TLR4-dependant immune response, but not hepatitis B virus reactivation, is important in radiation-induced liver disease of liver cancer radiotherapy.

Toll-like receptor 4 (TLR4) is an important trigger of the immune response against hepatitis B virus (HBV) infection and liver injuries. The roles of HBV reactivation versus TLR4-dependant immune response may be critical factors in preventing radiation-induced liver diseases (RILDs) after liver cancer radiotherapy. This study consists of three phases. In the primary phase, livers of mutant TLR4 (TLR4(-)) mice were irradiated with 30 Gy in either the absence or presence of HBV infection. The latter was done by introduction of plasmid pAAV/HBV 1.2. In the advanced phase, RILDs were compared in normal TLR4 (TLR4(+)) versus TLR4(-) mice. In the validation phase, 28 liver cancer patients who had undergone radiotherapy before hepatectomy were enrolled. Liver biopsies near tumors, irradiated with 35-48 Gy, were used to construct tissue microarrays. HBV reactivation, TLR4 expression, and severity of RILDs were studied in both mouse and human. More HBV reactivation, without significant RILD, was observed in irradiated versus unirradiated TLR4(-) mice. RILD scores of TLR4(+) mice were higher than TLR4(-) mice. In humans, serious RILDs tended to develop in patients with high TLR4 expression, but not in patients with low TLR4 or high HBV surface antigen expression. High TLR4 expression was seen in only 2 of 12 HBV-reactive patients, but in HBV-nonreactive patients, it was seen in 6 of 9 (P < 0.03). In summary, RILDs correlated with high TLR4 expression, but not with HBV reactivation, which is inhibited in liver with high TLR4 expression after liver cancer radiotherapy.

Hyper-activated IRF-1 and STAT1 contribute to enhanced interferon stimulated gene (ISG) expression by interferon alpha and gamma co-treatment in human hepatoma cells.

Previous reports suggest that type I and type II Interferon can co-operatively inhibit some virus replication, e.g. HCV, SARS-CoV, HSV-1. To find out the molecular mechanism underlying this phenomenon, we analyzed the transcription profile stimulated by IFN-alpha and IFN-gamma in Huh-7 cells and found that the transcription of a subset of IFN stimulated genes (ISGs) including BclG, XAF1, TRAIL and TAP1 was enhanced when IFN-alpha and gamma were both present. Promoter analysis of BclG revealed that IRF-1 and STAT1 were both required in this process. Enhanced IRF-1/DNA complex formation was observed in interferon co-treatment group by gel shift analysis. Furthermore, IRF-1 activation was found to be generally required in this cluster of ISGs. STAT1 tyrosine phosphorylation was elevated by IFN combination treatment, however, only the hyper-transactivation of GAS but not ISRE was observed. In conclusion, hyper-activation of IRF-1 and elevated STAT1 dimer formation may be two general switches which contribute to a much more robust antiviral symphony against virus replication when type I and type II IFNs are co-administered.

[The effects of CpG-oligodeoxynucleotides on airway remodeling in chronic asthmatic mice].

