Hyperfractionated Treatment with 177Lu-Octreotate Increases Tumor Response in Human Small-Intestine Neuroendocrine GOT1 Tumor Model

Radionuclide treatment of patients with neuroendocrine tumors has advanced in the last decades with favorable results using 177Lu-octreotate. However, the gap between the high cure rate in animal studies vs. patient studies indicates a potential to increase the curation of patients. The aim of this study was to investigate the tumor response for different fractionation schemes with 177Lu-octreotate. BALB/c mice bearing a human small-intestine neuroendocrine GOT1 tumor were either mock treated with saline or injected intravenously with a total of 30–120 MBq of 177Lu-octreotate: 1 × 30, 2 × 15, 1 × 60, 2 × 30, 1 × 120, 2 × 60, or 3 × 40 MBq. The tumor volume was measured twice per week until the end of the experiment. The mean tumor volume for mice that received 2 × 15 = 30 and 1 × 30 MBq 177Lu-octreotate was reduced by 61% and 52%, respectively. The mean tumor volume was reduced by 91% and 44% for mice that received 2 × 30 = 60 and 1 × 60 MBq 177Lu-octreotate, respectively. After 120 MBq 177Lu-octreotate, given as 1–3 fractions, the mean tumor volume was reduced by 91–97%. Multiple fractions resulted in delayed regrowth and prolonged overall survival by 20–25% for the 120 MBq groups and by 45% for lower total activities, relative to one fraction. The results indicate that fractionation and hyperfractionation of 177Lu-octreotate are beneficial for tumor reduction and prolongs the time to regrowth.

Determination of CYP450 Expression Levels in the Human Small Intestine by Mass Spectrometry-Based Targeted Proteomics

The human small intestine can be involved in the first-pass metabolism of drugs. Under this condition, members of the CYP450 superfamily are expected to contribute to drug presystemic biotransformation. The aim of this study was to quantify protein expression levels of 16 major CYP450 isoforms in tissue obtained from nine human organ donors in seven subsections of the small intestine, i.e., duodenum (one section, N = 7 tissue samples), jejunum (three subsections (proximal, mid and distal), N = 9 tissue samples) and ileum (three subsections, (proximal, mid and distal), N = 9 tissue samples), using liquid chromatography tandem mass spectrometry (LC-MS/MS) based targeted proteomics. CYP450 absolute protein expression levels were compared to mRNA levels and enzyme activities by using established probe drugs. Proteins corresponding to seven of sixteen potential CYP450 isoforms were detected and quantified in various sections of the small intestine: CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, CYP3A5 and CYP4F2. Wide inter-subject variability was observed, especially for CYP2D6. CYP2C9 (p = 0.004) and CYP2C19 (p = 0.005) expression levels decreased along the small intestine. From the duodenum to the ileum, CYP2J2 (p = 0.001) increased, and a trend was observed for CYP3A5 (p = 0.13). CYP3A4 expression was higher in the jejunum than in the ileum (p = 0.03), while CYP4F2 expression was lower in the duodenum compared to the jejunum and the ileum (p = 0.005). CYP450 protein levels were better correlated with specific isoform activities than with mRNA levels. This study provides new data on absolute CYP450 quantification in human small intestine that could improve physiologically based pharmacokinetic models. These data could better inform drug absorption profiles while considering the regional expression of CYP450 isoforms.

Enterotoxigenic Escherichia coli degrades the host MUC2 mucin barrier to facilitate critical pathogen-enterocyte interactions in human small intestine.

Enterotoxigenic Escherichia coli (ETEC) are a genetically diverse pathologic variant of E. coli defined by the production of heat-labile (LT) and/or heat-stable (ST) toxins. ETEC are estimated to cause hundreds of millions of cases of diarrheal illness annually. However, it is not clear that all strains are equally equipped to cause disease and asymptomatic colonization with ETEC is common in low-middle income regions lacking basic sanitation and clean water where ETEC are ubiquitous. Recent molecular epidemiology studies have revealed a significant association between strains which produce EatA, a secreted autotransporter protein, and the development of symptomatic infection. Here, we demonstrate that LT stimulates production of MUC2 mucin by goblet cells in human small intestine, enhancing the protective barrier between pathogens and enterocytes. In contrast, using explants of human small intestine as well as small intestinal enteroids, we show that EatA counters this host defense by engaging and degrading the MUC2 mucin barrier to promote bacterial access to target enterocytes and ultimately toxin delivery suggesting that EatA plays a crucial role in the molecular pathogenesis of ETEC. These findings may inform novel approaches to prevention of the acute diarrheal illness as well as the sequelae associated with ETEC and other pathogens that rely on EatA and similar proteases for efficient interaction with their human hosts.

