E7 as a Promising Biomarker for Monitoring the Progression of Cervical Lesions in Patients with HPV 16/18 Infections.

Objective: To investigate the expression of E7 protein and its relationship with the progression and prognosis of cervical pre-cancerous lesions in patients with human papillomavirus (HPV) 16/18 infections. Methods: A total of 211 patients with positive HPV 16/18 were included in this study. Patients were categorized into three groups based on colposcopy results: NILM (Negative for Intraepithelial Lesion or Malignancy), LSIL (Low-Grade Squamous Intraepithelial Lesion), and HSIL (High-Grade Squamous Intraepithelial Lesion). E7 protein levels were quantified using Immunochromatographic Assay and compared using ANOVA. Cervical E7 protein levels were assessed before and one year after cervical cone biopsy in the HSIL group. Results: Among HPV 16/18-positive patients with normal Cervical Thinprep Cytologic Test (TCT) results, E7 protein content exhibited abnormal and significant values (P = .001). Mean E7 protein levels for the NILM, LSIL, and HSIL groups were 44.52 ng/mL, 114.60 ng/mL, and 389.20 ng/mL, respectively, and showed statistical significance (P = .000). In the HSIL group, E7 protein levels in HPV-negative patients were significantly lower one year after cervical cone biopsy compared to before (P = .001). However, HPV-positive patients displayed no significant alteration in E7 protein levels before and after biopsy (P = .08). Conclusions: E7 protein levels in detached cervical cells are closely associated with the severity and prognosis of cervical pre-cancerous lesions, suggesting their potential role as a biomarker for monitoring cervical lesion development.

HPV18 E6 inhibits α-ketoglutarate-induced pyroptosis of esophageal squamous cell carcinoma cells via the P53/MDH1/ROS/GSDMC pathway.

Oncogene E6 plays a critical role in the development and progression of esophageal cancer caused by human papillomavirus (HPV) infection. Alpha-ketoglutarate (AKG) is a key metabolite in the tricarboxylic acid cycle and has been widely used as a dietary and anti-ageing supplement. In this study, we found that treating esophageal squamous carcinoma cells with a high dose of AKG can induce cell pyroptosis. Furthermore, our research confirms that HPV18 E6 inhibits AKG-induced pyroptosis of esophageal squamous carcinoma cells by lowering P53 expression. P53 downregulates malate dehydrogenase 1 (MDH1) expression; however, MDH1 downregulates L-2-hydroxyglutarate (L-2HG) expression, which inhibits a rise in reactive oxygen species (ROS) levels-as L-2HG is responsible for excessive ROS. This study reveals the actuating mechanism behind cell pyroptosis of esophageal squamous carcinoma cells induced by high concentrations of AKG, and we posit the molecular pathway via which the HPV E6 oncoprotein inhibits cell pyroptosis.

Deteccion del virus del papiloma humano en biopsias de cancer de mama mediante PCR e inmunohistoquimica en el Hospital Santa Rosa durante el 2019 / Detection of human papilloma virus in breast cancer biopsies using PCR and immunohistochemistry at the Santa Rosa Hospital during 2019

El objetivo de este estudio fue determinar la presencia del Virus Papiloma Humano (VPH) tipo 16 y 18 en biopsias de tejido mamario parafinado de pacientes con diagnóstico clínico de cáncer de mama. Se analizaron 32 biopsias de cáncer de mama embebidas en parafina para detectar el ADN de VPH mediante PCR en tiempo real, los iniciadores estuvieron dirigidos al gen E6. Se evaluaron el tipo histológico, grado histológico y la sobreexpresión de C-erB2 y Ki-67 mediante inmunohistoquímica. El 84,38% (27) fueron positivos para VPH, el 25% (8) fueron positivos para VPH-16 y el 59,38% (19) para VPH-18. El 15,63% (5) de las muestras presentaron infección mixta. Se evidenció la sobrexpresión de C-erbB2 y Ki-67 en 6,25% (2) de las muestras positivas para VPH-16 y 15,63% (5) de las muestras positivas para VPH-18. Se detectó ADN de VPH-16 y VPH-18 en las muestras de biopsias analizadas mediante PCR en tiempo real.

