Nanomicellar Curcumin Supplementation Improves the Clinical Manifestations of HAM/TSP Patients.

BACKGROUND: HTLV-1 infection causes a chronic, progressive, demyelinating, neuroinflammatory disease called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Treatment of HAM/TSP patients which have high levels of proviral load and pro-inflammatory markers is a challenge for clinicians. Therefore, we aimed to investigate the immunomodulatory, anti-inflammatory, and antiviral effects of curcumin in HAM/TSP patients. METHODS: In this study, 20 newly diagnosed HAM/TSP patients (2 men and 18 women) were enrolled and evaluated for clinical symptoms, HTLV-1 proviral load, Tax and HBZ expression, neopterin serum concentration, and complete blood count (CBC) before and 12 weeks after treatment with nanomicellar curcumin (80 mg/day, orally). RESULTS: Clinical symptoms such as the mean Osame Motor Disability Score and Ashworth Spasticity Scale Score were significantly improved after the treatment (P = 0.001 and P = 0.001). Sensory symptoms such as pain and paresthesia were significantly decreased in all of the patients (P = 0.001). Furthermore, urinary disorders, including urinary frequency, incontinence, and the feeling of incomplete bladder emptying, were significantly improved (P = 0.001, P = 0.003, and P = 0.03). However, the mean HTLV-1 proviral load (P = 0.97) and CBC were similar, whereas Tax, HBZ, and neopterin levels tend to increase after the treatment (P = 0.004, P = 0.08, and P = 0.04). CONCLUSION: Results suggest that curcumin can safely improve the clinical symptoms of HAM/TSP patients but has no observable positive effects on the HTLV-1 proviral load, Tax, and HBZ expression. Therefore, prolonged use or the use of curcumin with antiviral agents in addition to clinical signs and symptoms can reduce the HTLV-1 proviral load and the expression of functional viral factors such as Tax and HBZ.

Silver nanoparticles exhibit size-dependent differential toxicity and induce expression of syncytin-1 in FA-AML1 and MOLT-4 leukaemia cell lines.

Human endogenous retrovirus (HERV) sequences make up ~8% of the human genome and increased expression of some HERV proteins has been observed in various pathologies including leukaemia and multiple sclerosis. However, little is known about the function of these HERV proteins or environmental factors which regulate their expression. Silver nanoparticles (AgNPs) are used very extensively as antimicrobials and antivirals in numerous consumer products although their effect on the expression of HERV gene products is unknown. Cell proliferation and cell toxicity assays were carried out on human acute T lymphoblastic leukaemia (MOLT-4) and Fanconi anaemia associated acute myeloid leukaemia (FA-AML1) cells treated with two different sizes of AgNPs (7nm and 50nm diameter). Reverse-transcriptase polymerase chain reaction and western blotting were then used to the assess expression of HERV-W syncytin-1 mRNA and protein in these cells. FA-AML1 cells were more sensitive overall than MOLT-4 to treatment with the smaller 7nm sized AgNp's being the most toxic in these cells. MOLT-4 cell were more resistant and showed no evidence of differential toxicity to the different sized particles. Syncytin-1 mRNA and protein were induced by both 7 and 50nm AgNPs in both cell types yet with different kinetics. In summary, the observation that AgNPs induce expression of syncytin-1 in FA-AML1 and MOLT-4 cells at doses as little as 5 µg/ml is grounds for concern since this protein is up-regulated in both malignant and neurodegenerative diseases. Considering the widespread use of AgNPs in the environment it is clear that their ability to induce syncytin-1 should be investigated further in other cell types.

Increased prevalence of JC polyomavirus in cervical carcinomas from women infected with HIV.

