40.08 Glutathione sp and Metabolic Signatures of NAFLD/NASH in the Rodent.

Posted on January 18, 2018

M. Schade1, J. Sanabria1, R. Aguilar1, M. Andryka1, A. Mallick1, J. Fannin1, J. Sanabria1,2  1Marshall University Schoool Of Medicine,Department Of Surgery,Huntington, WV, USA 2Case Western Reserve University School Of Medicine,Surgery, Nutrition And Preventive Medicine,Cleveland, OH, USA

Introduction:
Non Alcoholic Liver Disease (NAFLD) and its complications, NASH, ESLD and HCC have become the second most common cause for liver transplantation in the West. Although major advances have been done in the understanding of fatty liver disease, the metabolic disturbances that occur in the normal cell from NAFLD to NASH remains to be determined.

Methods:
C57Bl 6J mice (female: n=7/male: n=7 per each time point) were exposed to normal chow (control), high fat diet + fructose (HFD, Western diet) and methyl choline deficient diet (MCD). Liver and plasma were collected at weeks 7, 12, 16, 20, 24 and 48. Body weight, total body water, lean mass and fat compartment were determined by MRI spectroscopy. The proportion of cells undergoing mitosis or cell arrest were determined against liver apoptotic index and collagen deposition by standard stains using morphometric techniques. Quantitative protein expression of genes involved in cell metabolism or cell senescence (p53, p21, p16, mTOR1, STAT33, SIRT7, FOX01, Grb2) were determined by Western Blots.   In addition, Na/K-exchange pump regulation was determined by SRC expression.  While liver oxidative stress was determined by glutathione sp, hepatic mitochondrial ß -lipid oxidation function was determined by octanoate/butyrate ratio. Metabolites (n=852) were measured in treated plasma by LC/MS-MS. Principal component analyses (PCA) was conducted to detect metabolite differences among groups.  

Results:
The total body weight corrected for aging increased significantly in the HFD due to an increase in the fat compartment with similar lean mass and total body water (HFD vs normal chaw, p<005 by ANOVA).  The animals in the MCD group had a significant decrease in their body weight due to decreased fat, lean and total body water (MCD vs normal chaw, p<005 by ANOVA). Nonetheless, both diets induced NAFL/NASH. An increased proportion of cells in senescence was observed with decreased mitotic index and increased apoptotic activity (HFD and MCD vs normal chaw, p<0.05 by ANOVA). Morphological changes correlated with gene expression. Significant decreased in both mitochondrial ß-lipid oxidation function and Na/K-exchange pump activity after censored for aging (HFD and MCD vs normal chaw, p<0.05, by ANOVA). Glutathione ratio reduced/oxidized was significantly reduced animals with fatty liver (HFD and MCD vs normal chaw, p<0.05, ANOVA). Metabolic signatures of NAFLD and NASH after censored for aging on cell aging demonstrated disturbances in the lipid and carbohydrate metabolism.

Conclusion:

HFD and MCD generates NAFLD/NASH with decreased cellular redox status and mitochondrial ß-lipid oxidation, manifested by metabolic changes that are seen in accelerated cell senescence with characteristic plasma metabolic signatures. Described metabolic changes are of interest as therapeutic targets for arrest or reversibility of disease progression.   

 

40.09 A Novel Safer Hsp90 Inhibitor to Overcome Drug Resistant Melanomas

Posted on January 18, 2018

J. N. Sanchez1,2, C. Subramanian2, K. J. Kovatch3, M. S. Cohen1,2  1University Of Michigan,Department Of Pharmacology,Ann Arbor, MI, USA 2University Of Michigan,Department Of General Surgery,Ann Arbor, MI, USA 3University Of Michigan,Department Of Otolaryngology Head And Neck Surgery,Ann Arbor, MI, USA

Introduction:  The 90kDa heat shock protein (Hsp90) and its clients are key regulators of multiple signaling pathways in eukaryotic cells.  Its expression levels are increased 10 fold in malignancies like melanomas compared to normal tissue. Melanoma treatment has shifted to immunotherapies or targeted therapy with inhibitors targeting the MAP-kinase pathway. One challenge is that resistance to therapy especially with BRAF inhibitors occurs in a majority of patients. Since Hsp90 functions to facilitate the activation of several kinases involved in BRAF inhibitor resistance pathways, there is a high rationale that inhibition of Hsp90 would simultaneously inhibit multiple resistance pathways leading to antitumor efficacy. We have developed novel Hsp90 inhibitors that target the carboxy-terminus of the chaperone and do NOT induce the heat shock response. This response leads to dose-limiting toxicities plaguing all other Hsp90 inhibitors tested in clinical trials. We hypothesize that C-terminal inhibition of Hsp90 will simultaneously inhibit resistance pathways to rescue drug resistant melanoma cell lines.

Methods:  Vemurafenib resistance was induced in validated human melanoma cell lines. Control (Mel 11) and vemurafenib-resistant (Mel 11 VeR) cell lines were treated with a Hsp90 inhibitor (KU757) or BRAF inhibitor (vemurafenib), at range of concentrations for 24hrs. Following 24hr treatment with either inhibitor, induction of apoptosis was characterized by using a caspase 3/7 luciferase reporter assay and further confirmed using flow cytometry. Flow cytometry was used for cell cycle analysis. Additionally, key resistance pathway and heat shock proteins as well as cell cycle modulators were evaluated by Western blots.

Results: Cell viability was tested in Mel 11 and Mel 11 VeR. In both cell lines, KU757 treated cells showed a similar dose-dependent inhibition of proliferation irrespective of BRAF resistance with an IC50 level of 1μ M. Next the reporter assay found that there was an equal 15 fold increase in caspase 3/7 activity for both cell lines.  Further analysis of apoptosis by flow cytometry inidicated cell death by necrosis in addition to increase in caspase 3/7 activity. Analysis of cell cycle modulation using flow cytometry showed that in both cell lines, the percent of cells in G0/G1 decreased to 2.6% and 11% in Mel 11 and Mel 11 VeR respectively. Finally, Western blot analysis showed a 5 fold decrease in Akt and Erk in both cell lines with treatment compared to no treatment (p<0.01), as well as a 2 fold increase in Raf1 in vemurafenib treated cells. There was no compensatory heat shock response shown KU757 as indicated by no modulation Hsp70, Hsp32, or HSF1.

Conclusion: KU757 is equally effective in both normal and resistant melanoma cell lines inducing apoptosis, inhibiting cell growth and knocking down BRAF-inhibitor resistance pathways without inducing the heat shock response. Future translational validation in vivo will better define its clinical applications.  

