Bunkerd Kongyingyoes

Differential methylation of E2 binding sites in episomal and integrated HPV 16 genomes in preinvasive and invasive cervical lesions

Enhanced expression of the HPV 16 E6‐E7 oncogenes may trigger neoplastic transformation of the squamous epithelial cells at the uterine cervix. The HPV E2 protein is a key transcriptional regulator of the E6‐E7 genes. It binds to four E2 binding sites (E2BSs 1–4) in the viral upstream regulatory region (URR). Modification of E2 functions, for example, by methylation of E2BSs is hypothesized to trigger enhanced expression of the viral E6‐E7 oncogenes. In the majority of HPV‐transformed premalignant lesions and about half of cervical carcinomas HPV genomes persist in an extra‐chromosomal, episomal state, whereas they are integrated into host cells chromosomes in the remaining lesions. Here we compared the methylation profile of E2BSs 1–4 of the HPV 16 URR in a series of 18 HPV16‐positive premalignant lesions and 33 invasive cervical cancers. CpGs within the E2BSs 1, 3, and 4 were higher methylated in all lesions with only episomal HPV16 genomes compared with lesions displaying single integrated copies. Samples with multiple HPV16 integrated copies displayed high methylation levels for all CpGs suggesting that the majority of multiple copies were silenced by extensive methylation. These data support the hypothesis that differential methylation of the E2BSs 1, 3 and 4 is related to the activation of viral oncogene expression in cervical lesions as long as the viral genome remains in the episomal state. Once the virus becomes integrated into host cell chromosomes these methylation patterns may be substantially altered due to complex epigenetic changes of integrated HPV genomes.

Tetrahydrocurcumin alleviates hypertension, aortic stiffening and oxidative stress in rats with nitric oxide deficiency.

Tetrahydrocurcumin (THC), a major metabolite of curcumin, possesses strong antioxidant and cardioprotective properties. However, the activities of THC in hypertension and its associated complications remain unknown. The aim of this study was to investigate the effect of THC on hemodynamic status, aortic elasticity and oxidative stress in rats with N-nitro-L-arginine methyl ester (L-NAME)-induced hypertension. Hypertension was induced in male Sprague-Dawley rats by administration of L-NAME (50 mg kg−1 body weight) in drinking water for 5 weeks. THC at a dose of 50 or 100 mg kg−1 per day was administered daily during the fourth and fifth weeks when the hypertensive state had been established. The effects of THC on hemodynamics, aortic elasticity, endothelial nitric oxide synthase (eNOS) protein expression and oxidative stress markers were assessed. Marked increases in blood pressure, peripheral vascular resistance, aortic stiffness and oxidative stress were found in rats after L-NAME administration. THC significantly reversed these deleterious effects by reducing aortic wall thickness and stiffness. These effects were associated with increased aortic eNOS expression, elevated plasma nitrate/nitrite, decreased oxidative stress with reduced superoxide production and enhanced blood glutathione. Our results provide the first evidence that THC attenuates the detrimental effect of L-NAME by improving the hemodynamic status and aortic elasticity concomitant with reduction of oxidative stress. The present study suggests that THC might be used as a dietary supplement to protect against cardiovascular alterations under nitric oxide-deficient conditions.

E2 proteins of high risk human papillomaviruses down-modulate STING and IFN-κ transcription in keratinocytes.

