04.09 Pancreatic Cancer Resistance to MEK Inhibition is Mediated by IL-1α-Dependent STAT3 Reactivation

Posted on January 25, 2017

A. A. Gaidarski1, C. Roberts1, M. VanSaun1, N. Nagathihalli1, J. Castellanos2, N. Merchant1  1University Of Miami,Department Of Surgery,Miami, FL, USA 2Vanderbilt University Medical Center,Department Of Surgery,Nashville, TN, USA

Introduction: Chemotherapy against Pancreatic Ductal Adenocarcinoma (PDAC) is daunted by innate and inducible drug resistance. Attempts to target downstream effectors of KRAS, the hallmark oncogenic mutation in PDAC, are plagued by activation of resistance pathways. We previously identified Signal Transducer and Activator of Transcription 3 (STAT3) as a mediator of resistance to MEK inhibition. However, the mechanism of STAT3 activation in response to MEK inhibition is not known. We observed decreased Interleukin 1 (IL-1) levels in response to MEK inhibition. IL-1 is a pro-inflammatory cytokine that has been broadly implicated in carcinogenesis. The full scope of IL-1 activity and its role in tumor development is not understood due the complex, pleiotropic, and often opposing effects of the distinct subtypes IL-1α  and IL-1β . In PDAC, IL-1α  stimulates both the MAPK and the NF-kB pathways, key drivers of proliferation, invasion, and drug resistance. We now demonstrate that IL-1α  down-regulates STAT3 activity. Loss of this regulation could account for development of resistance in response to MEK inhibition.

 

Methods: A cytokine array of pancreatic tumors from Ptf1acre/+;LSL-KrasG12D;Tgfbr2fl/fl mice identified reduced levels of IL-1α  and IL-1β  in response to MEK inhibition. In order to investigate whether MEK inhibition effected IL-1 signaling in pancreatic tumor cells, cultures of Panc1, a human pancreatic cancer cell line, were stimulated with recombinant IL-1α  or IL-1β  alone and treated with AZD6244, an inhibitor of MEK. Western blot analysis was performed for protein mediators of tumor proliferation and drug resistance, including phospho-NF-kB, phospho-ERK1/2, and phospho-STAT3. Gene expression of NF-kB and MAPK transcriptional targets including IL-1α , IL-1β,  and IL-6, were assayed using quantitative real-time PCR.

 

Results: Stimulation of human pancreatic cancer cell line Panc1 with IL-1α , but not IL-1β , induced phosphorylation of NF-kB and ERK1/2, consistent with established signal transduction pathways. ERK1/2 activation was effectively reduced in the presence of the MEK inhibitor. IL-1α  reduced phosphorylation of STAT3 at the classical activating tyrosine (Y705), while concurrently increasing phosphorylation of a non-classical serine (S757) moiety, which has been implicated in gating the duration of active pSTAT3-Y705. IL-1α  stimulation induced gene expression of IL-1α , IL-1β  and IL-6, which was reduce by MEK inhibition.

 

Conclusions: Our results confirm that IL-1α  and not IL-1β  signaling in pancreatic tumor cells activates NF-kB and MAPK. Additionally, we identify regulatory S757 phosphorylation of STAT3 as a novel downstream target of IL-1α . Hence, we propose that development of resistance to MEK inhibition is mediated by a loss of IL-1α -mediated regulation of STAT3, permitting reactivation of target genes and tumor growth.

04.08 The Immunomodulatory Effect of Vesicular Stomatitis Virus on Peritoneal Surface Malignancy.

Posted on January 25, 2017

G. Davis2, S. A. Northrup1, M. Westcott2, J. H. Stewart1,2  1Duke University Medical Center,Surgical Oncology,Durham, NC, USA 2Wake Forest University School Of Medicine,Winston-Salem, NC, USA

Introduction:  Oncolytic virotherapy (OV) using vesicular stomatitis virus (VSV) has become an attractive therapeutic strategy for treating peritoneal surface dissemination of colorectal cancer (PSD of CRC) because it selectively induces apoptosis in malignant cells while sparing non-malignant cells. OV’s also modulate the immune environment to induce a systemic anti-tumor response. The goal of this project was to analyze the immunomodulatory and oncolytic functions of VSV using an established murine model of PSD from CRC. We used an 8-marker flow cytometry panel to characterize the cell types and cytokine environment of the peritoneal cavity in VSV treated and control mice.  

Methods:  This study utilized an established syngeneic murine model with CT26 cell line coexpressing luciferase and GFP. In this model, 5-6 week old balb/c mice are injected intraperitonealy with 1×106 luciferase/GFP-expressing CT26 cells. On day 4, the mice were imaged utilizing an IVIS system to determine the establishment of tumor. On day 5, mice were injected with either PBS or 1×108 PFU M51R-VSV.  Mice were sacrificed at 1, 3, or 7 days after virus/mock treatment, and a peritoneal lavage was performed on each mouse to collect peritoneal exudate cells (PEC) from the cavity. Cells from each sample were processed for flow cytometry, and supernatants from each sample were collected for a multiplex cytokine bead analysis. PECs were immunotyped using an 8-color panel, supernatants were assessed using a Biolegend 13-plex Mouse Inflammation Panel, and all samples were analyzed with a BD Facs Canto. 

Results: The results from this study show VSV-mediated alterations in the peritoneal cavity as early 24 hours post virus treatment. At this early time point, M51R-VSV treated animals have significantly fewer CD4+ T cells, CD8a+ T cells, and CD19+ CD11b+ B-1 B Cells. Additionally, M51R treatment altered the cytokine microenvironment; M51R-treated cavities contained less MCP-1 than mock-treated animals. On day 7, M51R-treated mice contained approximately 75% more CD4+ T cells than their PBS-treated cohorts. There were also significant differences in CD11b+ myeloid lineages. These changes in cellular composition were accompanied by marked changes in the local concentration of IL-6 in mock-treated vs M51R-treated animals. 

Conclusion: The results from this study suggest that VSV modulates the immune response through a number of key players and facilitates its therapeutic effect by altering the innate response at early time points, in order to produce an anti-tumor adaptive response at later time points. In our model, alterations in early recruitment of T-cells and myeloid cells, along with cytolysis of tumor cells resulted in a robust CD4 helper T cell response and a marked reduction in pro-tumor myeloid lineages 7 days after virus treatment.
 

04.07 Interferon-gamma enhances monocyte function and increases PD-L1 expression.

Posted on January 25, 2017

S. P. Walker1, N. Galbraith1, C. Bishop1, M. Reid1, S. Gardner1, H. Polk1  1University Of Louisville,Department Of Surgery,Louisville, KY, USA

Introduction:

Major infection and trauma lead to poorly understood pro-inflammatory and compensatory anti-inflammatory immunological responses. Some patients have resulting monocyte impairment which is predictive of nosocomial infection and death. Attempts to augment host defenses in surgical patients, such as interferon-gamma (IFN-γ), have thus far failed to improve mortality. An incomplete understanding exists of the underlying immunological mechanisms, including the role of programmed cell death-ligand 1 (PD-L1), a negative co-stimulatory molecule. The purpose of this study was to determine the effects of IFN-γ on monocyte function and PD-L1 expression.        

