Advances in imaging specific mediators of inflammatory bowel disease

Published: 21 August 2018Inflammatory bowel disease (IBD) is characterized by chronic remitting and relapsing inflammation of the lower gastrointestinal tract. The etiology underlying IBD remains unknown, but it is thought to involve a hypersensitive immune response to environmental antigens, including the microbiota. Diagnosis and monitoring of IBD is heavily reliant on endoscopy, which is invasive and does not provide information regarding specific mediators. This review describes recent developments in imaging of IBD with a focus on positron emission tomography (PET) and single-photon emission computed tomography (SPECT) of inflammatory mediators, and how these developments may be applied to the microbiota.Nicole Dmochowska, Hannah R. Wardill and Patrick A. Hughe

Nanoparticles Targeted to Fibroblast Activation Protein Outperform PSMA for MRI Delineation of Primary Prostate Tumors

OnlinePublAccurate delineation of gross tumor volumes remains a barrier to radiotherapy dose escalation and boost dosing in the treatment of solid tumors, such as prostate cancer. Magnetic resonance imaging (MRI) of tumor targets has the power to enable focal dose boosting, particularly when combined with technological advances such as MRI-linear accelerator. Fibroblast activation protein (FAP) is overexpressed in stromal components of >90% of epithelial carcinomas. Herein, the authors compare targeted MRI of prostate specific membrane antigen (PSMA) with FAP in the delineation of orthotopic prostate tumors. Control, FAP, and PSMA-targeting iron oxide nanoparticles were prepared with modification of a lymphotropic MRI agent (FerroTrace, Ferronova). Mice with orthotopic LNCaP tumors underwent MRI 24 h after intravenous injection of nanoparticles. FAP and PSMA nanoparticles produced contrast enhancement on MRI when compared to control nanoparticles. FAP-targeted MRI increased the proportion of tumor contrast-enhancing black pixels by 13%, compared to PSMA. Analysis of changes in R2 values between healthy prostates and LNCaP tumors indicated an increase in contrast-enhancing pixels in the tumor border of 15% when targeting FAP, compared to PSMA. This study demonstrates the preclinical feasibility of PSMA and FAP-targeted MRI which can enable targeted image-guided focal therapy of localized prostate cancer.Nicole Dmochowska, Valentina Milanova, Ramesh Mukkamala, Kwok Keung Chow, Nguyen T. H. Pham, Madduri Srinivasarao, Lisa M. Ebert, Timothy Stait-Gardner, Hien Le, Anil Shetty, Melanie Nelson, Philip S. Low, and Benjamin Thierr

Transcriptome Analysis of the Arabidopsis Megaspore Mother Cell Uncovers the Importance of RNA Helicases for Plant Germline Development

Germ line specification is a crucial step in the life cycle of all organisms. For sexual plant reproduction, the megaspore mother cell (MMC) is of crucial importance: it marks the first cell of the plant “germline” lineage that gets committed to undergo meiosis. One of the meiotic products, the functional megaspore, subsequently gives rise to the haploid, multicellular female gametophyte that harbours the female gametes. The MMC is formed by selection and differentiation of a single somatic, sub-epidermal cell in the ovule. The transcriptional network underlying MMC specification and differentiation is largely unknown. We provide the first transcriptome analysis of an MMC using the model plant Arabidopsis thaliana with a combination of laser-assisted microdissection and microarray hybridizations. Statistical analyses identified an over-representation of translational regulation control pathways and a significant enrichment of DEAD/DEAH-box helicases in the MMC transcriptome, paralleling important features of the animal germline. Analysis of two independent T-DNA insertion lines suggests an important role of an enriched helicase, MNEME (MEM), in MMC differentiation and the restriction of the germline fate to only one cell per ovule primordium. In heterozygous mem mutants, additional enlarged MMC-like cells, which sometimes initiate female gametophyte development, were observed at higher frequencies than in the wild type. This closely resembles the phenotype of mutants affected in the small RNA and DNA-methylation pathways important for epigenetic regulation. Importantly, the mem phenotype shows features of apospory, as female gametophytes initiate from two non-sister cells in these mutants. Moreover, in mem gametophytic nuclei, both higher order chromatin structure and the distribution of LIKE HETEROCHROMATIN PROTEIN1 were affected, indicating epigenetic perturbations. In summary, the MMC transcriptome sets the stage for future functional characterization as illustrated by the identification of MEM, a novel gene involved in the restriction of germline fate

Immuno-PET of Innate Immune Markers CD11b and IL-1β Detects Inflammation in Murine Colitis