Although subclinical persistent infections with the human polyomaviruses BKV and JCV are ubiquitous worldwide, these are known to vary in relation to diseases present and geographical location. DNAs from 220 cervical smears and 109 invasive cervical carcinomas obtained from HIV positive and HIV negative Kenyan women of known HPV status were analyzed by nested endpoint PCR for BKV and JCV. BKV-JCV DNA was detected in 5/105 (4.7%) of cervical smears and in 6/37 (16%) of cervical carcinomas from women infected with HIV whereas 9/115 (7.8%) of the cervical smears and 4/72 (5.5%) of the carcinomas were positive in HIV negative women. Nested PCR showed that all 24 samples were positive for JCV and not BKV. JCV was not more prevalent in either HPV positive (P = 0.438) or HPV negative women (P = 0.392). However, 37% of carcinomas and smears which were positive for JCV were also positive for a "high-risk" oncogenic HPV. Comparison of the incidence of JCV in cervical smears and cervical carcinomas showed a ∼3-fold increase in samples from HIV positive women with cervical carcinoma (P = 0.025) whereas no significant difference was found between cervical smears and cervical carcinomas from HIV negative women (P = 0.553). These results suggest that JCV may combine with high-risk HPV infection in women infected with HIV to influence the rate of progression to invasive cervical carcinoma.

Effects of the Prophylactic HPV Vaccines on HPV Type Prevalence and Cervical Pathology.

Vaccination programs with the current prophylactic HPV vaccines started in most countries around 2008 with introduction of the bivalent Cervarix HPV16/18 vaccine, rapidly followed by Gardasil (HPV6/11/16/18) and, finally, Gardasil 9 (HPV6/11/16/18/31/33/45/52/58), from 2015. Many studies have now confirmed their ability to prevent infection with vaccine-covered HPV types, and the subsequent development of either genital warts and/or cervical neoplasia, although this is clearly more effective in younger women vaccinated prior to sexual debut. Most notably, reductions in the prevalence of vaccine-covered HPV types were also observed in unvaccinated women at the same geographical location, presumably by sexual dissemination of these changes, between vaccinated and unvaccinated women. Furthermore, there are several studies that have demonstrated vaccine-associated HPV type-replacement, where vaccine-covered, high-risk HPV types are replaced by high-risk HPV types not covered by the vaccines, and these changes were also observed in vaccinated and unvaccinated women in the same study population. In light of these observations, it is not entirely clear what effects vaccine-associated HPV type-replacement will have, particularly in older, unvaccinated women.

Combining metabolic fingerprinting and footprinting to understand the phenotypic response of HPV16 E6 expressing cervical carcinoma cells exposed to the HIV anti-viral drug lopinavir.

Recently, it has been reported that the anti-viral drug, lopinavir, which is currently used as a human immunodeficiency virus (HIV) protease inhibitor, could also inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells. In this study, C33A parent control cells and HPV16 E6-transfected cells were exposed to lopinavir at concentrations ranging from 0 to 30 microM. The phenotypic response was assessed by Fourier transform infrared (FT-IR) spectroscopy directly on cells (the metabolic fingerprint) and on the cell growth medium (the metabolic footprint). Multivariate analysis of the data using both principal components analysis (PCA) and canonical variates analysis (PC-CVA) showed trends in scores plots that were related to the concentration of the drug. Inspection of the PC-CVA loadings vector revealed that the effect was not due to the drug alone and that several IR spectral regions including proteins, nucleotides and carbohydrates contributed to the separation in PC-CVA space. Finally, partial least squares regression (PLSR) could be used to predict the concentration of the drug accurately from the metabolic fingerprints and footprints, indicating a dose related phenotypic response. This study shows that the combination of metabolic fingerprinting and footprinting with appropriate chemometric analysis is a valuable approach for studying cellular responses to anti-viral drugs.

Raman chemical mapping reveals site of action of HIV protease inhibitors in HPV16 E6 expressing cervical carcinoma cells.

It has been shown that the HIV protease inhibitors indinavir and lopinavir may have activity against the human papilloma virus (HPV) type 16 inhibiting HPV E6-mediated proteasomal degradation of p53 in cultured cervical carcinoma cells. However, their mode and site of action is unknown. HPV-negative C33A cervical carcinoma cells and the same cells stably transfected with E6 (C33AE6) were exposed to indinavir and lopinavir at concentrations of 1 mM and 30 µM, respectively. The intracellular distribution of metabolites and metabolic changes induced by these treatments were investigated by Raman microspectroscopic imaging combined with the analysis of cell fractionation products by liquid chromatography-mass spectrometry (LC-MS). A uniform cellular distribution of proteins was found in drug-treated cells irrespective of cell type. Indinavir was observed to co-localise with nucleic acid in the nucleus, but only in E6 expressing cells. Principal components analysis (PCA) score maps generated on the full Raman hypercube and the corresponding PCA loadings plots revealed that the majority of metabolic variations influenced by the drug exposure within the cells were associated with changes in nucleic acids. Analysis of cell fractionation products by LC-MS confirmed that the level of indinavir in nuclear extracts was approximately eight-fold greater than in the cytoplasm. These data demonstrate that indinavir undergoes enhanced nuclear accumulation in E6-expressing cells, which suggests that this is the most likely site of action for this compound against HPV.

