The Influence of Psychological Stress on HPV Infection Manifestations and Carcinogenesis.

Psychological stress is an important factor involved in disease manifestations of human papillomavirus (HPV) infection, and it can participate in HPV-associated carcinogenesis. The impact or effect which stress can have (exert) depends on a person's genetic pool, experiences and behaviors. Due to inconsistencies in some study results, this issue remains a subject of research. Concerning the course of HPV manifestations, it has been observed that a higher number of life stressors in at least the previous 6 months, the absence of social support and the types of personal coping mechanisms employed, all influence HPV progression. In women with cervical dysplasia, a connection between greater stress experiences and dysregulation of specific immune responses has been observed. Once HPV enters a cell via the α6 integrin there are three possible sequences: latent infection, subclinical infection, and clinically manifest disease. HPV proliferation in differentiated epithelial cells induces morphologically cytopathic changes (koilocytosis, epidermal thickening, hyperplasia, hyperkeratosis). Oncogenic transformation requires the integration of the virus genome into the host genome. In doing so, DNA in the E1 region of E2 breaks down, leading to transcription disorders of E6 and E7. For the formation of irreversible malignancy, the following sequence is necessary: initial expression of E6 and E7 genes followed by suppression of apoptosis and the stabile expression of E6 and E7 proteins that protect transformed cells from apoptosis. A successful immune response is characterized by a strong, local cell-mediated immune response. Several factors are important for the regression of HPV manifestation/infection, among which is psychological stress which can prolong the duration and severity of HPV disease. Stress hormones may reactivate latent tumor viruses, stimulate viral oncogene expression, and inhibit antiviral host responses. In the regression of HPV infection, increased activity of Th1 cells was observed. However, during psychosocial stress, a decrease in the Th1 type of immune response is seen, and there is a shift towards a Th2 response. Understanding perceived stress and biological changes in stress, as well as the evaluation of immune parameters, gives researchers a better picture of how stress influences HPV infections and how to improve disease management and outcomes.

Human papilloma virus correlates of high grade cervical dysplasia in HIV-infected women in Mombasa, Kenya: a cross-sectional analysis.

BACKGROUND: Women living with HIV are at increased risk to be co-infected with HPV, persistent high-risk (HR) human papillomavirus (HPV) infection and increased HR HPV viral load, which make them more at risk for cervical cancer. Despite their inherent vulnerability, there is a scarcity of data on potential high risk (pHR) and HR HPV genotypes in HIV- infected women with cervical dysplasia and HPV-type specific viral load in this population in Sub Saharan Africa. The aim of this analysis of HIV-infected women was to explore the virological correlates of high-grade cervical dysplasia (CIN 2+) in HIV-infected women, thereby profiling HPV genotypes. METHOD: This analysis assesses baseline data obtained from a cohort study of 74 HIV-infected women with abnormal cytology attending a Comprehensive Care Centre for patients with HIV infection in Mombasa, Kenya. Quantitative real-time PCR was used for HPV typing and viral load. RESULTS: CIN 2 was observed in 16% (12/74) of women, CIN 3 in 23% (17/74), and, invasive cervical carcinoma (ICC) in 1% (1/74) of women. In women with CIN 3+, HPV 16 (44%), HPV 56 (33%), HPV 33 and 53 (HPV 53 (28%) were the most prevalent genotypes. HPV 53 was observed as a stand-alone HPV in one woman with ICC. A multivariate logistic regression adjusting for age, CD4 count and HPV co-infections suggested the presence of HPV 31 as a predictor of CIN 2+ (adjusted odds ratio [aOR]:4.9; p = 0.05; 95% (Confidence Interval) [CI]:1.03-22.5). Women with CIN2+ had a significantly higher viral log mean of HPV 16, (11.2 copies/ 10,000 cells; 95% CI: 9.0-13.4) than with CIN 1. CONCLUSION: The high prevalence of HPV 53 in CIN 3 and as a stand-alone genotype in the patient with invasive cervical cancer warrants that its clinical significance be further revisited among HIV-infected women. HPV 31, along with elevated means of HPV 16 viral load were predictors of CIN 2 + .

