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OBJECTIVE： To establish a method for simultaneous determination of contents of β-pinene， linalool， L-camphor， L-borneol， β-caryophyllene and xanthoxylin in the oil of Blumea balsamifera. METHODS： GC method was adopted. The determination was performed on RTX-1701 capillary column （programmed temperature）. The FID detector was controlled at 240 ℃. The inlet temperature was set at 240 ℃. The carrier gas was high-purity nitrogen 3 mL/min. The the sample size was 0.5 μL， and split ratio was 50 ∶ 1. RESULTS： The linear range of β-pinene， linalool， L-camphor， L-borneol， β-caryophyllene and xanthoxylin were 0.029 7-0.267 1 mg/mL （r＝0.999 9）， 0.024 3-0.218 9 mg/mL （r＝0.999 9）， 0.126 0-1.134 0 mg/mL （r＝0.999 9）， 0.217 2-1.954 8 mg/mL （r＝0.999 9）， 0.136 3-1.226 9 mg/mL （r＝0.999 9）， 0.044 5-0.400 3 mg/mL（r＝0.999 5）， respectively. The limits of quantitation were 0.028 5， 0.008 7， 0.018 6， 0.016 8， 0.014 5， 0.042 1 mg/mL； the limits of detection were 0.009 4， 0.002 9， 0.006 1， 0.005 5， 0.004 8， 0.013 9 mg/mL， respectively. RSDs of precision， stability， reproducibility and durability tests were all lower than 3％. The average recoveries were 98.13％-101.30％（RSD＝1.20％，n＝9），98.44％-101.81％（RSD＝1.28％，n＝9），98.26％-101.05％（RSD＝1.19％，n＝9），99.08％-101.58％（RSD＝0.89％，n＝9），98.66％-101.66％（RSD＝1.17％，n＝9），98.84％-103.60％（RSD＝0.96％，n＝9）， respectively. The contents of 6 components in the sample were 14.552-46.766， 16.951-22.096， 80.597-113.115， 205.224-242.537， 47.761-135.697， 26.493-45.771 mg/g， respectively. CONCLUSIONS： The established method is simple， accurate， precise and reproducible， which can be used for simultaneous determination of contents of 6 components in the oil of B. balsamifera. It can provide reference for comprehensive evaluation and extraction technology study of the oil of B. balsamifera.
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Objective To evaluate the therapeutic effect of antibiotics combined with recombinant tuberculosis vaccine AEC/BC02 on Mycobacterium tuberculosis infection in guinea pigs. Methods Two weeks after guinea pigs were challenged subcutaneously with a high dose of Mycobacterium tuberculosis,the guinea pigs with the positive skin test responses to the recombinant ESAT6-CFP10 allergen were randomly di-vided into four groups:normal saline (NS),AEC/BC02,antibiotics and antibiotics+AEC/BC02. In antibiotics+AEC/BC02 group,guinea pigs firstly received isoniazid ( INH) and rifapentine ( RFT) treatment once a week for a total of three times,and then were immunized with a single dose of AEC/BC02 vaccine six times at 10-day intervals. Guinea pigs in AEC/BC02 and antibiotics groups were respectively vaccinated with AEC/BC02 vaccine and given INH and RFT treatment at the same dose and frequency as given to antibiotics+ AEC/BC02 group. Thirteen weeks after challenge,all guinea pigs were sacrificed for necropsy. Results The gross pathological scores of NS,AEC/BC02,antibiotics and antibiotics+AEC/BC02 groups were 83±8,77± 22,45±28 and 19±14,respectively. Antibiotics+AEC/BC02 group had a significantly lower gross pathological score than antibiotics,AEC/BC02 and NS groups (P<0. 05,P<0. 01,P<0. 01,respectively). Moreover,the gross pathological scores of antibiotics and AEC/BC02 groups were significantly decreased as compared with that of NS group (both P<0. 01). However,there was no significant difference between AEC/BC02 and NS groups. The spleen bacterial load of antibiotics+AEC/BC02 group was (2. 50±1. 26) lg CFU,which was sig-nificantly lower than those of NS [(4. 92+0. 52) lg CFU],AEC/BC02 [(4. 78+0. 84) lg CFU] and antibi-otics [(4. 39+0. 50) lg CFU] groups (P<0. 01). Compared with NS group,antibiotic and AEC/BC02 groups showed no significant difference in spleen bacterial load. Histopathological changes indicated different levels of granulomatous lesions appeared in lung tissues of all groups and the most severe change was ob-served in AEC/BC02 group,followed by that in NS,antibiotics and antibiotics+AEC/BC02 groups. Conclu-sion INH and RFT treatment in combination with AEC/BC02 vaccine in the treatment of guinea pigs with Mycobacterium tuberculosis infection was superior to either treatment alone as it significantly alleviated organ lesions and lowered the bacterial loads in spleen and lung.
