Publications by authors named "Daniel P Regan"

21 Publications

  • Page 1 of 1

Transcriptional regulation of alcohol induced liver fibrosis in a translational porcine hepatocellular carcinoma model.

Biochimie 2021 Mar 12;182:73-84. Epub 2021 Jan 12.

Department of Radiology, University of Illinois at Chicago, United States; National Center for Supercomputing Applications, University of Illinois at Urbana-Champaign, United States; Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, United States. Electronic address:

Hepatocellular carcinoma (HCC) is the 5th most common and 2nd deadliest cancer worldwide. HCC risk factors include alcohol induced liver cirrhosis, which prompts hepatic inflammation, cell necrosis, and fibrosis deposition. As 25% of HCC cases are associated with alcohol induced liver disease, understanding the effects of the cirrhotic liver microenvironment on HCC tumor biology and therapeutic responses are critical. This study utilized the Oncopig Cancer Model-a transgenic pig model that recapitulates human HCC through induced expression of KRAS and TP53 driver mutations-to investigate the molecular mechanisms underlying alcohol induced liver disease. Oncopigs (n = 5) underwent fibrosis induction via infusion of ethanol and ethiodized oil (1:3 v/v dosed at 0.75 mL/kg) into the hepatic arterial circulation. Eight-weeks post induction, liver tissue samples from fibrotic and age-matched control (n = 5) Oncopigs were collected for histological evaluation and transcriptional profiling. Increased hepatic inflammation and fibrosis was observed in fibrotic Oncopigs via pathological assessment. Transcriptional profiling (RNA-seq) resulted in the identification of 4387 differentially expressed genes between Oncopig fibrotic and control livers. GO term enrichment analysis identified pathway alterations associated with cirrhosis progression in humans, including cell proliferation, angiogenesis, extracellular matrix deposition, and oxidation-reduction. Key alterations include activation of hepatic stellate cells, increased matrix metalloproteinase production, and altered expression of ABC and SLC transporter genes involved in transport of anticancer drugs.These results demonstrate Oncopig liver fibrosis recapitulates transcriptional hallmarks of human cirrhosis, making the Oncopig an ideal model for studying the effects of the cirrhotic liver microenvironment on HCC tumor biology and therapeutic response.
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http://dx.doi.org/10.1016/j.biochi.2020.12.022DOI Listing
March 2021

First-in-Class Inhibitors of Oncogenic CHD1L with Preclinical Activity against Colorectal Cancer.

Mol Cancer Ther 2020 08 4;19(8):1598-1612. Epub 2020 Jun 4.

The Skaggs School of Pharmacy and Pharmaceutical Sciences, Department of Pharmaceutical Sciences, The University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Since the discovery of CHD1L in 2008, it has emerged as an oncogene implicated in the pathology and poor prognosis of a variety of cancers, including gastrointestinal cancers. However, a mechanistic understanding of CHD1L as a driver of colorectal cancer has been limited. Until now, there have been no reported inhibitors of CHD1L, also limiting its development as a molecular target. We sought to characterize the clinicopathologic link between CHD1L and colorectal cancer, determine the mechanism(s) by which CHD1L drives malignant colorectal cancer, and discover the first inhibitors with potential for novel treatments for colorectal cancer. The clinicopathologic characteristics associated with CHD1L expression were evaluated using microarray data from 585 patients with colorectal cancer. Further analysis of microarray data indicated that CHD1L may function through the Wnt/TCF pathway. Thus, we conducted knockdown and overexpression studies with CHD1L to determine its role in Wnt/TCF-driven epithelial-to-mesenchymal transition (EMT). We performed high-throughput screening (HTS) to identify the first CHD1L inhibitors. The mechanism of action, antitumor efficacy, and drug-like properties of lead CHD1L inhibitors were determined using biochemical assays, cell models, tumor organoids, patient-derived tumor organoids, and pharmacokinetics and pharmacodynamics. Lead CHD1L inhibitors display potent antitumor activity by reversing TCF-driven EMT. The best lead CHD1L inhibitor possesses drug-like properties in pharmacokinetic/pharmacodynamic mouse models. This work validates CHD1L as a druggable target and establishes a novel therapeutic strategy for the treatment of colorectal cancer.
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http://dx.doi.org/10.1158/1535-7163.MCT-20-0106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665848PMC
August 2020

Identification of a Small-Molecule Inhibitor That Disrupts the SIX1/EYA2 Complex, EMT, and Metastasis.

Cancer Res 2020 06 27;80(12):2689-2702. Epub 2020 Apr 27.

Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFβ signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFβ signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510951PMC
June 2020

Vascularized Polymers Spatially Control Bacterial Cells on Surfaces.

Adv Biosyst 2020 01 9;4(1):e1900216. Epub 2019 Dec 9.

Department of Chemical and Biomedical Engineering, University of Maine, 5737 Jenness Hall, Orono, ME, 04469, USA.

