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The rising incidence of oropharyngeal cancer caused by high-risk Human Papillomavirus (HPV) type 16 and HPV18 in the U.S and other developed countries is an important public health issue. This has been attributed to changes in sexual behavior, including the practice of oral sex, which may expose individuals to increased risk of acquiring oral HPV infection. The incidence of oral HPV infections highlights the role of the oral cavity as an important anatomical site in the acquisition and transmission of high-risk HPVs. Generally, the use of mouthwash/oral rinses have focused on targeting the oral bacteriome, and could additionally be formulated for managing the oral virome. Here, we examined virucidal properties of common over-the-counter antibacterial mouthwash products against native HPV16 and HPV18 virion in vitro, and downstream modification of virus infectivity. We tested oral rinses containing essential oils/alcohol, hydrogen peroxide, and cetylpyridinium chloride. Our results demonstrated greater than 90% efficacy against HPV16 inactivation, but comparatively with less efficacy against HPV18. Overall, hydrogen peroxide containing oral rinses demonstrated the best efficacy against both high-risk types, albeit with lower efficacy against HPV18. Prophylactic virucidal oral rinses targeted towards high-risk HPVs could be beneficial in reducing incidental oral HPV load, prevalence, and persistent infections.

Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.

The cleavage of viral surface proteins by furin is associated with some viruses’ high virulence and infectivity. The human papillomavirus (HPV) requires the proteolytic processing of its capsid proteins for activation before entry. Variability in reactivity with furin and other proprotein convertases (PCs) among HPV types was investigated. HPV16, the most prevalent and carcinogenic HPV type, reacted with PCs with the broadest selectivity compared to other types in reactions of pseudoviral particles with the recombinant PCs, furin, PC4, PC5, PACE4, and PC7. Proteolytic preactivation was assessed using a well-established entry assay into PC-inhibited cells based on the green fluorescent protein as a reporter. The inhibition of the target cell PC activity with serpin-based PC-selective inhibitors also showed a diversity of PC selectivity among HPV types. HPV16 reacted with furin at the highest rate compared to the other types in time-dependent preactivation reactions and produced the highest entry values standardized to pseudoviral particle concentration. The predominant expression of furin in keratinocytes and the high reactivity of HPV16 with this enzyme highlight the importance of selectively targeting furin as a potential antiviral therapeutic approach.

Intrahepatic cholestasis of pregnancy (ICP) appears in the second or third trimester of pregnancy and is characterized by pruritus and elevated blood bile acid (BA) levels. Complications from these symptoms may include preterm birth, fetal distress, or stillbirth. Although the precise causes of ICP are unknown, genetic, hormonal, and environmental variables may be involved. First-line treatment for ICP is ursodeoxycholic acid (UDCA), which improves bile flow and consequently lowers BA levels and pruritus. The objective of this study is to investigate the impact of UDCA therapy on maternal and fetal outcomes in women with ICP. This was a prospective observational study of 123 pregnant women with ICP, aged between 20 and 45 years who were diagnosed clinically (pruritus) supported by abnormal laboratory results including elevated serum BA levels, and abnormalities in liver function tests, over the course of three years, from July 2021 to June 2024. Every patient received UDCA, commencing at 10-15 mg/kg/day and being titrated according to clinical guidelines. Maternal and fetal outcomes were tracked for the duration of the pregnancy, with data being collected at baseline (15 ± 1 weeks) and every two weeks until delivery. The mean age of the study participants was 29.6 ± 5.4 years, with the youngest patient being 20 years and the oldest being 45 years. Most women were multipara 65.9%, and the mean BMI was 27.8 ± 3.5 kg/m ². The mean time of gestational age at ICP diagnosis was 31.2 ± 2.7 weeks, and the time of gestational age at delivery was 37.1 ± 2.4 weeks. On average, the serum BA level at diagnosis was 23.5 ± 8.1 µmol/L. In the majority of ICP patients with good fetal outcomes, UDCA not only normalizes serum BA levels but also reduces maternal symptoms. In addition to addressing patient response variability to this therapy and optimizing dissemination procedures, the researchers expect that the results of this study will support the continued use of UDCA as first-line treatment for ICP, at least until more evidence becomes available.

