Endothelial AHR activity prevents lung barrier disruption in viral infection.

Disruption of the lung endothelial-epithelial cell barrier following respiratory virus infection causes cell and fluid accumulation in the air spaces and compromises vital gas exchange function1. Endothelial dysfunction can exacerbate tissue damage2,3, yet it is unclear whether the lung endothelium promotes host resistance against viral pathogens. Here we show that the environmental sensor aryl hydrocarbon receptor (AHR) is highly active in lung endothelial cells and protects against influenza-induced lung vascular leakage. Loss of AHR in endothelia exacerbates lung damage and promotes the infiltration of red blood cells and leukocytes into alveolar air spaces. Moreover, barrier protection is compromised and host susceptibility to secondary bacterial infections is increased when endothelial AHR is missing. AHR engages tissue-protective transcriptional networks in endothelia, including the vasoactive apelin-APJ peptide system4, to prevent a dysplastic and apoptotic response in airway epithelial cells. Finally, we show that protective AHR signalling in lung endothelial cells is dampened by the infection itself. Maintenance of protective AHR function requires a diet enriched in naturally occurring AHR ligands, which activate disease tolerance pathways in lung endothelia to prevent tissue damage. Our findings demonstrate the importance of endothelial function in lung barrier immunity. We identify a gut-lung axis that affects lung damage following encounters with viral pathogens, linking dietary composition and intake to host fitness and inter-individual variations in disease outcome.

Mirror-symmetric microtubule assembly and cell interactions drive lumen formation in the zebrafish neural rod.

By analysing the cellular and subcellular events that occur in the centre of the developing zebrafish neural rod, we have uncovered a novel mechanism of cell polarisation during lumen formation. Cells from each side of the neural rod interdigitate across the tissue midline. This is necessary for localisation of apical junctional proteins to the region where cells intersect the tissue midline. Cells assemble a mirror-symmetric microtubule cytoskeleton around the tissue midline, which is necessary for the trafficking of proteins required for normal lumen formation, such as partitioning defective 3 and Rab11a to this point. This occurs in advance and is independent of the midline cell division that has been shown to have a powerful role in lumen organisation. To our knowledge, this is the first example of the initiation of apical polarisation part way along the length of a cell, rather than at a cell extremity. Although the midline division is not necessary for apical polarisation, it confers a morphogenetic advantage by efficiently eliminating cellular processes that would otherwise bridge the developing lumen.

Development of the cerebellum: simple steps to make a 'little brain'.

The cerebellum is a pre-eminent model for the study of neurogenesis and circuit assembly. Increasing interest in the cerebellum as a participant in higher cognitive processes and as a locus for a range of disorders and diseases make this simple yet elusive structure an important model in a number of fields. In recent years, our understanding of some of the more familiar aspects of cerebellar growth, such as its territorial allocation and the origin of its various cell types, has undergone major recalibration. Furthermore, owing to its stereotyped circuitry across a range of species, insights from a variety of species have contributed to an increasingly rich picture of how this system develops. Here, we review these recent advances and explore three distinct aspects of cerebellar development - allocation of the cerebellar anlage, the significance of transit amplification and the generation of neuronal diversity - each defined by distinct regulatory mechanisms and each with special significance for health and disease.

Independently specified Atoh1 domains define novel developmental compartments in rhombomere 1.

The rhombic lip gives rise to neuronal populations that contribute to cerebellar, proprioceptive and interoceptive networks. Cell production depends on the expression of the basic helix-loop-helix (bHLH) transcription factor Atoh1. In rhombomere 1, Atoh1-positive cells give rise to both cerebellar neurons and extra-cerebellar nuclei in ventral hindbrain. The origin of this cellular diversity has previously been attributed to temporal signals rather than spatial patterning. Here, we show that in both chick and mouse the cerebellar Atoh1 precursor pool is partitioned into initially cryptic spatial domains that reflect the activity of two different organisers: an isthmic Atoh1 domain, which gives rise to isthmic nuclei, and the rhombic lip, which generates deep cerebellar nuclei and granule cells. We use a combination of in vitro explant culture, genetic fate mapping and gene overexpression and knockdown to explore the role of isthmic signalling in patterning these domains. We show that an FGF-dependent isthmic Atoh1 domain is the origin of distinct populations of Lhx9-positive neurons in the extra-cerebellar isthmic nuclei. In the cerebellum, ectopic FGF induces proliferation while blockade reduces the length of the cerebellar rhombic lip. FGF signalling is not required for the specification of cerebellar cell types from the rhombic lip and its upregulation inhibits their production. This suggests that although the isthmus regulates the size of the cerebellar anlage, the downregulation of isthmic FGF signals is required for induction of rhombic lip-derived cerebellar neurons.

