Neuroanatomical distribution of fluorophores within adult RXFP3 Cre-tdTomato/YFP mouse brain.

Relaxin-family peptide 3 receptor (RXFP3) is activated by relaxin-3 in the brain to influence arousal and related functions, such as feeding and stress responses. Two transgenic mouse lines have recently been developed that co-express different fluorophores within RXFP3-expressing neurons: either yellow fluorescent protein (YFP; RXFP3-Cre/YFP mice) or tdTomato (RXFP3-Cre/tdTomato mice). To date, the characteristics of neurons that express RXFP3-associated fluorophores in these mice have only been investigated in the bed nucleus of the stria terminalis and the hypothalamic arcuate nucleus. To better determine the utility of these fluorophore-expressing mice for further research, we characterised the neuroanatomical distribution of fluorophores throughout the brain of these mice and compared this to the published distribution of Rxfp3 mRNA (detected by in situ hybridisation) in wildtype mice. Coronal sections of RXFP3-Cre/YFP (n = 8) and RXFP3-Cre/tdTomato (n = 8) mouse brains were imaged, and the density of fluorophore-expressing cells within various brain regions/nuclei was qualitatively assessed. Comparisons with our previously reported RXFP3 mRNA distribution revealed that of 212 brain regions that contained either fluorophore or RXFP3 mRNA, approximately half recorded densities that were within two qualitative measurements of each other (on a 9-point scale), including hippocampal dentate gyrus and amygdala subregions. However, many brain areas with likely non-authentic, false-positive, or false-negative fluorophore expression were also detected, including the cerebellum. Therefore, this study provides a guide to which brain regions should be prioritized for future study of RXFP3 in these mice, to better understand the neuroanatomy and function of this intriguing, neuronal peptide receptor.

Immunization with a whole cell vaccine reduces pneumococcal nasopharyngeal density and shedding, and middle ear infection in mice.

Pneumococcal Conjugate Vaccines (PCVs) have substantially reduced the burden of disease caused by Streptococcus pneumoniae (the pneumococcus). However, protection is limited to vaccine serotypes, and when administered to children who are colonized with pneumococci at the time of vaccination, immune responses to the vaccine are blunted. Here, we investigate the potential of a killed whole cell pneumococcal vaccine (WCV) to reduce existing pneumococcal carriage and mucosal disease when given therapeutically to infant mice colonized with pneumococci. We show that a single dose of WCV reduced pneumococcal carriage density in an antibody-dependent manner. Therapeutic vaccination induced robust immune responses to pneumococcal surface antigens CbpA, PspA (family 1) and PiaA. In a co-infection model of otitis media, a single dose of WCV reduced pneumococcal middle ear infection. Lastly, in a two-dose model, therapeutic administration of WCV reduced nasal shedding of pneumococci. Taken together, our data demonstrate that WCV administered in colonized mice reduced pneumococcal density in the nasopharynx and the middle ear, and decreased shedding. WCVs would be beneficial in low and middle-income settings where pneumococcal carriage in children is high.

Pre-pregnancy obesity is associated with greater systemic inflammation and increased risk of antenatal depression.

