78.17 A Novel Role of TLR9 in Regulation of Peritoneal B Cell Differentiation in Polymicrobial Sepsis

Posted on January 26, 2017

L. Xu1,2, M. Deng1, H. Xu1, R. Hoffman1, P. Loughran1, T. Billiar1  1University Of Pittsburgh School Of Medicine,Department Of Surgery,Pittsburgh, PA, USA 2Union Hospital, TongJi Medical College?Huazhong University Of Science And Technology,Department Of Emergency,Wuhan, HUBEI, China

Introduction: Dysregulation of the inflammatory response against infection contributes to mortality in sepsis. Toll like receptor 9 (TLR9) is expressed in the cytoplasmic in immune and senses viral and bacterial DNA to initiate immune responses. However, the mechanism underlying the effects of TLR9 in sepsis has not been elucidated clearly.

Methods: To investigate the role TLR9 in sepsis, TLR9 mutation (TLR9CpG1/CpG1) and TLR9 global KO mice were subjected to cecal ligation and puncture (CLP) with their control strains WT (C57BL/6) for 18 hours. 

Results:Bacterial load in peritoneum and blood was significantly lower in TLR9CpG1/CpG1 and TLR9 KO septic mice than in their control strain at 18 hours after CLP. CLP-induced systemic cytokines, such as IL-6, IL-1β, IL-10, chemokines, such as MCP-1 and KC, and histones, was significantly lower in TLR9 deficiency mice than in WT mice. Interestingly, the peritoneal B cells number assessed by flow cytometry in both TLR9 deficient mice strains was significantly higher than in their control strain at baseline and after CLP. Importantly, the percent of B1 cells, which have phagocytosis ability, was significantly higher in the TLR9 deficient strains than in the WT mice.

Conclusion:Our data suggests an unrecognized role of TLR9 in regulation of host defense via regulation of peritoneal B cell function. However, the mechanism of how TLR9 regulation the peritoneal B cell function needs to be further elucidated.
 

78.15 Downregulation of the Tumor Supressor RASSF2 in Colorectal Cancer Cell Lines

Posted on January 26, 2017

J. Carter1, J. Burton1, N. Galbraith1, M. R. Eichenberger1, S. Galandiuk1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction:
We have previously identified increased expression of all members of the miR-200 family in colorectal cancer (CRC) as compared to normal colon epithelium cell lines. The members of the miR-200 family are highly enriched in epithelial tissues and they have been linked to several cancers including CRC. Using our screening data, pathway analysis software identified all members of the miR-200 family to have a validated regulatory role upstream of Ras proteins in the MAPK canonical pathway. Further analysis of the miR-200 family confirmed validated target interaction with Ras associated domain-containing protein 2 (RASSF2). RASSF2 is one of six proteins in the RASSF family that is encoded by the RASSF2 gene. RASSF2 is a negative regulator of Ras and binds directly to K-Ras within the Ras effector domain in a GTP-dependent process. RASSF2 has previously been shown to promote apoptosis, cause cell cycle arrest and is regarded as a novel K-Ras-specific effector and potential tumor suppressor. Therefore, we hypothesize that miR-200 family and RASSF2 expression will differ between CRC cell lines and a normal colon epithelial cell line.

Methods:
Four CRC cell lines (SW116, SW480, HT29, T84) and one normal colon epithelial cell line (CCD841) were acquired for use (ATCC®, Manassas, VA). Total RNA was converted to cDNA. Specific TaqMan® miRNA primers and probes for the miR-200 family and RASSF2 mRNA were used to bind to complementary sequences on target cDNA during qRT-PCR (Life Technologies, Carlsbad, CA). All reactions were completed in duplicate. Statistical analysis was performed using a two sample t-test comparing each CRC cell line to the normal colon epithelial cell line. Western blots were performed on cell lysates to identify RASSF2 protein relative concentrations.

Results:
Data are shown in Figure 1. We observed up-regulation of all members of the miR-200 family in CRC cell lines as compared to the normal colon epithelial cell line (p<0.001). Conversely, RASSF2 mRNA was found to be down-regulated in CRC cell lines compared to the normal colon epithelial cell line (p<0.001). RASSF2 protein was absent in 3 of the 4 CRC cell lines and down-regulated in the remaining CRC cell line (Dukes' B, SW480) compared to the normal colon epithelial cell line.

Conclusions:
As hypothesized, miR-200 expression was increased and RASSF2 mRNA and protein was decreased in CRC cell lines, suggesting a potential association between the miR-200 family and RASSF2. Further study into the gain and loss of function of the miR-200 family should be investigated to determine whether the miR-200 directly targets RASSF2 and influences CRC tumorigenesis.

78.14 Proximal Esophageal Contraction in Stroke and Head and Neck Cancer Using High Resolution Manometry

Posted on January 26, 2017

D. Lukato1, C. Jones1, S. Rosen1, C. Walczak1, T. McCulloch1  1University Of Wisconsin – Madison,Otolaryngology/Surgery,Madison, WI, USA

Introduction:
Patients with stroke or head and neck cancer (HNCa) often experience long-term effects of dysphagia. Along the swallowing pathway, the proximal esophagus exhibits a unique contractile property compared to the rest of the esophagus. There is currently no clear understanding of how the proximal esophageal contraction is altered in a pathological state. The purpose of this study is to evaluate the effects of stroke and HNCa on swallowing-related proximal esophageal pressure.

Methods:
We analyzed retrospective swallowing pressure data from 62 healthy adults, 9 patients with stroke, and 17 patients with HNCa. Each participant performed three swallows of 1-3 mL of normal saline. High resolution manometry was used to measure four variables: proximal esophageal contraction amplitude; contraction duration; pharyngo-esophageal transition time from the velopharynx; and pharyngo-esophageal transition time from 1 cm above the upper esophageal sphincter (UES). A one-way analysis of variance (ANOVA) was done to compare variables between the three groups. We hypothesize that patients with stroke and HNCa have longer pharyngo-esophageal transition time, shorter contraction duration, and lower proximal esophageal contraction amplitude than in healthy people.

Results:
Patients with stroke experienced significantly longer pharyngo-esophageal transition time from the velopharynx than healthy participants and HNCa patients (p < 0.01). Patients with stroke exhibited longer pharyngo-esophageal transition time from 1cm above the UES than both controls and HNCa patients (p < 0.01). HNCa patients had significantly longer pharyngo-esophageal transition time from 1 cm above the UES than healthy participants (p <0.05). The control group experienced significantly longer contraction duration than both stroke and HNCa groups (p < 0.05 and p < 0.01, respectively). No significant differences in contraction amplitude were observed (p > 0.05).

Conclusion:
Patients with stroke and HNCa experience delayed onset of proximal esophageal contraction and shorter contraction duration. These findings may be helpful to clinicians for understanding the effects of stroke and HNCa on swallowing. Future directions to address our limitations include evaluation of the proximal esophageal contraction with different bolus volumes, consistency, and swallowing techniques.
 