 

40.06 Prevention of early liver metastasis after pancreatectomy by NF-kB inhibitor

Posted on January 18, 2018

N. SAITO1, T. Uwagawa1, H. Shiba1, R. Hamura1, N. Takada1, H. Sugano1, T. Horiuchi1, Y. Shirai1, T. Ohashi1, K. Yanaga1, K. Yanaga1  1Jikei University School Of Medicine,Department Of Surgery,Minato-ku, TOKYO, Japan

Introduction:  Liver metastasis is a significant clinical problem early after resection of pancreatic cancer. Kocherization during early phase of the operation for pancreatic cancer prohibits the use of no-touch isolation, when tumor cells may dislodge, implant and settle in the liver through the portal vein. Matrix metalloproteinase (MMP)-2/9 degrades basement membrane and makes the tumor more susceptible to infiltration. FUT 175, a serine protease inhibitor, is known to inhibit MMP-2/9 by suppressing the NF-kB signal. Therefore, we hypothesized that preoperative treatment by FUT 175 may prevent hepatic metastasis and prolong postoperative survival of patients with post-resectional pancreatic cancer. 

Methods:  In vitro, we used a mouse pancreatic cancer cell line (PAN02). Between control and FUT 175 treatment groups, cell viability was examined by MTT assay, and the levels of cytoplasm MMP-2/9 were evaluated by Western blotting. Furthermore, activation levels of MMP-2/9 in supernatant of cultured cells were investigated by gelatin zymography. In vivo, 1´106 PAN02 cells were injected into the spleen of the mouse (C57BL6) to produce liver metastases. In treatment group, PAN02 cells were treated with FUT 175, as compared to vehicle alone in control group. The volume of liver metastasis was assessed by MRI once a week. After sacrifice, metastatic tumors were evaluated by various assays. Also, survival rates were compared.  

Results: FUT 175 did not suppress the viability of PAN02 cells for the first 24 h of treatment (p>0.05). However, the levels of cytoplasmic MMP-2/9 were down-regulated by FUT 175 after 24 h of treatment. In vivo, for control group, all animals died within 7 weeks after injection (n=10), for which MRI revealed rapid growth of metastatic tumors. On the contrary, the tumor growth rates of treatment group were very slow, and 4 of 10 animals (40%) were alive for over 7 weeks. 

Conclusion: Because of suppression of the level of MMP-2/9 in vitro and prolonged survival of liver metastasis of pancreatic cancer in mice, FUT 175 may prevent liver recurrence after pancreatectomy.

 

40.07 Glutathione sp and Metabolic Signatures of Physiologic Liver Senescence in the Rodent.

Posted on January 18, 2018

M. Schade1, J. A. Sanabria1, R. Aguilar1, M. Andryka1, A. Mallick1, J. Fannin1, J. Sanabria1,2  1Marshall University Schoool Of Medicine,Department Of Surgery,Huntington, WV, USA 2Case Western Reserve University School Of Medicine,Surgery, Nutrition And Preventive Medicine,Cleveland, OH, USA

Introduction:
Human life expectancy has increased to over 80years in the last decade and more than half of the Western population will be over the age of 50 in 2020. Surgical procedures are expected to increase in number and complexity in the older. The understanding of the aging process and its metabolic implications on cell energy requirements is of paramount importance. Nevertheless, biological markers of cell stress and/or healing reservoir on the aging liver remains unclear.

Methods:
C57Bl 6J mice (female: n=7/male: n=7 per each time point) and db/db (diabetic, female=7) were exposed to normal chow. Livers and plasma were collected at different stages of animal growth and aging (weeks 7, 12, 16, 20, 24 and 48). Body weight, total body water, lean mass and fat compartment were determined by MRI spectroscopy. Cardiac function was followed by echocardiography. The proportion of cells on mitosis or in senescence was determined against liver apoptotic index and collagen deposition by standard stains using morphometric techniques. Quantitative protein expression of genes involved in cell metabolism or cell senescence (p53, p21, p16, mTOR1, STAT33, SIRT7, FOX01, Grb2) were determined by Western Blots.   In addition, Na/K-exchange pump regulation was determined by SRC expression.  While liver oxidative stress was determined by glutathione sp, hepatic mitochondrial ß -lipid oxidation function was determined by octanoate/butyrate ratio. Metabolites (n=852) were measured in treated plasma by LC/MS-MS. Principal component analyses (PCA) and partial least square discriminant analysis (PLS-DA) were conducted to detect metabolite differences among groups.

Results:
The total body weight increases with aging manly due to an increase in the fat compartment with decreased lean mass and total body water (W7vsW24vsW48, p<005 by ANOVA).  LVEF decreased and increased LV wall with age. An increased proportion of cells in senescence was observed with decreased mitotic index and increased apoptotic activity (W7vsW16vsW24vsW48, p<0.05 by ANOVA). Morphological changes correlated with expected gene expression and significant decreased in both mitochondrial ß-lipid oxidation function and Na/K-exchange pump activity (p<0.05, by ANOVA). Glutathione ratio reduced/oxidized was reduced with aging (p<0.05, ANOVA). These findings were more accentuated in the diabetic mice. Metabolic prints on cell aging showed disturbances of not only lipid but carbohydrate metabolism.

Conclusion:
Body aging under normal diet is characterized by an increase fat compartment with decreased lean mass and total body water. Changes that correlate with a decreased in liver mitochondrial function, Na/K-exchange pump activity and cell redox status, conferring metabolic signatures of liver aging. These changes must be taking into account on judging liver response to injury, liver disease development and progress and early detection of liver malignancy.
 

40.04 Diagnosis of Pancreatic Adenocarcinoma via Protein Signatures from Fine Needle Aspirations

Posted on January 18, 2018

M. H. Gerber1, D. Delitto1, B. DiVita1, S. Han1, R. M. Thomas1, J. Trevino1, S. J. Hughes1  1University Of Florida,Department Of Surgery,Gainesville, FL, USA

Introduction:  Cytological analysis of fine needle aspiration (FNA) of pancreatic lesions fails to confirm pancreatic ductal adenocarcinoma (PDAC) in up to one third of patients. We discovered that a 4-analyte protein signature from tumor lysates distinguishes PDAC from other lesions including chronic pancreatitis, thus raising the notion protein signatures could prove superior to FNA cytological analysis in the diagnosis of PDAC.  Here, we aim to translate this observation to the FNA biopsy platform.

Methods:  At time of operation, a “virtual” FNA biopsy of various pancreatic pathologies was obtained using a 19-gauge needle with six passes through the intestinal wall into the region of interest. Biopsy samples were placed in various quantities of lysis buffer and protein concentrations of each sample determined. IL-6 concentrations (one of the informative analytes in the diagnostic protein signature) were determined for each FNA biopsy sample, with and without spiking of “contaminant” plasma.