Methods:

Venous blood was taken using EDTA tubes from healthy volunteers and stimulated ex-vivo with 100 ng/mL of lipopolysaccharide (LPS) for 18 hours in the presence or absence of recombinant IFN-γ (100 ng/mL).  The monocyte inflammatory response was measured using quantitative measurements of surface protein expression of human leukocyte antigen-DR (HLA-DR) and PD-L1 on CD14 positive monocytes using flow cytometry.  Cytokine responses including tumor necrosis factor-alpha (TNF-α) were measured by ELISA. Statistical analyses was determined using Paired T-test or Wilcoxon signed-rank test as appropriate, with significance set at 0.05. Six donors were used for each experiment.     

Results:

Exposure to LPS led to an increase in HLA-DR expression and increased TNF-α production. IFN-γ alone offered minimal effects on HLA-DR or TNF-α levels.  The addition of the immunoadjuvant IFN-γ augmented HLA-DR expression and TNF-α levels at 18 h (p<0.05). Monocyte PD-L1 expression was low at baseline (0 h) in healthy volunteers, but increased in the presence of either LPS or IFN-γ alone (p<0.05). IFN-γ treatment in combination with LPS led to a synergistic increase in PD-L1 expression at 6 h and 18 h after stimulation (p<0.05).

Conclusion:

In this study we found that IFN-γ acts to increase monocyte responsiveness as measured by increased HLA-DR expression and TNF-α production in the presence of LPS. Monocyte PD-L1, a negative co-stimulatory molecule, increased in the presence of LPS. Adjuvant IFN-γ treatment further increased PD-L1 expression. These results suggest that IFN-γ therapy stimulates a negative feedback loop through the PD-1-PD-L1 axis, which could have therapeutic implications in approaching immunoadjuvant therapy in the “high risk” patient. 

04.06 Topical Imiquimod for Cutaneous In-Transit Metastases of Melanoma

Posted on January 25, 2017

K. M. Leick1, E. P. Salerno1, J. M. Obeid1, D. H. Deacon1, C. L. Slingluff1  1University Of Virginia,Surgery,Charlottesville, VA, USA

Introduction: Topical imiquimod, a TLR7 agonist, has been reported to induce regression of some cutaneous in-transit metastases of melanoma, yet rates of tumor control and predictors of response are not established. We have reported that melanoma metastases may be grouped into Immunotypes representing patterns of immune cell infiltration: A (none), B (perivascular cuffing), and C (diffuse). The goals of this study were to provide pilot data toward testing the following hypotheses: 1) imiquimod may induce complete regressions of cutaneous melanoma metastases, 2) imiquimod extends time to major surgery, and 3) clinical responses to imiquimod are more frequent when the metastases have pre-existing immune cell infiltrates.

Methods: The UVA melanoma database was searched for patients with in-transit metastases treated with imiquimod (n=12) and matched for control patients with in-transit metastases who were not treated with imiquimod (n=11). Biopsies of metastases (IRB #10598 and 10803) were evaluable for 9 treated patients prior to initiating imiquimod. Immunohistochemistry staining (IHC) was performed on formalin-fixed paraffin-embedded sections of those tumors, using antibodies to CD45 and CD34 to assess patterns of immune cell infiltrates (Immunotypes).

Results:Among 12 patients treated with topical imiquimod, 2 (17%) had complete regressions of the metastases, while 6 (50%) had mixed responses. Complete or mixed responses typically enabled patients to delay or to avoid major surgical resections or limb perfusion. Complete responses were all in the tumors with Immunotypes B and C (2/7), and none were in those with Immunotype A (0/2). Median time to surgery for the imiquimod group (357 days) trended longer than for controls (49 days, p=0.076). For the evaluable imiquimod-treated patients, higher Immunotype (B and C vs. A) was associated with improved survival (p=0.0019).

Conclusion:These pilot data demonstrate that a small but meaningful subset of patients experience complete regressions of skin metastases with imiquimod alone. Further, the findings suggest that imiquimod therapy may provide sufficient control to avoid or to delay major surgical resections. Both complete responses were in patients whose tumors had significant immune cell infiltrates prior to initiating therapy. Overall, metastases with higher Immunotype (B or C) who were treated with imiquimod had enhanced survival. These pilot data support use of imiquimod for patients with superficial metastatic disease who are suboptimal candidates for major surgical resection or aggressive systemic therapy. These data also suggest that T cell infiltration in the metastases may be associated with better outcomes for patients treated with imiquimod.

 

04.05 Radiation and Natural Killer Cell Therapy in Canine Sarcomas: Initial Results of a Co-Clinical Trial

Posted on January 25, 2017

J. S. Park1, Z. Wang2, S. K. Grossenbacher2, J. I. Luna2, I. Sturgill2, S. S. Withers3, M. S. Kent3, M. Chen4, W. T. Culp3, R. Rebhun3, A. M. Monjazeb5, W. J. Murphy2, R. J. Canter1  1UC Davis,Surgical Oncology/Surgery,Sacramento, CA, USA 2UC Davis,Dermatology,Sacramento, CA, USA 3UC Davis School Of Veterinary Medicine,Surgical And Radiological Sciences,Davis, CA, USA 4UC Davis,Pathology And Laboratory Medicine,Sacramento, CA, USA 5UC Davis,Radiation Oncology,Sacramento, CA, USA

Introduction: We have previously shown that radiotherapy (RT) increases natural killer (NK) cytotoxicity and homing in pre-clinical models of human solid cancers, including sarcomas. Since canine clinical trials are a valuable resource for novel immunotherapy protocols and sarcomas commonly afflict dogs, we hypothesized that dog PBMC-derived NK cells would be effective in canine models of sarcoma, including adoptive transfer in a canine RT/NK clinical trial.

Methods: Canine NK cells were isolated from 15 mls of fresh whole blood using Ficoll separation and CD5 depletion. Isolated NK cells were then expanded with irradiated K562c9IL21 for 2-3 weeks. Using 6-month metastasis-free survival as the primary endpoint, we are evaluating RT and adoptive NK immunotherapy in a canine clinical trial. For this trial, treatment consists of palliative RT weekly x4 followed by two intra-lesional injections of autologous NK cells. In correlative studies, including dog patient-derived xenografts (PDX), we assessed NK homing using eFluor 670 cell proliferation dye and NK function by expression of activation markers IFNγ, granzyme B, and perforin.

Results: We have treated 8 of planned 14 dogs with osteosarcoma on protocol with a median of 76×10^6 cells (92% viable). Of 3 evaluable dogs who have reached the 6-month primary endpoint, we have observed 1 partial response and 2 are metastasis-free, including 1 dog with complete resolution of a suspicious 3 mm pulmonary nodule. In dog patients on trial, phenotyping of expanded NK cells from all patients showed > 90% granzyme B and IFNγ expression prior to adoptive transfer. Tagging experiments 1 week after intratumoral injection revealed that 11 – 60% of CD45+ cells are eFluor 670 positive, confirming persistence of injected NK cells post injection. Analysis of unactivated circulating PBMCs post-injection demonstrated a significant increase in granzyme B expression (2.25X ± 0.42, P<0.01). Dog PDX studies demonstrate that focal RT increases NK homing to sarcomas on average 3.8X±0.3 (P<0.001) compared to unirradiated controls. Immunohistochemical analysis of tissue samples post RT shows a significant increase in CD3+ tumor-infiltrating lymphocytes post RT (P<0.05, see figure). Co-culture experiments of dog PDX sarcomas ex vivo with allogeneic NK cells shows RT-induced sensitization to NK killing at doses of 10 – 20 Gy (P<0.01)

Conclusion: RT and NK immunotherapy appear synergistic in dog models of sarcoma. Preliminary results from a canine clinical trial of palliative RT and autologous NK transfer for osteosarcoma are promising, including possible abscopal effects. Further evaluation of this novel radio-immunotherapy approach is warranted.

