MicroRNA expression associated with low-grade cervical intraepithelial neoplasia outcomes.

PURPOSE: Only a fraction of low-grade cervical intraepithelial neoplasia (CIN) progresses to high-grade CIN; however, the biological processes that differentiate progressive CIN from CIN that resolves naturally are poorly understood. MicroRNAs (miRNAs) are important epigenetic regulators of gene expression and thus, miRNA expression profiling can reveal the dysregulated biology underlying disease processes. The purpose of this case-control study was to reveal miRNA expression patterns and predict the underlying biological pathways that are associated with clinical outcomes of low-grade CIN. METHODS: Women with low-grade CIN diagnosis and definitive clinical outcomes (n = 51) were identified retrospectively using electronic clinical records. Comprehensive miRNA expression profiling was performed on the low-grade CIN diagnostic cervical biopsies retrieved from pathology archives. Differential miRNA expression was analyzed by comparing women with CIN that progressed to women with CIN that resolved naturally. RESULTS: Differential expression of 29 miRNAs was observed in low-grade CIN that progressed to high-grade compared to low-grade CIN that resolved. Of these, 24 were significantly downregulated in progressive CIN, including miR-638, miR-3196, miR-4488, and miR-4508, while 5 miRNAs, including miR-1206a, were significantly upregulated. Computational gene ontology analysis based on the discovered miRNAs and their putative mRNA targets revealed biological processes associated with oncogenic phenotypes. CONCLUSION: Distinct miRNA expression profiles are associated with clinical outcomes of low-grade CIN. The functional effects of the differentially expressed miRNAs may be biological determinants of CIN progression or resolution.

Antioxidants Associated With Oncogenic Human Papillomavirus Infection in Women.

BACKGROUND: Human papillomavirus (HPV) infection is a major cause of cervical cancer. Studies showed the onset of HPV carcinogenesis may be induced by oxidative stress affecting the host immune system. The association between antioxidants and oncogenic HPV remains unclear. In this study, we aim to identify antioxidants associated with vaginal HPV infection in women. METHODS: The associations between the 15 antioxidants and vaginal HPV infection status (no, low-risk [LR], and high-risk [HR] HPV) were evaluated using 11 070 women who participated in the 2003-2016 National Health and Nutrition Examination Survey (NHANES). RESULTS: We identified serum albumin and 4 dietary antioxidants (vitamin A, B2, E, and folate) inversely associated with HR-HPV infection. Women with a low level of albumin (≤39 g/L) have a significantly higher risk of HR-HPV (odds ratio [OR] = 1.4, P = .009 vs >44 g/L). A Nutritional Antioxidant Score (NAS) was developed based on these 4 dietary antioxidants. The women with the lowest quartile NAS had a higher chance of HR-HPV (OR = 1.3, P = .030) and LR-HPV (OR = 1.4, P = .002) compared with the women with the highest quartile NAS. CONCLUSIONS: We identified 5 antioxidants negatively associated with vaginal HR-HPV infection in women. Our findings provide valuable insights into understanding antioxidants' impact on HPV carcinogenesis.

Parents' receptiveness to oral health clinic-based vaccination.

In the United States, utilization of the human papillomavirus (HPV) vaccine has lagged far below public health goals for achieving satisfactory population-level protection against HPV associated cancers. Oral health professionals such as dentists and dental hygienists are important stakeholders in primary prevention of HPV-associated oropharyngeal cancer. We surveyed parents accompanying children to a local pediatric oral health clinic to ascertain their receptiveness to engaging their child's oral health team in their child's immunizations. Parents were generally receptive (86%) to discussing vaccines available for their children with both their child's dentist and dental hygienist. The majority of parents (79%) reported that they would allow their child's dentist to administer a vaccine to their child. Oral health providers are trusted healthcare professionals poised to make a positive impact on adolescent vaccination programs and they should be included in efforts to improve HPV vaccination rates.

