Risk factors associated with oral Human Papillomavirus (HPV) prevalence within a young adult population.

BACKGROUND: The prevalence of, and risk factors for, genital Human Papillomavirus (HPV) infections within the young adult population are well-established; the same is not known for oral HPV. This observational study aimed to determine oral HPV prevalence and abundance within a UK young adult population, and examine if sexual practices and established risk factors of oropharyngeal squamous cell carcinomas (OPSCCs) (such as smoking and alcohol consumption) influenced HPV prevalence. METHODS: Convenience sampling was used to recruit a small sample of 452 UK-based young adults studying at a higher education (HE) institution to the study; the study was not powered. A highly sensitive real-time PCR HPV screening method was developed for the detection of multiple HPV subtypes from oral swabs. HPV-positive samples were subsequently screened by qPCR for viral subtypes HPV-6, HPV-11, HPV-16, HPV-18. Results were analysed by univariate and multivariate methods and stratified for gender, with lifestyle behaviour data collected via questionnaire. Socio-economic status was not captured within the questionnaire. RESULTS: We found a high oral HPV prevalence of 22.79%, with a dominance of high-risk viral type HPV-16 (prevalence 19.12%; abundance average 1.08 × 105 copies/million cells) detected within healthy young adults. Frequent smoking (p = .05), masturbation (p = .029), and engagement in multiple sexual activities (p = .057), were found to be associated with oral HPV prevalence, and HPV-16 prevalence, whilst behaviours traditionally associated with genital HPV were not. CONCLUSIONS: Our results strengthen the link between sexual practices and oral HPV transmission. We suggest that young adults should be considered high-risk for the contraction of oral HPV, although acknowledge that this sample of HE students may not be representative of the wider population. We show that high-risk HPV-16 is prevalent in the healthy population, as well as dominating within OPSCC; this study is one of the first to determine the dominance of oral HPV-16 prevalence and abundance within this population, presenting a clear need for greater awareness of oral HPV infections, and the risk factors for HPV-positive OPSCC within young adults.

DUSP10 Negatively Regulates the Inflammatory Response to Rhinovirus through Interleukin-1ß Signaling.

Rhinoviral infection is a common trigger of the excessive inflammation observed during exacerbations of asthma and chronic obstructive pulmonary disease. Rhinovirus (RV) recognition by pattern recognition receptors activates the mitogen-activated protein kinase (MAPK) pathways, which are common inducers of inflammatory gene production. A family of dual-specificity phosphatases (DUSPs) can regulate MAPK function, but their roles in rhinoviral infection are not known. We hypothesized that DUSPs would negatively regulate the inflammatory response to RV infection. Our results revealed that the p38 and c-Jun N-terminal kinase (JNK) MAPKs play key roles in the inflammatory response of epithelial cells to RV infection. Three DUSPs previously shown to have roles in innate immunity (DUSPs 1, 4, and 10) were expressed in primary bronchial epithelial cells, and one of them, DUSP10, was downregulated by RV infection. Small interfering RNA-mediated knockdown of DUSP10 identified a role for the protein in negatively regulating inflammatory cytokine production in response to interleukin-1ß (IL-1ß), alone and in combination with RV, without any effect on RV replication. This study identifies DUSP10 as an important regulator of airway inflammation in respiratory viral infection.IMPORTANCE Rhinoviruses are one of the causes of the common cold. In patients with asthma or chronic obstructive pulmonary disease, viral infections, including those with rhinovirus, are the commonest cause of exacerbations. Novel therapeutics to limit viral inflammation are clearly required. The work presented here identifies DUSP10 as an important protein involved in limiting the inflammatory response in the airway without affecting immune control of the virus.

The role of protein kinase A regulation of the E6 PDZ-binding domain during the differentiation-dependent life cycle of human papillomavirus type 18.

