Percent duration of heart rate acceleration within the respiratory cycle: a novel approach to assess heart rate asymmetry.

Accelerations and decelerations of heart rate are nonsymmetrical in the magnitude and number of beat-to-beat changes. The asymmetric features of heart rate variability are related to respiratory durations. To explore the link between respiration and heart rate asymmetry (HRA), we evaluated 14 seated, healthy young adults who breathed with nine combinations of inspiration duration (TI) and expiration duration (TE), chosen respectively from 2, 4, and 6 s. A 5-min R-R interval (RRI) time series was obtained from each study period to construct an averaged pattern waveform relative to the respiratory cycle. We observed that the time interval between inspiration onset and RRI minimum progressively lengthened as TI and TE increased. The time interval between expiration onset and RRI maximum also lengthened when TE increased but shortened when TI increased. Consequently, TI and TE had different effects on the acceleration time (AT; from RRI maximum to RRI minimum) and deceleration time (DT; from RRI minimum to RRI maximum). The percentage of AT within the respiratory cycle showed a strong correlation with traditional Guzik's (r = 0.862, P < 0.001) and Porta's (r = 0.878, P < 0.001) indexes of HRA assessed in a Poincaré plot analysis. These findings suggest that, in addition to considering the magnitude and number of beat-to-beat changes, HRA can also be assessed based on another aspect: the duration of consecutive changes. The stepwise link between the duration of heart rate change and respiratory duration provides insight into the mechanisms connecting respiration to HRA.NEW & NOTEWORTHY In healthy adults who regulated their breathing across nine combinations of inspiration and expiration durations, we used averaged pattern waveform technique to quantify the durations of heart rate acceleration and deceleration within the respiratory cycle. The percent duration of acceleration showed a strong correlation with traditional heart rate asymmetry indexes, which evaluate the magnitude and number of beat-to-beat changes. This new approach opens a window to explore the asymmetric features of heart rate variability.

Different effects of inspiratory duration and expiratory duration on heart rate deceleration capacity and heart rate asymmetry.

PURPOSE: Low values of heart rate deceleration capacity (DC) and heart rate asymmetry (HRA) are associated with cardiovascular risks. Slow respiration has been proven to enhance the magnitudes of these indexes, but individual inspiratory (TI) and expiratory (TE) durations were not controlled in most studies. This study aims to examine whether the effects of TI and TE on these indexes would be the same and, if not, how to adjust TI and TE to maximize the effect of slow respiration. METHODS: We evaluated 14 seated healthy young adults who randomly controlled their breathing to nine combinations of TI and TE, each chosen respectively from 2, 4, and 6 s. A 5-min R-R interval time series was obtained from each study period for further analysis. RESULTS: The magnitude of DC increased when TI or TE increased, while that of acceleration capacity (AC) remained almost unchanged by TI. We further defined a new index as 100 × DC2/(DC2 + AC2) and found it to be correlated with conventional Guzik's (r = 0.94) and Porta's (r = 0.99) indexes of HRA during different combinations of TI and TE. Increasing TI and increasing TE both enhanced the magnitudes of HRA indexes, with TI taking effect when ≤ 4 s, and TE taking effect when > 4 s. DC and HRA indexes were maximized with a TI of 4 s and a TE of 6 s. CONCLUSION: We suggest that a TI of 3-4 s with a TE of 7-6 s is an appropriate standard for slow respiration.

Radiographic chest wall abnormalities in primary spontaneous pneumothorax identified by artificial intelligence.