Results: Samples were collected from 27 consecutive patients undergoing pancreatectomy (PDAC n = 18; normal n = 3; pancreatitis n = 6). Protein concentrations from FNA biopsy samples diluted in 150 ul of lysis buffer ranged from 1.8 –  260.8 mg/ml with a median of 17.1 mg/ml (Interquartile range (IQR): 11.5 – 32.6 mg/ml). IL-6 levels in the FNA samples ranged from 185 – 2941 pg/ml with a median of 486 pg/ml (IQR: 247 – 740 pg/ml). Plasma concentrations of IL-6 from the same patients ranged from 59 – 312 pg/ml with a median of 111 pg/ml (IQR:  65 – 203 pg/ml).  When patient FNA samples were compared to plasma samples, FNA samples had IL-6 concentrations 3.1 to 14.9 times higher than the matched patient plasma concentration.

Conclusion: Pancreatic FNA biopsy produces adequate quantities of protein for multiple replications on a variety of protein assay platforms. Normalization of cytokine concentrations to total protein may be subject to blood/plasma contamination inherent to the FNA procedure; thus alternative normalization to a resident, soluble protein may be necessary.  Soluble protein analysis of pancreatic FNA biopsy samples remains a realistic supplement to cytology in the diagnosis of PDAC.
 

40.05 Sphingosine Kinase Type 1 and Type 2 Works Differently in Pancreatic Cancer

Posted on January 18, 2018

M. Nakajima1, K. Yuza1, J. Tsuchida1, Y. Hirose1, K. Miura1, H. Ichikawa1, Y. Shimada1, T. Kobayashi1, J. Sakata1, H. Kameyama1, M. Abe2, K. Sakimura2, T. Wakai1, M. Nagahashi1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata City, NIIGATA, Japan 2Brain Research Institute, Niigata University,Department Of Cellular Neurobiology,Niigata, NIIGATA, Japan

Introduction: Pancreatic cancer is one of the most lethal diseases known, and it is important to develop new therapeutic agents. Sphingosine-1-phosphate (S1P) is a pleiotropic lipid mediator that regulates cell survival, migration, angiogenesis and lymphangiogenesis, which are all factors involved in cancer progression. S1P, which functions intra- and extracellularly, is generated inside the cell by two sphingosine kinases (SphK1 and SphK2). We have reported that SphK1 plays an important role in S1P secretion (J Biol Chem 2010) and cancer progression (Cancer Res 2012, J Surg Res 2016), and that SphK2 has a unique role in regulating cellular functions in the liver (Hepatology 2015). Little is known, however, about the role of SphK1 and SphK2 in pancreatic cancer progression. The aim of this study is to investigate the role of SphK1 and SphK2 in pancreatic cancer progression using SphK-knockout (KO) cells generated by CRISPR/Cas9 technology.

Methods: We generated Pan02 murine pancreatic cancer cell lines with a CRISPR/Cas9 mediated targeted deletion of the SphK1 or SphK2 gene. To investigate the role of SphK1 or SphK2 in cellular proliferation, we assessed cell growth by a spectrophotometric technique using the water-soluble tetrazolium salt, WST-8. Cell migration was measured by an in vitro scratch assay. In the animal experiments, we assessed the prognosis of C57BL/6 mice injected with the SphK1 KO or SphK2 KO Pan02 cells described above intraperitoneally.

Results: SphK2 KO Pan02 cells were significantly less proliferative than WT cells. Unexpectedly, SphK1 KO cells were significantly more proliferative than WT cells. The in vitro scratch assay indicated that SphK2 KO cells were less migratory than WT cells, and that SphK1 KO cells had greater migratory ability than WT cells. Furthermore, the animal experience showed that mice injected with SphK1 KO cells had shorter prognosis than those injected with SphK1 WT cells, while mice injected with SphK2 KO cells had longer prognosis than those injected with SphK2 WT cells. These results indicate that SphK2, rather than SphK1, may have important roles in proliferation and migratory behavior in pancreatic cancer cell lines and pancreatic cancer progression. On the other hand, SphK1 KO cells treated with gemcitabine had more survival rate than WT cells and SphK2 KO cells treated with gemcitabine had less survival rate than WT cells. These results indicate that SphK1 may have important roles in resistance against chemotherapy.

Conclusion: Our findings indicate that S1P produced by SphK1 and SphK2 may have different functions in pancreatic cancer cell. Targeting both SphK1 and SphK2 signaling pathways may be a potential strategy for pancreatic cancer treatment.

 

40.03 IL-23 Plays an Important Role in Short-Term Survival after Pancreatic Adenocarcinoma Resection

Posted on January 18, 2018

B. A. Krasnick1, S. M. Husain2, Y. Bi1, P. V. Dickson2,3, J. Deneve2,3, D. Shibata2,3, R. Fields1, W. G. Hawkins1, E. S. Glazer2,3  1Washington University,Surgery,St. Louis, MO, USA 2University Of Tennessee Health Science Center,Surgery,Memphis, TN, USA 3University Of Tennessee West Cancer Center,Surgery,Memphis, TN, USA

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) carries the highest case fatality rate of any cancer and will likely become the second leading cause of death by 2020.  TGF-ß has a paradoxical relationship with survival in PDAC patients where it is a tumor suppressor in early stage PDAC and a tumor promotor in late stage PDAC. While TGF-ß is known to drive inflammation in the tumor microenvironment (TME), the role of interleukins in the pancreatic TME is not well understood.  We hypothesized that IL23, a pro-inflammatory interleukin associated with suppression of cytotoxic T cells, is associated with survival in PDAC.

Methods:  24 long-term survivors (>30 months) and 24 short-term survivors (<12 months) with resected PDAC were identified. Tumor sections were taken from formalin fixed, paraffin embedded blocks and protein expression of IL-23 and TGF-ß were independently investigated with immunohistochemistry utilizing quantitative analysis with CellProfiler image analysis software. Immunohistochemistry expression of IL23 or TGF-ß was determined to be high (highest quartile), low (lowest quartile), or median (within the interquartile range) based on 5 representative images of each tumor section. Comparisons with clinical outcomes were investigated with Student’s t-test, ANOVA, or multivariate regression.

Results: There was no significant difference in the average age (66 ± 12 years), gender (44% male), or clinical stage between the two groups. Patients with low TGF-ß protein expression were more likely to be in the short-term survival group (OR=2.2, P=0.018). Tumors from short-term survivors were significantly more likely to have low IL23 expression (OR=0.48, P=0.019).  In long-term survivors, neither TGF-ß nor IL-23 protein expression was associated with survival (P>0.05). There was no difference in IL23 expression in low or median TGF-ß expressing tumors (P>0.5), however, in high TGF-ß expressing tumors, long-term survivors were associated with 20% higher IL23 protein expression (P=0.008). Multivariate analysis demonstrated that long-term survival was linearly associated with increasing IL23 expression (OR=3, P=0.001) but not TGF-ß expression (P=0.07). Overall, we also found a statistical association between IL23 expression and TGF-ß expression (P<0.001) and that long-term survival was associated with a higher ratio of IL23 / TGF-ß expression (P=0.04).