Nature uses vascular systems to permit large-area control over the functionality of surfaces that lie above them. In this work, the application of this concept to the control of a hybrid living-nonliving system is demonstrated. Defined arrangements of vascular channels are created in agar using a fugitive ink printing method. The antibiotic gentamicin is then introduced into the vascular network where it diffuses to the surface and interacts with a model system of Escherichia coli cells. The cells either live or die depending on their distance from the underlying channels, permitting spatial control over the biological system. Using single-channel systems to define critical parameters, a theoretical model is developed to define the final surface pattern based solely on the arrangement of the underlying vascular channels. The model is then successfully used to create more complex arrangements of cells at the surface. Finally, by introducing different types of active compounds into separate vascular channels, a mixture of bacterial species is separated and localized at defined points. This work demonstrates the ability of bioinspired embedded vascular systems to predictably control a biological system at a surface, laying the groundwork for future spatially and temporally controlled biointerfaces in both industry and medicine.
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http://dx.doi.org/10.1002/adbi.201900216DOI Listing
January 2020

Combining the geometry of folded paper with liquid-infused polymer surfaces to concentrate and localize bacterial solutions.

Biointerphases 2019 08 20;14(4):041005. Epub 2019 Aug 20.

Department of Chemical and Biomedical Engineering, University of Maine, 5737 Jenness Hall, Orono, Maine 04469.

Point-of-care (POC) detection and diagnostic platforms provide critical information about health and safety conditions in austere and resource-limited settings in which medical, military, and disaster relief operations are conducted. In this work, low-cost paper materials commonly used in POC devices are coated with liquid-infused polymer surfaces and folded to produce geometries that precisely localize complex liquid samples undergoing concentration by evaporation. Liquid-infused polymer surfaces were fabricated by infusing silicone-coated paper with a chemically compatible polydimethylsiloxane oil to create a liquid overlayer. Tests on these surfaces showed no remaining bacterial cells after exposure to a sliding droplet containing a concentrated solution of Escherichia coli or Staphylococcus aureus, while samples without a liquid layer showed adhesion of both microdroplets and individual bacterial cells. Folding of the paper substrates with liquid-infused polymer surfaces into several functional 3D geometries enabled a clean separation and simultaneous concentration of a liquid containing rhodamine dye into discrete, predefined locations. When used with bacteria, which are known for their ability to adhere to nearly any surface type, functional geometries with liquid-infused polymer surfaces concentrated the cells at levels significantly higher than geometries with dry control surfaces. These results show the potential of synergistically combining paper-based materials with liquid-infused polymer surfaces for the manipulation and handling of complex samples, which may help the future engineering of POC devices.
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http://dx.doi.org/10.1116/1.5114804DOI Listing
August 2019

High-fat diet induced central adiposity (visceral fat) is associated with increased fibrosis and decreased immune cellularity of the mesenteric lymph node in mice.

Eur J Nutr 2020 Jun 4;59(4):1641-1654. Epub 2019 Jun 4.

Department of Food Science and Human Nutrition, Colorado State University, 1571 Campus Delivery, 500 West Lake Street, Fort Collins, CO, 80523, USA.

Purpose: Accumulation of visceral, but not subcutaneous, adipose tissue is highly associated with metabolic disease. Inflammation inciting from adipose tissue is commonly associated with metabolic disease risk and comorbidities. However, constituents of the immune system, lymph nodes, embedded within these adipose depots remain under-investigated. We hypothesize that, lymph nodes are inherently distinct and differentially respond to diet-induced obesity much like the adipose depots they reside in.

Methods: Adipose tissue and lymph nodes were collected from the visceral and inguinal depots of male mice fed 13 weeks of standard CHOW or high fat diet (HFD). Immune cells were isolated from tissues, counted and characterized by flow cytometry or plated for proliferative capacity following Concanavalin A stimulation. Lymph node size and fibrosis area were also characterized.

Results: In HFD fed mice visceral adipose tissue accumulation was associated with significant enlargement of the lymph node encased within. The subcutaneous lymph node did not change. Compared with mice fed CHOW for 13 weeks, mice fed HFD had a decline in immune cell populations and immune cell proliferative ability, as well as, exacerbated fibrosis accumulation, within the visceral, but not subcutaneous, lymph node.

Conclusions: Obesity-induced chronic low-grade inflammation is associated with impaired immunity and increased susceptibility to disease. Excessive visceral adiposity and associated inflammation driven by diet likely leads to obesity-induced immune suppression by way of lymph node/lymphatic system pathophysiology.
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http://dx.doi.org/10.1007/s00394-019-02019-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891137PMC
June 2020

The Angiotensin Receptor Blocker Losartan Suppresses Growth of Pulmonary Metastases via AT1R-Independent Inhibition of CCR2 Signaling and Monocyte Recruitment.

J Immunol 2019 05 10;202(10):3087-3102. Epub 2019 Apr 10.

