Ipr1 modified BCG as a novel vaccine induces stronger immunity than BCG against tuberculosis infection in mice.

Tuberculosis is a major challenge to global public health. However, the Bacille Calmette­Guérin (BCG), the only vaccine available against tuberculosis, has been questioned for the low protective effect. The present study used the mouse gene intracellular pathogen resistance I (Ipr1) gene to alter the current BCG vaccine and evaluated its immunity effect against tuberculosis. This study also investigated the intrinsic relationships of Ipr1 and innate immunity. The reformed BCG (BCGi) carrying the Ipr1 gene was constructed. The mice were intranasally challenged with the M. tuberculosis H37Rv strain after vaccination with BCGi. Protection efficacy of the vaccine was assessed by the organ coefficient, bacterial load and pathological changes in the lung. The differential expression of 113 immune­related genes between BCGi and BCG groups were detected by an oligo microarray. According to the results of organ coefficient, bacterial load and pathological changes in the organization, BCGi had been shown to have stronger protective effects against M. tuberculosis than BCG. The oligo microarray and reverse transcription­quantitative polymerase chain reaction further revealed that the Ipr1 gene could upregulate the expression of 13 genes, including a >3­fold increase in Toll­like receptor (TLR)4 and 10­fold increase in surfactant protein D (sftpd). The two genes not only participate in innate immunity against pathogens, but also are closely interrelated. Ipr1 could activate the TLR4 and sftpd signaling pathway and improve the innate immunity against tuberculosis, therefore Ipr1 modified BCG may be a candidate vaccine against M. tuberculosis.

S-phase arrest after vincristine treatment may promote hepatitis B virus replication.

AIM: To observe the effect of vincristine on hepatitis B virus (HBV) replication in vitro and to study its possible mechanisms. METHODS: Vincristine was added to the cultures of two cell lines stably expressing HBV. Then, the levels of hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and hepatitis B core antigen (HBcAg) in the supernatants or cytoplasm were examined using by enzyme-linked immunosorbent assay and Western blot. The HBV pregenome RNA (pgRNA) was detected using reverse transcription-PCR and real-time fluorescent quantitative PCR (RT-qPCR), and viral DNA was detected using Southern blot and RT-qPCR. Cell proliferation after drug treatment was detected using the BrdU incorporation test and the trypan blue exclusion assay. Cell cycle and cell apoptosis were examined using flow cytometry and Western blot. RESULTS: Vincristine up-regulated HBV replication directly in vitro in a dose-dependent manner, and 24-h exposure to 0.1 µmol/L vincristine induced more than 4-fold and 3-fold increases in intracellular HBV DNA and the secretion of viral DNA, respectively. The expression of HBV pgRNA, intracellular HBsAg and HBcAg, and the secretion of HBeAg were also increased significantly after drug treatment. Most importantly, vincristine promoted the cell excretion of HBV nucleocapsids instead of HBV Dane particles, and the nucleocapsids are closely related to the HBV pathogenesis. Furthermore, vincristine inhibited the proliferation of cells stably expressing HBV. The higher the concentration of the drug, the more significant the inhibition of the cell proliferation and the stronger the HBV replication ability in cells. Flow cytometry indicated that cell cycle arrest at S-phase was responsible for the cell proliferation inhibition. CONCLUSION: Vincristine has a strong stimulatory effect on HBV replication and induces cell cycle arrest, and cell proliferation inhibition may be conducive to viral replication.

Epirubicin directly promotes hepatitis B virus (HBV) replication in stable HBV-expressing cell lines: a novel mechanism of HBV reactivation following anticancer chemotherapy.