Conclusion: We found that IL23 tumor expression is associated with survival after PDAC resection in short-term survivors and statistically related to TGF-ß expression for all patients.  While other groups have shown that TGF-ß is associated with survival in PDAC, we demonstrated that IL23 may be a critical component to understanding the relationship between TGF-ß expression and survival in patients with survival less than 12 months.

 

40.01 Modeling of Early Pancreatic Neoplasia Transcriptional Regulation in Mice

Posted on January 18, 2018

J. K. Thompson1, H. Crawford3,4, M. Pasca Di Magliano1,2, F. Bednar1  1University Of Michigan,Surgery,Ann Arbor, MI, USA 2University Of Michigan,Cell And Developmental Biology,Ann Arbor, MI, USA 3University Of Michigan,Molecular And Integrative Physiology,Ann Arbor, MI, USA 4University Of Michigan,Internal Medicine,Ann Arbor, MI, USA

Introduction: KRAS is the primary oncogenic driver in pancreatic ductal adenocarcinoma (PDA). Pancreatic acinar cells are most susceptible to transformation by Kras in mouse models of PDA. The earliest stage of transformation consists of conversion of the acinar cells to a duct-like progenitor phenotype in a process called acinar-ductal metaplasia (ADM). Networks of developmental transcription factors (TFs) are involved in fate specification and maintenance of acinar cells. We hypothesize that oncogenic Kras alters these networks to establish the neoplastic cell state within the pancreas. In this work, we aimed to validate a genetically engineered mouse model that will allow us to analyze changes in gene regulatory networks driven by oncogenic Kras in early pancreatic neoplasia.

Methods: Mice with a pancreatic acinar cell-specific, tamoxifen inducible Cre recombinase (Ela-CreER) were bred with strains containing the oncogenic Kras G12D allele and the fluorescent protein tdTomato in the Rosa26 locus. To activate the oncogenic Kras and the tdTomato lineage tracer, Ela-CreER; Kras G12D/+; R26 tdTomato mice and littermates lacking oncogenic Kras, were treated with daily gavages of tamoxifen (4mg/day) for five consecutive days. Mice were sacked one week after the initial tamoxifen gavage. We analyzed the expression of tdTomato by fluorescence microscopy and fluorescence-activated cell sorting (FACS). Total cell RNA was isolated from the sorted cells using the RNeasy Micro Kit (QIAGEN) and complementary DNA (cDNA) was synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). We utilized a panel of 26 pancreatic developmental TFs and three acinar and duct markers (amylase, elastase, keratin 19 – CK19) with specific quantitative PCR (qPCR) TaqMan probes to characterize the isolated cells. Differences in TF expression between the wildtype and oncogenic Kras-containing mice were analyzed with a Kruskal-Wallis rank test. Statistical significance was set at p<0.05.

Results: Tamoxifen gavage induced high level of tdTomato expression in Ela-CreER mice. Fluorescence microscopy confirmed that the tdTomato+ cells were also amylase positive. RT-qPCR analysis after FACS sorting confirmed high amylase, high elastase, and low CK19 expression in the tdTomato+ cells. We also consistently found measurable levels of 23/26 (88%) of pancreatic developmental TFs in the isolated acinar cells. Preliminary analysis did not reveal significant differences in TF levels in oncogenic Kras-expressing pancreata versus wildtype controls.

Conclusion: We established a genetically engineered mouse model of early pancreatic neoplasia, which allows for specific isolation of acinar cells and their progeny. Early analysis of the model suggests that one-week activation of oncogenic Kras does not yet lead to significant developmental TF expression changes in the adult pancreas.

 

40.02 HO-1 Polymorphism and Acute Necrotic Pancreatitis Through V-cam and E-selectin Expression

Posted on January 18, 2018

A. K. Gulla1,2, A. Gulbinas3, G. Barauskas3, Z. Dambrauskas3  1Georgetown University Medical Center,Department Of Surgery,Washington, DC, USA 2Vilnius University Hospital, Santaros Clinics,Department Of Surgery,Vilnius, SANTARISKIU 2, Lithuania 3Lithuanian University Of Health Sciences, Kaunas Clinics,Department Of Surgery,Kaunas, , Lithuania

Introduction:  Acute pancreatitis is a severe and frequently a life-threatening disease, which can lead to pancreatic necrosis, acute lung injury, SIRS and MODS. The inducible enzyme heme oxygenase-1 (HO-1) is an anti-oxidative, anti-inflammatory, and cytoprotective enzyme that is induced in response to cellular stress. The HO-1 promoter contains (GT)n dinucleotide repeats and is highly polymorphic in the population. In this study, we hypothesized that the number of GT repeats in HO-1 promoter can influence the occurrence of acute necrotic pancreatitis through v-cam and e-selectin expression due to its protective function. Patients with acute pancreatitis are more likely to have long repeats than controls.

Methods: Acute pancreatitis patients (n=135) and age- and sex-matched healthy controls (n=33) were studied. Peripheral blood samples from pancreatitis patients were collected on admission. Genomic DNA was extracted from the blood samples of patient and control groups. The HO-1 promoter region with the GT repeats was PCR amplified with fluorescent tagged primers and levels of cytokines were measured.

The PCR products were analyzed by ABI 3130 genetic analyzer and the exact size of the PCR products was determined by GeneMapper software.  A short allele was defined as containing 27 GT repeats or fewer, whereas a long allele was more than 27 repeats.

Results: The subjects were categorized into 3 groups based on the genotype

Results: one short and one long alleles (S/L), two short alleles (S/S) and two long alleles (L/L). The presence of S/L was similar between the patient group (41.2%) and the controls (39.4%). Interestingly, 46.6% of patients were carriers of two long repeats (L/L) while 11.1% v-cam and 11.1 % e-selectins levels (p<0.05) vs 24.2% of control subjects, whereas 12.2% of patients were carriers of two short repeats vs 36.4% of control population. 

Conclusion: Our data demonstrate a strong bias toward longer alleles and higher levels of v-cam and e-selectins among patients with acute pancreatitis. Thus, polymorphism of the GT repeats in the HO-1 promoter region may be a risk factor for developing acute pancreatitis. Further studies are now underway to analyze the pancreatic levels of HO-1 protein in acute pancreatitis patients and controls and to determine whether the presence of the short alleles facilitate HO-1 upregulation and consequently promote its protective anti-inflammatory function in acute pancreatitis.

4.14 Antibiotic Coated VICRYL Prevents P. aeruginosa Colonization in vitro Better than VICRYL Plus®

Posted on January 18, 2018

J. Baker1, E. Gulbins1,2, M. J. Edwards1, A. Seitz1  1University Of Cincinnati,Department Of Surgery,Cincinnati, OH, USA 2University Of Duisburg-Essen,Department Of Molecular Biology,Essen, , Germany

Introduction:  Surgical site infections affect up to 5% of patients undergoing inpatient surgery with a cost of $10 billion annually. Pseudomonas aeruginosa (P. aeruginosa) is the most common gram-negative pathogen and accounts for approximately 9% of SSIs. Triclosan coated suture (VICRYL plus) was introduced by Ethicon and has demonstrated in vitro and in vivo inhibition of bacterial growth against multiple pathogens.  We aimed to test our antibiotic coated suture versus VICRYL plus® suture against P. aeruginosa in vitro. We hypothesized that our suture would significantly decrease bacterial colonization compared to VICRYL plus®.