Potential Effects of Human Papillomavirus Type Substitution, Superinfection Exclusion and Latency on the Efficacy of the Current L1 Prophylactic Vaccines.

There are >200 different types of human papilloma virus (HPV) of which >51 infect genital epithelium, with the ~14 of these classed as high-risk being more commonly associated with cervical cancer. During development of the disease, high-risk types have an increased tendency to develop a truncated non-replicative life cycle, whereas low-risk, non-cancer-associated HPV types are either asymptomatic or cause benign lesions completing their full replicative life cycle. HPVs can also be present as non-replicative so-called "latent" infections and they can also show superinfection exclusion, where cells with pre-existing infections with one type cannot be infected with a different HPV type. Thus, the HPV repertoire and replication status present in an individual can form a complex dynamic meta-community which changes with respect to both time and exposure to different HPV types. In light of these considerations, it is not clear how current prophylactic HPV vaccines will affect this system and the potential for iatrogenic outcomes is discussed in light of recent outcome data.

Potential risk factors associated with the use of cidofovir to treat benign human papillomavirus-related disease.

BACKGROUND: Cidofovir is currently being used off-licence to treat different viral infections, such as benign low-risk human papillomavirus (HPV)-related recurrent respiratory papillomatosis (RRP). There are concerns over the safety of this practice as rat studies demonstrated a high malignant transformation rate. As yet, there are no clinical reports of cidofovir-induced malignant changes in humans. METHODS: Telomerase immortalised human keratinocytes (hTert) stably expressing E6 proteins from either low-risk HPV6b or high-risk HPV16 and vector control cells were treated with either low-dose (5 microg/ml) or higher dose (30 microg/ml) cidofovir for 2 days and the effects evaluated by clonogenic survival assays. Based on these results, gene expression microarray analysis was performed on cidofovir-treated low-risk E6 and vector cells before, during and after drug treatment, and the results verified by real-time PCR. RESULTS: Both low-risk and high-risk E6-expressing cells show significantly improved long-term survival compared with vector control cells when exposed to 5 microg/ml cidofovir for 2 days, (hTert T6E6 P=0.0007, hTert T16E6 P=0.00023 and hTert vector control P=0.62). Microarray and real-time PCR analyses of low-dose cidofovir-treated low-risk E6-expressing cells revealed changes in gene expression that are known to be associated with malignant progression, which were not observed in drug-treated vector control cells. CONCLUSIONS: This is the first report that cidofovir can both increase cell survival and induce alterations in gene expression that are known to be associated with malignant transformation in cells transduced only with the E6 gene from low-risk HPV. It is our belief that these data provide cause for concern over the off-license use of this drug to treat RRP.

Specific HIV protease inhibitors inhibit the ability of HPV16 E6 to degrade p53 and selectively kill E6-dependent cervical carcinoma cells in vitro.

Although HIV protease inhibitor (PI) drugs predominantly target HIV proteases 1 and 2, it is also known that part of their efficacy is due to selective inhibition of the proteasome. The pathogenicity of high-risk human papilloma virus (HPV) is dependent on expression of viral E6 proteins which inappropriately activate the 26S proteasome to degrade p53 and other cellular proteins that are detrimental to viral replication. Comparison of the ability of the PIs indinavir, ritonavir, amprenavir, lopinavir, atazanavir, nelfinavir and saquinavir to inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells showed that 15 microM lopinavir, 1 mM indinavir or 125 microM ritonavir treatment for 24 h produced a stable increase in the level of nuclear p53 in these cells with minimal cell death. After 4 h exposure of HPV16+ve SiHa cells to 15 microM lopinavir, a transient increase in wild-type p53 expression was observed associated with a 7% reduction in the chymotryptic activity of the 205 proteasome and apoptosis after 24h. Comparison of growth rates of PI treated SiHa, CaSki, C33A, C33A-E6 and non-transformed NIH/3T3 cells showed that SiHa were the most sensitive, whereas NIH/3T3 were least affected. In conclusion, these data show that specific HIV PIs such as lopinavir and possibly indinavir, can induce selective toxicity of HPV-transformed cervical carcinoma cells expressing wild-type p53 and may form the basis of a topically applied alternative to surgery for the treatment of HPV-related premalignant lesions of the cervix.