The aim of this study was to determine the presence of Human Papillomavirus (HPV) type 16 and 18 in biopsies of paraffin-embedded breast tissue from patients with clinically diagnosed breast cancer. 32 paraffin-embedded breast cancer biopsies were analyzed in order to detect HPV DNA by real-time PCR, the primers were directed at the E6 gene. The histological type, histological grade and overexpression of C-erB2 and Ki-67 were evaluated by immunohistochemistry. 84.38% (27) of the samples were positive for HPV, 25% (8) were positive for HPV-16 and 59.38% (19) were positive for HPV-18. Mixed infection was found in 15.63% (5) of the samples. Overexpression of C-erbB2 and Ki-67 was seen in 6.25% (2) of the samples positive for HPV-16 and in 15.63% (5) samples positive for HPV-18. HPV-16 and HPV-18 DNA was detected in the biopsy samples analyzed by real-time PCR.

Results from a cervical cancer screening program in Samsun, Turkey.

BACKGROUND: Cervical cancer is a preventable disease. This study aimed to share the results of the national cervical cancer screening program performed in primary health care institutions in Samsun between 2015 and 2019. METHODS: Women aged 30-65 years who were screened for cervical cancer in screening centers of Samsun between January 01, 2015, and December 31, 2019, were included in this descriptive study. The data were obtained from the automation program of the "National Human Papilloma Virus (HPV) Laboratory Application" used by the Provincial Directorate of Health Cancer Unit through filtering the completion time of the tests, and all results were evaluated without sampling. Thus, data were presented using descriptive statistics. RESULTS: The mean age of 89,302 women included in the cervical cancer screening program was 45.9 ± 9.0 years. Of the samples obtained from the participants, 1.0% were determined as insufficient material, 94.1% as HPV-negative, and 4.9% as HPV-positive. The most common HPV genotypes were 16, 51, 31, and 52. Of the 4337 HPV-positive women, 74.7% of the pap smear results were negative (including infection, 36.5%), and the most common premalignant lesions were atypical squamous cells of undetermined significance in 7.1% and low-grade squamous intraepithelial lesions in 6.9%. HPV 16/18 was also observed in 31.7% of HPV-positive women. Seven hundred ninety-five women were referred to a specialist physician for further examination and treatment within the scope of the screening algorithm. CONCLUSION: Detecting HPV-positivity by reaching more women within the national cervical cancer screening program's scope is vital in fighting against this disease. The effectiveness of cancer screening programs should be increased by ensuring community participation through awareness activities.

Changes in Cervical Cytology Results and Human Papillomavirus Types Among Persons Screened for Cervical Cancer, 2007 and 2015-2017.

OBJECTIVES: Since 2006, the US human papillomavirus (HPV) vaccination program has led to decreases in HPV infections caused by high-risk vaccine-targeted HPV types (HPV 16/18). We assessed differences in high-risk HPV prevalence by cervical cytology result among 20- to 24-year-old persons participating in routine cervical cancer screening in 2015-2017 compared with 2007. MATERIALS AND METHODS: Residual routine cervical cancer screening specimens were collected from 20- to 24-year-old members of 2 integrated healthcare delivery systems as part of a cross-sectional study and were tested for 37 HPV types. Cytology results and vaccination status (≥1 dose) were extracted from medical records. Cytology categories were normal, atypical squamous cells of undefined significance, low-grade squamous intraepithelial lesions (SIL), or high-grade SIL/atypical squamous cells cannot exclude high-grade SIL. Prevalences of HPV categories (HPV 16/18, HPV 31/33/45/52/58, HPV 35/39/51/56/59/66/68) were estimated by cytology result for 2007 and 2015-2017. RESULTS: Specimens from 2007 (n = 4046) were from unvaccinated participants; 4574 of 8442 specimens (54.2%) from 2015-2017 were from vaccinated participants. Overall, HPV 16/18 positivity was lower in 2015-2017 compared with 2007 in all groups: high-grade SIL/atypical squamous cells cannot exclude high-grade SIL, 16.0% vs 69.2%; low-grade SIL, 5.4% vs 40.1%; atypical squamous cells of undefined significance, 5.0% vs 25.6%; and normal, 1.3% vs 8.1%. Human papillomavirus 31/33/45/52/58 prevalence was stable for all cytology groups; HPV 35/39/51/56/59/66/68 prevalence increased among low-grade SIL specimens (53.9% to 65.2%) but remained stable in other groups. CONCLUSIONS: Prevalence of vaccine-targeted high-risk HPV types 16/18 was dramatically lower in 2015-2017 than 2007 across all cytology result groups while prevalence of other high-risk HPV types was mainly stable, supporting vaccine impact with no evidence of type replacement.