Methods:  Single stranded 2-0 VICRYL suture was coated with various antibiotics (levofloxacin, chloramphenicol, doxycycline, erythromycin, and vehicle control) using a proprietary method. P. aeruginosa was incubated overnight on tryptic soy broth (TSB) agar plates.  Sterile cotton tip applicators were used to transfer bacteria to TSB media and were incubated for 1 hour under agitation at 125 RPM into log growth phase. An inoculate of 1000 cfu in 1000 μL was prepared by measuring the optical density at 550 nm and calculating dilutions based upon a previously prepared standardization curve. Bacterial solution was placed in each well of a 24 well plate along with a 2 cm length of coated suture. The plate was then incubated at 37oC , at 100% humidity for 24 hours. The sutures were then removed and placed in 10 mL of PBS, sonicated in a 37oC water bath for 10 minutes to release adherent bacteria. Bacteria were then quantified by the plate dilution method.

Results: Doxycycline, chloramphenicol, and levofloxacin coated VICRYL significantly reduced P. aeruginosa colonization compared to vehicle coated VICRYL (99.97%, 99.06%, and 99.99%, respectively; p<0.05) and VICRYL plus® (99.95%, 98.50%, and 99.0%, respectively; p<0.05). There was no significant difference between vehicle coated VICRYL and VICRYL plus® (37.13%; p 0.11) against P. aeruginosa.

Conclusion: Our method of antibiotic coating significantly reduced P. aeruginosa colonization of VICRYL suture compared to both vehicle coated suture and VICRYL plus®. Our novel antibiotic coated suture is an effective tool to reduce bacterial colonization in vitro, and may prove better at preventing SSIs than commercially available VICRYL plus®. 

 

4.15 Tissue-engineered Human Spleen can be Generated in a Mouse Model

Posted on January 18, 2018

M. Isani1, X. Hou1, A. Fode1, C. Schlieve1, T. C. Grikscheit1  1Children’s Hospital Los Angeles,Los Angeles, CA, USA

Introduction:
Splenectomy leaves patients susceptible to overwhelming post-splenectomy infection (OPSI), which results in high mortality. The spleen is remarkably regenerative as demonstrated by the phenomenon of splenosis that was originally noted after autotransplantation, necrosis, and then growth of splenic tissue in the peritoneum of patients after traumatic splenic rupture.  Although splenosis sometimes generates functional spleen, it is built on a construct of necrotic tissue and has variable protection against OPSI. We hypothesized that omental implantation of human multicellular clusters of spleen stem/progenitor cells or splenic units (SU) on a biodegradable polymer scaffold could generate controlled functional tissue-engineered human spleen. 

Methods:
After appropriate IACUC and IBC approval, human spleen samples from patients undergoing splenectomy were washed with Hank’s balanced salt solution (HBSS), and mechanically and chemically digested into SU in a variation of a murine protocol we have previously described. SU were loaded onto a 2mm biodegradable polyglycolic/poly-L lactic acid scaffold and implanted either into the omentum or subcutaneously in NOD/SCID mice. After four weeks, implants were harvested, stained with hemotoxylin and eosin, and immunostained with a specific human anti-lamin antibody, a nuclear envelope marker and anti-PCNA, proliferating cell nuclear antigen. 

Results:
Omental implants generated visible human tissue-engineered spleen in mice (n=3). H&E staining revealed evidence of splenic structural elements. Immunofluorescence staining for human anti-lamin antibody confirmed that cells in TES derive from the human donor cells.  Furthermore, the PCNA costain identified actively proliferating human cells (nuclear stain DAPI in blue, Figure 1A, anti-lamin in green, Figure 1B, anti-PCNA in pink, Figure 1C, and Figure 1D is a merged image with inset showing proliferating human cell, all images at 40x with 50μm scale bar).

Conclusion:
These experiments demonstrate growth of human donor cells into tissue-engineered spleen from splenic units implanted onto a biodegradable construct in mice. Additionally, proliferation of these proven human cells is recorded.  Proliferation is fundamental to establishing, growing, and maintaining tissue-engineered organs.  This approach may therefore prove beneficial in future protection against OPSI via direct implantation of human splenic units in a trauma setting or to control splenic regeneration in failed partial splenectomy.
 

4.12 Human Intestinal Preservation: Histological Comparison between UW and HTK in Hypothermic Storage

Posted on January 18, 2018

M. Finotti1,2, T. Lysyy1, R. Morotti1, A. S. Munoz-Abraham1, A. Bertacco2, R. Agarwal1, C. Ibarra1, R. Patron1, A. Alkukhun1, F. D’Amico1,2, M. Rodriguez-Davalos1, D. Mulligan1, J. Geibel1  1Yale University School Of Medicine,Surgery,New Haven, CT, USA 2University Of Padua,Transplantation &Hepatobiliary/Surgery,Padua, PADUA, Italy

Introduction:

Human intestinal grafts intended for transplantation require optimal preservation to reduce ischemic injury. Hypothermic preservation is crucial in decreasing ischemia, and the type of solution used in cold preservation could influence graft survival. In this study, we used the Park/Chiu histological damage score to compare the efficacy of two solutions (UW and HTK solutions) in preserving human intestine under two different conditions: static storage and continuous hypothermic perfusion using an intestinal perfusion unit (IPU).

Methods: 18 human intestines were used, 14 were procured by our surgical team, with a Cold Ischemia Time (CIT) between 1-4 hours; and 4 were shipped with a CIT between 5-9 hours. The type of solution was randomized: UW or HTK was used to perfuse the intestine graft.  In 4 intestines Lactate Ringer (LR) was used to flush the luminal intestine after harvesting, then the graft were stored in static preservation solution. Duplicate samples from the jejunum and ileum were collected before the initiation of cold storage (T0 samples). After mean 8.6 hours of static (T8) or continuous perfusion preservation (PT8), we sampled each intestinal section (jejunum and ileum) in duplicates (total of 208 samples collected).

Results:

HTK and UW were used for the initial luminal flush and subsequent cold storage in six and eight intestines respectively. In four intestines, LR was used to flush the lumen and then specimens were stored in a solution of UW. At T0 there was no difference between solutions. The jejunum had a mean score of 1.06, 1.75 and 0.87 with UW, HTK and UW+LR respectively. In the ileum, the mean ischemic scores were 0.93, 0.83 and 1.33 with UW, HTK and UW+LR respectively. At T8 jejunum maintained in UW had a lower histological damage score in comparison to HTK (mean 1.75 with UW vs 3.16 with HTK, p<0.0193). The same pattern occurred when the IPU was used (PT8): the mean ischemic score in jejunum was 1.5 with UW vs 3 with HTK (p<0.0287). In the ileum, no statistically significant difference was seen at T0, T8 and PT8.