Flint Animal Cancer Center, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523;

Inflammatory monocytes have been shown to play key roles in cancer metastasis through promotion of tumor cell extravasation, growth, and angiogenesis. Monocyte recruitment to metastases is mediated primarily via the CCL2-CCR2 chemotactic axis. Thus, disruption of this axis represents an attractive therapeutic target for the treatment of metastatic disease. Losartan, a type I angiotensin II receptor (AT1R) antagonist, has been previously shown to have immunomodulatory actions involving monocyte and macrophage activity. However, the exact mechanisms accounting for these effects have not been fully elucidated. Therefore, we investigated the effects of losartan and its primary metabolite on CCL2-mediated monocyte recruitment and CCR2 receptor function using mouse tumor models and in vitro human monocyte cultures. We show, in this study, that losartan and its metabolite potently inhibit monocyte recruitment through the noncompetitive inhibition of CCL2-induced ERK1/2 activation, independent of AT1R activity. Studies in experimental metastasis models demonstrated that losartan treatment significantly reduced the metastatic burden in mice, an effect associated with a significant decrease in CD11b/Ly6C-recruited monocytes in the lungs. Collectively, these results indicate that losartan can exert antimetastatic activity by inhibiting CCR2 signaling and suppressing monocyte recruitment and therefore suggest that losartan (and potentially other AT1R blocker drugs) could be repurposed for use in cancer immunotherapy.
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http://dx.doi.org/10.4049/jimmunol.1800619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504574PMC
May 2019

NMR-based metabolic profiling of urine, serum, fecal, and pancreatic tissue samples from the Ptf1a-Cre; LSL-KrasG12D transgenic mouse model of pancreatic cancer.

PLoS One 2018 17;13(7):e0200658. Epub 2018 Jul 17.

Department of Chemistry & Biochemistry, Miami University, Oxford, Ohio, United States of America.

Pancreatic cancer is the third leading cause of cancer deaths in the United States with more than 53,000 expected to be diagnosed with the disease in 2018. The median survival time after diagnosis is four to six months. The poor survival statistics are due in part to the fact that pancreatic cancer is typically asymptomatic until it reaches advanced stages of the disease. Although surgical resection provides the best chance of survival, pancreatic cancer is rarely detected when surgery is still possible due, in part, to lack of effective biomarkers for early detection. The goal of the research reported here was to determine if it was possible to identify metabolic biomarkers for detection of pre-cancerous pancreatic intraepithelial neoplasia (PanIN) that precede pancreatic adenocarcinoma. The transgenic Ptf1a-Cre; LSL-KrasG12D mouse strain was used as a model of pancreatic cancer progression. Nuclear magnetic resonance (NMR) spectroscopy was employed to compare metabolic profiles of urine, sera, fecal extracts, and pancreatic tissue extracts collected from control and study mice aged 5, 11, and 15 months, including 47 mice with tumors. We were able to identify the following potential biomarkers: decreased 3-indoxylsulfate, benzoate and citrate in urine, decreased glucose, choline, and lactate in blood, and decreased phenylalanine and benzoate and increased acetoin in fecal extracts. Potential biomarkers were validated by p-values, PLS-DA VIP scores, and accuracies based on area under ROC curve analyses. Essentially, all of the metabolic profiling changes could be explained as being associated with the consequences of bicarbonate wasting caused by a complete substitution of the normal pancreatic acinar tissue by tissue entirely composed of PanIN. Given the nature of the mouse model used here, our results indicate that it may be possible to use NMR-based metabolic profiling to identify biomarkers for detection of precancerous PanIN that immediately precede pancreatic cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200658PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049928PMC
January 2019

Characterization of an Inducible Alcoholic Liver Fibrosis Model for Hepatocellular Carcinoma Investigation in a Transgenic Porcine Tumorigenic Platform.

J Vasc Interv Radiol 2018 08 7;29(8):1194-1202.e1. Epub 2018 Jun 7.

Department of Radiology, University of Illinois Health, 1740 West Taylor Street, MC 931, Chicago, Illinois, 60612. Electronic address:

Purpose: This study used the Oncopig Cancer Model (OCM) to develop alcohol-induced fibrosis in a porcine model capable of developing hepatocellular carcinoma.

Materials And Methods: Liver injury was induced in 8-week-old Oncopigs (n = 10) via hepatic transarterial infusion of 0.75 mL/kg ethanol-ethiodized oil (1:3 v/v). Feasibility was assessed in an initial Oncopig cohort (n = 5) by histologic analysis at 8 weeks after induction, and METAVIR results were compared to age- and sex-matched healthy controls (n = 5). Liver injury was then induced in a second OCM cohort (n = 5) for a time-course study, with post-induction disease surveillance via biweekly physical exam, lab analysis, and liver biopsies until 20 weeks after induction.

