Publications

@article{magnusson_exposure_2024,

title = {Exposure of Colon-Derived Epithelial Monolayers to Fecal Luminal Factors from Patients with Colon Cancer and Ulcerative Colitis Results in Distinct Gene Expression Patterns},

author = {Maria K. Magnusson and Anna Bas Forsberg and Alexandra Verveda and Maria Sapnara and Julie Lorent and Otto Savolainen and Yvonne Wettergren and Hans Strid and Magnus Simrén and Lena Öhman},

url = {https://www.mdpi.com/1422-0067/25/18/9886},

doi = {10.3390/ijms25189886},

issn = {1422-0067},

year  = {2024},

date = {2024-09-13},

urldate = {2024-09-13},

journal = {Int. J. Mol. Sci.},

volume = {25},

number = {18},

pages = {9886},

abstract = {Microbiota and luminal components may affect epithelial integrity and thus participate in the pathophysiology of colon cancer (CC) and inflammatory bowel disease (IBD). Therefore, we aimed to determine the effects of fecal luminal factors derived from patients with CC and ulcerative colitis (UC) on the colonic epithelium using a standardized colon-derived two-dimensional epithelial monolayer. The complex primary human stem cell-derived intestinal epithelium model, termed RepliGut® Planar, was expanded and passaged in a two-dimensional culture which underwent stimulation for 48 h with fecal supernatants (FS) from CC patients (n = 6), UC patients with active disease (n = 6), and healthy subjects (HS) (n = 6). mRNA sequencing of monolayers was performed and cytokine secretion in the basolateral cell culture compartment was measured. The addition of fecal supernatants did not impair the integrity of the colon-derived epithelial monolayer. However, monolayers stimulated with fecal supernatants from CC patients and UC patients presented distinct gene expression patterns. Comparing UC vs. CC, 29 genes were downregulated and 33 genes were upregulated, for CC vs. HS, 17 genes were downregulated and five genes were upregulated, and for UC vs. HS, three genes were downregulated and one gene was upregulated. The addition of FS increased secretion of IL8 with no difference between the study groups. Fecal luminal factors from CC patients and UC patients induce distinct colonic epithelial gene expression patterns, potentially reflecting the disease pathophysiology. The culture of colonic epithelial monolayers with fecal supernatants derived from patients may facilitate the exploration of IBD- and CC-related intestinal microenvironmental and barrier interactions.},

note = {Publisher: Multidisciplinary Digital Publishing Institute},

keywords = {colon cancer, epithelial barrier, epithelial monolayers, inflammatory bowel disease, intestinal microenvironment},

pubstate = {published},

tppubtype = {article}

}

@article{debad_trust_2024,

title = {Trust your gut: Establishing confidence in gastrointestinal models – An overview of the state of the science and contexts of use},

author = {Susan Debad and David Allen and Omari Bandele and Colin Bishop and Michaela Blaylock and Paul Brown and Maureen K. Bunger and Julia Y. Co and Lynn Crosby and Amber B. Daniel and Steve S. Ferguson and Kevin Ford and Gonçalo Gamboa da Costa and Kristin H. Gilchrist and Matthew W. Grogg and Maureen Gwinn and Thomas Hartung and Simon P. Hogan and Ye Eun Jeong and George E. N. Kass and Elaina Kenyon and Nicole C. Kleinstreuer and Ville Kujala and Patrik Lundquist and Joanna Matheson and Shaun D. McCullough and Angela Melton-Celsa and Steven Musser and Ilung Oh and Oluwakemi B. Oyetade and Sarita U. Patil and Elijah J. Petersen and Nakissa Sadrieh and Christie M. Sayes and Benjamin S. Scruggs and Yu-Mei Tan and Bill Thelin and M. Tyler Nelson and José V. Tarazona and John F. Wambaugh and Jun-Young Yang and Changwoo Yu and Suzanne Fitzpatrick},

