A variety of topics in basic science research are being pursued in the IBD Center.
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Investigators: John Hegarty, David Stewart
Hypothesis: Dequalinium analogues are a reliable transporter for antisense nucleotides designed to treat C. difficile infection, while having limited toxicity on non-pathogenic bacteria.
Summary: This study involves the synthesis of dequalinium analogues as potential transporters for antisense molecules (both phosphorothioates and phosphorodiamidate morpholinos). Complexed dequalinium analogue-antisense molecule complexes will be tested on cultures of C. difficile to evaluate the ability of these complexes to kill C. difficile, while sparing non-difficile organisms.
Collaborators: Volkmar Weissig, PhD (Midwestern University), Arun Sharma, PhD (Organic Synthesis Core), Jacek Krzeminski, PhD (Organic Synthesis Core)
Funding: Clinical and Translational Science Institute (CTSI) Grant, Penn State University.
Investigators: John Hegarty, David Stewart
Hypothesis: Fidaxomicin has the least detrimental effect on the microbiome of the human gut, and microbial co-occurrence patterns of fidaxomicin treated patients will be more like CDI-negative patients than those subjects treated with either metronidazole or vancomycin
Summary: This study involves the collection of human stools from C. difficile positive and negative patients, with in vitro treatment of stool aliquots from each subject using metronidazole, vancomycin and fidaxomicin. Microbiota testing using 16s rRNA and ITS sequencing will be performed to evaluate the effect these antibiotics have on the community of bacteria and fungi present in the human gut, as well as evaluating changes in community co-occurrences which might have a causal role in recurrent C. difficile infection.
Collaborators: Gina Lamendella, PhD (Juniata College)
Extramural funding: Merck
Investigators: John Hegarty, David Stewart
Hypothesis: Creation of the first atomic-scale structural models of C. difficile bacteriocins in their pre- and post-contracted states.
Summary: As a potential alternative agent for treating CDI, our group reported the species-specific in vitro antibacterial activity of R-type contractile bacteriocins from C. difficile. The first cryo-EM atomic-scale model of similar contractile structures was recently described (Nat Struct Mol Biol. 2015 May;22(5):377-82). We have successfully developed a novel preparation method to achieve the high-quality, concentrated C. difficile bacteriocins required for these studies. These samples are currently being analyzed by direct-electron detection cryo-electron microscopy to generate data sets for 3D reconstruction.
Collaborators: Kristin Shingler and Susan Hafenstein PhD Depts. Medicine, Microbiology and Immunology, James Conway PhD (University of Pittsburgh)
Extramural funding: Barsumian Trust
Investigators: Leonard R. Harris III, Sue Deiling, Walter Koltun
Hypothesis: Disease activity, including malignant degeneration will be associated with changes in serum proteins that may be used as less invasive and more accurate measures of disease activity.
Summary: Proteomics measures the concentrations of serum cytokines and other biologically active proteins that may play a role in disease processes. Proteomics may thus be used to possibly identify biomarkers that correlate with disease activity, complications or relapse. Serum was collected preop (within 7 days prior to surgery) and postop (1 month, 3 month, 6 month, 12 month) from patients receiving total proctocolectomy for Ulcerative Colitis, including those who developed dysplasia or malignancy due to their colitis. Serum was subsequently run on series of gels and the specific proteome of that individual including quantification of minor proteins, their isomers and post translational modification was analyzed with the goal of identifying protein markers for disease activity and dysplasia and/or cancer in UC patients, by comparing pre- and post-colectomy proteasomes.
Collaborators: David S. Phelps, PhD (Pediatrics), Todd M. Umstead (Pediatrics)
Funding: Peter and Marshia Carlino Fund for IBD research
Investigators: Joel Coble, Gregory Yochum, Walter Koltun
Hypothesis: Diverticular disease (DD) is a complex disease with both environmental but also significant genetic components. The identification of the genetic factors involved in disease pathogenesis may assist in the medical and surgical management of DD patients.