Conclusion:Small intestine ischemic damage during the hypothermic preservation before transplantation may depend on the choice of preservation solutions. In the present study, we demonstrated that in the jejunum, UW solution is associated with less ischemic damage in comparison to HTK after 8 hours of storage, both in the static and in continuous perfusion. In the ileum, there is no significant difference in ischemic damage in specimens preserved with  UW and HTK. Furthermore, we showed that at the time of intestinal harvest, LR is viable option to flush, clean, and prepare the intestine for transport with no apparent increased histological damage. These results remain consistent whether the intestine is continuously perfused or maintained in a static 4oC environment.

4.13 Screening Waitlisted Transplant Patients for Swine MHC Class II Antibodies

Posted on January 18, 2018

J. M. Ladowski1, G. Martens1, L. Reyes1, Z. Wang1, M. Tector1, A. J. Tector1  1University Of Alabama at Birmingham,Birmingham, Alabama, USA

Introduction: Xenotransplantation, using genetically-modified swine, represents a solution to the organ shortage and is rapidly approaching clinical trials. Avoiding recipients with donor-specific major histocompatibility complex (MHC) antibodies greatly extends graft life. There exist many tools to screen allotransplant recipients for the presence, specificity, and consequence of antibodies but few reagents to screen potential xenotransplant recipients. Therefore, the prevalence, specificity, and immunogenic potential of anti-swine MHC class II antibodies remains unknown. 

Methods: Broadly positive swine MHC class II cells were generated by transfection with a human class II transactivator transgene and single antigen cells were made by expression of swine class II heavy chains. Serum from 237 waitlisted patients were screened using a flow cytometry crossmatch. Positive samples were further assessed for complement activation with a complement dependent cytotoxicity assay. 

Results: The screening flow cytometry crossmatch revealed 30/237 (12.6%) samples with potential anti-pig MHC class II antibodies. The 30 sera were tested on single allele swine cells and showed binding predominately to swine DQ alleles. Antibodies were able to activate complement and showed an increased killing of MHC-DQ relative to MHC-DR positive cells.      

Conclusion: Reagents to screen potential xenotransplant recipients for the presence, specificity, and consequence of antibodies were produced. The methods detected many patients with haplotype specific anti-swine MHC class II antibodies capable of activating complement, especially against swine-DQ alleles. Lessons from allotransplantation show importance of screening and avoiding patients with these donor-specific antibodies.  

 

4.10 Characterization of Porcine Xenoantibody Response In Vitro

Posted on January 18, 2018

K. Pham1, N. Wilson-Schlei1, R. Redfield1  1University Of Wisconsin,Department Of Surgery,Madison, WI, USA

Introduction:  One possible solution to the current shortage of donor organs is the use of xenografts. A potential model for viable xenotransplantation has been porcine organs but the associated immune barriers has delayed its transition to clinical practice. A key component to this barrier is the difference in carbohydrate structures expressed on porcine cells, which serve as antigens to humans. Even with the use of carbohydrate pathway knock-out pigs in combination with immunosuppression, there are still issues with long term graft survival and function in non-human primates. The goal of our experiment is to characterize the pre-existing immune response of porcine xenotransplantation in vitro. 

Methods:  Using six samples of naïve rhesus macaque serum, we performed a flow crossmatch assay with porcine peripheral blood mononuclear cells (PBMC). Porcine PBMC’s were incubated with three different groups: media alone (used as a negative control), porcine plasma (self), and rhesus macaque plasma (the experimental group). We analyzed IgG and IgM binding between lymphocytes and macrophages using flow cytometry. Results were reported as median fluorescence intensity (MFI) and unpaired two-tailed T test was used for statistical analysis.

Results: It was found that naturally occurring anti-pig IgG binding had significantly increased MFI in the six rhesus macaque plasma samples when incubated with porcine PBMC (P value < 0.01). Anti-pig IgG binding was also found to be most prevalent in cells outside the lymphocyte gate.  In terms of IgG binding, MFI did not change for lymphocytes when compared to negative control (self and no plasma). IgG binding to non-lymphoid cells resulted in an average Δ MFI of 100 when compared to negative controls. Interestingly, there appeared to be a heterogeneous response when comparing individual amounts of anti-pig IgG and IgM. Ranges for IgG were of 737 to 1046 MFI. As for IgM, MFI ranged from 291 to 430.

Conclusion: With this experiment, we confirm previous reports that naturally occurring antibodies do bind to xenoantigens (Burlak, 2014, Ezzelarab, 2006, Rood, 2006). There appears to be an increased presence of IgG binding, particularly among cells that may be macrophages. This is consistent with previous results that implicated cells which appeared to be macrophages and xenoantibody of the IgG isotype. However, we need to analyze the data to determine whether the cells in this gate, in which most binding occurred, bind CD68 to show they are indeed macrophages. Future considerations will involve a more in-depth investigation of the role of macrophages in this flow crossmatch assay and also if PBMC’s represent a valid model for xenograft failure. 

 

4.11 Assessing Pollution Samples for Autoimmune Promoting Activity In Vitro

Posted on January 18, 2018

H. Tran1, C. O’Driscoll1, E. Hoffmann1, J. Fechner1, J. Mezrich1  1University Of Wisconsin,Transplant/Surgery/SMPH,Madison, WI, USA

Introduction: Autoimmune diseases have been dramatically increasing in incidence worldwide with over 80 recognized diseases. Particulate matter (PM) from airborne pollution has been strongly associated with aggravating autoimmunity in humans, but it has been difficult to characterize mechanisms for aggravation of disease and identify which exposures are most pathological. Our group studies the role the aryl hydrocarbon receptor (AHR) on T-cell differentiation and effector function. Our previous studies suggest that inhalation of polycyclic aromatic hydrocarbons (PAHs) found in PMs may impact autoimmune disease through the AHR. We hypothesized that different pollution samples will induce varying, yet predictable effects on autoimmune disease depending on the balance of the fractions of chemicals in the sample. To characterize this, mice were exposed to inhaled pollution samples (urban dust particle (UDP) and two diesel emission particles (DEP1 and DEP2)) and control two weeks prior to induction of experimental autoimmune encephalomyelitis (EAE). Inhalation of both diesel samples significantly aggravated EAE, while exposure to UDP trended towards ameliorating disease. In this study we explored in vitro assays that could characterize the pathologic effects of different exposures.

Methods:  Splenocytes of 2D2 mice, transgenic for a T-cell receptor specific to MOG (the antigen that mice respond to in EAE), were stimulated with MOG peptide and LPS (to mature DCs). Dilutions of the three samples were added to the cells. The cells were cultured for 4-5 days and the supernatants of the cultures were harvested. ELISA was used to measure IFNg and IL-17 levels, as measures of immune activation. Cytokine bead arrays were also used to measure additional cytokines. We cultured bone-marrow derived dendritic cells (BMDC) in another assay, exposed them in culture for 24 hours with pollutants or control, and assayed them for cytokine production.

