4.03 Scaffold-free 3D-Bioprinting (3DBP) of A Porcine Liver Model

Posted on January 18, 2018

C. A. Vega1, L. J. Smith2,3, K. A. Altman4, P. Li4, B. Ekser4  1Indiana University School Of Medicine,Indianapolis, IN, USA 2Indiana University – Purdue University Indianapolis,3D-Bioprinting Core,Indianapolis, IN, USA 3Indiana University School Of Medicine,Department Of Radiology And Imaging Sciences,Indianapolis, IN, USA 4Indiana University School Of Medicine,Division Of Transplant Surgery, Department Of Surgery,Indianapolis, IN, USA

Introduction: Xenotransplantation (XTx) could be the solution to the lack of transplantable organs. Recently, great progress has been made in XTx research and there are currently >26 different genetically-engineered (GE) pigs. The most complex GE pig contains six different gene knock-outs or -ins. However, no researcher knows what would be the best genetic combination for XTx. The production of GE pigs is very expensive and would be quite time consuming for each genetic combination. Capitalizing on a new 3D bioprinting technology, we propose to use GE cells to generate a scaffold-free 3D pig liver tissue constructs, which can be used to study human immunological and coagulative responses in a time and cost effective way.                                                   

Methods: A step-wise model for 3DBP was developed which included (i) determination of optimal size of spheroids, (ii) determination of optimal time of spheroid formation and stability, (iii) designing and printing of the most suitable 3D structure, and (iv) determination of viability of the 3D-bioprinted structure. The optimization of 3DBP was also based on (i) porcine hepatocyte isolation; (ii) cell aggregate (spheroid) diameter, roundness, smoothness, durability, stability, and viability; (iii) the ratio and combination of different cell types; (iv) and the assembly (printing) of tissue constructs using spheroids.  

 

Results: Using a combination of porcine fetal fibroblasts and liver derived cells (LDCs, CD31+) (10:1 ratio), 450-500µm diameter spheroids were generated after 2-3 days of plating and successfully used to print 3 preliminary constructs (Fig.1). These 2 cell-type 3D-bioprinted constructs were later tested for their viability/structure after 1, 2, and 3 weeks of printing (Fig.1). Next, a combination of fibroblasts, hepatocytes, and LDCs (2:1:0.1 ratio) formed spheroids 5 days after plating and were then used to print our first 3 cell-type construct. Most of the spheroids remained intact in this 3 cell-type constructs. After incubation for an additional 5 days, this 3-cell construct fused and formed a stable 3D structure. Histology (H&E staining) shows promising results.

Conclusion: We have successfully printed a scaffold-free 3D Bioprinted Porcine Liver Model containing hepatocytes, liver derived endothelial cells, and fibroblasts. Further cell ratio optimization is required to produce functional spheroids capable of forming a more stable construct in the future. Using CRISPR-Cas9 technology, desired GE pig cells will be printed for future constructs which will allow us to explore the immunogenic and coagulative properties of pig tissues in the human body. 

4.04 Discordant Impact on Coagulation Between HTK and UW

Posted on January 18, 2018

A. Kam1, H. B. Moore1, C. B. Walker1, J. L. Dexter-Meldrum1, M. Chapman1, T. Johnson1, M. A. Adams1, T. Pshak1, I. Kam1, E. Pomfret1, T. L. Nydam1  1University Of Colorado Denver,Surgery,Aurora, CO, USA

Introduction:  Less than optimal outcomes following donation after cardiac death (DCD) donation are in part attributed to the development of microthrombi within the microvasculature of the graft. Using thromboelastography (TEG) during DCD recovery we have found numerous donors retain full clotting strength despite heparinization. We therefore sought to determine the effects of two commonly used preservation solutions, University of Wisconsin (UW) solution and Histidine-Tryptophan-Ketoglutarte (HTK), on clot formation and fibrinolysis. We hypothesize HTK will have a greater effect on promoting hypocoagulability due to its lower viscosity.

Methods:  Coagulation was assessed by TEG using whole blood diluted to 25%, 50%, and 75% with each preservative solution including HTK, UW and normal saline (NS). NS was used as a control to adjust for dilutional effects. The following TEG parameters were recorded: clot initiation (R time), fibrin polymerization (angle), and clot strength [maximum amplitude (MA) and clot lysis 30 minutes after reaching MA (LY30)].  For each solution, the dilution concentration was correlated to TEG indices with Spearman’s Rho test.  Solutions were contrasted between each other at set concentrations using a Friedman test.  Data was described as the median.

Results: Eight healthy volunteers donated blood. The serial dilution of whole blood with NS correlated with a prolonged R time (0.527 p=0.008), shallow angle (-0.693 p<0.001), lower MA (-0.811 p<0.001), and lower LY30 (-0.482 p=0.017).  This was similar to HTK (R time 0.564 p=0.004; angle -0.752 p<0.001; MA -0.818 P<0.001 and LY30 -0.643 p=0.001).  UW dilution had similar correlations with the exception that LY30 had a positive correlation with dilution (0.410 p=0.047). When contrasting TEG indices at different dilutions, no significant differences were seen until the dilution reached 75%.  Whole blood diluted with 75% HTK had significantly reduced fibrin polymerization compared to NS and UW (17° vs 32° vs 28° p=0.005 Figure). Whereas UW had significantly increased LY30 compared to NS and HTK (1.9% vs 0% vs 0% p<0.001).

Conclusion: Different preservation solutions have similar effects on coagulation as normal saline until 75% of whole blood has been replaced.  However, at 75% dilution, these solutions have different contributions to disturbances in TEG indices promoting hypocoagulability. HTK promotes a weaker clot from impaired fibrin polymerization, while UW promotes fibrinolysis.  These data support tailoring preservation solution to the pre-existing hypercoagulabilty seen in donors in order to optimize organ recovery.
 

4.01 Conserved FcRN-albumin interaction: implications for porcine-to-human renal xenotransplantation.

Posted on January 18, 2018

J. R. Butler1,2, M. R. Greg2, J. Ladowski2, Z. Wang2, J. L. Estrada2, M. Tector2, A. Tector2  1Indiana University School Of Medicine,Indianapolis, IN, USA 2University Of Alabama at Birmingham,Birmingham, Alabama, USA

Introduction: Historically, animal models of renal xenotransplantation have been challenged by acute humoral rejection. When studied in pig-to-primate models, there have been inconsistent reports of postoperative proteinuria; because proteinuria is a hallmark of complement-mediated graft damage, it remains unclear whether renal handling of serum macromolecules represents a physiologic barrier to clinical application in the absence of immune-mediated injury. Recently, serum albumin has been identified as a renal filtrate; serum proteostasis is dependent on post-glomerulus albumin absorption. In particular, the neonatal Fc receptor (FcRn) has been identified as a primary driver of renal proximal tubule albumin absorption in humans. The object of this study was to query the potential for porcine FcRn to promote absorption of human albumin in the absence of immunologic injury.

