Different Grades Of Cervical Lesions Research Articles

HnRNP K induces HPV16 oncogene expression and promotes cervical cancerization.

This study aims to explore the expression of hnRNP K in cervical carcinogenesis and to investigate the regulatory role of hnRNP K on HPV16 oncogene expression as well as biological changes in cervical cancer cells. In total 1042 subjects, including 573 with the normal cervix and 469 with different grades of cervical lesions were enrolled in this study to explore the association between hnRNP K and HPV16 oncogene expression in cervical carcinogenesis. Additionally, the Gene Omnibus (GEO) database was used to analyze hnRNP K mRNA expression in cervical cancerization. Meanwhile, the effects of hnRNP K on cell biological functions and HPV16 oncogene expression were investigated in Siha cells. Moreover, Function analyses were conducted using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) databases after ChIP-seq. hnRNP K was highly expressed in cervical cancer and precancerous lesions, and positively correlated with HPV16 E6, but negatively correlated with HPV16 E2 and HPV16 E2/E6 ratio. hnRNP K induced cell proliferation, inhibited apoptosis and caused cell cycle arrest in the S phase, and particularly increased HPV16 E6 protein expression. This study revealed that hnRNP K overexpression has important warning significance for the malignant transformation of cervical lesions, and could be used as a potential therapeutic target for inhibiting the carcinogenicity of HPV16 and prevention of cervical carcinogenesis.

HnRNP E1 Regulates HPV16 Oncogene Expression and Inhibits Cervical Cancerization.

hnRNP E1 (heterogeneous nuclear ribonucleoprotein E1) is an important RNA-binding protein (RBPs) that plays a vital role in tumor development. Human papillomavirus 16 (HPV16) contains numerous sites that can bind to RNA/DNA and may be modified by multiple RBPs, which contribute to HPV gene expression and HPV-associated cancer development. However, the effects of hnRNP E1 on HPV16 oncogenes in the development of cervical lesions remain unclear. A total of 816 participants with different grades of cervical lesions were enrolled in a community-based cohort established in Shanxi Province, China. The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases were used to analyze the association between hnRNP E1 mRNA expression and cervical lesions. Cells with up_ and down_regulated hnRNP E1 were established. hnRNP E1 functions were evaluated using cell counting kit-8, flow cytometry analyses, and chromatin immunoprecipitation sequencing. Our results showed that hnRNP E1 expression was linearly dependent on the severity of the cervical lesions. Low expression of HPV16 E2, high expression of E6, and a low ratio of E2 to E6 could increase the risk of cervical lesions. hnRNP E1 expression was correlated with HPV16 oncogene expression. hnRNP E1-relevant genes were involved in the dopaminergic synapses, Wnt signaling pathway, gnRH secretion, and mTOR signaling pathway. hnRNP E1 significantly inhibited cell proliferation, induced apoptosis, arrested the cell cycle at the G0/G1 stage, and decreased HPV16 E6 expression. Our results indicate that hnRNP E1 could downregulate HPV16 E6 oncogene expression and inhibit cervical cancerization, which sheds new light on preventing the carcinogenicity of HPV across a range of diseases by regulating RNA-binding proteins.

MicroRNA Levels in Cervical Cancer Samples and Relationship with Lesion Grade and HPV Infection.