78.13 Characterization of MicroRNA Expression in Colorectal Cancer Cell Lines

Posted on January 26, 2017

J. Burton1, J. Carter1, N. Galbraith1, M. R. Eichenberger1, S. Galandiuk1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction:
Cell lines are a common experimental model used to study biological mechanisms involved in various diseases. In-vitro cell line studies allow for the investigation of signaling pathways, functional processes, identification of molecular markers of disease, and testing of cancer therapeutics. microRNAs (miRNAs) affect gene expression by negative regulation of target mRNA and are dysregulated in a variety of cancers including colorectal cancer (CRC). They exhibit a variety of regulatory functions related to cell growth, development, and differentiation. miRNAs are inherently stable accounting for their emerging use as biomarkers for human disease and as targets for disease intervention. We chose to characterize the differential expression of miRNAs in CRC as compared to a normal colon epithelial cell line in order to identify potential therapeutic targets for intervention.

Methods:
Sporadic CRC cell lines (SW116, SW480, HT29, T84)  representing locally confined to metastatic disease (modified Dukes’ A-D staging) and a normal colon epithelial cell line (CCD841) were acquired (ATCC®, Manassas, VA). Total RNA was extracted from cells and RNA quantity and purity determined. For each cell line, RT and qRT-PCR was performed. The expression levels of 380 miRNAs were examined using microfluidic array technology (Life Technologies, Carlsbad, CA). miRNA expression of each CRC cell line was compared to the miRNA expression of the normal colon epithelial cell line. Statistical analysis was performed using a two sample t-test and results were regarded significant when p<0.05.

Results:
Each cell line was screened 4 times. Comparison of miRNA expression in non-metastatic CRC cell lines (Dukes’ A, SW116, and Dukes’ B, SW480) compared to normal colon epithelium (CCD841) identified 190 significantly dysregulated miRNAs, 36 up-regulated and 139 down-regulated miRNA (p<0.05). Five of the 8 most significantly upregulated miRNAs were members of the miR-200 family (p<0.0001, fold-change 2688-18830). Comparison of metastatic CRC cell lines (Dukes’ C, HT29, and Dukes’ D, T84) as compared to normal colon epithelium identified 231 significantly dysregulated miRNAs, 39 up-regulated and 192 down-regulated miRNA (p<0.05). Five of the 6 most up-regulated miRNAs were all members of the miR-200 family (p<0.0001, fold-change 4171-35668). Single assay confirmation of individual miR-200 family members was performed in each cell line.

Conclusions:
We have characterized the miRNA expression in CRC cell lines as compared to a normal colon epithelial cell line. Identification of dysregulated miRNAs permits identification and investigation of clinically relevant miRNAs as potential targets for disease intervention in CRC. Members of the miR-200 family were highly expressed in all tested CRC cell lines. This provides an opportunity to study the miR-200 family already known to play a role in tumor progression and cancer metastasis.

78.12 High Levels of Ceramide in Breast Cancer and Its Microenvironment

Posted on January 26, 2017

K. Moro1, J. Tsuchida1, A. Ohtani1, M. Endo1, T. Niwano1, K. Tatsuda1, C. Toshikawa1, M. Hasegawa1, M. Ikarashi1, M. Nakajima1, Y. Koyama1, J. Sakata1, T. Kobayashi1, H. Kameyama1, K. Takabe2, T. Wakai1, M. Nagahashi1  1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA

Introduction: Sphingolipids, including sphingosine-1-phosphate (S1P) and ceramide, have
emerged as key regulatory molecules that control various aspects of cell growth and survival in cancer. Despite their critical roles, the levels of sphingolipids have never been measured in patients due to lack of methods to precisely quantify them until recently. We have recently published high levels of S1P not only in breast tumor, but also in tumor microenvironment, such as tumor interstitial fluid (IF), and reported that S1P plays pivotal roles in breast cancer progression. On the other hand, the levels of ceramide, a bioactive metabolite of S1P, in breast cancer patients have not yet well investigated to date. The aim of this study is to determine the levels of ceramide in the tumor and its microenvironment by mass spectrometry utilizing surgical specimens obtained from breast cancer patients.

Methods: Surgical specimens were obtained from 45 breast cancer patients who underwent mastectomy in Niigata University Medical and Dental Hospital. Tissue samples from 1) breast cancer (tumor), 2) peri-tumor normal breast defined as tissue within 1 cm from the gross edge of cancer and 3) normal breast distant from the cancer were collected from the surgical specimens. IF from the tumor, peri-tumor and normal tissue was also collected by a centrifugation method. Sphingolipids, including ceramides (C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0, C26:0) and their metabolites of monohexosylceramides and sphingomyelin, in the tissue samples and the IF were determined by mass spectrometry. Results were analyzed for statistical significance with the Kruskal-Wallis test.

Results:Levels of all species of the ceramides, except for C26:0-ceramide, were significantly higher in tumor tissue than those in peri-tumor and those in normal breast tissue. Similarly, all species of the monohexosylceramides, except for C26:0-monohexosylceramide, were significantly higher in tumor tissue than those in peri-tumor and those in normal breast tissue. All of the sphingomyelin were also significantly higher in tumor tissue than those in peri-tumor and those in normal breast tissue. Importantly, ceramide levels; C14:0, C16:0, C18:1, C18:0, C22:0, C24:1 and the total ceramides, in IF from tumor tissue were significantly higher than those in IF from peri-tumor and from normal breast tissue.

Conclusion:This is the first report that not only the levels of ceramides in tumor tissue, but also those in the IF from tumor tissue shows higher than those in the tissue and IF from peri-tumor and normal breast tissue. At this point, it is unclear whether ceramides in IF is from dead cancer cells, stromal cells, inflammatory cells or adipocytes. Given the fact that ceramides are known to play major role in cancer cell survival, further studies are warranted to elucidate the mechanism.

 

78.11 Multivariate Analysis Derived Biomediator Panel to Predict Survival in Critically Ill Patients

Posted on January 26, 2017

M. Malig1, C. N. Jenne1, D. J. Roberts1, C. G. Ball1, Z. Xiao1, A. W. Kirkpatrick1  1University Of Calgary,Calgary, AB, Canada

Introduction:  To date no single biomarker of systemic inflammation clearly predicts either outcomes or monitors therapy responses.  However, biomediator panels may provide a more comprehensive model of patient variation, and may reveal novel underlying relationships between mediators.  Selection of open abdomen (OA) management of critically ill patients practically designates a critically ill population with markedly increased complexity and potentially catastrophic complications.  The randomized-controlled human trial (Peritoneal-VAC) examining active negative pressure peritoneal therapy provided a rich repository of biomediator samples in such an OA population, although no individual biomarker assessed was predictive of survival.  Thus, a novel biomediator panel was created and tested using multivariate analysis techniques in an attempt to examine predictive values in this extreme cohort.