BACKGROUND: Pre-pregnancy obesity is an emerging risk factor for perinatal depression. However, the underlying mechanisms remain unclear. We investigated the association between pre-pregnancy body mass index (BMI) and perinatal depressive symptoms in a large population-based pre-birth cohort, the Barwon Infant Study. We also assessed whether the levels of circulating inflammatory markers during pregnancy mediated this relationship. METHODS: Depressive symptoms were assessed in 883 women using the Edinburgh Postnatal Depression Scale (EPDS) and psychological stress using the Perceived Stress Scale (PSS) at 28 weeks gestation and 4 weeks postpartum. Glycoprotein acetyls (GlycA), high-sensitivity C-reactive protein (hsCRP) and cytokines were assessed at 28 weeks gestation. We performed regression analyses, adjusted for potential confounders, and investigated mediation using nested counterfactual models. RESULTS: The estimated effect of pre-pregnancy obesity (BMI ≥ 30 kg/m2) on antenatal EPDS scores was 1.05 points per kg/m2 increase in BMI (95% CI: 0.20, 1.90; p = 0.02). GlycA, hsCRP, interleukin (IL) -1ra and IL-6 were higher in women with obesity, compared to healthy weight women, while eotaxin and IL-4 were lower. Higher GlycA was associated with higher EPDS and PSS scores and partially mediated the association between pre-pregnancy obesity and EPDS/PSS scores in unadjusted models, but this association attenuated upon adjustment for socioeconomic adversity. IL-6 and eotaxin were negatively associated with EPDS/PSS scores, however there was no evidence for mediation. CONCLUSIONS: Pre-pregnancy obesity increases the risk of antenatal depressive symptoms and is also associated with systemic inflammation during pregnancy. While discrete inflammatory markers are associated with antenatal depressive symptoms and perceived stress, their role in mediating the effects of pre-pregnancy obesity on antenatal depression requires further investigation.

Zebrafish Models of Paediatric Brain Tumours.

Paediatric brain cancer is the second most common childhood cancer and is the leading cause of cancer-related deaths in children. Despite significant advancements in the treatment modalities and improvements in the 5-year survival rate, it leaves long-term therapy-associated side effects in paediatric patients. Addressing these impairments demands further understanding of the molecularity and heterogeneity of these brain tumours, which can be demonstrated using different animal models of paediatric brain cancer. Here we review the use of zebrafish as potential in vivo models for paediatric brain tumour modelling, as well as catalogue the currently available zebrafish models used to study paediatric brain cancer pathophysiology, and discuss key findings, the unique attributes that these models add, current challenges and therapeutic significance.

A Review of Emerging Tear Proteomics Research on the Ocular Surface in Ocular Allergy.

Ocular allergy is an immunoglobulin E-mediated Type I hypersensitivity reaction localised to the ocular surface and surrounding tissues. Primary signs and symptoms of ocular allergy include itching, redness, irritation and inflammation. Eye-rubbing caused by itching has been shown to alter ocular surface protein concentrations in conditions linked to ocular allergy such as keratoconus. In keratoconus, the cornea begins to thin and sag over time, leading to progressive vision loss and blindness in severe conditions. Due to the high incidence of ocular allergy sufferers rubbing their eyes in response to symptoms of itching, the protein landscape of the ocular surface may be significantly altered. Differential protein expression caused by long-term inflammation and eye-rubbing may lead to subsequent changes in ocular surface structure and function over time. This review aims to summarise and explore the findings of current ocular allergy proteome research conducted using techniques such as gel electrophoresis, mass spectrometry and lab-on-a-chip proteomics. Proteins of interest for this review include differentially expressed immunoglobulins, mucins, functional proteins, enzymes and proteins with previously uncharacterised roles in ocular allergy. Additionally, potential applications of this research are addressed in terms of diagnostics, drug development and future research prospects.

Augmented reality in medical education: students' experiences and learning outcomes.

Augmented reality (AR) is a relatively new technology that allows for digitally generated three-dimensional representations to be integrated with real environmental stimuli. AR can make use of smart phones, tablets, or other devices to achieve a highly stimulating learning environment and hands-on immersive experience. The use of AR in industry is becoming widespread with applications being developed for use not just for entertainment and gaming but also healthcare, retail and marketing, education, military, travel and tourism, automotive industry, manufacturing, architecture, and engineering. Due to the distinct learning advantages that AR offers, such as remote learning and interactive simulations, AR-based teaching programs are also increasingly being adopted within medical schools across the world. These advantages are further highlighted by the current COVID-19 pandemic, which has caused an even greater shift towards online learning. In this review, we investigate the use of AR in medical training/education and its effect on students' experiences and learning outcomes. This includes the main goals of AR-based learning, such as to simplify the delivery and enhance the comprehension of complex information. We also describe how AR can enhance the experiences of medical students, by improving knowledge and understanding, practical skills and social skills. These concepts are discussed within the context of specific AR medical training programs, such as HoloHuman, OculAR SIM, and HoloPatient. Finally, we discuss the challenges of AR in learning and teaching and propose future directions for the use of this technology in medical education.

