Organoid Modeling of the Tumor Immune Microenvironment.

In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.

Zika Virus Infection during Pregnancy in Mice Causes Placental Damage and Fetal Demise.

Zika virus (ZIKV) infection in pregnant women causes intrauterine growth restriction, spontaneous abortion, and microcephaly. Here, we describe two mouse models of placental and fetal disease associated with in utero transmission of ZIKV. Female mice lacking type I interferon signaling (Ifnar1(-/-)) crossed to wild-type (WT) males produced heterozygous fetuses resembling the immune status of human fetuses. Maternal inoculation at embryonic day 6.5 (E6.5) or E7.5 resulted in fetal demise that was associated with ZIKV infection of the placenta and fetal brain. We identified ZIKV within trophoblasts of the maternal and fetal placenta, consistent with a trans-placental infection route. Antibody blockade of Ifnar1 signaling in WT pregnant mice enhanced ZIKV trans-placental infection although it did not result in fetal death. These models will facilitate the study of ZIKV pathogenesis, in utero transmission, and testing of therapies and vaccines to prevent congenital malformations.

Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages.

Alternative (M2) activation of macrophages driven via the α-chain of the receptor for interleukin 4 (IL-4Rα) is important for immunity to parasites, wound healing, the prevention of atherosclerosis and metabolic homeostasis. M2 polarization is dependent on fatty acid oxidation (FAO), but the source of the fatty acids that support this metabolic program has not been clear. We found that the uptake of triacylglycerol substrates via the scavenger receptor CD36 and their subsequent lipolysis by lysosomal acid lipase (LAL) was important for the engagement of elevated oxidative phosphorylation, enhanced spare respiratory capacity (SRC), prolonged survival and expression of genes that together define M2 activation. Inhibition of lipolysis suppressed M2 activation during infection with a parasitic helminth and blocked protective responses to this pathogen. Our findings delineate a critical role for cell-intrinsic lysosomal lipolysis in M2 activation.

TLR-driven early glycolytic reprogramming via the kinases TBK1-IKKɛ supports the anabolic demands of dendritic cell activation.

The ligation of Toll-like receptors (TLRs) leads to rapid activation of dendritic cells (DCs). However, the metabolic requirements that support this process remain poorly defined. We found that DC glycolytic flux increased within minutes of exposure to TLR agonists and that this served an essential role in supporting the de novo synthesis of fatty acids for the expansion of the endoplasmic reticulum and Golgi required for the production and secretion of proteins that are integral to DC activation. Signaling via the kinases TBK1, IKKɛ and Akt was essential for the TLR-induced increase in glycolysis by promoting the association of the glycolytic enzyme HK-II with mitochondria. In summary, we identified the rapid induction of glycolysis as an integral component of TLR signaling that is essential for the anabolic demands of the activation and function of DCs.

Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix. However, how the altered cell/extracellular-matrix signalling contributes to the PDAC tumour phenotype has been difficult to dissect. Here we design and engineer matrices that recapitulate the key hallmarks of the PDAC tumour extracellular matrix to address this knowledge gap. We show that patient-derived PDAC organoids from three patients develop resistance to several clinically relevant chemotherapies when cultured within high-stiffness matrices mechanically matched to in vivo tumours. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, matrix-induced chemoresistance occurs within a stiff environment due to the increased expression of drug efflux transporters mediated by CD44 receptor interactions with hyaluronan. Moreover, PDAC chemoresistance is reversible following transfer from high- to low-stiffness matrices, suggesting that targeting the fibrotic extracellular matrix may sensitize chemoresistant tumours. Overall, our findings support the potential of engineered matrices and patient-derived organoids for elucidating extracellular matrix contributions to human disease pathophysiology.

Metabolic Reprogramming Mediated by the mTORC2-IRF4 Signaling Axis Is Essential for Macrophage Alternative Activation.

