Biologicals in the prevention and treatment of intestinal graft rejection: The state of the art: Biologicals in Intestinal Transplantation.

Intestinal transplantation is the standard treatment for patients with intestinal failure with severe complications due to parenteral nutrition; however, rejection leads to graft failure in approximately half of both adult and pediatric recipients within 5 years of transplantation. Although intensive immunosuppressive therapy is used in an attempt to reduce this risk, commonly used treatment strategies are generally practice- and/or expert-based, as head-to-head comparisons are lacking. In this ever-developing field, biologicals designed to prevent or treat rejection are used increasingly, with both infliximab and vedolizumab showing potential in the treatment of acute cellular rejection in individual cases and in relatively small patient cohorts. To help advance progress in clinical care, we review the current use of biologicals in intestinal transplantation, and we provide future perspectives to guide this progress.

Gut mucosa dissociation protocols influence cell type proportions and single-cell gene expression levels.

Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of the cellular landscape of organs. Most single-cell protocols require fresh material, which limits sample size per experiment, and consequently, introduces batch effects. This is especially true for samples acquired through complex medical procedures, such as intestinal mucosal biopsies. Moreover, the tissue dissociation procedure required for obtaining single cells is a major source of noise; different dissociation procedures applied to different compartments of the tissue induce artificial gene expression differences between cell subsets. To overcome these challenges, we have developed a one-step dissociation protocol and demonstrated its use on cryopreserved gut mucosal biopsies. Using flow cytometry and scRNA-seq analysis, we compared this one-step dissociation protocol with the current gold standard, two-step collagenase digestion, and an adaptation of a recently published alternative, three-step cold-active Bacillus licheniformus protease digestion. Both cell viability and cell type composition were comparable between the one-step and two-step collagenase dissociation, with the former being more time-efficient. The cold protease digestion resulted in equal cell viability, but better preserves the epithelial cell types. Consequently, to analyze the rarer cell types, such as glial cells, larger total biopsy cell numbers are required as input material. The multi-step protocols affected cell types spanning multiple compartments differently. In summary, we show that cryopreserved gut mucosal biopsies can be used to overcome the logistical challenges and batch effects in large scRNA-seq studies. Furthermore, we demonstrate that using cryopreserved biopsies digested using a one-step collagenase protocol enables large-scale scRNA-seq, FACS, organoid generation and intraepithelial lymphocyte expansion.

Inflammation status modulates the effect of host genetic variation on intestinal gene expression in inflammatory bowel disease.

More than 240 genetic risk loci have been associated with inflammatory bowel disease (IBD), but little is known about how they contribute to disease development in involved tissue. Here, we hypothesized that host genetic variation affects gene expression in an inflammation-dependent way, and investigated 299 snap-frozen intestinal biopsies from inflamed and non-inflamed mucosa from 171 IBD patients. RNA-sequencing was performed, and genotypes were determined using whole exome sequencing and genome wide genotyping. In total, 28,746 genes and 6,894,979 SNPs were included. Linear mixed models identified 8,881 independent intestinal cis-expression quantitative trait loci (cis-eQTLs) (FDR < 0.05) and interaction analysis revealed 190 inflammation-dependent intestinal cis-eQTLs (FDR < 0.05), including known IBD-risk genes and genes encoding immune-cell receptors and antibodies. The inflammation-dependent cis-eQTL SNPs (eSNPs) mainly interact with prevalence of immune cell types. Inflammation-dependent intestinal cis-eQTLs reveal genetic susceptibility under inflammatory conditions that can help identify the cell types involved in and the pathways underlying inflammation, knowledge that may guide future drug development and profile patients for precision medicine in IBD.

Understanding human gut diseases at single-cell resolution.

Our understanding of gut functioning and pathophysiology has grown considerably in the past decades, and advancing technologies enable us to deepen this understanding. Single-cell RNA sequencing (scRNA-seq) has opened a new realm of cellular diversity and transcriptional variation in the human gut at a high, single-cell resolution. ScRNA-seq has pushed the science of the digestive system forward by characterizing the function of distinct cell types within complex intestinal cellular environments, by illuminating the heterogeneity within specific cell populations and by identifying novel cell types in the human gut that could contribute to a variety of intestinal diseases. In this review, we highlight recent discoveries made with scRNA-seq that significantly advance our understanding of the human gut both in health and across the spectrum of gut diseases, including inflammatory bowel disease, colorectal carcinoma and celiac disease.

Predicted efficacy of a pharmacogenetic passport for inflammatory bowel disease.

BACKGROUND: High inter-individual variability in therapeutic response to drugs used in the management of Inflammatory Bowel Disease (IBD) leads to high morbidity and high costs. Genetic variants predictive of thiopurine-induced myelosuppression, thiopurine-induced pancreatitis and immunogenicity of Tumour Necrosis Factor alpha (TNFα) antagonists have been identified, but uptake of pre-treatment pharmacogenetic testing into clinical guidelines has been slow. AIM: To explore the efficacy of a pharmacogenetic passport for IBD that includes multiple pharmacogenetic predictors of response. METHODS: Patients with IBD exposed to thiopurines and/or TNFα antagonists were retrospectively evaluated for the presence of thiopurine toxicity and/or immunogenicity of TNFα antagonists. All patients were genotyped using both whole-exome sequencing and the Illumina Global Screening Array. An in-house-developed computational pipeline translated genetic data into an IBD pharmacogenetic passport that predicted risks for thiopurine toxicity and immunogenicity of TNFα antagonists per patient. Using pharmacogenetic-guided treatment guidelines, we calculated clinical efficacy estimates for pharmacogenetic testing for IBD. RESULTS: Among 710 patients with IBD exposed to thiopurines and/or TNFα antagonists, 150 adverse drug responses occurred and our pharmacogenetic passport would have predicted 54 (36%) of these. Using a pharmacogenetic passport for IBD that includes genetic variants predictive of thiopurine-induced myelosuppression, thiopurine-induced pancreatitis, and immunogenicity of TNFα antagonists, 24 patients need to be genotyped to prevent one of these adverse drug responses. CONCLUSIONS: This study highlights the clinical efficacy of a pharmacogenetic passport for IBD. Implementation of such a pharmacogenetic passport into clinical management of IBD may contribute to a reduction in adverse drug responses.