In the early stages of human papillomavirus (HPV) infection, the viral proteins elicit specific immune responses that can participate to regression of ano-genital lesions. HPV E6 protein for instance can reduce type I interferon (IFN) including IFN-κ that is involved in immune evasion and HPV persistence. To evaluate the role of E2 protein in innate immunity in HPV16-associated cervical lesions, genome-wide expression profiling of human primary keratinocytes (HPK) transduced by HPV16 E2 was investigated using microarrays and innate immunity associated genes were specifically analyzed. The analyses showed that the expression of 779 genes was modulated by HPV16E2 and 92 of them were genes associated with innate immunity. Notably IFN-κ and STING were suppressed in HPK expressing the E2 proteins of HPV16 or HPV18 and the trans-activation amino-terminal domain of E2 was involved in the suppressive effect. The relationship between STING, IFN-κ and interferon stimulated genes (ISGs) in HPK was confirmed by gene silencing and real time PCR. The expression of STING and IFN-κ were further determined in clinical specimens by real time PCR. STING and IFN-κ were down-modulated in HPV positive low grade squamous intraepithelial lesions compared with HPV negative controls. This study demonstrates that E2 proteins of high risk HPV reduce STING and IFN-κ transcription and its downstream target genes that might be an immune evasion mechanism involved in HPV persistence and cervical cancer development.

Association of human papillomavirus type 16 long control region mutation and cervical cancer

BackgroundThe variation of human papillomavirus (HPV) genes or HPV variants demonstrates different risks of cervical cancer. Mutation in the long control region (LCR) at YY1-motifs is one of the mechanisms for enhancing viral oncogene expression during the course of cancer cell progression. In Thai women, cervical cancers are almost always associated with HPV16 variant sub-lineage Asian (HPV16As); however, the mechanism involved remains elusive. The aim of this study was to understand further the oncogenic potential of HPV16As.MethodsA total of 82 HPV16-positive specimens from Thai women were selected from formalin-fixed paraffin-embedded cervical tissues, and the full length E6 gene of each specimen was amplified and sequenced. LCRs of the HPV16As-positive cases were amplified and sequenced to analyze their polymorphisms. Transcriptional activities of the HPV16As LCRs were then compared with sub-lineage European (EUR), sub-lineage Asian-American 1 (AA1) and HPV16 prototype by insertion of the LCRs into the pGL3-Basic vector.ResultsThe HPV16 DNA sequences were classified as HPV16 prototype (18.3%), Asian (As, 61%), Asian American-1 (AA1, 8.5%), European (EUR, 7.3%), Asian African-2 (AFR2, 3.7%) and Java-135C (J135C, 1.2%). The prevalence of HPV16As was 30% in low-grade squamous intraepithelial lesion (LSIL), while that in high-grade squamous intraepithelial lesion (HSIL) and squamous cell cervical carcinoma (SCC) were 63.9% and 66.7%, respectively, which demonstrates a significant association of HPV16As with the disease severity. LCR polymorphisms from 43 HPV16As positive cases were analyzed by PCR-sequencing. Thirty-eight nucleotide variation positions spanned nucleotide positions 7157–82. Ten new mutations found in the HPV16As LCRs were located predominantly at the enhancer and proximal to the 3’-end of the early promoter. The LCRs of the common HPV16As, EUR and AA1 showed 5, 13 and 23-fold higher activity than the HPV16 prototype LCR, while those of the new nucleotide variations of As showed 19 (As-sv1) and 30 (As-sv14) -fold higher activity than the HPV16 prototype.ConclusionsHPV16As DNA sequence variation, especially at the proximal to early promoter in the LCR, enhances transcriptional activity. This could be one of the possible mechanisms for HPV16As-associated cervical cancer development.

Prevalence of human papillomavirus type 16 and its variants in abnormal squamous cervical cells in Northeast Thailand.