OBJECTIVE: To investigate if CpG-oligodeoxynucleotides (CpG-ODN) intervention has inhibitory effects on the development of airway remodeling in an ovalbumin (OVA)-sensitized mouse model of chronic asthma. METHODS: Forty female C57BL/6 mice were randomly divided into four groups (n = 10): (1) Group A (chronic asthma model): mice were sensitized by intraperitoneal injection of OVA (10 microg) precipitated with aluminium hydroxide (100 microg) on days 1 and 14. From day 21, the mice were challenged by nebulized 2.5% OVA solution (30 min/d, three times a week for 8 weeks). (2) Group B (CpG-ODN intervention group): mice were sensitized and challenged as above, and were given 60 microg CpG-ODN by intraperitoneal injection for once every two weeks. (3) Group C (GpG-ODN control): Mice were given GpC-ODN instead of CpG motifs, other treatments same as Group B. (4) Group D (saline control): mice were sensitized and challenged by saline. All mice were killed 24 h after the final OVA challenge. Blood was obtained for eosinophil counts and measurement of serum IgE by enzyme-linked immunoabsorbent assay (ELISA). Bronchoalveolar lavage fluid (BALF) was collected for total and differential counts. The concentration of interleukin-13 (IL-13) and transforming growth factor-beta1 (TGF-beta(1)) in BALF was measured by ELISA. The left lung was isolated for pathological examination. Lung sections were stained with hematoxylin and eosin (HE), and Masson's trichrome. Other sections were prepared for immunohistochemistry using monoclonal antibodies against alpha-smooth muscle actin (alpha-SMA) and TGF-beta(1). RESULTS: The eosinophil count [(89 +/- 10) x 10(4)/ml], serum IgE [(279 +/- 53) ng/ml], BALF eosinophils [(6.30 +/- 1.30) x 10(5)/ml] and the concentrations of BALF IL-13 [(4 015 +/- 361) pg/ml] and TGF-beta(1) [(356 +/- 64) pg/ml] in the OVA-sensitized mice (Group A) showed significant difference as compared with those in the NS control group (Group D, t values are 24.0, 15.7, 14.7, 18.4, 12.0 and 18.9 respectively, all P < 0.01). In Group A, the percentages of positive staining area in Masson's trichrome, alpha-SMA staining and TGF-beta(1) staining were (29.7 +/- 4.2)%, (45 +/- 7)% and (34 +/- 4)% respectively. These percentages were significantly different from those in the NS control group (Group D, t values are 18.0, 15.6 and 17.9 respectively, all P < 0.01). In mice treated with CpG-ODN (Group B), the percentages of positive staining area in Masson's trichrome, alpha-SMA staining and TGF-beta(1) staining were (13.8 +/- 3.2)%, (24.7 +/- 3.1)%, (18 +/- 4)% respectively, which were significantly different from those in Group A (t values are 9.5, 8.9 and 9.8 respectively, all P < 0.05). CONCLUSIONS: This study demonstrated that CpG-ODN could prevent Th2 responses, eosinophilic inflammation and the development of airway remodeling. Its inhibitory effect on airway remodeling might, in part, be due to inhibition of the expression of cytokines such as TGF-beta(1) and IL-13.

[Therapeutic effects and mechanisms of CpG oligodeoxynucleotides in mice infected with Mycobacterium tuberculosis].

OBJECTIVE: To investigate the protective effects and mechanisms of CpG oligodeoxynucleotides (CpG ODN) in mice infected with Mycobacterium tuberculosis. METHODS: Ninety-six female BALB/c mice were randomized into 4 groups, namely CpG ODN treatment (A group), control ODN treatment (B group), infection control (C group) and healthy control (D group) (n = 24 each). A group and B group were treated once with CpG ODN or control ODN (30 microg/mouse) intraperitoneally 2 weeks before infection with Mycobacterium tuberculosis. 1 x 10(6) bacili in a volume of 300 microl saline was injected into the tail vein of mice from A group, B group and C group. Twelve mice from each group were sacrificed at 3 weeks postinfection. Pathologic changes in lung tissues were observed. The expression of Toll-like receptor 9 (TLR9) mRNA and cytokine mRNA was detected by RT-PCR. The numbers of viable bacteria in lung and spleen were counted. The rest 12 mice from each group were monitored for 60 days to observe the mortality. RESULTS: CpG ODN was shown to increase the survival time of mice infected with Mycobacterium tuberculosis. The body weights of mice from A group [(20.37 +/- 1.12) g] were higher than those of B group [(17.50 +/- 0.62) g] and C group [(17.15 +/- 0.97) g, P < 0.01]. The lung weights of mice from A group [(0.25 +/- 0.02) g] were similar to those of B group [(0.27 +/- 0.34) g, P > 0.05], but less than those of C group [(0.28 +/- 0.26) g, P < 0.01]. The spleens of mice from A group [(0.63 +/- 0.37) g] were larger than those of B group [(0.39 +/- 0.05) g] and C group [(0.38 +/- 0.02) g, P < 0.01]. The inflammation in lung tissue of mice from A group was less than that of B group and C group. There was no mycobacterial outgrowth in lungs and spleens of mice from A group. The expression of TLR9 mRNA in lungs and spleens of mice from A group (0.61 +/- 0.29 and 0.72 +/- 0.48) was similar to that in B group (0.58 +/- 0.35 and 0.64 +/- 0.28) and C group (0.60 +/- 0.32 and 0.65 +/- 0.31, P > 0.05), but higher than that in D group (0.11 +/- 0.08 and 0.26 +/- 0.22, P < 0.01). CpG ODN did not affect the expression of TLR9 mRNA. The expression of IFN-gamma mRNA in lungs and spleens of mice from A group (0.44 +/- 0.07 and 0.76 +/- 0.09) was higher than that in B group (0.19 +/- 0.05 and 0.22 +/- 0.05) and C group (0.16 +/- 0.04 and 0.18 +/- 0.08, P < 0.01). The expression of IL-6 mRNA in lungs and spleens of mice from A group (1.56 +/- 0.29 and 8.21 +/- 0.82) was higher than that in B group (0.86 +/- 0.55 and 0.16 +/- 0.09) and C group (0.78 +/- 0.21 and 0.06 +/- 0.04, P < 0.01). The expression of IL-4 mRNA in lungs and spleens of mice from A group (0.18 +/- 0.05 and 0.06 +/- 0.02) was lower than that in B group (0.31 +/- 0.06 and 1.22 +/- 0.01) and C group (0.35 +/- 0.04 and 1.31 +/- 0.31, P < 0.01). The expression of IL-10 mRNA in spleens of mice from A group (0.05 +/- 0.02) was lower than that in B group (0.57 +/- 0.09) and C group (0.65 +/- 0.15, P < 0.01). CONCLUSION: CpG ODN could increase the immunity of mice against tuberculosis through up-regulation of Th1 immunity and down-regulation of Th2 immunity.