Macrophage activation status is intrinsically linked to metabolic remodeling. Macrophages stimulated by interleukin 4 (IL-4) to become alternatively (or, M2) activated increase fatty acid oxidation and oxidative phosphorylation; these metabolic changes are critical for M2 activation. Enhanced glucose utilization is also characteristic of the M2 metabolic signature. Here, we found that increased glucose utilization is essential for M2 activation. Increased glucose metabolism in IL-4-stimulated macrophages required the activation of the mTORC2 pathway, and loss of mTORC2 in macrophages suppressed tumor growth and decreased immunity to a parasitic nematode. Macrophage colony stimulating factor (M-CSF) was implicated as a contributing upstream activator of mTORC2 in a pathway that involved PI3K and AKT. mTORC2 operated in parallel with the IL-4Rα-Stat6 pathway to facilitate increased glycolysis during M2 activation via the induction of the transcription factor IRF4. IRF4 expression required both mTORC2 and Stat6 pathways, providing an underlying mechanism to explain how glucose utilization is increased to support M2 activation.

Type 1 Interferons Induce Changes in Core Metabolism that Are Critical for Immune Function.

Greater understanding of the complex host responses induced by type 1 interferon (IFN) cytokines could allow new therapeutic approaches for diseases in which these cytokines are implicated. We found that in response to the Toll-like receptor-9 agonist CpGA, plasmacytoid dendritic cells (pDC) produced type 1 IFNs, which, through an autocrine type 1 IFN receptor-dependent pathway, induced changes in cellular metabolism characterized by increased fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS). Direct inhibition of FAO and of pathways that support this process, such as fatty acid synthesis, prevented full pDC activation. Type 1 IFNs also induced increased FAO and OXPHOS in non-hematopoietic cells and were found to be responsible for increased FAO and OXPHOS in virus-infected cells. Increased FAO and OXPHOS in response to type 1 IFNs was regulated by PPARα. Our findings reveal FAO, OXPHOS and PPARα as potential targets to therapeutically modulate downstream effects of type 1 IFNs.

Correction: Time to revisit the endpoint dilution assay and to replace the TCID50 as a measure of a virus sample's infection concentration.

[This corrects the article DOI: 10.1371/journal.pcbi.1009480.].

Implementation and participation of an online nutrition curriculum for pediatric gastroenterology fellows.

Our team of nutrition experts developed an online nutrition curriculum consisting of 21 modules to serve as a resource for a stand-alone nutrition curriculum or as a supplement to existing nutrition electives during the Pediatric Gastroenterology fellowship. From April 2020 through January 2023, 2090 modules were completed by 436 fellows from 75 different programs across North America. The program was accessed most during tight restrictions on in-person learning during the COVID-19 pandemic. Overall, participants posttest scores improved from baseline pretest scores indicating retention of information from the modules. The overall success of this program suggests that there should be continued efforts to develop and offer online learning opportunities in clinical nutrition. There is an opportunity to expand the audience for the curriculum to include pediatric gastroenterologists from across the globe.

Electronic health record time-tracking provides real-time data to measure and benchmark dietitian productivity.

BACKGROUND: Traditional methods for benchmarking dietitian productivity are time-consuming and fail to accurately measure the total time spent providing nutrition care. An electronic health record (EHR)-based tool that allows for daily tracking of both face-to-face and patient care coordination time for dietitians was created. We assessed whether it provided consistent, continuous measurement of time and productivity. METHODS: This tool was created in an independent paediatric academic healthcare system in the USA. Time spent by dietitians in face-to-face settings and care coordination were tracked. Changes in time spent between the years 2013-2016 versus 2018-2019 were also analysed. RESULTS: The outpatient dietitian spent a mean total of 66.4 min per patient (37.8 ± 6.0 min in face-to-face care and 28.6 ± 5.2 min in care coordination). The total times and fractions spent on face-to-face and care coordination time varied by specialty. Comparison of the two periods of time revealed 75% more productivity on average of dietitians in different outpatient settings after including care coordination tracking. In addition, dietitians were more likely to document time spent in 5-min increments after the institution of this methodology as opposed to 15-min increments. CONCLUSIONS: An EHR-based tool that facilitates the documentation of both face-to-face time and patient care coordination time is feasible and enables consistent, continuous measurement of time and productivity. The real-time data from this tool can be used to support adequate dietitian staffing and be used to create a multicentre database to measure the actual time dietitians need to provide care and generate consistent staffing benchmarks.

