Prevalence and genotype distribution of human papillomavirus infection in Harbin, Northeast China.

This study aimed to investigate the overall prevalence of human papillomavirus (HPV) infection among women examined at a hospital in Harbin and to evaluate the impact of HPV types on the natural outcome and state of cervical cytology. A total of 2,938 female outpatients from the affiliated hospital of Harbin Medical University were enrolled. Rapid hybridization gene chip and liquid-based cytology tests were used to detect HPV genotypes and cervical cytology. The overall prevalence of HPV in women who came to this hospital was 36.45 %. The majority were infected with a single strain, and the high-risk HPV (HR-HPV) type constituted the largest proportion. HPV16 and 58 were the most common types, while the genotypes of single low-risk HPV (LR-HPV) were not the same in different age groups. HPV53, 16 and 81 were the most common types in multiple HR-HPV infection; HR-HPV16, 33, 81 and LR-HPV 6, 44, 43 were the most common in HR and LR-HPV infection. In total, 44.1 % of the women with HSIL and 44.0 % with ASCUS were positive for HR-HPV16. Multiple HPV infections and single HPV infections had no effect on the natural outcome after half a year. HPV16, 81 and 35 had a better natural outcome, followed by HPV52 and 53, but HPV58, 59 and 18 had a bad outcome after half a year. This is the first study to show that the distribution of HPV types is different in Harbin than it is in other regions. These findings will provide guidance for the vaccination program in this area.

Metadherin confers chemoresistance of cervical cancer cells by inducing autophagy and activating ERK/NF-κB pathway.

Overexpression of metadherin (MTDH) has been reported in many solid tumors and implicated in chemoresistance. This study aimed to examine MTDH expression in cervical cancer tissues and explore its role in chemoresistance of cervical cancer. MTDH expression in cervical cancer biopsies and several cervical cancer cell lines was detected by immunoblotting and immunohistochemisty. MTDH expression level was experimentally modulated in HeLa cells to determine the effects on chemoresistance to cisplatin. The results showed that MTDH expression was higher in tissues from both cervical squamous carcinoma and cervical adenocarcinoma, compared to normal cervical tissues. MTDH expression was not correlated to patient age or cervical cancer grade, although nuclear MTDH expression was correlated with poor differentiation of cervical cancer. In SiHa, HeLa, CasKi, and C33A cells, MTDH expression level was positively correlated with chemoresistance to cisplatin. MTDH increased autophagy in HeLa cells, which was associated with decreased cleavage of Caspase-3 and the activation of EER/NF-κB pathway. In conclusion, MTDH expression is high in cervical cancer, and it contributes to chemoresistance of cervical cancer. MTDH could be utilized as a therapeutic target to overcome chemoresistance of cervical cancer.

[Effect of polishing method and time on the fitness of CAD/CAM zirconia ceramic crown].

PURPOSE: To compare the effect of different polishing methods and time treatment on the fitness of CAD/CAM zirconia ceramic crowns. METHODS: Sixteen intact maxillary premolars were randomly divided into two groups, group A was treated with silicon carbide burs, while group B was treated with tungsten steel burs. At different polishing time points of the same tooth, digital impressions of each group were obtained, which were used to manufacture CAD/CAM zirconium ceramic crowns. After trial fitting, the gap impressions were obtained by using silicone rubber replication method, and the marginal and internal discrepancies were assessed. The data were statistically analyzed with SPSS 21.0 software package. RESULTS: The difference between the gap values of the marginal and internal markers of group A and group B was not statistically significant(P＞0.05). Compared with the no-polishing process, the differences of the marginal gap (39.67±8.35) µm and internal gap (45.18±7.16) µm of group A polished for 4 min, and the marginal gap (51.25±14.73) µm, and internal gap (48.56±6.45) µm of group B polished for 3 min, as well as the marginal gap (48.87±8.90) µm, and internal gap (45.99±7.12) µm of group B polished for 4 min, were all significant(P＜0.05). CONCLUSIONS: CAD/CAM zirconia ceramic crowns treated with silicon carbide bur for polishing 4 min and tungsten steel for 3 min has the best fitness.

