Sensitivity, Specificity, and Safety of a Novel ESAT6-CFP10 Skin Test for Tuberculosis Infection in China: 2 Randomized, Self-Controlled, Parallel-Group Phase 2b Trials.

BACKGROUND: Diagnostics to identify tuberculosis infection are limited. We aimed to assess the diagnostic accuracy and safety of ESAT6-CFP10 (EC) skin test for tuberculosis infection in Chinese adults. METHODS: We conducted 2 randomized, parallel-group clinical trials in healthy participants and tuberculosis patients. All participants were tested with the T-SPOT.TB test, then received an EC skin test and tuberculin skin test (TST). The diameter of skin indurations and/or redness at injection sites were measured at different time periods. A bacillus Calmette Guerin (BCG) model was established to assess the diagnosis of tuberculosis infection using an EC skin test. RESULTS: In total, 777 healthy participants and 96 tuberculosis patients were allocated to receive EC skin test at 1.0 µg/0.1 mL or 0.5 µg/0.1 mL. The area under the curve was 0.95 (95% confidence interval [CI], .91-.97) for the EC skin test at 1.0 µg/0.1 mL at 24-72 hours. Compared with the T-SPOT.TB test, the EC skin test demonstrated similar sensitivity (87.5, 95% CI, 77.8-97.2 vs 86.5, 95% CI, 79.5-93.4) and specificity (98.9, 95% CI, 96.0-99.9 vs 96.1, 95% CI, 93.5-97.8). Among BCG vaccinated participants, the EC skin test had high consistency with the T-SPOT.TB test (96.3, 95% CI, 92.0-100.0). No serious adverse events related to the EC skin test were observed. CONCLUSIONS: The EC skin test demonstrated both high specificity and sensitivity at a dose of 1.0 µg/0.1 mL, comparable to the T-SPOT.TB test. The diagnostic accuracy of the EC skin test was not impacted by BCG vaccination. CLINICAL TRIALS REGISTRATION: NCT02389322 and NCT02336542.

Efficacy and safety of recombinant Mycobacterium tuberculosis ESAT-6 protein for diagnosis of pulmonary tuberculosis: a phase II trial.

BACKGROUND: This study aimed to determine the efficacy and safety of recombinant Mycobacterium tuberculosis ESAT-6 protein for diagnosis of pulmonary tuberculosis (TB). MATERIAL AND METHODS: A phase II trial was performed in 158 patients with pulmonary TB (145 initially-treated and 13 re-treated) and 133 healthy subjects. Skin testing was carried out by injecting purified protein derivative (PPD) (on left forearm) or recombinant ESAT-6 protein at a dosage of 2, 5, or 10 µg/mL (on the right forearm) in each subject. Reaction activity and adverse events were monitored at 24, 48, and 72 h following the injection. Receiver operating characteristic curves were plotted to determine the areas under the curves (AUCs) and the cut-off induration diameters for the optimal diagnostic performance. RESULTS: The reaction activity was significantly increased upon recombinant ESAT-6 injection in pulmonary TB patients compared with healthy subjects. In pulmonary TB patients, the reaction was dose-dependent, and at 48 h, 10 µg/mL recombinant ESAT-6 produced a reaction similar to that produced by PPD. The AUCs for a 10 µg/mL dosage were 0.9823, 0.9552, and 0.9266 for 24 h, 48 h, and 72 h, respectively, and the induration diameters of 4.5-5.5 mm were the optimal trade-off values between true positive rates and false positive rates. No serious adverse events occurred in any subjects. CONCLUSIONS: Recombinant ESAT-6 protein is efficacious and safe for diagnosing pulmonary TB. Based on the reaction, performance, safety, and practicability, we recommend that 10 µg/mL at 48 h with an induration cut-off value of 5.0 mm be used.

Method validation of a bridging immunoassay in combination with acid-dissociation and bead treatment for detection of anti-drug antibody.