The maternal gut microbiome during pregnancy and offspring allergy and asthma.

Environmental exposures during pregnancy that alter both the maternal gut microbiome and the infant's risk of allergic disease and asthma include a traditional farm environment and consumption of unpasteurized cow's milk, antibiotic use, dietary fiber, and psychosocial stress. Multiple mechanisms acting in concert may underpin these associations and prime the infant to acquire immune competence and homeostasis following exposure to the extrauterine environment. Cellular and metabolic products of the maternal gut microbiome can promote the expression of microbial pattern recognition receptors, as well as thymic and bone marrow hematopoiesis relevant to regulatory immunity. At birth, transmission of maternally derived bacteria likely leverages this in utero programming to accelerate postnatal transition from a TH2- to TH1- and TH17-dominant immune phenotype and maturation of regulatory immune mechanisms, which in turn reduce the child's risk of allergic disease and asthma. Although our understanding of these phenomena is rapidly evolving, the field is relatively nascent, and we are yet to translate existing knowledge into interventions that substantially reduce disease risk in humans. Here, we review evidence that the maternal gut microbiome impacts the offspring's risk of allergic disease and asthma, discuss challenges and future directions for the field, and propose the hypothesis that maternal carriage of Prevotella copri during pregnancy decreases the offspring's risk of allergic disease via production of succinate, which in turn promotes bone marrow myelopoiesis of dendritic cell precursors in the fetus.

Antibodies, Nanobodies, or Aptamers-Which Is Best for Deciphering the Proteomes of Non-Model Species?

This planet is home to countless species, some more well-known than the others. While we have developed many techniques to be able to interrogate some of the "omics", proteomics is becoming recognized as a very important part of the puzzle, given how important the protein is as a functional part of the cell. Within human health, the proteome is fairly well-established, with numerous reagents being available to decipher cellular pathways. Recent research advancements have assisted in characterizing the proteomes of some model (non-human) species, however, in many other species, we are only just touching the surface. This review considers three main reagent classes-antibodies, aptamers, and nanobodies-as a means of continuing to investigate the proteomes of non-model species without the complications of understanding the full protein signature of a species. Considerations of ease of production, potential applications, and the necessity for producing a new reagent depending on homology are presented.

Phenotyping neurons activated in the mouse brain during restoration of salt debt.

Salt overconsumption contributes to hypertension, which is a major risk factor for stroke, heart and kidney disease. Characterising neuronal pathways that may control salt consumption is therefore important for developing novel approaches for reducing salt overconsumption. Here, we identify neurons within the mouse central amygdala (CeA), lateral parabrachial nucleus (LPBN), intermediate nucleus of the solitary tract (iNTS), and caudal NTS (cNTS) that are activated and display Fos immunoreactivity in mice that have consumed salt in order to restore a salt debt, relative to salt replete and salt depleted controls. Double-label immunohistochemical studies revealed that salt restoring mice had significantly greater densities of activated enkephalin neurons within the CeA and iNTS, while statistically significant changes within the LPBN and cNTS were not observed. Furthermore, within the CeA, restoration of salt debt conferred a significant increase in the density of activated calretinin neurons, while there was no change relative to control groups in the density of activated neurons that co-expressed protein kinase C delta (PKC-δ). Taken together, these studies highlight the importance of opioid systems within the CeA and iNTS in neuronal processes associated with salt restoration, and may aid the development of future pharmacological and other strategies for reducing salt overconsumption.

Effect of MUC1 length polymorphisms on the NLRP3 inflammasome response of human macrophages.