Objective: To estimate the association of umbilical cord lactate levels and pH with Hypoxic-Ischemic Encephalopathy in neonates born with intrapartum asphyxia. Study Design: Comparative cross-sectional study. Place and Duration of study: Pediatrics Department, Pak Emirates Military Hospital, Rawalpindi Pakistan, from February 2021 to April 2022. Methodology: The study was conducted on neonates born with suspicion of intrapartum asphyxia at our hospital. Umbilical cord lactate levels and capillary pH were performed within the first 30 minutes of birth. Then, neonates were admitted to the neonatal intensive care unit and followed up for three days to look for the presence and severity of Hypoxic-Ischemic Encephalopathy (HIE). Association was ascertained between raised lactate levels and negative base deficit with HypoxicIschemic Encephalopathy. Results: A total of 1000 neonates were included in the final analysis. From 1000 neonates with intrapartum asphyxia, 790(79%) did not develop any grade of hypoxic-ischemic encephalopathy within the first three days of birth, while 210(21%) developed either Grade-I, II or III encephalopathies. Raised serum lactate and base deficit were significantly associated with the presence and severity of hypoxic-ischemic encephalopathy in neonates born with intrapartum asphyxia (p-value<0.001). Conclusion: Both umbilical artery lactate levels and capillary pH performed within the first 30 minutes of birth were associated with the presence and severity of Hypoxic-Ischemic Encephalopathy in neonates included in our study. These results favour lactate levels as a predictor of Hypoxic-Ischemic Encephalopathy.

Human papillomavirus (HPV) 16 capsids have been chosen as a DNA delivery vehicle in many studies. Our preliminary studies suggest that HPV58 capsids could be better vehicles than HPV16 capsids to deliver encapsidated DNA in vitro and in vivo. In the current study, we compared HPV16, HPV58, and the cottontail rabbit papillomavirus (CRPV) capsids either as L1/L2 VLPs or pseudoviruses (PSVs) to deliver externally attached GFP-expressing DNA. Both rabbit and human cells were used to test whether there was a species-specific effect. DNA delivery efficiency was determined by quantifying either GFP-expressing cell populations or mean fluorescent intensities (MFI) by flow cytometry. Interestingly, CRPV and 58-VLPs and PSVs were significantly more efficient at delivering attached DNA when compared to 16-VLPs and PSVs. A capsid/DNA ratio of 2:1 showed the highest efficiency for delivering external DNA. The PSVs with papillomavirus DNA genomes also showed higher efficiency than those with irrelevant plasmid DNA. HPV16L1/58L2 hybrid VLPs displayed increased efficiency compared to HPV58L1/16L2 VLPs, suggesting that L2 may play a critical role in the delivery of attached DNA. Additionally, we demonstrated that VLPs increased in vivo infectivity of CRPV DNA in rabbits. We conclude that choosing CRPV or 58 capsids to deliver external DNA could improve DNA uptake in in vitro and in vivo models.

Genetic and biochemical analyses of human papillomavirus type 16 (HPV16) capsids have shown that certain conserved L1 cysteine residues are critical for capsid assembly, integrity, and maturation. Since previous studies utilized HPV capsids produced in monolayer culture-based protein expression systems, the ascribed roles for these cysteine residues were not placed in the temporal context of the natural host environment for HPV, stratifying and differentiating human tissue. Here we extend upon previous observation, that HPV16 capsids mature and become stabilized over time (10-day to 20-day) in a naturally occurring tissue-spanning redox gradient, by identifying temporal roles for individual L1 cysteine residues. Specifically, the C175S substitution severely undermined wild-type titers of the virus within both 10 and 20-day tissue, while C428S, C185S, and C175,185S substitutions severely undermined wild-type titers only within 20-day tissue. All mutations led to 20-day virions that were less stable than wild-type and failed to form L1 multimers via nonreducing SDS-PAGE. Furthermore, Optiprep-fractionated 20-day C428S, C175S, and C175,185S capsids appeared permeable to endonucleases in comparison to wild-type and C185S capsids. Exposure to an oxidizing environment failed to enhance infectious titers of any of the cysteine mutants over time as with wild-type. Introduction of these cys mutants results in failure of the virus to mature.