miR-21, miR-214 and miR-let-7a are three validated and well-known miRNAs. miR-21 is described as an "oncomir" while miR-214 and miR-let-7a are described mainly as tumor suppressors. The role of these miRNAs remains unclear in cervical cancer, an important malignancy among women worldwide and responsible for many deaths every year. The objective of this study is to describe the expression profile of miR-21, miR-214 and miR-let-7a in plasma and in cervical scraping from a control group and patients with different grades of cervical lesions and invasive cervical cancer and correlate with HPV infection groups. Plasma and cervical scraping were submitted to DNA and RNA extraction. HPV detection and typing were performed by conventional PCR followed by PAGE to amplicons interpretation. The miRNA relative expression in plasma and cervical scraping samples was performed by real time PCR using specific TaqMan probes. miR-21 (p=0.0277) and miR-214 (p=0.0151) were up-regulated in cervical scraping samples of invasive cervical cancer (ICC) group. However, miR-214 was also up-regulated in the LSIL group (p=0.0062). Both miRNAs were not related to HPV infection. However, miR-let-7a was higher in HPV positive plasma samples (p=0.0433) than in HPV negative plasma samples and the correlation analysis confirmed the association between the levels of this miRNA with the presence of HPV (p=0.0407; r=0.3029), but not with lesion grade (p>0.05). Our results suggest that miR-21 is related to cervical cancer progression and miR-214 appears to have an ambiguous role in cervical lesions. miR-let-7a may be upregulated at a systemic level in patients with HPV infection.

Quick assessment of DNA damage in cervical epithelial cells using a chromatin dispersion test.

This study was aimed to quantify genomic DNA breakages in the cervical epithelium cells of patients diagnosed with different grades of cervical lesions using a quick test based on chromatin dispersion after controlled protein depletion. The association between the progressive stages of cervical dysplasia and the levels of DNA damage, taking into account the presence of papillomavirus human (HPV) infection, was investigated. A hospital-based unmatched case-control study was conducted during 2018 with a sample of 78 women grouped according to histological diagnosis as follows: 23 women with low grade-squamous intraepithelial lesion (LG-SIL), 34 women with high grade- squamous intraepithelial lesion (HG-SIL), and three women with cervical carcinoma (CC). In parallel, 15 women without cervical lesions were included as a Control cohort. DNA damage levels in cervical epithelial cells were assessed using the chromatin dispersion test (CDT) and controlled in parallel with DNA breakage detection coupled with florescent in situ hybridization (DBD‒FISH) using whole genomic DNA probes. CDT produces different morphotypes in the cervical epithelium that can be associated with the level of DNA breakage revealed with DBD‒FISH. A significant increase of DNA damage was correlated with the histological progression of the patients and human papillomavirus (HPV) infection. The CDT is a simple, accurate and inexpensive morphological bioassay to identify different levels DNA damage that can be associated with the level of abnormal cells present in the cervical epithelium in patients who commonly present HPV infection.

Characteristics of cervical microecology in late reproductive-age women with different grades of cervical lesions

To analyze the characteristics of cervical microecology in late reproductive-age women with cervical lesions and explore new methods for preventing cervical lesions. Cervical smears were obtained from a total of 147 women of late reproductive age, including 24 with high-risk HPV infection (HR-HPV), 27 with low-grade squamous intra-epithelial lesions (LSIL), 36 with high-grade squamous intra-epithelial lesions (HSIL), 35 with cervical cancer (CC) and 25 healthy women. llumina MiSeq sequencing of V3-V4 region of the 16S rRNA gene amplicons was used to characterize the vaginal microbiota of the women. OTUs analysis of the valid data was performed, and the α-diversity (Chao1, Simpson's Index and Shannon Index) and β-diversity (T-test, weighted UniFrac β diversity, and MetaStat analysis) were evaluated. Dilution curve and species accumulation boxplot validated the quality of the samples. OTUs analysis of the 5 groups demonstrated that cervical bacterial genus consisted primarily of Lactobacillus, Garrotella and Prussiella. With the aggravation of the lesions, the expression abundance of Lactobacillus was decreased, and Gardnerella and Prussiella were increased. The Chao1, Simpson and Shannon indexex showed no significant difference. T test indicated that 9 to 15 genera from 4 groups showed significant difference from the healthy control group. In all but the LSIL group, Lactobacillus (P1-2=0.025, P1-3=0.025, P1-4 < 0.001), Gardiner (P1-2=0.01, P1-3=0.001, P1-4 < 0.001), and Pruella (P1-2=0.047, P1-3=0.023, P1-4=0.048) showed the highest abundance in the cervical smears. The abundance of Gardiner (P1-3=0.021), Ignatius (P1-3=0.015) and Streptococcus (P1-3=0.041) was the highest in women with LSIL as compared with healthy women. In all the 5 groups, MetaStat analysis showed that lactobacillus (P1-4=0.025), gardnella (P1-2=0.004, P1-4=0.002, P1-5=0.001) and proctella (P3-5=0.005) had the highest abundance in the cervical flora. The abundance of Lactobacillus, Gardnella and Proctella is the highest in cervical bacteria at the genus level and may vary with disease progression. The α-diversity does not differ significantly, suggesting that apart from pathological factors, physiological factors also contribute to the difference in α-diversity. Women with LSIL have the most similar cervical flora to healthy women, which is consistent with the prognosis of the disease and confirms that the expression of cervical microecology is related to disease prognosis and may serve as a biological indicator for favoralble prognosis.