Mucin 1 is a cell-membrane associated mucin, expressed on epithelial and immune cells that helps protect against pathogenic infections. In humans, MUC1 is highly polymorphic, predominantly due to the presence of a variable number tandem repeat (VNTR) region in the extracellular domain that results in MUC1 molecules of typically either short or long length. A genetic link is known between these MUC1 polymorphisms and inflammation-driven diseases, although the mechanism is not fully understood. We previously showed that MUC1 on murine macrophages specifically restricts activation of the NLRP3 inflammasome, thereby repressing inflammation. This study evaluated the effect of MUC1 VNTR polymorphisms on activity of the NLRP3 inflammasome in human macrophages, finding that long MUC1 alleles correlated with increased IL-1ß production following NLRP3 inflammasome activation. This indicates that the length of MUC1 can influence IL-1ß production, thus providing the first evidence of an immune-modulatory role of MUC1 VNTR polymorphisms in human macrophages.

The Role of the Cell Surface Mucin MUC1 as a Barrier to Infection and Regulator of Inflammation.

The family of cell surface (cs-) mucins are constitutively expressed at the cell surface by nearly all epithelial cells, beneath the gel-mucin layer. All cs-mucin family members have structural features that enable them to act as a releasable decoy barrier to mucosal pathogens, by providing ligands for pathogen binding and the ability to shed the bound extracellular domain. Due to the towering structure of cs-mucins at the surface, binding of mucosal pathogens can also sterically block binding to underlying cellular receptors. The cytoplasmic tail domain of cs-mucins are capable of initiating signal transduction cascades and due to their conservation across species, may play an important biological role in cellular signaling. MUC1 is one of the most extensively studied of the cs-mucin family. With respect to its physiological function in the mucosal environment, MUC1 has been demonstrated to play a dynamic role in protection of the host from infection by a wide variety of pathogens and to regulate inflammatory responses to infection. This review briefly summarizes the current knowledge and new findings regarding the structural features relating to the function of MUC1, its role as a protective barrier against pathogen invasion and mechanisms by which this cs-mucin regulates inflammation.

Mucin 1 protects against severe Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a bacterial pathogen that commonly resides in the human nasopharynx, typically without causing any disease. However, in some cases these bacteria migrate from the nasopharynx to other sites of the body such as the lungs and bloodstream causing pneumonia and sepsis, respectively. This study used a mouse model of infection to investigate the potential role of Mucin 1 (MUC1), a cell membrane-associated glycoprotein known for playing a key barrier role at mucosal surfaces, in regulating this process. Wildtype (WT) and MUC1-deficient (Muc1-/-) mice were infected intranasally with an invasive strain of S. pneumoniae and bacterial loads in the nasopharynx, lungs, and blood were analyzed. Lungs were graded histologically for inflammation and cytokine profiles in the lungs analyzed by ELISA. While there was no difference in pneumococcal colonization of the nasopharynx between WT and Muc1-/- mice, infected Muc1-/- mice showed high pneumococcal loads in their lungs 16 hours post-infection, as well as bacteremia. In contrast, infected WT mice cleared the pneumococci from their lungs and remained asymptomatic. Infection in Muc1-/- mice was associated with an elevation in lung inflammation, with cellular recruitment especially of monocytes/macrophages. While MUC1-deficiency has been shown to increase phagocytosis of Pseudomonas aeruginosa, macrophages from Muc1-/- mice exhibited a reduced capacity to phagocytose S. pneumoniae indicating diverse and bacterial-specific effects. In conclusion, these findings indicate that MUC1 plays an important role in protection against severe pneumococcal disease, potentially mediated by facilitating macrophage phagocytosis.

Protease-activated Receptor 1 Plays a Proinflammatory Role in Colitis by Promoting Th17-related Immunity.

