Early expression of mature αß TCR in CD4-CD8- T cell progenitors enables MHC to drive development of T-ALL bearing NOTCH mutations.

During normal T cell development in mouse and human, a low-frequency population of immature CD4-CD8- double-negative (DN) thymocytes expresses early, mature αß T cell antigen receptor (TCR). We report that these early αß TCR+ DN (EADN) cells are DN3b-DN4 stage and require CD3δ but not major histocompatibility complex (MHC) for their generation/detection. When MHC - is present, however, EADN cells can respond to it, displaying a degree of coreceptor-independent MHC reactivity not typical of mature, conventional αß T cells. We found these data to be connected with observations that EADN cells were susceptible to T cell acute lymphoblastic leukemia (T-ALL) transformation in both humans and mice. Using the OT-1 TCR transgenic system to model EADN-stage αß TCR expression, we found that EADN leukemogenesis required MHC to induce development of T-ALL bearing NOTCH1 mutations. This leukemia-driving MHC requirement could be lost, however, upon passaging the tumors in vivo, even when matching MHC was continuously present in recipient animals and on the tumor cells themselves. These data demonstrate that MHC:TCR signaling can be required to initiate a cancer phenotype from an understudied developmental state that appears to be represented in the mouse and human disease spectrum.

Presentation design and delivery to improve knowledge translation in a remote world.

The practical dissemination of new knowledge is not given adequate attention despite large investment in undertaking high-quality research and the desire for evidence-based practice. It is important that those involved in knowledge translation and continuing medical education understand the fundamental principles of effective presentations, whether at scientific conferences, workshops or group teaching sessions. The switch to remote presentations has made this a more challenging endeavour. We describe established presentation techniques that improve knowledge translation and how to use them in both face-to-face and remote settings.

CD4 T-cell immune stimulation of HER2 + breast cancer cells alters response to trastuzumab in vitro.

INTRODUCTION: The HER2 + tumor immune microenvironment is composed of macrophages, natural killer cells, and tumor infiltrating lymphocytes, which produce pro-inflammatory cytokines. Determining the effect of T-cells on HER2 + cancer cells during therapy could guide immunogenic therapies that trigger antibody-dependent cellular cytotoxicity. This study utilized longitudinal in vitro time-resolved microscopy to measure T-cell influence on trastuzumab in HER2 + breast cancer. METHODS: Fluorescently-labeled breast cancer cells (BT474, SKBR3, MDA-MB-453, and MDA-MB-231) were co-cultured with CD4 + T-cells (Jurkat cell line) and longitudinally imaged to quantify cancer cell viability when treated with or without trastuzumab (10, 25, 50 and 100 µg/mL). The presence and timing of T-cell co-culturing was manipulated to determine immune stimulation of trastuzumab-treated HER2 + breast cancer. HER2 and TNF-α expression were evaluated with western blot and ELISA, respectively. Significance was calculated using a two-tailed parametric t-test. RESULTS: The viability of HER2 + cancer cells significantly decreased when exposed to 25 µg/mL trastuzumab and T-cells, compared to cancer cells exposed to trastuzumab without T-cells (p = 0.01). The presence of T-cells significantly increased TNF-α expression in trastuzumab-treated cancer cells (p = 0.02). Conversely, cancer cells treated with TNF-α and trastuzumab had a similar decrease in viability as trastuzumab-treated cancer cells co-cultured with T-cells (p = 0.32). CONCLUSIONS: The presence of T-cells significantly increases the efficacy of targeted therapies and suggests trastuzumab may trigger immune mediated cytotoxicity. Increased TNF-α receptor expression suggest cytokines may interact with trastuzumab to create a state of enhanced response to therapy in HER2 + breast cancer, which has potential to reducing tumor burden.

Anti-HER2 induced myeloid cell alterations correspond with increasing vascular maturation in a murine model of HER2+ breast cancer.