Cytology and human papillomavirus co-test results preceding incident high-grade cervical intraepithelial neoplasia.

OBJECTIVE: High-risk HPV (hrHPV) and cytology co-testing is utilized for primary cervical cancer screening and for enhanced follow-up of women who are hrHPV-positive, cytology negative. However, data are lacking on the utility of this method to detect pre-cancer or cancer in community-based clinical practice. This study describes cytology and hrHPV results preceding high-grade cervical intraepithelial neoplasia, adenocarcinoma in situ, or cervical cancer (i.e., CIN2+) in an integrated health system employing routine co-testing among women aged 30 years and older. METHODS: We conducted a cross-sectional analysis of adult female members of Kaiser Permanente Northern California (KPNC) with incident CIN2+ between July 2008 and June 2009. The primary outcome was the proportions of cytologic diagnoses and hrHPV co-test results preceding a diagnosis of CIN2+. Cervical cytology and hrHPV testing results were abstracted from electronic medical records. RESULTS: Of 1283 CIN2+ cases among adult women, 880 (68.5%) were among women aged 30 years and older and 145/880 (16.5%, 95% CI 14.1-19.1) had only normal cytology during the 12 months prior to diagnosis. Furthermore, 133/880 (15.1%, 95% 12.9-17.7) were preceded by only normal cytology and persistent hrHPV infection (at least 2 positive hrHPV tests) during the 6-36 months preceding CIN2+ diagnosis. CONCLUSIONS: Incident CIN2+ is frequently preceded by normal cytology and persistent hrHPV infection among women aged 30 years and older; screening strategies that employ HPV testing and cytology may improve the detection of CIN2+ compared with cytology alone.

Pharmacotherapy of recurrent respiratory papillomatosis: an expert opinion.

BACKGROUND: Recurrent respiratory papillomatosis is caused by the human papillomavirus types (HPV) 6 and 11. It affects both children and adults. In a small number of cases, the disease can be very aggressive causing significant morbidity and possibly death. Surgical therapy is the primary treatment but in patients with aggressive disease, adjunctive therapy is initiated. The majority of these adjuncts center on immunomodulation, disruption of molecular signaling cascades or interruption of viral replication to help decrease the severity of the disease. Recently, a preventative vaccine has become available but data on its effectiveness will be at least a decade away. In the mean time, researchers are examining other vaccination strategies in the fight against HPV disease. OBJECTIVE: We will review the following pharmacotherapies used in the adjunct treatment of RRP: interferon, acyclovir, ribivirin, cidofovir, COX-2 inhibitors, retinoids, anti-reflux medications, zinc, indole-3-carbinol, therapeutic/preventative vaccines. METHODS: This is a review paper. Utilizing Medline and Pubmed from 1966 to present, the key words as well as the above listed adjunct treatments were searched for relevant papers. CONCLUSION: The evidence supporting each of these adjuncts varies with a majority having only case reports or cases-series to support their use. However, there is hope on the horizon with regard to the HPV vaccine and its potential to prevent future transmission of this disease.

Production of human papillomavirus type 16 virus-like particles in transgenic plants.