BACKGROUND: Proteolytic cleavage of protease-activated receptor 1 (PAR1) can result in potent downstream regulatory effects on inflammation. Although PAR1 is expressed throughout the gastrointestinal tract and activating proteases are increased in inflammatory bowel disease, the effect of PAR1 activation on colitis remains poorly understood, and has not previously been studied in pediatric disease. METHODS: Expression of PAR1 and inflammatory cytokines in colonic biopsies from pediatric patients with Crohn's disease exhibiting active moderate to severe colitis was measured by quantitative PCR. The functional relevance of these clinical data was further studied in a mouse model of Citrobacter rodentium-induced colitis. RESULTS: PAR1 expression was significantly upregulated in the inflamed colons of pediatric patients with Crohn's disease, with expression levels directly correlating to disease severity. In patients with severe colitis, PAR1 expression uniquely correlated with Th17-related (IL17A, IL22, and IL23A) cytokines. Infection of PAR1-deficient (PAR1) and wildtype mice with colitogenic C. rodentium revealed that disease severity and colonic pathology were strongly attenuated in mice lacking PAR1. Furthermore, Th17-type immune response was completely abolished in the colons of infected PAR1 but not wildtype mice. Finally, PAR1 was shown to be essential for secretion of the Th17-driving cytokine IL-23 by C. rodentium-stimulated macrophages. CONCLUSIONS: This study demonstrates a strong link between PAR1 expression, Th17-type immunity, and disease severity in both pediatric patients with Crohn's disease and C. rodentium-induced colitis in mice. The data presented suggest PAR1 exerts a proinflammatory role in colitis in both humans and mice by promoting a Th17-type immune response, potentially by supporting the production of IL-23.

How host regulation of Helicobacter pylori-induced gastritis protects against peptic ulcer disease and gastric cancer.

The bacterial pathogen Helicobacter pylori is the etiological agent of a range of gastrointestinal pathologies including peptic ulcer disease and the major killer, gastric adenocarcinoma. Infection with this bacterium induces a chronic inflammatory response in the gastric mucosa (gastritis). It is this gastritis that, over decades, eventually drives the development of H. pylori-associated disease in some individuals. The majority of studies investigating H. pylori pathogenesis have focused on factors that promote disease development in infected individuals. However, an estimated 85% of those infected with H. pylori remain completely asymptomatic, despite the presence of pathogenic bacteria that drive a chronic gastritis that lasts many decades. This indicates the presence of highly effective regulatory processes in the host that, in most cases, keeps a check on inflammation and protect against disease. In this minireview we discuss such known host factors and how they prevent the development of H. pylori-associated pathologies.

The MUC1 mucin protects against Helicobacter pylori pathogenesis in mice by regulation of the NLRP3 inflammasome.

OBJECTIVES: The mucin MUC1, best known for providing an epithelial barrier, is an important protective host factor in both humans and mice during Helicobacter pylori pathogenesis. This study aimed to identify the long-term consequences of MUC1 deficiency on H. pylori pathogenesis and the mechanism by which MUC1 protects against H. pylori gastritis. DESIGN: Wildtype and Muc1(-/-) mice were infected for up to 9 months, and the gastric pathology, immunological response and epigenetic changes assessed. The effects of MUC1 on the inflammasome, a potent inflammatory pathway, were examined in macrophages and H. pylori-infected mice deficient in both MUC1 and inflammasome components. RESULTS: Muc1(-/-) mice began to die 6 months after challenge, indicating Muc1 deficiency made H. pylori a lethal infection. Surprisingly, chimaeric mouse infections revealed MUC1 expression by haematopoietic-derived immune cells limits H. pylori-induced gastritis. Gastritis in infected Muc1(-/-) mice was associated with elevated interleukin (IL)-1ß and epigenetic changes in their gastric mucosa similar to those in transgenic mice overexpressing gastric IL-1ß, implicating MUC1 regulation of an inflammasome. In support of this, infected Muc1(-/-)Casp1(-/-) mice did not develop severe gastritis. Further, MUC1 regulated Nlrp3 expression via an nuclear factor (NF)-κB-dependent pathway and reduced NF-κB pathway activation via inhibition of IRAK4 phosphorylation. The importance of this regulation was proven using Muc1(-/-)Nlrp3(-/-) mice, which did not develop severe gastritis. CONCLUSIONS: MUC1 is an important, previously unidentified negative regulator of the NLRP3 inflammasome. H. pylori activation of the NLRP3 inflammasome is normally tightly regulated by MUC1, and loss of this critical regulation results in the development of severe pathology.