Immuno-responsive tissue engineered oral mucosal equivalents containing macrophages

Macrophages play a key role in orchestrating the host immune response towards invading organisms or non-self molecules in the oral mucosa. Three-dimensional (3D) oral mucosal equivalents (OME) containing oral fibroblasts and keratinocytes are used extensively to mimic the human oral mucosa where they have been employed to examine innate immune responses to both bacterial and fungal pathogens as well as to biomaterials. Although the presence of immune cells is critical in generating an immune response, very few studies have incorporated leukocytes into OME and to date none have contained primary human macrophages. Here we report the generation of an immuno-competent OME to investigate immune responses toward bacterial challenge. Primary human monocyte-derived macrophages (MDM) were as responsive to bacterial lipopolysaccharide (LPS) challenge when cultured within a 3D hydrogel in terms of pro-inflammatory cytokine (IL-6, CXCL8 and TNF-α) gene expression and protein secretion as compared to culture as 2D monolayers. MDM were incorporated into a type 1 collagen hydrogel along with oral fibroblasts and the apical surface seeded with oral keratinocytes to generate a MDM-containing OME. Full-thickness MDM-OME displayed a stratified squamous epithelium and a fibroblast-populated connective tissue containing CD68-positive MDM that could be readily isolated to a single cell population for further analysis by collagenase treatment followed by flow cytometry. When stimulated with LPS, MDM-OME responded with increased pro-inflammatory cytokine secretion, most notably for TNF-α that increased 12-fold when compared to OME alone. Moreover, this pro-inflammatory response was inhibited by pre-treatment with dexamethasone, showing that MDM-OME are also amenable to drug-treatment. Dual-labelled immunofluorescence confocal microscopy revealed that MDM were the sole source of TNF-α production within MDM-OME. These data show functional activity of MDM-OME and illustrate their usefulness for investigations aimed at monitoring the immune response of the oral mucosa to pathogens, biomaterials, and for tissue toxicity and anti-inflammatory drug delivery studies

Increased abundance of tumour-associated neutrophils in HPV-negative compared to HPV-positive oropharyngeal squamous cell carcinoma is mediated by IL-1R signalling

The incidence of human papillomavirus (HPV)-associated cancer is increasing and HPV is now implicated in the aetiology of more than 60% of all oropharyngeal squamous cell carcinomas (OPSCC). In OPSCC, innate immune cells such as neutrophils and macrophages generally correlate with poor prognosis, whilst adaptive immune cells, such as lymphocytes, tend to correlate with improved prognosis. This may, in part, be due to differences in the immune response within the tumour microenvironment leading to the recruitment of specific tumour-associated leukocyte sub-populations. In this study, we aimed to examine if differences exist in the levels of infiltrated leukocyte sub-populations, with particular emphasis on tumour-associated neutrophils (TAN), and to determine the mechanism of chemokine-induced leukocyte recruitment in HPV-positive compared to HPV-negative OPSCC. Immunohistochemical analysis showed that HPV-negative OPSCC contained significantly more neutrophils than HPV-positive tumours, whilst levels of CD68+ macrophages and CD3+ lymphocytes were similar. Using a 3D tissue culture model to represent tumour-stromal interactions, we demonstrated that HPV-negative tumour-stromal co-cultures expressed significantly higher levels of CXCL8, leading to increased neutrophil recruitment compared to their HPV-positive counterparts. HPV-negative OPSCC cells have previously been shown to express higher levels of IL-1 than their HPV-positive counterparts, indicating that this cytokine may be responsible for driving increased chemokine production in the HPV-negative 3D model. Inhibition of IL-1R in the tumour-stromal models using the receptor-specific antagonist, anakinra, dramatically reduced chemokine secretion and significantly impaired neutrophil and monocyte recruitment, suggesting that this tumour-stromal response is mediated by the IL-1/IL-1R axis. Here, we identify a mechanism by which HPV-negative OPSCC may recruit more TAN than HPV-positive OPSCC. Since TAN are associated with poor prognosis in OPSCC, our study identifies potential therapeutic targets aimed at redressing the chemokine imbalance to reduce innate immune cell infiltration with the aim of improving patient outcome

Childhood health motivation and adult cardiometabolic health in the Childhood Determinants of Adult Health (CDAH) study

