Postnatal development of the mucosal immune system and consequences on health in adulthood

The intestinal microbiota is a dynamic multifaceted ecosystem which has evolved a complex and mutually beneficial relationship with the mammalian host. The contribution to host fitness is evident, but in recent years it has become apparent that these commensal microorganisms may exert far more influence over health and disease than previously thought. The gut microbiota are implicated in many aspects of biological function, such as metabolism, angiogenesis and immune development: disruption, especially during the neonatal period, which may impose life-long penalty. Elimination of the microbiota appears difficult, but manipulation of the ratios and dominance of composite populations can be achieved by alterations in diet, rearing environment, antibiotics and/or probiotics. Components of the intestinal microbiota are frequently documented to affect normal function of the mucosal immune system in experimental animals and in domesticated, agricultural species. However, it is not always clear that the effects described are sufficiently well understood to provide a sound basis for commercial intervention. Some microbial interventions may be beneficial to the host under particular circumstances, while detrimental during others. It is essential that we further our understanding of the complex and intricate host-commensal relationship to avoid causing more long-term damage than advantag

Examining the Relationship between Religiosity, Alcohol Consumption and Violent Behaviour in Young Adults in North West England

Existing literature on the topic of religiosity and violent behaviour is still underdeveloped and much of the research is concentrated on Christian religiosity. Most of the literature on the topic suggests that religiosity acts as a protective factor for violent behaviour. The mechanisms through which this effect is seen however, are still poorly understood, though there are a wealth of criminological theories to provide a foundation upon which to hypothesize. The present study employs a quantitative approach to investigate the relationship between religiosity, alcohol consumption and violent behaviour. Explanatory, self-report questionnaires were distributed to 226 undergraduate students at one university in North West England in the year 2016 and unlike most previous studies on the topic, the present study did not exclude participants based on their religion, additionally including those who do not have a religion to act as a control group of sorts. Overall, findings were largely consistent with those of previous research, with religiosity being found to have negative correlations with both violent behaviour and alcohol consumption. The relationships between religiosity and nine risk factors for violent behaviour and were additionally investigated, the results of which were varied. Furthermore, the present study concludes that religiosity does not have consistent effects across all religious groups, and that any findings concerning religiosity cannot be generalised to any religious group other than those investigated

Cytomegalovirus viral load within blood increases markedly in healthy people over the age of 70 years

Background Cytomegalovirus (CMV) is a highly prevalent herpesvirus, which maintains lifelong latency and places a significant burden on host immunity. Infection is associated with increased rates of vascular disease and overall mortality in the elderly and there is an urgent need for improved understanding of the viral-host balance during ageing. CMV is extremely difficult to detect in healthy donors, however, using droplet digital PCR of DNA from peripheral blood monocytes, we obtained an absolute quantification of viral load in 44 healthy donors across a range of ages. Results Viral DNA was detected in 24 % (9/37) of donors below the age of 70 but was found in all individuals above this age. Furthermore, the mean CMV load was only 8.6 copies per 10,000 monocytes until approximately 70 years of age when it increased by almost 30 fold to 249 copies in older individuals (p < 0.0001). CMV was found within classical CD14+ monocytes and was not detectable within the CD14-CD16+ subset. The titre of CMV-specific IgG increased inexorably with age indicating that loss of humoral immunity is not a determinant of the increased viral load. In contrast, although cellular immunity to the structural late protein pp65 increased with age, the T cell response to the immediate early protein IE1 decreased in older donors. Conclusion These data reveal that effective control of CMV is impaired during healthy ageing, most probably due to loss of cellular control of early viral reactivation. This information will be of value in guiding efforts to reduce CMV-associated health complications in the elderly

The Role of Human Papillomaviruses and Polyomaviruses in BRAF-Inhibitor Induced Cutaneous Squamous Cell Carcinoma and Benign Squamoproliferative Lesions

Background: Human papillomavirus (HPV) has long been proposed as a cofactor in the pathogenesis of cutaneous squamous cell carcinoma (cSCC). More recently, the striking clinico-pathological features of cSCCs that complicate treatment of metastatic melanoma with inhibitors targeting BRAF mutations (BRAFi) has prompted speculation concerning a pathogenic role for oncogenic viruses. Here, we investigate HPV and human polyomaviruses (HPyV) and correlate with clinical, histologic, and genetic features in BRAFi-associated cSCC. Materials and Methods: Patients receiving BRAFi treatment were recruited at Barts Health NHS Trust. HPV DNA was detected in microdissected frozen samples using reverse line probe technology and degenerate and nested PCR. HPV immunohistochemistry was performed in a subset of samples. Quantitative PCR was performed to determine the presence and viral load of HPyVs with affinity for the skin (HPyV6, HPyV7, HPyV9, MCPyV, and TSPyV). These data were correlated with previous genetic mutational analysis of H, K and NRAS, NOTCH1/2, TP53, CDKN2A, CARD11, CREBBP, TGFBR1/2. Chromosomal aberrations were profiled using single nucleotide polymorphism (SNP) arrays. Results: Forty-five skin lesions from seven patients treated with single agent vemurafenib in 2012–2013 were analyzed: 12 cSCC, 19 viral warts (VW), 2 actinic keratosis (AK), 5 verrucous keratosis/other squamoproliferative (VK/SP) lesions, one melanocytic lesion and 6 normal skin samples. Significant histologic features of viral infection were seen in 10/12 (83%) cSCC. HPV DNA was detected in 18/19 (95%) VW/SP, 9/12 (75%) cSCC, 4/5 (80%) SP, and 3/6 (50%) normal skin samples and in 1/12 cases assessed by immunohistochemistry. HPyV was co-detected in 22/30 (73%) of samples, usually at low viral load, with MCPyV and HPyV7 the most common. SNP arrays confirmed low levels of chromosomal abnormality and there was no significant correlation between HPV or HPyV detection and individual gene mutations or overall mutational burden. Conclusion: Despite supportive clinicopathologic evidence, the role for HPV and HPyV infection in the pathogenesis of BRAFi-induced squamoproliferative lesions remains uncertain. Synergistic oncogenic mechanisms are plausible although speculative. Nonetheless, with the prospect of a significant increase in the adjuvant use of these drugs, further research is justified and may provide insight into the pathogenesis of other BRAFi-associated malignancies