Results: In Cohort 1, 8-week post-induction liver histologic analysis revealed median METAVIR F3 (range, F3-F4) fibrosis, A2 (range, A2-A3) inflammation, and 15.3% (range, 5.0%-22.9%) fibrosis. METAVIR and inflammation scores were generally elevated compared to healthy controls (F0-F1, P = 0.0013; A0-A1, P = .0013; median percent fibrosis 8.7%, range, 5.8%-12.1%, P = .064). In Cohort 2, histologic analysis revealed peak fibrosis severity of median METAVIR F3 (range, F2-F3). However, lack of persistent alcohol exposure resulted in liver recovery, with median METAVIR F2 (range, F1-F2) fibrosis at 20 weeks after induction. No behavioral or biochemical abnormalities were observed to indicate liver decompensation.

Conclusions: This study successfully validated a protocol to develop METAVIR F3-F4 fibrosis within 8 weeks in the OCM, supporting its potential to serve as a model for hepatocellular carcinoma in a fibrotic liver background. Further investigation is required to determine if repeated alcohol liver injury is required to develop an irreversible METAVIR grade F4 porcine cirrhosis model.
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http://dx.doi.org/10.1016/j.jvir.2018.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6388685PMC
August 2018

Adipose tissue extrinsic factor: Obesity-induced inflammation and the role of the visceral lymph node.

Physiol Behav 2018 06 1;190:71-81. Epub 2018 Mar 1.

Department of Food Science and Human Nutrition, USA. Electronic address:

Obesity-related adverse health consequences occur predominately in individuals with upper body fat distribution commonly associated with increased central adiposity. Visceral adipose tissue accumulation is described to be the greatest driver of obesity-induced inflammation, however evidence also supports that the intestines fundamentally contribute to the development of obesity-induced metabolic disease. The visceral adipose depot shares the same vasculature and lymph drainage as the small intestine. We hypothesize that the visceral lymph node, which drains adipose tissue and the gastrointestinal tract, is central to the exacerbation of systemic pro-inflammation. Male C57BL/6 mice were fed CHOW or high fat diet (HFD) for 7 weeks. At termination the mesenteric depot, visceral lymph node and ileum, jejunum and Peyer's patches were collected. Cytokine concentration was determined in adipose tissue whereas immune cell populations where investigated in the visceral lymph node and intestinal segments by flow cytometry. Visceral adipose tissue and the gastrointestinal tract mutually influence immune cells enclosed within the visceral lymph node. HFD increased visceral lymph node immune cell number. This likely resulted from 1.) an increase in immune cells migration from the small intestines likely from activated dendritic cells that travel to the lymph node and 2.) cytokine effluent from visceral adipose tissue that promoted expansion, survival and retention of pro-inflammatory immune cells. Overall, the visceral lymph node, the immune nexus of visceral adipose tissue and the small intestines, likely plays a fundamental role in exacerbation of systemic pro-inflammation by HFD-induced obesity. The research of Tim Bartness greatly enhanced the understanding of adipose tissue regulation. Studies from his laboratory significantly contributed to our awareness of extrinsic factors that influence body fatness levels. Specifically, the work he produced eloquently demonstrated that adipose tissue was more complex than an insulating storage center; it was connected to our brains via the sympathetic and sensory nervous system. Mapping studies demonstrated that adipose tissue both receives and sends information to the brain. Further, his lab demonstrated that nervous system connections contributed to lipolysis, thermogenesis and adipocyte proliferation and growth. The work of Tim Bartness will continue to influence adipose tissue research. As such, Tim Bartness directly inspired the following research. Adipose tissue extrinsic factors are not limited to the peripheral nervous system. The lymphatic system is an additional extrinsic factor that cross talks with adipose tissue, however its role in this context is under emphasized. Here we begin to elucidate how the lymphatic system may contribute to the comorbidities associated with visceral adipose tissue accumulation.
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http://dx.doi.org/10.1016/j.physbeh.2018.02.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461448PMC
June 2018

Canine sarcomas as a surrogate for the human disease.

Pharmacol Ther 2018 08 9;188:80-96. Epub 2018 Mar 9.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA; Flint Animal Cancer Center, Colorado State University, Fort Collins, CO 80523, USA; University of Colorado Cancer Center, Anschutz Medical Campus, Aurora, CO 80045, USA.

