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Type 1 diabetes is a complex disease characterized by the lack of endogenous insulin secreted from the pancreatic β-cells. Although β-cell targeted autoimmune processes and β-cell dysfunction are known to occur in type 1 diabetes, a complete understanding of the cell-to-cell interactions that support pancreatic function is still lacking. To characterize the pancreatic endocrine compartment, we studied pancreata from healthy adult donors and investigated a single cell surface adhesion molecule, desmoglein-2 (DSG2). Genetically-modified mice lackingDsg2were examined for islet cell mass, insulin production, responses to glucose, susceptibility to a streptozotocin-induced mouse model of hyperglycaemia, and ability to cure diabetes in a syngeneic transplantation model. Herein, we have identified DSG2 as a previously unrecognized adhesion molecule that supports β-cells. Furthermore, we reveal thatDSG2is within the top 10 percent of all genes expressed by human pancreatic islets and is expressed by the insulin-producing β-cells but not the somatostatin-producing δ-cells. In aDsg2loss-of-function mice (Dsg2lo/lo), we observed a significant reduction in the number of pancreatic islets and islet size, and consequently, there was less total insulin content per islet cluster.Dsg2lo/lomice also exhibited a reduction in blood vessel barrier integrity, an increased incidence of streptozotocin-induced diabetes, and islets isolated fromDsg2lo/lomice were more susceptible to cytokine-induced β-cell apoptosis. Following transplantation into diabetic mice, islets isolated fromDsg2lo/lomice were less effective than their wildtype counterparts at curing diabetes. In vitro assays using the Beta-TC-6 murine β-cell line suggest that DSG2 supports the actin cytoskeleton as well as the release of cytokines and chemokines. Taken together, our study suggests that DSG2 is an under-appreciated regulator of β-cell function in pancreatic islets and that a better understanding of this adhesion molecule may provide new opportunities to combat type 1 diabetes.

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Leucine-rich repeat containing G-protein coupled receptor (LGR5) is stem cell marker that potentiates canonical signaling in the Wnt/β-catenin signaling pathway (Barker et al. 2007). LGR5 is known to be expressed on several tissues; particularly the stem cells within intestinal crypts and the stem cell niche at the base of hair follicles (Barker et al. 2007 and Jaks et al. 2008). The plasticity and stem phenotype of LGR5+ cells has led to their identification as a cancer stem cell marker for some solid tumors including colorectal cancer (CRC) (Barker et al. 2009). This presents LGR5 as a potential target molecule for CAR-T cell directed specific tumor cell killing. For treatment of CRC with LGR5-directed CAR-T cells, it is critical to gain an understanding of the level of LGR5 expression on normal tissue compared with cancerous tissue. The aim of this study was to develop a screening technique for patient selection, and importantly identify potential responders to LGR5-targeting CAR-T cell therapy.Utilizing commercially purchased tissue microarrays a cohort of patient primary tumor biopsies (n=472) were assessed for LGR5 expression; ~60% displayed moderate to high level LGR5 positivity, with a trend toward higher grade, later stage tumors having higher levels of LGR5. LGR5 expression levels on lymph node metastases showed a positive correlation to LGR5 expression on patient-matched primary tumor tissue. Seven out of ten liver biopsies from metastatic colon cancer lesions showed a medium to high level LGR5 staining, indicating this may be used as a screening tool for metastatic CRC patients. We also observed restricted expression of LGR5 across the many normal tissues, suggesting limited off-target effects of LGR5-targeting CAR-T cells.The data obtained in this study provides evidence that LGR5 IHC of biopsies could be used as a diagnostic screening tool of CRC patients for inclusion into a Phase1a/2 clinical trial. Barker N, et al., 2007, Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449: 1003–1007 Barker N. et al. 2009, Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 457: 608–611Jaks V. et al. 2008, Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet. Nov;40(11):1291-9. Citation Format: Emma J. Thompson, Veronika Bandara, Batjargal Gundsambuu, Silvana Napoli, Stuart Mills, Jade Foeng, Dylan McPeake, Timona Tyllis, Caitlin Abbott, Allison Cowin, Timothy Sadlon, Shaun McColl, Carmela Ricciardelli, Simon C. Barry, Claudine S. Bonder. Assessing LGR5 expression levels on colorectal cancer tissue samples for use in LGR5-targeting CAR-T cell therapy clinical trial. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5545.

