An improved method for primary culture of normal cervical epithelial cells and establishment of cell model in vitro with HPV-16 E6 gene by lentivirus.

Normal human cervical epitheliums infected with HPVs gene in vitro are underlying molecular models to investigate physiological mechanisms of cervical epithelia and cervical disease. The current study aimed to establish a modified culture method for cervical epithelium and explore the feasibility of transfection with HPV-16 E6 gene mediated by lentivirus in primary cervical cells. The cells were dissociated enzymatically using Dispase II combined with 0.25% Trypsin-0.01% ethylenediamine tetracetic acid (EDTA) or Collagenase I. The detached effectiveness of Dispase II at different times was compared. Isolated cells were cultured and subcultured in modified keratinocyte serum-free medium (K-SFM) supplemented with 5% fetal bovine serum (FBS) or K-SFM alone. Cytokeratin was used as the identification of cervical epitheliums. Proliferative capacity and growth curve of cervical epitheliums were evaluated by cell counting kit-8 (CCK-8). The cells at passages 3 were used to infect with HPV-16 E6 gene by lentivirus. The expression of green fluorescent protein (GFP) presented in the infected cells was observed via fluorescence microscopy and the levels of E6 mRNA were detected by quantitative real-time PCR (qRT-PCR). The results indicate that cervical epithelial cells can be isolated successfully by Dispase II combined with 0.25% Trypsin-0.01% EDTA method for 20 hr and maintained for five or six passages in K-SFM medium with 5% FBS. The present study proposed a brief and high-yield protocol for isolation and culture of human cervical epitheliums. Moreover, an infected cell model with HPV-16 E6 gene mediated by lentivirus was established which can do duty for studies in vitro on the carcinogenic mechanism of HR-HPVs.

Detection of cervical intraepithelial neoplasia with HPVE6/E7 mRNA among women with atypical squamous cells of unknown significance.

OBJECTIVE: To compare E6/E7 mRNA and HPV DNA assays for evaluating women with atypical cells of undetermined significance (ASCUS). METHODS: The present prospective study enrolled patients with ASCUS undergoing HPV testing at Third Affiliated Hospital of Zhengzhou University, China, between September 1, 2013, and January 31, 2016. Patients with positive HPV DNA test results underwent screening by E6/E7 mRNA assay, and the accuracy of HPV DNA and E6/E7 mRNA testing were compared, with histology used for definitive diagnoses. RESULTS: In total, 591 patients with ASCUS underwent HPV DNA screening, with 455 and 136 having positive and negative results, respectively; 252 patients with positive results and 66 with negative results underwent biopsy and histology testing and were included in the study. The sensitivity of the E6/E7 mRNA assay was similar to that of HPV DNA testing (88.2%, 95% confidence interval [CI] 77.6-94.4 vs 90.7%, 95%CI 81.2-95.9; P=0.636) for the detection of cervical intraepithelial neoplasia grade 2+, and the specificity was higher (36.4%, 95%CI 29.6-43.9 vs 24.3%, 95%CI 19.1-30.3; P=0.006). The area under the receiver operating characteristic curve was greater for E6/E7 mRNA testing compared with HPV DNA testing (0.658 vs 0.588). CONCLUSION: The higher specificity of the E6/E7 mRNA assay means it could be a promising technique in the management of women with ASCUS.

MiRNAs Predict the Prognosis of Patients with Triple Negative Breast Cancer: A Meta-Analysis.