Coinfection of High-Risk Human Papillomavirus and Lower Genital Tract Pathogens in the Development of High-Grade Cervical Lesions.

Purpose This study investigated the infection status and relationship between other common lower genital tract infectious pathogens and high-risk human papillomavirus (HR-HPV) in the high-grade cervical lesions. Methods Overall, 882 patients were enrolled in this retrospective study, of which 339 patients (≥HSIL group) were confirmed with high-grade squamous intraepithelial lesions (HSIL) or cervical squamous cell carcinoma (SCC), while 543 patients (≤LSIL group) were diagnosed with low-grade squamous intraepithelial lesions (LSIL) or normal cervical pathology diagnosis. Cervical swab specimens were tested for HPV, pathogenic bacteria (PB), U. urealyticum (UU), M. hominis (MH), and C. trachomatis (CT) in both groups. Results The infection rates of HR-HPV, PB, UU (at high density), and CT were higher in the ≥HSIL group than in the ≤LSIL group (P < 0.001); however, higher infection rates with MH were not observed (P > 0.05). PB, UU, and CT were associated with HR-HPV infection (P < 0.001). The PB and UU infection rates in the ≥HSIL group were significantly different from those in the ≤LSIL group, regardless of whether there was an HR-HPV infection at the same time (P < 0.05). However, this was not the case for the CT (P > 0.05). Furthermore, 259 pathogenic bacterial strains were detected in 882 cases. The difference in the distribution of pathogenic bacterial flora in the different grades of cervical lesions had no statistical significance, which was prioritized over Escherichia coli (P > 0.05). Conclusion PB, UU, and CT infection is associated with susceptibility to HR-HPV, HR-HPV coinfection with these pathogens might increase the risk of high-grade cervical lesions, and PB and UU might be independent risk factors for cervical lesions.

Association between interleukin-2, interleukin-10, secretory immunoglobulin A and immunoglobulin G expression in vaginal fluid and human papilloma virus outcome in patients with cervical lesions.

The present study was designed to investigate the association between a change in vaginal local immunity and human papilloma virus (HPV) infection outcome in patients with cervical lesions, through the study of the expression of vaginal local immune factors, interleukin (IL)-2, IL-10, secretory immunoglobulin A (sIgA) and IgG, in patients with different grades of cervical lesions and different degrees of cervical lesions caused by HPV infection prior to and following treatment. The experimental group comprised 136 patients with low-grade squamous intraepithelial lesions, 236 patients with high-grade squamous intraepithelial lesions and 133 patients with cervical squamous cell carcinoma, while the control group comprised 100 time- and location-matched healthy women. The concentrations of sIgA, IgG, IL-2 and IL-10 in the vaginal lavage fluid, were detected using ELISA prior to treatment and at 3, 6 and 12 months after treatment. Prior to treatment, differences in HPV infection rate and changes in vaginal immune factors between patients with all grades of lesions and controls were statistically significant (P<0.05). Furthermore, IL-2 and IL-10 expression levels and the IL-2/IL-10 ratio in patients with different grades of lesions, with or without seroconversion, were significantly different to those in controls (P<0.05). However, the differences between changes in IgG and sIgA expression between patients with HPV seroconversion and patients with persistent HPV infection were not statistically significant (P>0.05). The results of the present study suggest that the restoration of humoral immune function promotes HPV seroconversion, and that IL-2 and IL-10 levels and their ratio may reflect the severity of cervical lesions and treatment effects to a certain extent.