BACKGROUND: Therapy targeted to the human epidermal growth factor receptor type 2 (HER2) is used in combination with cytotoxic therapy in treatment of HER2+ breast cancer. Trastuzumab, a monoclonal antibody that targets HER2, has been shown pre-clinically to induce vascular changes that can increase delivery of chemotherapy. To quantify the role of immune modulation in treatment-induced vascular changes, this study identifies temporal changes in myeloid cell infiltration with corresponding vascular alterations in a preclinical model of HER2+ breast cancer following trastuzumab treatment. METHODS: HER2+ tumor-bearing mice (N = 46) were treated with trastuzumab or saline. After extraction, half of each tumor was analyzed by immunophenotyping using flow cytometry. The other half was quantified by immunohistochemistry to characterize macrophage infiltration (F4/80), vascularity (CD31 and α-SMA), proliferation (Ki67) and cellularity (H&E). Additional mice (N = 10) were used to quantify differences in tumor cytokines between control and treated groups. RESULTS: Immunophenotyping showed an increase in macrophage infiltration 24 h after trastuzumab treatment (P ≤ 0.05). With continued trastuzumab treatment, the M1 macrophage population increased (P = 0.02). Increases in vessel maturation index (i.e., the ratio of α-SMA to CD31) positively correlated with increases in tumor infiltrating M1 macrophages (R = 0.33, P = 0.04). Decreases in VEGF-A and increases in inflammatory cytokines (TNF-α, IL-1ß, CCL21, CCL7, and CXCL10) were observed with continued trastuzumab treatment (P ≤ 0.05). CONCLUSIONS: Preliminary results from this study in a murine model of HER2+ breast cancer show correlations between immune modulation and vascular changes, and reveals the potential for anti-HER2 therapy to reprogram immunosuppressive components of the tumor microenvironment. The quantification of immune modulation in HER2+ breast cancer, as well as the mechanistic insight of vascular alterations after anti-HER2 treatment, represent novel contributions and warrant further assessment for potential clinical translation.

Diagnosis of appendicitis in the paediatric emergency department: an update.

PURPOSE OF REVIEW: Concern regarding appendicitis is a common reason for presentation to the paediatric emergency department. We review recent progress in the use of biomarkers, imaging and clinical scoring systems in improving diagnostic accuracy in suspected appendicitis in children. RECENT FINDINGS: Use of ultrasound, often performed at the bedside, is becoming more widespread with a parallel reduction in computed tomography (CT) use. Protocols for image acquisition and interpretation have been shown to improve diagnostic accuracy. Novel biomarkers have been explored and clinical diagnostic algorithms refined but none have achieved the level of diagnostic accuracy required. SUMMARY: Appendicitis remains a clinical diagnosis. Point of care ultrasound is increasingly available and offers higher diagnostic accuracy than several routinely performed laboratory investigations. Recent publications provide support for increased use of clinician performed ultrasound, increased use of MRI, less use of CT, less emphasis on basic laboratory investigation and a renewed respect for the value of serial examination, particularly early in the course of illness.

Everything is awesome: Don't forget the Lego.

AIM: Children frequently ingest coins (generally with minimal reported side effects); however, the ingestion of other items has been subject to less academic study. Parental concern regarding ingestion applies across a range of materials. In this study, we aimed to determine typical transit times for another commonly swallowed object: a Lego figurine head. METHODS: Six paediatric health-care professionals were recruited to swallow a Lego head. Previous gastrointestinal surgery, inability to ingest foreign objects and aversion to searching through faecal matter were all exclusion criteria. Pre-ingestion bowel habit was standardised by the Stool Hardness and Transit (SHAT) score. Participants ingested a Lego head, and the time taken for the object to be found in the participants stool was recorded. The primary outcome was the Found and Retrieved Time (FART) score. RESULTS: The FART score averaged 1.71 days. There was some evidence that females may be more accomplished at searching through their stools than males, but this could not be statistically validated. CONCLUSIONS: A toy object quickly passes through adult subjects with no complications. This will reassure parents, and the authors advocate that no parent should be expected to search through their child's faeces to prove object retrieval.

The Role of Ultrasound in the Diagnosis of Pediatric Nasal Fractures.