OBJECTIVES To investigate the prevalence of HPV, HPV16, and HPV16 variants in scraped cervical cells cytologically diagnosed as normal cervical cell and in formalin-fixed, paraffin-embedded tissues of cervical intraepithelial neoplasia II-III and squamous cervical carcinoma in Northeast Thailand. METHODS All samples were subjected to PCR using consensus GP5+/GP6+ primers. HPV16 was genotyped by Southern blot hybridization and reverse line blot hybridization. The HPV16 E6 gene was amplified and sequenced. RESULTS HPV infections were found in 33.8% of normal cervical cells, 97.3% of cervical intraepithelial neoplasia II-III, and 100% of squamous cervical carcinomas. The prevalence of HPV16 increased significantly with histological grade (normal cervical cell, 16.7%; cervical intraepithelial neoplasia II-III, 38.9%; squamous cervical carcinoma, 75%). The most common variant found was the Asian (As) (58.7%) followed by the European (E) lineage (41.3%). The HPV16 As lineages showed a risk association in 73.9% of squamous cervical cancer and 57.1% of cervical intraepithelial neoplasia II-III, while no increased risk was observed in the E lineages. CONCLUSION Our study demonstrates that HPV16, in particular the As variant, was the major causative agent associated with cervical cancer in Northeast Thailand, and our study suggests that some mutations of the E6 gene in this variant, which leads to amino acid changes, may be more carcinogenic.

Rice Bran Protein Hydrolysates Improve Insulin Resistance and Decrease Pro-inflammatory Cytokine Gene Expression in Rats Fed a High Carbohydrate-High Fat Diet

A high carbohydrate-high fat (HCHF) diet causes insulin resistance (IR) and metabolic syndrome (MS). Rice bran has been demonstrated to have anti-dyslipidemic and anti-atherogenic properties in an obese mouse model. In the present study, we investigated the beneficial effects of rice bran protein hydrolysates (RBP) in HCHF-induced MS rats. After 12 weeks on this diet, the HCHF-fed group was divided into four subgroups, which were orally administered RBP 100 or 500 mg/kg, pioglitazone 10 mg/kg, or tap water for a further 6 weeks. Compared with normal diet control group, the MS rats had elevated levels of blood glucose, lipid, insulin, and HOMA-IR. Treatment with RBP significantly alleviated all those changes and restored insulin sensitivity. Additionally, RBP treatment increased adiponectin and suppressed leptin levels. Expression of Ppar-γ mRNA in adipose tissues was significantly increased whereas expression of lipogenic genes Srebf1 and Fasn was significantly decreased. Levels of mRNA of proinflammatory cytokines, Il-6, Tnf-α, Nos-2 and Mcp-1 were significantly decreased. In conclusion, the present findings support the consumption of RBP as a functional food to improve insulin resistance and to prevent the development of metabolic syndrome.

Atypical bacterial pathogen infection in children with acute bronchiolitis in northeast Thailand

BACKGROUND Atypical bacterial pathogens--including Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Chlamydia trachomatis--are important infectious agents of the respiratory system. Most current information pertains to adults and little is known about the role of these organisms in lower respiratory tract infections among young children with acute bronchiolitis. METHODS This study detected these pathogens in the nasopharyngeal secretions of children between 1 month and 2 years of age admitted with acute bronchiolitis to hospitals in Khon Kaen, northeast Thailand. The M pneumoniae and C pneumoniae in the nasopharyngeal secretions were detected using multiplex and nested-polymerase chain reaction (PCR), whereas PCR and restriction fragment length polymorphism were used to investigate C trachomatis. These samples were also tested by multiplex reverse transcriptase PCR for respiratory viruses, including respiratory syncytial virus (RSV), influenza A, influenza B, and human metapneumovirus. RESULTS Of the 170 samples taken from hospitalized children with acute bronchiolitis, 12.9% were infected with atypical bacteria and 85.3% with respiratory viruses. RSV was the most common causative viral agents found in 64.7% of the samples. M pneumoniae was the most common atypical bacterial pathogen (14/170, 8.2%) and most of the patients infected with it were between 6 and less than 12 months of age (71 cases). Of the infected cases in this age group, 7 of 14 were infected with M pneumoniae and 4 of 4 with C pneumoniae. Both M pneumoniae (13/14) and C pneumoniae (4/4) had etiologies indicating viral coinfections. Four (2.4%) of all of the cases had C trachomatis infections and all of these were infected with RSV, including three patients less than 6 months of age. CONCLUSION These results suggest that in children with virus-induced acute bronchiolitis coinfection with M pneumoniae, C pneumoniae, or C trachomatis can be expressed differently in each age group. These atypical bacteria may be the important infectious agents that induce severe illness of acute bronchiolitis.