Hypoxia increases heparanase-dependent tumor cell invasion, which can be inhibited by antiheparanase antibodies.

The beta-endoglucuronidase heparanase plays an important role in tumor invasion, a process that is significantly enhanced by hypoxia. We have used a strategy of stable transfection with antisense to derive ovarian carcinoma cell lines that express different levels of heparanase and used these to demonstrate that invasion correlates with heparanase activity. Secreted heparanase activity was increased by reduction, hypoxia, and growth of cells under reduced oxygen (1%) augmented heparanase activity and invasion, both of which are inhibited by treatment with antiheparanase antibodies. This is the first demonstration that heparanase activity may be regulated by microenvironmental redox conditions, which influence invasion, and that invasion can be blocked with specific heparanase-neutralizing antibodies.

Analysis of the Prevalence of HTLV-1 Proviral DNA in Cervical Smears and Carcinomas from HIV Positive and Negative Kenyan Women.

The oncogenic retrovirus human T-cell lymphotropic virus type 1 (HTLV-1) is endemic in some countries although its prevalence and relationship with other sexually transmitted infections in Sub-Saharan Africa is largely unknown. A novel endpoint PCR method was used to analyse the prevalence of HTLV-1 proviral DNA in genomic DNA extracted from liquid based cytology (LBC) cervical smears and invasive cervical carcinomas (ICCs) obtained from human immunodeficiency virus-positive (HIV+ve) and HIV-negative (HIV-ve) Kenyan women. Patient sociodemographic details were recorded by structured questionnaire and these data analysed with respect to HIV status, human papillomavirus (HPV) type (Papilocheck(®)) and cytology. This showed 22/113 (19.5%) of LBC's from HIV+ve patients were positive for HTLV-1 compared to 4/111 (3.6%) of those from HIV-ve women (p = 0.0002; odds ratio (OR) = 6.42 (2.07-26.56)). Only 1/37 (2.7%) of HIV+ve and none of the 44 HIV-ve ICC samples were positive for HTLV-1. There was also a significant correlation between HTLV-1 infection, numbers of sexual partners (p < 0.05) and smoking (p < 0.01). Using this unique method, these data suggest an unexpectedly high prevalence of HTLV-1 DNA in HIV+ve women in this geographical location. However, the low level of HTLV-1 detected in HIV+ve ICC samples was unexpected and the reasons for this are unclear.

A Single-Arm, Proof-Of-Concept Trial of Lopimune (Lopinavir/Ritonavir) as a Treatment for HPV-Related Pre-Invasive Cervical Disease.

BACKGROUND: Cervical cancer is the most common female malignancy in the developing nations and the third most common cancer in women globally. An effective, inexpensive and self-applied topical treatment would be an ideal solution for treatment of screen-detected, pre-invasive cervical disease in low resource settings. METHODS: Between 01/03/2013 and 01/08/2013, women attending Kenyatta National Hospital's Family Planning and Gynaecology Outpatients clinics were tested for HIV, HPV (Cervista®) and liquid based cervical cytology (LBC-ThinPrep®). HIV negative women diagnosed as high-risk HPV positive with high grade squamous intraepithelial lesions (HSIL) were examined by colposcopy and given a 2 week course of 1 capsule of Lopimune (CIPLA) twice daily, to be self-applied as a vaginal pessary. Colposcopy, HPV testing and LBC were repeated at 4 and 12 weeks post-start of treatment with a final punch biopsy at 3 months for histology. Primary outcome measures were acceptability of treatment with efficacy as a secondary consideration. RESULTS: A total of 23 women with HSIL were treated with Lopimune during which time no adverse reactions were reported. A maximum concentration of 10 ng/ml of lopinavir was detected in patient plasma 1 week after starting treatment. HPV was no longer detected in 12/23 (52.2%, 95%CI: 30.6-73.2%). Post-treatment cytology at 12 weeks on women with HSIL, showed 14/22 (63.6%, 95%CI: 40.6-82.8%) had no dysplasia and 4/22 (18.2%, 95%CI: 9.9-65.1%) were now low grade demonstrating a combined positive response in 81.8% of women of which 77.8% was confirmed by histology. These data are supported by colposcopic images, which show regression of cervical lesions. CONCLUSIONS: These results demonstrate the potential of Lopimune as a self-applied therapy for HPV infection and related cervical lesions. Since there were no serious adverse events or detectable post-treatment morbidity, this study indicates that further trials are clearly justified to define optimal regimes and the overall benefit of this therapy. TRIAL REGISTRATION: ISRCTN Registry 48776874.