Objective: This research examined if childhood health motivation was associated with adult health behaviors and objectively measured health outcomes.Method: Data were from the Childhood Determinants of Adult Health study. Children aged 9 to 15 years in 1985 completed a questionnaire with health motivation items. In 2004-2006, when aged 26 to 36, participants completed assessments of health behaviors (smoking, diet, alcohol consumption, and physical activity) and cardiometabolic outcomes (body mass index, carotid intima-media thickness from ultrasound, and HOMA insulin resistance from fasting blood samples). Structural path regression analyses examined pathways from health motivation in childhood to adult cardiometabolic outcomes, mediated via adult health behaviors measured concurrently, controlling for age, sex and socioeconomic position.Results: There were 6,230 (49% female) children with data on health motivation. There were two latent constructs: health motivation (4 items: visiting a dentist, visiting a doctor, knowing about your body, and eating a good diet) and risk motivation (3 items: not being a smoker, not being fat, and not drinking alcohol). Greater health motivation was directly associated with nonsmoking, lower carotid intima-media thickness, and lower body mass index in adulthood. Greater risk motivation was directly associated with smoking, higher alcohol consumption, and poorer diets in adulthood. It was also indirectly associated with higher carotid intima-media thickness and higher HOMA insulin resistance via poorer health behaviors.Conclusions: Health motivation during childhood appears important to maintain health across the life course. It could be a target for interventions to improve cardiovascular health in children and adults

Electrospun patch delivery of anti-TNFα F(ab) for the treatment of inflammatory oral mucosal disease

Chronic ulcerative oral mucosal inflammatory diseases, including oral lichen planus and recurrent aphthous stomatitis, are painful and highly prevalent, yet lack effective clinical management. In recent years, systemic biologic therapies, including monoclonal antibodies that block the activity of cytokines, have been increasingly used to treat a range of immune-mediated inflammatory conditions such as rheumatoid arthritis and psoriasis. The ability to deliver similar therapeutic agents locally to the oral epithelium could radically alter treatment options for oral mucosal inflammatory diseases, where pro-inflammatory cytokines, in particular tumour-necrosis factor-α (TNFα), are major drivers of pathogenesis. To address this, an electrospun dual-layer mucoadhesive patch comprising medical-grade polymers was investigated for the delivery of F(ab) biologics to the oral mucosa. A fluorescent-labelled F(ab) was incorporated into mucoadhesive membranes using electrospinning with 97% v/v ethanol as a solvent. The F(ab) was detected within the fibres in aggregates when visualised by confocal microscopy. Biotinylated F(ab) was rapidly eluted from the patch (97 ± 5% released within 3 h) without loss of antigen-binding activity. Patches applied to oral epithelium models successfully delivered the F(ab), with fluorescent F(ab) observed within the tissue and 5.1 ± 1.5% cumulative transepithelial permeation reached after 9 h. Neutralising anti-TNFα F(ab) fragments were generated from whole IgG by papain cleavage, as confirmed by SDS-PAGE, then incorporated into patches. F(ab)-containing patches had TNFα neutralising activity, as shown by the suppression of TNFα-mediated CXCL8 release from oral keratinocytes cultured as monolayers. Patches were applied to lipopolysaccharide-stimulated immune-competent oral mucosal ulcer equivalents that contained primary macrophages. Anti-TNFα patch treatment led to reduced levels of active TNFα along with a reduction in the levels of disease-implicated T-cell chemokines (CCL3, CCL5, and CXCL10) to baseline concentrations. This is the first report of an effective device for the delivery of antibody-based biologics to the oral mucosa, enabling the future development of new therapeutic strategies to treat painful conditions

Childhood health motivation and adult cardiometabolic health in the Childhood Determinants of Adult Health (CDAH) study.

OBJECTIVE:This research examined if childhood health motivation was associated with adult health behaviors and objectively measured health outcomes. METHOD:Data were from the Childhood Determinants of Adult Health study. Children aged 9 to 15 years in 1985 completed a questionnaire with health motivation items. In 2004-2006, when aged 26 to 36, participants completed assessments of health behaviors (smoking, diet, alcohol consumption, and physical activity) and cardiometabolic outcomes (body mass index, carotid intima-media thickness from ultrasound, and HOMA insulin resistance from fasting blood samples). Structural path regression analyses examined pathways from health motivation in childhood to adult cardiometabolic outcomes, mediated via adult health behaviors measured concurrently, controlling for age, sex and socioeconomic position. RESULTS:There were 6,230 (49% female) children with data on health motivation. There were two latent constructs: health motivation (4 items: visiting a dentist, visiting a doctor, knowing about your body, and eating a good diet) and risk motivation (3 items: not being a smoker, not being fat, and not drinking alcohol). Greater health motivation was directly associated with nonsmoking, lower carotid intima-media thickness, and lower body mass index in adulthood. Greater risk motivation was directly associated with smoking, higher alcohol consumption, and poorer diets in adulthood. It was also indirectly associated with higher carotid intima-media thickness and higher HOMA insulin resistance via poorer health behaviors. CONCLUSIONS:Health motivation during childhood appears important to maintain health across the life course. It could be a target for interventions to improve cardiovascular health in children and adults. (PsycINFO Database Record (c) 2019 APA, all rights reserved)