Relationship between cervical lesions and the type-specific viral load of high risk HPV reflected by the Ct value of Cobas 4800 HPV system

Objective: To explore the relationship between cervical lesions and high risk HPV (HR-HPV) viral load reflected by the cycle threshold (Ct) values of Cobas 4800 HPV (Cobas 4800) system. Methods: From August 2016 to September 2017, 7 000 women from Shenzhen, were recruited for cervical cancer screening with Cobas 4800 system and cytology co-test. Colposcope biopsies were performed on women who were positive of HPV 16, 18, and positive of HPV types other than 16,18 with cytology [≥ atypical squamous cell of undetermined signification (ASCUS)], or HPV negative but abnormal of cytology [≥ low grade squamous intraepithelial lesion (LSIL)]. The Ct values of HPV 16, 18 and all combined other types coming from Cobas 4800 system were used as an indicator of viral load to analyze the relationship between type-specific HPV load and the cervical lesions. Results: (1) Among the 7 000 screening women, 370 cases were positive for cervical cancer screening, 325 of them underwent colposcope biopsies, and coloposcopy referred rate was 87.8% (325/370). Among 325 women undergoing cervical biopsy, pathological diagnosis was 119 cases of normal cervical cervix, 151 cases of LSIL, and 55 cases of high-grade squamous intraepithelial lesion (HSIL) and above (HSIL(+); including 53 cases of HSIL, 1 case of cervical adenocarcinoma, and 1 case of cervical squamous cell carcinoma). (2) The Ct value of HPV 16 was inversely correlated with the upgrading of the lesions (r=-0.617, P=0.000), and significant different among normal cervix,LSIL and HSIL(+) (35.4±4.5 vs 31.0±6.0 vs 26.5±4.0; F=25.537, P=0.000). There was no correlation between Ct value of HPV 18 and cervical lesions (r=-0.021, P=0.902). The Ct value of other 12 HPV types was statistically difference among normal normal cervix, HSIL(+) and cervicitis (33.0±5.3 vs 29.9±7.2 vs 29.8±5.8; F=5.087, P=0.007). Among them, LSIL and HSIL(+) were significantly lower than normal cervix (P<0.05), but there was no significant difference between LSIL and HSIL(+) (P>0.05). Conclusion: The Ct value of HPV 16 detecting in Cobas 4800 system as an indicator of virus load obviously correlates with different grades of cervical lesions, therefore could be a reference of cervical lesion existence and an indicator of lesion prognosis.

Detection and analysis of FAM19A4 promoter methylation in cervical exfoliated cells

Objective: To investigate the cinical value of FAM19A4promoter methylation in cervicalexfoliated cells for triage of cervical cancer. Methods: A total of 162 high-risk HPV-infected patients who were pathologically confirmed as different cervical lesions from August 2017 to December 2017 were collected in Guangdong Women and Children Hospital. Taqman probe-based quantitative PCR (qPCR) was used to detect the methylation of FAM19A4 promoterin different grades of cervical lesions, and the value of FAM19A4 methylation in predicting cervical HSIL and the above lesions was calculated by diagnostic test. Results: (1)The positive rates of FAM19A4 methylation in cervical exfoliated cells increased with the severity of cervical lesions, which were 7.69% (4/52) , 34.62% (9/26) , 55.56% (20/36) , 95.83% (46/48) in normal cervix/cervicitis, cervical LSIL, HSIL, and cervical cancer, respectively(P<0.05).(2)There was no significant difference in the detection rates of FAM19A4 methylation between different age groups, pathological types, clinical stage, tumor size and lymph node metastasis status (P>0.05). (3) The specificity and positive predictive value of FAM19A4 methylation in detecting cervical HSIL alone and ≥HSIL lesions were the optimal, with the AUC of 0.69 and 0.84, respectively. When combined with HPV16/18 genotyping, the sensitivity was significantly improved. Conclusions: The detection of FAM19A4 promoter methylation in cervical exfoliated cells has a high clinical value of discriminating ≥HSIL lesions; and the cotest of methylated FAM19A4 and HPV16/18 genotyping can identify ≥HSIL lesions more sensitively.