Age of Onset of Functional Constipation.

In a review of 538 children with functional constipation, we analyzed ages of presentation and onset, symptom duration, and behavioral/developmental problems. We divided the subjects into quartiles (Q1-Q4) based on age of onset. Median onset age was 2.3 years. The oldest group had the shortest symptom duration before referral at 1.8 ±â€Š1.8 years (compared with Q3 to Q1, P = 0.039, P = 0.001, P < 0.001, respectively). Of the Q4 subjects, 22% had a behavioral/developmental problem (P < 0.001 compared with Q1-Q3). We conclude that most children develop functional constipation as infants and toddlers, but those with later onset are more likely to have behavioral/developmental issues and see a specialist sooner.

A Comparison of Antibiotic Serum Concentrations Drawn Simultaneously from Peripherally Inserted Central Catheters and Peripheral Veins in Children with Respiratory Infection.

The purpose of the study was to evaluate serum concentration of antibiotics drawn from a peripherally inserted central catheter (PICC) compared with a peripheral venipuncture. This prospective comparative study included patients with ages 1month to 21years admitted with a respiratory infection requiring IV vancomycin or IV tobramycin via a newly placed PICC. The difference between the antibiotic levels from the venipuncture and PICC samples was statistically significant for both the peak and trough levels. However, the difference in values was not enough to impact antibiotic dosing and therefore was not clinically significant.

Reducing Unnecessary Transfusions of RBCs in Inpatients Admitted Across Niagara Health Community Hospitals.

BACKGROUND AND OBJECTIVES: Blood products are scarce resources. Audits on the use of red blood cells (RBCs) in tertiary centers have repeatedly highlighted inappropriate use. Earlier retrospective audit at our local community hospitals has demonstrated that only 85% and 54% of all requests met Choosing Wisely Canada guidelines for pre-transfusion hemoglobin (Hb) of 80 g/L or less and single unit, respectively.We sought to improve RBC utilization by 15% over a period of 12 months (meeting Choosing Wisely Canada criteria of pre-transfusion Hb ≤80g/L by >80% and single-unit transfusion by >65%). METHODS: Following repeated PDSA (Plan-Do-Study-Act) cycles, we implemented educational strategies, prospective transfusion medicine (TM) technologist-led screening of orders, and an RBC order set. RESULTS: The 3-month median percentages of appropriate RBC use for pre-transfusion Hb and single unit (September-November 2021) across all 3 hospitals were 90% and 71%, respectively. Overall, the rate of appropriate RBCs based on pre-transfusion Hb remained above target (>80%), with minimal improvement across all hospitals (median percentage at pre- and post-technologist screening periods of 87% and 90%, respectively). The median percentage of appropriate RBCs based on single-unit transfusion orders has improved across all Niagara Health hospitals with sustained targets (3-month median percentage at pre- and post-technologist screening and most recent time periods of 54%, 56%, and 71%, respectively). CONCLUSIONS: We have taken a collaborative, multifaceted approach to optimizing utilization of RBCs across the Niagara Health hospitals. The rates of appropriate RBC use were comparable with the provincial and national accreditation benchmark standards. In particular, the TM technologist-led screening was effective in producing sustained improvement with respect to single-unit transfusion. One of the balancing outcomes was increasing workload on technologists. Local and provincial efforts are needed to facilitate recruitment and retention of laboratory technologists, especially in community hospitals.

Spatial Architecture of Myeloid and T Cells Orchestrates Immune Evasion and Clinical Outcome in Lung Cancer.