Anti-drug antibody (ADA) positivity is correlated with disease relapse risk when treated with monoclonal antibody (mAb) therapeutics. ADA evaluation can assist with interpreting pharmacokinetic, pharmacological, and toxicology results. Here, we established an ADA assay based on two steps of acid dissociation combined with a bridging immunoassay to provide a comprehensive validation strategy. The three-tiered sample analysis process included screening, confirmation, and titration assays using therapeutic HLX26 (targeting lymphocyte activation gene-3 [LAG-3]) as an example. The cut points were determined by testing 50 individual normal human serum samples, including screening cut point (SCP) (SNR: 1.08), confirmatory cut point (CCP) (% inhibition: 12.65), and titration cut point (TCP) (sample-to-noise ratio [SNR]: 1.17). The assay sensitivity, low positive control (LPC), and high positive control (HPC) titer acceptable range were also set up as 33.0 ng/mL, 41.0 ng/mL, and 320-1280, respectively. After full validation, both the intra-assay and inter-assay precision testing passed with coefficient of variations (CVs) < 20%. The assay enabled excellent drug tolerance up to 768.0 µg/mL at the HPC level and 291.0 µg/mL at the LPC level, while the tolerance of target interference was up to 74.0 ng/mL of soluble LAG3. Moreover, no false-positive results were observed in the presence of 5% hemolyzed serum samples and 150 mg/dL of triglyceride in the serum samples, no hook effect was observed, and the stability performed normally under room temperature for 24 h, 2-8 °C for 7 d, and six freeze/thaw cycles. In summary, this ADA assay is feasible and could be used for evaluating the immunogenicity of HLX26 in clinical trials.

[The effect of various antigen compounds of tuberculosis composite antigen vaccine on the immune function in mice].

OBJECTIVE: To study the immune function of mice immunized by different combinations of antigen 85b (Ag85b), fusion protein culture filtered protein 10 (CFP-10), early secreted antigenic target 6 kDa protein (ESAT-6) and heat shock protein X (Hsp X) with combined adjuvants of Bacille Calmette-Guerin (BCG) CpG and aluminum. METHODS: According to antigen combinations, 48 BALB/c mice were divided into 8 groups: (1) group A: Ag85b + CFP-10/ESAT-6 + HspX + adjuvant; (2) group B: CFP-10/ESAT-6 + HspX + adjuvant; (3) group C: Ag85b + HspX + adjuvant; (4) group D: Ag85b + CFP-10/ESAT-6 + adjuvant; (5) group E: Ag85b + adjuvant; (6) group F: CFP-10/ESAT-6 + adjuvant; (7) group G: HspX + adjuvants; (8) control group: saline (6 mice per group). The mice were subcutaneously immunized 3 times. One week after the third subcutaneous immunization, spleens were collected for enzyme-linked immunospot (ELISPOT) assay to detect IFN-γ and IL-4 secretion, and for the lymphocyte proliferation assay to observe antigen-specific lymphocyte proliferation. Serum samples were separated for enzyme-linked immunosorbent assay (ELISA) to detect the titers of antigen-specific IgG, IgG(1) and IgG(2a) antibodies. RESULTS: The amount of IFN-γ spots in Group E [median(quartile), 122.8 (78.4 - 184.4)] was significantly more than that in group C [14.3 (6.5 - 14.6)] and the control group [0.5 (0.5 - 1.3)] (u = 0.0, P < 0.01). The amount of IL-4 spots in Group D stimulated with Ag85b and CFP-10/ESAT-6 [173.5 (78.8 - 233.4), 132.8 (50.3 - 159.4)] were significantly more than those in the control group [0.5 (0.5 - 1.3), 5.3 (2.9 - 6.5)] (u = 0.0, P < 0.01). The level of stimulation index of lymphocyte proliferation in Group A, C, D, E (2.42 ± 0.50, 2.18 ± 0.37, 2.86 ± 0.51, 2.70 ± 0.15) was significantly higher than that of the control group (1.11 ± 0.13) (F = 20.96, P < 0.01). The level of antigen-specific IgG, IgG(1), IgG(2a) antibody titers induced by Hsp X [lg(antibody dilution degree), 3.90 - 5.21] was significantly higher than those induced by Ag85b (3.30 - 4.51) and CFP-10/ESAT-6 (3.10 - 4.05) (F = 63.8 - 70.4, P < 0.01). CONCLUSIONS: With the use of adjuvants, different antigen combinations showed different influences on the immune function in mice. A combination of 3 antigens did not elicit the best immune effect, suggesting that the interaction among antigens may affect their immunity.

[A guinea pig model of latent Mycobacterium tuberculosis H37Rv infection].

