[The effect of CD4+ CD25+ regulatory T cell inactivation combined with levofloxacin on murine tuberculosis].

OBJECTIVE: To investigate the effects of inactivation of CD(4)(+)CD(25)(+) regulatory T cells (Treg) combined with the administration of levofloxacin (LFX) on the cellular immune response of murine tuberculosis. METHODS: Inactivation of Treg was achieved by intraperitoneal injection of anti-CD(25), clone PC61. Female C57BL/6 mice were divided into 4 groups, PC61 alone, LFX alone, PC61 plus LFX, and the control, with 19 mice in each group. The LFX group and the control group were treated with rat-IgG isotope control. Mice were inoculated with H(37) Rv (1 x 10(6) CFU) via the tail vein 3 days later. From the 2nd day, the LFX group and the PC61 plus LFX group received intragastric administration of LFX at 200 mg x kg(-1) x d(-1) per mouse for 45 days. Blood and spleen Tregs were measured by flow cytometry. The cellular immune response and pulmonary histopathology at different time points were also evaluated after infection. RESULTS: At the 10th and 30th day, the ratio of CD(4)(+)CD(25)(+)/CD(4)(+)T cells in the spleen was (30 +/- 4)% and (17.3 +/- 1.6)% respectively in the control group, (21 +/- 4)% and (16.1 +/- 1.3)% respectively in the PC61 group, (44 +/- 6)% and (24.7 +/- 2.0)% respectively in the LFX group, (24 +/- 3)% and (10.4 +/- 1.0)% respectively in the PC61 plus LFX group. The differences were significant between groups (q = 3.62 - 5.56, P < 0.05), but the difference between the PC61 plus LFX group and the PC61 group at the 10th day. Same results were obtained from the peripheral blood. PC61 plus LFX therapy resulted in BCG specific cytokine response (IL-17, IFN-gamma, TNF-alpha) from murine spleen cells at the 10th and the 30th day, and also in milder pathologic changes and the lowest mortality. CONCLUSIONS: The cellular immune response was enhanced by Treg inactivation and LFX therapy, which decreased the pathologic changes and the mortality of murine tuberculosis.

[Study on specific cellular immunity of enhance intracellular survival antigen of Mycobacterium tuberculosis in patients with pulmonary tuberculosis].

OBJECTIVE: To investigate the level of specific cellular immunity in patients with active pulmonary tuberculosis and its potential relationship with severity of the disease. METHODS: Thirty active pulmonary tuberculosis patients with positive tubercle bacilli in sputum were enrolled for the study. Immune responses including lymphocytes proliferation enhanced by enhance intracellular survival (EIS) antigen and cytokine production including interferon-gamma (IFN-gamma) and interleukin-10 (IL-10), were assayed. Cell proliferation was determined by methyl thiazolyl tetrazolium (MTT), while cytokine production was quantified by enzyme linked immunoadsorbent assay (ELISA). The results were compared to those of 20 healthy individuals and 16 persons recovered from tuberculosis. RESULTS: Cell proliferation response and IFN-gamma production were significantly higher in patients convalescent from tuberculosis compared to patients with active pulmonary tuberculosis, EIS antigen was found to elicit a dominant Th2 cytokine response. CONCLUSION: Impaired Th1 immune response to EIS is observed in patients with active pulmonary tuberculosis. Induction of imbalance of Th1/Th2 immune response may be the main action of EIS, which may be a factor of pathogenesis of tuberculosis.

[Evaluation of the accuracy of domestic commercial HBV DNA real-time polymerase chain reaction kits using COBAS TaqMan HBV Test as reference].

OBJECTIVE: To evaluate of the accuracy of domestic commercial HBV DNA real-time polymerase chain reaction kits. METHODS: Using COBAS TaqMan HBV Test as reference, we evaluate the accuracy of a domestic commercial HBV DNA real-time polymerase chain reaction kit (PG). RESULTS: Among the samples with viral load at the range of 10(1), 10(2), 10(3), 10(4), 10(5), 10(6), 10(7) (IU/mL), the Coefficient of Correlation(r) between the result determined by domestic kit (PG) and those of Roche COBAS TaqMan HBV Test were: -0.08011, -0.05056, 0.105642, 0.312181, 0.908046, 0.866175, -0.23295, respectively; the percentage of false negative results were 60%, 30%, 33.3%, 8.3%, 0, 0, 0, respectively. Among the samples with viral load over than 10(7) (IU/ml), the result determined by PG is significantly lower. CONCLUSION: The accuracy of PG is not satisfied, especially in those samples with viral load less than 10(4) (IU/ml). A implication from these observation is that samples from patients received antiviral treatment should be tested by Roche COBAS TaqMan HBV Test.

[Immunosuppression induced by measles virus in adult patients is not related to CD4+ CD25+ regulatory T cell induction].