Human papillomavirus type 16 variant analysis of E6, E7, and L1 [corrected] genes and long control region in [corrected] cervical carcinomas in patients in northeast China.

Human papillomavirus type 16 (HPV 16) plays a cardinal role in the pathogenesis of cervical cancer. HPV 16 has intratypic variants which show different geographical distributions and different oncogenic potentials. To analyze the presence of sequence variations of HPV 16 variants in northeast China, 71 cervical carcinomas were identified by HPV typing. HPV 16-positive specimens were analyzed by PCR-directed sequencing in the E6, E7, and L1 genes and the LCR (long control region). The variation data were compared with those of neighboring districts. In this hospital-based study, HPV 16 was the most common type (73.24%). In HPV 16-positive specimens, 67.31% belonged to the European (E) lineage, while 32.69% were Asian (As) variants. The Asian-American (AA), African-1 (Af-1), African-2 (Af-2), and northern American (NA) lineages were not detected. The most frequently observed variation sites were T178G (32.69%) in E6; A647G (34.62%), G666A (38.46%), and T846C (32.69%) in E7; C6826T (36.17%) and G7060A (61.70%) in L1; and G7521A (98.08%) in the LCR. The most prevalent amino acid variations were D25E in E6 and N29S in E7. In addition, 28 novel variations of HPV 16 were reported. Some covariations between different genes were obtained. In this study, HPV 16 variants belonged to the European lineage and the Asian lineage. Compared with neighboring districts, the distribution of HPV 16 variants in northeast China had a typical pattern. As the first report on HPV 16 variants in northeast China, it should be helpful for designing a HPV vaccine and HPV vaccination program in China.

Low-serum culture system improves the adipogenic ability of visceral adipose tissue-derived stromal cells.

In obese adipose tissue, infiltrating macrophages release proinflammatory cytokines that trigger insulin resistance. An adipocyte-based platform from visceral fat would be useful to elucidate the pathology of adipose inflammation and to develop therapeutic drugs for insulin resistance. ADSCs (adipose tissue-derived mesenchymal stromal cells) expanded from subcutaneous fat are intensively studied as sources for regenerative medicine. However, the adipocyte culture system from visceral fat tissue has not been utilized yet. We aimed to establish the bioactive adipocyte platform using ADSCs from visceral fat pad. Stromal vascular fractions were processed from epididymal fat pads of Sprague-Dawley rats and three human omental fat pads, and the ADSCs were expanded using a low-serum culture method. The responses of ADSCs and ADSC-adipocytes (their adipogenic lineages) to pioglitazone, a therapeutic drug for diabesity, were evaluated by gene expression and ELISA. ADSCs (1×108) were expanded from 10 g of rat epididymal fat pads or human omental fat pads over five passages. Cell surface marker expressions revealed that visceral ADSCs were equivalent to mesenchymal stem cells. ADSC-adipocytes expanded in the low-serum culture system significantly showed higher expression of adipogenic markers [PPAR (peroxisome proliferator-activated receptor) γ, LPL (lipoprotein lipase) and FABP4 (fatty acid-binding protein 4)] and adipocytokines [adiponectin, resistin, leptin, PAI-1 (plasminogen-activator inhibitor 1) and IL (interleukin)-10] than those expanded in a high-serum culture system. Pioglitazone accelerated the adipogenic induction and increased adiponectin expression in human ADSCs by 57.9±5.8-fold (mean±S.E.M.) relative to control cells (P<0.001). Both in rat and human ADSC-adipocytes, TNF-α significantly induced proinflammatory cytokines [MCP-1 (monocyte chemoattractant protein-1) and IL-6] and suppressed adiponectin expression, while pioglitazone antagonized these effects. The present findings suggest that visceral ADSC-adipocytes expanded in low-serum culture would be useful for adiposcience and pharmacological evaluations.

[Application of orthogonal analysis to the optimization of HPV16 E2 protein expression].