Distribution of high-risk human papillomavirus genotype prevalence and attribution to cervical precancerous lesions in rural North China.

ObjectivePrecise prevention is more desired for cervical cancer due to the huge population, high prevalence of human papillomavirus (HPV) infection in China and the vision of screen-and-treat strategies in low- and middle-income countries (LMICs). Considerations of combining type-specific prevalence and attribution proportion to high-grade cervical intraepithelial neoplasia are informative to more precise and effective region-specific cervical cancer prevention and control programs. The aim of the current study was to determine the genotype distribution of HPV and attribution to cervical precancerous lesions among women from rural areas in North China.MethodsA total of 9,526 women participated in the cervical cancer screening project in rural China. The samples of women who tested positive for HPV were retested with a polymerase chain reaction (PCR)-based HPV genotyping test. The attribution proportion of specific high-risk human papillomavirus (HR-HPV) types for different grades of cervical lesions was calculated by using the type contribution weighting method.ResultsA total of 22.2% (2,112/9,526) of women were HR-HPV positive and HPV52 (21.7%) was the most common HR-HPV genotype, followed by HPV58 (18.2%), HPV53 (18.2%) and HPV16 (16.2%). The top three genotypes detected in HR-HPV-positive cervical intraepithelial neoplasia (CIN)1 were HPV16 (36.7%), HPV58 (20.4%), HPV56 (15.3%). Among CIN2+, the most frequent genotypes were HPV16 (75.6%), HPV52 (17.8%), HPV58 (16.7%). HPV16, 56, 58, 53, 52, 59, 68, and 18 combined were attributed to 84.17% of all CIN1 lesions, and HPV16, 58, and 52 combined were attributed to 86.98% of all CIN2+ lesions.ConclusionsThe prevalence of HR-HPV infection among women from rural areas in North China was high and HPV16, HPV58, HPV52 had paramount attributable fraction in CIN2+. Type-specific HPV prevalence and attribution proportion to cervical precancerous lesions should be taken into consideration in the development of vaccines and strategy for screening in this population.

High-risk human papillomavirus genotype distribution and attribution to cervical cancer and precancerous lesions in a rural Chinese population.

ObjectiveTo explore the genotype distribution of high-risk human papillomavirus (HR-HPV) and its attribution to different grades of cervical lesions in rural China, which will contribute to type-specific HPV screening tests and the development of new polyvalent HPV vaccines among the Chinese population.MethodsOne thousand two hundred ninety-two subjects were followed based on the Shanxi Province Cervical Cancer Screening Study I (SPOCCS-I), and screened by HPV DNA testing (hybrid capture® 2 [HC2]), liquid-based cytology (LBC), and if necessary, directed or random colposcopy-guided quadrant biopsies. HPV genotyping with linear inverse probe hybridization (SPF10-PCR-LiPA) was performed in HC2 positive specimens. Attribution of specific HR-HPV type to different grades of cervical lesions was estimated using a fractional contribution approach.ResultsAfter excluding incomplete data, 1,274 women were included in the final statistical analysis. Fifteen point two percent (194/1,274) of women were HR-HPV positive for any of 13 HR-HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) and the most common HR-HPV types were HPV16 (19.1%) and HPV52 (16.5%). The genotypes most frequently detected in HR-HPV-positive cervical intraepithelial neoplasia grade 1 (CIN1) were HPV52 (24.1%), HPV31 (20.7%), HPV16 (13.8%), HPV33 (13.8%), HPV39 (10.3%), and HPV56 (10.3%); in HR-HPV-positive cervical intraepithelial neoplasia grade 2 or worse (CIN2+): HPV16 (53.1%), HPV58 (15.6%), HPV33 (12.5%), HPV51 (9.4%), and HPV52 (6.3%). HPV52, 31, 16, 33, 39, and 56 together contributed to 89.7% of HR-HPV-positive CIN1, and HPV16, 33, 58, 51, and 52 together contributed to 87.5% of CIN2+.ConclusionIn summary, we found substantial differences in prevalence and attribution of CINs between different oncogenic HPV types in a rural Chinese population, especially for HPV16, 31, 33, 52, and 58. These differences may be relevant for both clinical management and the design of preventive strategies.