OBJECTIVE: To establish the guinea pig model of latent Mycobacterium tuberculosis (MTB) H37Rv infection, and to study the multiplication dynamics of MTB in vivo, and the relationship between latent MTB infection and PPD skin test. METHODS: Sixty-two guinea pigs were randomly divided into the model group (n = 42) and the control group (n = 20), and the model group was subdivided into a 4 weeks group (n = 12), an 8 weeks group (n = 21) and a 12 weeks group (n = 9), challenged by 500 CFU H37Rv with restored toxicity. After 2 weeks challenge, the model groups were treated with isoniazid (INH, 10 mg/kg) + pyrazinamidum aldinamide (PZA, 40 mg/kg) for 4 weeks, 8 weeks and 12 weeks respectively. The natural recurrence of tuberculosis was observed in the model 4 weeks group, and the natural and induced recurrence by dexamethasone was observed in the model 8 weeks group and 12 weeks group. PPD skin test, the pathologic changes, and MTB quantity of organs were observed. RESULTS: In the control group, the average MTB quantity of spleen was 3.3 lg CFU after 2 weeks challenge, and the average MTB quantity of spleen and lung in guinea pigs were 4.5 lg CFU and 1.8 lg CFU respectively after 6 weeks challenge, and they reached 5.3 lg CFU and 5.4 lg CFU at 18 weeks respectively. The latent MTB infection of the model 4 weeks group recurred naturally 12 weeks after stopping treatment. The latent MTB infection of the model 8 weeks group recurred naturally and by dexamethasone treatment. The latent MTB infection of the model 12 weeks group did not recur naturally, but dexamethasone induced recurrence. The positive PPD response correlated with recurrence. CONCLUSIONS: A latent MTB infection model was established successfully by H37Rv challenge and treatment with INH and PZA. The latent MTB infection may recur naturally or by induction. The PPD response was related to tuberculosis recurrence.

A Computational Method for the Identification of Endolysins and Autolysins.

BACKGROUND: Cell lytic enzyme is a kind of highly evolved protein, which can destroy the cell structure and kill the bacteria. Compared with antibiotics, cell lytic enzyme will not cause serious problem of drug resistance of pathogenic bacteria. Thus, the study of cell wall lytic enzymes aims at finding an efficient way for curing bacteria infectious. Compared with using antibiotics, the problem of drug resistance becomes more serious. Therefore, it is a good choice for curing bacterial infections by using cell lytic enzymes. Cell lytic enzyme includes endolysin and autolysin and the difference between them is the purpose of the break of cell wall. The identification of the type of cell lytic enzymes is meaningful for the study of cell wall enzymes. OBJECTIVE: In this article, our motivation is to predict the type of cell lytic enzyme. Cell lytic enzyme is helpful for killing bacteria, so it is meaningful for study the type of cell lytic enzyme. However, it is time consuming to detect the type of cell lytic enzyme by experimental methods. Thus, an efficient computational method for the type of cell lytic enzyme prediction is proposed in our work. METHODS: We propose a computational method for the prediction of endolysin and autolysin. First, a data set containing 27 endolysins and 41 autolysins is built. Then the protein is represented by tripeptides composition. The features are selected with larger confidence degree. At last, the classifier is trained by the labeled vectors based on support vector machine. The learned classifier is used to predict the type of cell lytic enzyme. RESULTS: Following the proposed method, the experimental results show that the overall accuracy can attain 97.06%, when 44 features are selected. Compared with Ding's method, our method improves the overall accuracy by nearly 4.5% ((97.06-92.9)/92.9%). The performance of our proposed method is stable, when the selected feature number is from 40 to 70. The overall accuracy of tripeptides optimal feature set is 94.12%, and the overall accuracy of Chou's amphiphilic PseAAC method is 76.2%. The experimental results also demonstrate that the overall accuracy is improved by nearly 18% when using the tripeptides optimal feature set. CONCLUSION: The paper proposed an efficient method for identifying endolysin and autolysin. In this paper, support vector machine is used to predict the type of cell lytic enzyme. The experimental results show that the overall accuracy of the proposed method is 94.12%, which is better than some existing methods. In conclusion, the selected 44 features can improve the overall accuracy for identification of the type of cell lytic enzyme. Support vector machine performs better than other classifiers when using the selected feature set on the benchmark data set.