Are dietitian recommendations followed? A descriptive study of paediatric hospitalised and ambulatory patients.

BACKGROUND: The primary objective of this cross-sectional retrospective study was to describe the implementation of dietitian prescribed nutrition recommendations in malnourished paediatric patients in the hospital and ambulatory settings. We also aimed to investigate other characteristics that could be associated with differences in implementation. METHODS: Data were collected from 186 hospitalised and 565 ambulatory patients between February 2020 and January 2021. Data included age, hospital or ambulatory specialty departments, primary diagnosis, malnutrition status, hospital length of stay (LOS), and medical nutrition therapy recommendations. Implementation by the medical team in the hospital setting and adherence by the family in the outpatient setting were categorised as "Full", "Partial" or "None". "Partial" and "None" were combined for analysis. RESULTS: Dietitian prescribed recommendations were implemented in 79.6% of hospitalised patients. In the ambulatory population, 46.4% of patients were adherent with nutrition recommendations. Within the hospital, there was a significant difference in implementation of nutrition recommendations based on age (p = 0.047), hospital department (p = 0.002) and LOS (p = 0.04), whereas, in the ambulatory population, there were no significant differences in the rate of adherence among any of the studied characteristics. CONCLUSIONS: Dietitian recommendations are frequently implemented in the hospital, whereas adherence to such recommendations is poor in the outpatient population. Interventions to improve adherence to nutrition recommendations in the ambulatory setting are needed.

Adverse events associated with umbilical vascular catheters in the neonatal intensive care unit: A retrospective cohort study.

BACKGROUND: Umbilical catheters are commonly inserted in newborns in the neonatal intensive care unit (NICU) yet are associated with serious adverse events (AEs) such as malposition, migration, infection, thrombosis, hepatic complications, cardiac effusion, and cardiac tamponade. There is a need to determine the incidence and risk factors for AEs to inform safe practice. OBJECTIVES: The objective of this study was to determine the incidence and risk factors for AEs (all-cause and individual types) associated with umbilical venous catheters (UVCs) and umbilical arterial catheters (UACs) in the NICU. METHODS: A retrospective cohort study was conducted in an Australian level-VI NICU over a 3-year period. Any newborn who had both a UVC and UAC insertion attempt was included. RESULTS: There were 236 neonates who had 494 catheters (245 UVCs and 249 UACs). Of these, 71% of UVCs (95% confidence interval [CI]: 65.6-76.9%; incidence rate: 181.1-237.3 per 1000 catheter days) and 43.8% of UACs (95% CI: 38-50.5%; incidence rate: 102.0-146.3 per 1000 catheter days) were associated with an AE. The most common AE was malposition on first X-ray for UVCs (60.1%, 95% CI: 55.1-67.3) and UACs (32.6%, 95% CI: 26.8-39.6). A dwell time of ≥7 days was a significant predictor of UAC failure (incidence risk ratio: 1.5, 95% CI: 1.1-2.1, p = 0.006) and migration of the UVC (incidence risk ratio: 3.5, 95% CI: 1.0-11.5, p = 0.043). CONCLUSION: Adverse events related to insertion occurred in a relatively high percentage of umbilical catheters placed. Increased dwell time remains a significant risk factor for catheter migration and overall failure. Practice change and consideration of risk factors for both individual and overall AE risk are necessary to reduce complications.

COVID-19 virtual patient cohort suggests immune mechanisms driving disease outcomes.

To understand the diversity of immune responses to SARS-CoV-2 and distinguish features that predispose individuals to severe COVID-19, we developed a mechanistic, within-host mathematical model and virtual patient cohort. Our results suggest that virtual patients with low production rates of infected cell derived IFN subsequently experienced highly inflammatory disease phenotypes, compared to those with early and robust IFN responses. In these in silico patients, the maximum concentration of IL-6 was also a major predictor of CD8+ T cell depletion. Our analyses predicted that individuals with severe COVID-19 also have accelerated monocyte-to-macrophage differentiation mediated by increased IL-6 and reduced type I IFN signalling. Together, these findings suggest biomarkers driving the development of severe COVID-19 and support early interventions aimed at reducing inflammation.

Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways.

Nonresolving inflammation expands a heterogeneous population of myeloid suppressor cells capable of inhibiting T cell function. This heterogeneity has confounded the functional dissection of individual myeloid subpopulations and presents an obstacle for antitumor immunity and immunotherapy. Using genetic manipulation of cell death pathways, we found the monocytic suppressor-cell subset, but not the granulocytic subset, requires continuous c-FLIP expression to prevent caspase-8-dependent, RIPK3-independent cell death. Development of the granulocyte subset requires MCL-1-mediated control of the intrinsic mitochondrial death pathway. Monocytic suppressors tolerate the absence of MCL-1 provided cytokines increase expression of the MCL-1-related protein A1. Monocytic suppressors mediate T cell suppression, whereas their granulocytic counterparts lack suppressive function. The loss of the granulocytic subset via conditional MCL-1 deletion did not alter tumor incidence implicating the monocytic compartment as the functionally immunosuppressive subset in vivo. Thus, death pathway modulation defines the development, survival, and function of myeloid suppressor cells.

Memory CD8(+) T cells use cell-intrinsic lipolysis to support the metabolic programming necessary for development.

Generation of CD8(+) memory T cells requires metabolic reprogramming that is characterized by enhanced mitochondrial fatty-acid oxidation (FAO). However, where the fatty acids (FA) that fuel this process come from remains unclear. While CD8(+) memory T cells engage FAO to a greater extent, we found that they acquired substantially fewer long-chain FA from their external environment than CD8(+) effector T (Teff) cells. Rather than using extracellular FA directly, memory T cells used extracellular glucose to support FAO and oxidative phosphorylation (OXPHOS), suggesting that lipids must be synthesized to generate the substrates needed for FAO. We have demonstrated that memory T cells rely on cell intrinsic expression of the lysosomal hydrolase LAL (lysosomal acid lipase) to mobilize FA for FAO and memory T cell development. Our observations link LAL to metabolic reprogramming in lymphocytes and show that cell intrinsic lipolysis is deterministic for memory T cell fate.

Ectoparasites associated with the Bushveld gerbil (Gerbilliscus leucogaster) and the role of the host and habitat in shaping ectoparasite diversity and infestations.

Rodents are known hosts for various ectoparasite taxa such as fleas, lice, ticks and mites. South Africa is recognized for its animal diversity, yet little is published about the parasite diversity associated with wild rodent species. By focusing on a wildlife-human/domestic animal interface, the study aims to record ectoparasite diversity and levels of infestations of the Bushveld gerbil, Gerbilliscus leucogaster, and to establish the relationship between ectoparasite infestation parameters and host- and habitat factors. Rodents (n = 127) were trapped in 2 habitat types (natural and agricultural) during 2014­2020. More than 6500 individuals of 32 epifaunistic species represented by 21 genera and belonging to 5 taxonomic groups (fleas, sucking lice, ticks, mesostigmatan mites and trombiculid mites) were collected. Mesostigmatan mites and lice were the most abundant and fleas and mesostigmatan mites the most prevalent groups. Flea and mesostigmatan mite numbers and mesostigmatan mite species richness was significantly higher on reproductively active male than female rodents. Only ticks were significantly associated with habitat type, with significantly higher tick numbers and more tick species on rodents in the natural compared to the agricultural habitat. We conclude that the level of infestation by ectoparasites closely associated with the host (fleas and mites) was affected by host-associated factors, while infestation by ectoparasite that spend most of their life in the external environment (ticks) was affected by habitat type.

The North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition Position on the Role of the Registered Dietitian Nutritionist in the Care of the Pediatric Patient With Chronic Gastrointestinal Diseases.

The optimization of nutrition is essential for the growth and development of all children, including those with gastrointestinal (GI) conditions that can variably affect nutrient intake, absorption, or metabolism. Registered Dietitian Nutritionists (RDNs) are essential partners in delivering high quality care for pediatric GI disorders, but limited evidence is available to support the role of the RDN in the care of these patients. This position paper outlines the evidence supporting the role of the RDN in the management of chronic pediatric GI issues in both inpatient and outpatient settings. Gaps in the literature, opportunities for future research, and barriers to RDN access are discussed.