Phytoconstituents of traditional Himalayan Herbs as potential inhibitors of Human Papillomavirus (HPV-18) for cervical cancer treatment: An In silico Approach.

Human papillomavirus (HPV) induced cervical cancer is becoming a major cause of mortality in women. The present research aimed to identify the natural inhibitors of HPV-18 E1 protein (1R9W) from Himalayan herbs with lesser toxicity and higher potency. In this study, one hundred nineteen phytoconstituents of twenty important traditional medicinal plants of Northwest Himalayas were selected for molecular docking with the target protein 1R9W of HPV-18 E1 Molecular docking was performed by AutoDock vina software. ADME/T screening of the bioactive phytoconstituents was done by SwissADME, admetSAR, and Protox II. A couple of best protein-ligand complexes were selected for 100 ns MD simulation. Molecular docking results revealed that among all the selected phytoconstituents only thirty-five phytoconstituents showed the binding affinity similar or more than the standard anti-cancer drugs viz. imiquimod (-6.1 kJ/mol) and podofilox (-6.9 kJ/mol). Among all the selected thirty-five phytoconstituents, eriodictyol-7-glucuronide, stigmasterol, clicoemodin and thalirugidine showed the best interactions with a docking score of -9.1, -8.7, -8.4, and -8.4 kJ/mol. Based on the ADME screening, only two phytoconstituents namely stigmasterol and clicoemodin selected as the best inhibitor of HPV protein. MD simulation study also revealed that stigmasterol and clicoemodin were stable inside the binding pocket of 1R9W, Stigmasterol and clicoemodin can be used as a potential investigational drug to cure HPV infections.

Immunogenicity and safety of the human papillomavirus vaccine in young survivors of cancer in the USA: a single-arm, open-label, phase 2, non-inferiority trial.

BACKGROUND: Young survivors of cancer are at increased risk for cancers that are related to human papillomavirus (HPV), primarily caused by oncogenic HPV types 16 and 18. We aimed to examine the immunogenicity and safety of the three-dose series of HPV vaccine in young survivors of cancer. METHODS: We conducted an investigator-initiated, phase 2, single-arm, open-label, non-inferiority trial at five National Cancer Institute-designated comprehensive cancer centres in the USA. Eligible participants were survivors of cancer who were HPV vaccine-naive, were aged 9-26 years, in remission, and had completed cancer therapy between 1 and 5 years previously. Participants received three intramuscular doses of either quadrivalent HPV vaccine (HPV4; enrolments on or before March 1, 2016) or nonavalent HPV vaccine (HPV9; enrolments after March 1, 2016) over 6 months (on day 1, at month 2, and at month 6). We also obtained data from published clinical trials assessing safety and immunogenicity of HPV4 and HPV9 in 9-26-year-olds from the general population, as a comparator group. The primary endpoint was antibody response against HPV types 16 and 18 at month 7 in the per-protocol population. A response was deemed non-inferior if the lower bound of the multiplicity-adjusted 95% CI was greater than 0·5 for the ratio of anti-HPV-16 and anti-HPV-18 geometric mean titres (GMTs) in survivors of cancer versus the general population. Responses were examined separately in male and female participants by age group (ie, 9-15 years and 16-26 years). Safety was assessed in all participants who received at least one vaccine dose and for whom safety data were available. This study is registered with ClinicalTrials.gov, NCT01492582. This trial is now completed. FINDINGS: Between Feb 18, 2013, and June 22, 2018, we enrolled 453 survivors of cancer, of whom 436 received one or more vaccine doses: 203 (47%) participants had survived leukaemia, 185 (42%) were female, and 280 (64%) were non-Hispanic white. Mean age at first dose was 15·6 years (SD 4·6). 378 (83%) of 453 participants had evaluable immunogenicity data; main reasons for exclusion from per-protocol analysis were to loss to follow-up, patient reasons, and medical reasons. Data were also obtained from 26 486 general population controls. The ratio of mean GMT for anti-HPV types 16 and 18 in survivors of cancer versus the general population was more than 1 for all subgroups (ie, aged 9-15 years, aged 16-26 years, male, and female groups) in both vaccine cohorts (ranging from 1·64 [95% CI 1·12-2·18] for anti-HPV type 16 in female participants aged 9-15 years who received HPV9, to 4·77 [2·48-7·18] for anti-HPV type 18 in male participants aged 16-26 years who received HPV4). Non-inferiority criteria were met within each age and sex subgroup, except against HPV type 18 in female participants aged 16-26 years receiving HPV9 (4·30 [0·00-9·05]). Adverse events were reported by 237 (54%) of 435 participants; injection site pain was most common (174 [40%] participants). One serious adverse event (ie, erythema nodosum) was possibly related to vaccine (HPV9; 16-26 year female cohort). INTERPRETATION: Immunogenicity and safety of HPV vaccine three-dose series in survivors of cancer is similar to that in the general population, providing evidence for use in this clinically vulnerable population. FUNDING: US National Cancer Institute, Merck, Sharp & Dohme, and American Lebanese Syrian Associated Charities.