BACKGROUND: Nasal fractures are the most commonly encountered facial fracture in children presenting to emergency departments. Though plain radiographs have long been used to aid the diagnosis of fractures, its limited diagnostic accuracy has led to the increasing use of ultrasound. Ultrasound offers a cheap, safe, and readily available imaging modality. Evidence in the adult population has shown ultrasound to be far more accurate in identifying nasal fractures. The efficacy of ultrasound in the pediatric setting though remains uncertain. METHODS: A systematic review of the Pubmed and EmBase databases was undertaken. The search terms (nose OR nasal) AND (fracture) AND (ultrasound OR ultrasonography OR sonography) and associated MeSH terms were searched. The search was limited to those <18 years of age. RESULTS: Following review and exclusion, 3 papers met the inclusion criteria. All 3 studies showed ultrasound was able to detect nasal fractures in children. Two studies showed that ultrasound diagnosed fractures with a greater accuracy than plain radiographs. One study used ultrasound alone and reported a sensitivity of 75% and specificity as 92.3%. CONCLUSION: With the limited evidence to date in the pediatric population, ultrasound appears to offer a more accurate radiological investigation in nasal fractures. It could be considered diagnostically superior to plain radiographs and reduces radiation exposure in children. Further work is required to better determine its true utility and improve its diagnostic accuracy.

An update in the initial management of paediatric status epilepticus.

PURPOSE OF REVIEW: Over the last 2 years, algorithms for the optimal management of status epilepticus have changed, as the medical community has recognized the need to terminate seizures in status in a timely manner. Recent research has evaluated the different choices of benzodiazepine and has given consideration to second-line treatment options. RECENT FINDINGS: There has been a move to examine alternatives to phenytoin (such as levetiracetam and lacosamide) as second-line agents. Valproate should be used cautiously in view of the potential side effects. Three ongoing trials [Established Staus Epilepticus Treatment Trial (ESETT), Convulsive Status Epilepticus Paediatric Trial (ConSEPT), and emergency treatment with levetiracetam or phenytoin in status epilepticus in children (EcLiPSE)] are comparing the efficacy of levetiracetam and phenytoin. SUMMARY: Benzodiazepines remain the first-line agent of choice, although there is ongoing discussion about the mode of administration and the best drug to choose. The results of ESETT, ConSEPT, and EcLiPSE will affect our future management of status, as we give consideration to levetiracetam as an alternative to phenytoin. Other medications such as lacosamide may emerge in future algorithms too.

Utility of blood cultures in children admitted to hospital with community-acquired pneumonia.

AIM: The aim of the study was to assess the utility of blood cultures in children admitted to hospital with community-acquired pneumonia. The primary outcome was the number of positive blood culture results, and secondary outcomes included the effect of positive blood culture results on management, and the identification of other clinical/biochemical variables that could predict blood culture results or the course of illness. METHODS: A retrospective data analysis was carried out on all children admitted to Gosford Hospital during the 2-year period from July 2013 to June 2015. Included were patients under 16 years old who had a diagnosis-related group code of pneumonia. A review of blood culture results, chest X-ray, serology, C-reactive protein and white cell count and clinical outcomes were analysed. RESULTS: There were 215 paediatric admissions with a diagnosis of pneumonia during the 2-year study period. A blood culture was collected in 82.3% (177/215). Although seven had a positive blood culture, only two of these were finally reported as true positives and both were Streptococcus pneumoniae. Both patients were treated with a cephalosporin and demonstrated clinical improvement. No changes were made to their treatment based on the blood culture results. CONCLUSION: Blood cultures have a low yield and do not appear to be helpful when collected in all patients admitted to hospital with community-acquired pneumonia.

Maternal IL-6 can cause T-cell-mediated juvenile alopecia by non-scarring follicular dystrophy in mice.

Aiming to decipher immunological mechanisms of the autoimmune disorder alopecia areata (AA), we hypothesized that interleukin-6 (IL-6) might be associated with juvenile-onset AA, for which there is currently no experimental model. Upon intramuscular transgenesis to overexpress IL-6 in pregnant female C57BL/6 (B6) mice, we found that the offspring displayed an initial normal and complete juvenile hair growth cycle, but developed alopecia around postnatal day 18. This alopecia was patchy and reversible (non-scarring) and was associated with upregulation of Ulbp1 expression, the only mouse homolog of the human AA-associated ULBP3 gene. Alopecia was also associated with inflammatory infiltration of hair follicles by lymphocytes, including alpha-beta T cells, which contributed to surface hair loss. Despite these apparently shared traits with AA, lesions were dominated by follicular dystrophy that was atypical of human AA disease, sharing some traits consistent with B6 alopecia and dermatitis. Additionally, juvenile-onset alopecia was followed by complete, spontaneous recovery of surface hair, without recurrence of hair loss. Prolonging exposure to IL-6 prolonged the time to recovery, but once recovered, repeating high-dose IL-6 exposure de novo did not re-induce alopecia. These data suggest that although substantial molecular and cellular pathways may be shared, functionally similar alopecia disorders can occur via distinct pathological mechanisms.