A Unique Panel of Patient-Derived Cutaneous Squamous Cell Carcinoma Cell Lines Provides a Preclinical Pathway for Therapeutic Testing

Background: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. Methods: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. Results: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. Conclusions: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation

Immune responses in pigs vaccinated with adjuvanted and non-adjuvanted A(H1N1)pdm/09 influenza vaccines used in human immunization programmes.

Following the emergence and global spread of a novel H1N1 influenza virus in 2009, two A(H1N1)pdm/09 influenza vaccines produced from the A/California/07/09 H1N1 strain were selected and used for the national immunisation programme in the United Kingdom: an adjuvanted split virion vaccine and a non-adjuvanted whole virion vaccine. In this study, we assessed the immune responses generated in inbred large white pigs (Babraham line) following vaccination with these vaccines and after challenge with A(H1N1)pdm/09 virus three months post-vaccination. Both vaccines elicited strong antibody responses, which included high levels of influenza-specific IgG1 and haemagglutination inhibition titres to H1 virus. Immunisation with the adjuvanted split vaccine induced significantly higher interferon gamma production, increased frequency of interferon gamma-producing cells and proliferation of CD4(-)CD8(+) (cytotoxic) and CD4(+)CD8(+) (helper) T cells, after in vitro re-stimulation. Despite significant differences in the magnitude and breadth of immune responses in the two vaccinated and mock treated groups, similar quantities of viral RNA were detected from the nasal cavity in all pigs after live virus challenge. The present study provides support for the use of the pig as a valid experimental model for influenza infections in humans, including the assessment of protective efficacy of therapeutic interventions

Transcriptomic analysis of cutaneous squamous cell carcinoma reveals a multi-gene prognostic signature associated with metastasis

Background: Metastasis of cutaneous squamous cell carcinoma (cSCC) is uncommon. Current staging methods are reported to have sub-optimal performances in metastasis prediction. Accurate identification of patients with tumours at high risk of metastasis would have a significant impact on management.Objective: To develop a robust and validated gene expression profile (GEP) signature for predicting primary cSCC metastatic risk using an unbiased whole transcriptome discovery-driven approach.Methods: Archival formalin-fixed paraffin-embedded primary cSCC with perilesional normal tissue from 237 immunocompetent patients (151 non-metastasising and 86 metastasising) were collected retrospectively from four centres. TempO-seq was used to probe the whole transcriptome and machine learning algorithms were applied to derive predictive signatures, with a 3:1 split for training and testing datasets.Results: A 20-gene prognostic model was developed and validated, with an accuracy of 86.0%, sensitivity of 85.7%, specificity of 86.1%, and positive predictive value of 78.3% in the testing set, providing more stable, accurate prediction than pathological staging systems. A linear predictor was also developed, significantly correlating with metastatic risk.Limitations: This was a retrospective 4-centre study and larger prospective multicentre studies are now required.Conclusion: The 20-gene signature prediction is accurate, with the potential to be incorporated into clinical workflows for cSCC

Neonatal Colonisation Expands a Specific Intestinal Antigen-Presenting Cell Subset Prior to CD4 T-Cell Expansion, without Altering T-Cell Repertoire

Interactions between the early-life colonising intestinal microbiota and the developing immune system are critical in determining the nature of immune responses in later life. Studies in neonatal animals in which this interaction can be examined are central to understanding the mechanisms by which the microbiota impacts on immune development and to developing therapies based on manipulation of the microbiome. The inbred piglet model represents a system that is comparable to human neonates and allows for control of the impact of maternal factors. Here we show that colonisation with a defined microbiota produces expansion of mucosal plasma cells and of T-lymphocytes without altering the repertoire of alpha beta T-cells in the intestine. Importantly, this is preceded by microbially-induced expansion of a signal regulatory protein α-positive (SIRPα+) antigen-presenting cell subset, whilst SIRPα−CD11R1+ antigen-presenting cells (APCs) are unaffected by colonisation. The central role of intestinal APCs in the induction and maintenance of mucosal immunity implicates SIRPα+ antigen-presenting cells as orchestrators of early-life mucosal immune development

Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma

Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF MAPK pathway activation. Here, we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFb Smad signaling which is localised to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through BRafV600E or KRASG12D knockin) and TGFb signaling ablation (through Tgfbr1 deletion) in Lgr5+ve stem cells enables rapid cSCC development in the mouse. Mutation of TP53 (which is commonly mutated in sporadic cSCC) coupled with TGFbR1 deletion in Lgr5+ve cells also results in cSCC development. These findings indicate that Lgr5+ve stem cells can act as a cell of origin for cSCC and that RAS-RAF-MAPK pathway hyperactivation or TP53 mutation, coupled with loss of TGFb signaling, are driving events of skin tumorigenesis