Viral load and integration status of HPV58 associated with cervical lesion severity in women of Northeast China.

We aimed to evaluate the viral load and integration status of human papillomavirus 58 in women with different grades of cervical lesions to determine whether viral load and integration status are related to malignant transformation in HPV58-infected women. A total of 212 cervical specimens were collected from women in Northeast China who had undergone human papillomavirus genotyping and were HPV58-positive. The HPV58 viral load was determined using real-time polymerase chain reaction, and the integration status was discriminated using the ratio of HPV58 E2 gene copy number to E6 gene copy number. The median HPV58 viral load in women with normal cervix or cervicitis, low-grade squamous cell intraepithelial lesion, high-grade squamous cell intraepithelial lesion and cervical cancer was 352.12, 864.21, 1199.75 and 693.04 copies/genome, respectively. High significance was obtained when comparing the viral load of infected women presenting normal/cervicitis with that of the women either with precancerous cervical lesions or cervical cancer (P < 0.05). The HPV58 genome was in the episomal form in 35 samples (16.5%), mixed episomal and integrated forms in 165 (77.8%) samples, and completely integrated into the host genome in 12 (5.7%) samples. The HPV58 E2/E6 copy number ratio in the cervical cancer group was significantly lower than that in the other groups (P < 0.01). The HPV58 viral load in patients with precancerous cervical lesions or cervical cancer increases significantly with disease progression. The HPV58 E2/E6 copy number ratio in patients with cervical cancer is lower than that for less severe cervical lesions, suggesting a high degree of viral integration may be a considerable risk factor for cervical cancer.

Correlation of plasma nitrite/nitrate levels and inducible nitric oxide gene expression among women with cervical abnormalities and cancer

Cervical cancer is caused by infection with high risk human papillomavirus (HR-HPV). Inducible nitric oxide synthase (iNOS), a soluble factor involved in chronic inflammation, may modulate cervical cancer risk among HPV infected women. The aim of the study was to measure and correlate plasma nitrite/nitrate levels with tissue specific expression of iNOS mRNA among women with different grades of cervical lesions and cervical cancer. Tissue biopsy and plasma specimens were collected from 120 women with cervical neoplasia or cancer (ASCUS, LSIL, HSIL and invasive cancer) and 35 women without cervical abnormalities. Inducible nitric oxide synthase (iNOS) mRNA from biopsy and plasma nitrite/nitrate levels of the same study subjects were measured. Single nucleotide polymorphism (SNP) analysis was performed on the promoter region and Ser608Leu (rs2297518) in exon 16 of the iNOS gene. Differences in iNOS gene expression and plasma nitrite/nitrate levels were compared across disease stage using linear and logistic regression analysis. Compared to normal controls, women diagnosed with HSIL or invasive cancer had a significantly higher concentration of plasma nitrite/nitrate and a higher median fold-change in iNOS mRNA gene expression. Genotyping of the promoter region showed three different variations: A pentanucleotide repeat (CCTTT) n, −1026T > G (rs2779249) and a novel variant −1153T > A. These variants were associated with increased levels of plasma nitrite/nitrate across all disease stages. The higher expression of iNOS mRNA and plasma nitrite/nitrate among women with pre-cancerous lesions suggests a role for nitric oxide in the natural history of cervical cancer.