Identifying autism early: The Toddlers at Risk of Autism Clinic model.

AIM: This paper describes the Toddlers at Risk of Autism Clinic (TRAC), which utilises the Social Attention and Communication Study (SACS) and/or Autism Detection in Early Childhood (ADEC) play-based assessments to facilitate the early diagnosis of autism. METHODS: A retrospective audit was conducted of all 42 children assessed over a 3-year period in the TRAC. A semi-structured interview and play-based assessment (SACS and ADEC) were used to aid experienced clinicians in diagnosing autism. Intervention was recommended, and families were routinely followed up. Analysis was conducted on the tools used, the outcomes of assessment, diagnosis and stability of diagnosis on follow-up. RESULTS: During this period, 35 boys and 7 girls were assessed, with a mean age of 25 months. The average waiting time for clinic was 11.6 weeks. Twenty-five patients were diagnosed with autism; 90.5% of toddlers given an initial diagnosis retained that diagnosis at follow-up. Out of the 17 children who were not diagnosed with autism in the TRAC, one child was later diagnosed with autism. CONCLUSION: Experienced clinicians can use the SACS and/or ADEC to assist with a Diagnostic and Statistical Manual diagnosis of autism in toddlers.

Signalling protein complexes isolated from primary human skin-resident T cells can be analysed by Multiplex IP-FCM.

Studying signal transduction in skin-resident T cells (sr-T cells) can be limited by the small size of clinical biopsies. Here, we isolated sr-T cells from clinical samples and analysed signalling protein complexes by multiplex immunoprecipitation detected by flow cytometry (mIP-FCM). In samples from two independent donors, antigenic stimulation induced signalling proteins to join shared complexes that were observed in seven pairwise combinations among five proteins. This demonstrates that sr-T cells isolated from small clinical samples provide sufficient material for mIP-FCM-based analysis of signalling-induced protein complexes. We propose that this strategy may be useful for gaining improved mechanistic insight of sr-T cell signal transduction associated with dermatological disease.

Toward T cell protein-protein interaction activity relevant to alopecia areata.

Development of better therapies for the T cell-mediated autoimmune disease alopecia areata (AA) could be expedited by an improved understanding of the immunologic signals underlying its pathogenesis. To approach this, our group is mounting a new technological and analytical platform, multiplex immunoprecipitation detected by flow cytometry (MIF). MIF is designed to allow analysis of collections of protein-protein interactions that participate in T cell signaling webs. Early experiments suggest that MIF can detect the increased protein-protein interaction network activity that occurs under conditions of T cell antigenic stimulation. Future experiments will focus on application of MIF to T cells isolated from AA or control patient samples, to identify critical T cell signaling complexes associated with the disorder.

Categorising paediatric prescribing errors by junior doctors through prescribing competency assessment: does assessment reflect actual practice?

PURPOSE: It is recognised that paediatric prescribing errors are prevalent, and that most are made by junior doctors; however, detecting errors in order to demonstrate actual error rates can be difficult. There is evidence to suggest that dosing errors are the most common type of prescribing error in practice, but there has been little research on whether prescribing assessments are an effective reflection of actual practice.This article aims to determine if prescribing error types in a paediatric prescribing competency assessment reflects error types seen in actual practice. METHODS: This study was conducted in Royal Manchester Children's Hospital (RMCH) and the participants were junior doctors working at RMCH in 2010-2011. The intervention was a prescribing competency assessment package at RMCH.The main outcome measurement was the category and rate of prescribing errors. Results were taken from the junior doctors' prescribing competency assessment. The assessment papers were analysed for errors and the errors were then broken down into pre-defined categories. RESULTS: Rates of prescribing errors in the competency assessment are higher than published results shown in practice (23.1 %). The most common type of prescribing error (incorrect calculation of dose) reflects results seen in actual practice. CONCLUSION: The types of prescribing errors made in the competency assessment are reflective of errors made in actual practice. Prescribing teaching can be tailored according to the types of errors noted; and the prescribing competency package as a whole can be used to educate junior doctors on good prescribing practice and reduce prescribing errors.