The Effect of Indian Fig Fruit Extract on Human Papilloma Virus containing Cervical Cancer Cells (HeLa) by Decreasing the HPV18 L1 Gene Load.

BACKGROUND: Global trend is moving towards the use of natural phytochemicals to fight against pathogens. Human cervical cancer is directly associated with onco-potent type of Human Papilloma Virus (HPV). There is no known medicine for clearance of HPV type whose persistence is the cause of occurrence and re-occurrence of cervical cancer. The different species of fig fruit and their latex are reported to have HPV associated genital warts clearance capability. METHODS: In the current investigation, the effect of the methanol extract of Ficus benghalensis L. fruits on HPV type18 viral load in HeLa cell line was tested by doing PCR using HPV L1 primers (MY09/My011) and the cytotoxicity was also analysed by MTT assay. The induction of apoptotic activity in terms of DNA fragmentation and hyper-chromic effects of DNA was analysed. RESULTS: The PCR results showed a reduction in the HPV18 DNA and also the treatment exhibited a promising cytotoxicity with IC50 value at 211.86 µg/ml. The DNA samples from treated HeLa cells showed DNA shearing and laddering as a mark of apoptotic DNA fragmentation (Fig. 2) and the UV absorbance value at 260 nm was found to be significantly (p <0.01) higher in the DNA sample treated with fruit extract compared to the untreated DNA sample. CONCLUSION: The Ficus benghalensis L. fruit extract reduced the HPV viral load in HPV18 containing HeLa cells and showed an effective cytotoxicity on HeLa cell line. It also could induce the apoptotic activity in HeLa cell line and this study results suggest that the Ficus benghalensis L. fruits can be used to fight against cervical carcinoma, acting on HPV load.

Oncogenic HPV promotes the expression of the long noncoding RNA lnc-FANCI-2 through E7 and YY1.

Long noncoding RNAs (lncRNAs) play diverse roles in biological processes, but their expression profiles and functions in cervical carcinogenesis remain unknown. By RNA-sequencing (RNA-seq) analyses of 18 clinical specimens and selective validation by RT-qPCR analyses of 72 clinical samples, we provide evidence that, relative to normal cervical tissues, 194 lncRNAs are differentially regulated in high-risk (HR)-HPV infection along with cervical lesion progression. One such lncRNA, lnc-FANCI-2, is extensively characterized because it is expressed from a genomic locus adjacent to the FANCI gene encoding an important DNA repair factor. Both genes are up-regulated in HPV lesions and in in vitro model systems of HR-HPV18 infection. We observe a moderate reciprocal regulation of lnc-FANCI-2 and FANCI in cervical cancer CaSki cells. In these cells, lnc-FANCI-2 is transcribed from two alternative promoters, alternatively spliced, and polyadenylated at one of two alternative poly(A) sites. About 10 copies of lnc-FANCI-2 per cell are detected preferentially in the cytoplasm. Mechanistically, HR-HPVs, but not low-risk (LR)-HPV oncogenes induce lnc-FANCI-2 in primary and immortalized human keratinocytes. The induction is mediated primarily by E7, and to a lesser extent by E6, mostly independent of p53/E6AP and pRb/E2F. We show that YY1 interacts with an E7 CR3 core motif and transactivates the promoter of lnc-FANCI-2 by binding to two critical YY1-binding motifs. Moreover, HPV18 increases YY1 expression by reducing miR-29a, which targets the 3' untranslated region of YY1 mRNA. These data have provided insights into the mechanisms of how HR-HPV infections contribute to cervical carcinogenesis.