[Effect of Mycobacterium smegmatis vaccine on the level of nitric oxide produced by peritoneal macrophages in mice].

OBJECTIVE: To study the effect of Mycobacterium smegmatis vaccine on the level of nitric oxide (NO) produced by peritoneal macrophages in immunized mice. METHODS: Balb/c mice were randomized into low-dose, middle-dose, and high-dose groups (injected with different doses of Mycobacterium smegmatis vaccine) and a control group (injected with normal saline). Then the peritoneal macrophages were cultured with lipopolysaccharide in vitro. The supernatants were collected and the concentrations of NO were analyzed through the reaction with Griess reagents. RESULTS: The levels of NO produced by the peritoneal macrophages in the control group, low-dose group, middle-dose group, and high-dose group were (3.50 +/- 3.11), (16.63 +/- 6.47), (13.97 +/- 6.20), and (7.55 +/- 2.26) ng/ml, respectively. The levels of NO in all dosing groups were significantly different from that in control group (P < 0.01). CONCLUSION: Mycobacterium smegmatis vaccine can promote the peritoneal macrophages to produce NO in mice.

[Preparation of two antigens--Ag85b and HspX of Mycobacterium tuberculosis H37Rv and the effects of their co-administration with adjuvants in mice].

OBJECTIVE: To synthesize two antigens-Ag85b and HspX of Mycobacterium tuberculosis H37Rv with molecular biological methods and to observe their biologic activity after co-administration of adjuvants (aluminum and/or CpG) in mice. METHODS: Recombinant expression plasmids pET30a-Ag85b and pET30a-HspX were constructed. The objective DNA fragments was characterized with restriction enzyme. Then the recombinant plasmids were transformed into E. coli BL-21, and two proteins were expressed by induction of isopropyl beta-D-1-thiogalactopyranoside. After purification with anion exchange column Source30, QHP, and hydrophobic chromatography column, two proteins were identified by amino acid sequencing. After the successful preparation of these two antigens, they were co-administered in mice with adjuvants of aluminum and/or CpG (Ag85b, Ag85b + Al, Ag85b + CpG, Ag85b + Al + CpG; HspX, HspX + Al, HspX + CpG, HspX + Al + CpG); one group received normal saline and served as the control. Splenic lymphocytes were isolated for enzyme-linked immunosorbent spot assay to detect the secreted specific interferon-gamma (IFN-gamma); in addition, lymphocytes proliferation test was performed to observe lymphocytes proliferation after in vitro stimulated with two antigens. RESULTS: The purity of two proteins reached 95% after purification. The N-terminal amino acid sequence (15 aa) of the purified proteins was same as the target sequence. For Ag85b, the secreted specific IFN-gamma from isolated splenic lymphocytes after having been stimulated in vitro with Ag85b (80 microg/ml) remarkably increased in Ag85b + CpG group, Ag85b + Al group, and Ag85b + CpG + Al group; the changes were significantly different between these three groups and control group (P < 0.05). For HspX, the changes were significantly different between HspX + Al + CpG group and normal sodium group, although remarked increase of IFN-gamma was also observed in HspX group, HspX + Al group, and HspX + CpG group. CONCLUSIONS: Ag85b and HspX were successfully expressed and purified. A cell-mediated immunity may be induced when the antigens are co-administered with adjuvants of aluminum and/or CpG in mice, indicating that the recombinant proteins are bioactive.

Ag85b/ESAT6-CFP10 adjuvanted with aluminum/poly-IC effectively protects guinea pigs from latent mycobacterium tuberculosis infection.

The high global burden of tuberculosis (TB) underscores the urgent need for an effective TB vaccine since the only licensed Bacillus Calmette-Guérin (BCG) vaccine is ineffective in preventing adult pulmonary TB and affords no protection against latent TB infection (LTBI). Herein we investigated the potential of Mycobacterium tuberculosis (Mtb) antigen proteins AEC comprised of Ag85b and ESAT6-CFP10 proteins in conjunction with aluminum (Al) and polyriboinosinic-polyribocytidylic acid (poly-IC) as a novel subunit vaccine against TB. The immunogenicity and protection induced by the adjuvanted vaccine were evaluated in two animal models. Mice vaccinated with AEC/Al/poly-IC exhibited significant antigen-specific humoral immune responses and cell-mediated immunity as determined by immunoassay and multicolor flow cytometric assay, and the protective effect of the vaccine was demonstrated in a guinea pig model of latent Mtb infection. Compared to the control group, the mean pathological scores and bacterial loads in lungs and spleens of AEC/Al/poly-IC-immunized guinea pigs were significantly reduced. These data indicate that the AEC/Al/poly-IC is highly immunogenic in mice and can effectively protect guinea pigs against latent Mtb infection; it may represent a promising candidate vaccine for the control of latent TB.