Risk of abnormal cervical cytology in HIV-infected women testing positive for both human papillomavirus and Epstein-Barr virus in genital tract specimens.

PURPOSE: Although infection with high-risk human papillomavirus (HPV) is a prerequisite for cervical cancer development, HPV infection is not sufficient to promote cancer in the majority of infected women. We tested the hypothesis that human herpesviruses might cooperate with HPV to promote the development of cervical dysplasia, an early indicator of cervical cancer development. METHODS: This study used archived specimens from a cohort of human immunodeficiency virus (HIV)-seropositive women seeking gynecological care at the Medical Center of New Orleans, Louisiana. Viral DNA was detected by PCR amplification and risk of abnormal cervical cytology was determined in relation to virus test results. RESULTS: Consensus human herpesvirus PCR with herpes speciation by restriction endonuclease digestion revealed Epstein-Barr virus (EBV) to be the most prevalent herpesvirus in cervicovaginal lavage specimens. Further analysis using an EBV-specific PCR assay and cervical swab specimens demonstrated an approximately fourfold increased risk of abnormal cervical cytology in women testing positive for cervical EBV and high-risk HPV compared to women testing positive for high-risk HPV alone. This relationship was independent of markers of advancing HIV disease. CONCLUSION: Cervical shedding of EBV appears to predict a greater risk of cervical dysplasia in HIV-infected women with a high-risk HPV infection.

HPV-Associated Oropharyngeal Cancer in the HIV/AIDS Patient.

Since their discovery as the etiologic agents of cervical cancer in the mid-1970s, human papillomaviruses (HPVs) have been linked with a growing number of epithelial-derived tumors, including head and neck squamous cell carcinomas. HPV demonstrates a particular predilection for causing tumors of the oropharynx, with the majority of cases involving infection with high-oncogenic risk HPV-16. People living with HIV are at increased risk of infection with HPV- and HPV-related oral complications even with adequate control of their HIV infection with antiretroviral therapy. In this chapter, we discuss the molecular mechanisms that underlie HPV-mediated oncogenesis in the oropharynx. We also describe the progress that has been made in understanding the epidemiology of oral HPV infection and the determinants of oral HPV-related pathology. Finally, we examine what can be done to treat and prevent oral HPV infection, benign lesions, and cancer, particularly in the context of the HIV-positive patient.

Detection of human papillomavirus DNA in formalin-fixed, paraffin-embedded squamous papillomas of the oral cavity.

BACKGROUND: Squamous papillomas are exophytic proliferations of surface oral epithelium. Human papillomavirus (HPV) infection is widely accepted as the etiology of squamous papillomas however the virus cannot be detected in a significant percentage of lesions. MATERIAL AND METHODS: Using polymerase chain reaction (PCR), we tested 35 formalin-fixed paraffin-embedded (FFPE) squamous papillomas for the presence of HPV DNA. RESULTS: Six papillomas (17%) tested positive for HPV DNA; four contained HPV-6 and two contained HPV-11. Given that ß-globin DNA was only identified in half of the samples, DNA degradation appears to have significantly impacted the results. CONCLUSIONS: The results likely represent an underestimation of the true number of HPV-positive specimens in our study. Potential explanations for HPV-negative squamous papillomas include transient HPV infection, failure of the experiment to detect HPV if present, or the possibility that some lesions may not result from HPV infection. Key words:HPV, PCR, FFPE, papilloma, oral.

Epstein-Barr Virus, High-Risk Human Papillomavirus and Abnormal Cervical Cytology in a Prospective Cohort of African Female Sex Workers.

