Prostaglandin E2 promotes intestinal repair through an adaptive cellular response of the epithelium

Adaptive cellular responses are often required during wound repair. Following disruption of the intestinal epithelium, wound‐associated epithelial (WAE) cells form the initial barrier over the wound. Our goal was to determine the critical factor that promotes WAE cell differentiation. Using an adaptation of our in vitro primary epithelial cell culture system, we found that prostaglandin E2 (PGE (2)) signaling through one of its receptors, Ptger4, was sufficient to drive a differentiation state morphologically and transcriptionally similar to in vivo WAE cells. WAE cell differentiation was a permanent state and dominant over enterocyte differentiation in plasticity experiments. WAE cell differentiation was triggered by nuclear β‐catenin signaling independent of canonical Wnt signaling. Creation of WAE cells via the PGE (2)‐Ptger4 pathway was required in vivo, as mice with loss of Ptger4 in the intestinal epithelium did not produce WAE cells and exhibited impaired wound repair. Our results demonstrate a mechanism by which WAE cells are formed by PGE (2) and suggest a process of adaptive cellular reprogramming of the intestinal epithelium that occurs to ensure proper repair to injury

A stem-cell-derived platform enables complete Cryptosporidium development in vitro and genetic tractability

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using air-liquid interface (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion \u3e 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions

Optimized methods to measure acetoacetate, 3-hydroxybutyrate, glycerol, alanine, pyruvate, lactate and glucose in human blood using a centrifugal analyser with a fluorimetric attachment

Optimized methods are described for the analysis of glucose, lactate, pyruvate, alanine, glycerol, D-3-hydroxybutyrate and acetoacetate in perchloric acid extracts of human blood using the Cobas Bio centrifugal analyser. Glucose and lactate are measured using the photometric mode and other metabolites using the fluorimetric mode. The intra-assay coefficients of variation ranged from 0.7 to 4.1%, except with very low levels of pyruvate and acetoacetate where the coefficients of variation were 7.1 and 12% respectively. All seven metabolites can be measured in a perchloric acid extract of 20 μl of blood. The methods have been optimized with regard to variation in the perchloric acid content of the samples. These variations arise from the method of sample preparation used to minimize changes occurring in metabolite concentration after venepuncture

Vocal Fatigue als Indikator für komplexe Stimmstörungen – eine diagnostische und therapeutische Herausforderung

Eine alltagseinschränkende, pathologische Stimmermüdung (Vocal Fatigue) wird v. a. bei BerufssprecherInnen (z. B. LehrerInnen) zunehmend beobachtet und als Indikator für komplexe Stimmstörungen betrachtet. Da bislang wenig systematische Studien zu diesem Phänomen existieren, fehlt bislang eine einheitliche Diagnostik. Anhand der Kasuistik eines Berufssprechers werden Möglichkeiten einer strukturierten Erhebung von Vocal Fatigue vorgestellt und im Hinblick auf ihre laryngoskopischen, instrumentell akustischen („performance fatigue“) und subjektiven Merkmale („perceived fatigue“) diskutiert. Zudem wird eine Abgrenzung zu den Modellen Vocal Effort, Vocal Demand und Vocal Demand Response nach Hunter et al. (2020) vorgenommen. = Vocal fatigue is increasingly observed among professional voice users (e.g., teachers). This pathologic state is associated with restrictions in everyday life and is considered to be an indicator of complex voice disorders. Since there are few systematic studies on the phenomenon, there is currently no uniform definition or diagnostic workup. Based on the case study of a professional speaker, possibilities for a structured vocal fatigue assessment are presented. These are discussed with regard to their laryngoscopic, instrumental acoustic ("performance fatigue") and subjective symptom-related characteristics ("perceived fatigue"). In addition, a differentiation from the vocal effort, vocal demand, and vocal demand response models according to Hunter et al. (2020) is made. Keywords: Muscle tension dysphonia; Vocal Fatigue Index (VFI-D)

Neonatal mouse gut metabolites influence Cryptosporidium parvum infection in intestinal epithelial cells

The protozoan parasit

HOIL1 regulates group 2 innate lymphoid cell numbers and type 2 inflammation in the small intestine

Patients with mutations in HOIL1 experience a complex immune disorder including intestinal inflammation. To investigate the role of HOIL1 in regulating intestinal inflammation, we employed a mouse model of partial HOIL1 deficiency. The ileum of HOIL1-deficient mice displayed features of type 2 inflammation including tuft cell and goblet cell hyperplasia, and elevated expression of Il13, Il5 and Il25 mRNA. Inflammation persisted in the absence of T and B cells, and bone marrow chimeric mice revealed a requirement for HOIL1 expression in radiation-resistant cells to regulate inflammation. Although disruption of IL-4 receptor alpha (IL4Rα) signaling on intestinal epithelial cells ameliorated tuft and goblet cell hyperplasia, expression of Il5 and Il13 mRNA remained elevated. KLRG

A novel strategy to increase the proliferative potential of adult human β-cells while maintaining their differentiated phenotype

Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human β-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases β-catenin nuclear translocation and β-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human β-cell proliferation while maintaining a β-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human β-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human β-cell proliferation while maintaining the β-cell phenotype

Long-term culture captures injury-repair cycles of colonic stem cells

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hop