Inflammatory bowel disease (IBD) is a chronic relapsing and remitting inflammatory disease of the gastrointestinal tract. The diagnosis and monitoring of IBD are reliant on endoscopy, which is invasive and does not provide information on specific mediators. Symptom flare in IBD is associated with increased activation of innate immune pathways. Immuno-PET approaches have previously demonstrated the ability to detect colitis; however, a direct comparison of antibodies targeted to innate immune mediators and cells has not been done. We aimed to compare immuno-PET of antibodies to IL-1β and CD11b against standard 18F-FDG and MRI approaches to detect colonic inflammation. Methods: Colonic concentrations of IL-1β and myeloperoxidase were determined by ELISA, and colonic infiltration by CD11b-positive CD3-negative innate immune cells was determined by flow cytometry and compared between healthy and dextran sodium sulphate-treated colitic mice. PET of 89Zr-lα-IL-1β, 89Zr-α-CD11b, and 18F-FDG was compared by volume-of-interest analysis and with MRI by region-of-interest analysis. Imaging results were confirmed by ex vivo biodistribution analysis. Results: Colonic inflammation was associated with impaired colonic epithelial barrier permeability, increased colonic IL-1β and myeloperoxidase concentrations, and increased CD11b-positive CD3-negative innate immune cell infiltration into the colon. 89Zr-α-IL-1β and 89Zr-α-CD11b immuno-PET detected colonic inflammation, as did 18F-FDG, and all PET tracers were more sensitive than MRI. Although 18F-FDG volumes of interest correlated with colitis severity and a strong trend was observed with 89Zr-α-IL-1β, no correlation was observed for 89Zr-α-CD11b or MRI. 89Zr-α-IL-1β was distributed mainly to the gastrointestinal tract, whereas 89Zr-α-CD11b was distributed to more tissue types. Conclusion: Immuno-PET using antibodies directed to innate immune markers detected colonic inflammation, with 89Zr-α-IL-1β providing a more tissue-specific signal than 89Zr-α-CD11b. Development of these technologies for human subjects will potentially provide a less invasive approach than endoscopy for diagnosing and monitoring IBD.Nicole Dmochowska, William Tieu, Marianne D. Keller, Hannah R. Wardill, Chris Mavrangelos, Melissa A. Campaniello, Prab Takhar and Patrick A. Hughe

(89)Zr-pro-MMP-9 F(ab')(2) detects colitis induced intestinal and kidney fibrosis

Intestinal fibrosis is a common complication of inflammatory bowel disease but remains difficult to detect. Matrix metalloproteases (MMPs) have key roles in fibrosis and are therefore potential targets for fibrosis detection. We determined whether immunoPET of F(ab')2 antibody fragments targeting MMPs detects colitis induced colonic fibrosis. Mice were administered 2% dextran sulfate sodium treated water for 1 cycle (inflamed) or 3 cycles (fibrotic), or were untreated (control). Colonic and kidney collagen, innate cytokine, MMPs and fecal MPO concentrations were analyzed by multiplex/ELISA. α-pro-MMP-9 F(ab')2 fragments were engineered and conjugated to 89Zr for PET imaging, ex-vivo Cherenkov analysis and bio-distribution. Colonic innate cytokine concentrations and fecal myeloperoxidase were increased in inflamed mice but not fibrotic mice, while collagen concentrations were increased in fibrotic mice. MMPs were increased in inflamed mice, but only pro-MMP-9 remained increased in fibrotic mice. 89Zr-pro-MMP-9 F(ab')2 uptake was increased in the intestine but also in the kidney of fibrotic mice, where collagen and pro-MMP-9 concentrations were increased. 89Zr-pro-MMP-9 F(ab')2 detects colitis induced intestinal fibrosis and associated kidney fibrosis.Nicole Dmochowska, William Tieu, Marianne D. Keller, Courtney A. Hollis, Melissa A. Campaniello, Chris Mavrangelos, Prab Takhar and Patrick A. Hughe

Acute colitis drives tolerance by persistently altering the epithelial barrier and innate and adaptive immunity

BACKGROUND:Inflammatory bowel disease (IBD) has a remitting and relapsing disease course; however, relatively little is understood regarding how inflammatory damage in acute colitis influences the microbiota, epithelial barrier, and immune function in subsequent colitis. METHODS:Mice were administered trinitrobenzene sulphonic acid (TNBS) via enema, and inflammation was assessed 2 days (d2) or 28 days (d28) later. Colitis was reactivated in some mice by re-treating at 28 days with TNBS and assessing 2 days later (d30). Epithelial responsiveness to secretagogues, microbiota composition, colonic infiltration, and immune activation was compared between all groups. RESULTS:At day 28, the distal colon had healed, mucosa was restored, and innate immune response had subsided, but colonic transepithelial transport (P = 0.048), regulatory T-cell (TREG) infiltration (P = 0.014), adherent microbiota composition (P = 0.0081), and responsiveness of stimulated innate immune bone marrow cells (P < 0.0001 for IL-1β) differed relative to health. Two days after subsequent instillation of TNBS (d30 mice), the effects on inflammatory damage (P < 0.0001), paracellular permeability (P < 0.0001), and innate immune infiltration (P < 0.0001 for Ly6C+ Ly6G- macrophages) were reduced relative to d2 colitis. However, TREG infiltration was increased (P < 0.0001), and the responsiveness of stimulated T cells in the mesenteric lymph nodes shifted from pro-inflammatory at d2 to immune-suppressive at d30 (P < 0.0001 for IL-10). These effects were observed despite similar colonic microbiota composition and degradation of the mucosal layer between d2 and d30. CONCLUSIONS:Collectively, these results indicate that acute colitis chronically alters epithelial barrier function and both innate and adaptive immune responses. These effects reduce the consequences of a subsequent colitis event, warranting longitudinal studies in human IBD subjects.Hannah R Wardill, Jocelyn M Choo, Nicole Dmochowska, Chris Mavrangelos, Melissa A Campaniello, Joanne M Bowen, Geraint B Rogers, Patrick A Hughe

Fecal supernatants from active ulcerative colitis patients impair colonic epithelial barrier integrity and activate pelvic mucosal extrinsic afferent nerves

Hannah R Wardill, Nicole Dmochowska, Melissa A Campaniello, Chris Mavrangelos, Joanne M Bowen, Jane M Andrews, Sam P Costello, and Patrick A Hughe

Proteolytic activation of IL-1 beta impairs epithelial barrier integrity in the ulcerative colitis lumen

AbstractP A Hughes, H R Wardill, N Dmochowska, C Mavrangelos, M Campaniello, J M Bowen, S Smid, S P Costello, J M Andrew