BACKGROUND: High-oncogenic-risk human papillomavirus (hrHPV) is necessary, although insufficient, to promote cervical cancer. Like HPV, Epstein-Barr virus (EBV) is a common pathogen with the capacity to promote epithelial neoplasms. We examined the association between cervical EBV, hrHPV, and cytology in female sex workers in Nairobi, Kenya. METHODS: Women (n = 332) with known cervical cytology and hrHPV mRNA results were evaluated for cervical EBV DNA by conventional polymerase chain reaction. Prevalence ratios (PRs) were calculated to assess the relationships between EBV, hrHPV, and cervical cytology. Prospective analyses used risk ratios and time-to-event analyses to determine the association of EBV with hrHPV clearance and with abnormal cytology outcomes. RESULTS: Baseline prevalence of hrHPV and EBV was 29% and 19%, respectively. Higher EBV prevalence was found among women with older age, HIV, hrHPV, abnormal cytology, Mycoplasma genitalium infection, smoking habits, younger age at sexual debut, and less frequent condom use. At baseline, women with EBV had a higher prevalence of hrHPV infection than did EBV-negative women (52% vs. 24%; HIV-adjusted PR [95% confidence interval], 1.8 [1.3-2.6]). Epstein-Barr virus-positive women had a higher prevalence than did EBV-negative women of high-grade precancer (15% vs. 2%) and abnormal cytology (37% vs. 15%), although HIV- and hrHPV-adjusted associations were not significant (high-grade precancer: PR, 2.0 [0.7-5.9]; abnormal cytology: PR, 1.4 [0.9-2.2]). In prospective analyses, a marginal association was observed between baseline EBV detection and delayed hrHPV clearance. CONCLUSIONS: Our data support a possible role for EBV as a high-risk marker or cofactor for HPV-mediated cervical cancer development.

Differential regulation of microRNA-146a and microRNA-146b-5p in human retinal pigment epithelial cells by interleukin-1ß, tumor necrosis factor-α, and interferon-γ.

PURPOSE: The inflammatory response of the retinal pigment epithelium (RPE) is implicated in the pathogenesis of age-related macular degeneration. The microRNAs miR-146a and miR-146b-5p can regulate the inflammatory process by attenuating cytokine signaling via the nuclear factor-κB pathway. The aim of the present study is to investigate the expression of miR-146a and miR-146b-5p in human RPE cells and their response to proinflammatory cytokines. METHODS: Confluent cultures of RPE cells established from adult human donor eyes were treated with the proinflammatory cytokines interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-1ß. The expression of microRNAs was analyzed by real-time PCR using total RNA fraction. The retinal pigment epithelial cell line ARPE-19 was employed to analyze the promoter activity of the genes encoding miR-146a and miR-146b-5p. STAT1-binding activity of oligonucleotides was analyzed by electrophoretic mobility shift assay. ARPE-19 cells were transiently transfected with miR-146a and miR-146b-5p mimics for the analysis of IRAK1 expression by western immunoblotting. RESULTS: Real-time PCR analysis showed that miR-146a and 146b-5p are expressed in RPE cells. The cells responded to proinflammatory cytokines (IFN-γ + TNF-α + IL-1ß) by highly increasing the expression of both miR-146a and miR-146b-5p. This was associated with an increase in the expression of transcripts for CCL2, CCL5, CXCL9, CXCL10, and IL-6, and a decrease in that for HMOX1. The miR-146a induction was more dependent on IL-1ß, since its omission from the cytokine mix resulted in a greatly reduced response. Similarly, the induction of miR-146b-5p was more dependent on IFN-γ, since its omission from the cytokine mix minimized the effect. In addition, the increase in MIR146B promoter activity by the cytokine mix was effectively blocked by JAK inhibitor 1, a known inhibitor of the JAK/STAT signaling pathway. The expression of IRAK1 protein was decreased when ARPE-19 cells were transiently transfected with either miR-146a mimic or miR-146b-5p mimic. CONCLUSIONS: Our results clearly show that both miR-146a and miR-146b-5p are expressed in human RPE cells in culture and their expression is highly induced by proinflammatory cytokines (IFN-γ + TNF-α + IL-1ß). The induction of miR-146a showed a dependency on IL-1ß, while that of miR-146b-5p on IFN-γ. Our results show for the first time that miR-146b-5p expression is regulated by IFN-γ, potentially via the JAK/STAT pathway. These two microRNAs could play a role in inflammatory processes underlying age-related macular degeneration or other retinal degenerative diseases through their ability to negatively regulate the nuclear factor-κB pathway by targeting the expression of IRAK1.