An overview of early investigational drugs for the treatment of human papilloma virus infection and associated dysplasia.

INTRODUCTION: High-risk HPV (HR-HPV) related invasive cervical cancer (ICC) causes >270,000 deaths per annum world-wide with over 85% of these occurring in low-resource countries. Ablative and excisional treatment modalities are restricted for use with high-grade pre-cancerous cervical disease with HPV infection and low-grade dysplasia mostly managed by a watch-and-wait policy. AREAS COVERED: Various pharmacological approaches have been investigated as non-destructive alternatives for the treatment of HR-HPV infection and associated dysplasia. These are discussed dealing with efficacy, ease-of-use (physician or self-applied), systemic or locally applied, side-effects, cost and risks. The main focus is the perceived impact on current clinical practice of a self-applied, effective and safe pharmacological anti-HPV treatment. EXPERT OPINION: Current prophylactic HPV vaccines are expensive, HPV type restricted and have little effect in already infected women. Therapeutic vaccines are under development but are also HPV type-restricted. At present, the developed nations use national cytology screening and surgical procedures to treat only women identified with HPV-related high-grade dysplastic disease. However, since HPV testing is rapidly replacing cytology as the test-of-choice, a suitable topically-applied and low-cost antiviral treatment could be an ideal solution for treatment of HPV infection per se with test-of-cure carried out by repeat HPV testing. Cytology would only then be necessary for women who remained HPV positive. Although of significant benefit in the developed countries, combining such a treatment with self-sampled HPV testing could revolutionise the management of this disease in the developing world which lack both the infrastructure and resources to establish national cytology screening programs.

Using HIV drugs to target human papilloma virus.

Over the past decade it has been demonstrated that HIV protease inhibitors have various off-target activities that has enabled them to be repositioned as treatments for a range of other pathologies. Human papilloma virus and related malignancies have been shown to be susceptible to these agents and current progress with this indication is summarized here together with a discussion of the rationale for the off-target effects of these compounds.

A metabolomics investigation into the effects of HIV protease inhibitors on HPV16 E6 expressing cervical carcinoma cells.

Recently, it has been reported that anti-viral drugs, such as indinavir and lopinavir (originally targeted for HIV), also inhibit E6-mediated proteasomal degradation of mutant p53 in E6-transfected C33A cells. In order to understand more about the mode-of-action(s) of these drugs the metabolome of HPV16 E6 expressing cervical carcinoma cell lines was investigated using mass spectrometry (MS)-based metabolic profiling. The metabolite profiling of C33A parent and E6-transfected cells exposed to these two anti-viral drugs was performed by ultra performance liquid chromatography (UPLC)-MS and gas chromatography (GC)-time of flight (TOF)-MS. Using a combination of univariate and multivariate analyses, these metabolic profiles were investigated for analytical and biological reproducibility and to discover key metabolite differences elicited during anti-viral drug challenge. This approach revealed both distinct and common effects of these two drugs on the metabolome of two different cell lines. Finally, intracellular drug levels were quantified, which suggested in the case of lopinavir that increased activity of membrane transporters may contribute to the drug sensitivity of HPV infected cells.

HIV Infection Alters the Spectrum of HPV Subtypes Found in Cervical Smears and Carcinomas from Kenyan Women.