Summary: Increasing evidence suggest as much as 50% of diverticular disease (DD) has a genetic basis, yet to date little research has been done to identify the possible genetic contributors to disease. We have recruited a family with strong penetrance of DD at a young age and performed total exome sequencing of 5 of the family members, 3 affected and 2 unaffected. By identifying the variants found only in the affected individuals and triaging those, we have found highly suggestive variants in several genes, including one in a Laminin gene, LAMB4 and another in the COL1A1 gene. By then specifically sequencing both these genes in 160 other individuals with ‘sporadic’ diverticular disease we found further allelic variants in both LAMB4 and COL1A1. Using immunofluorescent techniques, we have shown a significant decrease in the expression of LAMB4 in the myenteric plexus of patients with diverticular disease compared to controls. This lack of expression may affect the development of the enteric nervous system, which is known to have altered morphology in DD patients. Similarly additional variants have also been found in the COL1A1 gene and staining has shown a significant increase in this collagen type in DD patients, suggesting possible defects in structural integrity that could lead to the development of diverticuli. This research is the first to provide possible genetic and molecular mechanisms by which DD can develop and may help to identify patients who may be predisposed to DD.
Collaborators: Dr. James Broach (Biochemistry & Molecular Biology), Dr. Glenn Gerhard (Temple), Dr. Feng Yue (Biochemistry & Molecular Biology/Bioinformatics), Dr. Francesca Ruggiero (Pathology)
Funding: Peter and Marshia Carlino Fund for IBD research
Investigators: Katie Schieffer, Matthew Coates, Gregory Yochum, Walter Koltun
Hypothesis: Having an animal model of a common surgical procedure for children with severe IBD is critical to understand differences in surgical outcomes, to study tissue responses over time, and to test responses to given therapeutics.
Summary: In severe pediatric cases of ulcerative colitis, diseased tissue is removed and the small intestine is joined to the anus during ileal-anal anastomosis surgery. For reasons not fully defined, persistent inflammation hinders recovery in some patients. We have generated a model of this surgery in rats and will use it to further understand underlying mechanisms of inflammation, and interventions that promote inflammatory resolution over time.
Funding: Peter and Marshia Carlino Fund for IBD research
Investigators: Katie Schieffer, Leonard Harris, Sue Deiling, Gregory Yochum, Walter Koltun
Hypothesis: Host genetics and microbiome differences may explain why one of two siblings FAP siblings developed pouchitis following IPAA surgery.
Summary: Total proctocolectomy with ileal pouch-anal anastomosis (IPAA) is a common surgical method for treatment of patients with familial adenomatous polyposis (FAP) and ulcerative colitis (UC). Inflammation of the ileal pouch reservoir, termed pouchitis, is common in UC patients, but rare in FAP. In a family with FAP, two siblings received IPAA surgery with one subsequently developing pouchitis while the other was spared. We are using 16s rRNA gene sequencing to interrogate the mucosal bacteria at the time of surgery and identifying IBD associated SNPs using our custom array to determine whether differences in host genetics and the microbiome can explain the differential susceptibility to the onset of pouchitis.
Collaborators: Regina Lamendella, Justin R. Wright (Juniata College)
Funding: Peter and Marshia Carlino Fund for IBD research
Investigators: Matthew Coates, MD, PhD
Summary: Many individuals with inflammatory bowel disease (IBD) experience chronic abdominal discomfort, even during seemingly quiescent phases of their disease, and no one is sure why. This proposal is designed to investigate the potential role that intestinal serotonin plays in this condition.
Potential Mechanism: NIH K23
Investigators: Katie Schieffer, Sue Deiling, Leonard Harris, Christine Choi, Gregory Yochum, Walter Koltun
Hypothesis: Differentially expressed genes will demonstrate pathophysiologic mechanisms underlying early onset diverticulitis.
Summary: While there are several environmental and genetic factors recognized to contribute to the progression of diverticulosis to diverticulitis, causative genes have not been identified. Recognizing RNA-Seq as the current approach to investigate gene expression, we sought to compare RNA expression between full-thickness sigmoid specimens among patients with distinct clinical characteristics: young patients with family history, elderly patients without a family history, and control patients with no evidence of diverticulosis. Additional validation will be performed using SYBR green to analyze gene expression of individual genes that are suggested by the RNA-Seq data analysis.
Funding: Peter and Marshia Carlino Fund for IBD research
Investigators: Joel Coble, Christine Choi, Walter Koltun
Hypothesis: Causative variants of Crohn’s Disease will be discovered by exome sequencing and SNP analysis in a 3-generation family in which the first affected patient, a fraternal twin, has affected offspring while the unaffected twin does not.