Current challenges for CAR-T therapies in solid tumors relate to target specificity and T cell penetration of the tumor. A key determinant in CAR-T selection for clinical trials is the potency and persistence of the selected CAR-T to kill cancerous cells. This is often difficult to determine across different donors and batches. Additionally, these characteristics can be difficult to determine in vitro in a rapid, high-throughput manner. In this study we have utilized real-time impedance cytotoxicity assays to screen LGR5-targeting CAR-T cells. LGR5 is a well described cancer stem cell antigen implicated in tumor initiation and metastasis, and prognosis of colon, ovarian and neuroblastoma cancers. Impedance measurements are taken continuously, providing comprehensive real-time information about CAR-T cell efficiency over time, which is not achievable with single timepoint cytotoxicity assays. Through this validation process we have been able to concurrently test different Effector to Target (E:T) ratios and different CAR constructs to accurately and directly compare CAR-T killing efficiency. This readout is a key indicator of CAR-T potency. Similar to inhibitory concentration 50 (IC50) or lethal dose 50 (LD50) data, CAR-T potency can be expressed in a measure as kill time 50 (KT50), and represents the time required for the CAR-T to stimulate cytolysis in 50% of the target cancer cells. KT50 data was key to the selection of our lead clinical candidate; CNA3103. In addition to selection of our lead candidate, real-time impedance technology has allowed us to screen a broad range of LGR5-expressing cancer types. Our data indicates that CNA3103 can effectively target colon, neuroblastoma, and ovarian cancer cells in vitro. Real-time impedance assay has allowed us to analyze cancer cells with different levels of LGR5 expression to confirm the specificity of our CAR-T cells and to develop an understanding of how LGR5 expression levels might impact cancer cell killing. The data also indicates that our LGR5 targeting CAR-T cells have the potential to repeatedly kill, i.e. the same CAR-T cell is able to move on from one cancer cell to another and remain cytotoxic. Taken together, real-time cytotoxicity assays provide valuable pre-clinical data, enabling the selection of CNA3103 as our LRG5-CAR-T clinical candidate. This technology may also assist in patient and dose selection within a clinical trial. Citation Format: Emma J. Thompson, Veronika Bandara, Timothy Sadlon, Batjargal Gundsambuu, Lih Yin Tan, Carmela Ricciardelli, Simon C. Barry, Claudine S. Bonder. Real-time cytotoxicity assays as a pre-clinical screening tool for LGR5-targeting CAR-T cells for treatment of solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5184.

The growth of solid tumours relies on an ever-increasing supply of oxygen and nutrients that are delivered via vascular networks. Tumour vasculature includes endothelial cell lined angiogenesis and the less common cancer cell lined vasculogenic mimicry (VM). To study and compare the development of vascular networks formed during angiogenesis and VM (represented here by breast cancer and pancreatic cancer cell lines) a number of in vitro assays were utilised. From live cell imaging, we performed a large-scale automated extraction of network parameters and identified properties not previously reported. We show that for both angiogenesis and VM, the characteristic network path length reduces over time; however, only endothelial cells increase network clustering coefficients thus maintaining small-world network properties as they develop. When compared to angiogenesis, the VM network efficiency is improved by decreasing the number of edges and vertices, and also by increasing edge length. Furthermore, our results demonstrate that angiogenic and VM networks appear to display similar properties to road traffic networks and are also subject to the well-known Braess paradox. This quantitative measurement framework opens up new avenues to potentially evaluate the impact of anti-cancer drugs and anti-vascular therapies., Fouladzadeh, Dorraki and colleagues investigate the development of angiogenic networks for in vitro cancer cell lines. They demonstrate that during the growth stages of vasculogenic mimicry, the number of edges and vertices decreases but the edge length increases resulting in improved network efficiency.

This study investigated factors associated with acute stress symptoms in parents of seriously ill children across a range of illnesses and treatment settings within a pediatric hospital setting. It was hypothesized that psychosocial variables would be more strongly associated with acute stress responses than demographic and child illness variables. Participants were 115 mothers and 56 fathers of children treated within the oncology, cardiology, and intensive care departments of a pediatric hospital. Acute stress, psychosocial, demographic, and medical data were collected within the first 4 weeks of the child's hospital admission. A robust hierarchical regression model revealed that psychosocial factors significantly explained 36.8% of the variance in parent acute stress responses (p < .001); demographic variables significantly added a further 4.5% (p = .022), but illness-related factors did not contribute to the model. Findings support the implementation of a general psychosocial screening approach for parents across the wider hospital system, and that psychosocial risk factors may be targeted in interventions across different illnesses and treatment settings to improve parent outcomes.