Modulation of lung inflammation by the Epstein-Barr virus protein Zta.

Several studies have implicated gamma-herpesviruses, particularly Epstein-Barr virus (EBV), in the progression of idiopathic pulmonary fibrosis. The data presented here examine the possible role that EBV plays in the potentiation of this disease by evaluating the pulmonary response to expression of the EBV lytic transactivator protein Zta. Expression of Zta in the lungs of mice via adenovirus-mediated delivery (Adv-Zta) produced profibrogenic inflammation that appeared most pronounced by day 7 postexposure. Relative to mice exposed to control GFP-expressing adenovirus (Adv-GFP), mice exposed to Adv-Zta displayed evidence of lung injury and a large increase in inflammatory cells, predominantly neutrophils, recovered by bronchoalveolar lavage (BAL). Cytokine and mRNA profiling of the BAL fluid and cells recovered from Adv-Zta-treated mice revealed a Th2 and Th17 bias. mRNA profiles from Adv-Zta-infected lung epithelial cells revealed consistent induction of mRNAs encoding Th2 cytokines. Coexpression in transient assays of wild-type Zta, but not a DNA-binding-defective mutant Zta, activated expression of the IL-13 promoter in lung epithelial cells, and detection of IL-13 in Adv-Zta-treated mice correlated with expression of Zta. Induction of Th2 cytokines in Zta-expressing mice corresponded with alternative activation of macrophages. In cell culture and in mice, Zta repressed lung epithelial cell markers. Despite the profibrogenic character at day 7, the inflammation resolves by 28 days postexposure to Adv-Zta without evidence of fibrosis. These observations indicate that the EBV lytic transactivator protein Zta displays activity consistent with a pathogenic role in pulmonary fibrosis associated with herpesvirus infection.

Development and validation of a HPV-32 specific PCR assay.

BACKGROUND: Human Papillomavirus-32 (HPV-32) has traditionally been associated with focal-epithelial-hyperplasia (FEH). It is also present in 58% of oral warts of HIV-positive individuals whose prevalence is increasing. Current methods for the detection of HPV-32 are labor-intensive and insensitive so the goal of this work was to develop a highly sensitive and easy to use specific polymerase chain reaction (PCR) assay. MATERIALS AND METHODS: An HPV-32 L1 specific PCR assay was developed and optimized. The sensitivity and specificity was compared to previous assays utilized for detection (PGMY and MY09/11 PCR with dot blot hybridization) using cloned HPV-32 L1, the closely related HPV-42 L1 as well as clinical samples (oral swabs and fluids from 89 HIV-positive subjects). RESULTS: The HPV-32 specific PCR assay showed improved sensitivity to 5 copies of HPV-32 as compared to the PGMY PCR, MY09/11 PCR and dot blot which had a limit of detection of approximately 3,000 copies. Using the HPV-32 dot blot hybridization assay as the gold standard, the HPV-32 specific PCR assay has a sensitivity of 95.8% and 88.9% by sample and subject, respectively, and specificity was 87.8% and 58.8% by sample and subject, respectively. The low sensitivity is due to the HPV-32 specific PCR assays ability to detect more HPV-32 positive samples and may be the new gold standard. CONCLUSION: Due to the ease, sensitivity, and specificity the HPV-32 specific PCR assay is superior to previous assays and is ideal for detection of HPV-32 in large cohorts. This assay provides an excellent tool to study the natural history of HPV-32 infection and the development of oral warts.

Epstein-Barr virus growth/latency III program alters cellular microRNA expression.

The Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cancers. Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs. Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis. The extent to which EBV exploits cellular miRNAs is unknown. Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155. In contrast, miR-28 expression was found to be lower in type III latency. The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers.

Oral HPV complications in HIV-infected patients.