Methods: Intravital 2-photon microcopy has been instrumental in the study of renal albumin handling. Though this process has identified albumin as a renal filtrate in rat, mouse, and human, this is the first reported use of dual photon microcopy to assess the porcine kidney. After mapping the path of albumin across the filtration barrier in the porcine kidney, we established a fluorescence-based in vitro model of cellular uptake to query the ability of porcine proximal tubule cells to effectively absorb human albumin. With an established model to asses cross-species albumin-epithelial interactions at the cellular level, we sought to better understand the relationship at the molecular level. To this end we used a Biacore sensorgram to establish kenetics of the FcRn-albumin interaction. Finally, at the organismal level, we utilized a rejection free pig-to-primate model of life-sustaining renal transplant to analyze the potential loss of serum protein macromolues.

Results: Intravital 2-photon microcopy successfully followed labeled human albumin through the porcine kidney. Similar to results from other animal models, albumin is filtered at the porcine glomerulus. An in vitro cellular model of albumin uptake supports the ability of porcine renal proximal tubal cells to absorb human albumin. At the molecular level, porcine FcRn binds human albumin with the same affinity it does autologous porcine albumin; KD values of 7.22e-7 vs 3.92e-7 respectively (p= 0.21). Finally, when studied in vivo in a porcine to primate model free of immune-mediated graft damage the porcine kidney does not produce significant proteinuria.

Conclusion: Despite historical concern that renal loss of albumin would cause clinically significant proteinuria in porcine-to-primate xenotransplantation, this is not observed in the absence of immune-mediated graft injury. At the organ, cellular, and molecular level the porcine kidney process human albumin in a physiologic manner. Proteinuria does not represent an independent barrier to the clinical application of porcine-to-human xenotransplantation.

4.02 Automated Quantification of Soft Endpoints in Wound Healing Analysis

Posted on January 18, 2018

S. Mascharak1, A. L. Moore1,2, B. Duoto1, D. S. Foster1, R. E. Jones1,3, G. Wernig4, M. T. Longaker1  1Stanford University,Dept. Surgery,Palo Alto, CA, USA 2Brigham And Women’s Hospital,Dept. Surgery,Boston, MA, USA 3University Of Texas Southwestern Medical Center,Dept. Surgery,Dallas, TX, USA 4Stanford University,Dept. Pathology,Palo Alto, CA, USA

Introduction:  Studies on dermal wound healing and scarring commonly rely on qualitative assessments of morphological and histological characteristics. These can be time consuming and prone to individual observer bias. Furthermore, it is not always feasible to visually ascertain metrics of scar organization on the micron scale. We have developed a package of image processing methods to automate quantification of morphological and histological dermal scar features. Using this tool, we elucidate the effects of an anti-scarring treatment on collagen content, fiber organization, and hair follicle neogenesis after dermal wounding. 

Methods:  Two stented dermal wounds were made on the dorsa of C57BL/6 mice and injected with various concentrations (PBS, 0.1, 2, 20 mg/mL) of a potential anti-scarring drug (Doxycycline). Histology (Masson’s Trichrome, Picrosirius Red) and hair follicle neogenesis were assessed at 14 and 28 days, respectively, and analyzed with Matlab 2017a. Histological features were measured using a combination of color deconvolution, adaptive filtering, and skeletonization of individual collagen fibers. Hair follicles were segmented using top-hat and shape filters.

Results: Automated quantification of color-deconvoluted histology images showed a significant decrease in collagen deposition for wounds treated with anti-scarring drug (*p = 0.036, 2 mg/mL vs PBS, Fig. 1A-C). Color deconvolution also segmented mature (red) and immature (green) collagen fibers in birefringent Picrosirius Red-stained samples, revealing a concomitant increase in immature collagen deposition (*p = 0.018, 2 mg/mL vs PBS, Fig. 1D Picro Green). Next, images of collagen fibers were skeletonized to trace individual tracks, revealing significant decreases in mature fiber length (*p = 0.042, 2 mg/mL vs PBS), number (*p = 0.036), and branching (*p = 0.038) after treatment (Fig. 1E-F). Strikingly, alignment of mature collagen fibers decreased after treatment (*p = 0.048, 2 mg/mL vs PBS), indicating basket-weave organization reminiscent of unwounded skin. Shape filtration segmented hair follicles, dramatically speeding up the counting process (Fig. 1G). A significant decrease in follicles was observed after treatment (*p = 0.025, 2 mg/mL vs PBS), though no change in morphology was observed (Fig. 1H). 

Conclusion: Automated image processing facilitates histological and morphological study of scars. Additionally, analysis of collagen fibers reveals differences in scar organization after treatment with Doxycycline, that would not be apparent to the naked eye. Based on these findings, we will expand the use of image processing tools to models of hypertrophic scarring and fetal wound healing, towards the goal of new quantitative benchmarks for scar analysis. 

3.20 Large-field of View Laparoscopic Visualization Utilizing Multiple Miniaturized Cameras

Posted on January 18, 2018

J. Kim1, A. Watras1, H. Liu1, Z. Zeng1, J. A. Greenberg2, C. P. Heise2, Y. Hu1, H. Jiang1  1University Of Wisconsin-Madison,Electrical And Computer Engineering,Madison, WI, USA 2University Of Wisconsin School Of Medicine And Public Health,Department Of Surgery,Madison, WI, USA

Introduction: Laparoscopic surgery has numerous clinical benefits for patients including decreased postoperative pain, decreased wound morbidity, earlier recovery, and improved cosmesis. The quality and extent of intra-abdominal visualization is crucial during laparoscopic procedures. However, the current system that uses a single camera has many drawbacks in terms of the quality and extent of visualization as well as operative efficiency. We created a multi-camera visualization system with a larger field of view (FoV) compared to standard laparoscopic cameras for improved operative efficiency.

Methods: Our multi-camera visualization system utilizes four cameras with a specially designed trocar and a real-time video stitching program. We performed a bean drop task in a commercially available simulator box to test our large-FoV visualization system. Our video stitching program used the video data from four individual cameras and combined these four videos in real-time to provide large FoV. A projective transformation was calculated using matched feature points for mapping each image from different cameras into a single coordinate system. One of the four cameras was considered to provide the main view and then the required transformations were computed to map the images from four different cameras to the coordinate system of the main view.

