CRISPR/Cas9-Mediated Targeting of BPV-1-Transformed Primary Equine Sarcoid Fibroblasts

Equine sarcoids (EqS) are fibroblast-derived skin tumors associated with bovine papillomavirus 1 and 2 (BPV-1 and -2). Based on Southern blotting, the BPV-1 genome was not found to be integrated in the host cell genome, suggesting that EqS pathogenesis does not result from insertional mutagenesis. Hence, CRISPR/Cas9 implies an interesting tool for selectively targeting BPV-1 episomes or genetically anchored suspected host factors. To address this in a proof-of-concept study, we confirmed the exclusive episomal persistence of BPV-1 in EqS using targeted locus amplification (TLA). To investigate the CRISPR/Cas9-mediated editing of BPV-1 episomes, primary equine fibroblast cultures were established and characterized. In the EqS fibroblast cultures, CRISPR-mediated targeting of the episomal E5 and E6 oncogenes as well as the BPV-1 long control region was successful and resulted in a pronounced reduction of the BPV-1 load. Moreover, the deletion of the equine Vimentin (VIM), which is highly expressed in EqS, considerably decreased the number of BPV-1 episomes. Our results suggest CRISPR/Cas9-based gene targeting may serve as a tool to help further unravel the biology of EqS pathogenesis

COVID-19 partial school closures and mental health problems: A cross-sectional survey of 11,000 adolescents to determine those most at risk.

Funder: NIHR Applied Research Collaboration Oxford and Thames ValleyFunder: The Westminster FoundationBACKGROUND: Understanding adolescents' mental health during lockdown and identifying those most at risk is an urgent public health challenge. This study surveyed school pupils across Southern England during the first COVID-19 school lockdown to investigate situational factors associated with mental health difficulties and how they relate to pupils' access to in-school educational provision. METHODS: A total of 11,765 pupils in years 8-13 completed a survey in June-July 2020, including questions on mental health, risk indicators and access to school provision. Pupils at home were compared to those accessing in-school provision on risk and contextual factors and mental health outcomes. Multilevel logistic regression analyses compared the effect of eight risk and contextual factors, including access to in-school provision, on depression, anxiety and self-reported deterioration in mental wellbeing. RESULTS: Females, pupils who had experienced food poverty and those who had previously accessed mental health support were at greatest risk of depression, anxiety and a deterioration in wellbeing. Pupils whose parents were going out to work and those preparing for national examinations in the subsequent school year were also at increased risk. Pupils accessing in-school provision had poorer mental health, but this was accounted for by the background risk and contextual factors assessed, in line with the allocation of in-school places to more vulnerable pupils. CONCLUSIONS: Although the strongest associations with poor mental health during school closures were established risk factors, further contextual factors of particular relevance during lockdown had negative impacts on wellbeing. Identifying those pupils at greatest risk for poor outcomes is critical for ensuring that appropriate educational and social support can be given to pupils either at home or in-school during subsequent lockdowns

What outcomes are important to patients with mild cognitive impairment or Alzheimer's disease, their caregivers, and health-care professionals? A systematic review

Introduction: Clinical trials involving patients with Alzheimer’s disease (AD) continue to try to identify disease-modifying treatments. Although trials are designed to meet regulatory and registration requirements, many do not measure outcomes of the disease most relevant to key stakeholders. Methods: A systematic review sought research that elicited information from people with AD, their caregivers, and health-care professionals on which outcomes of the disease were important. Studies published in any language between 2008 and 2017 were included. Results: Participants in 34 studies described 32 outcomes of AD. These included clinical (memory, mental health), practical (ability to undertake activities of daily living, access to health information), and personal (desire for patient autonomy, maintenance of identity) outcomes of the disease. Discussion: Evidence elicited directly from the people most affected by AD reveals a range of disease outcomes that are relevant to them but are not commonly captured in clinical trials of new treatments.</br

Papillomavirus-like Particles in Equine Medicine

Papillomaviruses (PVs) are a family of small DNA tumor viruses that can induce benign lesions or cancer in vertebrates. The observation that animal PV capsid-proteins spontaneously self-assemble to empty, highly immunogenic virus-like particles (VLPs) has led to the establishment of vaccines that efficiently protect humans from specific PV infections and associated diseases. We provide an overview of PV-induced tumors in horses and other equids, discuss possible routes of PV transmission in equid species, and present recent developments aiming at introducing the PV VLP-based vaccine technology into equine medicine

Loneliness and self-harm in adolescents during the first national COVID-19 lockdown: results from a survey of 10,000 secondary school pupils in England