Genetic characterization and clinical implications of human papillomavirus type 16 (HPV16) variants from northeastern Argentina

BackgroundHuman papillomavirus type 16 (HPV16) plays a central role in the development of cervical cancer. Worldwide studies indicate the existence of HPV16 variants that show different geographic distributions and oncogenic potential. ObjectiveOur goal was to describe the genetic variation of HPV16 isolates identified in urban women with different grades of cervical lesions living in northeastern Argentina. Study designWe analyzed 116 HPV16-positive cervical samples (16 NLIM, 62 L-SIL, 16 H-SIL and 22 cervical cancer) from patients attending health centers in Misiones (Argentina) during 2006–13. HPV16 isolates were genetically characterized through PCR amplification and direct sequencing of 364bp within the long control region, and the resulting sequences classified into variants based on phylogenetic analysis (lineages A, B, C and D). A potential association between HPV16 variants and lesion grade was evaluated through an odds ratio (OR) test. A temporal framework for the origin of HPV16 variants was assessed through coalescence analysis (BEAST v 1.7.5). ResultsPhylogenetic analysis of HPV16 sequences showed that 92.1% of the samples clustered with lineage A, and 6.9% to lineage D. HPV16 variants from lineage D were more frequently associated with high-grade lesions and cancer (HSIL+) than lineage A variants at an OR of 13.8 (1.6–117.0). The time to most common recent ancestor (tMCRA) of all variants was 119,103years before present (HPD 95%=48,486–197,239), a date consistent with the time frame for modern human evolution. ConclusionOur results suggest that HPV16 variants from lineage D may represent an additional risk factor for the development of cervical cancer in women living in northeastern Argentina. This study provides new information about viral isolates present in Argentina that will contribute to the monitoring of HPV16 infection in the vaccine era.

Distribution of HPV-16 variants among isolates from Paraguayan women with different grades of cervical lesion

ObjectiveTo determine the distribution of HPV-16 variants among Paraguayan women with different grades of cervical lesions. MethodsSixty-seven HPV-16-positive cervical samples obtained from women attending health centers in Paraguay between March 2007 and April 2009 were examined, including 29 low-grade squamous intraepithelial lesion (LSIL), 29 high-grade squamous intraepithelial lesion (HSIL), 4 cervical cancer, and 5 normal cytology samples. The specimens were analyzed by PCR-directed sequencing of a 364-bp fragment of the long control region of HPV-16, and a phylogenetic tree was compiled with MEGA 5.0 software. ResultsMost HPV-16 variants belonged to the European branch (82%); these variants were detected among 25 of 29 women with LSIL, 22 of 29 women with HSIL, 3 of 4 women with cervical cancer, and all women with normal cytology. Two isolates yielded new variants of the European branch with nucleotide substitutions at positions A7752C and A7810T. Non-European variants, such as African type 1 (1.5%) and Asian-American (16.5%), were detected only among women with cervical lesions (4/29, LSIL; 6/29, HSIL; 1/29, cervical cancer). These variants had at least 6 nucleotide substitutions adjacent to or within transcription factor binding sites. ConclusionAll branches of HPV-16 variants were detected among Paraguayan women with cervical lesions.

The HPV16 transcriptome in cervical lesions of different grades

Infections with high-risk human papillomaviruses (HPV), mainly HPV type 16, can cause malignant transformation of the human cervical epithelium and cervical cancer (CxCa). Very little is known about the quantitative expression of HPV16 transcripts in cervical lesions of different grades. We have analysed the viral transcriptome in 80 HPV16 DNA positive cervical smears including lesions of different cytological grades, using nucleic acid sequence-based amplification (NASBA)–Luminex hybridisation assays quantifying spliced and unspliced HPV16 transcripts. Based on the quantitative analysis of single transcripts, highly significant changes in transcript levels were observed between different grades of cervical lesions. In conclusion, quantitative expression changes of HPV16 transcript markers may be involved in tumour progression. This study provides a basis for selection of candidate RNA markers for diagnostics of HPV16-related disease.