Chronic hepatitis B virus (HBV) infection is a worldwide problem and HBV reactivation following anticancer chemotherapy has become an emerging clinical challenge. However, the mechanisms of HBV reactivation following chemotherapy remain unclear. Epirubicin is an anthracycline drug used in chemotherapy to treat numerous types of malignancy, including breast cancer, acute leukemia, malignant lymphoma, lung cancer, ovarian cancer and stomach cancer. Epirubicin acts by intercalating DNA strands and inhibiting DNA and RNA synthesis. In this study, it was demonstrated that epirubicin directly upregulated the levels of in vitro HBV replication in a concentration-dependent manner. Exposure to epirubicin for 24 h induced >11- and 6-fold increases in the levels of intracellular and secreted HBV DNA, respectively. In concordance with the elevated levels of HBV DNA, the expression levels of HBV pregenomic RNA, intracellular HBV surface and HBV core antigens, and secreted HBV e antigen were significantly increased by treatment with 0.5 µM epirubicin. Notably, epirubicin promoted cellular excretion of HBV nucleocapsids, which are closely associated with the pathological effects of HBV, including acute liver failure. In conclusion, epirubicin exhibited a direct stimulatory effect on HBV replication and this may be a novel mechanism of HBV reactivation following cytotoxic anticancer chemotherapy.

[Analysis of hydrolytic enzyme activities on sludge aerobic/anoxic digestion after ultrasonic pretreatment].

In order to evaluate the function of sludge aerobic/anoxic digestibility by ultrasonic pretreatment. The SS, VSS and hydrolytic enzyme activities (amylase, glucosidase, protease, phosphatase) were measured before and after ultrasonic pretreatment (28 kHz, 0.15 kW x L(-1), 10 min). The results showed that the performances of aerobic/anoxic were greatly improved after ultrasonic pretreatment, the removal efficiency of VSS went to 44.3%, 7.8% better than of traditional aerobic/anoxic digestion. The variational trend of sludge hydrolytic enzyme activities increased firstly and then fell off during 13d digestion, the maximum of amylase activity and glucosidase activity in ultrasonic sludge, appeared in the 5 d, amylase activity was 0.104 micromol x g(-1) and glucosidase activity was 0.637 (micromol x g(-1). The maximum of intracellular protease activity and extracellular proteases activity in ultrasonic sludge, appeared in the 7 d, intracellular protease activity was 23.68 micromol x g(-1), higher than extracellular proteases activity, and it was playing a leading role in sludge digestion. The acid phosphatase activity of ultrasonic sludge was higher than the control sludge, and the alkaline phosphatase was sensitive to environment. So the alkaline phosphatase activity reduced when the internal properties of sludge was changed.

[Analysis of community structure on sludge aerobic/anoxic digestion after ultrasonic pretreatment].

The microbial community structure of sludge aerobic/anoxic digestion after ultrasonic pretreatment was studied by PCR amplification and DGGE based on 16S rDNA. The genomic DNA of sludge at different stages was extracted with SDS cell lysate method. After purification of DNA, the 16S rDNA genes (V3 region) were amplified by using the universal primers (F357GC and R518). The results of agarose gel (1.5%) electrophoresis showed that the PCR products were about 190 bp in length. The amplified DNA fragments were separated by paralleled DGGE with the denaturant (urea and acrylamide) from 30% to 60%. The sequences were used for homology analysis and phylogenetic trees were constructed. The DGGE profiles showed that the change of microbial diversity was in correspondence to different periods. Compared with 0 d, the diversities of microorganisms were 61.2%, 48.2%, 46.4%, 42.6% and 41.7%, respectively after 5 d, 10 d, 15 d, 20 d, 25 d. Shannon density index of bacteria experienced a process from a gradual reduction to stable state. This suggested that ultrasonic pretreatment had a significant impact on bacterial community structures. Cluster analysis of DGGE by UPGMA (unweighted air group method, arithmetic mean) divided all lanes into three clusters, which corresponded to different periods during the whole experiment. The sequences indicated that Firmicute, Genuscitrobacter, Bacilli, alpha-Proteobacteria, beta-Proteobacteria were the predominant microbial populations in the process of sludge aerobic/anoxic digestion after ultrasonic pretreatment.