Methods:  Partial least squares regression with discriminant analysis (PLS-DA) was used to develop a biomediator panel modeled at baseline, 24, and 48 hours after OA management using Peritoneal-VAC plasma samples. Eight cytokines/acute phase proteins (APP) were used to generate an eight-component biomediator panel to predict overall patient outcome. Components for the panel were selected based on literature research and included: TNF-α, IL-1β, IL-4, IL-6, IL-7, IL-8, IL-10, and fibrinogen. 

Results: The generated models had R2 values ranging from 0.213-0.541, whereas Q2 ranged from 0.007-0.13. The biomediator panel modeled at 24 hours was most predictive for patient outcome (Q2 = 0.13), and accounted for over 50% of the observed experimental variance.

Conclusion: The proposed biomediator panel was more predictive of patient outcome when modeled at certain time points than others; however, overall the panel was a poor predictor of patient outcome. Thus, more research is required to determine more informative biomediators and the most appropriate time frame in which the biomediator storm may best predict outcome in the critically ill/injured.

 

78.09 SphKs/S1P Activate Erk, p38 MAP Kinases, and PI3K/AKT/mTOR Pathways for Cancer Cell Survival

Posted on January 26, 2017

A. MAITI1, K. Takabe1, N. C. Hait1  1Roswell Park Cancer Institute,Division Of Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA

Introduction:   Cell proliferation and migration are essential components of cancer progression. Mechanism responsible for highly proliferative cancer cells can be specific targets for the treatments of cancer patients. PI3K/AKT/mTOR signaling pathways are involved in crosstalk with RAS/RAF/MEK/ERK and p38 MAPK pathways, which are considered the master controller of proliferation, migration and cell survival. A bioactive sphingolipid signaling mediator sphingosine-1-phosphate (S1P) has emerged as a key regulatory molecule in cancer progression. S1P is generated intracellularly by two isoenzymes, SphK1 and SphK2. Compared to cytosolic SphK1, SphK2 is mainly localized in the nucleus in many cells. S1P is critically involved in the major pathways in oncogenesis including promotion of cellular survival, proliferation and transformation, prevention of apoptosis, stimulation of angiogenesis and metastasis. Here we hypothesized that SphKs/S1P activate ERK, p38 MAP kinases, and PI3K/AKT/mTOR pathways to regulate cancer cell proliferation and migration.

Methods:  Experiments were done in human breast cancer cells, MCF7 and MDA-MB-453 cells. Serum starved cell were treated with epidermal growth factor (EGF) and S1P. Endogenous activities of SphKs was inhibited with N,N-Dimethylsphingosine (DMS) and either of SphK1 or SphK2 were ectopically over expressed and treated with EGF. Western blots analysis were done to examine protein expression. Cell migration assay was done by DMS pretreatment followed by EGF and S1P treatment. In order to downregulate one particular SphK, we used specific siRNAs in breast cancer cells and treated with chemotherapeutic drug, doxorubicin to measure cell proliferation, apoptosis, and ceramide levels.

Results:  Treatment of cells with S1P from outside activates S1PRs, enhances phosphorylation of ERK, p38, AKT and p70 S6 Kinase1 (SK6). Interestingly, ectopic expression of SphK1 drastically enhanced basal as well as EGF-mediated phosphorylation of those signaling molecules in cultured cells.  Down regulation of SphK2 with siRNA, reduced EGF-mediated SphK2 activity and drastically reduced basal as well as EGF-mediated cell proliferation. Further, inhibiting SphKs activities with DMS reduced S1P mediated P-ERK, P-AKT levels. In addition, DMS significantly reduced basal as well as EGF and S1P-mediated cell migration. Downregulation of individual SphK with specific siRNA, enhanced ceramide levels and sensitizes breast cancer cells to doxorubicin mediated apoptosis.

Conclusion:  Our data strongly suggested that SphK1, SphK2 and generated S1P are playing critical roles to regulate AKT, ERK and p38 signaling pathways to regulate cancer cell proliferation and migration by using different mechanisms based on their cellular location. This study warrants immediate attention to develop specific inhibitors of each SphK for therapy.

 

78.10 Inhibition of Cell Proliferation of Pancreatic Cancer Cells by Prostaglandins E1, A2, and B2

Posted on January 26, 2017

J. Yu1, R. Damoiseaux2, S. Liu1, R. Sanchez1, F. C. Brunicardi1  1University Of California – Los Angeles,Department Of Surgery,Los Angeles, CA, USA 2University Of California – Los Angeles,Department Of Molecular And Medical Pharmacology,Los Angeles, CA, USA

Introduction:
The prostaglandins are a family of lipid compounds, found in most human tissues with diverse hormone-like effects. We have recently identified prostaglandins E1, A2, and B2 (PGE1, PGA2, and PGB2) as potential inhibitors of pancreatic cancer proliferation via high-throughput drug screening of small molecules. However, the molecular mechanisms of how prostaglandins inhibit pancreatic cancer cell proliferation are poorly understood.

Methods:
A dilution series for a final concentration from 1mM to 1pM were used to determine the concentration required for 50% inhibition (IC50) for PGA2, PGB2 and PGE1 on human pancreatic cancer cell line MiaPaca2. To further determine whether PGA2, PGB2 and PGE1 inhibit pancreatic cancer cell growth via shared molecular targets or via independent mechanisms, RNA sequencing was applied to pancreatic cancer cell line MiaPaca2 with or without treatment of 5mM PGA2, PGB2 and PGE1. There were 3 replicates per each treatment group. Total RNAs were extracted 72 h post-treatment using Qiagen RNeasy columns. Sequencing reads were obtained using the Illumina HiSeq2000 platform. Differentially expressed genes from treated versus non-treated pancreatic cancer cells were calculated using “Cuffdiff” program from the Cufflinks suites and the top differentially expressed genes were then validated using QPCR.

Results:
The dose-response curve of PGE1 showed that IC50 of PGE1 on Mia Paca2 cell proliferation was 550nM, which was lower than the IC50 for PGA2 (1260nM) and PGB2 (1600nM). Giving a cut-off at log2 fold-change>0.5 and P<0.05, 205, 237 and 284 up-regulated genes and 289, 278 and 355 down-regulated genes were identified associated with PGA2, PGB2, and PGE1 treatment, respectively. Among these genes, only 31 genes in common were up regulated after treatments of all 3 prostaglandins. Gene ontology analysis revealed that these 31 up-regulated genes were enriched in genes of DNA damage response and apoptosis (P=0.05). In contrast, 93 genes were found significantly inhibited in all 3 prostaglandin-treated cells, including Tetraspanin 1 (TSPAN1), Sulforaphane (SFN) and Keratin 15 (KRT15). Gene ontology analysis revealed that these 93 down-regulated genes were enriched in genes involved in epidermal ectoderm development (P=2.9E-6) and cell proliferation (P=0.02). QPCR proved the significant reduction of gene expression for TSPAN1, SFN and KRT15, which have been associated with epidermal ectoderm development and oncogenesis.