Pet dogs are becoming increasingly recognized as a population with the potential to inform medical research through their treatment for a variety of maladies by veterinary health professionals. This is the basis of the One Health initiative, supporting the idea of collaboration between human and animal health researchers and clinicians to study spontaneous disease processes and treatment in animals to inform human health. Cancer is a major health burden in pet dogs, accounting for approximately 30% of deaths across breeds. As such, pet dogs with cancer are becoming increasingly recognized as a resource for studying the pharmacology and therapeutic potential of anticancer drugs and therapies under development. This was recently highlighted by a National Academy of Medicine Workshop on Comparative Oncology that took place in mid-2015 (http://www.nap.edu/21830). One component of cancer burden in dogs is their significantly higher incidence of sarcomas as compared to humans. This increased incidence led to canine osteosarcoma being an important component in the development of surgical approaches for osteosarcoma in children. Included in this review of sarcomas in dogs is a description of the incidence, pathology, molecular characteristics and previous translational therapeutic studies associated with these tumors. An understanding of the patho-physiological and molecular characteristics of these naturally occurring canine sarcomas holds great promise for effective incorporation into drug development schemas, for evaluation of target modulation or other pharmacodynamic measures associated with therapeutic response. These data could serve to supplement other preclinical data and bolster clinical investigations in tumor types for which there is a paucity of human patients for clinical trials.
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http://dx.doi.org/10.1016/j.pharmthera.2018.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6432917PMC
August 2018

Mesenchymal Stem Cells Recruit CCR2 Monocytes To Suppress Allergic Airway Inflammation.

J Immunol 2018 02 19;200(4):1261-1269. Epub 2018 Jan 19.

Center for Immune and Regenerative Medicine, Department of Clinical Sciences, Colorado State University, Fort Collins, CO 80523.

Mesenchymal stem cells (MSC) exert immune modulatory properties and previous studies demonstrated suppressive effects of MSC treatment in animal models of allergic airway inflammation. However, the underlying mechanisms have not been fully elucidated. We studied the role of MSC in immune activation and subsequent recruitment of monocytes in suppressing airway hyperresponsiveness and airway inflammation using a mouse model of allergic airway inflammation. MSC administration prior to or after allergen challenge inhibited the development of airway inflammation in allergen-sensitized mice. This was accompanied by an influx of CCR2-positive monocytes, which were localized around injected MSC in the lungs. Notably, IL-10-producing monocytes and/or macrophages were also increased in the lungs. Systemic administration of liposomal clodronate or a CCR2 antagonist significantly prevented the suppressive effects of MSC. Activation of MSC by IFN-γ leading to the upregulation of CCL2 expression was essential for the suppressive effects, as administration of wild-type MSC into IFN-γ-deficient recipients, or IFN-γ receptor-deficient or CCL2-deficient MSC into wild-type mice failed to suppress airway inflammation. These results suggest that MSC activation by IFN-γ, followed by increased expression of CCL2 and recruitment of monocytes to the lungs, is essential for suppression by MSC in allergen-induced airway hyperresponsiveness and airway inflammation.
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http://dx.doi.org/10.4049/jimmunol.1700562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809262PMC
February 2018

The Mouse Model of Pancreatic Cancer Atlas (MMPCA) for classification of pancreatic cancer lesions: A large histological investigation of the Ptf1aCre/+;LSL-KrasG12D/+ transgenic mouse model of pancreatic cancer.

PLoS One 2017 9;12(11):e0187552. Epub 2017 Nov 9.

Department of Chemistry & Biochemistry, Miami University, Oxford, Ohio, United States of America.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading forms of cancer related deaths in the United States. With limited treatment options and unreliable diagnostic methods, long-term survival rates following a diagnosis of pancreatic cancer remain poor. Pancreatic intraepithelial neoplasia (PanIN) are precancerous lesions that precede progression towards PDAC. PanIN occur in increasing complexity as the disease progresses and the description of PanIN plays a critical role in describing, staging and diagnosing PDAC. Inconsistencies in PanIN classifications exist even amongst leading pathologists. This has led to debate and confusion among researchers and pathologists involved in pancreatic cancer research, diagnosis and treatment. We have sought to initiate a discussion with leading pathologists with a goal of increasing consensus in the interpretation of PanIN and associated structures within the precancerous pancreas. Toward achieving this goal, we are in the process of conducting an extensive study of over 1000 male and female pancreata in varying stages of PanIN progression isolated from the Ptf1aCre/+;LSL-KrasG12D/+ transgenic mouse model of pancreatic cancer. Using this extensive database, we have established the Mouse Model of Pancreatic Cancer Atlas (MMPCA) to serve as a platform for meaningful and interactive discussion among researchers and pathologists who study pancreatic disease. We hope that the MMPCA will be an effective tool for promoting a more consistent and accurate consensus of PanIN classifications in the future.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187552PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679567PMC
November 2017

Immunization against full-length protein and peptides from the Lutzomyia longipalpis sand fly salivary component maxadilan protects against Leishmania major infection in a murine model.

Vaccine 2017 12 24;35(48 Pt B):6611-6619. Epub 2017 Oct 24.

Department of Clinical Sciences, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, United States; Department of Microbiology, Immunology, Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, United States.