url = {https://altex.org/index.php/altex/article/view/2787},

doi = {10.14573/altex.2403261},

issn = {1868-8551},

year  = {2024},

date = {2024-07-16},

urldate = {2024-07-16},

journal = {ALTEX},

volume = {41},

number = {3},

pages = {402–424},

abstract = {The webinar series and workshop titled “Trust Your Gut: Establishing Confidence in Gastrointestinal Models – An Overview of the State of the Science and Contexts of Use” was co-organized by NICEATM, NIEHS, FDA, EPA, CPSC, DoD, and the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and hosted at the National Institutes of Health in Bethesda, MD, USA on October 11-12, 2023. New approach methods (NAMs) for assessing issues of gastrointestinal tract (GIT)- related toxicity offer promise in addressing some of the limitations associated with animal-based assessments. GIT NAMs vary in complexity, from two-dimensional monolayer cell line-based systems to sophisticated 3-dimensional organoid systems derived from human primary cells. Despite advances in GIT NAMs, challenges remain in fully replicating the complex interactions and pro­cesses occurring within the human GIT. Presentations and discussions addressed regulatory needs, challenges, and innovations in incorporating NAMs into risk assessment frameworks; explored the state of the science in using NAMs for evaluating systemic toxicity, understanding absorption and pharmacokinetics, evaluating GIT toxicity, and assessing potential allergenicity; and discussed strengths, limitations, and data gaps of GIT NAMs as well as steps needed to establish confidence in these models for use in the regulatory setting. 

Plain language summaryNon-animal methods to assess whether chemicals may be toxic to the human digestive tract promise to complement or improve on animal-based methods. These approaches, which are based on human or animal cells and/or computer models, are faced with their own technical challenges and need to be shown to predict adverse effects in humans. Regulators are tasked with evaluating submitted data to best protect human health and the environment. A webinar series and workshop brought together scientists from academia, industry, military, and regulatory authorities from dif­ferent countries to discuss how non-animal methods can be integrated into the risk assessment of drugs, food additives, dietary supplements, pesticides, and industrial chemicals for gastrointestinal toxicity.},

keywords = {Adverse events, Co-culture model, Enteroendocrine Cells, epithelial barrier, Gut barrier function, Gut liver microphysiological system, in vitro models, intestinal barrier, microphysiological system, microphysiological systems, organ-on-chips},

pubstate = {published},

tppubtype = {article}

}

@article{sarma_design_2023,

title = {Design of 8-mer Peptides that Block Clostridioides difficile Toxin A in Intestinal Cells},

author = {Sudeep Sarma and Carly M. Catella and Ellyce T. San Pedro and Xingqing Xiao and Deniz Durmusoglu and Stefano Menegatti and Nathan Crook and Scott T. Magness and Carol K. Hall},

doi = {10.1101/2023.01.10.523493},

year  = {2023},

date = {2023-01-12},

urldate = {2023-01-12},

pages = {2023.01.10.523493},

abstract = {Clostridioides difficile ( C. diff .) is a bacterium that causes severe diarrhea and inflammation of the colon. The pathogenicity of C. diff . infection is derived from two major toxins, toxins A (TcdA) and B (TcdB). Peptide inhibitors that can be delivered to the gut to inactivate these toxins are an attractive therapeutic strategy. In this work, we present a new approach that combines a pep tide b inding d esign algorithm (PepBD), molecular-level simulations, rapid screening of candidate peptides for toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block the glucosyltransferase activity of TcdA by targeting its glucosyltransferase domain (GTD). Using PepBD and explicit-solvent molecular dynamics simulations, we identified seven candidate peptides, SA1-SA7. These peptides were selected for specific TcdA GTD binding through a custom solid-phase peptide screening system, which eliminated the weaker inhibitors SA5-SA7. The efficacies of SA1-SA4 were then tested using a trans-epithelial electrical resistance (TEER) assay on monolayers of the human gut epithelial culture model. One peptide, SA1, was found to block TcdA toxicity in primary-derived human jejunum (small intestinal) and colon (large intestinal) epithelial cells. SA1 bound TcdA with a K D of 56.1 ± 29.8 nM as measured by surface plasmon resonance (SPR). 