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Objective To identify the cross-reactive antigens shared by Mycobacteria smegmatis(MS) and Mycobacteria tuberculosis(MTB) and to analyze their antigenicity.Methods Bacterial antigens were extracted from strains of MS and MTB by ultrasonication.Western blot assay was performed to analyze common antigens that reacted with both of the antiserum samples against MS and MTB.The extracted bacterial antigens were mixed with incomplete Freund′s adjuvant and then were injected into muscles of mice.Cytokines secreted by murine spleen lymphocytes following stimulation with various antigens of MS and MTB were determined by ELISPOT and flow cytometry on the 7th day.IgG levels in serum samples were detected by ELISA 7 days after injection.Results There were cross-reactive antigens shared by MS and MTB.Potent humoral immune responses and cellular immunity against both MS and MTB could be induced by those cross-reactive antigens after sensitization the mice by either MS or MTB antigens.Cytokines of IL-2 and IFN-γ in CD4+ and CD8+T cells of mice stimulated with MS or MTB antigens were significantly increased as compared with those of non-sensitization group and those of Brucella antigens stimulation group.ConclusionCross-reactive antigens shared by MS and MTS can effectively promote specific immune reactions to the infection of MTB, which provides a scientific basis for the development of tuberculosis vaccines.
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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 establish a suitable guinea pig model for evaluating the protective effects of new therapeutic TB vaccines in preclinical study .Methods The guinea pigs were subcutaneously injected with single-cell suspension of multi-drug resistant M.tuberculosis at a dose of 1000 CFU, 0.2 ml per animal.The study was divided into experimentⅠand experimentⅡ.In experimentⅠ, the guinea pigs were given immuno-therapy and/or chemical treatment on day 3 after infection .Four guinea pigs in each group were dissected at weeks 5, 7 and 9 after infection for evaluating lesion scores and histopathology changes of liver , spleen and lung, as well as bacterial load in spleen .In experimentⅡ, the guinea pigs were given immunotherapy and/or chemical treatment with different doses on day 14 after infection .All guinea pigs were dissected at week 5 after infection for evaluating lesion scores of liver , spleen and lung , as well as bacterial load in spleen .Results In experimentⅠ, all of the three treatments including immunotherapy combined with chemotherapy , immunotherapy alone and chemotherapy alone could effectively prevent organ lesion and reduce bacterial load in spleen , which were significantly different from negative control group .The immunotherapy combined with chemotherapy showed better treatment effects than other two treatments .Along with a prolonged period after drug withdrawal , the de-gree of organ lesion in immunotherapy group and chemotherapy group rebounded sharply , but only slightly in im-munotherapy combined with chemotherapy group .In experimentⅡ, animals in all treatment groups showed alle -viated organ lesion and reduced bacterial load in spleen .A relatively better treatment effect was observed in im-munotherapy combined with chemotherapy group .Conclusion The established guinea pig model of M.tubercu-losis infection showed an advantage of good repeatability .It might be used to evaluate the protective effects of new therapeutic vaccines against tuberculosis in preclinical study .