Results: The four cameras are deployed and retrieved by the mechanical system of the trocar. The working port is not occupied by the cameras during operation and allows insertion of surgical instruments. Therefore, our system can reduce the number of ports or free up a surgical port, a potential advantage of our system. As a demonstration, we successfully performed a bean drop task in the trainer box by using our large-FoV visualization system. The figure below shows that a bean was picked up by a grasper and dropped into the hole of an inverted cup. The red dotted circles are traces of a moving bean picked by a grasper. The real-time stitched video can track the complete motion of the bean without any physical camera maneuver (Figure (a, b)), which is another clear advantage over current laparoscopic cameras that need to be operated separately by an assistant. In contrast, an image from a single camera cannot track the whole trajectory of the bean and cover the beans and the cup simultaneously (Figure (c, d)).

Conclusion: Our multi-camera visualization system provides a large FoV, frees up a surgical port, and eliminates the needs of physical maneuvering of the laparoscopic camera (thus the camera operating assistant). This system can provide larger extent of intra-abdominal visualization and may lead to higher efficiency of operation.

 

3.19 Effects of Exogenous Del1 Protein on Human Skeletal Stem Cell Proliferation

Posted on January 18, 2018

T. Boyko1,2, O. Marecic1, M. Lopez1, C. Chan1, M. T. Longaker1, G. P. Yang1  1Stanford University School Of Medicine,Surgery,Stanford, CALIFORNIA, USA 2University At Buffalo,Surgery,Buffalo, NY, USA

Introduction:  Millions of people fracture their bones yearly, 5% of which fail to heal properly. Risk factors for this include old age, osteoporosis and diabetes. Failure of bony fusion also complicates thousands of orthopedic, spinal and maxillofacial surgeries including joint replacements, spinal fusion and dental implants resulting in significant morbidity and increased costs. Skeletal progenitor cells have recently been described. They give rise to three main tissues of the skeletal system: bone, cartilage and stroma, and are responsible for bone healing. We have previously shown that the extracellular matrix protein Del1 acts to promote proliferation and prevents apoptosis of the subpopulation of mouse skeletal progenitor cells involved in fracture healing – theBone Cartilage Stroma Progenitor cells (BCSPs). Del1 knockout mice heal fractures with less bone, and the healed bone is weaker than that of wild type control fractures. We hypothesized that addition of exogenous Del1 protein could promote proliferation of human skeletal stem cell (HSSC).

Methods:  Femur heads of three patients undergoing hip replacement surgery, age 70, 71 and 75, were obtained. Following mechanical and enzymatic digestion, HSSCs were isolated via FACS, using previously identified cell surface markers. HSCCs were plated in vitro with or without the addition of human Del1 protein. Proliferation was measured daily over 5 days.

Results: The addition of exogenous human Del1 protein to cultured HSSCs led to increased proliferation rates compared to untreated HSSCs. Cell numbers reached statistical significance on the third day of treatment (p<0.05) and remained statistically significant over the remaining days cells were counted in all three human samples.

Conclusion: Del1 knockout mice do not have a significant phenotype without injury. Following fracture, knockout mice are able to heal, but with significantly less bone. We have shown that the underlying mechanism consists of Del1 acting as a pro-proliferative and anti-apoptotic factor for mouse skeletal progenitor cells. In this study, we demonstrate that exogenous human Del1 can promote proliferation of human skeletal progenitor cells. This suggests that Del1 has the potential for therapeutic use to reduce morbidity from failure of bone fracture healing or inadequate bone fixation of an implant.

 

3.18 Diversity of Enterococcus faecalis in the Early Microbiota of Rats

Posted on January 18, 2018

P. T. Delaplain1, J. Wang1, B. Bell1, A. Grishin1, H. Ford1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA

Introduction:
Although necrotizing enterocolitis (NEC) is believed to be associated with bacterial colonization of the neonatal gut, the exact nature of the relationship between NEC and specific groups of bacteria or characteristics of bacterial populations remains largely elusive. Analysis of bacterial populations using high throughput sequencing of 16S RNA brought only limited success, as the same bacterial species were found in sick and healthy individuals. Our previous studies implicated a clinically relevant strain of Cronobacter muytjensii, but not several other strains of this species, as a causative agent of NEC. This lead us to hypothesize that NEC is caused by a plethora of opportunistic pathogens, which may belong to a variety of bacterial species, whereas colonization with non-pathogenic bacteria may be innocuous or even protective. In the framework of this hypothesis, we are characterizing multiple isolates of one of the most common first colonizers, Enterococcus faecalis, and their role in the pathogenesis of experimental NEC.

Methods:
Neonates obtained from timed pregnant rats sourced from Charles River or Harlan were either breast fed or formula fed. E. faecalis bacteria were isolated from the intestines of 4-day-old rats based on their thermotolerance, ability to grow in the presence of azide, characteristic appearance upon Gram staining, and 16S RNA sequencing. The isolates were further characterized for colony morphology, hemolysis, gelatin liquefaction, sugar utilization, antibiotic resistance, and spectra of DNA HindIII restriction fragments. Isolates that possessed different combinations of the corresponding phenotypic traits were considered different strains. The strains identified were examined for their ability to activate the pro-inflammatory transcription factor NF-κ B in IEC-6 enterocytes using Western blotting with anti-Iκ Bα  antibodies. 

Results:
21 different strains were identified among the 146 independent isolates of E. faecalis. DNA restriction enzyme digestion revealed two unique genomic patterns. Some, but not all strains activated NF-κ B in IEC-6 cells. In addition, small colony size on standard media seemed to correlate with a preference for anaerobic conditions. 

Conclusion:
There is a considerable diversity of E. faecalis even in animals coming from the same specific pathogen-free environment. Some bacterial phenotypes, specifically hemolysis, proteolysis, antibiotic resistance, and ability to trigger inflammatory response in enterocytes, may be causatively associated with pathogenicity in NEC. Further characterization of these bacteria may identify both effective first colonizers as well as potentially pathogenic or protective strains.
 