 

04.04 Myeloid Derived Suppressor Cells in the Inflammatory Response of Pancreatitis and Pancreatic Cancer

Posted on January 25, 2017

N. E. Cieza Rubio1, R. L. Heimark1,2  1University Of Arizona,Department Of Surgery,Tucson, AZ, USA 2The Arizona Cancer Center,Tucson, AZ, USA

Introduction:

Tumor-infiltrating myeloid-derived suppressor cells (MDSCs), are important mediators of a tumor-permissive microenvironment that contributes to tumor growth and could account for the limited success of immunotherapeutic strategies. MDSCs suppress adaptive immunity by blocking T cell activation, inducing Treg accumulation, and inhibiting natural killer cell cytotoxicity against tumor cells. Aim: We investigated the roles of MDSCs in the regeneration of the exocrine pancreas associated with acute pancreatitis and the progression of acinar to ductal metaplasia. 

Methods:  

Model of Acute Pancreatitis: Pancreatitis was induced using a regimen of 7 hourly intraperitoneal injections of caerulein (50 g/kg) followed by a 48-hour rest period and a second regimen of 7 hourly intraperitoneal injections of caerulein before sacrifice at different time points (1, 12, 24, 48 and 72 hours after last caerulein injection). 

Model of MDSCs Depletion: Wild type mice were treated with bi-daily intraperitoneal doses of CXCR2a (SB-265610) (4 mg/kg). Treatment started one day before induction of pancreatitis with caerulein and continued until animal euthanasia.

Positive Selection of gMDSCs: After preparation of a single-cell suspension from pancreas or spleen, CD11b+Gr1+ cells were indirectly magnetically labeled with Anti-Ly-6G-Biotin and Anti-Biotin Microbeads.The magnetically labeled Ly6G+ cells, also known as positively selected cell fraction, were retained within the column and collected once the column is removed from the magnetic field.

Cell surface marker analysis was performed by flow cytometry using the BD FACSCANTO II.

Results:

Acute pancreatitis was induced in wild type and P48+/Cre;LSL-KRASG12D mice using caerulein and an early influx of MDSCs into the pancreas was observed flow cytometry and immunocytochemistry. Numbers of Gr1(+)CD11b(+) MDSCs increased over 20-fold in pancreata of mice with acute pancreatitis to account for nearly 15% of intrapancreatic leukocytes and have T cell suppressive properties. This marked accumulation of MDSCs returned to normal values within 24 hours of the insult in wild type mice; however, in the oncogenic KRAS mice, MDSCs levels remained elevated. When intrapancreatic MDSCs were depleted by administration of a CCR2 antagonist (SB265610) in wild type mice the severity of acinar damage was increased. This was also accompanied by a delayed regeneration determined morphologically and with the mitotic immunomarker phospho-histone H3. Isolated intrapancreatic MDSCs from treated mice induce naïve acinar cells to undergo acinar ductal metaplasia when co-cultured in collagen 3D cultures. Purified splenic MDSCs failed to induce the phenotypic transdifferentiation.

Conclusion:

MDSCs are required for adequate pancreatic regeneration in wild type mice with acute pancreatitis and their persistent elevation in oncogenic KRAS mice is not only associated with immune-evasion, but may also function as direct enhancer of malignant proliferation.

04.03 Loss of Class I MHC Adds Prognostic Value to PD-L1 Expression in Non-Small Cell Lung Cancer Patients

Posted on January 25, 2017

J. M. Obeid1, G. Erdag4, R. D. Gentzler2, M. G. Brown5, J. V. Cross3, D. H. Deacon1, T. N. Bullock3, C. L. Slingluff1  1University Of Virginia,Surgery Department,Charlottesville, VA, USA 2University Of Virginia,Heme/Onc / Medicine Department,Charlottesville, VA, USA 3University Of Virginia,Pathology Department,Charlottesville, VA, USA 4Johns Hopkins University School Of Medicine,Pathology Department,Baltimore, MD, USA 5University Of Virginia,Immunology,Charlottesville, VA, USA

Introduction:  Expression of PD-L1 on tumor cells reflects the presence of an active immune response, and its increased expression is associated with improved patient survival in several cancers. In Non-small cell lung cancer (NSCLC), however, this association is less clear. Effective immune responses may be inhibited in the tumor microenvironment by downregulating class I MHC (MHC-I) expression on cancer cells. Thus, we hypothesized that low MHC-I expression may add additional negative prognostic value in NSCLC with low PD-L1 expression.

Methods:  A tissue microarray (TMA) was constructed from 151 NSCLC specimens (90 adenocarcinoma (AdCA), 58 squamous cell (SQCC), 3 mixed histology). Patients had stage I (61%), II (22%), III (14%) and IV (3%) disease that was resected between 2011 and 2014 (median follow up: 27 months). The TMA was analyzed by immunohistochemistry for PD-L1 (clone 5H1) and MHC-I heavy chains (clone HC-10). PD-L1 expression was assessed as a percentage of cancer cells staining positive, with 5% and 50% thresholds for positivity. Expression of the MHC-I was reported as a score of 1 to 5, accounting for staining intensity and percent of tumor cells staining. Kaplan Meier curves were used to plot survival, and a log rank test was used to test associations with patient survival. 

Results: Low PD-L1 expression, below a 5% threshold, was associated with worse overall survival (p=0.02) but the association was not significant with a 50% threshold (p=0.06). Low MHC-I expression on tumor cells (score ≤ 3) was not significantly associated with worse survival (p=0.14). However, when combined, the tumors with both low MHC-I and low PD-L1 expression (<5% and <50%) were significantly associated with worse patient outcomes (p=0.002 (Figure) and p=0.004 respectively), similar results were obtained in SQCC and AdCA subsets separately, and when controlling for stage.

Conclusion: Co-occurrence of low PD-L1 expression and MHC-I downregulation in tumor cells, is associated with a significant decrease in patient survival for patients with NSCLC. When MHC-I expression is high, PD-L1 expression loses its prognostic significance. This finding may help to clarify the role of pre-existing immunity to NSCLC, and may help to develop a useful biomarker combination. By characterizing the immune profile of NSCLC, modifications of this analysis should be explored for their ability to predict patient response to immune therapy.