This study was aimed to identify pET21b-HPV16E2/BL21(DE3) strain and to optimize the expression of human papillomavirus type 16 (HPV16) E2 protein by orthogonal analysis. Four influence factors on two levels were selected to increase the target protein quantity. The four factors were induction time, induction temperature, inductor concentration and cell density. The quantity of HPV16 E2 protein was used as the evaluation parameter. Induced by IPTG, HPV16 E2 protein was analyzed by SDS-PAGE and Western Blot. Target protein was analyzed by GIS imaging system to quantify the protein level. SPSS13. 0 software was applied to analyze the result. Data showed that the expression strain pET211rHPV16 E2/BL21(DE3) was identified correctly. HPV16 E2 protein expressed mainly at insoluble form. The 42KD protein band was identified by SDS-PAGE and Western blot. Orthogonal test was applied on influence factor analysis and expression optimization successfully. Main influence factors were inductor concentration and induction temperature. The optimimum condition of maximum expression quantity was 37 degrees C, 7h, 1.0 mmol/L IPTG and OD600 1.0. In this experiment, orthogonal test could not only be used to analyze the influential factors and promote the target protein expression, but also be used to provide a better experiment method for molecular biological study.

Investigation and analysis of antibody levels of hepatitis A among children before and after implementing the Expanded National Immunization Program in China.

OBJECTIVE: To analyze changes of hepatitis A antibody levels and immunization coverage of HAV vaccine among children before and after implementing the Expanded National Immunization Program in five counties of China, and to provide evidence for developing hepatitis A vaccine immunization strategies. METHODS: 449 children born in 2001, 2005 and 2009 were selected from five counties for an immunization coverage and a sero-prevalence survey of hepatitis A. The chemiluminescence microparticle immunoassays (CMIA) were used to detect hepatitis A IgG antibody and analyzed the factors which influenced the immunization coverage and positive rates. RESULTS: Among 449 subjects of children born in 2001, 2005 and 2009, the immunization coverage were 53.02%, 78.52% and 99.34% (χ2 = 91.285, P < 0.001). The positivity rates of hepatitis A IgG antibody were 61.07%, 81.21%, 95.36% (χ2 = 54.198, P < 0.001), respectively. The immunization coverage and positivity rate significantly increased with the delay of birth year. Children accepting different doses of HA vaccines are significantly different in positive rates of HA antibody, while there are no significant differences of different genders, years of birth, residence types, or types of registered permanent residence in different regions. The positivity rate increased significantly with administration of hepatitis A vaccine and shorter intervals between sample collection and HAV immunization. CONCLUSIONS: After the Expanded National Immunization Program was implemented, hepatitis A antibody levels were significantly increased in five counties, which indicates a successful result of EPI.

Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C.

AIM: To construct eukaryotic expression plasmids of full-length Hepatitis B Virus (HBV) genotype C genome, which contain lamivudine-resistant mutants (YIDD, YVDD) or wild-type strain (YMDD), and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg)] of the recombinant plasmids in HepG2 cells. METHODS: Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD, pMD18T-HBV/YVDD and pMD18T-HBV/YMDD, using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1 (+), between the EcoRI and HindIII sites. After being characterized by restriction endonuclease digestion, and DNA sequence analysis, the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection, the levels of intracellular viral DNA replication were detected by real-time PCR, and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA. RESULTS: Restriction endonuclease digestion and DNA sequence analysis confirmed that the three recombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells, high levels of intracellular viral DNA replication were observed, and HBsAg and HBeAg were secreted into the cell culture supernatant. CONCLUSION: Eukaryotic expression plasmids pcDNA3.1 (+)-HBV/YIDD, pcDNA3.1 (+)-HBV/YVDD or pcDNA3.1 (+)-HBV/YMDD, which contained HBV genotype C full-length genome, were successfully constructed. After transfection into HepG2 cells, the recombinant plasmids efficiently expressed HBV DNA, HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.

[A meta-analysis on the relations between Helicobacter pylori infection and chronic urticaria].