3.16 Acta2, Tnc, and Col24a1 Expression are Associated with Fibroblast Formation of Abdominal Adhesions

Posted on January 18, 2018

D. Foster1,2, C. D. Marshall1,2, R. C. Ransom2, A. Manjunath3, G. Gulati3, M. S. Hu2, C. C. Chan3, W. T. Leavitt2, A. L. Moore2, L. A. Barnes2, M. Murphy2, M. T. Longaker1,2,3  1Stanford University,Department Of Surgery,Stanford, CA, USA 2Stanford University,Hagey Laboratory For Pediatric Regenerative Medicine,Stanford, CA, USA 3Stanford University,Institute For Stem Cell Biology And Regenerative Medicine,Stanford, CA, USA

Introduction:  After surgical interventions or secondary to fibrotic disease, intra-abdominal adhesions can form. The presence of adhesions makes further surgery challenging and can have other negative consequences such as infertility, bowel obstructions and chronic pain. Adhesions are thought to form by fibroblast collagen deposition, similar to cutaneous scar formation. The mechanisms underlying adhesion formation, including origin of the fibroblasts involved or signaling process governing this phenomenon, however, remain poorly characterized. Currently, there is no effective therapy to prevent or treat adhesive disease.  

Methods:  Intra-abdominal adhesions were established between the bowel and the peritoneal lining of the abdominal sidewall in PDGFRα-GFP mice, in which PDGFRα (platelet derived growth factor receptor alpha), a pan-fibroblast marker, is tagged with green fluorescent protein (GFP) [Fig. 1A]. Sham-surgery PDGFRα-GFP mice were used for comparison. Once formed, the adhesion, abdominal wall and bowel wall tissues were extracted and digested with collagenase. FACS sorting and quantitative PCR confirmed fibroblast identity via expression of known fibroblast genes. Bulk RNA sequencing was conducted on sorted fibroblasts and gene expression was compared between the adhesion and sham-surgery cohorts. For the next aim of this project, human abdominal adhesion tissue is being harvested from ileostomy takedown patients. All experiments were approved by Stanford University’s IRB or APLAC, as applicable. 

Results: FACS sorting of mouse adhesion tissue showed consistent expression of fibroblast genes including PDGFRα, Vim (vimentin), and Col1a2 (encodes for collagen 1). This was confirmed with quantitative PCR. RNA sequencing showed significantly higher expression of relevant fibrosis-associated genes in both male and female mice from the adhesion cohort including Acta2, Tnc, and Col24a1, compared to sham [Fig. 1B]. Acta2 encodes for smooth muscle α-2-actin (αSMA) and is expressed by myofibroblasts and smooth muscle cells. Tnc encodes for tenascin C, an extracellular matrix protein, and Col24a1 is a member of the collagen gene family involved in type 1 collagen regulation. Human adhesion tissue will be sorted and sequenced, and gene expression will be analyzed and compared with mouse results. The results of our human adhesion tissue experiments are forthcoming. 

Conclusion: Identification of this gene expression pattern in adhesions presents the opportunity for possible therapeutic targets. Comparison of our results from mouse experiments with cellular activity and gene expression patterns in human tissue will bring us closer to developing a potential therapy to combat adhesive disease. 

3.17 Anti-fibrosis Effect of Novel Oridonin Analog CYD0618 via Suppression of NF-κB Pathway

Posted on January 18, 2018

C. B. Cummins1, X. Wang1, J. Xu2, Y. Ding2, H. Chen2, J. Zhou2, R. Radhakrishnan1  1University Of Texas Medical Branch,Department Of Surgery,Galveston, TX, USA 2University Of Texas Medical Branch,Department Of Pharmacology And Toxicology,Galveston, TX, USA

Introduction:
Liver fibrosis is characterized as excessive deposition of the extracellular matrix (ECM) proteins, especially collagen type I. Activated hepatic stellate cells (HSCs) are the primary cell type responsible for ECM deposition, and NF-κB signal has been reported as one of major mediators of HSC activation. Previously, our team reported oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, exhibited anti-hepatic fibrogenetic activity In vitro. In the present study, we examined the effects of its novel derivative CYD0618 on HSC viability, apoptosis and NF-κB signaling.  

Methods:
The proliferation effects of CYD0618 treatment on activated human and rat HSC cell lines LX-2 and HSC-T6 were measured by Alamar Blue Assay. Apoptosis was measured by Cell Death ELISA. Cellular proteins were determined by Western blots and immunofluorescence.

Results:
CYD0618 significantly inhibited LX-2 cells proliferation in a dose-dependent manner with an IC50 value of ~0.45 μM after 48 hours treatment, this was ~15 fold more potent than the parent compound oridonin. Similar effects were seen in HSC-T6 cells with an IC50 of ~0.75 μM. Cell apoptosis was induced by CYD0618 in both cell lines. CYD0618 treatment increased cell cycle inhibitory protein p21, p27, and induced apoptosis marker cleaved poly (ADP-ribose) polymerase (c-PARP), while significantly suppressing the expression of collagen type I. Notably, CYD0618 blocked lipopolysaccharides(LPS)-induced NF-κB p65 nuclear translocation and DNA binding activity, in addition to preventing LPS-induced NF-κB inhibitory protein IκBα phosphorylation and degradation. LPS-stimulated NF-κB downstream target cytokines IL-6, MCP-1 were also attenuated by CYD0618. It has been reported that phosphorylation of the NF-κB p65 on the serine 536 residue affects its nuclear translocation and transcription of target genes. Our data showed that endogenous NF-κB p65 S536 phosphorylation was inhibited by CYD0618 treatment in a time-dependent fashion. Importantly, NF-κB specific chemical inhibitor Bay-11-0781 was found to inhibit the proliferation, and promote apoptosis in both LX-2 and HSC-T6 cells.

Conclusion:
The potent anti-hepatic fibrogenetic effect of CYD0618 may be mediated via suppression of the NF-κB pathway.
 

3.15 Investigating the Integrin α3β1–dependent Secretome in Keratinocytes: Implications for Wound Healing

Posted on January 18, 2018

L. A. DeFreest1, L. Van De Water2, C. DiPersio2  1Albany Medical College,Department Of Surgery, Division Of General Surgery, Section Of Trauma And Critical Care,Albany, NY, USA 2Albany Medical College,Department Of Surgery And Department Of Regenerative & Cancer Cell Biology,Albany, NY, USA

Introduction: Integrin receptors expressed in epidermal keratinocytes have been shown to have important roles in cutaneous wound healing including paracrine stimulation of angiogenesis, regulation of keratinocyte proliferation and migration, and production and remodeling of the extracellular matrix (ECM) to which they adhere. Our laboratory has investigated the roles of the laminin-binding integrin α3β1 in keratinocytes using both in vivo and in vitro models of wound healing. Our previous data supports a role for α3β1 in modulation of these processes by regulating the secretion of proteins into the extracellular milieu.  Our long term goal is to elucidate the α3β1-dependent paracrine and autocrine signaling mechanisms that modulate the wound microenvironment to promote efficient wound healing.