Cervical cancer is linked to infection with human papillomaviruses (HPV) and is the third most common cancer among women worldwide. There is a strong demand for the development of an HPV preventive vaccine. Transgenic plants expressing the HPV major capsid protein L1 could be a system to produce virus-like particles for prophylactic vaccination or could even be used as edible vaccines to induce an L1-specific prophylactic immune response. Here, we describe the generation of transgenic tobacco and potato plants carrying the HPV type 16 major structural gene L1 under the control of the cauliflower mosaic virus 35S promoter. All attempts to express either the original, unmodified L1 gene or an L1 gene with a codon usage optimized for expression in plants failed. Surprisingly, small amounts of the protein were detected using an L1 gene optimized for expression in human cells. However, Northern blot analysis revealed that most of the L1 transcripts were degraded. Introduction of the translational enhancer Omega derived from the tobacco mosaic virus strongly increased transcript stability and resulted in accumulation of L1 protein to approximately 0.5 to 0.2% of total soluble protein in transgenic tobacco and potato plants, respectively. The plant-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles. Furthermore, we did not find any indications of protein modification of the L1 protein produced in plants. Plant-derived L1 was as immunogenic as L1 expressed in baculovirus-infected insect cells. Feeding of tubers from transgenic potatoes to mice induced an anti-L1 antibody response in 3 out of 24 mice, although this response was only transient in two of the mice. Our data, however, indicate that an anti-L1 response was primed in about half of the 24 animals.

[Antimitogenic effect of Pygeum africanum extracts on human prostatic cancer cell lines and explants from benign prostatic hyperplasia]. / Efecto antimitogénico de extractos de Pygeum africanum sobre líneas celulares de cáncer de próstata humana y explantes de hiperplasia benigna de próstata.

OBJECTIVES: To analyze the effect of Pygeum africanum extracts on the in vitro proliferation of human prostate cells. METHODS: Prostate cancer cell lines and benign prostatic hyperplasia derived epithelial cells were cultured and treated with P. africanum extracts. The effect on cell proliferation was monitored by H3-thymidine and bromodeoxyuridine uptake and flow cytometry assays. RESULTS: The incubation with P. africanum extracts, with or without addition of amino acids, significantly and in a dose-dependent manner inhibits the proliferation of prostate cancer derived cells LnCaP, PZ-HPV-7, and CA-HPV-10. In the PZ-HPV-7 cells P. africanum extracts counteract the mitogenic action of EGF and block the transition from G1 to S in the cell cycle. P. africanum extracts also exert a potent antimitogenic action on the epithelial cells derived from benign prostatic hyperplasia explants. CONCLUSION: The ethanolic P. africanum extracts have an antimitogenic effect on prostate cancer cells and benign prostatic hyperplasia epithelial cells. Such effect is associated with the inhibition of the mitogenic action of EGF, and it is accompanied by a decrease of cells entering the S Phase of the cell cycle.

Displacement of YY1 by differentiation-specific transcription factor hSkn-1a activates the P(670) promoter of human papillomavirus type 16.

Transcription from human papillomavirus type 16 (HPV16) P(670), a promoter in the E7 open reading frame, is repressed in undifferentiated keratinocytes but becomes activated upon differentiation. We showed that the transient luciferase expression driven by P(670) was markedly enhanced in HeLa cells cotransfected with an expression plasmid for human Skn-1a (hSkn-1a), a transcription factor specific to differentiating keratinocytes. The hSkn-1a POU domain alone, which mediates sequence-specific DNA binding, was sufficient to activate the expression of luciferase. Electrophoretic mobility shift assay revealed the presence of two binding sites, sites 1 and 2, upstream of P(670), which were shared by hSkn-1a and YY1. Site 1 bound more strongly to hSkn-1a than site 2 did. YY1 complexing with a short DNA fragment having site 1 was displaced by hSkn-1a, indicating that hSkn-1a's affinity with site 1 was stronger than YY1's. Disrupting the binding sites by nucleotide substitutions raised the basal expression level of luciferase and decreased the enhancing effect of hSkn-1a. In HeLa cells transfected with circular HPV16 DNA along with the expression plasmid for hSkn-1a, the transcript from P(670) was detectable, which indicates that the results obtained with the reporter plasmids are likely to have mimicked the regulation of P(670) in authentic HPV16 DNA. The data strongly suggest that the transcription from P(670) is repressed primarily by YY1 binding to the two sites, and the displacement of YY1 by hSkn-1a releases P(670) from the repression.

Arsenic trioxide induces apoptosis of HPV16 DNA-immortalized human cervical epithelial cells and selectively inhibits viral gene expression.