Primary spontaneous pneumothorax (PSP) primarily affects slim and tall young males. Exploring the etiological link between chest wall structural characteristics and PSP is crucial for advancing treatment methods. In this case-control study, chest computed tomography (CT) images from patients undergoing thoracic surgery, with or without PSP, were analyzed using Artificial Intelligence. Convolutional Neural Network (CNN) model of EfficientNetB3 and InceptionV3 were used with transfer learning on the Imagenet to compare the images of both groups. A heatmap was created on the chest CT scans to enhance interoperability, and the scale-invariant feature transform (SIFT) was adopted to further compare the image level. A total of 2,312 CT images of 26 non-PSP patients and 1,122 CT images of 26 PSP patients were selected. Chest-wall apex pit (CAP) was found in 25 PSP and three non-PSP patients (p < 0.001). The CNN achieved a testing accuracy of 93.47 % in distinguishing PSP from non-PSP based on chest wall features by identifying the existence of CAP. Heatmap analysis demonstrated CNN's precision in targeting the upper chest wall, accurately identifying CAP without undue influence from similar structures, or inappropriately expanding or minimizing the test area. SIFT results indicated a 10.55 % higher mean similarity within the groups compared to between PSP and non-PSP (p < 0.001). In conclusion, distinctive radiographic chest wall configurations were observed in PSP patients, with CAP potentially serving as an etiological factor linked to PSP. This study accentuates the potential of AI-assisted analysis in refining diagnostic approaches and treatment strategies for PSP.

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.

Modeling Methodology for Site Selection Evaluation of Underground Coal Gasification Based on Combination Weighting Method with Game Theory.

The lack of systematic geological work is an essential reason why underground coal gasification (UCG) has not been industrialized for a long time. Building a scientific index system and favorable area evaluation technology for the UCG site selection is the key to breaking through the geological bottleneck. Aiming at the problems of the single index weight determination method, intense subjectivity, and poor reliability of current evaluation models, we put forward an evaluation modeling methodology for the UCG site selection using the combination weighting method with the game theory. The factors of coal resource conditions associated with the potential risk of UCG are systematically analyzed. From the six dimensions of the geological structure, hydrogeology, seam occurrence, coal properties, reserves, and roof lithology, 23 key factors were selected as evaluation indexes to construct a hierarchical model composed of the target layer, category index layer, and index layer. The influence of each index on UCG and its reasonable value range were systematically analyzed. The evaluation index system for UCG site selection was formed. The improved analytic hierarchy process (AHP) was adopted to sequence indices and determine their subjective weight. And the variability, conflict, and information amount of the index data were analyzed by the CRiteria Importance Through Intercriteria Correlation (CRITIC) method to calculate the objective weight. Then, the subjective and objective weights were combined through game theory. On this basis, fuzzy theory was employed to calculate the membership of indices and construct the fuzzy comprehensive judgment matrix. The evaluation model of the UCG site selection was applied to the suitability evaluation of resource conditions of UCG pilot projects at Zhongliangshan (ZLS), Huating (HT), and Shanjiaoshu (SJS) mines in China. The result shows that the resource conditions of HT are the best, followed by ZLS and, finally, SJS, which are consistent with the actual running effects of the three UCG pilot projects. It indicates that the evaluation model can provide a scientific theoretical basis and reliable technical support for the UCG site selection.

[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.

[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.

[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.

[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.

[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.

[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.

[A study of standardization to the rapid detection of Mycobacterium tuberculosis based on phage amplified biologically assay].

OBJECTIVE: To evaluate the phage amplified biologically (PhaB) assay in the rapid detection of Mycobacteria tuberculosis in samples. METHODS: The conditions of the PhaB assay, including various infection times prior to addition of virucide and the effect of the inactivation agents which could inactive the extracellular phages, were investigated and compared. The sensitivity, specificity and accuracy of PhaB assay were tested when it was used in rapid detection of Mycobacterium tuberculosis. Some agents of the method, including Mycobacteriophage, virucide and help cells (Mycobacterium smegmatis) were investigated at different times when they were preserved at 4 degrees C. RESULTS: (1) The optimal infection time prior to addition of virucide was between 3 and 4 hours. Four percent FAS (ferrous ammonium sulphate) could inactive 1 x 10(9) PFU (plaque-forming unit) in five minutes. (2) The samples were positive when 80 - 200 CFU of Mycobacterium tuberculosis were present. However, the positive rate of non-Tuberculosis Mycobateria (NTM) was varied. All bacteria lived in the respiratory tract were negative. (3) The important agents used in this test showed optical effect when they were preserved at 4 degrees C. CONCLUSIONS: The method based on mycobacteriophage-amplified biologically assay could rapidly detect Mycobacterium tuberculosis, and it was effective, accurate, and simple to perform. It was appropriate for using in developing countries, compared with a variety of molecular techniques.