Understanding the role of the tumor microenvironment (TME) in lung cancer is critical to improving patient outcomes. We identified four histology-independent archetype TMEs in treatment-naïve early-stage lung cancer using imaging mass cytometry in the TRACERx study (n = 81 patients/198 samples/2.3 million cells). In immune-hot adenocarcinomas, spatial niches of T cells and macrophages increased with clonal neoantigen burden, whereas such an increase was observed for niches of plasma and B cells in immune-excluded squamous cell carcinomas (LUSC). Immune-low TMEs were associated with fibroblast barriers to immune infiltration. The fourth archetype, characterized by sparse lymphocytes and high tumor-associated neutrophil (TAN) infiltration, had tumor cells spatially separated from vasculature and exhibited low spatial intratumor heterogeneity. TAN-high LUSC had frequent PIK3CA mutations. TAN-high tumors harbored recently expanded and metastasis-seeding subclones and had a shorter disease-free survival independent of stage. These findings delineate genomic, immune, and physical barriers to immune surveillance and implicate neutrophil-rich TMEs in metastasis. SIGNIFICANCE: This study provides novel insights into the spatial organization of the lung cancer TME in the context of tumor immunogenicity, tumor heterogeneity, and cancer evolution. Pairing the tumor evolutionary history with the spatially resolved TME suggests mechanistic hypotheses for tumor progression and metastasis with implications for patient outcome and treatment. This article is featured in Selected Articles from This Issue, p. 897.

The role of APOBEC3B in lung tumor evolution and targeted cancer therapy resistance.

In this study, the impact of the apolipoprotein B mRNA-editing catalytic subunit-like (APOBEC) enzyme APOBEC3B (A3B) on epidermal growth factor receptor (EGFR)-driven lung cancer was assessed. A3B expression in EGFR mutant (EGFRmut) non-small-cell lung cancer (NSCLC) mouse models constrained tumorigenesis, while A3B expression in tumors treated with EGFR-targeted cancer therapy was associated with treatment resistance. Analyses of human NSCLC models treated with EGFR-targeted therapy showed upregulation of A3B and revealed therapy-induced activation of nuclear factor kappa B (NF-κB) as an inducer of A3B expression. Significantly reduced viability was observed with A3B deficiency, and A3B was required for the enrichment of APOBEC mutation signatures, in targeted therapy-treated human NSCLC preclinical models. Upregulation of A3B was confirmed in patients with NSCLC treated with EGFR-targeted therapy. This study uncovers the multifaceted roles of A3B in NSCLC and identifies A3B as a potential target for more durable responses to targeted cancer therapy.

Gastric adenocarcinoma metastasis to the rectum causing complete obstruction, a case report.

Gastric adenocarcinoma is a leading cause of mortality worldwide. The most common sites of metastases are the liver, peritoneum, lungs, and bones. Cases have been described in the colon and rectum, but are very rare. This case report describes a patient in remission from diffuse signet ring cell type gastric adenocarcinoma with resection and chemoradiation close to 10 years prior to presenting with a near-obstructing rectal mass that was consistent with metastatic gastric adenocarcinoma. Gastric cancer spreads via hematogenous, lymphatic, peritoneal seeding, or local recurrence pathways. Given the length of time between initial presentation and eventual metastasis, the theory of dormancy is discussed and proposed as a possible cause in the delay of metastasis to the rectum. This highlights the importance of maintaining a high index of suspicion for recurrence and metastasis in a patient with a history of gastric cancer, who presents with a new obstructing rectal mass.

Defining the identity and the niches of epithelial stem cells with highly pleiotropic multilineage potency in the human thymus.

Thymus is necessary for lifelong immunological tolerance and immunity. It displays a distinctive epithelial complexity and undergoes age-dependent atrophy. Nonetheless, it also retains regenerative capacity, which, if harnessed appropriately, might permit rejuvenation of adaptive immunity. By characterizing cortical and medullary compartments in the human thymus at single-cell resolution, in this study we have defined specific epithelial populations, including those that share properties with bona fide stem cells (SCs) of lifelong regenerating epidermis. Thymic epithelial SCs display a distinctive transcriptional profile and phenotypic traits, including pleiotropic multilineage potency, to give rise to several cell types that were not previously considered to have shared origin. Using here identified SC markers, we have defined their cortical and medullary niches and shown that, in vitro, the cells display long-term clonal expansion and self-organizing capacity. These data substantively broaden our knowledge of SC biology and set a stage for tackling thymic atrophy and related disorders.

Vitamin B5 supports MYC oncogenic metabolism and tumor progression in breast cancer.

