Transcriptome and Proteome Profiling of Primary Human Gastric Interstitial Cells of Cajal Predicts Pacemaker Networks.

Background/Aims: Interstitial cells of Cajal (ICC) are specialized gastrointestinal (GI) pacemaker cells required for normal GI motility. Dysfunctions in ICC have been reported in patients with GI motility disorders, such as gastroparesis, who exhibit debilitating symptoms and greatly reduced quality of life. While the proteins, calcium-activated chloride channel anoctamin-1 (ANO1) and the receptor tyrosine kinase (KIT), are known to be expressed by human ICC, relatively little is known about the broad molecular circuitry underpinning human ICC functions. The present study therefore investigates the transcriptome and proteome of ANO1-expressing, KITlow/CD45-/CD11B- ICC obtained from primary human gastric tissue. Methods: Excess human gastric tissue resections were obtained from sleeve gastrectomy patients. ICC were purified using fluorescence-activated cell sorting (FACSorting). Then, ICC were characterized by using immunofluorescence, real-time polymerase chain reaction, RNA-sequencing and mass spectrometry. Results: Compared to unsorted cells, real-time polymerase chain reaction showed the KITlow/CD45-/CD11B- ICC had: a 9-fold (P < 0.05) increase in ANO1 expression; unchanged KIT expression; and reduced expression for genes associated with hematopoietic cells (CD68, > 10-fold, P < 0.001) and smooth muscle cells (DES, > 4-fold, P < 0.05). RNA-sequencing and gene ontology analyses of the KITlow/CD45-/CD11B- cells revealed a transcriptional profile consistent with ICC function. Similarly, mass spectrometry analyses of the KITlow/CD45-/CD11B- cells presented a proteomic profile consistent with ICC activities. STRING-based protein interaction analyses using the RNA-sequencing and proteomic datasets predicted protein networks consistent with ICC-associated pacemaker activity and ion transport. Conclusion: These new and complementary datasets provide a valuable molecular framework for further understanding how ICC pacemaker activity regulates smooth muscle contraction in both normal GI tissue and GI motility disorders.

Single-cell RNA sequencing predicts motility networks in purified human gastric interstitial cells of Cajal.

BACKGROUND: Gastrointestinal (GI) motility disorders affect millions of people worldwide, yet they remain poorly treated in part due to insufficient knowledge of the molecular networks controlling GI motility. Interstitial cells of Cajal (ICC) are critical GI pacemaker cells, and abnormalities in ICC are implicated in GI motility disorders. Two cell surface proteins, KIT and ANO1, are used for identifying ICC. However, difficulties accessing human tissue and the low frequency of ICC in GI tissues have meant human ICC are insufficiently characterized. Here, a range of characterization assays including single-cell RNA sequencing (scRNA-seq) was performed using KIT+ CD45- CD11B- primary human gastric ICC to better understand networks controlling human ICC biology. METHODS: Excess sleeve gastrectomy tissues were dissected; ICC were analyzed by immunofluorescence, fluorescence-activated cell sorting (FACSorting), real-time PCR, mass spectrometry, and scRNA-seq. KEY RESULTS: Immunofluorescence identified ANO1+ /KIT+ cells throughout the gastric muscle. Compared to the FACSorted negative cells, PCR showed the KIT+ CD45- CD11B- ICC were enriched 28-fold in ANO1 expression (p < 0.01). scRNA-seq analysis of the KIT- CD45+ CD11B+ and KIT+ CD45- CD11B- ICC revealed separate clusters of immune cells and ICC (respectively); cells in the ICC cluster expressed critical GI motility genes (eg, CAV1 and PRKG1). The scRNA-seq data for these two cell clusters predicted protein interaction networks consistent with immune cell and ICC biology, respectively. CONCLUSIONS & INFERENCES: The single-cell transcriptome of purified KIT+ CD45- CD11B- human gastric ICC presented here provides new molecular insights and hypotheses into evolving models of GI motility. This knowledge will provide an improved framework to investigate targeted therapies for GI motility disorders.

Understanding the Biology of Human Interstitial Cells of Cajal in Gastrointestinal Motility.

Millions of patients worldwide suffer from gastrointestinal (GI) motility disorders such as gastroparesis. These disorders typically include debilitating symptoms, such as chronic nausea and vomiting. As no cures are currently available, clinical care is limited to symptom management, while the underlying causes of impaired GI motility remain unaddressed. The efficient movement of contents through the GI tract is facilitated by peristalsis. These rhythmic slow waves of GI muscle contraction are mediated by several cell types, including smooth muscle cells, enteric neurons, telocytes, and specialised gut pacemaker cells called interstitial cells of Cajal (ICC). As ICC dysfunction or loss has been implicated in several GI motility disorders, ICC represent a potentially valuable therapeutic target. Due to their availability, murine ICC have been extensively studied at the molecular level using both normal and diseased GI tissue. In contrast, relatively little is known about the biology of human ICC or their involvement in GI disease pathogenesis. Here, we demonstrate human gastric tissue as a source of primary human cells with ICC phenotype. Further characterisation of these cells will provide new insights into human GI biology, with the potential for developing novel therapies to address the fundamental causes of GI dysmotility.

Institutional review of patients presenting with suspected appendicitis.

BACKGROUND: Appendicitis is a common gastrointestinal surgical emergency. Treatment balances the risks of negative appendicectomy (NA) against potential complicated appendicitis in determining clinical priority. This study reviewed the population characteristics, results of the diagnostic modalities and Alvarado score (AlvS) of patients with suspected appendicitis. METHODS: A clinical audit of emergency appendicectomies was performed. Generalized linear models with a binomial distribution were used to evaluate the association between the age groups, gender, white cell count (WCC), neutrophil count (NC) and C-reactive protein (CRP) levels versus NAs and the different types of appendicitis. The utilization and accuracy of preoperative ultrasound and computed tomography (CT) and a preliminary analysis of AlvS were also evaluated. RESULTS: Patients 17 to 24 years old had significantly higher odds of NA but lower odds of complicated appendicitis compared with patients above 40 years. Adult women and men had significantly higher odds of NA and suppurative appendicitis (SA), respectively. Only adults with SA and acute appendicitis had significantly higher odds of raised WCC, NC and CRP. The sensitivity of CT for adult females was high (100%). Patients who had CT and an AlvS of more than 7 did not have NAs. CONCLUSION: Elevated WCC, NC and CRP were all associated with acute appendicitis and SA in adults only. CT is useful for refining the diagnosis in adult females. A combination of inflammatory markers, ultrasound and AlvS may be used selectively to complement or maximize the advantages of CT.