Methods: Using a cell culture model of activated mouse keratinocytes (MK cells), we have prepared medium conditioned by MK cells that are homozygous for a null mutation in the gene that encodes the α3 integrin subunit (i.e. lacking α3β1 integrin), or MK cells stably transfected with human α3 (i.e. expressing α3β1 integrin). These conditioned media were analyzed by mass spectrometry to identify proteins secreted by MK cells in an α3β1–dependent manner, with a focus on those proteins that were upregulated at least four-fold in the presence of α3β1. Selected proteins in both media and lysates from MK cells were then evaluated by western blot to confirm and quantitate the secreted proteins.

Results: Of the >7000 proteins identified by mass spectrometry, nineteen were upregulated more than four-fold in medium from α3β1–expressing mouse keratinocytes. These proteins corresponded to a wide variety of keratinocyte functions including ECM, pro-angiogenic, and immunological species.  Interestingly, lactotransferrin (LTfn) secretion was up-regulated 18-fold in α3β1-expressing cells.  Western blot confirmed a significant upregulation of LTfn secretion as well as production of the protein in cell lysates.

Conclusion: Proteomic analysis of conditioned media from MK cell lines identified numerous secreted proteins that are upregulated by α3β1 indicating an important role for this integrin in controlling the keratinocyte secretome. Lactotransferrin has previously been shown to have a role in wound healing and has anti-microbial and immunomodulatory properties making it an interesting candidate for further study. Interestingly, LTfn is known to be expressed in response to estrogen and under the control of the estrogen receptor, suggesting an intersection of signaling pathways downstream of integrins and the estrogen receptor which has not been previously studied in detail. Elucidation of the signaling mechanisms that result in α3β1–dependent production of LTfn may lead to novel therapeutics to promote closure of surgical wounds.
 

3.12 FXR May Be Necessary for Experimental Murine LPS Induced Peritonitis Intestinal Damage

Posted on January 18, 2018

M. U. Mallicote1, O. Escobar1, C. Gayer1  1Children’s Hospital Los Angeles,Los Angeles, CA, USA

Introduction: Bile acids are known to stimulate the Farnesoid X receptor (FXR) pathway in the intestines; however, there are discrepancies in the literature about the effects of FXR activation on the intestinal barrier. FXR activation has been reported to decrease gut permeability in chronic injury models, although FXR activation is known to decrease intestinal cell proliferation. Given these inconsistencies, we hypothesize that FXR activation is detrimental to the intestinal barrier in an acute injury model.

 

Methods: In vivo, wild-type (WT) and global FXR knock-out (FXR-KO) mice were injected with lipopolysaccharide (LPS) or saline via intra-peritoneal route to induce intestinal injury and gavage-fed FITC-dextran. Animals were sacrificed at 16 hours. Barrier function was assessed by serum FITC levels and mRNA expression of IL-6, IL-10, TNF, and IL1b was assessed by RT-PCR.

 

Results: Barrier permeability in WT mice was significantly increased in LPS versus saline controls. This effect, however, was significantly attenuated in FXR-KO mice. LPS increased FXR mRNA expression in WT but not in FXR-KO. Of the cytokines tested, only TNF was elevated in both WT and FXR-KO mice injected with LPS versus saline controls. FXR-KO mice injected with LPS also showed highly elevated levels of IL-6 and IL1b whereas the WT mice did not. Interestingly, the FXR-KO mice also exhibited high levels of IL-10 an anti-inflammatory cytokine.

 

Conclusions: In WT mice, LPS-induced inflammation mainly leads to elevated levels of TNF. In FXR-KO mice there is elevation of IL-6, TNF, and IL1b, however intestinal barrier damage appears to be attenuated. Of note, in FXR-KO mice there was elevation of the anti-inflammatory cytokine IL-10 which may be the cause of the attenuated barrier damage we are seeing. Determining why the absence of FXR leads to IL-10 induction may lead to the development of novel therapies for regulating the intestinal epithelium during acute injury and may help prevent gut-origin sepsis.

3.14 C-Jun Induction Leads to Increased Scar Formation and Fibrosis in Mice

Posted on January 18, 2018

B. Duoto1,2, A. Moore1,3, D. Foster1, R. E. Jones1,4, S. Mascharak1, G. Wernig5, M. Longaker1  1Stanford Univserity,Surgery,Stanford, CALIFORNIA, USA 2San Jose State University,Biology,San Jose, CALIFORNIA, USA 3Brigham And Women’s HospitalBrigham And Women’s Hospital,Surgery,Boston, MASSACHUSSETTS, USA 4University Of Texas Southwestern Medical Center,Surgery,Dallas, TX, USA 5Stanford University,Pathology,Stanford, CALIFORNIA, USA

Introduction:

Fibrosis and scar formation are major clinical issues which result in disfigurement and permanent functional loss. In both adults and children, excessive fibrosis after surgery or injury can result in complications that are difficult to treat, often recur, and have few effective therapeutic options. Additionally, the only animal models that exist to simulate these processes in humans include the red Duroc pig and rabbit ear, which are expensive, difficult to use, and do not provide transgenic modeling. Recently, a paper describing a transgenic mouse strain that utilizes over-expression of c-Jun, an AP-1 transcription factor, to induce global tissue fibrosis was published. We hypothesize that local induction of c-Jun in the same transgenic mouse would result in increased scarring and fibrosis.

Methods:
            Stented excisional dorsal wounding was performed on c-JuntetO; R26-M2rtTA mice along with injections of phosphate buffered saline (PBS), or with c-Jun inducing agent doxycycline at 0.1mg/mL, and 2mg/mL concentrations. Induction and dressing changes were performed every other day until wounds were completely healed. Wounds were then harvested and stained with hematoxylin and eosin for scar thickness and trichrome for collagen deposition. These assays quantify stain-specific wound characteristics.

Results:
            Preliminary histological staining data uncovered a significantly increased scar thickness in the c-JuntetO R26-M2rtTA mice as compared to the C57BL/6J PBS control mice (*p<0.0001) and the C57BL/6J 2mg/mL doxycycline control mice (*p<0.0001). Comparatively, in regards to the amount of scar collagen deposition, no significance was found between the c-JuntetO R26-M2rtTA mice and the C57BL/6J PBS control mice (p=0.0832) as well as the C57BL/6J 2mg/mL doxycycline control mice (p=0.1692).

Conclusion:

Histological stains of c-JuntetO R26-M2rtTA dorsal wounds support that there are distinct differences in scar formation in the c-Jun transgenic model as compared to controls. These data support that this novel mouse model can be developed to study the molecular pathways which lead to, and inhibit, fibrosis. In future studies we will investigate novel inhibitors of fibrosis in this animal model, study the scar forming fibroblasts in greater detail, and compare our results to human specimens. 