 

04.02 The inflammatory environment of the gut in the surgical Crohn’s patient

Posted on January 25, 2017

M. Laffin2, T. Perry2, B. Dicken2, R. Fedorak3, K. Madsen3  1University Of Alberta,Edmonton, AB, Canada 2University Of Alberta,Department Of Surgery,Edmonton, AB, Canada 3University Of Alberta,Department Of Medicine,Edmonton, AB, Canada

Introduction: Crohn’s disease (CD), is a type of inflammatory bowel disease, defined by transmural inflammation of the alimentary tract. Complications of CD leads to intestinal resection in the majority of patients during their lifetime. While CD can affect any segment of the intestinal tract, it is most often found in the ileocecal region, making Ileocolic resection the most common procedure performed.  Unfortunately, after resection of the diseased segment, inflammation almost inevitably returns proximal to the surgical anastomosis in the neo-terminal ileum. Studying this recurrence may offer a glimpse into the pathophysiology and development of the disease. To date, few published results examine the inflammatory milieu of the peri-operative CD patient and how that profile relates to recurrence.

The objective of our study is to define the immunologic environment of the gut at the time of surgery which and identified how it is associated with post-operative recurrence.

Methods: 26 CD patients were recruited at the time of Ileocolic resection and followed prospectively. Mesenteric lymph node (MLN) and mucosal samples from the terminal ileum were snap frozen in the operating theatre. All patients received follow-up ileocolonoscopy six months post-operatively, at which time recurrence scores in the neo-terminal ileum were recorded using the Rutgeert’s scale. Endoscopic recurrence was defined as a score ≥ 2. A comprehensive multiplex immune assay utilizing the MesoScale Discovery platform measured tissue concentrations of 31 cytokines and chemokines. Known clinical characteristics associated with recurrence were tested using the Fisher’s exact test. Individual analytes were compared using the Mann-Whitney-U test.

Results: 30% of patients had endoscopic disease recurrence at follow up. Mean time to ileocolonoscopy was 209 days in the recurrence group vs. 176 days in the remission group. Clinical parameters including fistulizing disease, previous surgery, and smoking were not significantly associated with recurrence after 6 months in our cohort. When patients were stratified based on these clinical parameters mucosal IL-6 was associated with stricturing disease, smoking was associated with elevated mucosal IL-6 and IL-1α, and perianal disease was associated with elevated mucosal TNF-α, IL-6, IL-1β, and IFN-γ.

When analytes were evaluated by post-operative recurrence, elevated MCP-1 in the mucosa of surgical specimens was associated with disease recurrence. An elevated level of IL-5 and IL-16 was seen in the MLN of patients who recurred.

Conclusion: Results from this study suggest that chemokines are a factor in post-operative recurrence as MCP-1, IL-5 and IL-16 all act as prominent chemoattractants to various immune cell types. These observations highlight the need for further study in cell recruitment to the gut, and its relationship to post-operative recurrence.
 

03.20 Timing is Everything: Relationship between Platelet Dysfunction and Metabolic Status

Posted on January 25, 2017

L. J. Schaub1, R. M. Paredes1, A. R. Macko1, J. J. Glaser1, H. B. Moore2,3, E. E. Moore2,3, F. R. Sheppard1  1Naval Medical Research Unit-San Antonio,Expeditionary And Trauma Medicine,Ft. Sam Houston, TX, USA 2Denver Health Medical Center,Surgery,Aurora, CO, USA 3University Of Colorado Denver,Surgery,Aurora, CO, USA

Introduction: Platelet dysfunction (PD) is a significant component of trauma induced coagulopathy (TIC).  The conditions under which PD occurs and the role of platelet transfusion remain to be elucidated. We hypothesized that correction of PD may be determined by the metabolic state during resuscitation.

Methods: NHPs (n=8) underwent poly-traumatic (soft tissue injury and femur fracture) severe hemorrhagic shock (MAP=20mmHg). Resuscitation was accomplished via a combination of fresh whole blood (WB) and normal saline (NS), with resuscitation volume determined by shed blood volume (SBV). Arterial blood gas, complete blood count (CBC), lactate and  Multiplate® platelet aggregation analyses were performed at baseline (BSLN), end of shock (EOS), end of resuscitation (EOR) and T=360min after initiation of shock. Multiplate platelet aggregation was determined by area under the aggregation curve in units (U) over a 6min measurement period and normalized to actual platelet count. Results reported as mean±SEM. Statistical analysis was performed by Spearmen correlation and one-way ANOVA with p<0.05 considered significant.

Results: Lactate and base deficit (BD) levels increased at EOS compared to BSLN.  Lactate levels returned to BSLN levels by EOR.  BD improved from a nadir at EOS but did not return to BSLN levels by T=360min. (Fig 1). Platelet aggregation decreased in response to adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA), most notably following the initiation of resuscitation (Fig 1).  Significant correlations were observed between platelet aggregation and BD: ADP (r=0.7070, p<0.0001), COL (r=0.6194, p=0.0002), and AA (r=0.4831, p=0.0051).  In contrast, no significant correlations were observed between platelet aggregation and lactate:  ADP (r=-0.2298, p=0.2058), COL (r=-0.3111, p=0.0830), and AA (r=-.0923, p=0.6156).      

Conclusion: In this study, PD was observed at EOS with significant PD in response to all platelet agonists identified at EOR.  Based on correction of lactate, by EOR aerobic metabolism was restored rapidly by resuscitation; however BD buffering was delayed. Platelet function correlated strongly with BD but not lactate, and the administration of platelets in WB did not correct PD. These data indicate that administration of platelets prior to correction of BD may be ineffective. This may be related to a residual byproduct of the shock state; e.g. metabolites from anaerobic metabolism, suggesting that the timing of platelet therapy may be important for optimal effectiveness. 

 

03.19 Pre-Existing Malignancy Induces a Highly Suppressive T Regulatory Cell Phenotype in Mice with Sepsis

Posted on January 25, 2017

K. M. Ramonell1, C. Chen1, Z. Liang1, C. M. Coopersmith1, M. L. Ford1  1Emory University School Of Medicine,Department Of Surgery,Atlanta, GA, USA

Introduction:  Patients with pre-existing malignancy are nearly ten times more likely to develop sepsis than the general population and mice with pre-existing tumors exhibit worsened survival following cecal ligation and puncture (CLP) as compared to previously healthy controls. Here, we aimed to determine whether alterations in T regulatory (Treg) cell frequency or suppressive capacity play a role in the increased mortality observed in the setting of cancer and sepsis.

Methods:  Cancer was induced via subcutaneous injection of 125,000 Lewis lung cancer cells into the right inner thigh of C57BL/6 mice, which were then followed for three weeks for the development of palpable tumors. Mice with cancer and previously healthy mice were then made septic via 2x25g CLP or underwent sham laparotomy. Spleens were harvested 24hr post-sepsis induction and splenocytes were stained with fluorescently conjugated mAbs specific for CD3, CD4, CD8, CD25, FoxP3, TIGIT, and Helios. Tregs were defined as CD3+CD4+CD25+FoxP3+ cells. Data was collected on an LSRII Flow Cytometer and analyzed with One-Way ANOVA testing. For all data points, n=5-10 mice/group.