[Application of multiplex polymerase chain reaction in rapid identification of Mycobacterium bovis BCG].

OBJECTIVE: To establish a method for the rapid identification of Mycobacterium bovis BCG. METHODS: A genomic region designated RD1 was found to be deleted from BCG strains, but present in other strains of Mycobacterium bovis and other members of the Mycobacterium tuberculosis complex (MTC) including Mycobacterium tuberculosis, Mycobacterium africanum, and Mycobacterium microti. With this information, a multiplex PCR method, developed to detect the deletion of RD1, was used to differentiate BCG strains from other strains of Mycobacterium bovis and other members of MTC. RESULTS: RD1 was shown to be absent in 5 BCG vaccine strains and 2 BCG strains isolated from an infant who died of systemic disseminated infection induced by BCG vaccination, but it was present in 3 Mycobacterium bovis standard strains, 6 Mycobacterium bovis strains isolated from diseased cows, deer or patients with pulmonary tuberculosis and other MTC strains including Mycobacterium tuberculosis H(37)Rv and H(37)Ra strains, 48 Mycobacterium tuberculosis strains isolated from patients with pulmonary tuberculosis, and 3 Mycobacterium africanum standard strains. CONCLUSION: The multiplex PCR method is simple, rapid, and specific for the identification of BCG among strains of MTC, and is applicable in clinical laboratories.

[Studies on the relationship between the level of cytokine and liver function in patients with clonorchiasis sinensis].

OBJECTIVE: To explore the relationship between cytokine level and liver function among patients infected with Clonorchis sinensis. METHODS: 47 patients were divided into three groups according to the degree of Child-Pugh liver function grade: 20 in group A (3-4 scores), 15 in group B (5-6 scores) and 12 in group C (7-9 scores). Interleukin 2 (IL-2), soluble IL-2 receptor (sIL-2R), interleukin 8 (IL-8) and tumor necrosis factor (TNF-alpha) were examined by radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Automatic biochemical analyzer was employed for the determination of serum level of total bilirubin (TBL), albumin (ALB) and alanine aminotransferase (ALT). Data were analyzed with SAS statistic software. RESULTS: Serum levels of sIL-2R, IL-8 and TNF-alpha from patients were significantly higher than those obtained from healthy people (P<0.05, P<0.05, P<0.01), whereas the IL-2 level was significantly lower than the former (P<0.01). With the affected degree of the liver, serum levels of sIL-2R, IL-8 and TNF-alpha increased, in contrast to the decrease of IL-2 level. The differences were significant between groups A and C (P<0.05). The level of sIL-2R and TNF-alpha directly correlated with that of TBL (r=0.331 P<0.05, r=0.518 P<0.01) and ALT (r=0.475 P<0.01, r=0.285 P<0.05) respectively, but inversely correlated with the level of ALB (r=-0.319 P<0.05, r=-0.665 P<0.01). CONCLUSION: The infection of Clonorchis sinensis results in the reduction of cellular immune function of the patients. Certain relationship exists between serum cytokine level and liver function. Two cytokines, sIL-2R and TNF-alpha, are involved in the process of pathology.

Preclinical study and phase I clinical safety evaluation of recombinant Mycobacterium tuberculosis ESAT6 protein.