Quantitative sequencing clarifies the role of disruptor taxa, oral microbiota, and strict anaerobes in the human small-intestine microbiome

Background Upper gastrointestinal (GI) disorders and abdominal pain afflict between 12 and 30% of the worldwide population and research suggests these conditions are linked to the gut microbiome. Although large-intestine microbiota have been linked to several GI diseases, the microbiota of the human small intestine and its relation to human disease has been understudied. The small intestine is the major site for immune surveillance in the gut, and compared with the large intestine, it has greater than 100 times the surface area and a thinner and more permeable mucus layer. Results Using quantitative sequencing, we evaluated total and taxon-specific absolute microbial loads from 250 duodenal-aspirate samples and 21 paired duodenum-saliva samples from participants in the REIMAGINE study. Log-transformed total microbial loads spanned 5 logs and were normally distributed. Paired saliva-duodenum samples suggested potential transmission of oral microbes to the duodenum, including organisms from the HACEK group. Several taxa, including Klebsiella, Escherichia, Enterococcus, and Clostridium, seemed to displace strict anaerobes common in the duodenum, so we refer to these taxa as disruptors. Disruptor taxa were enriched in samples with high total microbial loads and in individuals with small intestinal bacterial overgrowth (SIBO). Absolute loads of disruptors were associated with more severe GI symptoms, highlighting the value of absolute taxon quantification when studying small-intestine health and function. Conclusion This study provides the largest dataset of the absolute abundance of microbiota from the human duodenum to date. The results reveal a clear relationship between the oral microbiota and the duodenal microbiota and suggest an association between the absolute abundance of disruptor taxa, SIBO, and the prevalence of severe GI symptoms.

Enterotoxigenic Escherichia coli degrades the host MUC2 mucin barrier to facilitate critical pathogen-enterocyte interactions in human small intestine.

Enterotoxigenic Escherichia coli (ETEC) are a genetically diverse pathologic variant of E. coli defined by the production of heat-labile (LT) and/or heat-stable (ST) toxins. ETEC are estimated to cause hundreds of millions of cases of diarrheal illness annually. However, it is not clear that all strains are equally equipped to cause disease and asymptomatic colonization with ETEC is common in low-middle income regions lacking basic sanitation and clean water where ETEC are ubiquitous. Recent molecular epidemiology studies have revealed a significant association between strains which produce EatA, a secreted autotransporter protein, and the development of symptomatic infection. Here, we demonstrate that LT stimulates production of MUC2 mucin by goblet cells in human small intestine, enhancing the protective barrier between pathogens and enterocytes. In contrast, using explants of human small intestine as well as small intestinal enteroids, we show that EatA counters this host defense by engaging and degrading the MUC2 mucin barrier to promote bacterial access to target enterocytes and ultimately toxin delivery suggesting that EatA plays a crucial role in the molecular pathogenesis of ETEC. These findings may inform novel approaches to prevention of the acute diarrheal illness as well as the sequelae associated with ETEC and other pathogens that rely on EatA and similar proteases for efficient interaction with their human hosts.

A proximal-to-distal survey of healthy adult human small intestine and colon epithelium by single-cell transcriptomics

Background and Aims: Single-cell transcriptomics offer unprecedented resolution of tissue function at the cellular level, yet studies in healthy adult human small intestine and colon are sparse. Here, we present single-cell transcriptomics from 3 humans covering the duodenum, jejunum, ileum, and ascending, transverse, and descending colon. Methods: 12,590 single epithelial cells from three independently processed organ donors were evaluated for organ-specific lineage biomarkers, differentially regulated genes, receptors, and drug targets. Analyses focused on intrinsic cell properties and capacity for response to extrinsic signals along the gut axis across different humans. Results: Cells were assigned to 25 epithelial lineage clusters. Human intestinal stem cells (ISCs) are not specifically marked by many murine ISC markers. Lysozyme expression is not unique to Paneth cells (PCs), and PCs lack expression of expected niche-factors. BEST4 cells express NPY and show functional and maturational differences between SI and colon. Tuft cells possess a broad ability to interact with the innate and adaptive immune systems through previously unreported receptors. Some classes of mucins, hormones, cell-junction, and nutrient absorption genes show unappreciated regional expression differences across lineages. Differential expression of receptors and drug targets across lineages reveals biological variation and potential for variegated responses. Conclusions: Our study identifies novel lineage marker genes; covers regional differences; shows important differences between mouse and human gut epithelium; and reveals insight into how the epithelium responds to the environment and drugs. This comprehensive cell atlas of the healthy adult human intestinal epithelium resolves data gaps in anatomical regions along the gastrointestinal tract and advances our understanding of human intestinal physiology.