OBJECTIVE: To investigate the relationship between Helicobacter pylori (HP) infection and the development of chronic urticaria. METHODS: Published case-control studies which concerned HP infection related chronic urticaria were searched in Wanfang, CNKI, CQVIP Chinese databanks and PubMed. Meta-analysis was applied to analyze the pooled odds ratio (OR) and 95% confidence interval (CI). RESULTS: 37 studies which comprised 2 909 cases of chronic urticaria and 1 873 persons served as controls were enrolled. When compared with the controls, HP infection significantly increased the risk of chronic urticaria development with a pooled OR of 3.20 (95% CI: 2.31-4.43). Results from Meta-regression analyses showed that the distribution of residential areas and detection method being used were potential influential factors. CONCLUSION: HP infection seemed to be associated with an increased risk of developing the chronic urticaria.

Interleukin-12 gene adjuvant increases the immunogenicity of virus-like particles of human papillomavirus type 16 regional variant strain.

OBJECTIVES: To analyze the immunogenicity of virus-like particles (VLP) of human papillomavirus type 16 (HPV16) isolated in East China and the adjuvant potential of interleukin-12 (IL-12). METHODS: The variant HPV16 L1VLP expressed in sf9 insect cells were purified with cesium chloride gradient centrifugation. BALB/c mice were vaccinated with VLP (L1N), VLP with Freund's adjuvant (L1A) or VLP with IL-12 recombinant plasmid (L1P). HPV16 VLP specific IgG and IFN-γ level in the serum were detected by ELISA, and the percentage of CD4(+) and CD8(+) in spleen cells was detected with flow cytometry. RESULTS: The titers of serum IgG antibodies in vaccinated groups were higher than in negative control and the serum antibodies mainly recognized conformation-dependent HPV16 VLP epitopes. Splenic CD4(+) and CD8(+) T cell subsets increased after vaccination in every experimental group, and CD8(+) increased obviously in L1P group. The ratio of CD4(+)/CD8(+) decreased in L1P group and increased in the other two groups, compared to control group. Vaccination induced specific secretion of IFN-γ in the serum of vaccinated group (p<0.05), especially in the L1P group. CONCLUSIONS: VLP of HPV16 variant strain isolated in East China could induce humoral immunity and cellular immunity in mice, and IL-12 recombinant plasmid can enhance cellular immunity.

Serological data analyses show that adenovirus 36 infection is associated with obesity: a meta-analysis involving 5739 subjects.

OBJECTIVE: Serological studies on the relationship between adenovirus 36 (Ad36) and an increased risk of obesity development have shown conflicting results. We reviewed the published studies and carried out a meta-analysis to explore this relationship. METHODS: PubMed was searched until December 2012 for the relative references with sufficient information to estimate odds ratios (ORs) and 95% confidence intervals (CIs). A total of 11 case-control studies, including 2508 obese subjects and 3005 controls, were selected. RESULTS: Compared with nonobese controls, Ad36 infection significantly increased the obesity risk by a pooled OR of 1.60 (95% CI = 1.14-2.25; P < 0.01). Meta-regression showed that the types of subject and obesity assessments were potential risk factors. In the subgroup analysis, a significantly increased risk was found in children (OR = 1.95; 95% CI = 1.34-2.85; z = 3.45; P < 0.01) and those with an obesity assessment of BMI ≥ 30 kg/cm2 (OR = 1.89; 95% CI = 1.15-3.10; P < 0.05). CONCLUSIONS: Ad36 infection is associated with an increased risk of obesity development. To our knowledge, this is the first report to reveal the significant relationship in children with a serological data analysis.

Characterization of two new monoclonal antibodies against human papillomavirus type 16 L1 protein.

BACKGROUND: Human papillomavirus type 16 (HPV16) infection is implicated in cervical carcinogenesis. This study aimed to characterize two new monoclonal antibodies (mAbs) against HPV L1 protein. METHODS: The immunocompetence of AE3 and AG7 mAbs for HPV L1 protein was evaluated by Western blot analysis, immunostaining, hemagglutination inhibition assay, and ELISA. The heavy chain variable region (VH) and light chain variable region (VL) of AE3 and AG7 mAbs were sequenced and analyzed. RESULTS: Both mAbs specifically recognized HPV16 L1 and virus-like particles (VLPs). Both the affinity and the titer of AE3 mAb were higher than that of AG7. There were differences in sequences in the complementary determining regions (CDR) 2 and 3 of VL, as well as in the CDR1 and CDR3 of VH. The two mAbs have distinct predicted three-dimensional structures. CONCLUSIONS: We characterized two mAbs neutralizing antibodies for HPV L1 protein, which would help develop genetic-engineered neutralizing antibodies against HPV16 for diagnostic and therapeutic purposes.