Impact of Diversity in Training Resources on Self-Confidence in Diagnosing Skin Conditions Across a Range of Skin Tones: An International Survey.

Background: Medical images are invaluable in facilitating recognition of clinical signs. Recent studies highlight a lack of diversity of skin tone images used within medical education. However, there is a paucity of data on the impact of this on patient care. Aims: To investigate diversity in training resources used by users of an International online teaching platform and self-confidence in diagnosing skin conditions in all skin tones. Methods: Users of an online teaching platform (www.dftbskindeep.com) were invited to participate in a survey evaluating key points including geographical location, ethnicity, profession, specialty, years of experience, training resources and confidence in diagnosing skin conditions. Data analyses were performed using SPSS. Categorical variables were presented as proportions. Chi-squared or Fisher's exact tests were used to compare the distribution between groups as appropriate. Results: Of 600 participants, 74% reported training resources featuring predominantly white skin. Participants were "generally uncertain" in 43% cases, "sometimes uncertain but clinically safe" (52%), and "confident across a range of skin tones" in a minority (5%). Self-confidence was associated with location [higher in Africa (29%) and Latin America (11%), (p < 0.001)]; diversity of training resources [higher with a mix (10%) or darker tones (20%) (p < 0.001)]; clinical experience [6-10 (5%) or >10 years of practice (11%) (p < 0.001)] and specialty [highest in dermatologists (53%, p < 0.001)]. Self-confidence was lowest among pediatricians, emergency medicine and pediatric emergency medicine specialists (<5%). Conclusions: These data provide preliminary evidence that training resources used by healthcare professionals on a global scale may lack enough diversity on representation of skin images, and a lack of self-confidence in diagnosing pediatric skin conditions. Further work is needed to understand the impact on knowledge and patient care to ensure equitable healthcare for all.

Assessing Healthcare Professionals' Identification of Paediatric Dermatological Conditions in Darker Skin Tones.

The impacts of the lack of skin tone diversity in medical education images on healthcare professionals (HCPs) and patients are not well studied. The aim of this study was to assess the diagnostic knowledge of HCPs and correlate this with confidence and training resources used. An online multiple choice quiz was developed. The participants' demographics, training resources and self-confidence in diagnosing skin conditions were collected. The differences in the results between the subgroups and the correlations between the respondents' experience, self-reported confidence and quiz results were assessed. The mean score of 432 international participants was 5.37 (SD 1.75) out of a maximum of 10 (highest score). Eleven percent (n = 47) reached the 80% pass mark. Subanalysis showed no difference by the continent (p = 0.270), ethnicity (p = 0.397), profession (p = 0.599), training resources (p = 0.198) or confidence (p = 0.400). A significance was observed in the specialty (p = 0.01). A weak correlation between experience and confidence (Spearman's ρ = 0.286), but no correlation between scores and confidence or experience (ρ = 0.087 and 0.076), was observed. Of diagnoses, eczema was recognised in 40% and meningococcal rash in 61%. This is the first study assessing the identification of paediatric skin conditions in different skin tones internationally. The correct identification of common/important paediatric conditions was poor, suggesting a possible difference in knowledge across skin tones. There is an urgent need to improve the representation of all skin tones to ensure equity in patient care.

Quantifying Tumor Heterogeneity via MRI Habitats to Characterize Microenvironmental Alterations in HER2+ Breast Cancer.