Leishmaniasis is an arthropod vectored disease causing considerable human morbidity and mortality. Vaccination remains the most realistic and practical means to interrupt the growing number and diversity of sand fly vectors and reservoirs of Leishmania. Since transmission of Leishmania is achieved exclusively by sand fly vectors via immune-modulating salivary substances, conventional vaccination requiring an unmodified host immune response for success are potentially destined to fail unless immunomodulatory factors are somehow neutralized. Using cationic liposome DNA complexes (CLDC) as an adjuvant system along with Lu. longipalpis sand fly salivary component maxadilan (MAX) as antigen (Ag), we show that mice are protected from the MAX-induced exacerbation of infection with Leishmania major (Lm). The CLDC adjuvant and alum were comparable in terms of lesion induration and decreased parasite burden, however the alum adjuvant imposed more inflammation at the injection site. BALB/c, C3H and C57BL/6 mice vaccinated with MAX-CLDC containing either the full-length MAX or peptides spanning the N- and C-terminal regions of MAX are protected against footpad challenges with Lm co-injected with MAX. When compared to unvaccinated controls, all strains of mice immunized with CLDC containing either peptides encompassing the first 20 N-terminal AA or those spanning the last 15 AA of the C-terminal domain of MAX demonstrated decreased parasite burden after 9 or 18 weeks post challenge with Lm + MAX. MAX-CLDC immunized mice showed increased IFNγ-secreting and decreased IL-4-secreting CD4 cells in footpad-draining lymph nodes. Antisera from C-terminal peptide (P11) MAX-CLDC-vaccinated animals was capable of recognizing FL-MAX and its C-terminal domain and also blocked MAX-mediated reprogramming of bone marrow-derived dendritic cells (BM-DC) in vitro. This peptide vaccine targeting sand fly MAX, improves host immunity against MAX-mediated immunomodulation.
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http://dx.doi.org/10.1016/j.vaccine.2017.10.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5710984PMC
December 2017

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling.

J Vis Exp 2017 08 19(126). Epub 2017 Aug 19.

Department of Chemistry & Biochemistry, Miami University;

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1a;LSL-Kras) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.
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http://dx.doi.org/10.3791/55647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5614348PMC
August 2017

Suppression of Canine Dendritic Cell Activation/Maturation and Inflammatory Cytokine Release by Mesenchymal Stem Cells Occurs Through Multiple Distinct Biochemical Pathways.

Stem Cells Dev 2017 02 22;26(4):249-262. Epub 2016 Dec 22.

Department of Clinical Sciences, Center for Immune and Regenerative Medicine, College of Veterinary Medicine and Biomedical Sciences, Colorado State University , Fort Collins, Colorado.

Mesenchymal stem cells (MSC) represent a readily accessible source of cells with potent immune modulatory activity. MSC can suppress ongoing inflammatory responses by suppressing T cell function, while fewer studies have examined the impact of MSC on dendritic cell (DC) function. The dog spontaneous disease model represents an important animal model with which to evaluate the safety and effectiveness of cellular therapy with MSC. This study evaluated the effects of canine MSC on the activation and maturation of canine monocyte-derived DC, as well as mechanisms underlying these effects. Adipose-derived canine MSC were cocultured with canine DC, and the MSC effects on DC maturation and activation were assessed by flow cytometry, cytokine ELISA, and confocal microscopy. We found that canine MSC significantly suppressed lipopolysaccharide (LPS)-stimulated upregulation of DC activation markers such as major histocompatibility class II (MHCII), CD86, and CD40. Furthermore, pretreatment of MSC with interferon gamma (IFNγ) augmented this suppressive activity. IFNγ-activated MSC also significantly reduced LPS-elicited DC secretion of tumor necrosis factor alpha without reducing secretion of interleukin-10. The suppressive effect of IFNγ-treated MSC on LPS-induced DC activation was mediated by soluble factors secreted by both MSC and DC. Pathways of DC functional suppression included programmed death ligand-1 expression and secretion of nitrous oxide, prostaglandin E2, and adenosine by activated MSC. Coculture of DC with IFNγ-treated MSC maintained DC in an immature state and prolonged DC antigen uptake during LPS maturation stimulus. Taken together, canine MSC are capable of potently suppressing DC function in a potentially inflammatory microenvironment through several separate immunological pathways and confirm the potential for immune therapy with MSC in canine immune-mediated disease models.
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http://dx.doi.org/10.1089/scd.2016.0199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913781PMC
February 2017

Autophagy Inhibition Delays Early but Not Late-Stage Metastatic Disease.

J Pharmacol Exp Ther 2016 08 26;358(2):282-93. Epub 2016 May 26.

Department of Clinical Sciences, Colorado State University, Fort Collins, Colorado (R.A.B., D.P.R., R.J.H., D.L.G.); and Department of Pharmacology, University of Colorado School of Medicine, Aurora, Colorado (P.M., A.T.)