Lignan enriched fraction (LRF) of Phyllanthus amarus promotes apoptotic cell death in human cervical cancer cells in vitro.

Phyllanthus amarus is widely grown in this sub-continent and used traditionally to treat many common ailments. In the present study, lignan rich fraction of P. amarus extract was used on cervical cancer cell lines (HeLa, SiHa and C33A) to study it's mechanism of cell death induction. As the cells were treated with IC50 doses of LRF, characteristic apoptotic features were observed. Increased sub G0 population were observed both in Hela and C33 cells, while G1/S arrest was observed in SiHa cells than their untreated counterparts. Increased production of ROS and change in MMP were also detected in the treated cells. Presence of γH2AX, was observed by immunofluorescence. Reduced expression of HPV (16/18) as well as ET-1, an autocrine growth substance, were observed in the treated cells. Immunoblotting as well as ICFC studies showed enhanced expressions of BAX, Caspase 3 and PARP (cleaved) in the treated cells. A major lignan, phyllanthin was isolated from the chloroform fraction and showed strong irreversible affinities for viral E6 and MDM2 in in silico analysis. The study conclusively indicates that LRF has the potential to induce apoptotic cell death in cervical cancer cells by activation of p53 and p21 against DNA damage.

Treatment with Radix Euphorbiae Ebracteolatae Significantly Decreases the Expression of E6 and L1, and Increases the Expression of p53 and Rb in HPV18-infected Human Foreskin Keratinocytes.

BACKGROUND: Radix Euphorbiae Ebracteolatae (REE) was recently reported to be significantly superior to vitamin A acid ointment in treating multiple plantar warts. However, the effects of REE on HPV18 remain unclear. Therefore, the current study aimed to investigate the effects of REE on the proliferation of HPV18, and explore possible molecular mechanisms underlying the effects. METHODS: HFK and HFK-HPV18 were treated with water-extracted single or compound REE, ethanol-extracted single or compound REE, TNF-α and IFN for 3 days, respectively. In addition, the organotypic rafts containing HFK-HPV18 and HFK were treated with REE, IFN and TNF-α for 7 days, respectively. Cell proliferation rates were measured with Brdu. mRNA expression of E6, L1, p53 and Rb was detected by qPCR. Protein expression of p53, Rb and L1 was detected by Western blot. RESULTS: Compared to HFK group, HFK-HPV18 group had significantly higher expression of E6 and L1. Compared to the control group, HFK-HPV18 treated with REE, TNF-α and IFN displayed significantly lower proliferation rates. The mRNA expression of E6 was markedly lower, and mRNA expression of p53 and Rb was significantly higher after treatment of REE in HFK-HPV18 or in organotypic rafts containing HFK-HPV18. Treatment with REE markedly increased the protein expression of p53 and Rb, and decreased the protein expression of L1 in HFK-HPV18 or in organotypic rafts containing HFK-HPV18. Among all formula of REE, the inhibition of proliferation rates and expression of E6 and L1, and the increase in expression of p53 and Rb in HFK-HPV18 was highest in ethanol-extracted compound REE group. CONCLUSIONS: The proliferation rates are significantly lower in HFK-HPV18 treated with REE. The expression of E6 and L1 is markedly lower, and expression of p53 and Rb is significantly higher after REE treatment in HFK-HPV18 or organotypic rafts containing HFK-HPV18. Among all formula of REE, ethanol-extracted compound REE displays the highest protection against HPV18.

Long-distance communication: Looping of human papillomavirus genomes regulates expression of viral oncogenes.

High-risk human papillomaviruses (HPVs) are a major cause of cancers. HPVs infect epithelial cells, and viral oncogenes disrupt several cellular processes, including cell division, differentiation, and apoptosis. Expression of these oncogenes is relatively low in undifferentiated epithelial cells but increases in differentiating cells by unknown mechanisms. In a new study, Parish and colleagues unveil how two cellular proteins, CCCTC-binding factor (CTCF) and Yin Yang 1 (YY1), mediate looping of the HPV18 genome, which regulates expression of viral oncogenes in both dividing and differentiating epithelial cells.

[New strategies for the prevention and control of cervical cancer in Chile.] / Nuevas estrategias de prevención y control de cáncer de cuello uterino en Chile.