[Analysis of the genotype of intraspecies of common Mycobacteria based on 16S-23S rRNA gene internal transcribed spacer sequences].

OBJECTIVE: To analyze the genotypes of intraspecies of common mycobacteria. METHODS: The genotypes of 94 strains of mycobacteria from DSMZ, as compared with 12 reference strains, were studied by 16S-23S rRNA internal transcription space (ITS) sequence analysis. RESULTS: The sequencing of 16S-23S rRNA ITS of the 106 strains of common mycobacteria were completed. All the mycobacteria could be discriminated to several genotypes except 4 M. intracellulare, 4 M. avium, 6 M. marinum and 2 M. malmoense which were identical to their reference strains. CONCLUSIONS: 16S-23S rRNA ITS sequence analysis is a reliable method to discriminate mycobacteria in interspecies even in intraspecies.

[The animal experimental study on rifampicin-dependent Mycobacterium tuberculosis].

OBJECTIVE: To investigate the presence of rifampicin-dependent Mycobacterium tuberculosis strains by use of a guinea pig model of tuberculosis of rifampicin-dependent Mycobacterium tuberculosis. METHODS: Guinea pigs were randomly divided into groups of infection by rifampicin-dependent Mycobacterium tuberculosis strains (1130 strain, 1219 strain, b858 strain), rifampicin-resistant Mycobacterium tuberculosis strain (1290 strain) and ATCC 35810 strain and each group was further divided into an experimental group and a control group. The guinea pigs were challenged with 1130 strain, 1219 strain, b858 strain, 1290 strain and ATCC 35810 strain to establish the tuberculosis model. The experimental groups were treated with rifampicin. The parameters including macroscopic visceral pathological change index, visceral weight index (spleen, lungs and liver), the colony-forming units (CFU) quantity of visceral Mycobacterium tuberculosis culture (spleen, lungs) and tissue pathology of guinea pigs were observed. RESULTS: At the 7th week after challenged with 1130 strain, 1219 strain, 1290 strain and b858 strain, all animals were sacrificed. The macroscopic visceral pathological change indices of the experimental group were 68.7 +/- 13.8, 60.0 +/- 13.5, 70.0 +/- 5.8 and 23.8 +/- 18.9, whereas all those parameters of the control group were 76.2 +/- 18.9, 40.0 +/- 16.8, 63.8 +/- 10.3 and 22.5 +/- 15.5 respectively, and there was no significance between the experimental group and the control group (t = 0.64, 1.85, 0.35 and 0.10, all P > 0.05). The spleen weight indices of experimental group were 0.229 +/- 0.048, 0.256 +/- 0.067, 0.324 +/- 0.054 and 0.199 +/- 0.029, whereas all those parameters of control groups were 0.278 +/- 0.025, 0.216 +/- 0.076, 0.368 +/- 0.033 and 0.213 +/- 0.038 respectively, and there was no significance between the experimental group and the control group (t = 1.75, 0.79, 1.41 and 0.57, all P > 0.05). The CFU quantity of spleen Mycobacterium tuberculosis culture of the experimental group were 4.98 +/- 0.30, 4.68 +/- 1.26, 5.07 +/- 0.47 and 3.85 +/- 0.45, whereas all those parameters of control groups were 4.90 +/- 1.03, 4.79 +/- 0.45, 5.08 +/- 0.55 and 4.23 +/- 0.95 respectively, and there was no significance between the experimental group and the control group (t = 0.11, 0.15, 0.03 and 0.73, all P > 0.05); Moreover, the tissue pathology of both groups was similar. CONCLUSIONS: The tuberculosis model of rifampicin-dependent Mycobacterium tuberculosis strains was similar to the model of rifampicin-resistant Mycobacterium tuberculosis in guinea pigs. Rifampicin-dependency was not evident in this guinea pig tuberculosis model.