Arsenic trioxide (As2O3), a major ingredient of arsenic compounds in traditional Chinese medicine, exhibits anti-acute promyelocytic leukemic activity. Considering that over 80% of human malignant tumors derive from epithelial cells, we studied the effect of As2O3 on HPV 16 DNA-immortalized human cervical epithelial cells (HCE16/3 cells) in vitro. As2O3 reduced HCE16/3 cell survival, induced apoptosis at a low concentration and selectively inhibited expression of viral early genes. This effect was evidenced by a reduction of cell viability in the MTT assay, G1 arrest and significant apoptosis upon flow-cytometric analysis, presence of apoptotic bodies, formation of DNA ladders upon gel electrophoresis and inhibition of viral early gene expression by RT-PCR and Western blot. There was a good correlation between cell apoptosis and viral early gene inhibition after As2O3 treatment, suggesting that induction of apoptosis of HCE16/3 cells by As2O3 treatment might be associated with down-regulation of viral oncogene expression. In conclusion, our findings indicate that As2O3 induces apoptosis of HCE16/3 cells, which may provide a new approach for treating HPV-associated tumors.

Traditional therapies for the treatment of condylomata acuminata (genital warts).

The evaluation of therapies for condylomata acuminata (genital warts) is imprecise because it is not possible to distinguish between relapse (reappearance of previously treated warts) and reinfection (appearance of new warts in a new location). Published trials use diverse criteria for patient selection, therapeutic response and follow-up period. A further complication is that current treatments aim to clear visible lesions, which is no proof of cure as human papillomavirus may persist in a latent state. Within these limitations, the advantages and disadvantages of current therapies are reviewed, including first-line therapies such as podophyllin solution, podophyllotoxin alcohol solution or cream, cryotherapy with liquid nitrogen, laser therapy (both carbon dioxide and NdYag), and trichloracetic acid. Second-line therapies, such as electrosurgery, excision, 5-fluorouracil and interferons, are also reviewed. In general, all available treatments for genital warts are more or less unsatisfactory, with recurrence rates of 30-70% at 6 months follow-up periods.

A novel drug screening assay for papillomavirus specific antiviral activity.

Discovery and development of human papillomavirus (HPV) specific antiviral agents have been hampered by the lack of an in vitro assay permissive to HPV replication. An experimental assay system for monitoring HPV-11 DNA replication has been optimized for use as a papillomavirus antiviral drug screening tool. Cloned HPV DNA was introduced into SCC-4 cells by electroporation and viral DNA replication monitored by Southern blot. Kinetic studies demonstrated an increased HPV genome copy number with time. Viral DNA replicated as episomal, unit length genome and remained episomal after multiple passages. These data suggested the basis for an in vitro replication assay for evaluating the antiviral activity of potential chemotherapeutic agents directly on HPV. This model was used to investigate antiviral activities of current anti-HPV therapies such as 5-fluorouracil (5-FU) and alpha-interferon (alpha-IFN) and potential therapies such as sodium butyrate, 5-bromo-20-deoxyuridine (BrdU) and antisense oligonucleotides. HPV- 11 replication is significantly inhibited by BrdU and sodium butyrate; however 5-FU and alpha-IFN did not give consistent dose response results. Finally, ISIS 2105, a 20-mer phosphorothioate antisense oligonucleotide, which targets HPV-11 E2 gene product, showed potent antiviral activity in this assay with an IC50 of approximately 70 nM.

Transcription factor YY1 represses cell-free replication from human papillomavirus origins.