3.11 Ursodeoxycholic acid (UDCA) may protect against intestinal injury in mouse models of peritonitis

Posted on January 18, 2018

M. Nguyen1, O. Escobar1, C. Gayer1  1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CALIFORNIA, USA

Introduction:
Ursodeoxycholic acid (UDCA) is a secondary bile acid that, unlike many other bile acids, is hydrophilic and has anti-inflammatory properties. We have previously demonstrated that UDCA inhibits intestinal epithelial cell proliferation and stimulates migration in in vitro through an EGFR-dependent mechanism involving downstream COX-2 upregulation. We hypothesize that UDCA will protect the intestinal epithelial barrier in an acute model of intestinal injury via a pathway that requires EGFR, ERK, and COX-2.

Methods:
Wild-type C57BL/6 or Velvet (EGFR dominant-negative) mice underwent intraperitoneal injections of 30 mg/kg lipopolysaccharide (LPS) and oral gavage of 175 mg/kg fluorescein isothiocyanate (FITC)-dextran, with or without 100 mg/kg UDCA . Mice were sacrificed at 16 hours, and small intestine and serum were harvested. Phosphorylated ERK and COX-2 were measured by Western blot and immunofluorescent (IF) staining of intestine.  FITC-dextran levels were measured by fluorimetry of serum.

Results:
Mice treated with UDCA demonstrated decreased villus height loss and serum FITC-dextran levels, as previously reported. Western blot revealed that small intestine mucosal scrapings of these same mice contained increased phosphorylated ERK. This effect was attenuated in mice treated with UDCA + LPS, and further attenuated in mice treated with LPS alone. As expected, small intestine mucosal scrapings of mice treated with LPS alone contained increased COX-2. Conversely, COX-2 was decreased in mice treated with UDCA and further decreased in mice treated with UDCA + LPS, though the differences were very slight. IF staining demonstrated increased COX-2 expression in mice treated with UDCA + LPS, though there was no appreciable difference in COX-2 expression among mice undergoing other treatments.

Conclusion:
These data suggest that UDCA protects the intestinal epithelial barrier in part through an EGFR- and ERK-dependent mechanism. In the context of our previously reported in vitro data showing that UDCA increases intestinal epithelial cell migration through a mechanism dependent on EGFR, COX-2, and ERK, the data presented here further support that UDCA could be used to protect the intestine from acute injury.
 

3.09 Nanofiber Scaffold-Skin Composite for Treatment of Excisional Wounds in Diabetic Rats

Posted on January 18, 2018

J. A. Ungar1, L. Fu2, S. Aravind1, J. Xie3, M. Carlson1  1University Of Nebraska College Of Medicine,College Of Medicine,Omaha, NE, USA 2University of Nebraska Medical Center,Regenerative Medicine,Omaha, NE, USA 3University of Nebraska Medical Center,Surgery – Transplant,Omaha, NE, USA

Introduction: For dermal defects in diabetic subjects, both split thickness skin grafting (STSG) and dermal replacement products are minimally effective in preventing wound contraction. We hypothesized that treatment of full-thickness skin wounds in diabetic rats with a composite of autologous skin islands embedded in a nanofiber scaffold (NFS) would produce less wound contraction compared to standard skin grafting.

Methods: NFS were electrospun from PCL and gas-expanded to produce a 2mm thick microporous matrix arrayed with 1mm wells (skin island placement) spaced 3mm apart. Wistar rats (3mo/300g, N=35) with STZ-induced diabetes underwent dermal wounding (2cm circular excision, two per dorsum) with an encircling ring tattooed around each wound. Subjects were randomized to receive immediate application of a wound treatment: (1) gauze only; (2) meshed STSG; (3) NFS only; (4) 1mm skin islands, or SI, every 3mm; (5) NFS+SI. Gross wound area (WA) and tattoo area (TA) at days 0, 14, 28 and microscopic cross-sectional granulation tissue area (GTA) at days 14, 28 were determined with digital planimetry.

Results: WA and TA on day 0 were not different. At d14, WA in the SI group was less compared to the other groups (Table 1). At both d14 & 28, wounds treated with any NFS had greater GTA compared to other groups. At d28, wounds were 90+% closed, based on WA & GTA. The NFS+SI group demonstrated the least wound contraction at d28, having the greatest TA and the smallest change in WA. However, the NFS+SI group also had the greatest GTA at d28. On d28 H&E histology, the skin islands in the NFS+SI groups were viable, but the NFS was only minimally incorporated into the underlying wound.

Conclusion: Dermal excisional wounds in diabetic rats treated with a NFS-autologous skin composite had less wound contraction compared to other treatments, including STSG. However, the mechanism for this effect may involve physical splinting or some other effect, since the NFS-treated rats only had minimal incorporation of the synthetic construct into the wound, while having greater area of granulation tissue. In order to obtain a dermal replacement which both prevents wound contraction and strongly incorporates into the wound, NFS modification likely will be necessary.

 

3.10 Decreased Inflammation in Scarless Fetal Skin Wound Healing is Regulated by miR-146a

Posted on January 18, 2018

L. C. Dewberry1, M. M. Hodges1, C. Zgheib1, J. Xu1, S. A. Hilton1, J. Hu1, K. W. Liechty1,2  1University Of Colorado Denver,Department Of Surgery,Aurora, CO, USA 2Children’s Hospital Colorado,Pediatric Surgery,Aurora, CO, USA

Introduction:
We have previously shown fetal skin wounds heal without scar formation, however, if fetal wound size increases there is a transition from scarless regeneration to scar formation, similar to scar formation seen in the adult.  This transition from scarless regeneration to scar formation was also associated with an increased inflammatory response. Inflammation is regulated, in part, through gene expression of proinflammatory cytokines.  Previous work from our laboratory has demonstrated increased gene expression of these proinflammatory cytokines (IL-6, IL-8). An additional layer of regulation is provided by micrRNAs.  Specifically, miRNA-146a has been shown to act as a “molecular brake” on inflammation by inhibiting proinflammatory gene expression (specifically IL-6 and IL-8).  We hypothesized that the transition from scarless regeneration to scar formation is associated with increased inflammation, increased proinflammatory signaling, and decreased miRNA-146a expression.

Methods:
To test this hypothesis 2-mm and 8-mm dermal wounds were created in mid-gestation fetal sheep (70-75 days gestation). Wounds were harvested at 3 and 7 days post-wounding, total RNA extracted, and gene expression of miR-146a analyzed with real-time PCR.