The association between IgG1, IgG2 subclasses toward human papillomavirus 16-like particles in the serum and cervical intraepithelial neoplasia

Objective The paper aimed to study the relationship between the expressions of immunoglobulin G (IgG) subclasses toward human papillomavirus 16-like particles (HPV16VLPs) in the serum of patients and different grades of cervical lesions.

Genetic diversity of human papillomavirus type 16 E6, E7, and L1 genes in Italian women with different grades of cervical lesions

High-risk human papillomaviruses (HPVs) are risk factors for the development of cervical cancer. HPV 16 is the most common type, being present in about 60% of cervical cancers worldwide. Previous studies have reported upon the association between HPV 16 E6 variants and increased risk of cervical intraepithelial neoplasia and invasive cervical cancer. In this study, the presence of HPV 16 polymorphisms in the E6, E7, and L1 genes was investigated in relation to the presence of high-grade lesions. Sequencing of the E6 gene revealed the presence of nucleotide mutations resulting in 15 amino acid changes. Of these, the G134D and C136R fall within the CXXC zinger finger domain important for p53 binding. In the E7 gene, four nucleotide variations were identified with two leading to the amino acid substitutions L15V and S31R. The L1 gene showed 13 nucleotide changes leading to 11 amino acid substitutions. Among these, the R364C and N367D are located at the base of the HI-loop of the L1 protein, considered to be the immunodominant epitope of HPV 16. No significant relationship between HPV 16 variants and high-grade lesions was found. Phylogenetic analysis showed that all the HPV 16 variants identified belonged to the European lineage, except one which was of the Asian-American lineage. The European-350G variant was detected most frequently (22 of 34, 64.7%). The study provides some new data on the genetic diversity of HPV 16 which may help to understand the oncogenic potential of the virus and to improve management of patients.

Evidence of HPV16 integration in low- and high-grade cervical lesions that regress demonstrated by multiple displacement amplification and Southern blot hybridisation

The prevalence and significance of human papillomavirus (HPV) integration among different grades of cervical lesions is uncertain. In this study, HPV physical status was examined by the combination of multiple...

Constitutive activation of nuclear factor-kB: preferntial homodimerization of p50 subunits in cervical carcinoma

Infection of specific types of 'high risk' HPVs such as HPV 16 and HPV 18 has been associated with the development of cervical cancer. Deregulation of specific NF-kappaB members has also been implicated with the development of many cancers including cervical cancer. We have studied the expression and DNA-binding activity of NF-kappaB during the development of cervical cancer involving cervical precancer, cancer and control tissues with or without HPV infection. We observed constitutive activation of NF-kappaB to a significant level in squamous-cell carcinomas while no or negligible NF-kappaB binding activity was observed in normal controls or precancerous lesions. Interestingly, there is a gradual increase in binding activity and expression of NF-kappaB from low-grade squamous intraepithelial lesions (LSIL) to high-grade squamous intraepithelial lesion (HSIL) and to invasive squamous cell carcinoma (SCC). On further dissection of NF-kappaB complex, the p50 subunit which generally heterodimerizes with p65 to form an active form NF-kappaB appears to form a p50/p50 homodimer instead of conventional p50/p65 heterodimer. In situ analysis of expression of p50 and p65 subunits by immunohistochemistry in tissue sections from different grades of cervical lesions including invasive cancer also demonstrate a gradual increase in the expression and nuclear localization of p50 subunit only as the severity of lesions increases. We have observed a very high expression of nuclear p50 in HSIL and invasive cancers while the level of nuclear p65 is significantly lower or nil. We further observed that this activation is not dependent on HPV infection since both HPV positive and HPV negative tumors showed the same pattern of high binding activity and increased expression of NF-kappaB p50/p50 homodimer. Although nuclear translocation and localization of p65 was observed to a lesser extent in invasive tumor, p65 was not found to be involved in dimmer formation. Thus the gradual activation and expression of NF-kappaB as a function of severity of cervical lesions and the change in dimerization pattern in favor of p50/p50 homodimers appears to play an important role during the development of cervical carcinoma.