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Objective To establish a suitable guinea pig model for evaluating the protective effects of new TB vaccines in BCG prime-boost regimen .Methods Two different immunization strategies by using the recombinant TB vaccine were employed to boost BCG primed guinea pigs in this study .One was for short-term evaluation with 14 weeks interval between prime and boost immunization and another was for long -term evaluation with 54 weeks interval .In the short-term evaluation group , guinea pigs were boosted twice with the recombinant TB vaccine ( AEC/BC02 ) in every two weeks , while guinea pigs in the long-term evaluation group were boosted for three times with two weeks interval between each injection .A negative con-trol group ( NS→NS) and a BCG control group ( BCG→NS) were both set up in two evaluation groups .One week after the last immunization , all guinea pigs were challenged with M.tuberculosis.Six to seven weeks after bacteria challenge , all animals were euthanized and dissected to evaluate lesion scores of liver , spleen and lung, as well as the viable bacterial load in spleen .Results In the short-term evaluation group , the le-sion scores in those boosted with vaccine (3.33±5.00) was lower than that of BCG control group (5.56± 7.27) (P>0.05) and negative control group (47.00±28.11) (P=0.0001).The difference between BCG control group and negative control group in lesion score was also significant .The animals in vaccine boosted group had lower bacterial loads (0.78±1.55 log10 ) in spleen than that in BCG control group (1.06±1.87) (P>0.05) and negative control group (5.47±0.61) (P=0.0003).In the long-term evaluation group, the lesion score in those boosted with vaccine was lower (5.0±7.6) than that in BCG control group (14.4± 13.5) (P=0.0394) and negative control group (56.9±14.1) (P0.05) and negative control group (5.43±0.56) (P=0.01).There was a significant difference in bacterial load between BCG control group and negative group (P=0.0089).Conclusion The results suggest that the interval time between BCG-prime and boost immunization should be properly prolonged in the guinea pig model used for evaluating the protective effects of new TB vaccines in BCG prime -boost regimen .
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Objective To establish a guinea pig model of latent Mycobacterium tuberculosis infec-tion for evaluating the effects of therapeutic vaccines .Methods Guinea pigs were subcutaneously inocula-ted with 5.0×103 CFU Mtb.The skin test was performed with 0.5μg recombinant ESAT6-CFP10 protein to detect positive conversion rates at different time points .Two weeks after Mtb inoculation , guinea pigs in model group received 5 mg isoniazid treatment ( three times a week for four weeks ) by oral gavage , while those in control group received normal saline .At the sixth week after Mtb infection , guinea pigs with and without isoniazid treatment were dissected for pathology examination .The pathological scores of liver , spleen and lung, as well as bacteria loads in spleen were compared between two groups .The established guinea pig model of latent infection was then validated by testing two reference vaccines ( AEC/BC02 and AEC/BC03 ) . Results Two weeks after Mtb inoculation , all guinea pigs showed positive EC skin test with induration area of (19.9±3.0) mm.Upon four weeks of isoniazid treatment , the guinea pigs in model group showed no pathological changes with zero scores in the examined organs .No bacterium was detected in spleen of ani-mals from model group.However, the total pathological score was 38.8±16.5 and bacteria load in spleen was (5.1±0.3) Log10 CFU with the guinea pigs from control group .Natural recurrence of tuberculosis in model group was observed after drug withdrawal .The total pathological scores were 48.5±23.9 and 51.3± 23.41.The bacterial loads in spleen were (4.5±1.3) and (4.2±1.1) Log10 CFU and bacterial loads in lung were (4.1±1.2) and (3.4±1.3) Log10 CFU respectively as verified with reference vaccines of AEC /BC02 and AEC/BC03.Conclusion Isoniazid treatment inhibited the proliferation of inoculated Mtb in guinea pigs.A guinea pig model of latent Mycobacterium tuberculosis infection is successfully established with an advantage of good repeatability .Therefore, it can be used to evaluate the effects of therapeutic vaccines on latent Mycobacterium tuberculosis infection.