Results: In cancer septic mice, the frequency of Tregs was significantly increased compared to septic mice without cancer (14.5% vs 8.2%; p=0.02). Absolute counts followed a similar trend (4.7 x 105 vs. 3.2 x 105; p=0.05) but differences were not statistically significant. Additionally, cancer septic mice had the greatest frequency of Helios and FoxP3 double positive Tregs compared to non-cancer septic mice (12.5% vs. 8.0%; p=0.004) and cancer sham mice (12.5% vs. 10.0%; p=0.08). When further analyzing Helios+ Tregs, cancer septic mice exhibited greater frequencies of TIGIT+ cells compared to non-cancer septic mice (14.0% vs 7.1%; p<0.001) and cancer sham mice (11.5% vs. 14.0%; p=0.09). When analyzing non-Treg CD4 T cells, the frequency of Helios+TIGIT+ cells was significantly less compared to Helios+TIGIT+ Tregs (9.0% vs. 0.8%; p<0.001) isolating Helios and TIGIT expression on CD4 T cells to Tregs. 

Conclusion: Sepsis in the setting of cancer significantly increases the frequency of total Tregs and Helios+TIGIT+ Tregs. This phenotype has been previously reported to be highly suppressive and could contribute to the exaggerated mortality observed when these two conditions occur together. These findings help elucidate the specific derangements in the immune profile of co-existing cancer and sepsis in order to develop effective therapies for this vulnerable population.

03.18 Optimal Timing of Administration of Histone Deacetylase Inhibition for Prevention of ARDS

Posted on January 25, 2017

G. Kasotakis1, M. Galvan1, P. Osathanugrah1, N. Dharia1, L. Bufe1, Z. Breed1, D. Mabarak1, N. Singh1, E. Kintsurashvili1, D. Remick1  1Boston University,Dept Of Trauma, Acute Care Surgery & Surgical Critical Care,Boston, MA, USA

Introduction:  Acute Respiratory Distress Syndrome (ARDS) is a clinical syndrome characterized by uncontrolled inflammation of the lungs, after application of a severe inflammatory stimulus. We have previously demonstrated an ameliorated syndrome and improved survival in mice with the early administration (30 min) of valproic acid (VPA), a broad-spectrum Histone Deacetylase Inhibitor (HDACI), while studies in humans have shown no benefit when anti-inflammatories are administered late in the course of disease. With the current project we aim to identify the window of time within which, if an HDACI is administered, clinical and laboratory improvement can be seen.   

Methods:  Mice (C57BL/6, n=12/group) had 50×106 E. coli (strain 19138) instilled endotracheally and VPA (250mg/kg) administered intraperitoneally 3, 4, 6, and 9 hours later. These were compared with controls without VPA. Six hours after VPA administration, the animals were sacrificed, and bronchoalveolar lavage fluid (BAL) IL-6, TNF, neutrophils and macrophages, as well as the E.coli Colony Forming Units (CFU) were quantified. Plasma IL-6 was also measured. A separate groups of mice (n=12/group) were followed prospectively for 14 days to assess survival. Continuous variables were compared with a t-test and survival with the log-rank test. Statistical significance was declared at p<0.05. 

Results: BAL IL-6 and TNF were significantly lower in the animals administered VPA within 3h (p<0.05), but not when administered later (4, 6, 9h). There was no difference in the BAL E. coli CFU, macrophage or neutrophil numbers with any timing of administration studied in this project (3, 4, 6 or 9h). Survival improved only when VPA was administered up to 3h.

Conclusion: A narrow therapeutic window of opportunity exists in this clinically relevant murine model of gram negative pneumonia-induced ARDS, and likely explains the lack of response in studies with late administration of anti-inflammatory therapies in humans. Future directions include use of murine models with kinetics similar to those of ARDS development in humans, and study of narrow spectrum HDACI.

 

03.17 Impact of Tranexamic Acid on Posttraumatic Coagulation and Inflammation

Posted on January 25, 2017

M. D. Johnson1, R. Veile1, L. Friend1, H. Goetzman1, T. Pritts1, C. Caldwell1, A. Makley1, M. Goodman1  1University Of Cincinnati,College Of Medicine,Cincinnati, OH, USA

Introduction:  Posttraumatic coagulopathy and inflammation can exacerbate secondary cerebral damage after traumatic brain injury (TBI). Tranexamic acid (TXA) has been shown clinically to reduce mortality in hemorrhaging and head injured trauma patients, and has the potential to mitigate secondary brain injury with its reported anti-fibrinolytic and anti-inflammatory properties. We hypothesized that TXA would improve posttraumatic coagulation and inflammation in a murine model of TBI alone and in a combined injury model of TBI and hemorrhage (TBI/H).

Methods:  An established murine weight-drop model was used to induce a moderate TBI. Mice were administered intraperitoneal injections of 10mg/kg TXA or equivalent volume of saline 10 minutes after injury. An additional group of mice was subjected to TBI followed by hemorrhagic shock using a pressure-controlled model. TBI/H mice were given intraperitoneal injections of TXA or saline during resuscitation. Blood was collected at intervals after injury to assess coagulation by thromboelastometry (ROTEM®) and inflammation by Multiplex cytokine analysis. Soluble P-selectin, a biomarker of platelet activation, and serum neuron specific enolase, a biomarker of cerebral injury, were also measured at intervals. Brain tissue was analyzed for inflammatory changes by Mutiplex ELISA and splenic tissue was collected for splenic cell population assessment by flow cytometry.  

Results: There were no coagulation, serum or cerebral cytokine, P-selectin, or neuron specific enolase differences between mice treated with TXA or saline after TBI. Following the addition of hemorrhagic shock and resuscitation to TBI, TXA administration still did not affect coagulation parameters, systemic or cerebral inflammation, or platelet activation, as compared to saline alone. At 24 hours post-TBI, mice given TXA demonstrated lower splenic total cell counts (93.6 vs. 130.1, TXA vs. saline, p = 0.01), central memory CD8 (0.2 v. 0.3, TXA vs. saline, p = 0.001), effector CD8 (0.04 vs. 0.1, TXA vs. saline, p = 0.006), B cell (48.5 vs. 72.9, TXA vs. saline, p = 0.001), and increased naïve CD4 (14.6 vs. 11.5, TXA vs. saline, p = 0.04) cell populations. By contrast, TXA did not affect splenic leukocyte populations after combined TBI/H

Conclusion: Despite clinical data suggesting a mortality benefit, TXA did not modulate the coagulation effects, inflammatory changes, or biomarker generation in either the TBI or TBI/hemorrhage murine models. Administration of TXA following TBI alone altered splenic leukocyte populations, which may contribute to a change in posttraumatic immune status. Future studies should be done to investigate the role of TXA in the development of posttraumatic immunosuppression and risk of nosocomial infections.