This study identifies physiological habitats using quantitative magnetic resonance imaging (MRI) to elucidate intertumoral differences and characterize microenvironmental response to targeted and cytotoxic therapy. BT-474 human epidermal growth factor receptor 2 (HER2+) breast tumors were imaged before and during treatment (trastuzumab, paclitaxel) with diffusion-weighted MRI and dynamic contrast-enhanced MRI to measure tumor cellularity and vascularity, respectively. Tumors were stained for anti-CD31, anti-ɑSMA, anti-CD45, anti-F4/80, anti-pimonidazole, and H&E. MRI data was clustered to identify and label each habitat in terms of vascularity and cellularity. Pre-treatment habitat composition was used stratify tumors into two "tumor imaging phenotypes" (Type 1, Type 2). Type 1 tumors showed significantly higher percent tumor volume of the high-vascularity high-cellularity (HV-HC) habitat compared to Type 2 tumors, and significantly lower volume of low-vascularity high-cellularity (LV-HC) and low-vascularity low-cellularity (LV-LC) habitats. Tumor phenotypes showed significant differences in treatment response, in both changes in tumor volume and physiological composition. Significant positive correlations were found between histological stains and tumor habitats. These findings suggest that the differential baseline imaging phenotypes can predict response to therapy. Specifically, the Type 1 phenotype indicates increased sensitivity to targeted or cytotoxic therapy compared to Type 2 tumors.

Longitudinal FRET Imaging of Glucose and Lactate Dynamics and Response to Therapy in Breast Cancer Cells.

PURPOSE: The reprogramming of cellular metabolism is a hallmark of cancer. The ability to noninvasively assay glucose and lactate concentrations in cancer cells would improve our understanding of the dynamic changes in metabolic activity accompanying tumor initiation, progression, and response to therapy. Unfortunately, common approaches for measuring these nutrient levels are invasive or interrupt cell growth. This study transfected FRET reporters quantifying glucose and lactate concentration into breast cancer cell lines to study nutrient dynamics and response to therapy. PROCEDURES: Two FRET reporters, one assaying glucose concentration and one assaying lactate concentration, were stably transfected into the MDA-MB-231 breast cancer cell line. Correlation between FRET measurements and ligand concentration were measured using a confocal microscope and a cell imaging plate reader. Longitudinal changes in glucose and lactate concentration were measured in response to treatment with CoCl2, cytochalasin B, and phloretin which, respectively, induce hypoxia, block glucose uptake, and block glucose and lactate transport. RESULTS: The FRET ratio from the glucose and lactate reporters increased with increasing concentration of the corresponding ligand (p < 0.005 and p < 0.05, respectively). The FRET ratio from both reporters was found to decrease over time for high initial concentrations of the ligand (p < 0.01). Significant differences in the FRET ratio corresponding to metabolic inhibition were found when cells were treated with glucose/lactate transporter inhibitors. CONCLUSIONS: FRET reporters can track intracellular glucose and lactate dynamics in cancer cells, providing insight into tumor metabolism and response to therapy over time.

Medical Education Blog and Podcast Utilization During the COVID-19 Pandemic.

Introduction The coronavirus disease 2019 (COVID-19) pandemic disrupted traditional in-person learning models. Free Open Access Medical (FOAM) education resources naturally filled this void, so we evaluated how medical blog and podcast utilization changed during the early months of the pandemic. Methods Academic medical podcast and blog producers were surveyed on blog and podcast utilization immediately before (January-March 2020) and after (April-May 2020) the COVID-19 pandemic declaration and subsequent lockdown. Utilization is quantified in terms of blog post pageviews and podcast downloads. Linear regression was used to estimate the effect of publication during the COVID-19 period on 30-day downloads or pageviews. A linear mixed model was developed to confirm this relationship after adjustment for independent predictors of higher 30-day downloads or pageviews, using the podcast or blog as a random intercept. Results Compared to the pre-pandemic period, downloads and pageviews per unique blog and podcast publication significantly increased for blogs (median 30-day pageviews 802 to 1860, p<0.0001) but not for podcasts (median 30-day downloads 2726 to 1781, p=0.27). Publications that contained COVID-19 content were strongly associated with higher monthly utilization (ß=7.21, 95% CI 6.29-8.14 p<0.001), and even non-COVID-19 material had higher utilization in the early pandemic (median 30-day downloads/pageviews 868 to 1380, p<0.0001). Discussion The increased blog pageviews during the early months of the COVID-19 pandemic demonstrated the important role of blogs in rapid knowledge translation. Podcasts did not experience a similar increase in utilization.