OBJECTIVE: To discuss cervical cancer (CC), Human PapillomaVirus (HPV),CC control program and propose alternatives for Chile. MATERIALS AND METHODS: We analyzed the national program of CC 1966-2015 and the clinical CC guideline 2015-2020;HPV prevalence in women and in cases of CC; HPV infection and serology; the self-vaginal sample; the accuracy and cost-effectiveness of screening with HPV versus Papanicolaou,and triage options among HPV-AR positives. RESULTS: 600 women die of CC each year in Chile, mainly from low resources. Papanicolaou coverage is <70%; Papanicolaou sensitivity is much lowerthan HPV test.Change from Papanicolaou to HPV test is cost-effective. Since 2015, girls under 13 have been vaccinated against HPV. CONCLUSIONS: .There are the technical and economic conditions for a substantial improvement of CC in Chile: replacement of the Papanicolaou by HPV; screening every five years, with the option of self-sampling, and triage based on HPV 16/18 or Papanicolaou typing.

OBJETIVO: Discutir el cáncer cervicouterino (CC), el virus del papiloma humano (VPH),el programa de control del CC y proponer alternativas para Chile. MATERIAL Y MÉTODOS: Se analiza el programa nacional del CC 1966-2015 y la guía clínica 2015-2020, la prevalencia deVPH en mujeres y en casos de CC; la infección y serología deVPH; la autotoma; la precisión y rentabilidad del tamizaje con VPH contra el Papanicolaou y las opciones de triaje enVPH AR positivas. RESULTADOS: En Chile mueren 600 mujeres (principalmente de bajos recursos) al año por CC. La cobertura del Papanicolaou es <70%, sensibilidad muy inferior al test de VPH, por lo que el cambio esrentable.Desde 2015 se vacuna contraVPH a niñas menores de 13 años. CONCLUSIONES: Las condiciones técnicas y económicas existen en Chile para lograr una mejoría sustancial del CC:se sugiere el reemplazo del Papanicolaou por el examen deVPH;tamizaje cada cinco años con opción de autotoma; triaje con base en la tipificación deVPH 16/18 o Papanicolaou.

Identification of several high-risk HPV inhibitors and drug targets with a novel high-throughput screening assay.

Human papillomaviruses (HPVs) are oncogenic viruses that cause numerous different cancers as well as benign lesions in the epithelia. To date, there is no effective cure for an ongoing HPV infection. Here, we describe the generation process of a platform for the development of anti-HPV drugs. This system consists of engineered full-length HPV genomes that express reporter genes for evaluation of the viral copy number in all three HPV replication stages. We demonstrate the usefulness of this system by conducting high-throughput screens to identify novel high-risk HPV-specific inhibitors. At least five of the inhibitors block the function of Tdp1 and PARP1, which have been identified as essential cellular proteins for HPV replication and promising candidates for the development of antivirals against HPV and possibly against HPV-related cancers.

High sensitivity, loop-mediated isothermal amplification combined with colorimetric gold-nanoparticle probes for visual detection of high risk human papillomavirus genotypes 16 and 18.

High-risk human papillomavirus (HR-HPV) causes cervical cancer. HPV16 and HPV18 are the most prevalent strains of the virus reported in women worldwide. Loop-mediated isothermal amplification (LAMP) is an alternative method for DNA detection under isothermal conditions. However, it results in a turbid amplified product which is not easily detected by the naked eye. This study aimed to develop an improved technique by using gold nanoparticles (AuNPs) attached to a single-stranded DNA probe for the detection of HPV16 and HPV18. Detection of the LAMP product by AuNP color change was compared with detection by visual turbidity. The optimal conditions for this new LAMP-AuNP assay were an incubation time of 20min and a temperature of 65°C. After LAMP amplification was complete, its products were hybridized with the AuNP probe for 5min and then detected by the addition of magnesium salt. The color changed from red to blue as a result of aggregation of the AuNP probe under high ionic strength conditions produced by the addition of the salt. The sensitivity of the LAMP-AuNP assay was greater than the LAMP turbidity assay by up to 10-fold for both HPV genotypes. The LAMP-AuNP assay showed higher sensitivity and ease of visualization than did the LAMP turbidity for the detection of HPV16 and HPV18. Additionally, AuNP-HPV16 and AuNP-HPV18 probes were stable for over 1year. The combination of LAMP and the AuNP-probe colorimetric assay offers a simple, rapid and highly sensitive alternative diagnostic tool for the detection of HPV16 and HPV18 in district hospitals or field studies.