Evaluation of an electronic medical record-based Paediatric Nutrition Screening Tool.

BACKGROUND: Nutrition screening is recommended to identify children at risk for malnutrition. A unique screening tool was developed based on American Society for Parenteral and Enteral Nutrition (ASPEN) recommendations and embedded in the electronic medical record to assess for nutrition risk. METHODS: The components of the tool included the Paediatric Nutrition Screening Tool (PNST) and other elements recommended by ASPEN. To evaluate the screening tool, retrospective data were analysed on all patients admitted to acute care units of Children's Wisconsin in 2019. Data collected included nutrition screen results, diagnosis and nutrition status. All patients who received at least one full nutrition assessment by a registered dietitian (RD) were included in analysis. RESULTS: One thousand five hundred seventy-five patients were included in analysis. The following screen elements were significantly associated with a diagnosis of malnutrition: any positive screen (p < 0.001), >2 food allergies (p = 0.009), intubation (p < 0.001), parenteral nutrition (p = 0.005), RD-identified risk (p < 0.001), positive risk per the PNST (p < 0.001), BMI-for-age or weight-for-length z-score (p < 0.001), intake <50% for 3 days (p = 0.012) and NPO > 3 days (p = 0.009). The current screen had a sensitivity of 93.9%, specificity of 20.3%, positive predictive value (PPV) of 30.9% and negative predictive value (NPV) of 89.8%. This is compared with the PNST which had a sensitivity of 32%, specificity of 94.2%, PPV of 71% and NPV of 75.8% in this study population. CONCLUSION: This unique screening tool is useful for predicting nutrition risk and has a greater sensitivity than the PNST alone.

Best Practices and Educator Strategies for Facilitating a Flipped Classroom in Graduate Medical Education.

OBJECTIVE: Effective flipped classroom (FC) education fosters learner engagement, promoting higher-level cognitive skills. FC learning in graduate medical education (GME) has increased, but few educators have significant experience with FC facilitation. There are no evidence-based practices to support professional development of FC facilitation skills in GME. The objective of this study is to identify best practices for effective FC facilitation in GME. STUDY DESIGN: We conducted a mixed-methods, cross-sectional study of faculty educators who participated in a randomized controlled trial (RCT) using FC for physiology education in neonatal-perinatal medicine. Educators completed a 25-question survey about effective strategies for FC facilitation. A subset of educators participated in interviews to share their FC facilitation experiences and strategies to maximize learner engagement. Quantitative survey data were analyzed with descriptive statistics. Qualitative survey and interview data were coded and analyzed inductively to identify themes. RESULTS: Seventy-five educators completed the survey (75/136, 55% response rate), and 11 participated in semistructured interviews. While educators facilitated a median of two FC sessions (interquartile range: 1, 5) during the RCT, 43 (57%) had not received prior training in FC facilitation. Qualitative data analyses generated five themes that aligned with quantitative survey results: (1) educator preferences, (2) unique FC facilitation skills, (3) learning environment optimization, (4) subject matter expertise, and (5) learner behavior management. Sixty-two educators (83%) felt they were well prepared to lead FC sessions. Thirty-six educators (48%) reported that unprepared learners disrupt the learning environment, and the provision of clear expectations and adequate time to prepare for FCs improves learner preparation. Strategies to facilitate effective FC sessions included creating a safe learning environment and engaging learners in critical thinking. CONCLUSION: Educators highlighted faculty development needs, strategies, and actions to promote effective FC facilitation. Further exploration through learner interviews will provide additional evidence for the development of best practices and resources for FC facilitation. KEY POINTS: · Educators prefer the FC educational modality over traditional didactic lectures.. · Prior experiences in simulation debriefing provide foundational skills for new FC facilitators.. · Setting learner expectations and ensuring safe space in the classroom encourage learner engagement.. · Educator and learner preparation for FC is essential to optimize the learning experience.. · Unique approaches in facilitation are required to support all types of learners..