Gene expression profiles in peripheral blood mononuclear cells of SARS patients.

AIM: To investigate the role of inflammatory and anti-viral genes in the pathogenesis of SARS. METHODS: cDNA microarrays were used to screen the gene expression profiles of peripheral blood mononuclear cells (PBMCs) in two SARS patients (one in the acute severe phase and the other in the convalescent phase) and a healthy donor. In addition, real-time qualitative PCR was also performed to verify the reproducibility of the microarray results. The data were further analyzed. RESULTS: Many inflammatory and anti-viral genes were differentially expressed in SARS patients. Compared to the healthy control or the convalescent case, plenty of pro-inflammatory cytokines such as IL-1, TNF-alpha, IL-8, and MAPK signaling pathway were significantly upregulated in the acute severe case. However, anti-inflammatory agents such as IL-4 receptor, IL-13 receptor, IL-1Ra, and TNF-alpha-induced proteins 3 and 6 also increased dramatically in the acute severe case. On the contrary, a lot of IFN-stimulated genes like PKR, GBP-1 and 2, CXCL-10 and 11, and JAK/STAT signal pathway were downregulated in the acute severe case compared to the convalescent case. CONCLUSION: Gene expression in SARS patients mirrors a host state of inflammation and anti-viral immunity at the transcription level, and understanding of gene expression profiles may make contribution to further studies of the SARS pathogenesis.

High persistence rate of hepatitis B virus in a hydrodynamic injection-based transfection model in C3H/HeN mice.