Human papillomavirus (HPV) E6 proteins of high-risk alpha types target a select group of PSD95/DLG1/ZO1 (PDZ) domain-containing proteins by using a C-terminal PDZ-binding motif (PBM), an interaction that can be negatively regulated by phosphorylation of the E6 PBM by protein kinase A (PKA). Here, we have mutated the canonical PKA recognition motif that partially overlaps with the E6 PBM in the HPV18 genome (E6153PKA) and compared the effect of this mutation on the HPVl8 life cycle in primary keratinocytes with the wild-type genome and with a second mutant genome that lacks the E6 PBM (E6ΔPDZ). Loss of PKA recognition of E6 was associated with increased growth of the genome-containing cells relative to cells carrying the wild-type genome, and upon stratification, a more hyperplastic phenotype, with an increase in the number of S-phase competent cells in the upper suprabasal layers, while the opposite was seen with the E6ΔPDZ genome. Moreover, the growth of wild-type genome-containing cells was sensitive to changes in PKA activity, and these changes were associated with increased phosphorylation of the E6 PBM. In marked contrast to E6ΔPDZ genomes, the E6153PKA mutation exhibited no deleterious effects on viral genome amplification or expression of late proteins. Our data suggest that the E6 PBM function is differentially regulated by phosphorylation in the HPV18 life cycle. We speculate that perturbation of protein kinase signaling pathways could lead to changes in E6 PBM function, which in turn could have a bearing on tumor promotion and progression.

Caenorhabditis elegans, a model organism for investigating immunity.

The nematode Caenorhabditis elegans has been a powerful experimental organism for almost half a century. Over the past 10 years, researchers have begun to exploit the power of C. elegans to investigate the biology of a number of human pathogens. This work has uncovered mechanisms of host immunity and pathogen virulence that are analogous to those involved during pathogenesis in humans or other animal hosts, as well as novel immunity mechanisms which appear to be unique to the worm. More recently, these investigations have uncovered details of the natural pathogens of C. elegans, including the description of a novel intracellular microsporidian parasite as well as new nodaviruses, the first identification of viral infections of this nematode. In this review, we consider the application of C. elegans to human infectious disease research, as well as consider the nematode response to these natural pathogens.

Pellino-1 Regulates the Responses of the Airway to Viral Infection.

Exposure to respiratory pathogens is a leading cause of exacerbations of airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). Pellino-1 is an E3 ubiquitin ligase known to regulate virally-induced inflammation. We wished to determine the role of Pellino-1 in the host response to respiratory viruses in health and disease. Pellino-1 expression was examined in bronchial sections from patients with GOLD stage two COPD and healthy controls. Primary bronchial epithelial cells (PBECs) in which Pellino-1 expression had been knocked down were extracellularly challenged with the TLR3 agonist poly(I:C). C57BL/6 Peli1-/- mice and wild type littermates were subjected to intranasal infection with clinically-relevant respiratory viruses: rhinovirus (RV1B) and influenza A. We found that Pellino-1 is expressed in the airways of normal subjects and those with COPD, and that Pellino-1 regulates TLR3 signaling and responses to airways viruses. In particular we observed that knockout of Pellino-1 in the murine lung resulted in increased production of proinflammatory cytokines IL-6 and TNFα upon viral infection, accompanied by enhanced recruitment of immune cells to the airways, without any change in viral replication. Pellino-1 therefore regulates inflammatory airway responses without altering replication of respiratory viruses.

Airway Epithelial Cells Generate Pro-inflammatory Tenascin-C and Small Extracellular Vesicles in Response to TLR3 Stimuli and Rhinovirus Infection.

Viral infections are a common cause of asthma exacerbations, with human rhinoviruses (RV) the most common trigger. RV signals through a number of different receptors, including toll-like receptor (TLR)3. Tenascin-C (TN-C) is an immunomodulatory extracellular matrix protein present in high quantities in the airway of people with asthma, and expression is also upregulated in nasal lavage fluid in response to RV infection. Respiratory viral infection has been demonstrated to induce the release of small extracellular vesicles (sEV) such as exosomes, whilst exosomal cargo can also be modified in the bronchoalveolar lavage fluid of people with asthma. These sEVs may potentiate airway inflammation and regulate the immune response to infection. This study characterizes the relationship between RV infection of bronchial epithelial cells and the release of TN-C, and the release of sEVs following stimulation with the TLR3 agonist and synthetic viral mimic, poly(I:C), as well as the function of the released protein/vesicles. The BEAS-2B airway epithelial cell line and primary human bronchial epithelial cells (PBECs) from asthmatic and non-asthmatic donors were infected with RV or treated with poly(I:C). TN-C expression, release and localization to sEVs was quantified. TN-C expression was also assessed following intra-nasal challenge of C57BL/6 mice with poly(I:C). BEAS-2B cells and macrophages were subsequently challenged with TN-C, or with sEVs generated from BEAS-2B cells pre-treated with siRNA targeted to TN-C or control. The results revealed that poly(I:C) stimulation induced TN-C release in vivo, whilst both poly(I:C) stimulation and RV infection promoted release in vitro, with elevated TN-C release from PBECs obtained from people with asthma. Poly(I:C) also induced the release of TN-C-rich sEVs from BEAS-2B cells. TN-C, and sEVs from poly(I:C) challenged cells, induced cytokine synthesis in macrophages and BEAS-2B cells, whilst sEVs from control cells did not. Moreover, sEVs with ~75% reduced TN-C content did not alter the capacity of sEVs to induce inflammation. This study identifies two novel components of the inflammatory pathway that regulates the immune response following RV infection and TLR3 stimulation, highlighting TN-C release and pro-inflammatory sEVs in the airway as relevant to the biology of virally induced exacerbations of asthma.