OBJECTIVE: To investigate of the relationship of the immunosuppression induced by Measles virus in adult patients and CD4+ CD25+ regulatory T cell. METHODS: Thirty-four patients with measles and 27 healthy control subjects were included in this study. The whole blood was collected and CD4+ CD25+ cell and FoxP3+ cell were analyzed by flow cytometry, and CD4+ CD25- and CD4+ CD25+ T lymphocytes were isolated from PBMCs of patients with measles or healthy donors, CD4+ CD25- T cells were cultured in absence or presence of anti-CD3, or BCG, or live attenuated MV. The cell culture supernatant was collected after 72 hours and the concentration of IFN-gamma and IL-10 was determined. RESULTS: Compared to healthy donors, we observed a reduction of the number of white blood cells and lymphocytes in patients with measles, but there was not significantly different in the frequency of CD4+ CD25+ T cells and CD4+ CD25high T cells within the total CD4+ population in the blood. Treg from both measles patients and healthy controls significantly inhibited IFN-gamma production by CD4+ CD25- T cells in response to anti-CD3 stimulation. CONCLUSION: Induction and expansion of Treg may not represent a mechanism involved in the establishment of immune suppression by MV.

[Expressive features of HBsAg and HBcAg in the livers of chronic hepatitis B and its clinical significance].

OBJECTIVE: To investigate the necessity of detecting on the expressive intensity and pattern of HBsAg and HBcAg in the livers of chronic hepatitis B. METHODS: HBsAg and HBcAg were detected in paraffin-embedded liver tissue by EnVision immunohistochemistry. Serum hepatitis B virus DNA (HBV DNA) was tested by real-time quantitative polymerase chain reaction. The degrees of hepatic inflammatory activity (grade) and fibrosis (stage) of liver biopsies were determined according to the standard of the Chinese program of prevention and treatment of viral hepatitis. RESULTS: The expression of HBsAg was not correlated with the grade, the stage and the levels of serum HBV DNA (P > 0.05). Liver HBcAg expressive intensity was not correlated with the grade (r=0.02, P > 0.05), while negatively correlated with the stage (r=0.28, P < 0.01) and positively correlated with the serum HBV DNA levels (r=0.53, P < 0.01). Liver HBcAg expressive pattern was negatively correlated with the grade (r=-0.27, P < 0.01). The grade in cytoplasmic pattern group was higher than in nuclear pattern group and in mixed pattern group (P < 0.01), and that in mixed pattern group was higher in nuclear pattern group (P < 0.01). Liver HBcAg expressive pattern was negatively correlated with the stage (r=-0.23, P < 0.01). The stage in cytoplasmic pattern group was higher than in nuclear pattern group and in mixed pattern group (P < 0.05). Liver HBcAg expressive pattern was positively correlated with the levels of serum HBV DNA (r=0.22, P < 0.01). CONCLUSION: Distinguishing the expressive intensity and pattern of HBsAg and HBcAg in the liver of chronic hepatitis B may not help understand the degree of hepatic lesion. The detection of HBcAg in liver tissue of CHB may be beneficial for the antiviral therapy.

[Immunological and virological efficacy against HBV chronic infection of the therapeutic vaccine composed of HBV core plus PreS1 in HBV transgenic mice].

BACKGROUND: To investigate the immunological and virological efficacy of the therapeutic vaccine HBV CS1, a recombinant fusion protein which is composed of HBV core aa 1-155 plus PreS1 aa 3-55,against chronic HBV infection. METHODS: HBV transgenic mice were immunized with HBV CS1(5 ug) emulsified in equal volume of complete Freund adjuvant on day 0, followed by a second vaccination with HBV CS1(5 ug) emulsified with incomplete Freund adjuvant on days 21. Mice of control group were mock-vaccinated with PBS plus complete Freund adjuvant/incomplete Freund adjuvant. The splenocytes of individual mouse were subjected to T cell proliferation assays by using 3Hg thymidine, HBsAg and HBV DNA in sera of mice were detected by ELISA and quantitative PCR, respectively. RESULTS: HBV CS1 specific T cell response were induced in mice immunized with HBV CS1, with the titer of HBsAg and the level of HBV DNA decreased significantly after twice immunization with HBV CS1, while the control group almost remained the same. CONCLUSION: HBV CS1 has the immunological and virological efficacy against chronic HBV infection in HBV transgenic mice; HBV CS1 could represent candidate vaccine for further studies on its role as therapeutic vaccine against HBV chronic infection in human.

[Identification and molecular cloning and sequence analysis of a novel coronavirus from patients with SARS by RT-PCR].

OBJECTIVE: To investigate the etiologic agents of the SARS and develop diagnostic method for this disease. METHODS: Thirty-six nasopharyngeal aspirate specimens from 27 patients with SARS in Shenzhen were collected. The samples were aliquotted to three parts and subjected to molecular assays for human metapneumovirus, chlamydia and a novel coronavirus, which was reported recently to be the etiologic agent of SARS. Nested RT-PCR was used to amplify the RNA polymerase gene of the novel coronavirus and the PCR products were sequenced directly or after cloned to pMD18-T vector. RESULTS: Human metapneumovirus and chlamydia genes were detected in none of the specimens using the RT-PCR and nested-PCR, respectively. The novel coronavirus gene were amplified in 6 of 36 specimens, the sequence analysis indicated that this novel coronavirus is unrelated to any other coronavirus reported previously. The nucleotide and deduced amino acid alignment between this coronavirus and others was not more than 40% and 70% to 82%, respectively, while the nucleotide sequence cloned from the 6 patients were identical. CONCLUSIONS: The SARS patients in Shenzhen were infected with coronavirus and this novel coronavirus is associated with SARS. The sequence analysis indicated that the coronavirus from SARS patients in Shenzhen is the same as that identified from other areas such as Canada and Hong Kong. A specific diagnostic nested RT-PCR was developed to identify this novel coronavirus infection.