By age 16, 20% of children will suffer a concussion. Many experience persisting post-concussive symptoms (PCS), the cause(s) of which remain unclear. We mapped concussion recovery to 3 months post-injury and explored non-modifiable (e.g., age, sex, pre-injury factors, injury mechanism, acute PCS) and modifiable (post-acute child symptoms) predictors of persisting symptoms in order to identify opportunities for early intervention. We conducted a prospective, longitudinal study in the emergency department of a tertiary, pediatric hospital recruiting children within 48 h of concussion (T0), with follow-up at 2 days (T1), 2 weeks (T2), 1 month (T3), and 3 months (T4). Primary outcome was T2 clinician diagnosis. Clinical history, injury mechanism, acute symptoms, and physical and cognitive function were assessed. Parents rated child behavior and fatigue, and their mental health. We enrolled 256 participants, 72% males: 62 (24.3%) were symptomatic at T2. Recovered and symptomatic groups endorsed similar pre-injury PCS, but group differences were found at T1 across all PCS subscales, except Emotional, where symptoms were not evident until T2. By T2, there was significant PCS reduction, steepest in the "Recovered" group, which also had a lower rate of pre-injury psychiatric diagnoses, acute CT scans and less severe parent-rated PCS at T1 than the symptomatic group. They all demonstrated lower parent-rated PCS and less internalizing behaviors (all, p

Tumour vasculature supports the growth and progression of solid cancers with both angiogenesis (endothelial cell proliferation) and vasculogenic mimicry (VM, the formation of vascular structures by cancer cells themselves) predictors of poor patient outcomes. Increased circulating platelet counts also predict poor outcome for cancer patients but the influence of platelets on tumour vasculature is incompletely understood. Herein, we show with in vitro assays that platelets did not influence angiogenesis but did actively inhibit VM formation by cancer cell lines. Both platelet sized beads and the releasates from platelets were partially effective at inhibiting VM formation suggesting that direct contact maximises the effect. Platelets also promoted cancer cell invasion in vitro. B16F10 melanomas in Bcl-xPlt20/Plt20 thrombocytopenic mice showed a higher content of VM than their wildtype counterparts while angiogenesis did not differ. In a xenograft mouse model of breast cancer with low-dose aspirin to inactivate the platelets, the burden of MDA-MB-231-LM2 breast cancer cells was reduced and the gene expression profile of the cancer cells was altered; but no effect on tumour vasculature was observed. Taken together, this study provides new insights into the action of platelets on VM formation and their involvement in cancer progression.

Computerized neuropsychological tests (CNTs) are widely used internationally in concussion management. Their prognostic value for predicting recovery post-concussion is poorly understood, particularly in pediatric populations. The aim of the present study was to examine whether cognitive functioning (measured by CogSport) has prognostic value for predicting rapid versus slow recovery. This is a prospective longitudinal observational cohort study conducted at a state-wide tertiary pediatric hospital. Data were collected at 1-4, 14, and 90 days post-injury. Eligible children were aged ≥5 and18 years presenting to the Emergency Department having sustained a concussion within 48 h. Concussion was defined according to the Zurich/Berlin Consensus Statement on Concussion in Sport. Dependent variables were reaction times and error rates on the CogSport Brief Battery. In total, 220 cases were analyzed; 98 in a rapid recovery group (asymptomatic at 14 days post-injury, mean age 11.5 [3.2], 73.5% male) and 122 in a slow recovery group (symptomatic at 14 days post-injury, mean age 12.0 [3.1], 69.7% male). Longitudinal GEE analyses modeled the trajectories of both mean log

IntroductionThe majority of children who sustain a concussion will recover quickly, but a significant minority will experience ongoing postconcussive symptoms, known as postconcussion syndrome (PCS). These symptoms include emotional, behavioural, cognitive and physical symptoms and can lead to considerable disability. The neurobiological underpinnings of PCS are poorly understood, limiting potential clinical interventions. As such, patients and families frequently re-present to clinical services, who are often ill equipped to address the multifactorial nature of PCS. This contributes to the high cost of concussion management and the disability of children experiencing PCS. The aims of the present study are: (1) to plot and contrast recovery pathways for children with concussion from time of injury to 3 months postinjury, (ii) evaluate the contribution of acute biomarkers (ie, blood, MRI) to delayed recovery postconcussion and (3) estimate financial costs of child concussion to patients attending the emergency department (ED) of a tertiary children’s hospital and factors predicting high cost.Methods and analysisTake C.A.Re is a prospective, longitudinal study at a tertiary children’s hospital, recruiting and assessing 525 patients aged 5–Ethics and disseminationEthical approval has been obtained through the Royal Children’s Hospital Melbourne Human Research Ethics Committee (33122). We aim to disseminate the findings through international conferences, international peer-reviewed journals and social media.Trial registration numberACTRN12615000316505; Results.