Results: In the MOG assay, all three PMs led to decreased levels of IL-6. Diesel exposure increased IFNg, but decreased IL-10 expression, a cytokine known to suppress immune responses. In contrast, UDP had no effect on IFNg expression, but increased IL-17 and IL-10 expression. In the BMDC assay, exposure to UDP increased levels of IL-10 production while the diesel samples did not. UDP also increased IDO expression in DCs, an enzyme involved in the production of regulatory T cells.

Conclusion: The current data suggests that the in vitro assays may have potential for screening the autoimmune promoting activity in samples of pollution. UDP, which is made up of multiple different sources of pollution, is known to include a complex mixture of chemicals, which may affect many different cell-types, whereas diesel samples, derived from single sources of pollution, may have more predictable and specific consequences on T-cell differentiation and disease. Further characterization of this will allow improved strategies for avoidance and remediation of polluted environments.

 

4.09 Regional Differences in Elastic Strain Correlate with Altered Mechanotransduction and Keloid Progression

Posted on January 18, 2018

T. Dohi1, J. Padmanabhan1, P. Than1, S. Akaishi3, M. Terashima2, N. Matsumoto3, R. Ogawa3, G. C. Gurtner1  1Stanford University,Department Of Surgery,Palo Alto, CA, USA 2Stanford University,Department Of Civil & Environmental Engineering,Palo Alto, CA, USA 3Nippon Medical School,Department Of Plastic, Reconstructive And Aesthetic Surgery,Bunkyo, TOKYO, Japan

Introduction: There is high morbidity, mortality, and cost related to fibroproliferative disorders. Of these, keloids are unique in that they progress beyond the original site of injury and form large, linear scars often of significantly different shape from the initial wound. The unknown pathophysiology, high rate of recurrence, and lack of effective treatment modalities makes keloid disease a challenging clinical problem. Progression beyond the original injury site and extension into normal peripheral skin highlights the zone between the keloid and surrounding skin as a target for investigation.

Methods:  We analyzed posture-related changes in local strain in various human anatomic locations correlated with keloid formation (n=10). Additionally, we developed a finite element method (FEM) analytic model to study the effect of von Mises stress and true strain in keloid-containing regions of skin. We also performed targeted-molecular analysis of human keloid-containing skin samples to identify the key biochemical pathways involved in keloid progression (n=5). 

Results: Anatomic regions prone to keloid formation are subject to high-levels of multidirectional posture-related strain. Focusing on these areas we created a model using finite element analysis and demonstrated that multidirectional loading leads to high true strain in the peripheral tissue surrounding the keloid. Furthermore, comparative immunohistochemical staining of human keloid tissue, peripheral tissue and control skin revealed that the peripheral tissue exhibits high levels of proliferation and altered mechanotransduction components including HSP27 and NFkB.

Conclusion: In summary, we describe a novel mechanism through which keloid disease may progress beyond the original wound site. Specifically, we show that changes in human posture result in elevated true strain in tissues immediately surrounding keloids in anatomic regions that are prone to keloid formation. We found that key mechanotransduction signaling pathways are altered in these regions, which may underlie the unique spread of keloid disease beyond the original wound site. These findings identify several molecular targets for therapy with potential for rapid clinical translation.

 

4.08 Notch Activator Jagged1 Results in Increased Closure Rates in an Ex Vivo Murine Skin Wound Model 1

Posted on January 18, 2018

Z. Aburjania1, T. W. King1  1University Of Alabama at Birmingham,Plastic Surgery,Birmingham, Alabama, USA

Introduction:
Decreased rates of wound healing affect millions of patients annually. We are interested in discovering novel strategies to enhance the wound healing process in diabetic patients.  We have previously shown that inhibiting Notch inhibits wound healing. Based upon our previous work, we propose that upregulation of Notch would increase rates of wound healing.  JAG1 is a known activator of Notch. Therefore, we hypothesized that applying topical JAG1 to ex vivo excisional wounds on the backs of mice would result in increased Notch activity, and thus an increased wound healing rate as compared to untreated wounds.

Methods:
Skin biopsies from 12-week old, healthy mice, (1-cm2 full-thickness) were cultured ex vivo. A 4-mm wound was created in the center of the skin biopsy. A topical application onto the open wound bed of JAG1 (10 nM) or vehicle (PBS) was applied daily for 14 days. Digital photographs were taken daily and the skin was processed for histological and protein analysis on days 3, 7, 10, and 14. The wounds were analyzed using ImageJ software. Wound area was calculated as a percent area of the original wound size. Statistical significance was defined as p<0.05 using the students’ t-test.

Results:
Partial to complete re-epithelialization was seen in the wounded tissues over the experimental period in both the control & JAG1 treated groups. The mouse skin treated with topical JAG1 had an increased rate of wound closure when compared to wounds treated with PBS.

Conclusion:
JAG1 increases the rate of re-epithelialization of cutaneous wounds in an ex vivo murine wound-healing model, indicating that Notch signaling plays a crucial role in wound healing in mice. Based upon our findings, further study of Notch in wound healing should be conducted which may then lead to better therapeutics for the wound healing process in patients. 

1

4.07 A Novel Human Xenograft Model to study Strategies in Articular Cartilage Regeneration.

Posted on January 18, 2018

M. P. Murphy1, M. Lopez1, R. C. Ransom1, O. Marecic1, R. E. Brewer1, L. S. Koepke1, S. Mascharak1, C. F. Chan1, M. T. Longaker1  1Stanford University,Surgery,Palo Alto, CA, USA

Introduction:  Currently there are no effective strategies for regenerating articular cartilage in diseases such as osteoarthritis (OA). Our group has utilized the mouse model to study the effects of surgical, chemical and cellular manipulation in articular cartilage regeneration. We aim to understand the effects of surgical, chemical and cellular manipulation in articular cartilage regeneration in a novel xenograft human model.

Methods: We transplanted the phalanges of 18-week old fetal specimens subcutaneously in the dorsum of immuno-compromised NSG P3 mice. After we confirmed viabiliy of the human xenograft using MRI, microCT and on gross inspection we performed microfracture (MF) surgery on the articular joints. Histological composition was assessed using Movat’s Pentachrome stain and Safranin O/Fast green stain. Immunohistochemistry (IHC) was performed assessing levels of Col 1, 2, 10 and MMP13. Proliferation was assessed with EdU labelling in histology and intracellular FACS. 

Results: We successfully developed a model of investigating the effects of surgical manipulation on human articular cartilage regeneration. (Figure 1) Following 6 weeks of implantation the xenograft had grown in size. The microCT images show calcification and MRI show viable articular cartilage. We have found that similar to our mouse model, fibrocartilage forms after MF. On histology and IHC we have determined the difference between normal articular cartilage and MF tissue. Cellular proliferation increases following injury.