Human papillomavirus (HPV) infection and disease, including that of the oral cavity, has not dramatically declined since the introduction of potent combination therapy to control HIV. Two manifestations of HPV in the oral cavity that may be on the rise are HPV-32-associated oral warts and HPV-16-associated oral cancers. A current research focus is the natural history of HPV infections in the oral cavity. A better understanding of the natural history of oral HPV infections could lead to improved diagnosis and treatment for oral HPV complications in HIV-positive patients.

Epstein-Barr virus latent membrane protein 1 induces cellular MicroRNA miR-146a, a modulator of lymphocyte signaling pathways.

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is a functional homologue of the tumor necrosis factor receptor family and contributes substantially to the oncogenic potential of EBV through activation of nuclear factor kappaB (NF-kappaB). MicroRNAs (miRNAs) are a class of small RNA molecules that are involved in the regulation of cellular processes such as growth, development, and apoptosis and have recently been linked to cancer phenotypes. Through miRNA microarray analysis, we demonstrate that LMP1 dysregulates the expression of several cellular miRNAs, including the most highly regulated of these, miR-146a. Quantitative reverse transcription-PCR analysis confirmed induced expression of miR-146a by LMP1. Analysis of miR-146a expression in EBV latency type III and type I cell lines revealed substantial expression of miR-146a in type III (which express LMP1) but not in type I cell lines. Reporter studies demonstrated that LMP1 induces miR-146a predominantly through two NF-kappaB binding sites in the miR-146a promoter and identified a role for an Oct-1 site in conferring basal and induced expression. Array analysis of cellular mRNAs expressed in Akata cells transduced with an miR-146a-expressing retrovirus identified genes that are directly or indirectly regulated by miR-146a, including a group of interferon-responsive genes that are inhibited by miR-146a. Since miR-146a is known to be induced by agents that activate the interferon response pathway (including LMP1), these results suggest that miR-146a functions in a negative feedback loop to modulate the intensity and/or duration of the interferon response.

Human papillomavirus infection and disease in the HIV+ individual.

HPV infection of both the genital tract and oral cavity of HIV+ men and women is increased. HPV-related pathology is also increased in the HIV+ individuals, usually with further increases seen for those HIV+ individuals with lower CD4 cell counts. Fortunately, the rates of cervical cancer and anal cancer are relatively low and not related to CD4 cell count. Treatment of the HIV+ individual with HPV-related disease is challenging and requires close long-term follow-up to prevent recurrent disease. The mechanism of how HPV and HIV interact is still not known but is more likely to be linked to immune suppression rather than a direct interaction between viruses. The newly developed HPV vaccines will likely have a significant impact on HPV-related disease in immunocompetent individuals. It remains to be seen what impact these vaccine will have on the immune depressed.

The impact of highly active antiretroviral therapy and immunodeficiency on human papillomavirus infection of the oral cavity of human immunodeficiency virus-seropositive adults.

BACKGROUND: Prevalence of human papillomavirus (HPV)-associated oral condylomas has reportedly increased in HIV-infected individuals since the introduction of highly active antiretroviral therapy (HAART). The relationships between HIV therapy regimen, overall health, and subclinical oral HPV have not been examined. GOAL: To determine oral HPV genotype prevalence and the impact of HAART and health in the HIV+ population. STUDY: An L1 consensus-primer polymerase chain reaction and linear array assay were used to examine the prevalence of 27 HPV genotypes in saliva of 98 HIV+ individuals. Risk assessment variables were compared to oral HPV status. RESULTS: Oral HPV was detected in 37% of HIV+ African American individuals. Caucasians were at greater risk of oral HPV infection than African Americans. Markers of advanced HIV disease did not predict HPV status. Therapy status was associated with HPV detection. CONCLUSIONS: Treatment of HIV, rather than HIV immunosuppression, appears to play a role in oral HPV infections in HIV+ individuals.

Seroepidemiology of infection with human papillomavirus 16, in men and women attending sexually transmitted disease clinics in the United States.