The β common ([βc]/CD131) family of cytokines comprises granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5, all of which use βc as their key signaling receptor subunit. This is a prototypic signaling subunit-sharing cytokine family that has unveiled many biological paradigms and structural principles applicable to the IL-2, IL-4, and IL-6 receptor families, all of which also share one or more signaling subunits. Originally identified for their functions in the hematopoietic system, the βc cytokines are now known to be truly pleiotropic, impacting on multiple cell types, organs, and biological systems, and thereby controlling the balance between health and disease. This review will focus on the emerging biological roles for the βc cytokines, our progress toward understanding the mechanisms of receptor assembly and signaling, and the application of this knowledge to develop exciting new therapeutic approaches against human disease. Refereed/Peer-reviewed

Psychological and mental health factors are important areas of focus for clinicians when considering post-concussion management. Not everyone who experiences a concussion will experience mental health difficulties; however, for those who do, they can persist and significantly impact post-concussion symptoms. The presence of psychological and mental health factors post concussion can be extensively diverse, including mood and anxiety disorders, post-traumatic stress disorder, and fatigue-related disorders. Several factors have been shown to contribute toward how a child or adolescent experiences post-concussion symptoms that may include age, sex, personality traits, and family dynamics. This chapter is divided into sections outlining the common mental health difficulties experienced post concussion and the key personal factors to be considered in the post-concussion stage. At the conclusion of this chapter, a clinical case study of a high-school age female is presented that outlines the presence and resolution of post-concussion symptoms with a focus on psychological and mental health factors. Recommendations are made for future areas of research.

BACKGROUND: The Sport Concussion Assessment Tool version 3 (SCAT3) and its child version (ChildSCAT3) are composite physical and neuropsychological scoring systems used to assess athletes after sport-related concussion. Based on limited validation data, we aimed to evaluate the ability of SCAT3 and ChildSCAT3 to differentiate children aged 5 to 16 years with concussion from controls. METHODS: Prospective observational study of children in the emergency department with concussion (CONC group) and 2 control groups ([1] upper-limb injury [ULI] and [2] Well children) with equal-sized subgroups in 3 age bands of 5 to 8, 9 to 12, and 13 to 16 years. ChildSCAT3 was used for participants aged 5 to 12 years, and SCAT3 was used for participants aged 13 to 16 years. Differences between study groups were analyzed by using analysis of variance models, adjusting for age and sex. RESULTS: We enrolled 264 children (90 CONC, 90 ULI, and 84 Well) in equal-sized age bands. The number and severity of child- and parent-reported symptom scores were significantly higher in the CONC group than either control group (P < .001). Mean double (ChildSCAT3 P < .001) and tandem stance errors (both P ≤ .01) were also significantly higher, and immediate memory was significantly lower for the CONC group (P < .01). No statistically significant group differences were found for orientation and digit backward tasks. There were no significant differences between ULI and Well control groups. CONCLUSIONS: Overall, SCAT3 and ChildSCAT3 can differentiate concussed from nonconcussed patients, particularly in symptom number and severity.

Objective Posttraumatic stress disorder (PTSD) and posttraumatic stress symptoms (PTSSs) are common for parents of children with life-threatening illnesses or injuries. The impact of these psychological sequelae on parents' personal use of health services is unknown. The present study aimed to investigate whether PTSS severity prospectively predicts increased health service utilization (HSU), and to examine the relative importance of other predisposing and enabling factors in predicting HSU. Method The sample comprised 106 parents of children with various life threatening illnesses, who completed a resource use questionnaire at 19 months following their child's diagnosis or admission. HSU was assessed as reported general practitioner and psychologist visits. Results Parent PTSS severity at 7 months following their child's diagnosis or hospital admission predicted being in higher service utilization categories in the following 12 months; as PTSS score increased, the odds of being in higher categories increased. Hierarchical ordinal logistic regression procedures indicated predisposing and enabling factors failed to further explain HSU. Conclusions These findings highlight the importance of PTSSs to HSU and are consistent with studies of samples experiencing other forms of trauma, such as war or natural disaster. Our results also suggest that an individuals' need, in terms of the severity of their PTSSs, appears most important in predicting their health service engagement. Although this is positive, the effectiveness of this service use, in terms of cost and outcomes, remains unclear. Further, despite the levels of PTSSs observed in the present sample, a minority of individuals sought psychosocial care. (PsycINFO Database Record