We have established cell-free replication for the human papillomavirus type 18 (HPV-18) origin of replication (ori)-containing DNA by using purified HPV-18 E1 and E2 gene products expressed as fusion proteins in Escherichia coli. The transcription factor YY1 has been shown to regulate RNA transcription by binding to a sequence overlapping the putative E1 protein binding site in the HPV-18 ori. We show that exogenously added YY1 fusion protein inhibited HPV-18 ori replication. Cotransfection of YY1 expression vectors also inhibited transient replication in 293 cells. However, inhibition did not appear to be mediated by binding to its cognate site in the ori as YY1 also inhibited the replication of the HPV-11 ori, which does not have a known or suspected YY1 binding site. Moreover, inhibition was not alleviated by the inclusion of YY1 binding oligonucleotides in the replication reaction mixtures. Rather, we demonstrated a direct interaction between purified fusion E2 protein and fusion YY1 protein by the pull-down assay and a partial restoration of replication activity by an elevated E2 protein concentration. These results suggest that YY1 can inhibit HPV ori replication by interfering with E2 protein functions.

Suppression of tumorigenesis by transcription units expressing the antisense E6 and E7 messenger RNA (mRNA) for the transforming proteins of the human papilloma virus and the sense mRNA for the retinoblastoma gene in cervical carcinoma cells.

Human cervical carcinoma cell lines that harbor human papilloma virus (HPV) have been reported to express HPV E6 and E7 proteins at least in the beginning stages if not at all stages of the disease. The HPV E6 and E7 proteins bind to and inactivate the products of the p53 and retinoblastoma (Rb) tumor suppressor genes, which thereby allow the cervical carcinoma cells to circumvent the action of these tumor suppressor genes. We observed that the introduction of the antisense HPV 18 E6 and E7 sequences, as well as a sense cDNA for the human wild-type Rb gene into a human cervical carcinoma cell line (HeLa), which is positive for the HPV 18 provirus, decreased the in vitro and in vivo growth rate of the transfected cells if both antisense transcripts for the HPV 18 E6 and E7 and sense transcripts for human Rb were expressed. In addition, overexpression of a complementary DNA (cDNA) for the Rb messenger RNA was sufficient to slow the proliferation of HeLa cells, and the level of Rb cDNA expression was correlated with the degree to which the rate of growth of the tumor was slowed. The results of our experiments show that the presence of HPV E6 and E7 proteins and the resultant inactivation of Rb in cervical carcinoma cells contributes to the neoplastic phenotype even in highly evolved cervical carcinoma cell lines such as HeLa, which have been derived from a cervical carcinoma patient at an advanced stage of the disease process. These data suggest that the HPV proteins play a role not only at the beginning of cervical cancer, but also at advanced stages of this disease. These experiments may lead to genetic approaches to the control of this disease that involve antisense sequences that downregulate the E6 and E7 genes or lead to expression of the Rb gene.

[Immunochemical therapy of warts and growths due to papillomavirus]. / La thérapie immuno-chimique des verrues et végétations dues au virus papillomateux.

A complex immuno-chemical treatment (immuno-stimulation, blockage of virus replication, interferon induction) was applied to 23 patients with warts and vegetations with various localisations. Results confirmed the treatment efficiency.

HPV-18 immortalization of human keratinocytes.

The oncogenic potential of human papillomavirus type 18 which is found in a significant number of cervical and penile cancer biopsies was tested in primary human keratinocytes derived from neonatal foreskin. Viral DNA and a gene for resistance to neomycin were introduced into these cells by calcium phosphate transfection. Selection of cells in G418 led to the isolation of resistant colonies which were propagated in culture. Four cell lines termed FE-A, FEH 18L, FEP18-5, and FEP18-11 have been maintained in culture for 1 1/2-2 years and were selected for further analysis. In all cases the viral DNA was integrated into the cellular genome and the early genes were transcribed, including RNA complementary to the E2, E6, and E7 open reading frames. Radioimmunoprecipitation showed that all cell lines synthesized the E6 and E7 proteins. However, none of the cell lines tested were tumorigenic. The differentiation capacity of these cells was analyzed by assessing their ability to proliferate clonally after exposure to 1.2 mM calcium chloride. All four cell lines were resistant to this stimulus and formed colonies upon return to regular growth medium whereas normal cells differentiated terminally. K6a and K14 keratin RNA expression was down-regulated in the HPV immortalized cell lines compared to primary human epithelial cells.