Hyperthermia Selectively Targets Human Papillomavirus in Cervical Tumors via p53-Dependent Apoptosis.

Human papillomavirus (HPV) is associated with cervical cancer, the third most common cancer in women. The high-risk HPV types 16 and 18 are found in over 70% of cervical cancers and produce the oncoprotein, early protein 6 (E6), which binds to p53 and mediates its ubiquitination and degradation. Targeting E6 has been shown to be a promising treatment option to eliminate HPV-positive tumor cells. In addition, combined hyperthermia with radiation is a very effective treatment strategy for cervical cancer. In this study, we examined the effect of hyperthermia on HPV-positive cells using cervical cancer cell lines infected with HPV 16 and 18, in vivo tumor models, and ex vivo-treated patient biopsies. Strikingly, we demonstrate that a clinically relevant hyperthermia temperature of 42 °C for 1 hour resulted in E6 degradation, thereby preventing the formation of the E6-p53 complex and enabling p53-dependent apoptosis and G2-phase arrest. Moreover, hyperthermia combined with p53 depletion restored both the cell-cycle distribution and apoptosis to control levels. Collectively, our findings provide new insights into the treatment of HPV-positive cervical cancer and suggest that hyperthermia therapy could improve patient outcomes.

Non-clinical safety assessment of single and repeated intramuscular administration of a human papillomavirus-16/18 vaccine in rabbits and rats.

The human papillomavirus (HPV)-16/18 vaccine (Cervarix®) is a prophylactic vaccine for the prevention of cervical cancer. The vaccine contains recombinant virus-like particles assembled from the L1 major capsid proteins of the cervical cancer-causing viral types HPV-16 and HPV-18, and Adjuvant System 04 (AS04), which contains the immunostimulant MPL and aluminium salt. To evaluate potential local and systemic toxic effects of the HPV-16/18 vaccine or AS04 alone, three repeated-dose studies were performed in rabbits and rats. One rabbit study also included a single-dose evaluation. In rabbits (~2.5 kg), the full human dose (HD) of the vaccine was evaluated (0.5 ml per injection site), and in rats (~250 g), 1/5 HD of vaccine was evaluated, corresponding to ≥ 12 times the dosage in humans relative to body weight. In both animal models, the treatment-related changes included a slight transient increase in the number of circulating neutrophils as well as a local inflammatory reaction at the injection site. These treatment-related changes were less pronounced after four doses of AS04 alone than after four doses of the HPV-16/18 vaccine. Additional treatment-related changes in the rat included lower albumin/globulin ratios and microscopic signs of inflammation in the popliteal lymph nodes. In both animal models, 13 weeks after the fourth dose, recovery was nearly complete, although at the injection site in some animals there were signs of discoloration, muscle-fibre regeneration and focal points of macrophage infiltration. Therefore, in these non-clinical models, the single and repeated dose administrations of the HPV-16/18 vaccine or AS04 alone were safe and well tolerated.

Honeybee venom possesses anticancer and antiviral effects by differential inhibition of HPV E6 and E7 expression on cervical cancer cell line.

Bee venom (BV) therapy is a type of alternative medical treatment used to treat various diseases in oriental medicine. The mechanisms underlying the effects of BV remain poorly understood. In the present study, we evaluated the antiviral effect of BV on cervical carcinoma cell lines (CaSki, HeLa, C33A and TC-1). BV treatments resulted in a more significant suppression of cell growth in HPV 16-infected cells (CaSki) and a lesser suppression in HPV 18-infected cells (HeLa). However, less suppression was observed in HPV-negative C33A cells. In 10 µg/ml BV-treated CaSki cells, the mRNA expression and protein levels of HPV16 E6 and E7 were significantly decreased by BV, while HPV18 E6 and E7 mRNA expression levels were not significantly altered by 10 µg/ml BV-treated HeLa cells. The antitumor effects of BV were in accordance with in vitro data, in restricting tumor growth in vivo and were much more effective on the suppression of tumor growth. Furthermore, the mRNA and protein expression levels of HPV16 E6 and E7 were decreased by BV in TC-1 tumors. These findings demonstrated the antiviral effects of BV in HPV-infected cervical cancer cells and the anticancer effects of BV in HPV16 E6/E7-expressed TC-1 tumors. Collectively, BV plays a differential role in suppressing HPV16-infected cells (CaSki cells) and HPV18-infected cells (HeLa cells) by the downregulation of E6/E7 protein of HPV16/18.