Conclusion:
This analysis demonstrates the inhibitory effect of PGE1, PGA2, and PGB2 on pancreatic cancer cell proliferation in vitro. Moreover, by comparing the transcriptome of pancreatic cancer cells before and after prostaglandin treatments, the RNA Sequencing data revealed a list of shared gene targets including TSPAN1, SFN and KRT15, suggesting the mechanism of the inhibitory effect of PGE1, PGA2, and PGB2 on pancreatic cancer cells.
 

78.08 Precision Oncology At a Single-Cell Level: Quality Control for Single-cell Whole-Genome Amplification

Posted on January 26, 2017

S. Sho1, C. M. Court1, P. Winograd1, J. S. Tomlinson1  1University Of California – Los Angeles,General Surgery,Los Angeles, CA, USA

Introduction:

Obtaining circulating tumor cells from a peripheral blood draw opens the door to the possibility of applying precision oncology strategies from a “liquid biopsy.” Realization of this potential requires whole genome amplification (WGA) for genomic analysis of single cells. However, WGA remains prone to non-linear and uneven amplifications, resulting in inaccurate representation of the starting genome. WGA reactions are also known to be stoichastic, resulting in highly varied amplified DNA quality between samples. Thus, appropriate selection of the high-quality WGA product is needed to ensure the accuracy and reliability of downstream analysis. Currently, quality analysis methods for WGA products is not well defined, and no clear workflow exists for obtaining and selecting optimal WGA products for downstream molecular assay. 

Methods:
Laser microdissection (LMD) was used isolate pancreatic cancer cell line (HPAF-II) cells. Nine single-cells, six 5-cell groups and six 10-cell groups were obtained for WGA using multiple displacement amplification (MDA) method. Quality of WGA products was assessed by performing real-time quantitative (RT-qPCR) PCR for 8 cancer related genes. Quality score (QS) of 0-8 was assigned based on the ability of RT-qPCR to detect these cancer genes in the WGA DNA product (0: absence, 1: presence). Single-cell WGA with perfect QS of 8 and low QS of 1 were subjected to array comparative genomic hybridization (aCGH) and next generation sequencing (NGS), in order to assess if QS could predict the success of downstream analysis. WGA products of 5-cells and 10-cells with QS of 8 were also subjected to aCGH and NGS, in order to determine the impact of increasing number of starting cells on the quality of downstream analysis. 

Results:

Successful DNA amplification was noted in all samples undergoing WGA. However, only 22% (2 of 9) single-cell WGAs had the perfect QS of 8, with the rest of ranging between 1-3. For both 5- and 10-cell WGA, 50% (3 of 6) of the WGA products had QS of 8. When applied to aCGH and NGS, the single-cell WGA product with low QS of 1 resulted in poor quality data (Table 1). On the other hand, the single-cell WGA with the perfect QS of 8 resulted in a significantly higher quality aCGH and NGS data. When the WGA products of 5-/10-cells with perfect QS of 8 were subjected to aCGH and NGS, results were even better, approximating that of nonamplified DNA.

 

Conclusion:
WGA quality control by RT-qPCR offers a simple, inexpensive way of selecting optimal WGA products for downstream analysis. Furthermore, aCGH/NGS results approximating nonamplified DNA can be obtained by using high quality WGA product (QS of 8) obtained from using 5-10 cells as starting template. 

78.07 Pancreatic adenocarcinomas with mature blood vessels have better overall survival

Posted on January 26, 2017

K. Takabe1, E. Katsuta1, M. G. Dozmorov2, A. L. Olex5, S. N. Hochwald1, L. J. Fernandez4  1Roswell Park Cancer Institute,Department Of Surgical Oncology,Buffalo, NY, USA 2Virginia Commonwealth University,Department Of Biostatistics,Richmond, VA, USA 3Roswell Park Cancer Institute,Gastrointestinal Surgery, Department Of Surgical Oncology,Buffalo, NY, USA 4Virginia Commonwealth University,Division Of Surgical Oncology, Department Of Surgery,Richmond, VA, USA 5Virginia Commonwealth University,Wright Center For Clinical And Translational Research,Richmond, VA, USA

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) is known for its hypovascularity and hypoxic microenvironment compared to other types of cancers. Angiogenesis, generation of new blood vessels, is one of the hallmarks of cancer and is well established to contribute to tumorigenesis. However, some types of tumors with a rich vascular network have excellent survival. A phase 3 study showed no improvement in outcome with bevacizumab added to standard chemotherapy for PDAC. Therefore, we hypothesized that PDAC with relatively high vascularity may be associated with improved survival.

Methods:  Using Level 3 gene expression data from The Cancer Genome Atlas (TCGA), we performed survival analysis of genes related to angiogenesis, vascular endothelial cells and hypoxia. Data for n=178 patients was log2-transformed and analyzed using Kaplan-Meyer log-rank test. For each gene of interest, the cutoff was determined by automated scanning 25%-75% gene expression interval and selecting the threshold yielding the lowest p-value.

Results: First, we verified our dataset by confirming whether high expression of angiogenic factor and vascular signals associate with survival in colon cancer, which is known to respond to bevacizumab. High expression of angiogenic gene (VEGF-A) as well as marker gene for vascular endothelial cells (CD31) was associated with poor overall survival (OS) in colon cancer. In the PDAC cohort, the mean observation period was 451 days. High expression of CD31, which indicate presence of vascular endothelial cells, was significantly associated with better OS (p=0.019). Further, genetic markers of mature vessels, TIE1 and TIE2, which are endothelium-specific receptor tyrosine-kinases, and S1PR1, which play a critical role for acquisition of vascular stability, were all associated with better OS (p=0.008, 0.041, and 0.013, respectively). No angiogenic factors, VEGFs or Angiopoietins were associated with survival. Multivariate analysis using Cox proportional hazard regression demonstrated that residual tumor (R1, 2; p=0.020) and CD31 high expression (p=0.009) were the only independent factors that negatively impacted on OS. Additionally, CD31 expression demonstrated strongly positive correlation with not only vascular endothelial cell markers such as TAL1 (R=0.67), but also vascular stability related genes, such as JAM2 and CD93 (R=0.77 and 0.70), and vascular morphogenesis genes, RHOJ and ERG (R=0.74, 0.79). CD31 also demonstrated strong positive correlation with BCL6B (R=0.79), an anti-fibrogenic factor, which implies that less fibrosis in PDAC correlates with higher vascularity and better OS. Interestingly, there was no survival difference by expression of HIF1-alpha, an ischemia marker. 

Conclusion: PDAC with mature blood vessels, detected by high expression of CD31, TIE1, TIE2 or S1PR1 have better overall survival, independent of the presence of hypoxia.