SIGNIFICANCE STATEMENT: Infections by Clostridioides difficile , a bacterium that targets the large intestine (colon), impact a significant number of people worldwide. Bacterial colonization is mediated by two exotoxins: toxins A and B. Short peptides that can inhibit the biocatalytic activity of these toxins represent a promising strategy to prevent and treat C. diff . infection. We describe an approach that combines a Peptide B inding D esign (PepBD) algorithm, molecular-level simulations, a rapid screening assay to evaluate peptide:toxin binding, a primary human cell-based assay, and surface plasmon resonance (SPR) measurements to develop peptide inhibitors that block Toxin A in small intestinal and colon epithelial cells. Importantly, our designed peptide, SA1, bound toxin A with nanomolar affinity and blocked toxicity in colon cells.},

keywords = {Adverse events, epithelial barrier, In vitro model, inflammatory bowel disease, intestinal organoids, intestinal stem cells, microbiome},

pubstate = {published},

tppubtype = {article}

}

@article{speer_evaluation_2019,

title = {Evaluation of human primary intestinal monolayers for drug metabolizing capabilities},

author = {Jennifer E. Speer and Yuli Wang and John K. Fallon and Philip C. Smith and Nancy L. Allbritton},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829970/},

doi = {10.1186/s13036-019-0212-1},

issn = {1754-1611},

year  = {2019},

date = {2019-11-04},

urldate = {2019-11-04},

journal = {J. Biol. Eng},

volume = {13},

pages = {82},

abstract = {Background 

The intestinal epithelium is a major site of drug metabolism in the human body, possessing enterocytes that house brush border enzymes and phase I and II drug metabolizing enzymes (DMEs). The enterocytes are supported by a porous extracellular matrix (ECM) that enables proper cell adhesion and function of brush border enzymes, such as alkaline phosphatase (ALP) and alanyl aminopeptidase (AAP), phase I DMEs that convert a parent drug to a more polar metabolite by introducing or unmasking a functional group, and phase II DMEs that form a covalent conjugate between a functional group on the parent compound or sequential metabolism of phase I metabolite. In our effort to develop an in vitro intestinal epithelium model, we investigate the impact of two previously described simple and customizable scaffolding systems, a gradient cross-linked scaffold and a conventional scaffold, on the ability of intestinal epithelial cells to produce drug metabolizing proteins as well as to metabolize exogenously added compounds. While the scaffolding systems possess a range of differences, they are most distinguished by their stiffness with the gradient cross-linked scaffold possessing a stiffness similar to that found in the in vivo intestine, while the conventional scaffold possesses a stiffness several orders of magnitude greater than that found in vivo. 

Results 

The monolayers on the gradient cross-linked scaffold expressed CYP3A4, UGTs 2B17, 1A1 and 1A10, and CES2 proteins at a level similar to that in fresh crypts/villi. The monolayers on the conventional scaffold expressed similar levels of CYP3A4 and UGTs 1A1 and 1A10 DMEs to that found in fresh crypts/villi but significantly decreased expression of UGT2B17 and CES2 proteins. The activity of CYP3A4 and UGTs 1A1 and 1A10 was inducible in cells on the gradient cross-linked scaffold when the cells were treated with known inducers, whereas the CYP3A4 and UGT activities were not inducible in cells grown on the conventional scaffold. Both monolayers demonstrate esterase activity but the activity measured in cells on the conventional scaffold could not be inhibited with a known CES2 inhibitor. Both monolayer culture systems displayed similar ALP and AAP brush border enzyme activity. When cells on the conventional scaffold were incubated with a yes-associated protein (YAP) inhibitor, CYP3A4 activity was greatly enhanced suggesting that mechano-transduction signaling can modulate drug metabolizing enzymes. 

Conclusions 

The use of a cross-linked hydrogel scaffold for expansion and differentiation of primary human intestinal stem cells dramatically impacts the induction of CYP3A4 and maintenance of UGT and CES drug metabolizing enzymes in vitro making this a superior substrate for enterocyte culture in DME studies. This work highlights the influence of mechanical properties of the culture substrate on protein expression and the activity of drug metabolizing enzymes as a critical factor in developing accurate assay protocols for pharmacokinetic studies using primary intestinal cells. 

Graphical abstract},

keywords = {Absorptive Enterocyte Monolayers, Bioavailability, drug metabolising enzymes ({DME}), epithelial barrier, Fg, Intestinal Epithelial Cells, permeability},

pubstate = {published},

tppubtype = {article}

}