The autophagy pathway has been recognized as a mechanism of survival and therapy resistance in cancer, yet the extent of autophagy's function in metastatic progression is still unclear. Therefore, we used murine models of metastatic cancer to investigate the effect of autophagy modulation on metastasis development. Pharmacologic and genetic autophagy inhibition were able to impede cell proliferation in culture, but did not impact the development of experimentally induced 4T1 and B16-F10 metastases. Similarly, autophagy inhibition by adjuvant chloroquine (CQ) treatment did not delay metastasis in an orthotopic 4T1, tumor-resection model. However, neoadjuvant CQ treatment or genetic autophagy inhibition resulted in delayed metastasis development, whereas stimulation of autophagy by trehalose hastened development. Cisplatin was also administered either as a single agent or in combination with CQ. The combination of cisplatin and CQ was antagonistic. The effects of autophagy modulation on metastasis did not appear to be due to alterations in the intrinsic metastatic capability of the cells, as modulating autophagy had no impact on migration, invasion, or anchorage-independent growth in vitro. To explore the possibility of autophagy's influence on the metastatic microenvironment, bone marrow-derived cells (BMDCs), which mediate the establishment of the premetastatic niche, were measured in the lung and in circulation. Trehalose-treated mice had significantly more BMDCs than either vehicle- or CQ-treated mice. Autophagy inhibition may be most useful as a treatment to impede early metastatic development. However, modulating autophagy may also alter the efficacy of platinum-based therapies, requiring caution when considering combination therapies.
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http://dx.doi.org/10.1124/jpet.116.233908DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959099PMC
August 2016

Do Mesenchymal Stromal Cells Influence Microscopic Residual or Metastatic Osteosarcoma in a Murine Model?

Clin Orthop Relat Res 2016 Mar;474(3):707-15

Department of Clinical Sciences and School of Biomedical Engineering, Colorado State University, 300 W Drake Road, Fort Collins, CO, 80525, USA.

Background: Mesenchymal stromal cells (MSCs) have been shown in rodent models to promote primary and pulmonary metastatic sarcoma growth when injected in the presence of gross tumor. In theory, this would limit their use in a clinical setting after limb salvage treatment for osteosarcoma. Although concerning, these models do not translate to the clinical setting wherein MSCs could be used after primary tumor resection to aid in bone healing and incorporation of tumor endoprostheses. If we can determine whether the use of MSCs in this setting is safe, it might improve our ability to augment bone healing in patients undergoing limb salvage.

Questions/purposes: The purpose of this study was to determine (1) whether MSCs promote pulmonary metastatic disease progression in a murine osteosarcoma model; and/or (2) whether they affect local disease recurrence in the presence of microscopic residual osteosarcoma.

Methods: An orthotopic model of luciferase-expressing osteosarcoma was developed. At 10 days, resection of the primary tumor was performed. One hundred fourteen female C3H mice were inoculated with DLM8-luc osteosarcoma in the proximal tibia. Ninety-four mice developed orthotopic osteosarcoma with luciferase expression. Mice with bioluminescent evidence of a primary tumor received either a microscopically "clean" amputation at a time when residual microscopic metastatic disease was present in the lungs (pulmonary metastasis group; n = 65) or a "dirty" amputation (local recurrence group; n = 29). Mice were randomized to receive intravenous MSCs, MSCs at the surgical site, or no MSCs. Mice were monitored for development and progression of pulmonary metastasis and local recurrence by bioluminescence imaging and daily measurements at the surgical site. The number of pulmonary nodules, time to first evidence of metastasis, and size of recurrent tumor were compared using Kruskal-Wallis, analysis of variance, Welch's, t-tests, or Mann-Whitney tests as appropriate for the specific data sets with p < 0.05 considered significant.

Results: Mice receiving intravenous MSCs had a faster time to first detection of pulmonary metastasis (2.93 ± 1.90 days) compared with mice with local injection of MSCs (6.94 ± 6.78 days) or no MSCs (5.93 ± 4.55 days) (p = 0.022). MSC treatment did not influence whether mice developed local recurrence (p = 0.749) or size of recurrent tumors (p = 0.221).

Conclusions: MSCs delivered to the surgical site did not promote local recurrence or size of recurrent tumors, but intravenous injection of MSCs did hasten onset of detection of pulmonary metastatic disease. Although local administration of MSCs into a surgical site does not appear to promote either pulmonary metastatic disease or local recurrence, large variation within groups and small numbers diminished statistical power such that a Type II error cannot be ruled out.

Clinical Relevance: If MSCs are to be used to augment bone healing in the postlimb salvage setting in patients with osteosarcoma, it will be important to understand their influence, if any, on pulmonary micrometastsis or residual microscopic local disease. Although murine models do not completely recapitulate the clinical scenario, these results suggest that intravenous delivery of MSCs may promote micrometastatic pulmonary disease. Local administration into a surgical wound, even in the presence of residual microscopic disease, may be safe, at least in this murine model, but further investigation is warranted before considering the use of MSCs for clinical use in patients with osteosarcoma.
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http://dx.doi.org/10.1007/s11999-015-4362-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4746149PMC
March 2016

Primary primitive neuroectodermal tumors of the retina and ciliary body in dogs.