[Differentiation of infection with Mycobacterium tuberculosis by recombinant Mycobacterium tuberculosis 11000 protein].

OBJECTIVE: To study the differentiation effect of recombinant Mycobacterium tuberculosis 11000 protein on infection of Mycobacterium tuberculosis. METHODS: Guinea pigs were immunized with different strains of mycobacterium, and then all guinea pigs were given intradermal injections with recombinant Mycobacterium tuberculosis 11000 protein and purified protein derivative of tuberculin (PPD) or purified protein derived from M. intracellulare (PPD-B). Skin reactions defined with two transverse diameters were read double-blinded after 24 and (or) 48 hours, and the means of the two transverse diameters were counted as the reaction diameters. RESULTS: All guinea pigs immunized with different strains of Mycobacteria responded to PPD or PPD-B with positive skin reactions. The recombinant Mycobacterium tuberculosis 11000 protein elicited positive skin reactions in guinea pigs infected with live Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum and Mycobacterium kansasii, and the reaction diameters were (14.7 +/- 2.0) mm, (9.3 +/- 3.8) mm, (18.7 +/- 2.4) mm and (14.8 +/- 4.2) mm, respectively. But it failed to elicit positive skin reaction in guinea pigs immunized with killed Mycobacterium tuberculosis, live BCG and other MOTT (mycobacteria other than Mycobacterium tuberculosis). CONCLUSIONS: Recombinant Mycobacterium tuberculosis 11000 protein can differentiate infection with live Mycobacterium tuberculosis from immunization with killed Mycobacterium tuberculosis, live BCG or other MOTT.

Association of 22q11 deletion with isolated congenital heart disease in three Chinese ethnic groups.

BACKGROUND: Congenital heart disease (CHD) is the most common type of heart disease among children. About 75% of DiGeorge syndrome (DGS) and velo-cardio-facial syndrome (VCFS) includes CHD. A deletion within chromosome 22q11.2 has been identified in the majority of patients with DGS and VCFS. And 22q11.2 deletion has become one of the markers used to study CHD in these syndromes. Whether 22q11.2 deletion is associated with isolated CHD is not known and was the topic of this study. METHODS AND RESULTS: We studied the 22q11.2 deletion in three Chinese ethnic groups (Tai, Bai and Han people) with 19 sporadic, isolated CHD by genotype and haplotype analysis with D22S420 etc. 11 consecutive polymorphic microsatellite markers. Among 19 isolated CHD patients, four had Tetralogy of Fallot (TOF), five exhibited Ventricular Septal Defect (VSD), five showed Atrial Septal Defect (ASD) and 5 had Patent Ductus Arteriosus (PDA). In some isolated CHD patients, 3 Mb and 1.5 Mb deletion to chromosome 22q11.2 was found. 2 of 4 TOF (50%) and 1 of 5 VSD (20%) and 1 of 5 PDA (20%) respectively were found to have deletions at D22S944. CONCLUSIONS: 22q11.2 deletion can be detected in isolated TOF, VSD and PDA of three Chinese ethnic groups, without detectable 22q11.2 deletion in those isolated ASD patients examined thus far. Our finding may be the first to show the 22q11.2 deletion in sporadic, isolated PDA/VSD patients whose family members are without CHD. In addition, D22S420 etc. 11 consecutive polymorphic microsatellite markers are very useful for the determination of 22q11.2 deletion in isolated CHD in China.

[Clinical analysis of primary biliary cirrhosis: a report of 42 cases].

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 Sjogren 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), gamma-glutamyltranspeptidase (gamma-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-M(2) 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 gamma-GT levels together with positive AMA/AMA-M(2) 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.

[Effects of Mycobacterium smegmatis vaccine on cytokines production and Th1/Th2 responses in mice].