Tumors are intrinsically heterogeneous and it is well established that this directs their evolution, hinders their classification and frustrates therapy1-3. Consequently, spatially resolved omics-level analyses are gaining traction4-9. Despite considerable therapeutic interest, tumor metabolism has been lagging behind this development and there is a paucity of data regarding its spatial organization. To address this shortcoming, we set out to study the local metabolic effects of the oncogene c-MYC, a pleiotropic transcription factor that accumulates with tumor progression and influences metabolism10,11. Through correlative mass spectrometry imaging, we show that pantothenic acid (vitamin B5) associates with MYC-high areas within both human and murine mammary tumors, where its conversion to coenzyme A fuels Krebs cycle activity. Mechanistically, we show that this is accomplished by MYC-mediated upregulation of its multivitamin transporter SLC5A6. Notably, we show that SLC5A6 over-expression alone can induce increased cell growth and a shift toward biosynthesis, whereas conversely, dietary restriction of pantothenic acid leads to a reversal of many MYC-mediated metabolic changes and results in hampered tumor growth. Our work thus establishes the availability of vitamins and cofactors as a potential bottleneck in tumor progression, which can be exploited therapeutically. Overall, we show that a spatial understanding of local metabolism facilitates the identification of clinically relevant, tractable metabolic targets.

The Candida albicans toxin candidalysin mediates distinct epithelial inflammatory responses through p38 and EGFR-ERK pathways.

The fungal pathogen Candida albicans secretes the peptide toxin candidalysin, which damages epithelial cells and drives an innate inflammatory response mediated by the epidermal growth factor receptor (EGFR) and mitogen-activated protein kinase (MAPK) pathways and the transcription factor c-Fos. In cultured oral epithelial cells, candidalysin activated the MAPK p38, which resulted in heat shock protein 27 (Hsp27) activation, IL-6 release, and EGFR phosphorylation without affecting the induction of c-Fos. p38 activation was not triggered by EGFR but by two nonredundant pathways involving MAPK kinases (MKKs) and the kinase Src, which differentially controlled p38 signaling outputs. Whereas MKKs mainly promoted p38-dependent release of IL-6, Src promoted p38-mediated phosphorylation of EGFR in a ligand-independent fashion. In parallel, candidalysin also activated the EGFR-ERK pathway in a ligand-dependent manner, resulting in c-Fos activation and release of the neutrophil-activating chemokines G-CSF and GM-CSF. In mice, early clearance events of oral C. albicans infection required p38 but not c-Fos. These findings delineate how candidalysin activates the pathways downstream of the MAPKs p38 and ERK that differentially contribute to immune activation during C. albicans infection.

Effect of Echinostoma caproni on Presumptive Lactic Acid Bacteria Abundance and Salmonella enterica Serovar Typhimurium Colonization in the Mouse Gut.

Co-infections of mammalian hosts with intestinal helminths and bacterial pathogens are common, especially in areas with inadequate sanitation. Interactions between co-infecting species and host microbiota can cause significant changes in host immunity, disease severity, and pathogen transmission, requiring unique treatment for each case. A greater understanding of the influences of parasite-bacteria co-infections will improve diagnosis and therapeutic approaches to control infectious diseases. To study the influence of the trematode parasite Echinostoma caproni on commensal and pathogenic bacteria in the mouse gut, we examined the abundance of intestinal lactic acid bacteria and Salmonella enterica serovar Typhimurium in control mice not exposed to E. caproni (P-) or S. Typhimurium (S-), E. caproni-infected (P+S-), S. Typhimurium-infected (P-S+), and E. caproni-S. Typhimurium co-infected (P+S+) mice, and determined bacterial burdens in the livers and spleens of the P-S+ and P+S+ mice. We also examined a subset of P+S- and P+S+ mice for survival and the relative location of E. caproni in the small intestine. The numbers of presumptive lactic acid bacteria were significantly higher in the P+S+ and P-S+ mice compared to the uninfected mice, and S. Typhimurium colonization in the liver and spleen was significantly reduced in the P+S+ mice compared to the P-S+ mice. Echinostoma caproni were located anteriorly in the intestine of P+S- mice, while in the P+S+ mice, the parasites were distributed more posteriorly. Survival of E. caproni was unaffected in either group. The results of our study suggest that E. caproni facilitates a higher abundance of presumptive lactic acid bacteria in the mouse intestine and reduces colonization of S. Typhimurium in the liver and spleen of the co-infected host.