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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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Objective: To study the effect of helicobacter pylori on gastric mucosal cell proliferation in gastritis. Methods: Fifty-six gastritis patients with or without Helicobacter pylori infection (Hp+ 27; Hp- 29) were selected. The expression of proliferation cell nuclear antigen (PCNA), epidermal growth factor receptor (EGFR), transforming growth factor beta receptor type Ⅰand typeⅡ(TGF?RⅠ, TGF?RⅡ) in gastric mucosa were examined by immunohistochemical method. Results: The PCNA and EGFR were significantly higher in Hp positive chronic gastritis patients than in Hp negative ones(P
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Objective: To study the clinical features of primary biliary cirrhosis (PBC) in order to facilitate cognition of the disease. Methods: Clinical data of 42 patients clinically and/or histologically diagnosed with PBC were reviewed. Anti mitochondrial antibody (AMA) negative/positive patients as well as the patients who were/were not associated with Sj?gren Syndrome (SS) were compared in terms of clinical, biochemical and immunological features. Results: Among the 42 patients, 78.6%(33/42) of the cases were females; the mean age at diagnosis was (61.1?10.8) years. The most frequent symptoms were fatigue. Serum alkaline phosphatase (ALP), ?-glutamyltranspeptidase (?-GT) and total bile acid (TBA) levels were markedly elevated in the majority of the patients, whereas ALT and AST levels were mildly to moderately elevated. Thirty-one patients had a total bilirubin (TBil) level above normal. The levels of TBil and prothrombin time had positive correlationship with years of the course (P=0.000, r=0.696; P=0.005, r=0.424), whereas serum albumin level had negative correlationship with years of the course (P=0.002, r=-0.462). Thirty-seven patients had elevated serum IgM and 34 patients were AMA/AMA-M2 positive. AMA negative and AMA positive patients were similar in terms of clinical manifestations and liver biochemistries findings. Serum IgM and IgA levels were significantly lower, whereas total cholesterol level was higher in AMA negative patients when compared with AMA positive cases. Fifteen cases were associated with SS, which were similar in terms of clinical, biochemical and immunological features when compared with the PBC patients were not associated with SS. Conclusion: PBC is mostly found in middle aged and old women. Elevated serum ALP, TBA and ?-GT levels together with positive AMA/AMA-M2 can help to diagnose PBC. AMA negative PBC patients are characterized by relatively lower serum IgM and IgA levels and higher total cholesterol level. PBC patients who are associated with SS have not substantial differences in the clinical, biochemical and immunological spectra of the disease.
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Objective To study a new and rapid method for detection of anti-histoplasma antibody by dot immunogold filtration assay (DIGFA). Methods DIGFA was developed by coating purified protein derivative of histoplasmin (P-HTPM) on nitrocellulose membrane as membrane antigen and labeling SPA with colloidal gold. Anti-histoplasma antibodies in sera from mice immunized with Histoplasma were detected by DIGFA. Results All immunizedsera with Histoplasma were positive by DIGFA as well as the results detected by ELISA. The sera immunized respectively with Monilia and Penicillium marneffei were negtive by DIGFA while 1 of 8 immunizedsera with Blastomyces dermatitidis and 2 of 9 immunizedsera with Paracoccidioides brasiliensis were found to be positive. Conclusion DIGFA was a valuable and rapid method to detect histoplasmaantibody in serum.
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Objective Zinc sulfate has anti inflammatory action in many animal models, however, the effects of zinc in colitis remained uncertain. The present study was to evaluate the role of zinc sulfate in experimental colitis and probe into its underlying mechanisms. Methods Colitis was induced by administrating 2,4,6 trinitrobenzenesulphonic acid (TNB) rectally in Spragur Dawley female rats. Beginning at the first day of TNB colitis, the rats were treated with zinc sulfate enema once daily for 6 days. The rats were sacrificed at days 8. The effects of zinc sulfate were evaluated by examining mucosal lesion area, mucosal myeloperoxidase (MPO) and superoxide dismutase (SOD) activities, mucosal prostaglandin E 2(PGE 2) and leukotriene B 4(LTB 4) levels. Results TNB induced severe colitis as evidenced by increased mucosal lesion area, mucosal MPO activity and PGE 2 and LTB 4 levels. Six days after the application of the zinc sulfate enema, the mucosal lesion area, MPO activity, PGE 2 and LTB 4 levels were all decreased significantly, except mucosal SOD activity that was remained unchanged after zinc treatments. Conclusions The data suggest that zinc sulfate enemas have an anti inflammatory action on experimental colitis through the mechanism other than increasing SOD activity.
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Objective Effects of melatonin and serotonin on ethanol induced ulceration in the rat stomach were investigated. Methods Serotonin was injected subcutaneously, then melatonin solution and finally ethanol was put into the ex vivo gastric lumen to prepare the experimental models. Glandular mucosal blood flow (GMBF) and the gastric mucosal injury were observed. Results Melatonin and serotonin administration did not induce observable gastric mucosal of damage in the ex vivo stomach, but the serotonin reduced glandular mucosal blood flow (GMBF) with dose dependence in this organ. Ethanol reduced GMBF and induced visible glandular mucosal damage. The latter effect was prevented by melatonin pretreatment. Serotonin pretreatment aggravated the gastric mucosal injury and GMBF changes induced by ethanol, but these effects were partially reversed by melatonin. Conclusion The findings indicate that the GMBF and gastric injury are related, the reduction in GMBF, however, may not be the sole factor responsible for ulceration. The antagonistic effects of melatonin on serotonin action on the stomach suggest that melatonin may play a modulator role for serotonin action on the gastrointestinal tract.