Involvement of de novo ceramide synthesis in pro-inflammatory adipokine secretion and adipocyte-macrophage interaction.

Interaction between adipocytes and macrophages has been suggested to play a central role in the pathogenesis of obesity. Ceramide, a sphingolipid de novo synthesized from palmitate, is known to stimulate pro-inflammatory cytokine secretion from multiple types of cells. To clarify whether de novo synthesized ceramide contributes to cytokine dysregulation in adipocytes and macrophages, we observed cytokine secretion in mature 3T3-L1 adipocytes (L1) and RAW264.7 macrophages (RAW) cultured alone or co-cultured under the suppression of de novo ceramide synthesis. Palmitate enhanced ceramide accumulation and stimulated the expression and secretion of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in L1. The suppression of serine-palmitoyl transferase, a rate-limiting enzyme of de novo ceramide synthesis, by myriocin or siRNA attenuated those palmitate-induced alterations, and a ceramide synthase inhibitor fumonisin B1 showed similar results. In contrast, the inhibitor of sphingosine kinase or a membrane-permeable ceramide analogue augmented the cytokine secretion. Myriocin effects on the palmitate-induced changes were not abrogated by toll-like receptor-4 blockade. Although palmitate stimulated RAW to secrete tumor necrosis factor-α (TNF-α), it did not significantly increase ceramide content, and neither myriocin nor fumonisin B1 attenuated the TNF-α hypersecretion. The co-culture of L1 with RAW markedly augmented IL-6 and MCP-1 levels in media. Myriocin or fumonisin B1 significantly lowered these cytokine levels and suppressed the gene expression of TNF-α and MCP-1 in RAW and of IL-6 and MCP-1 in L1. In conclusion, de novo synthesized ceramide partially mediates the palmitate effects on pro-inflammatory adipokines and is possibly involved in the interaction with macrophages.

MiR-122 directly inhibits human papillomavirus E6 gene and enhances interferon signaling through blocking suppressor of cytokine signaling 1 in SiHa cells.

Human Papillomavirus (HPV) 16 infection is considered as one of the significant causes of human cervical cancer. The expression of the viral oncogenes like E6 and E7 play an important role in the development of the cancer. MiR-122 has been reported to exhibit a strong relationship with hepatitis viruses and take part in several tumor development, while the effects of miR-122 on HPV infection and the HPV viral oncogenes expression still remain unexplored. In this study, using RNAhybrid software, the potential binding sites between miR-122 and HPV16 E6 and E7 mRNAs were identified. Over and loss of miR-122 function showed that miR-122 could directly bind with HPV16 E6 mRNA and significantly inhibit its expression in SiHa cells, which was further confirmed by constructing the miR-122-E6-mu to eliminate the miR-122 binding effects with E6. The increase of the expression of type I interferon (IFN) and its classical effective molecules and the phosphorylation of signal transducers and activators of transcription (STAT1) protein indicated that miR-122 might enhance type I interferon in cervical carcinoma cells, which explained the significant reduction of HPV16 E7 and E6*I mRNA expression. This might be due to the binding between miR-122 and suppressor of cytokine signaling 1 (SOCS1) mRNA, which is the suppressor of interferon signaling pathway. Moreover, it was identified that the miR-122 binding position was nt359-nt375 in SOCS1 mRNA. Taken together, this study indicated that HPV16 could be effectively inhibited by miR-122 through both direct binding with E6 mRNA and promoting SOCS1-dependent IFN signaling pathway. Thus, miR-122 may serve as a new therapeutic option for inhibiting HPV infection.

[Expression and purification of HPV16 E2 protein and preparation of its antiserum].