 

03.16 Resuscitation of Hemorrhagic Shock Using Fresh Frozen Plasma Prevents Lung ICAM-1 Expression

Posted on January 25, 2017

L. M. Bond1, P. J. Matheson1,2, V. S. Graham1, M. A. Eid1,2, C. N. Kapsalis1,2, A. J. Matheson1,2, C. D. Downard1,2, J. W. Smith1,2  1University Of Louisville,Department Of Surgery,Louisville, KY, USA 2Robley Rex Veterans Affairs Medical Center,Research,Louisville, KY, USA

Introduction:  Hemorrhagic shock (HS) impairs vital organ perfusion to cause tissue hypoxia, compartmental fluid shifts, hypercoagulability, and inflammation. This increases lung neutrophil sequestration, which is partially mediated by altered ICAM-1 expression.  Resuscitation (RES) with blood and i.v. fluids (conventional resuscitation, CR) restores central hemodynamic parameters, but can progress to multiple organ failure and death.  Adding Fresh Frozen Plasma (FFP) to blood RES maintains arterial oxygen delivery to prevent these changes.  We hypothesized that RES using FFP might restore liver blood flow (LBF) and prevent increased ICAM-1 expression to minimize lung neutrophil infiltration.

Methods:  Anesthetized male Sprague-Dawley rats were randomized to groups (n=8/gp): 1) Sham, 2) Sham+FFP, 3) HS/CR, or 4) HS+FFP.  Sham animals underwent surgery, but no HS/CR.  HS was 40% MAP (60 min) and CR was return of shed blood + 2 volumes of i.v. normal saline.  HS+FFP received shed blood plus one volume of pre-warmed FFP.  Liver blood flow was by galactose clearance.  After 4 hours, samples were harvested. Lung pathology was graded by pathologist blinded to groups in H&E and lung ICAM-1 immunohistochemistry (IHC).  

Results: LBF was decreased in HS/CR compared to Sham (*P<0.05), but HS+FFP restored LBF to Sham levels (*P<0.05).  Lung pathology showed increased neutrophil infiltration in the HS/CR group compared to Sham, which was largely prevented in the HS+FFP group. Lung ICAM-1 expression was increased in HS/CR (*P<0.05) but decreased in HS+FFP (*P<0.05). Hematuria was noted in 4 out of 8 HS/CR rats that received FFP. 

Conclusion: Resuscitation with FFP when used with blood RES minimized inflammatory changes in the lung.  Liver blood flow was also improved by resuscitation with FFP, which might support improved lung status.  These results suggest that FFP can be used in place of aggressive intravenous saline resuscitation in hemorrhagic shock.  The use of FFP has found favor in human patients and these studies support that use.

 

03.15 Biologics-Supplemented Hemostatic Patch In A Nonsurvival Porcine Model Of Hepatic Resection

Posted on January 25, 2017

S. Aravind1,2, F. Fabian3, R. Spretz3, U. R. Yanala1,2, J. Ragusa3, A. E. Ismail3, G. Larsen ?3, W. Velander3, M. A. Carlson1,2  1University Of Nebraska College Of Medicine,General Surgery,Omaha, NE, USA 2VA Medical Center,General Surgery,Omaha, NE, USA 3University Of Nebraska – Lincoln,Bio-Medical Engineering,Lincoln, NE, USA

Introduction:

In this study we evaluated the acute efficacy of a hemostatic patch consisting of custom-made nano-engineered resorbable polycaprolactone (PCL) mesh embedded with human clotting factors in a nonsurvival porcine model of hepatic resection. Our overall goal with this work has been to develop a synthetic resorbable biologics-supplemented bandage for surgical bleeding. 

Methods:

Normovolemic normothermic domestic swine (male, N = 51, ~3 months, 33–40 kg) were anesthetized and splenectomized before they underwent nonanatomical scissor resection of the left medial liver lobe. Subjects were randomized among four treatment groups: group 1 (control) = cautery + suture ligation + Surgicel® application; group 2 = PCL mesh alone (16 x 8cm, six-ply); group 3 = PCL with fibrin sealant (FS = human plasma-derived fibrinogen (pdFI) + human recombinant thrombin (rFII) + human recombinant Factor XIII (rFXIII)); group 4 = PCL with rFII + rFXIII only. Test bandages were applied with 5 min of bimanual compression. Resuscitation was administered with warm Lactated Ringer's solution (10 mL/min; given for MAP <80% of pre-injury MAP). Subjects underwent vital sign monitoring and blood testing for 60 min, then necropsy with blood loss quantification. 

Results:

Pre-injury parameters (subject weight, vital signs, preparation blood loss, and baseline laboratory tests) were not different among the treatment groups. One anesthetic death occurred in group 3, leaving 50 subjects for analysis (Table 1). Blood loss was different among groups, with group 3 and 4 having the highest and lowest blood loss, respectively. Final plasma fibrinogen level also was different, with group 4 having the highest. Final MAP, hemoglobin, and protime were not different among the groups. 

Conclusion:

Resorbable PCL mesh, either alone or in combination with rFII + rFXIII, appeared to have at least equivalent hemostatic efficacy (with respect to 1 h blood loss) compared with traditional surgical technique in this model of porcine hepatic resection. Additional preclinical testing (including efficacy in different hemorrhage models, and toxicology) is planned.

 

03.14 Sepsis induces phenotypic changes upon both hepatic and migrating invariant Natural Killer T-cells

Posted on January 25, 2017

T. T. Chun1, E. Fallon1, W. G. Cioffi1, A. Ayala1, D. S. Heffernan1  1Brown University School Of Medicine,Division Of Surgical Research, Department Of Surgery,Providence, RI, USA

Introduction: Invariant Natural Killer T (iNKT)-cells are key regulators of the immune response to peritoneal sepsis. Following induction of sepsis, iNKT-cells become activated, leave the liver and migrate to the peritoneal cavity. Program Death Receptor-1 (PD-1), a co-inhibitory check point protein, has been shown to control the activation and migration of iNKT-cells. However, little is known about the expression of PD-1 or its ligands PD-L1 or PD-L2 upon iNKT-cells. Although a role for other co-stimulatory molecules, specifically CD25 and CD40 has been demonstrated in several chronic disease states, little is known about the expression of co-inhibitory/co-stimulatory molecules upon activated iNKT-cells following polymicrobial sepsis.

Methods: 8-12 week old male C57BL/6 mice underwent sepsis (cecal ligation and puncture (CLP)) or sham procedure. Liver and peritoneal cells were harvested for analysis by flow cytometery for early (4 hours) and later (12 hours) alterations. iNKT-cells were identified using CD1d-loaded tetramer from the NIH tetramer facility. iNKT-cells were further phenotyped using monoclonal antibodies to PD-1, PD-L1, PD-L2, CD25 and CD40.

Results:As we previously observed, 4 hours following sepsis by CLP, numbers of hepatic iNKT-cells were unchanged, whereas by 12 hours, the hepatic iNKT cell population had declined, corresponding to the emergence of iNKT cells within the peritoneal cavity. With respect to early hepatic iNKT phenotype changes, 4 hours following sepsis, there was no alteration in CD25, CD40 or PD-1/PD-L1/PD-L2 expression upon iNKT-cells. However, compared to sham, by 12 hours following sepsis, although PD-1+iNKT-cell populations remained unchanged in both the liver and peritoneal cavity, it was noted that within the liver there was a marked decline in both PD-L1+iNKT-cells (49% vs 32%;p<0.01) and PDL2+ iNKT-cells (48.1% vs 27%;p<0.001). This corresponded with an increase in both PD-L1 (41% vs 83%;p<0.01) and PD-L2 expression (49% vs 87%;p<0.01) among septic mouse peritoneal iNKTcells. With respect to other molecules, there was no change in CD40 expression upon iNKT cells (32.7% vs 31.9%;p=0.9), but by 12 hours following sepsis there was a decline in both hepatic CD25+iNKT cells (54% vs 41%;p=0.03) and peritoneal CD25+iNKT-cells (62% vs 37%;p=0.01).