This study investigated factors associated with acute stress symptoms in parents of seriously ill children across a range of illnesses and treatment settings within a pediatric hospital setting. It was hypothesized that psychosocial variables would be more strongly associated with acute stress responses than demographic and child illness variables. Participants were 115 mothers and 56 fathers of children treated within the oncology, cardiology, and intensive care departments of a pediatric hospital. Acute stress, psychosocial, demographic, and medical data were collected within the first 4 weeks of the child's hospital admission. A robust hierarchical regression model revealed that psychosocial factors significantly explained 36.8% of the variance in parent acute stress responses (p.001); demographic variables significantly added a further 4.5% (p = .022), but illness-related factors did not contribute to the model. Findings support the implementation of a general psychosocial screening approach for parents across the wider hospital system, and that psychosocial risk factors may be targeted in interventions across different illnesses and treatment settings to improve parent outcomes.

BackgroundHumans are by nature a social species, with much of human experience spent in social interaction. Unsurprisingly, social functioning is crucial to well-being and quality of life across the lifespan. While early intervention for social problems appears promising, our ability to identify the specific impairments underlying their social problems (eg, social communication) is restricted by a dearth of accurate, ecologically valid and comprehensive child-direct assessment tools. Current tools are largely limited to parent and teacher ratings scales, which may identify social dysfunction, but not its underlying cause, or adult-based experimental tools, which lack age-appropriate norms. The present study describes the development and standardisation of Paediatric Evaluation of Emotions, Relationships, and Socialisation(PEERS®), an iPad-based social skills assessment tool.MethodsThe PEERS project is a cross-sectional study involving two groups: (1) a normative group, recruited from early childhood, primary and secondary schools across metropolitan and regional Victoria, Australia; and (2) a clinical group, ascertained from outpatient services at The Royal Children’s Hospital Melbourne (RCH). The project aims to establish normative data for PEERS®, a novel and comprehensive app-delivered child-direct measure of social skills for children and youth. The project involves recruiting and assessing 1000 children aged 4.0–17.11 years. Assessments consist of an intellectual screen, PEERS® subtests, and PEERS-Q, a self-report questionnaire of social skills. Parents and teachers also complete questionnaires relating to participants’ social skills. Main analyses will comprise regression-based continuous norming, factor analysis and psychometric analysis of PEERS® and PEERS-Q.Ethics and disseminationEthics approval has been obtained through the RCH Human Research Ethics Committee (34046), the Victorian Government Department of Education and Early Childhood Development (002318), and Catholic Education Melbourne (2166). Findings will be disseminated through international conferences and peer-reviewed journals. Following standardisation of PEERS®, the tool will be made commercially available.

Circulating vascular progenitor cells contribute to the pathological vasculogenesis of cancer whilst on the other hand offer much promise in therapeutic revascularization in post-occlusion intervention in cardiovascular disease. However, their characterization has been hampered by the many variables to produce them as well as their described phenotypic and functional heterogeneity. Herein we have isolated, enriched for and then characterized a human umbilical cord blood derived CD133+ population of non-adherent endothelial forming cells (naEFCs) which expressed the hematopoietic progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ endothelial progenitor cells (EPCs). Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin, (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature endothelial cells increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human endothelial cells (ECs). Extensive genomic and proteomic analyses of the naEFCs showed that intercellular adhesion molecule (ICAM)-3 is expressed on their cell surface but not on mature endothelial cells. Furthermore, functional analysis demonstrated that ICAM-3 mediated the rolling and adhesive events of the naEFCs under shear stress. We suggest that the distinct population of naEFCs identified and characterized here represents a new valuable therapeutic target to control aberrant vasculogenesis. Refereed/Peer-reviewed

The recent article of Duke 1 on Meniere's syndrome caused by allergy prompts me to report another possible cause of this syndrome in the use of radium near the ears: REPORT OF CASE Miss S., aged 62, entered Hartford Hospital, Sept. 25, 1923, because of hyperthyroidism, and was given an eleven-hour treatment by radium. It was applied to twelve different places on the neck, two of them being over the tonsils externally, close to the ears. She left the hospital the next day, but has been under careful observation since. In the three weeks following the treatment, she remarked frequently that the only places where she "felt the radium" were the ears. She said that they seemed "unnatural," and sometimes described the feeling as a "flopping sensation." Just three weeks after the treatment, she had a sudden, severe attack of vertigo, which lasted forty-five minutes and left her with the