Results:
Large wounds were associated with increased inflammation and increased expression of proinflammatory cytokine expression.  In addition, miRNA-146a expression was significantly upregulated in day three small wounds compared to day three large wounds (Figure 1; p=0.03). There was no difference in gene expression between small and large wounds at day 7. 

Conclusion:
We conclude that inflammation and inflammatory signaling plays a role in scarless wound healing. In particular, upregulation of miRNA-146a in the smaller wounds may create an environment conducive to regeneration by decreasing the inflammatory response.   More studies are needed to further elucidate the mechanisms of inflammatory regulation in fetal regeneration.
 

3.07 Intestinal Epithelial Cell-Specific mTORC1 Regulates Food Intake

Posted on January 18, 2018

S. Tay2, J. Guo1, B. W. Warner1  1Washington University,Pediatric Surgery,St. Louis, MO, USA 2Washington University,School Of Medicine,St. Louis, MO, USA

Introduction:
Intestinal epithelial cell (IEC)-specific mammalian target of rapamycin complex 1 (i-mTORC1) regulates IEC cell growth and proliferation. Recently, we found that intestine-specific blockade of i-mTORC1 activity by disrupting Raptor gene expression after small bowel resection in mice led to significant loss of body weight. The purpose of this study was to determine a mechanism for this weight loss.

Methods:
Raptor knockout mice (Villin-CreER(+); Raptor(flox/flox) ) and wild-type littermate mice underwent intraperitoneal injection of tamoxifen to inducibly delete Raptor protein expression selectively in the intestinal epithelium of adult mice. We then placed 8 Raptor knockout and 8 control mice on high fat diet for 5 weeks and measured body composition, food intake, fasting blood glucose, lipid metabolism, and white and brown adipose tissue stores. 

Results:
Compared to control mice, Raptor-deficient mice had 46% less white adipose tissue, 30% lower total cholesterol, and 34% lower direct HDL (T test, P < 0.05). Further, the Raptor-deficient mice had 40% less food intake and gained only 14% of baseline weight over 5 weeks, compared to 40% weight gain in control mice. Interestingly, fecal fat content, fasting blood glucose, and brown adipose tissue were not affected. Our results suggest that the decreased body weight gain following i-mTORC1 inactivation is not due to glucose response or lipid absorption or excretion, instead it is strongly associated with reduced food intake. 

Conclusion:
The finding of reduced food intake due to an intestinal epithelial cell-specific gene disruption is novel. Prior studies have looked at satiety and adiposity signals such as cholecystokinin, insulin, and leptin, but the drastic decrease in food intake coupled with normal glucose response and lipid absorption observed in Raptor knockout mice could not be explained by either of these signals. While gut microbiota has been found to modulate gut-brain interaction and food intake, and we cannot rule out the effects of commensal bacteria, the remarkable suppression of food intake seen in Raptor knockout mice suggests that an internal pathway may underlie such phenomenon. Future studies are planned to investigate this pathway.
 

3.08 MiR-146a-Conjugated Cerium Oxide Nanoparticles Accelerate Diabetic Wound Healing by Promoting Angiogenesis

Posted on January 18, 2018

C. Zgheib1, J. Xu1, M. M. Hodges1, J. Hu1, L. C. Dewberry1, S. A. Hilton1, S. Seal2, K. W. Liechty1  1Laboratory For Fetal And Regenerative Biology, Department Of Surgery, School Of Medicine, University Of Colorado Denver – Anschutz Medical Campus And Colorado Children’s Hospital,Aurora, CO, USA 2Advanced Materials Processing And Analysis Centre, Nanoscience Technology Center (NSTC), Mechanical Materials Aerospace Eng, University Of Central Florida,Orlando, FL, USA

Introduction: Delayed wound healing is one of the most common complications associated with diabetes. These wounds do not heal properly due to chronic inflammation and impaired angiogenesis. We have previously shown that local delivery of the anti-inflammatory miR-146a using cerium oxide nanoparticles carrying miR-146a (CNPs+miR-146a) accelerates diabetic wound healing by promoting an anti-inflammatory M2 macrophage phenotype and reducing the inflammatory response. M2 macrophages have been shown to stimulate angiogenesis through the production of pro-angiogenic growth factors such as VEGF. Thus, we hypothesize that in addition to normalizing the inflammatory response, CNPs+miR-146a enhance diabetic wound healing through the correction of impaired angiogenesis.

Methods: To test this hypothesis, 8 mm full-thickness excisional wounds were created on the dorsal skin of diabetic mice and immediately treated with 10 uM of CNPs+miR-146a or PBS. Digital images of wounds were taken for calculation of wound surface area. Subsets of these wounds were harvested 7 days after wounding and miR-146a, miR-15b, VEGF, and BCL2 gene expression were analyzed.

Results: Our data show that at 7 days post-wounding, CNPs+miR-146a treatment resulted in improvement in wound closure over PBS-treated control wounds in diabetic mice. In addition, CNPs+miR-146a treatment rectified the compromised angiogenesis by increasing the levels of VEGF and BCL2. This is partly due to the downregulation of the levels of miR-15b, an anti-angiogenesis microRNA.

Conclusion: Our findings demonstrate that local delivery of miR-146a via conjugation with CNPs accelerates healing of diabetic wounds by normalizing inflammation and promoting angiogenesis. This CNP-based platform for miR-146a delivery is a promising effective adjunct therapy and warrants further studies prior to future clinical translation.

3.06 Hyperglycemia reduces long non-coding RNA Lethe expression through the ribosomal binding protein HuR

Posted on January 18, 2018

J. Xu1, J. Hu1, C. Zgheib1, M. M. Hodges1, K. W. Liechty1  1University Of Colorado-Anschutz Medical Campus,Department Of Surgery, Laboratory For Fetal And Regenerative Biology,Aurora, CO, USA

Introduction: Recent studies reveal that long non-coding RNAs (lncRNAs) play important regulatory roles in many biological processes. We have previously shown that the lncRNA Lethe is down-regulated in diabetic wounds and that Lethe is involved in the regulation of Reactive oxygen species (ROS) production in macrophages, indicating a potential role for Lethe in the pathogenesis of diabetic wounds. RNA binding protein HuR stabilizes mRNA by binding to 3’UTR of mRNA and inhibiting microRNA binding. We hypothesize that hyperglycemia reduces Lethe expression by alteration of HuR binding.

