Biology subjects

Crawford, S. E.

Publications and source records attributed to Crawford, S. E..

3 recordsLinked to original sources

bioRxiv2021

Drivers of Transcriptional Variance in Human Intestinal Epithelial Organoids

Background & AimsHuman intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. MethodsRNA-sequencing datasets from 251 experiments performed on 35 human enteroid and colonoid lines from 28 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and Gene Set Enrichment Analysis (GSEA) was used to identify differentially expressed genes and enriched of pathways. ResultsPERMANOVA, Pearson correlations, and dendrogram analysis of all data indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3D, transwell, and monolayer) had the largest effect (7,271-1,305 differentially expressed genes-DEGs); segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect (5,977-420 DEGs), and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell vs. monolayer cultures, and cholesterol biosynthesis genes enriched in Matrigel vs. collagen cultures. ConclusionsSurprisingly large differences in organoid transcriptome were driven by variations in culture methods such as format and substrate, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal organoids.

molecular biology↗

bioRxiv2021

TNF Blockade Reduces Prostatic Hyperplasia and Inflammation while Limiting BPH Diagnosis in Patients with Autoimmune Disease

Benign prostatic hyperplasia (BPH) is ostensibly linked to autoimmune (AI) diseases, but whether the prostate is a target of systemic inflammation associated with AI conditions is unknown. Prostatic inflammation is linked to fibrosis, hyperplasia, and reduced responses to BPH-related medical therapies. This study was conducted to determine if AI disease correlates with BPH diagnosis and whether systemic targeting of an inflammatory mediator limits prostatic inflammation and hyperplasia. Patient medical records (n=112,152) were evaluated to determine BPH prevalence among different AI diseases. Inflammatory cells from human BPH tissues were analyzed by single-cell (sc)RNA-seq and the tumor necrosis factor (TNF)-antagonist etanercept was tested in two murine models of prostatic enlargement. BPH prevalence was significantly higher among patients with AI disease compared to unaffected individuals. However, AI patients treated with TNF-antagonists had a significantly reduced incidence of BPH. Data from scRNA- seq identified macrophages as a dominant source of TNF and in vitro assays confirmed that TNF stimulates BPH-derived fibroblast proliferation. In the AI patient cohort and murine models, systemic treatment with TNF-antagonists decreased prostatic epithelial proliferation, macrophage infiltration, and epithelial NF{kappa}B activation compared to control tissues. These studies are the first to show that patients with AI diseases have a heightened susceptibility to BPH and that the TNF-signaling axis is important for BPH pathogenesis. Macrophage-secreted TNF may mechanistically drive BPH via chronic activation of the signaling axis and NF{kappa}B. TNF blockade appears to be a promising new pharmacological approach to target inflammation and suppress BPH. One sentence summaryPatient data and mouse models suggest that repurposing tumor necrosis factor alpha blockade reduces inflammation-mediated prostatic hyperplasia.

cell biology↗

bioRxiv2020

New Insights and Enhanced Human Norovirus Cultivation in Human Intestinal Enteroids

Human noroviruses (HuNoVs) are the leading cause of epidemic and sporadic acute gastroenteritis worldwide. We previously demonstrated human intestinal stem cell-derived enteroids (HIEs) support cultivation of several HuNoV strains. However, HIEs did not support virus replication from every HuNoV-positive stool sample, which led us to test and optimize new media conditions, identify characteristics of stool samples that allow replication, and evaluate consistency of replication over time. Optimization of our HIE-HuNoV culture system has shown that: 1) A new HIE culture media made with conditioned medium from a single cell line and commercial media promote robust replication of HuNoV strains that replicated poorly in HIEs grown in our original culture media made with conditioned media from 3 separate cell lines; 2) GI.1, eleven GII genotypes (GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII.8, GII.12, GII.13, GII.14 and GII.17) and six GII.4 variants, can be cultivated in HIEs; 3) successful replication is more likely with virus in stools with higher virus titers; 4) GII.4_Sydney_2012 virus replication was reproducible over three years; and 5) HuNoV infection is restricted to the small intestine, based on replication in duodenal and ileal HIEs but not colonoids from the same donors. These results improve the HIE culture system for HuNoV replication. Use of HIEs by several laboratories worldwide to study the molecular mechanisms that regulate HuNoV replication confirms the usefulness of this culture system and our optimized methods for virus replication will advance the development of effective therapies and methods for virus control. ImportanceHuman noroviruses (HuNoVs) are highly contagious and cause acute and sporadic diarrheal illness in all age groups. In addition, chronic infections occur in immunocompromised cancer and transplant patients. These viruses are antigenically and genetically diverse and there are strain-specific differences in binding to cellular attachment factors. In addition, new discoveries are being made on strain-specific differences in virus entry and replication and the epithelial cell response to infection in human intestinal enteroids. Human intestinal enteroids are a biologically-relevant model to study HuNoVs; however, not all strains can be cultivated at this time. A complete understanding of HuNoV biology thus requires cultivation conditions that will allow the replication of multiple strains. We report optimization of HuNoV cultivation in human intestinal enteroid cultures to increase the numbers of cultivatable strains and the magnitude of replication, which is critical for testing antivirals, neutralizing antibodies and methods of virus inactivation.

microbiology↗