BACKGROUND: To investigate the ability of rESAT6 to identify different mycobacteria-sensitized guinea pigs and its safety in preclinical and phase I clinical study. MATERIAL AND METHODS: Guinea pigs were sensitized with different Mycobacteria. After sensitization, all animals were intradermally injected with rESAT6 and either PPD or PPD-B. At 24 h after the injection, the erythema of the injection sites were measured using a double-blind method. For the preclinical safety study, different doses of rESAT6 and BSA were given 3 times intramuscularly to guinea pigs. On day 14 after the final immunization, the guinea pigs were intravenously injected with the same reagents in the hind legs and the allergic reactions were observed. A single-center, randomized, open phase I clinical trial was employed. The skin test was conducted in 32 healthy volunteers aged 19-65 years with 0.1 µg, 0.5 µg, and 1 µg rESAT6. Physical examination and laboratory tests were performed before and after the skin test and adverse reactions were monitored. The volunteers' local and systemic adverse reactions and adverse events were recorded for 7 days. RESULTS: Positive PPD or PPD-B skin tests were observed in all Mycobacteria-sensitized guinea pigs; the diameters of erythema were all >10 mm. The rESAT6 protein induced a positive skin test result in the guinea pigs sensitized with MTB, M. bovis, M. africanum and M. kansasii; the diameters of erythema were 14.7±2.0, 9.3±3.8, 18.7±2.4, and 14.8±4.2 mm, respectively. A negative skin test result was detected in BCG-vaccinated and other NTM-sensitized guinea pigs. The rESAT6 caused no allergic symptoms, but many allergic reactions, such as cough, dyspnea, and even death, were observed in the guinea pigs who were administered BSA. During the phase I clinical trial, no adverse reactions were found in the 0.1 µg rESAT6 group, but in the 0.5 µg rESAT6 group 2 volunteers reported pain and 1 reported itching, and in the 1 µg rESAT6 group there was 1 case of pain, 1 case of itching, and 1 case of blister. No other local or systemic adverse reactions or events were reported. CONCLUSIONS: The rESAT6 can differentiate effectively among MTB infection, BCG vaccination, and NTM infection and is safe in healthy volunteers.

[Observation on dynamic changes of SEA specific antibody in sera of BALB/c mice infected with Schistosoma japonicum].

OBJECTIVE: To investigate the dynamic changes of SEA-induced specific IgG, IgM in sera of BALB/c mice infected with Schistosoma japonicum in 18 weeks. METHODS: After mice were infected with S. japonicum cercarial for 2 weeks, the sera were collected from 2 to 18 weeks post-infection. The serum levels of SEA-specific IgG and IgM antibodies were measured respectively by ELISA, and the different fractions of IgG and IgM antibodies were identified by the Western blotting method. RESULTS: The ELISA results showed that the serum levels of SEA-specific IgG increased 5, 6, 9, 11 week, after the infection, and SEA-specific IgM increased obviously 5, 9 weeks after the infection. The Western blotting results showed that 140, 180 kDa molecules were recognized by IgG antibodies in the mouse sera 4 weeks after the infection. The specific IgG antibodies of 43, 50 kDa antigens appeared 5 weeks after the infection. 60-130 kDa fractions were recognized by IgG in the sera 6 weeks post-infection, and 38, 73 kDa proteins were recognized by IgG in the sera 9 weeks post-infection. The IgG antibodies of 26, 32, 35, 80 kDa molecules appeared 11 weeks post-infection and reacted strongly 12 weeks post-infection. The IgM antibodies of 100, 140, 180 kDa molecules appeared 3 weeks after the infection, and 73 kDa protein was recognized by sera 6 weeks after the infection, but the reaction became strong 9 weeks after the infection. The 38, 43, 50 kDa proteins induced IgM antibodies in 9-week-infection sera and the reaction became stronger 9 weeks after the infection. CONCLUSIONS: There is a dynamic change in the levels of specific IgG and IgM antibodies induced by S. japonica SEA and the appearance of the antibodies is related to different infection stages. The 43, 50, 10, 140, 180 kDa antigens might have the potential value of early immunodiagnosis. The 73 kDa antigen shows high diagnostic value in both acute and chronic schistosomiasis. The 28, 32, 35, 38, 80 kDa antigens are not only the diagnostic molecules for chronic schistosomiasis, but they may also have therapeutic effects, and in addition, they may be the candidate vaccines of the disease.