78.06 Acute Vitamin C exposure As A Potential Antitumorigenic Agent For Colon Cancer

Posted on January 26, 2017

M. M. Aldajani1, C. N. Vanicek1, N. Alhazzaa1, R. Agarwal1, J. P. Geibel1  1Yale University School Of Medicine,Surgery,New Haven, CT, USA

Introduction:

Colorectal cancer remains one of the leading causes of death in the United States and worldwide. Although early screening has played an important role in improving clinical outcomes resulting in a reduced death rate. There remain opportunities to develop new therapeutic agents and strategies to further improve survival odds. In this regiment of new methodologies one can look at agents that can also be taken prophylactically to reduce the risk of tumor formation. Typically for cancer to grow and invade healthy tissues, it must maintain a higher (more alkali) intracellular pH and a lower (more acidic) extracellular pH, in the immediate environment around the metaplastic cell. This leads to volume changes within the tumor and to destruction of the surrounding cells thereby aiding the tumor to expand in size. Recently there is evidence the chronic exposure to Vitamin C may lead to a reduction in tumor formation in the colon. In the present study we examined what acid base transport proteins are modulated by acute exposure to Vitamin C that result in enhanced proton extrusion, and elevation in intracellular pH.

Methods:

Following excision, rat colon was digested leaving single isolated crypts, which were then incubated with the pH sensitive dye BCECF. The crypts were then imaged while being perfused with solutions either containing or devoid of Vitamin C. The ratio image data was collected every 15 second to allow a direct real time measurement of the effects of Vitamin C on colonic hydrogen ion transport.

Results:Acute exposure to Vitamin C lead to a reduction in Na-dependent H ion extrusion (NHE). The effects of Vitamin C were dose dependent. In a separate series of studies, we investigated the role of glucose in the effect of Vitamin C. Removal of glucose from the solutions or addition of phlorizine had no effect on the Vitamin C of NHE activity.

Conclusion:

We found that acute exposure to Vitamin C can reduce NHE activity and that this effect is not glucose dependent. Our results show an important role for Vitamin C as an antitumorigenic agent under acute conditions. The glucose independence makes this an important nutraceutical therapy that can be use both in nondiabetic and diabetic patients.

 

78.05 Pre-treatment Serum Th1 Cytokines do not Predict Survival or Extent of Disease in Soft Tissue Sarcoma

Posted on January 26, 2017

S. J. Judge1, M. Yanagisawa1, T. Henderson1, S. Thorpe2, A. M. Monjazeb4, W. J. Murphy3, R. J. Canter1  1UC Davis,Surgical Oncology,Sacramento, CA, USA 2UC Davis,Orthopedic Oncology,Sacramento, CA, USA 3UC Davis,Dermatology, Laboratory Of Cancer Immunology,Sacramento, CA, USA 4UC Davis,Radiation Oncology,Sacramento, CA, USA

Introduction: With the expanding use of immunotherapy for cancer therapy, including soft tissue sarcomas (STS), there is a growing need for biomarkers of response and prognosis. We have previously demonstrated that changes in circulating levels of TNF-alpha (TNF-α), Interferon-gamma (IFN-γ), and Interleukin-6 (IL-6) predict metastasis-free survival in STS patients receiving preoperative sorafenib and radiotherapy (RT). The objective of the current study was to determine the impact of baseline cytokine expression on prognosis and risk of malignancy in STS.

Methods: From March 2013 to February 2015, 33 patients with suspicious soft tissue tumors underwent pre-treatment measurements of serum cytokine/chemokine expression, including IFN-γ, TNF-α, IL-2, IL-6, G-CSF, GM-CSF, EGF, IL-4, IL-8, IL-10, FGF, TGF-α, PDGF, TNF-?, sIL-2rα, and VEGF. Based on our previous data, we focused the analysis on IFN-γ, TNF-α, IL-2, IL-6 levels to predict risk of malignancy/stage (benign, low grade, high grade), metastasis free survival and overall survival. Parametric and non-parametric statistics were used as appropriate.

Results: Of the 33 patients, the majority was female (52%) and had retroperitoneal tumors (67%). Five patients (15%) had benign disease, 16 (48%) low grade STS, and 12 (36%) high grade STS. With a median follow up of 27 months, 9 (27%) experienced distant recurrence, and 4 (12%) died of disease. There were no significant differences in baseline cytokine levels between benign and malignant STS or between low grade and high grade malignant STS (See Figure). There were no significant differences in baseline cytokine levels between patients who developed metastases and those who did not, with the exception of IFN-gamma which was significantly higher in non metastatic STS compared to metastatic STS (mean ±SD: 125 ±216 vs 14 ±16 pg/mL; p = 0.05).

Conclusion: In this cohort of heterogeneous soft tissue tumor and STS patients, with the exception of IFN- γ, baseline levels of circulating Th1 cytokines do not predict risk of malignancy/extent of disease and do not predict with metastasis-free survival or overall survival. Although changes in cytokine levels with treatment may predict response to therapy/immunotherapy, better biomarkers of baseline prognosis are needed.

 

78.04 Vitamin C And Nitric Oxide: A Synergistic Effect On Colonic Crypts

Posted on January 26, 2017

C. N. Vanicek1, M. M. Aldajani1, N. Alhazzaa1, R. Agarwal1, J. P. Geibel1  1Yale University School Of Medicine,Surgery,New Haven, CT, USA

Introduction:

For colorectal cancer to grow and invade healthy tissues, it must maintain a higher (more alkali) intracellular pH and a lower (more acidic) extracellular pH, in the immediate environment around the metaplastic cell. For this reason, colorectal cancer remains as one of the leading causes of death in the United States and worldwide.

In a study presented at this meeting we provide evidence that acute Vitamin C exposure at high doses could down regulate Na-dependent acid efflux pathways in colonic crypt cells. In previous studies, Vitamin C was shown to increase nitric oxide production through stimulating nitric oxide synthase. The role of nitric oxide (NO) on cancer growth remains complex. Prolonged chronic exposure to NO ,which is typical during chronic inflammation, leads to gene mutations which were linked to cancer development. However, conversely it also has been shown that NO derived from macrophages, Kupffer cells, natural killer cells, and endothelial cells is a tumorcidal agent that results in cancer apoptosis and cessation of growth and metastasis. In this present study we examined the acute effects of Vitamin C exposure on nitric oxide

production and its effects on Na-dependent acid secretion.

Methods:

Following excision, rat colon was digested leaving single isolated crypts, which were then

incubated with the pH sensitive dye BCECF. The crypts were then imaged while being perfused

with solutions either containing or devoid of Vitamin C, L-NAME and L-Arginine. The ratio image

data was collected every 10 second. to allow a direct real time measurement of changes in pH All

data were then analyzed with GraphPad Prism.

 

 

Results:

By comparing rates of pH recovery under these various conditions we found that: 1) There was no difference in the Na-dependent H extrusion rate between Vitamin C and L-arginine. 2) Treatment of colonic crypts with L-NAME, a NO synthesis inhibitor, reversed the Vitamin C dependent NHE inhibition.

