Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem-cell self-renewal.

The canonical Wnt/ß-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling ß-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5+ intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/ß-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5+ ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5+ ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5+ ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

Changes in Antibiotic Treatment for Children With Lyme Meningitis 2015-2020.

OBJECTIVES: Although ceftriaxone has been the first-line treatment of Lyme meningitis, the 2020 Infectious Disease Society of America, American College of Rheumatology, and American Academy of Neurology guideline recommends either doxycycline or ceftriaxone. Our objective was to explore recent trends in enteral antibiotic treatment of children with Lyme meningitis. METHODS: Using International Classification of Diseases, 10th Revision codes for case identification, we performed a multicenter retrospective study of patients ≤21 years of age presenting to a pediatric hospital contributing to the Pediatric Health Information System who were diagnosed with Lyme meningitis from 2015 to 2020. Our primary outcome was treatment with parenteral antibiotics, defined by either a procedure code for indwelling catheter placement or >7 days of inpatient parenteral Lyme disease-specific therapy. To examine trends over the study period, we used a generalized estimating equation, with parenteral antibiotics as the dependent variable and study year as the independent variable, adjusted for patient age, sex, race, ethnicity, and insurance status clustered by hospital. RESULTS: We identified 239 patients with Lyme meningitis treated at 24 participating centers. Overall, 48 (20.0%) were treated with parenteral antibiotics, with a declining rate over the study period (43.8% in 2015 to 8.9% in 2020). After adjustment, the odds of treatment with a parenteral antibiotic decreased over the study period (adjusted odds ratio 0.57; 95% confidence interval 0.41-0.80). CONCLUSIONS: Parenteral antibiotic treatment of children with Lyme meningitis has been on the decline in advance of the published clinical guideline. Carefully designed comparative effectiveness studies are needed to examine the effect of oral versus parenteral treatment regimens on clinical outcomes.

Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity.

Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.