PURPOSE: miRNAs are stable and can be extracted from tissues, blood and other body fluid without degradation. miRNAs are abnormally expressed in the presence of a pathological status, including cancer. Therefore, miRNAs are ideal biomarkers for cancer diagnosis and prognosis. Patients with triple negative breast cancer (TNBC) suffer the worst prognosis, although great efforts have been made. Many studies have investigated the role of miRNAs in predicting the outcomes of TNBC patients for better adjustment of treatment. However, results were inconsistent. Thus, we performed a meta-analysis to summarize the published studies for conclusive results. METHODS: Eligible studies from different database were retrieved from the online databases, and we used STSTA 12.0 to analysis the prognostic role of miRNAs in triple negative breast cancer. RESULTS: Overall high miRNA expression indicated a worse survival with HR value of 1.78 (95% CI: 0.97-3.25). However, subtotal HRs of oncogenic miRNAs and tumor suppressive miRNAs were 2.73 (95% CI: 2.08-3.57; P<0.001) and 0.44 (95% CI: 0.21-0.90; P = 0.024), respectively, and no heterogeneity was observed within the subgroups. CONCLUSIONS: The miRNAs showed a slightly stronger prognostic value for disease-free survival, relapse-free survival and distant metastasis-free survival compared to the overall survival of TNBC patients. Circulating miRNAs could serve as potential biomarkers for the prognosis of TNBC patients and need further investigation.

Trimester- and Assay-Specific Thyroid Reference Intervals for Pregnant Women in China.

Objective. The guidelines of the American Thyroid Association (ATA) recommend an upper limit reference interval (RI) of thyroid stimulating hormone (TSH) of 2.5 mIU/L in the first trimester of pregnancy and 3.0 mIU/L in subsequent trimesters, but some reported ranges in China are significantly higher. Our study aimed to establish trimester- and assay-specific RIs for thyroid hormones in normal pregnant Chinese women. Methods. In this cross-sectional study, 2540 women with normal pregnancies (first trimester, n = 398; second trimester, n = 797; third trimester, n = 1345) and 237 healthy nonpregnant control subjects were recruited. Serum TSH, free thyroxin (FT4), thyroid peroxidase antibody (TPOAb), and thyroglobulin antibody (TgAb) levels were determined by automated chemiluminescence with an Immulite 2000 system (Siemens, Erlangen, Germany). After outliers were excluded, the 2.5-97.5th percentiles were used to define the RIs. Results. The RIs of thyroid function in the first, second, and third trimesters of pregnancy and in nonpregnant controls were 0.07-3.96, 0.27-4.53, 0.48-5.40, and 0.69-5.78 mIU/L for TSH and 9.16-18.12, 8.67-16.21, 7.80-13.90, and 8.24-16.61 pmol/L for FT4, respectively. Conclusion. The trimester- and assay-specific RIs of thyroid function during pregnancy differed between trimesters, which suggests that it is advisable to detect and avoid misclassification of thyroid dysfunction during pregnancy for women in Henan, China.

Performance of the HPV-16 L1 methylation assay and HPV E6/E7 mRNA test for the detection of squamous intraepithelial lesions in cervical cytological samples.

HPV-16 L1 methylation and E6/E7 mRNA have suggested that they had close relationship with cervical neoplastic progression. This study aimed to evaluate the clinical performance of the HPV-16 L1 methylation assay and E6/E7 mRNA test for detecting high-grade cervical lesions (CIN2+). A total of 81 women with liquid-based cytology (LBC) samples, histological results, and positive HPV-DNA test for HPV type 16 only were included in this study. HPV-16 L1 methylation and E6/E7 mRNA levels were measured using methylation-sensitive high resolution melting (MS-HRM) analysis and Quantivirus®HPV E6/E7 RNA 3.0 assay (bDNA), respectively, in the same residue of LBC samples. The current date showed a positive correlation between the HPV-16 L1 methylation and the E6/E7 mRNA levels. The L1 methylation and mRNA levels both increased with disease severity. The mRNA test method showed higher sensitivity and NPV (98.0 and 91.7% vs. 89.8 and 80.8%), while lower specificity and PPV (34.4 and 69.6% vs. 65.6 and 80.0%), than the L1 methylation assay for detecting histology-confirmed CIN2+. When using the detection method of mRNA test combined with L1 methylation assay, we obtained a sensitivity of 89.8% and a specificity of 71.9%. These findings suggest that assessment of HPV-16 L1 methylation testing combined with E6/E7 mRNA testing may be a promising method for the triage of women with HPV type 16 only.