Conclusion:

We found that exposure to Vitamin C gives a similar inhibition in acid secretion as Arginine a known NO producer. This further shows a tight link to Vitamin C and NO production and the ability of NO to significantly inhibit hydrogen ion excretion. In separate studies where crypts were given L-NAME the level of acid secretion returned to normal control rates. Our data suggest a tight link between Vitamin C exposure and a reduction of acid secretion via NO synthesis. Therefor the anti-tumorigenic effect equated to Vitamin C may be synergistic with the tumorcidal effect of NO found in other tissues.

78.03 NIR-Conjugated Humanized Anti-CEA Antibody to Target Colon Cancer in an Orthotopic Nude Mouse Model

Posted on January 26, 2017

J. C. DeLong1, T. Murakami1,2, P. J. Yazaki3, R. M. Hoffman1,2, M. Bouvet1  1University Of California – San Diego,Surgery,San Diego, CA, USA 2AntiCancer, Inc.,San Diego, CA, USA 3City Of Hope National Medical Center,Immunology,Duarte, CA, USA

Introduction:  The success of a curative surgery for cancer is dependent on the complete removal of all cancer cells, an R0 resection. Intraoperative verification of clear tumor margins is not possible by the surgeon and requires a surgical pathologist to analyze frozen sections of the resected tumor. Tumor visualization by the surgeon can be enhanced through fluorescence-guided surgery (FGS) by delivering labeled tumor-specific antibodies. We selected humanized anti-carcinoembryonic antigen (CEA) conjugated to a near-infrared (NIR) dye to target orthotopically implanted human colon cancer in nude mice.

Methods:  The HT-29 cell line for human colon cancer was grown in culture and subcutaneously injected subcutaneously in a nude mouse model. After 3 weeks of growth tumors were resected and cut into 2 mm3 fragments that were sutured to the cecum of 5 additional nude mice. The tumors were allowed to grow for 4 weeks at which point 3 had successful orthotopic tumor growth and were selected for injection of the humanized antibody for CEA that was convalently bound to the IR800 NIR dye (anti-CEA-IR800) through an ester reaction. Antibody-dye conjugate (75 μg ) was intravenously administered via tail vein injection. Images were taken with the Pearl Trilogy Small Animal Imaging System (Li-Cor, Lincoln, NE) pre-injection, 5 min post, 5 hours post, 24 hours post (with laparotomy views), and 48 hours post injection (with laparotomy views) with both 700 nm and 800 nm channels. Images were evaluated using Image Studio.

Results: Images taken at 5 min and 5 hours (through skin, no laparotomy) did not demonstrate appreciable accumulation in the tumor. At 24 hours laparotomy was performed and the tumors were strongly labeled with anti-CEA-IR800 when imaged through the 800 nm channel. At 48 hours laparotomy was repeated which again demonstrated strong labeling of the tumors through the 800 nm channel, but with a lower absolute intensity (in relative units), than at 24 hours for each of the 3 mice imaged. Normal bowel was fluorescent through the 700 nm channel due to the autofluorescence of plant chlorophylls in mouse chow.

Conclusion: Humanized anti-CEA-IR800 can rapidly and effectively label CEA-expressing human colon cancer in an orthotopic nude mouse model. Given the ability of this technology to target and label tumor with great specificity, anti-CEA-IR800 should be made available for clinical use for fluorescence guided surgery in the near future.

78.02 Altered Microbiome in Pancreatic Cancer and Chronic Pancreatitis

Posted on January 26, 2017

M. A. Mederos1, A. McElhany1, J. Petrosino2, N. J. Ajami2, N. Villafane1, S. Mohammed1, E. Oliva1, W. E. Fisher1, G. Van Buren1  1Baylor College Of Medicine,Department Of Surgery, Division Of Surgical Oncology,Houston, TX, USA 2Baylor College Of Medicine,Department Of Molecular Virology And Microbiology,Houston, TX, USA

Introduction:  Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease that lacks a method of early detection. Established risk factors for PDAC suggest an inflammatory mechanism of carcinogenesis. Recent epidemiological data portends that certain bacterial populations may increase PDAC susceptibility & progression by diverse mechanisms including modulating inflammation. We aim to characterize the microbiome of pancreatic tissue in those with PDAC & chronic pancreatitis (CP).

Methods:  Patients who underwent pancreas resection at our institution from 2004-2016 were identified from our database. Baseline demographics, co-morbid conditions, clinical characteristics, & outcome data were obtained from review of the database. In this cross-sectional study, we used next-generation sequencing protocols & high throughput 16S rRNA gene sequencing to characterize the microbiota in CP/PDAC & normal-adjacent tissue samples (n=6 matched pairs). Shannon diversity indices were used to compare taxonomic richness. Beta-diversity distance comparison was used to compare compositional differences between the two patient groups. P values were calculated using a Wilcoxon matched-pairs rank test & Kruskal-Wallis statistical tests with false discovery rate correction.

Results: Baseline demographics were similar between patient groups. Analysis shows that the microbiome in PDAC & CP tissue samples is dominated by species of lipopolysaccharide (LPS)-rich Proteobacteria followed by Firmicutes, Bacteroidetes, & Actinobacteria with relative abundance means of 53.65%, 31.55%, 5.56%, & 5.04%, respectively. We also observed lower bacterial richness in tumor-associated samples compared to normal-adjacent (p=0.65). This finding was accompanied by an overall increase of LPS-rich Enterobacter species in PDAC tissue compared to normal tissue (p=0.094).

Conclusion: Our analysis showed a dominance of LPS-rich Proteobacteria & a trend to lower microbial richness. Low microbial richness suggests the dominance of a single or few bacteria — a hallmark of microbe-induced inflammatory processes. The trend toward decreased diversity is possibly a result of a multi-factorial dysbiotic state & probably contributes to the pathogenesis of PDAC. Recent studies suggest a role for Proteobacteria in carcinogenesis through inflammatory processes mediated by TLR-activating molecules such as LPS.  There is an inherent basal diversity in the microbial structure across multiple body sites due to environmental & genetic factors, thus more specimens are needed to correlate microbial patterns with clinical outcomes. Further studies with more specimens are needed to detect a statistical difference of the microbiome between matched tissue pairs.

78.01 Comparison of pH­sensitive fluorescent nanoprobe to cetuximab­IRDye800 for realtime imaging of SCC

Posted on January 26, 2017

M. Tabata1, N. Nathan1, T. Teraphongphom1, K. Hettie2, J. Klockow2, S. Rogalla3, R. Ertsey1, E. Rosenthal1,2  2Stanford University,Radiology,Stanford, CA, USA 3Stanford University,Pediatrics,Palo Alto, CA, USA 1Stanford University,Otolaryngology – Head And Neck Cancer,Palo Alto, CA, USA

Introduction: Despite widespread acceptance of fluorescence imaging for several different types of cancer, the ideal optical imaging probe for intraoperative delineation of head and neck squamous cell carcinoma (HNSCC) margins has not yet been identified. Identification of this probe for detecting subclinical disease in tumor margins will improve oncologic surgical outcomes. This study compares a fluorescently labeled anti-­epidermal growth factor receptor (EGFR) antibody, Cetuximab-­IRDye800CW (IR800-CTM), with an ultra pH­-sensitive (UPS) fluorescent nanoprobe for detection of HNSCC.