Summary: Inflammatory bowel disease (IBD) is a complex genetic disease known to be associated with over 160 susceptibility genetic loci. However, the pathophysiologic impact of the majority of these genetic loci is unclear. Crohn’s disease has a recognized familial disposition and individual studies of families provides opportunity for novel discoveries of relevant genes. On such family pedigree included a family with fraternal twins with an affected twin had two affected offspring, while the unaffected twin did not have disease nor progeny with disease.(Figure) This study utilized two approaches; 1) use of our laboratory’s custom IBD microarray made which queries over 300 single nucleotide polymorphisms (SNPs) associated with IBD, and 2) Exome sequencing. We investigated whether the diseased and non-diseased family members shared SNP genotype patterns, and also utilized the exome sequences of 6 family members to investigate other potential causative genetic variants.
Collaborators: James Broach, PhD (Chair of the Department of Biochemistry and Molecular Biology, Director of the Penn State Institute for Personalized Medicine), Lisa Schneper, PhD (Research Associate, Department of Biochemistry and Molecular Biology), Arthur Berg, PhD (Associate Professor of Biostatistics and Bioinformatics, Director of the Bioinformatics Core, Director of the PhD program in Biostatistics, Leader of the Biomedical Informatics Program (CTSI)), Joel Coble (current PhD candidate)
Investigators: Katie Schieffer, Leonard Harris, Sue Deiling, John Hegarty, Christine Choi, Gregory Yochum, Walter Koltun
Hypothesis: Clinical disease characteristics, and prognosis including surgical outcomes can be correlated and predicted by patient genotyping of relevant genes, that will fundamentally assist with medical and surgical decision making.
Summary: This custom Illumina microarray consists of 324 single nucleotide polymorphisms that have been previously investigated to have association with inflammatory bowel disease (IBD). An additional 50 SNPs represent the variety of the lab’s interests in colorectal disease, including colorectal cancer, diverticulitis and infectious colitis. This microarray has been updated regularly by previous investigators under Dr. Koltun’s direction since 2009.
Collaborators: Arthur Berg, PhD (Associate Professor of Biostatistics and Bioinformatics, Director of the Bioinformatics Core, Director of the PhD program in Biostatistics, Leader of the Biomedical Informatics Program (CTSI))
Funding: Peter and Marshia Carlino Fund for IBD research
Investigators: Katie Schieffer, Leonard Harris, Sue Deiling, Gregory Yochum, Walter Koltun
Hypothesis: Diverticular disease is a complex genetic disorder that will be characterized in part by allele variants in various genes of immune function.
Summary: Analysis of single nucleotide polymorphisms (SNPs) has been instrumental in the investigation of several diseases. In 2012 Jostins et al identified several additional susceptible genetic loci associated with Inflammatory Bowel Disease (IBD) using the Immunochip, a microarray designed by Illumina which investigates over 250,000 SNPs many of which are associated with several immunologic disorders. Given that diverticulitis is an inflammatory disease characterized by the presence of infection in patients with diverticulosis, we will use the Immunochip to investigate categories of patients with diverticulitis, based on varying disease severity: 1) asymptomatic diverticulosis, 2) medically managed diverticulitis, 3) surgically managed diverticulitis, 4) no diverticulosis.
Collaborators: Arthur Berg, PhD (Associate Professor of Biostatistics and Bioinformatics, Director of the Bioinformatics Core, Director of the PhD program in Biostatistics, Leader of the Biomedical Informatics Program (CTSI)); James Broach, Institute for Personalized Medicine Director.
Funding: Peter and Marshia Carlino Fund for IBD research
Investigators: Melanie Eshelman, Walter Koltun, Gregory Yochum
Hypothesis: The TCF7L1 transcription factor promotes CRC growth by repressing expression of tumor suppressor genes.
Summary: Wnt/beta-catenin signaling controls proliferation in the intestinal crypt microenvironment. T-cell factor (TCF) transcription factors bind beta-catenin in the nucleus to activate target gene expression. We found that the TCF7L1 family member functions as an oncogene and we are currently identifying the direct target genes required for this phenotype.
Funding: Pending NIH grant