Conclusion: We believe that our surgical technique and topical factors including BMP2 and Avastin will provide regenerative surgeons with a new approach to treating OA. We will further augment these factors with induced SSC from human Adipose-derived Stromal Cells. Our findings provide us with a "preclinical" human model that can be utilised to effectively investigate strategies for articular cartilage regeneration. 

 

4.05 Human Small Intestine Transplantation: Jejunum is more Susceptible to Ischemia than Ileum

Posted on January 18, 2018

T. Lysyy1, M. Finotti1,2, R. Morotti1, A. S. Munoz-Abraham1, A. Bertacco2, R. Agarwal1, C. Ibarra1, R. Patron1, A. Alkukhun1, F. D’Amico1,2, M. Rodriguez-Davalos1, D. Mulligan1, J. Geibel1  1Yale University School Of Medicine,Surgery,New Haven, CT, USA 2University Of Padua,Transplantation And Heptobiliary/Surgery,Padua, PADUA, Italy

Introduction: The human small intestine is the most ischemia-sensitive organ among all transplanted abdominal organs. Intestinal transplantation (IT) is an effective alternative for total parenteral nutrition in patients with short-bowel syndrome or intestinal failure. Postoperative complications of IT (including graft failure, acute rejection, and necrosis) are correlated to poor preservation and prolonged ischemia time. In this study, we evaluated the sensitivity of human small intestinal grafts to ischemic injury in two different hypothermic preservation conditions, and compared the extent of ischemic damage in both proximal (jejunum) and distal (ileum) segments.

Methods: Eighteen human small intestines were procured: 14 grafts were obtained on site with cold ischemia time (CIT) range of 1-4 hours (avg. 2.03 hrs), and 4 were shipped to our institution with CIT range of 5-9 hours (avg. 7.56 hrs). Histological samples were collected from jejunum and ileum intestinal segments at different time points (2 samples per time point from each segment, n=208 samples). The first set of samples was obtained at the time of procurement/organ delivery (T0) (baseline histological assessment). A second set was obtained at T0 with samples stored at 4oC for mean 8.6 hours (T8). A third set was obtained after mean 8.6 hours of continuous hypothermic perfusion using an intestinal perfusion unit (IPU) (PT8). All samples were fixed with 10% formalin prior to a blinded histological evaluation. Samples were scored using the Park/Chiu system. Scores were analyzed with Graph Pad Prism 6 for each group.

Results:In the group with CIT 1-4 hrs at T0, there was no histological score difference between the jejunum and ileum (mean  score in proximal samples 0.928 vs distal 0.961).  However, at T8 (mean score in proximal was 2.10 vs distal 1.39, (p<0.0005)) and PT8 (mean proximal 1.89 vs distal 1.25, (p<0.0005)), there is a statistically significant difference in the ischemic scores of the jejunum and ileum. In the second group with CIT 5-9 hrs the mean ischemic scores at T0 were 2.38 in jejunum and 1 in ileum (p<0.0232). At T8, the mean scores were 3 for jejunum and 3.75 for ileum, with no statistical difference (p>0.05). Similarly, at PT8 (mean ischemic scores: proximal 3.12 vs distal 3.87), there was no difference in the degree of ischemia between the two segments, (p>0.05).

Conclusion:The proximal segment (jejunum) of the human intestine is more susceptible to ischemia than the distal segment (ileum). Prolonged CIT (5-9 hrs) increases the degree of ischemic damage, affecting the jejunum more than ileum. The early use of the intestinal perfusion unit after procurement (<5 hours) appeared to reduce ischemic injury in the jejunum compared to static cold storage. Furthermore, selective transplantation of the more distal segment of the small intestine (ileum) could reduce ischemia-related postoperative complications and improve clinical outcomes and patient survival.

4.06 Cellular and Mechanical Mechanisms Underlying Regeneration in Mandibular Distraction Osteogenesis

Posted on January 18, 2018

R. C. Ransom1,2, A. C. Carter3, A. Salhotra1,2, T. Leavitt1, O. C. Marecic1,2, M. Lopez1,2, M. Murphy1,2, C. K. Chan1,2, D. C. Wan1, H. Y. Chang3, M. T. Longaker1,2  1Hagey Laboratory For Pediatric Regenerative Medicine,Department Of Surgery, Division Of Plastic And Reconstructive Surgery, Stanford University School Of Medicine,Stanford, CA, USA 2Institute For Stem Cell Biology And Regenerative Medicine,Stanford University,Stanford, CA, USA 3Center For Personal Dynamic Regulomes,Stanford University,Stanford, CA, USA

Introduction:  Accumulated evidence indicates that mechanical cues, which include physical forces, alterations in extracellular matrix mechanics and changes in cell shape, are transmitted to the nucleus directly or indirectly to orchestrate transcriptional activities that are crucial for tissue regeneration. Although mechanotransduction is thought to occur via integration of multiple signaling pathways, the precise mechanism leading to downstream cellular responses is not well understood. We have developed a mouse model of mandibular distraction osteogenesis (DO) which allows for tracing of cell fate and genetic dissection of mechanotransduction during bone formation.

Methods:  We examined cell-type-specific responses to mechanical force within distinct subpopulations of the mouse skeletal stem cell (mSSC) hierarchy. After determining that bone, cartilage, and stromal tissue are clonally derived in mice from lineage-restricted stem and progenitor cells in vivo, we employed this strategy to purify specific skeletogenic populations during mandibular distraction osteogenesis by prospective isolation using FACS. We employed the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) to profile open chromatin landscapes in these cell populations to understand the epigenetic changes in response to distraction. To investigate the role of mechanotransduction via focal adhesion kinase (FAK) in distraction osteogenesis, we inhibited FAK using the small molecule PF57223, a specific and potent inhibitor of FAK signaling. Three-dimensional reconstruction of μCT images of gradually distracted specimens revealed disrupted bone formation under conditions of FAK inhibition. ATAC-seq was employed for determination of FAK-responsive regions of the epigenome within each subpopulation of the skeletal stem cell hierarchy.

Results: We show that mechanical force augments the numbers and function of multiple cell populations across the skeletal hierarchy, including mouse skeletal stem and progenitor cells and their differentiated subsets. Mechanistically, distraction induces robust cell-matrix interactions that are coupled to cell-specific transcriptional responses via epigenomic pathways and pharmacological inactivation of this pathway disrupts bone formation. 

Conclusion: Here we employ a rigorous mandibular DO model in mice that is genetically dissectable, allowing for detailed examination of the fundamental principles regulating de novo bone formation. The identification of the cellular source of regeneration, the timeline for progenitor cell response, and determination of how these cells transduce physical stimuli to enact a regenerative response may provide new and effective strategies for reconstruction of the craniofacial skeleton.

 

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