Age- and sex-related reference intervals of lymphocyte subsets in healthy ethnic Han Chinese children.

Immunophenotyping of blood lymphocytes has become an important tool in the diagnosis of immunologic and hematologic disorders such as immunodeficiencies, lymphoproliferative and autoimmune diseases. Lymphocyte subsets include total T-cells (CD3(+)), TH (T helper, CD3(+) CD4(+)), TC (cytotoxic T cells, CD3(+) CD8(+)), B-cells (CD3(-) CD19(+)), and NK-cells (CD3(-) CD16(+) CD56(+)). Specific lymphocyte subset reference intervals should be locally established for meaningful comparison and to obtain an accurate interpretation of the results. Reference intervals of lymphocyte subsets for Chinese children are scarce. We performed dual-platform flow cytometry to determine the reference intervals of the percentages and absolute counts of lymphocyte subsets, including total T-cells, TH cells, TC cells, B-cells, and NK-cells in 1,027 ethnic Han children aged 4 months to 7 years in Henan, China. The children were divided into seven age groups. The percentages and absolute counts differed significantly with age, with the percentages of TH cells and B cells and the CD4/CD8 ratio peaking during the first year, while the percentages of total T cells, TC cells, and NK cells were obviously increased with age; girls showed a trend toward having a higher percentage of TH cells and a higher CD4/CD8 ratio than boys. The absolute counts of lymphocyte subsets peaked during first year and then decreased steadily with age. The reference intervals of lymphocyte subsets among children from China differed from the reported values in Hong Kong, the United States, Cameroon, and Italy. The differences observed could be due to genetic and environmental factors, coupled with the methodology used. The reference intervals of lymphocyte subsets could be used as initial national reference ranges in guidelines for children aged 4 months to 7 years.

[Human leukocyte antigen-G 14 bp deletion polymorphism in severe pre-eclampsia].

OBJECTIVE: To investigate the relationship between human leukocyte antigen-G (HLA-G) gene Exon 8 14 bp deletion polymorphism and the pathogenesis of severe pre-eclampsia. METHODS: Forty-two pregnant women with severe pre-eclampsia, who admitted to the Third Affiliated Hospital of Zhengzhou University from October 2008 to February 2009, and their newborns were chosen as the severe pre-eclampsia group. Another 45 healthy gravidas at the third trimester and their newborns were chosen as the control. All gravidas in both groups were Han Nationality. HLA-G Exon 8 genotyping was detected by PCR in both groups and the allele frequencies and genotype frequencies were compared between the two groups. The genotype frequencies of maternal-neonatal pairs were also analyzed. RESULTS: (1) In the severe pre-eclampsia group, 14% of the maternal-neonatal pairs were homozygote of 14 bp deletion, and significantly higher frequency 33% (15/45) was found in the control group (P=0.038). (2) No significant difference was found in the allele frequencies and genotype frequencies of HLA-G 14 bp deletion polymorphism among all the mothers between the two groups (P>0.05). (3) The +14 bp and -14 bp allele frequencies of HLA-G 14 bp deletion polymorphism in newborns in the severe pre-eclampsia group were 44% (37/84) and 56% (47/84), respectively, and 30% (27/90) and 70% (63/90) in the control group. Although there was no significant difference between the two groups, but differences in trends was identified (chi2=3.678 P=0.055); The genotype (-14 bp/-14 bp) frequency of neonates in the severe pre-eclampsia group showed no difference compared with that in the control group [29% (12/42) vs 49% (22/45)], but differences in trends was also found (P=0.052). CONCLUSIONS: HLA-G 14 bp deletion polymorphism is associated with the susceptibility of severe pre-eclampsia in Chinese Han nationality. Maternal-fetal genotype pairs of -14 bp/-14 bp may have reduced risk of severe pre-eclampsia.