AIM: To optimize the viral persistence rate in a hydrodynamic injection (HI) based hepatitis B virus (HBV) transfection mouse model. METHODS: (1) 5-6-wk-old male C3H/HeN and C57BL/6 mice were hydrodynamically injected with 10 µg endotoxin-free pAAV/HBV1.2 plasmid DNA via the tail vein. Hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg) and HBV DNA, both in the serum and liver, were detected at different time points post HI by ELISA, immunohistochemical staining or quantitative polymerase chain reaction (PCR); (2) male C3H/HeN and C57BL/6 mice, either hydrodynamically injected mice at 10 wk post HI or naïve mice, were all immunized subcutaneously with 5 µg HBsAg formulated in complete Freund's adjuvant three times at a 2-wk interval. Two weeks after the final immunization, splenocytes were isolated for T cell function analysis by ELISPOT assay; and (3) five weeks post HI, C3H/HeN mice were intragastrically administered 0.1 mg/kg entecavir once a day for 14 d, or were intraperitoneally injected with 1 mg/kg interferon (IFN)-α twice a week for 2 wk, or were treated with PBS as controls. The sera were collected and assayed for HBV DNA on days 0, 7 and 14 after drug treatment. RESULTS: (1) Approximately 90% (22/25) of the injected C3H/HeN mice were still HBsAg-positive at 46 wk post HI, whereas HBsAg in C57BL/6 mice were completely cleared at 24 wk. Serum levels of HBeAg in C3H/HeN mice were higher than those in C57BL/6 mice from 4 wk to 46 wk. HBV DNA levels in the hydrodynamically injected C3H/HeN mice were higher than those in the C57BL/6 mice, both in the serum (from 4 wk to 46 wk) and in the liver (detected at 8 wk and 46 wk post HI). Histology showed that hepatitis B core antigen and HBsAg were expressed longer in the liver of C3H/HeN mice than in C57BL/6; (2) HBsAg specific T cell responses after HBsAg vaccination in hydrodynamically injected C3H/HeN and C57BL/6 mice, or naive control mice were detected by ELISPOT assay. After stimulation with HBsAg, the frequencies of IFN-γ producing splenocytes in the hydrodynamically injected C3H/HeN mice were significantly lower than those in hydrodynamically injected C57BL/6 mice, control C3H/HeN and control C57BL/6 mice, which were 0, 17 ± 7, 18 ± 10, and 41 ± 10 SFCs/10(6) splenocytes, respectively, and the mean spot sizes showed the same pattern. Even just stimulated with PMA and ionomysin, T-cell responses elicited in the vaccinated control C3H/HeN were much higher than those in hydrodynamically injected C3H/HeN mice; and (3) For drug treatment experiments on the hydrodynamically injected C3H/HeN mice, serum HBV DNA levels in the entecavir treatment group declined (131.2 folds, P < 0.01) on day 7 after treatment and kept going down. In the group of IFN-α treatment, serum HBV DNA levels declined to a lowest point (6.42 folds, P < 0.05) on 7 d after treatment and then rebounded. CONCLUSION: We have developed a novel HI-based HBV transfection model using C3H/HeN mice, which had a higher HBV persistence rate than the classic C57BL/6 mouse model.

siRNA-mediated inhibition of HBV replication and expression.

AIM: RNA interference (RNAi) is a newly discovered phenomenon provoked by dsRNA. The dsRNA is initially cleaved by Dicer into 21-23 nt small interfering RNA (siRNA) and can then specifically target homologous mRNA for degradation by cellular ribonucleases. RNAi has been successfully utilized to down-regulate the endogenous gene expression or suppress the replication of various pathogens in mammalian cells. In this study, we investigated whether vector-based siRNA promoted by U6 (pSilencer1.0-U6) could efficiently inhibit HBV replication in cell culture. METHODS: pSilencer vectors with inserts targeting on different regions of HBV genome were constructed. These plasmids were co-transfected with pHBV3.8 into Huh-7 cells via lipofection and viral antigens were measured by ELISA. Viral RNA was analyzed by Northern blot. The mRNA of MxA and 2'-5'OAS was reverse transcribed and quantified by real-time PCR. RESULTS: Vector-based siRNA could potently reduce hepatitis B virus antigen expression in transient replicative cell culture. Furthermore, Northern blot analysis showed that viral RNA was effectively degraded, thus eliminating the messengers for protein expression as well as template for reverse transcription. Real-time PCR analysis of cellular MxA and 2'-5'OAS gene expression revealed that vector-based siRNA did not provoke the interferon pathway which reassured the specificity of the vector-based RNA interference technique. CONCLUSION: Our results indicate that RNA interference may be a potential tool to control HBV infection.

Establishment of a cell-based assay system for hepatitis C virus serine protease and its primary applications.

AIM: To establish an efficient, sensitive, cell-based assay system for NS3 serine protease in an effort to study further the property of hepatitis C virus (HCV) and develop new antiviral agents. METHODS: We constructed pCI-neo-NS3/4A-SEAP chimeric plasmid, in which the secreted alkaline phosphatase (SEAP) was fused in-frame to the downstream of NS4A/4B cleavage site. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transient or stable expression cells. Stably expressing cell lines were obtained by G418 selection. Pefabloc SC, a potent irreversible serine protease inhibitor, was used to treat the stably expressing cell lines to assess the system for screening NS3 inhibitors. To compare the activity of serine proteases from 1b and 1a, two chimeric clones were constructed and introduced into both transient and stable expression systems. RESULTS: The SEAP activity in the culture media could be detected in both transient and stable expression systems, and was apparently decreased after Pefabloc SC treatment. In both transient and stable systems, NS3/4A-SEAP chimeric gene from HCV genotype 1b produced higher SEAP activity in the culture media than that from 1a. CONCLUSION: The cell-based system is efficient and sensitive enough for detection and comparison of NS3 protease activity, and screening of anti-NS3 inhibitors. The functional difference between NS3/4A from 1a and 1b subtypes revealed by this system provides a clue for further investigations.