Vet Ophthalmol 2013 Jul 15;16 Suppl 1:87-93. Epub 2013 May 15.

Department of Microbiology, Immunology, and Pathology, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, CO, USA.

We describe the clinical, histological, and immunohistochemical features of primary intraocular primitive neuroectodermal tumors in eight dogs. Four of eight tumors exhibited histological features similar to human retinoblastomas characterized by Flexner-Wintersteiner rosettes, and fleurettes, and demonstrated variable immunoreactivity for retinal markers opsin, S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP). All dogs with tumors displaying histological and immunohistochemical features of retinal differentiation were ≤2 years of age. All tumors diagnosed as medulloepitheliomas (n = 4) did not display histological and immunohistochemical features of retinal differentiation and were present in dogs 7 years or older. Age of onset, in conjunction with immunohistochemistry for opsin, S-Ag, and IRBP, is an important aid in the differentiation of primary, primitive neuroectodermal tumors arising within the canine ciliary body, retina, and optic papilla.
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http://dx.doi.org/10.1111/vop.12056DOI Listing
July 2013

Characterization of cytokines associated with Th17 cells in the eyes of horses with recurrent uveitis.

Vet Ophthalmol 2012 May 7;15(3):145-52. Epub 2011 Oct 7.

Department of Small Animal Medicine and Surgery, The University of Georgia College of Veterinary Medicine, 501 D.W. Brooks Drive, Athens, GA 30602, USA.

Objective: Equine recurrent uveitis (ERU) is a spontaneous disease that is the most common cause of blindness in horses, affecting up to 15% of the horse population. Th17 cells are a major cell population driving the pathogenesis in several mouse models of autoimmune inflammation, including experimental autoimmune uveitis. The purpose of this study is to investigate the role a Th17 cell-mediated response plays in the pathogenesis of ERU.

Procedure: Banked, Davidson's-fixed equine globes histopathologically diagnosed with ERU (n = 7) were compared immunohistochemically with healthy control globes (n = 7). Immunohistochemical staining was performed using a pan-Leptospira antibody and antibodies against IL-6, IL-17, and IL-23. Additionally, immunostaining was performed for T-cell (CD3) and B-cell (CD79α) markers. Specificity of immunoreactivity was confirmed by western blot analysis.

Results: Immunohistochemical staining was positive for IL-6, IL-17, and IL-23 within the cytoplasm of nonpigmented ciliary epithelial cells and mononuclear inflammatory cells infiltrating the iris, and ciliary body of ERU horses (n = 7) but negative in controls (n = 7). ERU-affected eyes were CD3 positive (n = 7) and CD79α negative (n = 7). Staining for Leptospira was negative in all ERU and control globes.

Conclusions: Strong immunoreactivity for IL-6, IL-17, and IL-23, in conjunction with the fact that T lymphocytes are the predominating inflammatory cells present in ERU, suggests that IL-17-secreting helper T-cells play a role in the pathogenesis of ERU. These findings suggest that horses with ERU may serve as a naturally occurring animal model for autoimmune uveitis.
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http://dx.doi.org/10.1111/j.1463-5224.2011.00951.xDOI Listing
May 2012

Clinicopathologic findings in a dog with a retrobulbar meningioma.

J Vet Diagn Invest 2011 Jul 13;23(4):857-62. Epub 2011 Jun 13.

Department of Small Animal Medicine, College of Veterinary Medicine, The University of Georgia, 501 D.W. Brooks Drive, Athens, GA 30602, USA.

An 11-year-old Fox Terrier dog was evaluated for a 10-month history of progressive exophthalmia and visual deficits in the right eye. Ophthalmologic examination revealed severe corneal fibrosis and pigmentation, which obscured examination of the anterior chamber of the right eye. There was decreased retropulsion of the right eye. Neurological examination revealed an absent menace response bilaterally. Pupillary light reflex was normal in the left eye. Due to the corneal pathology, pupillary light reflex was unable to be evaluated in the right eye. A retrobulbar mass with heterogeneous echotexture was identified using ultrasonography. Cytological evaluation of a fine-needle aspirate of the mass disclosed a neoplastic cell population consisting of round to polygonal cells with lightly basophilic to gray cytoplasm and round to ovoid nuclei having a coarse granular chromatin pattern. Magnetic resonance imaging disclosed a right-sided retrobulbar mass that extended through the optic canal and was contiguous with an extra-axial mass in the ventral right rostral and middle cranial fossae. The mass displayed homogenous and strong contrast enhancement. Following exenteration, histological examination of the retrobulbar mass was consistent with meningioma. Immunohistochemically, tumor cells stained positive for vimentin (cytoplasmic) and E-cadherin (membranous), and negative for S100, pancytokeratin, and cytokeratins AE1 and AE3.
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http://dx.doi.org/10.1177/1040638711408280DOI Listing
July 2011