Adolescents’ loneliness and self-harm have received considerable attention during the COVID-19 pandemic with concerns that the socioecological changes taking place would contribute to an escalation of both loneliness and self-harm. However, empirical evidence is scant. We estimated the prevalence of loneliness and self-harm in adolescent school pupils and investigated the association of loneliness and change in loneliness during the UK’s first lockdown with self-harm during lockdown in a cross-sectional school survey (OxWell) involving 10,460 12–18-year-olds from south England. Loneliness was measured with four items. Self-harm was ascertained through a detailed questionnaire. The prevalence of loneliness and self-harm were estimated applying post-stratification weights to account for differences between the study sample and the target population. The associations between indicators of loneliness and self-harm were examined using mixed effect models. 1,896 of 10,460 adolescents (18.1%) reported feeling lonely ‘often’ (weighted proportion 16.8%). 3,802/10,460 (36.4%; weighted proportion 35%) felt more lonely since lockdown. Self-harm during lockdown was reported by 787/10,460 adolescents (7.5%; weighted proportion 6.7%). Controlling for confounders, adolescents who reported feeling lonely ‘often’ [adjusted odds ratio (aOR) 2.8, 95% CI 2.1–3.9, p < 0.0001] or ‘sometimes’ (aOR 2.2, 95% CI 1.5–3.2, p < 0.0001) were more likely to self-harm during lockdown relative to adolescents who reported ‘never’ or ‘hardly ever’ feeling lonely. Exacerbation in loneliness during lockdown was associated with an increase in the odds of self-harm during lockdown. Loneliness, heightened loneliness and self-harm were common during lockdown and closely linked. It is important to support schools in address loneliness and self-harm as part of efforts to improve well-being as the long tail of the pandemic continues to impact on child and adolescent mental health. Understanding how loneliness and self-harm may co-vary could be important for future self-harm reduction strategies in young persons. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12144-022-03651-5

A chimeric 18L1-45RG1 virus-like particle vaccine cross-protects against oncogenic alpha-7 human papillomavirus types.

Persistent infection with oncogenic human papillomaviruses (HPV) types causes all cervical and a subset of other anogenital and oropharyngeal carcinomas. Four high-risk (hr) mucosal types HPV16, 18, 45, or 59 cause almost all cervical adenocarcinomas (AC), a subset of cervical cancer (CxC). Although the incidence of cervical squamous cell carcinoma (SCC) has dramatically decreased following introduction of Papanicolaou (PAP) screening, the proportion of AC has relatively increased. Cervical SCC arise mainly from the ectocervix, whereas AC originate primarily from the endocervical canal, which is less accessible to obtain viable PAP smears. Licensed (bivalent and quadrivalent) HPV vaccines comprise virus-like particles (VLP) of the most important hr HPV16 and 18, self-assembled from the major capsid protein L1. Due to mainly type-restricted efficacy, both vaccines do not target 13 additional hr mucosal types causing 30% of CxC. The papillomavirus genus alpha species 7 (α7) includes a group of hr types of which HPV18, 45, 59 are proportionally overrepresented in cervical AC and only partially (HPV18) targeted by current vaccines. To target these types, we generated a chimeric vaccine antigen that consists of a cross-neutralizing epitope (homologue of HPV16 RG1) of the L2 minor capsid protein of HPV45 genetically inserted into a surface loop of HPV18 L1 VLP (18L1-45RG1). Vaccination of NZW rabbits with 18L1-45RG1 VLP plus alum-MPL adjuvant induced high-titer neutralizing antibodies against homologous HPV18, that cross-neutralized non-cognate hr α7 types HPV39, 45, 68, but not HPV59, and low risk HPV70 in vitro, and induced a robust L1-specific cellular immune response. Passive immunization protected mice against experimental vaginal challenge with pseudovirions of HPV18, 39, 45 and 68, but not HPV59 or the distantly related α9 type HPV16. 18L1-45RG1 VLP might be combined with our previously described 16L1-16RG1 VLP to develop a second generation bivalent vaccine with extended spectrum against hr HPV

Tumor Cell Plasticity in Equine Papillomavirus-Positive Versus-Negative Squamous Cell Carcinoma of the Head and Neck

Squamous cell carcinoma of the head and neck (HNSCC) is a common malignant tumor in humans and animals. In humans, papillomavirus (PV)-induced HNSCCs have a better prognosis than papillomavirus-unrelated HNSCCs. The ability of tumor cells to switch from epithelial to mesenchymal, endothelial, or therapy-resistant stem-cell-like phenotypes promotes disease progression and metastasis. In equine HNSCC, PV-association and tumor cell phenotype switching are poorly understood. We screened 49 equine HNSCCs for equine PV (EcPV) type 2, 3 and 5 infection. Subsequently, PV-positive versus -negative lesions were analyzed for expression of selected epithelial (keratins, &beta;-catenin), mesenchymal (vimentin), endothelial (COX-2), and stem-cell markers (CD271, CD44) by immunohistochemistry (IHC) and immunofluorescence (IF; keratins/vimentin, CD44/CD271 double-staining) to address tumor cell plasticity in relation to PV infection. Only EcPV2 PCR scored positive for 11/49 equine HNSCCs. IHC and IF from 11 EcPV2-positive and 11 EcPV2-negative tumors revealed epithelial-to-mesenchymal transition events, with vimentin-positive cells ranging between &lt;10 and &gt;50%. CD44- and CD271-staining disclosed the intralesional presence of infiltrative tumor cell fronts and double-positive tumor cell subsets independently of the PV infection status. Our findings are indicative of (partial) epithelial&ndash;mesenchymal transition events giving rise to hybrid epithelial/mesenchymal and stem-cell-like tumor cell phenotypes in equine HNSCCs and suggest CD44 and CD271 as potential malignancy markers that merit to be further explored in the horse