BACKGROUND: The study sought to characterize the seroprevalence, seropersistence, and seroincidence of human papillomavirus (HPV)-16 antibody, as well as the behavioral risk factors for HPV-16 seropositivity. METHODS: Serologic data at baseline and at 6- and 12-month follow-up visits were used to examine the seroprevalence, seropersistence, and seroincidence of HPV-16 antibody in 1595 patients attending United States clinics treating sexually transmitted disease. Testing for antibody to HPV-16 was performed by capture enzyme-linked immunosorbent assay (ELISA) using viruslike particles. RESULTS: The seroprevalence of HPV-16 antibody was 24.5% overall and was higher in women than in men (30.2% vs. 18.7%, respectively). In those who were HPV-16 seropositive at baseline, antibody response persisted to 12 months in 72.5% of women and in 45.6% of men. The seroincidence of HPV-16 antibody was 20.2/100 person-years (py) overall, 25.4/100 py in women, and 15.7/100 py in men. In multivariate analysis, the seroprevalence of HPV-16 antibody was significantly associated with female sex, age >20 years, and the number of episodes of sex with occasional partners during the preceding 3 months, whereas the seroincidence of HPV-16 antibody was significantly associated with female sex, age >20 years, baseline negative ELISA result greater than the median value, and the number of episodes of unprotected sex with occasional partners during the preceding 3 months. CONCLUSION: Sex- and age-related differences in both the seropositivity and seroincidence of HPV-16 antibody persisted after adjustment for behavioral and sociodemographic risk factors, and behavioral risk factors during the preceding 3 months were stronger predictors of the seroprevalence and seroincidence of HPV-16 antibody than was lifetime sexual behavior.

Human papillomavirus infection and disease in HIV-infected individuals.

The possibility of increases in both oral and anogenital pathologic conditions due to human papillomavirus (HPV) in patients infected with the human immunodeficiency virus (HIV) is of concern and is the focus of numerous current research studies. HIV-infected women are at higher risk for cervical HPV detection, for infection with high-oncogenic-risk types of HPV, for persistent HPV infection, for cervical cytologic abnormalities, and for cervical intraepithelial neoplasms. HIV-infected men are at increased risk for anal HPV infection, for anal infection with high oncogenic-risk types of HPV, for persistent anal HPV infection, and for anal intraepithelial defects. Recent studies have shown an increased risk of oral warts in HIV-infected individuals despite treatment with highly active antiretroviral therapy (HAART). Oral HPV infection rates have not declined since the initiation of HAART, and evidence suggests that the rates may have actually increased in white HIV-infected males.

Human papillomavirus-specific antibody status in oral fluids modestly reflects serum status in human immunodeficiency virus-positive individuals.

Serological assays are valuable tools for studies of the epidemiology of human papillomaviruses (HPVs). The efficacy of a less invasive oral-fluid assay for detection of HPV antibodies was examined. Matched serum, saliva, and oral mucosal transudate (OMT) specimens collected from 150 human immunodeficiency virus-seropositive patients were tested for immunoglobulin G antibodies against HPV-6 and HPV-11 combined (HPV-6/11) and HPV-16 capsids. Antibodies to HPV were detected in both types of oral specimens. Seroprevalence rates were 55% for HPV-6/11 and 37% for HPV-16, whereas oral prevalence rates were significantly lower (for HPV-6/11 in saliva, 31%, and in OMT, 19%; for HPV-16 in saliva, 19%, and in OMT, 17%). HPV antibody detection in OMT more accurately reflected the presence of antibodies in serum than did HPV antibody detection in saliva. More stringent saliva assay cutpoints yielded stronger associations between oropositivity and seropositivity; less stringent OMT cutpoints yielded stronger associations between oropositivity and seropositivity. Although HPV antibodies were detected in oral fluids, further optimization of the assay is necessary before oral-fluid testing can be implemented as a reliable alternative to serum testing for HPV.