Association of Epstein-Barr virus infection with oral squamous cell carcinoma in a case–control study

BACKGROUND Besides the well-known risk factors, Epstein-Barr virus (EBV) might play a significant role in oral squamous cell carcinoma (OSCC). To explore the role of EBV in OSCC, the prevalence of EBV infection in oral exfoliated cells of OSCC cases and controls in northeastern Thailand was investigated, and the association of EBV in tumor lesion cells was further confirmed. METHODS Oral exfoliated cells were collected from OSCC cases and non-cancer controls. Cells from tumor lesions were taken from OSCC patients for further strong confirmation of the association of EBV with OSCC. EBV DNA was detected by polymerase chain reaction (PCR) using primers specific for EBV DNA polymerase. The EBV DNA positive samples were confirmed further by nested PCR. RESULTS Epstein-Barr virus was detected in the oral exfoliated cells of 45.05% of OSCC patients and 18.08% of the non-cancer control (P < 0.001). Similarly, EBV was detected in 32.5% of the tumor lesions. Betel quid chewing was statistically significantly associated with EBV prevalence (OR = 2.08), whereas no association with tobacco smoking and alcohol consumption. Alcohol consumption and betel quid chewing were significantly associated with OSCC (OR = 3.05 and OR = 5.05, respectively), but tobacco smoking was not associated. Interestingly, EBV was significantly associated with OSCC (OR = 3.76). CONCLUSIONS Epstein-Barr virus prevalence is associated with OSCC and seems to be enhanced by betel quid chewing, suggesting that EBV may, together with betel quid chewing, act as an important etiological risk factor of OSCC.

Up-Regulation of miR-21 Is Associated with Cervicitis and Human Papillomavirus Infection in Cervical Tissues

MicroRNA-21 (miR-21) is recognized as an oncomir and shows up-regulation in many types of human malignancy. The aim of this study was to investigate the association of miR-21 expression associated with HPV infection in normal and abnormal cervical tissues. Cervical tissue samples with different cytological or histopathological grades were investigated for HPV by PCR and for miR-21 and programmed cell death, protein 4 (PDCD4) expression using quantitative real-time PCR (qRT-PCR). Laser capture microdissection (LCM) of stromal and epithelial tissues and in situ hybridization (ISH) using locked nucleic acid (LNA) probes were performed on a subset of fixed specimens. Cell line experiments were conducted on fibroblasts stimulated in culture media from HeLa cells, which were then assessed for miR-21, PDCD4, IL-6 and α-SMA expression by qRT-PCR. Twenty normal cervical cell, 12 cervicitis, 14 cervical intraepithelial neoplastic I (CIN I), 22 CIN II-III and 43 cervical squamous cell carcinoma (SCC) specimens were investigated. miR-21 levels were significantly lower in normal than in abnormal tissues. The expression of miR-21 in HPV negative normal cytology was significantly lower than in HPV positive samples in abnormal tissue and SCC. The miR-21 expression was significantly higher in HPV negative cervicitis than HPV negative normal cells. LCM and ISH data showed that miR-21 is primarily expressed in the tumor-associated stromal cell microenvironment. Fibroblasts treated with HeLa cell culture media showed up-regulated expression of miR-21, which correlated with increased expression of α-SMA and IL-6 and with down-regulation of PDCD4. These results demonstrate that miR-21 is associated with HPV infection and involved in cervical lesions as well as cervicitis and its up-regulation in tumor-stroma might be involved in the inflammation process and cervical cancer progression.

The relationship of human papillomavirus (HPV) detection to pap smear classification of cervical-scraped cells in asymptomatic women in northeast Thailand.

Objective: To correlate the detection of human papillomavirus (HPV) with the Pap smear classification of cervical‐scraped cells from asymptomatic women living in northeast Thailand.