Methods: Thirteen immunodeficient mice were inoculated with HCT. Through tail vein injections, four mice were given 200 uL of IR800-CTM at 14uM. Three were given 100 uL of UPS nanoprobes, and three were given 200 uL, both at 0.1 mg/mL. Two were given 200 uL of saline, and one was given 200 uL of IRDye800 at 28uM. Images were acquired using the Pearl® Trilogy(LI-COR) in vivo optical imaging system at 8, 22, 26, 30, 46, and 72 hours along with the SPY Elite®(Novadaq) at 72 hours. Ex vivo images were acquired using the Pearl® and Odyssey®(LI-COR). Tumor­ to ­background ratios (TBR) were calculated by dividing the intensity of the fluorescence in the tumor by that of healthy flank tissue. TBRs of the UPS nanoprobe group and the IR800-CTM group were compared. An unpaired, two-­sided Student’s T-­test with unequal variance was used to test for statistical significance.

Results: There is no statistically significant difference between TBRs of UPS nanoprobes and IR800-CTM in vivo. We can successfully image tumors in vivo and obtain TBRs of 2.81 (±0.68 SD) with UPS nanoprobes and 5.27 (±1.85 SD) with IR800-CTM. Ex vivo histology confirms fluorescence in tumors. The TBR of the UPS nanoprobes reached a maximum at 22 hours and stayed above 83% of maximum until 72 hours. The lower dose of 100µL yields stronger and more specific signal than the 200µL dose. Both UPS nanoprobes and IR800-CTM localize in kidneys and liver, and IR800-CTM shows greater tumor:liver fluorescence ratio.

Conclusion: Both UPS nanoprobes and IR800-CTM are tools for intraoperative optical visualization of cancer. The earlier TBR peak of UPS nanoprobes is clinically advantageous. Currently, complete removal of HNSCC with minimal damage to other tissues cannot be guaranteed, and improved visualization of tumor margins would improve post-­surgery oncologic outcomes. We compare two methods for optical imaging of tumor margins in HNSCC. These outcomes will help guide further investigation of an optimal optical imaging agent for HNSCC. This methodology is reproducible for investigation in other tumor types.

71.08 Humans vs. Pigs vs. Rats; Native TEG Distribution Indicates Limitations of Animal Models of TIC

Posted on January 26, 2017

P. J. Lawson1, H. B. Moore1,2, G. R. Stettler1,2, A. L. Slaughter1,2, A. W. Bacon1,2, M. Fragoso1,2, A. Banerjee1, E. E. Moore1,2  1University Of Colorado Denver,Aurora, CO, USA 2Denver Health Medical Center,Aurora, CO, USA

Introduction:
Thrombelastography (TEG) has been used increasingly to characterize the coagulation changes associated with traumatic injury and hemorrhagic shock. However, animal models developed to investigate trauma induced coagulopathy (TIC) have failed to produce objective excessive bleeding. In patients activated TEGs (rapid and kaolin) are less sensitive in detecting hypercoaguable states following injury compared to a non-activated (native) TEG. We hypothesize that a native TEG will demonstrate marked differences in humans compared to these experimental models, which explains the difficulties in reproducing a clinically relevant coagulopathy in animal models.

Methods:
Whole blood was collected from 134 healthy human volunteers, 25 swine and 64 Sprague Dawley rats prior to experimentation. Citrated Native TEG’s were run on each whole blood sample within 2 hours of blood draw. The R-Time(min), Angle(degrees), MA(mm), and LY30(%) were analyzed and contrasted between species with data represented as the median and 25th to 75th quartile range.  Difference between species was conducted with a Kruskall Wallis test with alpha adjusted with a Bonferroni correction for multiple comparison (alpha = 0.016).

Results:
R-Time (clot initiation) was 17.9 min (15.0-21.1) for humans, 5.7 (4.9-8.8) for pigs, and 5.2 (4.4-6) for rodents. Humans had longer R-Times than both pigs (p<0.0001) and rats (p<0.0001); pigs were not different from rats. Angle (fibrin cross-linking) was 28.0 degrees (21.4-40.3) for humans, 71.7 (64.3-75.6) for pigs, and 61.8 (56.8-66.7) for rats. Humans had reduced Angle than both pigs (p<0.0001) and rats (p<0.0001); pigs were not different from rats. MA (clot strength) was 51.5 mm (47.4-55.0) for humans, 72.5 (70.4-75.5) for pigs, and 66.5 (56.5-68.6) for rats. Humans had reduced MA than both pigs (p<0.0001) and rats (p<0.0001); pigs were not different from rats. LY30 (fibrinolysis) was 1.2 % (0.6-2.2) for humans, 3.3 (1.9-4.3) for pigs, and 0.5 (0.1-1.2) for rats. Humans had a lesser LY30 than pigs (p=0.0006) and a greater LY30 than rats (p=0.0005), and pigs had a greater LY30 than rats (p<0.0001).

Conclusion:
Humans, swine, and rodents have distinctly different coagulation profiles when evaluated by native TEG. Animals are hypercoaguable with rapid clotting times and clots strengths nearly 50% stronger than humans. These coagulation differences indicate the limitations of previous models of TIC in producing coagulation abnormalities associated with increased bleeding. The inherent hypercoaguable baseline tendencies of these animals may result in subclinical biochemical changes that are not detected by conventional TEG and should be taken into consideration when extrapolated to clinical medicine.

71.07 Amitriptyline Treatment Improves Survival in a Murine Model of Sepsis

Posted on January 26, 2017

B. T. Xia1, Y. Kim1, E. Gulbins1,2, C. C. Caldwell1  1University Of Cincinnati College Of Medicine,Department Of Surgery,Cincinnati, OH, USA 2Universität Duisburg-Essen,Essen, NORTH RHINE-WESTPHALIA, Germany

Introduction:  During traumatic stress and sepsis, ceramide accumulates within the lipid bilayer, due to increased acid sphingomyelinase (Asm) and decreased acid ceramidase activities.  It has been demonstrated that ceramide augments the innate immune response, such as increased superoxide formation, which may damage tight junction proteins and disrupt epithelial barriers.  Disruption of epithelial barriers in the intestines and lungs has been shown to lead to edema, ischemia, and organ dysfunction.  We hypothesized that Asm inhibition will blunt the innate response and improve survival in a murine model of polymicrobial sepsis.

