen phosphate could obtain better bond durability with different type of resin cements.

Aluminum Oxide ; chemistry ; Ceramics ; chemistry ; Dental Bonding ; Glass ; chemistry ; Resin Cements ; chemistry

OBJECTIVETo evaluated the effect of curing modes and light-cure times on knoop hardness (KH) and microtensile bond strength (µTBS) of dentin adhesives in vitro.

METHODSTwenty molars were made into 80 dentin slices (about 1 mm thick). The dentin slices were prepared with an etch&rinse adhesive A (ONE-STEP PLUS) and a self-etch adhesive B (Clearfil SE Bond), and light-cured respectively under fast mode, i.e.1250 mW/cm(2) light intensity for 10 s, 15 s, 20 s, and ramp mode (soft start curing mode), i.e.initial 0 mW/cm(2) gradually increasing to 1250 mW/cm(2) in first 10 s, then steady for the next 10 s. The prepared dentin slices were kept in dark dry room for 24 h at 37°C, and KH were tested. The other 40 molars were flattened to expose coronal dentin, prepared with adhesives as above. Then the prepared teeth were restored with resin composites incrementally and cured under fast mode. The restored teeth were stored in water for 24 h at 37°C, and slowly sectioned to obtain multiple bonded beams. After 7 d water-storage, the samples received microtensile bond test, and the failure models of beams were observed under a stereomicroscope. Data were analyzed by ANOVA and LSD test (α = 0.05).

RESULTSNo statistical difference in KH [(28.20 ± 5.36), (29.13 ± 5.60), (28.13 ± 4.40), (27.06 ± 3.77) MPa] and µTBS [(22.30 ± 5.07), (22.73 ± 6.59), (26.32 ± 6.17), (25.67 ± 4.31) MPa] of adhesive A were found between four curing conditions (fast mode for 10 s, 15 s, 20 s and ramp mode for 20 s) (P > 0.05). In adhesive B, KH of Fast 20 s [(28.23 ± 3.67) MPa] were significantly higher than those of Fast 10 s [(14.15 ± 2.24) MPa] and Fast 15 s [(17.63 ± 2.17) MPa] (P < 0.05). The µTBS of Fast 20 s [(42.52 ± 3.59) MPa] were significantly higher than those of Fast 10 s [(24.21 ± 3.60) MPa], Fast 15 s [(22.25 ± 4.16) MPa] and Ramp 20 s [(31.12 ± 5.40) MPa] (P < 0.05). In Fast 20 s and Ramp 20 s modes, there were no statistical difference in KH of adhesive A and B, while µTBS of adhesive B were higher than that of adhesive A(P < 0.05).

CONCLUSIONSAs for different type dentin adhesives, the appropriate curing time in fast mode is different, and ramp mode (soft start curing mode) has no advantage over fast mode.

Dentin-Bonding Agents ; Hardness ; Humans ; In Vitro Techniques ; Light-Curing of Dental Adhesives ; methods ; Tensile Strength

OBJECTIVETo evaluate the short term clinical results of scaling and root planning (SRP) only, SRP combined with amoxicillin (AMX) and metronidazole (MTZ) after supragingival scaling or after SRP in the treatment of aggressive periodontitis (AgP).

METHODSA total of 45 patients with AgP were randomly divided into SRP group, SRP with AMX + MTZ after supragingival scaling group and AMX + MTZ after SRP group. Subgingival scaling and root planning were performed one week after supragingival scaling and finished within 1 month. AMX and MTZ were given for 7 days immediately after supragingival scaling or the last time of SPR. Clinical examinations including probing depth (PD), attachment level (AL) and bleeding index (BI) were performed at baseline and 8 weeks after non-surgical periodontal treatment by the same examiner.

RESULTSThere were more PD reduction and AL gain in both AMX + MTZ after supragingival scaling group and AMX + MTZ after SRP group compared with SRP group [2.5 (1.8, 3.3) mm, 2.3 (1.9, 2.7) mm vs. 1.8 (1.3, 2.1) mm, P < 0.05]; [0.9 (0.5, 1.4) mm, 0.8 (0.4, 1.3) mm vs. 0.4 (0.2, 1.0) mm, P < 0.05]. In sites PD ≥ 7 mm, PD reduction was more in AMX + MTZ after supragingival scaling group than AMX + MTZ after SRP group [4.0 (3.0, 5.0) mm vs. 4.0 (3.0, 4.0) mm, P < 0.05)].

CONCLUSIONSThe combined use of AMX and MTZ during non-surgical periodontal treatment for patients with AgP was effective in short term. In patients with most sites PD ≥ 7 mm, AMX and MTZ could be taken after supragingival scaling, but the long-term clinical effects needs further investigation.

Adult ; Aggressive Periodontitis ; drug therapy ; therapy ; Amoxicillin ; therapeutic use ; Anti-Bacterial Agents ; therapeutic use ; Anti-Infective Agents ; therapeutic use ; Combined Modality Therapy ; Dental Scaling ; Drug Therapy, Combination ; Female ; Humans ; Male ; Metronidazole ; therapeutic use ; Root Planing ; Time Factors ; Young Adult

OBJECTIVETo evaluate clinical therapeutic effect of acupuncture at Fengchi (GB 20) and Tianzhu (BL 10) on vertebrobasilar insufficiency (VBI).

METHODSOne hundred and sixteen cases of VBI were randomly divided into 2 groups, 58 cases in each group. The treatment group were treated with acupuncture at Fengchi (GB 20) and Tianzhu (BL 10), and the control group with oral administration of Nimodipine. Clinical symptoms, and the average blood flow rates of left vertebral artery (LVA), right vertebral artery (RVA) and basilar artery (BA) detected by transcranial Doppler's method (TCD) before and after treatment were investigated.

RESULTSThe total effective rate was 89.66% in the treatment group and 86.21% in the control group. Acupuncture had significantly therapeutic effect in improvement of clinical symptoms and the average blood flow rate of BA, better than Nimodipine.

CONCLUSIONAcupuncture at Fengch; (GB 20) and Tianzhu (BL 10) has obvious therapeutic effect on vertebrobasilar insufficiency.

Acupuncture Points ; Acupuncture Therapy ; Basilar Artery ; Humans ; Vertebral Artery ; Vertebrobasilar Insufficiency ; therapy