[Association between CD4+CD25+Foxp3+ regulatory T cells and serum transforming growth factor beta 1 in patients with chronic hepatitis B].

OBJECTIVE: To investigate whether the CD4+CD25+Foxp3+ regulatory T cells are associated with serum TGF beta 1 in patients with hepatitis B. METHODS: Patients with chronic hepatitis B (CHB), chronic asymptomatic carriers (AsC), normal subjects (NS) and the resolved from HBV infection (Resolved) were recruited in this study. Flow cytometric analysis was used to detect the frequency and phenotype of peripheral CD4+CD25+Foxp3+ T cells, and Foxp3 gene expression were examined by real time PCR. Serum TGF beta 1 levels were measured by ELISA (enzyme-linked immunosorbent assay). RESULTS: Patients with CHB or AsC exhibited significantly higher frequency of CD4+CD25+Foxp3+ T cells compared to healthy controls. CD4+CD25+ T cells derived from patients with CHB and AsC expressed higher level of Foxp3-mRNA. Furthermore, the frequency of CD4+CD25+Foxp3+ regulatory T cells was correlated with serum HBV DNA copy numbers in patients with CHB and AsC. Our results indicated that the serum TGF beta was increased in CHB and AsC patients compared to control patients, and that serum TGF beta was correlated with the expression of Foxp3-mRNA and the frequency of CD4+CD25+Foxp3+ regulatory T cells in patients with CHB and AsC. CONCLUSIONS: The findings have important implication in the understanding of the role and mechanism of aberrant CD4+CD25+Foxp3+ regulatory T cells in the maintenance of chronicity in hepatitis B patients.

[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.

[High throughput detection of drug-resistance gene mutations in HBV using MALDI-TOF mass spectrometry].

OBJECTIVE: To develop a high-throughput clinical method on drug-resistance gene mutations of HBV using MALDI-TOF-MS. METHOD: Using MassArray Assay Design software designed the iPLEX primers and followed the iPLEX instruction for amplification, SAP reaction, primer extenction, desalination, dispensing, MALDI-TOF-MS screening and data analysis of the gene mutation locus. 138 serum samples of chronic HBV patients with single drug-resistance or multiple drug-resistance on Lamivudin, adefovi, Entecavir were detected. RESULT: The HBV gene mutation platform was successfully developed and applied on the high-throughput dectection of clinical serum samples. It was also a high throughput assay which could be used to detect for more than 138 samples once. The MALDI-TOF-MS technology and the DNA sequencing simultaneously examine 33 samples, in which result of 10 sample is inconsistent, the including 2 samples by MALDI-TOF-MS technology has not tested, 1 sample has 2 inconsistent mutations. CONCLUSION: Detection of HBV gene mutations using MALDI-TOF-MS is highly-sensitive, highly-accurate, high-throughput, fast achieved and suitable to use in the diagnosis and monitoring of HBV.

[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.

[Investigation on activities of hammerhead ribozyme embedded in genomic RNA of hepatitis delta virus].

OBJECTIVE: To develop HDV as a vehicle to deliver hammerhead ribozyme into hepatocytes, the effects of modified HDV was assessed on the activity of embedded hammerhead ribozyme in vitro and in vivo. METHODS: In vitro activity of ribozyme or HDV-driven ribozyme was assessed by incubating with the [alpha-32 P]-ATP labeled HBV RNA substrates at different temperature. Huh-7 cells were cotransfected with ribozyme or HDV-ribozyme chimera and HBV genome, by which inhibition of ribozymes on HBV transcription in vivo were examined. RESULTS: The results indicated that both temperature and secondary structure influenced the cleavage activity of HDV-driven ribozyme significantly. When the factors were eliminated, the HDV-driven ribozyme could act as well as its counterpart naked ribozyme. While in cultured cells the HDV-driven ribozyme had higher inhibition to HBV gene expression than that of ribozyme alone. CONCLUSION: The results demonstrated that HDV may weaken the activity of embedded ribozyme in vitro, but make it enhanced in cultured cells. Thus, this study could provide a useful evidence to develop HDV as vector for liver-special delivery of ribozyme to against chronic HBV infection.

[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.