Frequent detection of high human papillomavirus DNA loads in oral potentially malignant disorders

Human papillomavirus (HPV) is estimated to be the cause of 40-80% of the squamous cell carcinoma of the oropharynx but only of a small fraction of the oral cavity cancers. The prevalence of oral HPV infection has significantly increased in the last decade, raising concerns about the HPV role in progression of oral potentially malignant disorders (OPMD) toward squamous cell carcinomas. We sought to study HPV infection in patients with oral lesions, and in control individuals, using non-invasive and site-specific oral brushing and sensitive molecular methods. HPV DNA positivity and viral loads were evaluated in relation to patient data and clinical diagnosis. We enrolled 116 individuals attending Dental Clinics: 62 patients with benign oral lesions (e.g. fibromas, papillomatosis, ulcers) or OPMD (e.g. lichen, leukoplakia) and 54 controls. Oral cells were collected with Cytobrush and HPV-DNA detected with quantitative real-time PCR (qPCR) for the more common high-risk (HR) and low-risk (LR) genotypes. HPV detection rate, percentage of HR HPVs and HPV-DNA loads (namely HPV16 and in particular, HPV18) were significantly higher in patients than in controls. Lichen planus cases had the highest HPV positive rate (75.0%), hairy leukoplakia the lowest (33.3%). This study detected unexpectedly high rates of HPV infection in cells of the oral mucosa. The elevated HR HPV loads found in OPMD suggest the effectiveness of qPCR in testing oral lesions. Prospective studies are needed to establish whether elevated viral loads represent a clinically useful marker of the risk of malignant progression

study on virological and immunological factors involved in determining the course of HPV anogenital infection

Infection by high-risk HPV types is necessary but not sufficient for progression to cancer. In fact, the majority of HPV infections do not lead to cytological anomalies or cancer but only a small percentage (10-15%) persist and promote the development of low and high-grade lesions. It is known that to persist HPV must escape the host immune system and persistence could be due to an impaired IFN-system activation in the infected individual. In literature, most studies were conducted in cell lines with constitutively oncogenes expression, whereas in vivo, the majority of HPV infections that resolve with time have a transient oncogene expression. To clarify the role of innate immune response, we analysed, for the first time, the expression of several TLRs (TLR2, TLR3, TLR4, TLR7 and TLR9), the IFN lambda pathways (INF-lambda 1, INF-lambda II, the IFN-lambda receptors), and the activation of several IFN induced genes (ISG15, MxA and p56) in HPV positive and negative cervical cells from 154 patients. The expression levels of TLR2, TLR3, TLR4 and TLR7 resulted highly variable both in HPV negative and in HPV positive patients. Differently, TLR9 was activated at different levels during HPV infection, with a tendency to be higher in LR-HPV infected women and significantly higher in patients with HPV persistent infection. Furthermore, we correlated TLRs expression with histological grade of lesions; TLR7 and TLR9 increased with the histopathological grade, but TLR9 in patients with persistent HPV infection was greater then in patients with CIN II or worse lesion, independently of lesions grade We hypothesize that the inability to clear HPV DNA after one year could be a risk factor to later develop high-grade lesions due to the persistence of the virus and the high levels of TLR9. IFN lambda 1 was expressed at higher level in patients with LR HPV infection compared to HR HPV and negative and we observed the same trend for IFN lambda 2/3. The elevated expression level of IL28R in patients with LR HPV infection respect to the other study groups confirmed the data obtained with IFN-lambda

study on virological and immunological factors involved in determining the course of HPV anogenital infection

Infection by high-risk HPV types is necessary but not sufficient for progression to cancer. In fact, the majority of HPV infections do not lead to cytological anomalies or cancer but only a small percentage (10-15%) persist and promote the development of low and high-grade lesions. It is known that to persist HPV must escape the host immune system and persistence could be due to an impaired IFN-system activation in the infected individual. In literature, most studies were conducted in cell lines with constitutively oncogenes expression, whereas in vivo, the majority of HPV infections that resolve with time have a transient oncogene expression. To clarify the role of innate immune response, we analysed, for the first time, the expression of several TLRs (TLR2, TLR3, TLR4, TLR7 and TLR9), the IFN lambda pathways (INF-lambda 1, INF-lambda II, the IFN-lambda receptors), and the activation of several IFN induced genes (ISG15, MxA and p56) in HPV positive and negative cervical cells from 154 patients. The expression levels of TLR2, TLR3, TLR4 and TLR7 resulted highly variable both in HPV negative and in HPV positive patients. Differently, TLR9 was activated at different levels during HPV infection, with a tendency to be higher in LR-HPV infected women and significantly higher in patients with HPV persistent infection. Furthermore, we correlated TLRs expression with histological grade of lesions; TLR7 and TLR9 increased with the histopathological grade, but TLR9 in patients with persistent HPV infection was greater then in patients with CIN II or worse lesion, independently of lesions grade We hypothesize that the inability to clear HPV DNA after one year could be a risk factor to later develop high-grade lesions due to the persistence of the virus and the high levels of TLR9. IFN lambda 1 was expressed at higher level in patients with LR HPV infection compared to HR HPV and negative and we observed the same trend for IFN lambda 2/3. The elevated expression level of IL28R in patients with LR HPV infection respect to the other study groups confirmed the data obtained with IFN-lambda

HPV16 E5 affects the KGFR/FGFR2b-mediated epithelial growth through alteration of the receptor expression, signaling and endocytic traffic