OBJECTIVE: To express and purify the human papillomavirus type 16 (HPV16) E2 protein in prokaryotic bacteria and prepare the antiserum of HPV16 E2. METHODS: After amplified by PCR, HPV16 E2 was inserted into pET21b vector. The recombinant pET21b-HPV16E2 vector was transfected into E.coli BL21 (DE3). Expression product was identified after induction. Through purification, denaturation and renaturation, soluble protein was obtained. With the HPV16 E2 protein, we immunized BALB/c mice and examined mouse IFN-γ, CD4(+); T cells, CD8(+); T cells, CD4/CD8 ratio and antiserum titer. RESULTS: Restriction digestion and DNA sequencing showed pET21b-HPV16E2 was constructed successfully. Relative molecular mass (Mr;) of HPV16 E2 was 42 000 in SDS-PAGE and the specificity of the protein was confirmed with Western blotting. The antiserum could specifically bind with HPV16 E2 protein. In the immunized BALB/c mice, antiserum titre, CD4(+); T cell count and CD4/CD8 ratio increased, while mouse IFN-γ did not change obviously. CONCLUSION: Soluble HPV16 E2 protein was obtained successfully. The antiserum of high titer against HPV16 E2 was prepared in mice.

Interleukin-12 gene adjuvant increases the immunogenicity of virus-like particles of human papillomavirus type 16 regional variant strain

Objectives: To analyze the immunogenicity of virus-like particles (VLP) of human papillomavirus type 16 (HPV16) isolated in East China and the adjuvant potential of interleukin-12 (IL-12). Methods: The variant HPV16 L1VLP expressed in sf9 insect cells were purified with cesium chloride gradient centrifugation. BALB/c mice were vaccinated with VLP (L1N), VLP with Freund's adjuvant (L1A) or VLP with IL-12 recombinant plasmid (L1P). HPV16 VLP specific IgG and IFN-γ level in the serum were detected by ELISA, and the percentage of CD4+ and CD8+ in spleen cells was detected with flow cytometry. Results: The titers of serum IgG antibodies in vaccinated groups were higher than in negative control and the serum antibodies mainly recognized conformation-dependent HPV16 VLP epitopes. Splenic CD4+ and CD8+ T cell subsets increased after vaccination in every experimental group, and CD8+ increased obviously in L1P group. The ratio of CD4+/CD8+ decreased in L1P group and increased in the other two groups, compared to control group. Vaccination induced specific secretion of IFN-γ in the serum of vaccinated group (p < 0.05), especially in the L1P group. Conclusions: VLP of HPV16 variant strain isolated in East China could induce humoral immunity and cellular immunity in mice, and IL-12 recombinant plasmid can enhance cellular immunity. .

Prevalence of occult hepatitis B virus infection among hepatopathy patients and healthy people in China.

OBJECTIVE: To investigate the prevalence of occult hepatitis B virus (HBV) infection among hepatopathy patients and healthy people in China. METHODS: The HBV DNA in 653 sera samples collected from cryptogenic chronic liver disease patients (159), hepatitis B surface antigen (HBsAg) negative hepatocellular carcinoma (HCC) patients (135) and HBsAg-negative healthy people (359) were tested by nested PCR using specific primers of the X region of the HBV genome. We performed real-time PCR to determine the levels of serum HBV-DNA. RESULTS: Prevalence of occult HBV infection was 28.3% (45/159), 70.4% (95/135) and 10.6% (38/359) in cryptogenic chronic liver disease patients, HBsAg-negative HCC patients and HBsAg-negative healthy people, respectively. The prevalence of occult HBV infection in IgG anti-HBc-positive subjects was 100% (45/45), 86.7% (85/98) and 33.3% (14/42) in cryptogenic chronic liver disease patients, HBsAg-negative HCC patients and HBsAg-negative healthy people, respectively. In all cases, viral loads were low (<10(4)viral copies/mL). CONCLUSION: The prevalence of occult HBV infection was significantly high among hepatopathy patients and healthy people in China. Thus, more meticulous attention should be given to prevent HBV transmission by blood transfusion or organ transplantation in endemic areas, and further studies on clinical implication and mechanism of occult HBV infection are required.