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Objective To comparatively evaluate the effects of a recombinant Mtb protein ESAT 6-CFP10 ( rESAT6-CFP10 ) and a purified protein derivative ( PPD ) as skin test reagents in guinea pigs . Methods Guinea pigs were sensitized with different Mycobacteria species .After sensitization , all guinea pigs were intradermally injected with rESAT6-CFP10 and PPD.At 48 h after the injection, the size of ery-thema at injection sites was measured by using a double-blind method .For guinea pigs sensitized with viable Mtb, the size of erythema at injection sites were measured at 24 h after the injection .The positive conversion rates of skin test with rESAT 6-CFP10 and PPD were calculated .Results The results of PPD skin test were positive in all guinea pigs sensitized with viable Mtb , killed Mtb and BCG with erythema diameters of (11.4 ±0.9) mm, (11.8±1.1) mm and (13.2±0.8) mm, respectively.Positive skin test with rESAT6-CFP10 was only observed in guinea pigs infected by viable Mtb-showing erythema diameters of (13.7±5.7) mm. The skin test with rESAT6-CFP10 was negative in guinea pigs sensitized by killed Mtb-and vaccinated by BCG.The skin tests by using rESAT6-CFP10 and PPD were performed on randomly selected guinea pigs at ninth day after infection by Mycobacterium tuberculosis H37Rv.At the 2nd week, totally 24 selected guinea pigs showed positive skin test results with rESAT6-CFP10 (24/24) with erythema diameters of (19.9± 3.0) mm, while only 15 out of 24 had positive PPD skin test with erythema diameters of (6.1±5.5) mm. At the 4th week, all guinea pigs showed positive PPD skin test (3/3) with erythema diameters of (12.7± 2.5) mm.Conclusion The skin test by using recombinant ESAT 6-CFP10 protein can effectively distin-guish viable Mtb infection from BCG vaccination and killed Mtb sensitization , which is a more suitable anti-gen than PPD for the early diagnosis of Mtb infection .
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ObjectiveTo investigate the learning curve of total thoracoscopicy cardiac surgery.MethodsClinical data of a succession of 125 patients undering total thoracoscopicy ASD and VSD repair between October 2004 to January 2010 were collected and reviewed.The procedure was perfomed by the same surgeon.The patients were divided equally into 5 groups (groupA,B,C,D and E,n =25 in each group ) according to the sequence of the operation.The operative time,extracorporeal circulation time,aortic cross-clamped time,the rate of conversion rate to thoracotomy and postoperative complications were compared between the 5 groups.ResultsThere were no statistically significant differences between the 5 groups with respect to age,gender,weight,dieases and surgical approach(P ＞ 0.05).The operative time,extracorporeal circulation time and aortic clump time in group A and group B significant longer then that in group C,group D and group E(P ＜0.05).Group A and group B was no statistically significant difference each other( P ＞ 0.05 ).Group C,D and E have no statistically significant differences between(P ＞0.05 ).The rate of conversion rate to thoracotomy and postoperative complications in 5 groups have no statistically significant differences comparative( P ＞ 0.05 ).ConclusionThe learning curve of total thoracoscopicy surgery is approximalely 50 cases.
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Objective To establish guinea pig model of type Ⅰ anaphylaxis, isolate the anaphy-lactic antibody(IgE and IgG) preliminarily from sera of sensitized guinea pigs, and investigate its functional characteristics. Methods Animal model was established by sensitizing guinea pigs with OVA and Al(OH)_3, level of antibody was determined by ELISA, IgE and IgG in sera were preliminarily isolated through saturated ammonium sulphate precipitate and affinity chromatograph of Protein A. A continuous passive cutaneous ana-phylaxis test (PCA) was performed by sensitizing guinea pigs individually with IgE and IgG and then challenging at different time , and the variation of blue spots in skin were observed after challenge . Results Concentration of IgE in model group and control group were 719.3750 ng/ml and 2.5250 ng/ml, the optical density of IgG in model group and control group were 0.9921 and 0.0174, the level of two antibodies in model group were significantly higher than that of control group (P<0.05). In 9 d continuous PCA test, the blue spot induced by IgE in skin lasted for 9 days and appeared the largest when challenged at day 5. The diameter of blue spot induced by IgG was the largest when challenged at day 2 and then decreased fast. Con-clusion Anaphylactic antibodies were successfully preliminarily isolated from sera of sensitizing guinea pig, both IgE and IgG play roles in type Ⅰ anaphylaxis of guinea pig, the hypersensitive reaction induced by IgG is fast and short than that induced by IgE, and IgG may become an important surrogate marker in immunotoxic-ity evaluation(type Ⅰ anaphylaxis)of vaccine.