OBJECTIVE: To validate the immunogenicity of Mycobacterium smegmatis and to study the immune modulatory function of Mycobacterium smegmatis vaccine made from Mycobacterium smegmatis by analyzing the effects of the vaccine on immune responses in mice. METHODS: Spleen cells and peritoneal macrophages from BALB/c mice which were randomized into a control group and Mycobacterium smegmatis vaccine groups (low, middle, and high doses) were cultured in vitro. Then the supernatants were collected and the concentrations of IL-2, IL-4, IL-12, and IFN-gamma were analyzed through ELISA. RESULTS: (1) IL-12 produced by the control mice and mice immunized with low, middle, high doses of Mycobacterium smegmatis vaccine was (32.6 +/- 22.7), (58.9 +/- 18.6), (77.3 +/- 38.0), (114.7 +/- 9.9) pg/ml respectively, and the middle and high dose group showed significant difference as compared with the control group (P < 0.05). (2) IL-2 produced by the control mice and mice immunized with low, middle, high dose of Mycobacterium smegmatis vaccine was (5.0 +/- 2.6), (13.4 +/- 9.23), (15.3 +/- 9.7), (22.6 +/- 7.5) pg/ml respectively, and the high dose group showed significant difference when compared with the control group (P < 0.01). (3) When the cells were stimulated with ConA in vitro, IFN-gamma produced by the control mice and mice immunized with middle dose of Mycobacterium smegmatis vaccine was (662 +/- 279) and (807 +/- 163) pg/ml, IL-4 produced by the two groups was (407 +/- 127) and (101 +/- 26) pg/ml, but the differences were not statistically significant (P > 0.05). When the cells were stimulated with Mycobacterium smegmatis-purified protein derivative (PPD) in vitro, IFN-gamma produced by the control mice and mice immunized with middle dose of Mycobacterium smegmatis vaccine was (14.0 +/- 6.31) and (55.3 +/- 32.4) pg/ml, the difference being statistically significant (P < 0.05), but IL-4 produced by the two groups was under the limit of detection. CONCLUSION: Mycobacterium smegmatis vaccine made from Mycobacterium smegmatis showed strong immunogenicity promoted Th1 responses and inhibited Th2 response in mice.

[The in vitro killing of intracellular Mycobacterium smegmatis by Mycobacteriophage].

OBJECTIVE: To study the effect of Mycobacteriophage on the lysis of intracellular Mycobacterium smegmatis. METHODS: Peritoneal macrophages from BALB/C mice were incubated with Mycobacterium smegmatis for 4 h, and the extracellular bacteria were removed. Then the infected macrophages were treated for 2 h with normal saline, or different doses of Mycobacteriophages (2.1 x 10(7) PFU, 2.1 x 10(6) PFU, and 2.1 x 10(5) PFU, respectively), all in a volume of 0.1 ml, and then the extracellular phages and Mycobacterium smegmatis were removed by washing. After incubation for 24 h, the number of viable intracellular bacteria was determined. The intracellular changes after infection of host bacteria by bacteriophages in the macrophages were observed by electron microscopy. RESULTS: The logarithm 10 of viable intracellular bacteria unit was 5.74 +/- 0.18 in the saline group, 4.77 +/- 0.08 in the high dose phage group (P < 0.01), 4.97 +/- 0.17 in the moderate dose phage group (P < 0.01), and 5.33 +/- 0.13 in the low dose phage group (P > 0.05). Electron microscopy confirmed the infection of intracellular bacteria by the bacteriophages and the production of filial bacteriophages. CONCLUSIONS: Mycobacteriophages phagocytosed by macrophages are capable of killing the infected mycobacteria. The result suggests that the use of Mycobacteriophages is a potentially novel strategy in the treatment of intracellular bacterial infection.

Recombinant tuberculosis vaccine AEC/BC02 induces antigen-specific cellular responses in mice and protects guinea pigs in a model of latent infection.

PURPOSE: To preliminarily evaluate the immunogenicity and efficacy of the recombinant tuberculosis vaccine AEC/BC02 in which Ag85b and fusion protein ESAT6-CFP10 were combined with bacillus Calmette-Guérin CpG and an aluminum salt-based adjuvant system. METHODS: Groups of BALB/c mice were immunized intramuscularly three times at 10-day intervals with AEC/BC02 or the adjuvant alone and the vaccine-induced cell-mediated immune responses were evaluated. The efficacy of AEC/BC02 was evaluated in two guinea pig models, one a model of prevention and the other a model of latent infection. RESULTS: The AEC/BC02 vaccine induced strong cellular immune responses characterized by a high frequency of antigen-specific interferon-γ-secreting T cells in mice at different time points after the last vaccination. In the preventive model of guinea pig, AEC/BC02 did not protect against Mycobacterium tuberculosis as a pre-exposure vaccine. However, in a latent infection model of guinea pig, it effectively controlled the reactivation of M. tuberculosis and lowered the bacterial load in the lung and spleen. CONCLUSION: These results indicate AEC/BC02 can protect against reactivation of latent infection and may function as a therapeutic vaccine.