[The emergence of novel GII.4 norovirus variant, Sydney_2012, in Shanghai, China].

To describe the epidemiological characteristics of norovirus (NOV) associated acute gastroenteritis in Shanghai and characterize the evolution pattern of circulating strains. From March 2012 to February 2013, 502 stool specimens were collected from adult (> or = 16 years) outpatients who visited either of the two sentinel hospitals in Shanghai for acute gastroenteritis. Molecular detection and genotyping of NoV were performed and the phylogenetic relationship of the circulating strains has also been comprehensively analyzed. The epidemics level of GI NoV was low throughout the surveillance period, with the positive rate of 3.78% (19 cases), and no seasonality of GI NoV infection could be distinguished. For GII genogroup, higher epidemics in adults in Shanghai, with the detection rate of 17.13% (86 cases), were observed. And relatively high epidemics of GII NoV infection were spotted between October and December in 2012. The frequency of NoV associated acute gastroenteritis in older people is significantly higher than that in young individuals (P < 0.05). Sequencing and genotyping analysis revealed that the high epidemics of GII NoV infection between October and December in 2012 is associated with the emergence of a novel GII.4 norovirus strain, termed Sydney_2012. Sequence analysis also demonstrated that this was a recombinant virus between a GII.e polymerase and GII.4 capsid, which has also been the dominant circulating strain in Shanghai. In 2012, a new GII.4 variant, termed Sydney_2012, emerged in Shanghai and caused high epidemics of acute gastroenteritis during late autumn and winter.

Rupintrivir is a promising candidate for treating severe cases of Enterovirus-71 infection.

AIM: To evaluate the suitability of rupintrivir against Enterovirus 71 (EV71) induced severe clinical symptoms using computational methods. METHODS: The structure of EV71 3C protease was predicted by homology modeling. The binding free energies between rupintrivir and EV71 3C and human rhinovirus 3C protease were computed by molecular dynamics and molecular mechanics Poisson-Boltzmann/surface area and molecular mechanics generalized-born/surface area methods. EV71 3C fragments obtained from clinical samples collected during May to July 2008 in Shanghai were amplified by reverse-transcription and polymerase chain reaction and sequenced. RESULTS: We observed that rupintrivir had favorable binding affinity with EV71 3C protease (-10.76 kcal/mol). The variability of the 3C protein sequence in isolates of various outbreaks, including those obtained in our hospital from May to July 2008, were also analyzed to validate the conservation of the drug binding pocket. CONCLUSION: Rupintrivir, whose safety profiles had been proved, is an attractive candidate and can be quickly utilized for treating severe EV71 infection.

[Construction of full-length complementary DNA of hepatitis C virus genome from an HCV infected patient].

BACKGROUND: To construct the full-length complementary DNA of HCV genome from an HCV infected patient. METHODS: Four HCV gene fragments (1.6, 3.5, 2.4 kb and 2.6 kb) were amplified by RT-PCR from serum of a Chinese patient and fused and connected together to produce a 9.2 kb subgenomic fragment, which was further cloned into a cassette vector with fixed 5-prime and 3-prime termini of HCV to make the full-length cDNA. The cDNA heterogeneity was analyzed by comparing the sequences of 4 isolated HVR1 regions. The prokaryotic expressed Core, NS3 protease, NS3 helicase were detected for their specific reactivities with patient serum by Western blot analysis. And the protease activity of NS3 was evaluated in a cell-based NS3/4A-SEAP expression system. RESULTS: The cDNA covered the near full-length of HCV genome from the patient's serum. The difference among HVR1 regions indicated no selection of HCV variants during RT-PCR and the quasi-species characteristic of the amplified cDNA. The prokaryotic expressed viral proteins could be identified by patient serum. In the NS3/4A-SEAP system, NS3 could cleave the 4A-4B site between NS4A and SEAP proteins and resulted in the secretion of SEAP in culture media. CONCLUSION: These results suggest that the cloned HCV cDNA encodes a complete and functional open reading frame and will be useful for further construction of infectious cDNA clone.