Early Detection of Autism (ASD) by a Non-invasive Quick Measurement of Markedly Reduced Acetylcholine & DHEA and Increased ß-Amyloid (1-42), Asbestos (Chrysotile), Titanium Dioxide, Al, Hg & often Coexisting Virus Infections (CMV, HPV 16 and 18), Bacterial Infections etc. in the Brain and Corresponding Safe Individualized Effective Treatment.

A brief historical background on Autism & some of the important symptoms associated with Autism are summarized. Using strong Electro Magnetic Field Resonance Phenomenon between 2 identical molecules with identical weight (which received U.S. Patent) non-invasively & rapidly we can detect various molecules including neurotransmitters, bacteria, virus, fungus, metals & abnormal molecules. Simple non- invasive measurement of various molecules through pupils & head of diagnosed or suspected Autism patients indicated that in Autism patients following changes were often found: 1) Acetylcholine is markedly reduced; 2) Alzheimer's disease markers (i.e. ß-Amyloid (1-42), Tau Protein, Apolipoprotein (Apo E4)) are markedly increased; 3) Chrysotile Asbestos is increased; 4) Titanium Dioxide (TiO2) is moderately increased; 5) Al is moderately increased; 6) Hg is moderately increased; 7) Dopamine, Serotonin & GABA are significantly reduced (up to about 1/10 of normal); 8) Often viral infections (such as CMV, HHV-6, HPV-16, HPV-18, etc.), and Bacterial infections (such as Chlamydia trachomatis, Mycobacterium TB, Borrelia Burgdorferi, etc.) coexist. Research by others on Autism spectrum disorder (ASD) shows that it is a group of complex neurodevelopmental disorders, with about 70% of ASD patients also suffering from gastro-intestinal problems. While Alzheimer disease (AD) is characterized by formation of 1) Amyloid plaques, 2) Neurofibrillary tangles inside of neurons, and 3) Loss of connections between neurons. More than 90% of AD develops in people over the age of 65. These 3 characteristics often progressively worsen over time. Although Autism Spectrum Disorder and Alzheimer's disease are completely different diseases they have some similar biochemical changes. Eight examples of such measurement & analysis are shown for comparison. Most of Autism patients improved significantly by removing the source or preventing intake of Asbestos, TiO2, Al & Hg or enhancing urinary output of above abnormal substances & coexisting infections, if treatment is given early. When HPV-16 & HPV-18 coexist, at triangular central area of the top of head, in addition to inability to talk, severe neuromuscular problems of lower extremity were found to also exist. However, if treatment is given 3-4 years after onset of Autism symptoms, even when successful biochemical reduction of above abnormal substances occurs, clinical improvement is less significant, since permanent damage in brain tissue seems to already exist. Therefore, early diagnosis & early treatment is very important for both Autism & Alzheimer's disease. In addition the optimal doses of Vitamin D3 and Taurine may play an important role in the future treatment of Autism, Alzheimer's Disease and memory disturbances by significantly increasing Acetylcholine and DHEA levels, enhancing the excretion of toxic substances in the urine, as well as having an anticancer effect.

Inhibitory activity of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (higher Basidiomycetes) on transformed cells by human papillomavirus.

In this study, we investigated the effects of the aqueous extracts of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum, obtained from three localities (China; and Morelos and Michoacan, Mexico) on cervical cells transformed by human papillomavirus (HeLa and SiHa) and C-33A cancer cells. The cells were plated in DMEM medium supplemented, and were incubated in the presence of different concentrations of G. lucidum for 24 h. Cell proliferation was determined by MTT colorimetric assay and viability by trypan blue assay. Inhibitory dose was determined (IC50) of the three different extracts of G. lucidum in the culture cell lines mentioned above. The apoptosis process was confirmed by nuclear DNA fragmentation and the cell cycle was determined by flow cytometry. The results showed that aqueous extracts G. lucidum obtained from three localities produced inhibition in the proliferation of VPH transformed cells; they also induced apoptosis and cell cycle arrest in HeLa, SiHa, and C-33A cancer cells. Therefore, it was found that aqueous extracts G. lucidum obtained from three different locations produced inhibitory effect on cancer cells and may have a potential therapeutic use for the prevention and treatment of this disease.