[Study on mutation of presenilin-1 gene in familial Alzheimer's disease].

OBJECTIVE: To explore the role of the mutation of presenilin-1 exon 6 in pathogenesis of Alzheimer's disease(AD) patients. METHODS: Exon 6 of presenilin-1 was analyzed by use of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA analyzer technique in 2 patients with familial AD, 53 patients with sporadic DA, 60 patients with vascular dementia(VD) and 90 normal controls. RESULTS: Mobility shift of SSCP in exon 6 of presenilin-1 was detected in 2 cases with FAD, 4 cases with SDA and 1 case with VD. Two missense mutations were found in the patients by DNA sequence analysis, one mutation was 1123 nt C-->G(Cys 23 Trp) and the other was 1300 nt A-->C(Asp 200 Ala). CONCLUSION: Mutations in exon 6 of presenilin-1 existed in the patients with FAD and SDA, and the two missense mutations were probably pathological by nature.

[Proliferative changes of human gastric mucosa cells in different pathological lesions and their clinical significance].

OBJECTIVE: To investigate the variation of cell proliferation and growth in different pathological lesions of gastric mucosa and to assess the possible roles of expression of epidermal growth factor receptor (EGFR), transforming growth factor beta receptor type I and type II (TGF(beta)RI, TGF(beta)RII). METHODS: The proliferating cell nuclear antigen (PCNA), EGFR, TGF(beta)RIand TGF(beta)RII were studied in chronic superficial gastritis (CSG, n = 30), chronic atrophic gastritis (CAG, n = 26), intestinal metaplasia (IM,n = 40), Dysplasia (DYS, n = 22), early gastric cancer (EGC, n = 22), advanced gastric cancer (AGC, n = 26) by immunohistochemical methods and their relations with carcinogenesis were analyzed. RESULTS: (1) In different gastric mucosa lesions (CSG, CAG, IM, DYS, EGC, AGC), there were significantly different expression of PCNA (chi2 = 91.06, P < 0.0001), EGFR (chi2 = 52.82, P < 0.0001), TGF(beta)RI (chi2 = 15.93, P = 0.007) and TGF(beta)RII (chi2 = 40.48, P < 0.0001), PCNA and EGFR were increased, TGF(beta)RI and TGF(beta)RII were decreased. (2) In DYS stage, PCNALI (40.00 +/- 16.34) was higher than in CSG (16.63 +/- 10.52), CAG (16.92 +/- 8.50) and IM (23.25 +/- 18.64), but lower than EGC (53.09 +/- 13.51) and AGC (57.54 +/- 16.88) (P < 0.0001); (3) EGFR expression in IM (55.0%) and DYS (72.7%) were higher than in CSG (10.0%) and CAG (3.8%) (P < 0.0001), but no different with EGC (59.1%) and AGC (73.1%). (4) TGF(beta)RI expression in EGC (50.0%) and AGC (30.8%) were lower than in CSG (73.3%) (P = 0.007). (5) TGF(beta)RII expression in AGC (26.9%) was lower than in CSG (83.3%), CAG (82.8%), IM (65.0%), DYS (54.5%) and EGC (45.5%) significantly (P < 0.0001). (6) The expression of EGFR had positive correlation with PCNA, TGF(beta)RI and TGF(beta)RII had negative correlation with PCNA respectively, TGF(beta)RI and TGF(beta)RII had positive correlation. CONCLUSIONS: DYS is the key link in the change of cell proliferation during gastric carcinogenesis; The increase of EGFR and the decrease of TGF(beta)R may play important roles in promoting gastric carcinogenesis by affecting gastric cell proliferation.

[Effect of helicobacter pylori on gastric mucosal cell proliferation in gastritis].

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 I and type II(TGFbetaRI, TGFbetaRII) 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<0.05); The TGFbetaRI(P=0.16) and TGFbetaRII(P=0.97) were lower. CONCLUSION: Hp infection promotes over proliferation of gastric mucosal cells.