Methods: To test our hypothesis, we incubated the murine macrophage cell line RAW264.7 with media containing 5 mM glucose (low glucose), or 25 mM glucose (high glucose) for 24 hours. RNA immunoprecipitation (RIP) was used to analyze HuR binding and Real-time PCR used to quantify relative gene expression.

Results: Lethe was significantly down-regulated in high glucose conditions.  Under low glucose conditions, the level of lethe in the anti-HuR immunoprecipitated lysate was significantly higher than in the IgG control group.  Under high glucose conditions, the level of lethe was not significantly different between the anti-HuR group and IgG control group, and was significantly lower compared to anti-HuR group under low glucose conditions.

Conclusion: These findings demonstrate a novel mechanism in the regulation of the effects of lncRNA Lethe expression, with HuR binding to lncRNA Lethe and hyperglycemia reduces HuR binding to Lethe to de-stabilize Lethe. Furthermore, these results may represent a potential novel therapeutic target to correct the impaired diabetic wound healing response.

 

3.04 The Role of microRNA-21 in the Regulation of Inflammation and Macrophage Polarization

Posted on January 18, 2018

C. E. Liechty1, J. Hu1, C. Zgheib1, K. W. Liechty1, J. Xu1  1University Of Colorado-Anschutz Medical Campus,Department Of Surgery, Laboratory For Fetal And Regenerative Biology,Aurora, CO, USA

Introduction: The diabetic wound healing impairment has been shown to be multifactorial. A central feature of diabetic wounds is the persistence of chronic inflammation, partly due to the prolonged presence of pro-inflammatory (M1) macrophages in diabetic wounds.  Persistence of the M1 macrophage phenotype, and failure to transition to the regenerative or pro-remodeling (M2) macrophage phenotype plays an indispensable role in diabetic wound impairment; however, the mechanism underlying this relationship remains unclear. Recently, microRNAs have also been shown to provide an additional layer of regulation of gene expression.  In particular, microRNA-21 (miR-21) is essential for inflammatory response. We hypothesized that miR-21 plays a role in regulating inflammation through promoting M1 macrophage polarization and the production of pro-inflammatory cytokines.

Methods: To test our hypothesis, we treated the mouse macrophage cell line RAW264.7 with LPS (10 pg/ml) and (20 ng/ml) for 24 h to generate M1 macrophages, or IL-4 (20 ng/ml) for 24 h to generate M2 macrophages after overnight serum starvation. MiR-21 overexpression was achieved by transfection of miR-21 mimic.  In order to examine the effect of inflammation on miR-21 expression, we treated the mouse macrophage cell line RAW264.7 with LPS; a known inducer of inflammation.  Cells were treated with 1, 10, or 100ng/ml LPS for 6 hours or 100ng/ml for 2, 4, 6 hours.  Real-time PCR analysis to quantify relative gene expression, using GAPDH or U6 as internal control for mRNA or miRNA expression.

Results: M1 polarized macrophages exhibited an upregulation of miR-21, as well as the M1 and pro-inflammatory markers IL-1b, TNFa, iNos, IL-6, and IL-8. Cells exposed to M2 conditions exhibited an upregulation of M2 markers Mrc1 and Arg1, and no significantly change of miR-21.  LPS stimulation of inflammation resulted in upregulation of miR-21, as well as the inflammatory markers TNFa and IL-6 in a dose and time dependent manner.  Overexpression of miR-21 in RAW264.7 macrophage cells resulted in an upregulation of miR-21 and also increased expression of the M1 markers IL-1b, TNFa, iNos, and IL-6.

Conclusion: These findings provide the first evidence that miR-21 is involved in the regulation of inflammation and promoting M1 macrophage polarization. Dysregulation of miR-21 in diabetic wounds may explain the abnormal inflammation and persistent M1 macrophage polarization seen in diabetic wounds, and may represent a potential role of miR-21 as a therapeutic target to counteract the impaired wound healing response in diabetic wounds.

 

3.05 Inflammatory Properties of Ileal Fluid from Patients with IBD

Posted on January 18, 2018

Y. Liu1, F. Kuehn1, R. Vasan1, E. Liu1, F. Adiliaghdam1, E. Samarbafzadeh1, R. Hodin1  1Massachusetts General Hospital,General And GI Surgery,Boston, MA, USA

Introduction:
Luminal contents play a crucial role in the induction and maintenance of intestinal inflammation. Patients with Crohn´s disease benefit from diversion of the fecal stream, with immediate recurrence of inflammation after restoration of intestinal continuity. Furthermore, pouchitis after ileo-anal anastomosis for ulcerative colitis does not occur prior to ostomy closure. These observations support the premise that exposure to factors within the fecal stream is a critical component in inciting phenotypic expression of inflammatory bowel disease (IBD). And, yet, the components within the fecal milieu that play a role in activating the inflammatory pathways still remain unknown. Recent data has demonstrated that levels of the anti-inflammatory mucosal defense factor Intestinal alkaline phosphatase (IAP) are reduced in colon biopsies of patients with IBD. The objectives are to examine the inflammatory properties of ileal fluid from patients with and without IBD. Our central hypothesis is that pro-inflammatory factors in intestinal contents activate inflammatory cascades in the intestinal epithelial lining and that targeting these factors/pathways can present novel therapeutic approaches to treat IBD.

Methods:
Ileal fluid samples from 46 ileostomy patients with and without IBD were collected in the surgical clinic at Massachusetts General Hospital, Boston. IAP Activity was measured using the para-Nitrophenylphosphate (pNPP) assay. The effluent was centrifuged and the supernatant assayed by ELISA for key pro-inflammatory cytokines. Human THP1 macrophages were exposed to the fluid from 23 consecutive patients and assayed for cytokine expression. 

Results: 

Ileal fluid from 46 patients (28 IBD, 18 non-inflammatory controls) with a median age of 58 years (range; 23-94) was collected and assayed. TNF-a levels were significantly higher in ileal fluid of IBD patients than in controls (33.4 ± 62.9pg/ml vs. 9.7 ± 7.6pg/ml; p < 0.05). IAP activity was significantly lower in patients with IBD compared to patients without underlying inflammatory disease (16.9 ± 6.1 U/mg protein vs. 21.0 ± 6.7 U/mg protein; p<0.05). The inflammatory response of THP1 cells exposed to ileal fluid supernatant showed an individual cytokine profile for each patient and did not correlate with the cytokine levels in the original sample or the underlying disease. 

Conclusion:
Analysis of Ileal luminal contents showed significantly higher TNFa levels and lower IAP activity in patients with IBD. The individual inflammatory response profile of each patient could serve as a basis for determining the risk for recurring disease or pouchitis in stoma patients with IBD. 
 

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