Human papillomavirus (HPV) capsids are composed of 72 pentamers of the major capsid protein L1, and an unknown number of L2 minor capsid proteins. An N-terminal \"external loop\" of L2 contains cross-neutralizing epitopes, and native HPV16 virions extracted from 20-day-old organotypic tissues are neutralized by anti-HPV16 L2 antibodies but virus from 10-day-old cultures are not, suggesting that L2 epitopes are more exposed in mature, 20-day virions. This current study was undertaken to determine whether cross-neutralization of other HPV types is similarly dependent on time of harvest and to screen for the most effective cross-neutralizing epitope in native virions. Neutralization assays support that although HPV16 L2 epitopes were only exposed in 20-day virions, HPV31 or HPV18 epitopes behaved differently. Instead, HPV31 and HPV18 L2 epitopes were exposed in 10-day virions and remained so in 20-day virions. In contrast, presumably due to sequence divergence, HPV45 was not cross-neutralized by any of the anti-HPV16 L2 antibodies. We found that the most effective cross-neutralizing antibody was a polyclonal antibody named anti-P56/75 #1, which was raised against a peptide consisting of highly conserved HPV16 L2 amino acids 56 to 75. This is the first study to determine the susceptibility of multiple, native high-risk HPV types to neutralization by L2 antibodies. Multiple anti-L2 antibodies were able to cross-neutralize HPV16, HPV31, and HPV18. Only neutralization of HPV16 depended on the time of tissue harvest. These data should inform attempts to produce a second-generation, L2-based vaccine.

Human papillomavirus (HPV) capsids are formed through a network of inter- and intra-pentameric hydrophobic interactions and disulfide bonds. 72 pentamers of the major capsid protein, L1, and an unknown amount of the minor capsid protein, L2, form the structure of the capsid. There are 12 conserved L1 cysteine residues in HPV16. While C175, C185, and C428 have been implicated in the formation of a critical inter-pentameric disulfide bond, no structural or functional roles have been firmly attributed to any of the other conserved cysteine residues. Here, we show that substitution of cysteine residues C161, C229, and C379 for serine hinders the accumulation of endonuclease-resistant genomes as virions mature within stratifying and differentiating human epithelial tissue. C229S mutant virions form, but are non-infectious. These studies add detail to the differentiation-dependent assembly and maturation that occur during the HPV16 life cycle in human tissue.

Background In normal cells proliferation and apoptosis are tightly regulated, whereas in tumor cells the balance is shifted in favor of increased proliferation and reduced apoptosis. Anticancer agents mediate tumor cell death via targeting multiple pathways of programmed cell death. We have reported that the non-pathogenic, tumor suppressive Adeno-Associated Virus Type 2 (AAV2) induces apoptosis in Human Papillomavirus (HPV) positive cervical cancer cells, but not in normal keratinocytes. In the current study, we examined the potential of AAV2 to inhibit proliferation of MCF-7 and MDA-MB-468 (both weakly invasive), as well as MDA-MB-231 (highly invasive) human breast cancer derived cell lines. As controls, we used normal human mammary epithelial cells (nHMECs) isolated from tissue biopsies of patients undergoing breast reduction surgery. Results AAV2 infected MCF-7 line underwent caspase-independent, and MDA-MB-468 and MDA-MB-231 cell lines underwent caspase-dependent apoptosis. Death of MDA-MB-468 cells was marked by caspase-9 activation, whereas death of MDA-MB-231 cells was marked by activation of both caspase-8 and caspase-9, and resembled a mixture of apoptotic and necrotic cell death. Cellular demise was correlated with the ability of AAV2 to productively infect and differentially express AAV2 non-structural proteins: Rep78, Rep68 and Rep40, dependent on the cell line. Cell death in the MCF-7 and MDA-MB-231 lines coincided with increased S phase entry, whereas the MDA-MB-468 cells increasingly entered into G2. AAV2 infection led to decreased cell viability which correlated with increased expression of proliferation markers c-Myc and Ki-67. In contrast, nHMECs that were infected with AAV2 failed to establish productive infection or undergo apoptosis. Conclusion AAV2 regulated enrichment of cell cycle check-point functions in G1/S, S and G2 phases could create a favorable environment for Rep protein expression. Inherent Rep associated endonuclease activity and AAV2 genomic hair-pin ends have the potential to induce a cellular DNA damage response, which could act in tandem with c-Myc regulated/sensitized apoptosis induction. In contrast, failure of AAV2 to productively infect nHMECs could be clinically advantageous. Identifying the molecular mechanisms of AAV2 targeted cell cycle regulation of death inducing signals could be harnessed for developing novel therapeutics for weakly invasive as well as aggressive breast cancer types.