Pellino-1 Regulates Immune Responses to Haemophilus influenzae in Models of Inflammatory Lung Disease.

Non-typeable Haemophilus influenzae (NTHi) is a frequent cause of lower respiratory tract infection in people with chronic obstructive pulmonary disease (COPD). Pellino proteins are a family of E3 ubiquitin ligases that are critical regulators of TLR signaling and inflammation. The aim of this study was to identify a role for Pellino-1 in airway defense against NTHi in the context of COPD. Pellino-1 is rapidly upregulated by LPS and NTHi in monocyte-derived macrophages (MDMs) isolated from individuals with COPD and healthy control subjects, in a TLR4 dependent manner. C57BL/6 Peli1-/- and wild-type (WT) mice were subjected to acute (single LPS challenge) or chronic (repeated LPS and elastase challenge) airway inflammation followed by NTHi infection. Both WT and Peli1-/- mice develop airway inflammation in acute and chronic airway inflammation models. Peli1-/- animals recruit significantly more neutrophils to the airway following NTHi infection which is associated with an increase in the neutrophil chemokine, KC, in bronchoalveolar lavage fluid as well as enhanced clearance of NTHi from the lung. These data suggest that therapeutic inhibition of Pellino-1 may augment immune responses in the airway and enhance bacterial clearance in individuals with COPD.

Mitotic control of human papillomavirus genome-containing cells is regulated by the function of the PDZ-binding motif of the E6 oncoprotein.

The function of a conserved PDS95/DLG1/ZO1 (PDZ) binding motif (E6 PBM) at the C-termini of E6 oncoproteins of high-risk human papillomavirus (HPV) types contributes to the development of HPV-associated malignancies. Here, using a primary human keratinocyte-based model of the high-risk HPV18 life cycle, we identify a novel link between the E6 PBM and mitotic stability. In cultures containing a mutant genome in which the E6 PBM was deleted there was an increase in the frequency of abnormal mitoses, including multinucleation, compared to cells harboring the wild type HPV18 genome. The loss of the E6 PBM was associated with a significant increase in the frequency of mitotic spindle defects associated with anaphase and telophase. Furthermore, cells carrying this mutant genome had increased chromosome segregation defects and they also exhibited greater levels of genomic instability, as shown by an elevated level of centromere-positive micronuclei. In wild type HPV18 genome-containing organotypic cultures, the majority of mitotic cells reside in the suprabasal layers, in keeping with the hyperplastic morphology of the structures. However, in mutant genome-containing structures a greater proportion of mitotic cells were retained in the basal layer, which were often of undefined polarity, thus correlating with their reduced thickness. We conclude that the ability of E6 to target cellular PDZ proteins plays a critical role in maintaining mitotic stability of HPV infected cells, ensuring stable episome persistence and vegetative amplification.

A two-gene balance regulates Salmonella typhimurium tolerance in the nematode Caenorhabditis elegans.

Lysozymes are antimicrobial enzymes that perform a critical role in resisting infection in a wide-range of eukaryotes. However, using the nematode Caenorhabditis elegans as a model host we now demonstrate that deletion of the protist type lysozyme LYS-7 renders animals susceptible to killing by the fatal fungal human pathogen Cryptococcus neoformans, but, remarkably, enhances tolerance to the enteric bacteria Salmonella Typhimurium. This trade-off in immunological susceptibility in C. elegans is further mediated by the reciprocal activity of lys-7 and the tyrosine kinase abl-1. Together this implies a greater complexity in C. elegans innate immune function than previously thought.