Methods:  Cecal ligation and puncture (CLP) was used to induce polymicrobial sepsis.  Mice were randomized to intraperitoneal injection of saline vehicle or amitriptyline (16 mg/kg) at the time of CLP.  Systemic and peritoneal bacterial load, immune response, and survival were the primary endpoints.

Results:  Septic mice treated with amitriptyline demonstrated improved survival compared to vehicle (p=0.03). Amitriptyline treatment resulted in decreased peritoneal neutrophil accumulation 16 hours post-CLP compared to vehicle (3.79 x106 cells/ml vs. 6.14 x106 cells/ml, p=0.03), and no changes in systemic and peritoneal bacterial burden. Of note, we did not observe changes in peritoneal neutrophil accumulation, oxidative burst, and systemic and peritoneal bacterial burden 6 hours after sepsis.

Conclusion:  Amitriptyline treatment improved survival in polymicrobial sepsis and decreased neutrophil accumulation after 16 hours.  We postulate that Asm inhibition may reduce epithelial exposure to activated neutrophils such that organ integrity and functionality are maintained.  Future experiments will be conducted to determine neutrophil infiltration, edema, and permeability in the lung and small bowel.  Altogether, we propose that ASM inhibition, in conjunction with antibiotics, is a promising therapeutic approach in the treatment of polymicrobial sepsis.
 

71.06 Remote Ischemic Conditioning in a Mouse Model of Traumatic Brain Injury

Posted on January 26, 2017

K. Ibraheem1, A. J. Sandweiss2, T. M. Largent-Milnes2, A. Azim1, N. Kulvatunyou1, L. Gries1, T. W. Vanderah2, P. Rhee3, T. O’Keeffe1, B. Joseph1  3Grady Memorial Hospital,Trauma And Surgical Critical Care/Department Of Surgery,Atlanta, GA, USA 1The University Of Arizona,Trauma,critical Care, Burn And Emergency Surgery/Department Of Surgery/School Of Medicine,Tucson, AZ, USA 2The University Of Arizona,Department Of Pharmacology,Tucson, AZ, USA

Introduction: Remote ischemic conditioning (RIC) has been shown to have protective effects in in a number of different clinical settings including coronary bypass surgery and kidney transplantations. There is recent evidence to suggest its beneficial role in preventing secondary brain insults in traumatic brain injury. The aim of our study was to examine the role of RIC in a mouse model of traumatic brain injury.

Methods: 100 male C57BL mice were subjected to a cortical controlled impact injury. Two hours after TBI, animals were allocated to RIC (n = 10) or Sham (n = 10). RIC was performed for 6 cycles of ischemia and reperfusion by clamping the femoral artery. Circulating levels of S100-B, neuron specific enolase (NSE) and Glial fibrillary acidic protein (GFAP) were serially measured as markers of damage to neurons and astrocytes at 2-, 6-, 24-, 48- and 120-hours post-intervention (RIC or Sham). Similarly, another 24 animals were subjected to a cortical controlled impact injury. Two hours after TBI, animals were allocated to RIC (n = 12) or Sham (n = 12) then assessed at 2-, 24-, 48-, 72- and 96-hours post-intervention (RIC or Sham) for cognitive performance using novel object recognition. The motor coordination was assessed by rotarod test at 24-, 48-, 72-, 96- and 120-hours post-intervention (RIC or Sham).

Results: There was no significant difference in systemic neuronal markers between RIC and Sham animals at 2-, 6-, and 24- hours. Serum GFAP trended down while serum NSE and S100-B trended up in both RIC and Sham animals. RIC animals had significantly higher recognition index than Sham at 24-, 48 and 72-hours post-intervention (Figure). Latency to fall was higher in RIC animals compared to Sham animals at all time points and statistically significant at 120-hours post-intervention. The RIC group demonstrated significantly improved cognitive function and motor coordination compared to the Sham group.

Conclusion: The application of remote ischemic conditioning 2 hours post-injury results in quicker recovery of cognitive functions and improved balance and motor coordination in a mouse model of traumatic brain injury despite no significant changes in systemic biomarkers of brain injury.

 

71.05 Inflammatory Regulation Promotes Angiogenesis in Chronic Renal Disease Model

Posted on January 26, 2017

X. Wang1, P. Duann1, C. Lu1, C. Moles1, M. Fahrenholtz1, M. Rae1, H. Li1, J. Cheng2, S. Balaji1, S. Keswani1  1Baylor College Of Medicine,Surgery,Houston, TX, USA 2Baylor College Of Medicine,Medicine,Houston, TX, USA

Introduction:
Tubulointerstitial fibrosis, compounded with peritubular capillary loss, is a common finding in progressive renal disorders. Treating progressive renal disorders patients’ costs over $40 billion annually in the US alone. Beyond dialysis and transplantation, novel therapies are needed. Previous reports suggest that there is a role for the anti-inflammatory cytokine, interleukin-10 (IL-10), in attenuating renal fibrosis in a disease murine model. We and others also showed that IL-10 can regulate angiogenesis and endothelial progenitor recruitment during dermal and ischemic cardiac tissue repair.  We hypothesize that IL-10 can promote angiogenesis and regulate TGFβ isoforms in unilateral urethral obstruction (UUO) murine model. 

Methods:
Primary fibroblasts (FB) were isolated from 8-10 week-old male C57BL/6J (WT) mice. IL-10 (50 ng/ml or 200 ng/ml) was added to cultures. VEGF and TGFβ-1 gene expressions were assessed by qPCR at 1, 2, 3 and 6h. Levels of TGFβ-1 and TGFβ-3 were determined at 48h by ELISA. Eight week-old WT and IL-10 null male mice were injected with lenti-IL-10/lenti-GFP (1×1010 IU) under kidney capsule. Three days after the injection, unilateral ureteral obstruction (UUO) was performed. UUO/sham kidneys and serum were collected at 14 days after UUO for RNA, ELISA and immunohistochemical analysis. Data presented as mean ± SD, n=3/treatment group. P value by ANOVA.

Results:

In primary FB culture, IL-10 treatment increased VEGF expression and altered the differential expression of TGFβ isoforms, with three-fold increment of ratio between TGFβ-3 to TGFβ-1. The role of IL-10 in regulatory angiogenesis was further validated in IL10-null mouse with UUO.  Lenti-Il10 treatment reduced intertubular fibrotic change (45±7%, p<0.05) and attenuated tubular dilatation in UUO (p<0.05, n=30/group). The CD31, an established endothelium marker, was essential to preserve tubular integrity, normally expressed in healthy tubules and abrogated after UUO. IL-10 null mice revealed a lower basal level of CD31 compared to WT mice (Fig.1). In both WT and IL-10 null mice, IL-10 treatment preserved CD31, suggested a potent capability to rescue peritubular capillary (Fig.1). 

Conclusion:

Our results indicate that IL-10 can effectively promote angiogenesis in vitro and prevent microvascular rarefaction in vivo. Taken together, our study might lead to a novel therapeutic for the treatment of CKD and associated angiogenic morbidity.

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