Infection with high risk HPV is implicated in pre-cancerous squamous intraepithelial lesions and their progression to cervical cancer. In the developed countries, infection with HPV 16 and 18 accounts for ~70% of cervical cancers, but it has been established that HPV type prevalence differs according to worldwide geographical location. In sub Saharan Africa infection with HPV is known to be augmented by HIV, which is endemic in this region. It is not yet clear, however, whether this ultimately influences progression to cervical cancer. Papillocheck(TM) and multiplex PCR were used to determine the range of HPV genotypes found in cervical smears and carcinomas from HIV positive and negative Kenyan women. Smear samples from HIV-positive women had a higher prevalence of: multiple HPV infections; high-risk HPVs 52, 58, 68, potential high risk 53/70, low-risk 44/55 and abnormal cytology compared to HIV-negative women. A low overall prevalence (~8%) of types 16/18 was found in all smear samples tested (n = 224) although this increased in invasive cervical carcinoma tissues to ~80% for HIV-negative and ~46% for HIV-positive women. Furthermore, HPV45 was more common in cervical carcinoma tissues from HIV-positive women. In summary HIV infection appears to alter the spectrum of HPV types found in both cervical smears and invasive cervical carcinomas. It is hypothesised there could be a complex interplay between these viruses which could either positively or negatively influence the rate of progression to cervical cancer.

Analysis of factors contributing to the low survival of cervical cancer patients undergoing radiotherapy in Kenya.

BACKGROUND: In contrast to the developed nations, invasive cervical cancer (ICC) is the most common womens malignancy in Kenya and many other locations in sub-Saharan Africa. However, studies on survival from this disease in this area of the world are severely restricted by lack of patient follow-up. We now report a prospective cohort study of ICC in Kenyan women analysing factors affecting tumour response and overall survival in patients undergoing radiotherapy. METHODS AND FINDINGS: Between 2008 and 2010, 355 patients with histologically confirmed ICC were recruited at the Departments of Gynaecology and Radiotherapy at Kenyatta National Hospital (KNH). Structured questionnaires were completed recording socio-demographics, tumour response and overall survival following treatment with combinations of external beam radiation (EBRT), brachytherapy and adjuvant chemotherapy. Of the 355 patients, 42% (146) were lost to follow-up while 18% (64) died during the two year period. 80.5% of patients presented with advanced stage IIB disease or above, with only 6.7% of patients receiving optimal combined EBRT, brachytherapy and adjuvant chemotherapy. Kaplan Meier survival curves projected two year survival at <20%. CONCLUSION: Cervical cancer is preventable yet poverty, poor education, lack of cancer awareness coupled with an absence of regular screening programs, late patient presentation, sub-optimal diagnosis and treatments are major factors contributing to the alarmingly low survival rate of cervical cancer patients in Kenya. It is concluded that simple cost-effective changes in clinical practice could be introduced which would have a marked impact on patient survival in this setting.

No biological evidence of XMRV infection in cervical smears from HIV/ HPV positive and negative Kenyan women.

BACKGROUND: XMRV (xenotropic murine leukaemia virus-related virus) is a gammaretrovirus first discovered in human prostate carcinomas and later linked to chronic fatigue syndrome (CFS). Emerging conflicting data and lack of reproducibility of results within the scientific community has now led to the association of XMRV with CFS being discounted. Indeed the case for an involvement with any human disease has been questioned with the suggestion that XMRV is a laboratory generated recombinant virus. The fact that not all published positive findings can be easily explained as contamination artefacts coupled with the observation that XMRV may have a sexually transmitted mode of infectivity and can be infectious for primates, where it preferential resides in cells of the reproductive tract, prompted us to look for evidence of XMRV in the cervical cells of a cohort of Kenyan women both with and without pre-existing HIV/HPV infections. RESULTS: Using a highly sensitive and selective triplex PCR approach we analysed DNA from the liquid based cytology (LBC) cervical smears of 224 Kenyan women. There was no evidence of XMRV expression in any of the sample population irrespective of HPV and/or HIV status. CONCLUSIONS: The data presented show no indication of XMRV infection in any of the cervical samples screened in this study. Approximately 50% of the women were HIV positive but this did not influence the findings signifying that XMRV does not act as an opportunistic infection in this cohort nor is it related to HPV status. Our results therefore support the findings that XMRV is confined to the laboratory and does not currently represent an infectious agent for humans, with a cautionary adage that such potential zoonotic viruses should be carefully monitored in the future.