Conclusion:We have previously identified a direct PD1:PDL1 ligation as a vital component to iNKT cell migration in response to sepsis. Our current data further supports this concept whereby iNKT cells which remained in the liver displayed decreased PDL1 and PDL2 ligation, potentially limiting their ability to migrate, whereas peritoneal iNKT cells which have migrated, displayed increased PDL1 and PDL2 expressions. Furthermore, the decline in CD25 expression potentially allowed for greater migration of iNKT-cells. This data further expands our understanding of the mechanism by which iNKT cells migrate into the peritoneal cavity in response to surgical sepsis. 

 

03.13 Reducing Hypercatecholaminemia After Trauma And Its Impact On The Erythropoietin Receptor

Posted on January 25, 2017

I. G. Alamo1, K. B. Kannan1, T. J. Loftus1, H. Ramos1, P. A. Efron1, A. M. Mohr1, A. M. Mohr1  1University Of Florida,Trauma,Gainesville, FL, USA

Introduction: Following severe injury, a norepinephrine (NE)-mediated hypercatecholamine state develops that is associated with dysfunctional erythroid maturation and manifests as a persistent injury-associated anemia. Previously, the reduction of the effects of NE, by propranolol (BB) or clonidine, has led to improvements in erythroid bone marrow function. The erythropoietin receptor (EPOr) is necessary for the maturation of early to late erythroid progenitor cells. In a clinically relevant rodent of lung contusion (LC)/hemorrhagic shock (HS)/chronic stress (CS), we hypothesize that both BB and clonidine improve erythroid maturation by modulating the expression of EPOr. 

Methods: Propranolol (BB) a non-selective beta adrenergic blocker, was given to competitively block NE binding following LCHS and LCHS/CS ± BB (10mg/kg) in male Sprague Dawley rodents. To centrally deplete NE release, clonidine, an alpha 2-agonist, was also given to rats following LCHS and LCHS/CS ± clonidine (75ug/kg). Animals underwent two hours of daily restraint stress until the day of sacrifice on day 7. Bone marrow EPOr mRNA and EPOr protein expression as well as bone marrow BFU-E and CFUE-E colony growth were assessed. Data was presented as mean±SD; *p<0.05 vs. untreated counterpart by t-test.

Results:Daily administration of BB following LCHS/CS improved bone marrow EPOr mRNA and EPOr protein expression by 94 and 96% (Figure). Similarly, the use of clonidine following LCHS/CS increased EPOr mRNA and EPOr protein expression by 90 and 96% (Figure). When BM EPOr expression was restored in the LCHS/CS+BB and LCHS/CS+clonidine groups, bone marrow BFU-E and CFU-E colony growth was similar to that of naïve animals (Figure). 

Conclusion:The addition of chronic stress to severe trauma negatively impacts bone marrow EPOr expression and erythroid colony growth. However, both daily propranolol and clonidine restore EPOr expression, which improves BFU-E and CFU-E colony growth.  Modulation of the hypercatecholaminemia state following severe trauma and chronic stress may provide a therapeutic alternative to improve erythroid maturation.  
 

03.12 Persistent Injury-Associated Anemia: The Role of the Bone Marrow Microenvironment

Posted on January 25, 2017

J. K. Millar1, K. B. Kannan2, T. J. Loftus2, I. G. Alamo2, J. Plazas3, P. A. Efron2, A. M. Mohr2  1University Of Florida,College Of Medicine,Gainesville, FL, USA 2University Of Florida,Department Of Surgery And Center For Sepsis And Critical Illness Research,Gainesville, FL, USA 3University Of Florida,Gainesville, FL, USA

Introduction: Following severe injury, a hypercatecholamine state develops that is associated with bone marrow dysfunction characterized by increased mobilization of hematopoietic progenitor cells to the peripheral blood and decreased growth of bone marrow progenitor cells. Bone marrow dysfunction clinically manifests as a persistent injury-associated anemia. Since conditions within the bone marrow microenvironment define the development of erythroid progenitor cells, we sought to determine the influence of lung contusion (LC), hemorrhagic shock (HS), and chronic stress (CS) on the hematopoietic cytokine response.

 

Methods: Bone marrow was obtained from male Sprague-Dawley rats (n=6/group) sacrificed seven days after lung contusion and hemorrhagic shock (LCHS) or LCHS followed by daily chronic restraint stress (LCHS/CS). End point polymerase chain reaction was performed for interleukin-1β (IL-1β), interleukin-10 (IL-10), stem cell factor (SCF), transforming growth factor-β (TGF-β), High Mobility Group Box-1 (HMGB-1), and B-cell lymphoma-extra large (Bcl-xL). Data were presented as mean ±standard deviation; *p<0.05 vs. naïve by t-test.  

 

Results: Seven days following LCHS and LCHS/CS, expression of hematopoietic cytokines (IL-1β, IL-10, SCF, and TGF-β) was decreased (Table). Seven days following LCHS and LCHS/CS, expression of HMGB-1, a mediator of granulocyte colony stimulating factor release, was significantly increased (Table). There were no statistically significant differences in cytokine expression between LCHS and LCHS/CS models. Bcl-xL was not affected by LCHS or LCHS/CS (naïve: 44 ±12, LCHS: 44 ±12, LCHS/CS: 37 ±1).     

 

Conclusions: The bone marrow microenvironment was significantly altered following severe trauma.  Hematopoietic cytokines were downregulated and the pro-inflammatory cytokine, HMGB-1, was upregulated.  Modulation of the bone marrow microenvironment may represent a therapeutic strategy following severe trauma to alleviate persistent injury-associated anemia.     

03.11 Alternative RNA Splicing in Critical Illness: Gene Expression Evidence

Posted on January 25, 2017

S. F. Monaghan1, K. J. Cygan2, J. Lomas-Neira1, C. Chun1, Y. Chen1, W. G. Cioffi1, W. G. Fairbrother2, A. Ayala1  1Brown University School Of Medicine,Surgery,Providence, RI, USA 2Brown University,Center For Computational Molecular Biology,Providence, RI, USA

Introduction: Previous work has focused on the regulation of transcription of DNA into RNA as it relates to critical illness. However, the next step in protein production, RNA splicing, has not received much attention. Over 90% of human genes with multiple exons have alternative splicing events. With such a high rate of variations from the transcribed gene to the protein, splicing must be under exquisite control; particularly when cellular resources are limited during critical illness. Here we hypothesize RNA splicing is altered in critical illness as evidenced by changes in the gene expression of RNA splicing protein. 

Methods: A murine model of acute respiratory distress syndrome (ARDS) was induced by subjecting mice to hemorrhagic shock followed by cecal ligation and puncture. Mice undergoing severe critical illness from this model were compared to sham controls. Blood and lung samples were collected and RNA was purified from the samples using the Globinclear (Ambion) and MasterPure RNA purification (Epicentre) kits. Next generation RNA sequencing was performed (Genewiz). Analysis of the RNA sequencing data was done using the STAR aligner and the GenomicAlignments package for R/Bioconductor. Genes were categorized as RNA splicing proteins using gene ontology (GO) terms. Alpha was set at 0.05 while adjusting for the number reads obtained.