The E5 oncoprotein of the human papillomavirus type 16 (HPV16 E5) cooperates in cervical carcinogenesis and in epithelial transformation deregulating cell growth, survival and differentiation through the modulation of growth factor receptors. Among the epithelial receptor tyrosine kinases, the keratinocyte growth factor receptor/fibroblast growth factor receptor 2b (KGFR/FGFR2b) is a major paracrine mediator of epithelial homeostasis and appears to have an unique and unusual role in epithelial tissues, exerting a tumor-suppressive function in vitro and in vivo. With the aim to better elucidate the molecular events involved in the pathological activity of 16E5, we investigated if the viral protein would be able to affect the KGFR expression, signaling and turnover by interference with its degradative and recycling endocytic pathways. Quantitative reverse transcriptase-PCR and biochemical approaches on human keratinocytes transfected with 16E5-HA showed that E5 protein is able to induce KGFR down-modulation at both transcript and protein levels. Immunofluorescence microscopy in double-transfected cells expressing both E5 and KGFR revealed that the viral protein alters the receptor endocytic trafficking and triggers its endosomal sorting to the indirect juxtanuclear recycling pathway. The shift from lysosomal degradation to recycling at the plasma membrane correlates with a reduced phosphorylation of the fibroblast growth factor receptor substrate-2α tyrosine 196, the major docking site for Grb2-Cbl complexes responsible for receptor ubiquitination and degradation. 5'-Bromo-deoxyuridine incorporation assay demonstrated that expression of 16E5 induces a decrease in the growth response to the receptor ligands as a consequence of KGFR down-modulation, suggesting that 16E5 might have a role on HPV infection in perturbing the KGFR-mediated physiological behavior of confluent keratinocytes committed to differentiation

Ocular side effects in chronic myeloid leukemia patients treated with imatinib.

Imatinib mesylate is a selective inhibitor of the bcr/abl, c-kit and PDGF receptor tyrosine kinases. Its ocular toxicity is little known with mild periorbital oedema being the most commonly reported side effect. We here describe our experience on ocular complications in imatinib treated Ph+ CML patients, which consisted of a wide spectrum of adverse effects ranging from periobital oedema to serious adverse events such as glaucoma

Interferon lambda 1 expression in cervical cells differs between low-risk and high-risk human papillomavirus-positive women

Persistent infection by high-risk (HR) human papillomavirus (HPV) types is a prerequisite for progression to cancer. HR-HPVs may lead to a deregulation of innate immunity by interfering with the epithelial type I interferon (IFN) response, whereas very little is known about type III IFNs, a key component of the mucosal antiviral response. This study reports a first attempt to evaluate the activation of type III IFN genes (IFN lambda 1-3), IFN lambda receptor genes (IFN-lambdaR1 and IL10R2), and IFN-induced genes (MxA, ISG15, ISG56) in HPV-positive and HPV-negative cervical cells from 154 women attending the gynecological unit of a university hospital in Rome. Despite an increased individual variability, a coordinated expression of several IFN lambda-related genes was observed. Furthermore, IFN lambda 1 and IFN-lambdaR1 genes were expressed at higher levels in cervical cells positive to low-risk (LR) HPV compared to HR-HPV and HPV-negative cells. Consistently, ISG15 expression was significantly higher in LR-HPV-infected women than in the other groups. Moreover, IFN lambda 1 expression decreased significantly with abnormal cytological results. This study is the first to show the activation of a type III IFN response in LR-HPV-positive cervical cells and suggests that the lack of this response in HR-HPV infection may be related to lesion progression

Insights into the role of innate immunity in cervicovaginal papillomavirus infection from studies using gene deficient mice

We demonstrate that female C57BL/6J mice are susceptible to a transient lower genital tract infection with MmuPV1 mouse papillomavirus and display focal histopathological abnormalities resembling human papillomavirus infection. We took advantage of strains of genetically deficient mice to study in vivo the role of innate immune signaling in control of papillomavirus. At 4 months, we sacrificed MmuPV1-infected mice and measured viral 757/3139 spliced transcripts by TaqMan RT-PCR, localization of infection by RNAscope in situ hybridization, and histopathological abnormities by H&E stain. Among mice deficient in receptors for pathogen-associated molecular patterns, MyD88-/- and STING-/- mice had 1350 and 80 copies of spliced transcripts/μg RNA, respectively, while no viral expression was detected in MAVS-/- and Ripk2-/- mice. Mice deficient in an adaptor molecule, STAT1-/-, for interferon signaling had 46,000 copies/μg RNA. Among mice with targeted deficiencies in the inflammatory response, IL-1R-/- and caspase-1-/- mice had 350 and 30 copies/μg RNA, respectively. Among mice deficient in chemokine receptors, CCR6-/- mice had 120 copies/μg RNA, while CXCR2-/- and CXCR3-/- mice were negative. RNAscope confirmed focal infection in MyD88-/-, STAT1-/- and CCR6-/- mice but was negative for other gene deficient mice. Histological abnormalities were seen only in the latter mice. Our findings and the literature support a working model of innate immunity to papillomaviruses involving activation of a MyD88 dependent pathway and IL-1 receptor signaling, control of viral replication by interferon-stimulated genes, and clearance of virus-transformed dysplastic cells by action of the CCR6/CCL20 axis.IMPORTANCE Papillomaviruses infect stratified squamous epithelia and the viral life cycle is linked to epithelial differentiation. Additionally, changes occur in viral and host gene expression and immune cells are activated to modulate the infectious process. In vitro studies with keratinocytes cannot fully model the complex viral and host response and do not reflect the contribution of local and migrating immune cells. We show that female C57BL/6J mice are susceptible to a transient papillomavirus cervicovaginal infection, and mice deficient in select genes involved in innate immune responses are susceptible to persistent infection with variable manifestations of histopathological abnormalities. The results of our studies support a working model of innate immunity to papillomaviruses, and the model provides a framework for more in depth studies. A better understanding of mechanisms of early viral clearance and development of approaches to induce clearance will be important for cancer prevention and treatment of HPV-related diseases