Therapy of human papillomavirus-related disease.

This chapter reviews the current treatment of chronic and neoplastic human papillomavirus (HPV)-associated conditions and the development of novel therapeutic approaches. Surgical excision of HPV-associated lower genital tract neoplasia is very successful but largely depends on secondary prevention programmes for identification of disease. Only high-risk HPV-driven chronic, pre-neoplastic lesions and some very early cancers cannot be successfully treated by surgical procedures alone. Chemoradiation therapy of cervical cancer contributes to the 66-79% cervical cancer survival at 5 years. Outlook for those patients with persistent or recurrent cervical cancer following treatment is very poor. Topical agents such as imiquimod (immune response modifier), cidofovir (inhibition of viral replication; induction apoptosis) or photodynamic therapy (direct damage of tumour and augmentation of anti-tumour immunity) have all shown some useful efficacy (~50-60%) in treatment of high grade vulvar intraepithelial neoplasia (VIN). Provider administered treatments of genital warts include cryotherapy, trichloracetic acid, or surgical removal which has the highest primary clearance rate. Patient applied therapies include podophyllotoxin and imiquimod. Recurrence after "successful" treatment is 30-40%. Further improvements could derive from a rational combination of current therapy with new drugs targeting molecular pathways mediated by HPV in cancer. Small molecule inhibitors targeting the DNA binding activities of HPV E1/E2 or the anti-apoptotic consequences of E6/E7 oncogenes are in preclinical development. Proteasome and histone deacetylase inhibitors, which can enhance apoptosis in HPV positive tumour cells, are being tested in early clinical trials. Chronic high-risk HPV infection/neoplasia is characterised by systemic and/or local immune suppressive regulatory or escape factors. Recently two E6/E7 vaccines have shown some clinical efficacy in high grade VIN patients and this correlated with strong and broad systemic HPV-specific T cell response and modulation of key local immune factors. Treatments that can shift the balance of immune effectors locally in combination with vaccination are now being tested. This article forms part of a special supplement entitled "Comprehensive Control of HPV Infections and Related Diseases" Vaccine Volume 30, Supplement 5, 2012.

Lopinavir up-regulates expression of the antiviral protein ribonuclease L in human papillomavirus-positive cervical carcinoma cells.

BACKGROUND: We have previously shown that the HIV protease inhibitor lopinavir has selective toxicity against human papillomavirus (HPV)-positive cervical carcinoma cells via an unknown mechanism. METHODS: SiHa cervical carcinoma cells were stably transfected with the proteasome sensor vector pZsProSensor-1 to confirm lopinavir inhibits the proteasome in these cells. The Panorama Xpress profiler 725 antibody array was then used to analyse specific changes in protein expression in lopinavir-treated versus control untreated SiHa cells followed by PCR and western blotting. Colorimetric growth assays of lopinavir-treated E6/E7 immortalised versus control human keratinocytes were performed. Targeted small interfering RNA gene silencing followed by growth assay comparison of lopinavir-treated/untreated SiHa cells was also used. RESULTS: Lopinavir induced an increase in the fluorescence of pZsProSensor-1 transfected SiHa cells, indicative of proteasomal inhibition. Ribonuclease L (RNASEL) protein was shown to be up-regulated in lopinavir-treated SiHa cells, which was confirmed by PCR and western blot. Targeted silencing of RNASEL reduced the sensitivity of SiHa cells to lopinavir. Selective toxicity against E6/E7 immortalised keratinocytes versus control cells was also seen with lopinavir and was associated with up-regulated RNASEL expression. CONCLUSIONS: These data are consistent with the toxicity of lopinavir against HPV-positive cervical carcinoma cells being related to its ability to block viral proteasome activation and induce an up-regulation of the antiviral protein RNASEL. This is supported by the drug's selective toxicity and up-regulation of RNASEL in E6/E7 immortalised keratinocytes combined with the increased resistance to lopinavir observed in SiHa cells following silencing of RNASEL gene expression.