Prospective multicentre cohort study of transthoracic echocardiography provision in the South West of the UK during the first wave of SARS-CoV-2 pandemic.

AIMS: It was predicted internationally that transthoracic echocardiography (TTE) would be vital during the SARS-CoV-2 outbreak. We therefore, designed a study to report the demand for TTE in two large District General Hospitals during the rise in the first wave of the SARS-CoV-2 pandemic in the UK. A primary clinical outcome of 30-day mortality was also assessed. METHODS: The TTE service across two hospitals was reconfigured to maximise access to inpatient scanning. All TTEs of suspected or confirmed SARS-CoV-2 patients over a 3-week period were included in the study. All patients were followed up until at least day 30 after their scan at which point the primary clinical outcome of mortality was recorded. Comparative analysis based on mortality was conducted for all TTE results, biochemical markers and demographics. RESULTS: 27 patients with confirmed SARS-CoV-2 had a TTE within the inclusion window. Mortality comparative analysis showed the deceased group were significantly older (mean 68.4, SD 11.9 vs 60.5, SD 13.0, p=0.03) and more commonly reported fatigue in their presenting symptoms (29.6% vs 71.4%, p=0.01). No other differences were identified in the demographic or biochemical data. Left ventricular systolic dysfunction was noted in 7.4% of patients and right ventricular impairment or dilation was seen in 18.5% patients. TTE results were not significantly different in mortality comparative analysis. CONCLUSION: This study demonstrates an achievable approach to TTE services when under increased pressure. Data analysis supports the limited available data suggesting right ventricular abnormalities are the most commonly identified echocardiographic change in SARS-CoV-2 patients. No association can be demonstrated between mortality and TTE results.

Anti-M-Induced Delayed Hemolytic Transfusion Reaction.

BACKGROUND: Anti-M is most often assumed to be naturally occurring and can be comprised of a mixture of predominantly immunoglobulin(Ig)M with a lesser IgG component. Anti-M-antibodies usually do not react at 37°C and therefore are considered to be of little clinical significance. METHODS: A 28-year-old man presented with hemorrhagic shock from numerous injuries sustained in a motor vehicle collision. The patient received several units of red blood cells (RBCs). The antibody screen, the direct antiglobulin test (DAT), and the RBC genotype were sent for laboratory evaluation. RESULTS: A total of 12 days after the first antibody screening result was negative (7 days after transfusion), the lowest hemoglobin value was 5.5 g per dL, and we observed a positive antibody screening result and DAT with immunoglobulin (Ig)G anti-M identified. After transfusion of 4 units of M antigen-negative RBC, the post-transfusion hemoglobin level increased to 8.9 g per dL. CONCLUSION: Obtaining M antigen-negative compatible RBCs is necessary in patients demonstrating IgG anti-M in plasma.

Does apolipoprotein E determine outcome of infection by varicella zoster virus and by Epstein Barr virus?

Over 90% of the population are infected with varicella zoster virus (VZV) but only some develop shingles - caused when the virus reactivates from latency, and only some shingles patients develop post-herpetic neuralgia (PHN), defined as pain continuing for more than about 4 months. Epstein Barr virus (EBV) similarly infects over 90% of the population; some of those infected during teenage or young adult years develop infectious mononucleosis (IM). The reason for these disparities between numbers infected and numbers affected by illness is unknown, but presumably reflects host factor(s). Our previous results showed that apolipoprotein E (APOE) genotype determines susceptibility to, or outcome of, infection in the case of several diseases of known infectious cause. Therefore, we investigated APOE genotypes of shingles, PHN, and IM patients. Our rationale for the previous studies and for investigating VZV was that these micro-organisms use for cell binding and entry the same sites in the cell surface as does the protein apoE, and that consequently, competition with apoE could affect the pathogen's extent of entry and hence extent of the damage caused. The APOE genotypes of shingles and PHN sufferers, and of IM sufferers were determined using restriction fragment length polymorphism. In females, epsilon4 homozygosity confers a risk of shingles and also of IM, and the APOE-epsilon4 allele is protective against PHN whereas APOE-epsilon3 allele is a risk. Our results showing that a host genetic factor influences the development of shingles and PHN in females have clinical significance: they could lead to identification of those (female) patients at greater risk of PHN, thus enabling these people to be targeted for treatment with the most effective drugs.