Results:Gene expression in mice with induced critical illness (ARDS) were compared to sham controls. More genes in the blood (17%) had significantly different expression levels between the groups (critically ill mouse vs sham control) when compared to the genes in the lung tissue (4%, p=0.0001). 618 genes had significant changes in expression in both the blood and lung tissue. Using GO terms, 17 RNA splicing genes were significantly changed in the blood and only one gene was identified in the lung (see table, in order of amount of change). Of the 17 genes with significantly different expression 5 showed an increased in expression while 12 showed a decrease in expression. Although fewer genes identified had more expression, they had a cumulative 86 fold change in expression compared to a 46 fold change in expression of the 12 genes that decreased. The one gene identified in the lung showed small decrease in expression.  

Conclusion:Numerous genes involved in RNA splicing show significant changes in expression, particularly in the blood, in an animal model system of critical illness. Since resources are limited in critical illness, the alterations in the expression of these RNA splicing proteins suggests there will be a change in the RNA processing during times of stress. Future work will assess changes in alternative splicing in critical illness and attempt to understand this process. 

 

03.10 Hemorrhagic Shock Drives the Plasma Metabolome in Trauma

Posted on January 25, 2017

G. R. Nunns1, N. Clenenden4,7, H. B. Moore1, A. D’Alessandro4, E. Gonzalez1, M. Fragoso2, E. Peltz1, C. C. Silliman5,6, A. Sauaia1, A. Banerjee1, E. E. Moore1,2  1University Of Colorado Denver,Surgery,Aurora, CO, USA 2Denver Health Medical Center,Surgery,Aurora, CO, USA 4University Of Colorado Denver,Department Of Biochemistry And Molecular Genetics,Aurora, CO, USA 5University Of Colorado Denver,Pediatrics,Aurora, CO, USA 6Bonfils Blood Center,Research Laboratory,Denver, CO, USA 7University Of Colorado Denver,Anesthesiology,Aurora, CO, USA

Introduction:

Succinate is a citric acid cycle intermediate that accumulates during ischemia, similar to lactate. Succinate has also been linked to reperfusion injury, platelet dysfunction and immunologic modulation. Mass spectrometry (MS) has recently provided detailed descriptions of the trauma plasma metabolome, elucidating many potential pathologic mediators including succinate. To guide therapeutics the relative contribution of isolated tissue injury (TI), isolated hemorrhagic shock (HS) and organ specific metabolopathy must be elucidated. We hypothesize that TI and HS will produce distinct metabolic signatures in plasma and that organ specific ischemia will delineate the dominant source of pathologic metabolites.

Methods:

A porcine model was used to determine the role of TI vs HS with 4 experimental groups: (1) sham (anesthesia, arterial line and tracheostomy), (2) TI (femur fracture/muscle crush, laparotomy/bowel crush, thoracotomy), (3) HS (hemorrhage to mean arterial pressure of 25mm Hg for 30 minutes, 30 minutes of descending thoracic aortic cross clamp followed by reperfusion), (4) combined TI + HS. Blood was collected at baseline, 30, 60 and 90 minutes after TI, for HS it was collected at baseline, 30, 60 minutes and 5 minutes after reperfusion. Organ specific ischemia was induced without TI or HS by arterial inflow occlusion (30 min) followed by reperfusion (spleen, kidney, liver and small bowel). Blood was drawn from the femoral vein and the venous outflow of each organ at baseline, 15 and 30 minutes of ischemia and after reperfusion. Plasma samples were analyzed by MS and interpreted using principal component analysis.

Results:

Lactate and succinate are not increased in plasma following multiple TI over sham levels. HS and TI + HS each increase plasma lactate compared to sham, 1.6 and 1.5 fold respectively. HS and TI + HS each produce a 3.8 fold increase in succinate compared to sham. All organs demonstrate uniform accumulation of lactate in response to ischemia, whereas succinate is increased disproportionately in the liver. At 30 minutes of ischemia, succinate levels in the liver are 6.5 x spleen, 8.2 x small bowel, 6 x renal and 2 x systemic levels.

Conclusion:

Multiple TI alone is insufficient to elevate plasma lactate or succinate. HS and HS+TI generate marked increases in succinate and lactate. Hemorrhagic shock associated hypoperfusion is the dominant stimuli for metabolic derangement associated with trauma. The liver is the main source of succinate production in response to ischemia. These data suggest that during trauma hepatic hypoperfusion is the primary source of plasma succinate with subsequent inflammatory, coagulopathy and immunomodulatory consequences.

03.09 Prospective Validation of the Sepsis MetaScore for Diagnosis of Infections Post Traumatic Injury

Posted on January 25, 2017

T. E. Sweeney1, A. Mansur2, J. Hinz2, P. Khatri1  1Stanford University,Institute Of Immunity, Transplantation And Infections,Palo Alto, CA, USA 2University Of Gottingen,Dept. Of Anesthesiology,Gottingen, LOWER SAXONY, Germany

Introduction:     

Severe traumatic injuries lead to immune dysregulation and a predisposal to hospital-acquired infections. While rapid diagnosis and treatment of infections improves outcomes, it is often difficult to clinically distinguish the signs and symptoms of infection from normal inflammation after major traumatic injury. We recently showed that our a set of 11 host genes (‘Sepsis MetaScore’, SMS) can diagnose infections after traumatic injury with high accuracy. Here we report interim results of a prospective cohort study designed to test the diagnostic power for infections of the SMS after blunt traumatic injury.

   

Methods:    

This is a prospective, non-blinded, cohort study of adults admitted to the University of Gottingen trauma intensive care unit (ICU) with severe blunt traumatic injury. Exclusion criteria were isolated head or spinal injuries. Blood was drawn at admission and every 48 hours thereafter for gene expression profiling. Procalcitonin was also drawn for comparison. To test diagnostic power, we compared mean scores taken between days 2-4 (the time of all infections) between infected and non-infected patients. This study is registered under ClinicalTrials.gov identifier NCT02656459, and was approved by both institutions’ ethics committees.

   

Results:    

At the interim, 16 patients were enrolled; with a mean age of 49 years (range 19-76), mean length of stay of 11.6 days (range 1-40), and of which 10 were male. Seven patients were determined to have infections during their ICU stay; all were diagnosed within days 2-4 post-injury. Comparing these patients to the four non-infected time-matched patients (five were discharged on days 1-2), the SMS showed an AUC of 0.82 for determination of infection.   Procalcitonin, when using the same procedure to test diagnostic power, showed an AUC of 0.44 for diagnosis of infections.

   

Conclusions:    

This interim report suggests that the SMS may have clinical utility for diagnosis of infections compared to other biomarkers such as procalcitonin. The present cohort has an unusually high percentage of patients with infections, but this is likely due to the very small number of enrolled participants to date.  We expect full recruitment within 6 months, at which time results in the entire cohort will be reported. 

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