46.06 Preoperative Anemia Predicts Poor Outcomes for Non-Metastatic RCC Patients with Venous Thrombus

Posted on December 22, 2014

K. E. Zorn1, W. P. Christensen1, V. Margulis3, T. M. Bauman1, C. G. Wood2, E. J. Abel1  1University Of Wisconsin,Urology,Madison, WI, USA 2University Of Texas MD Anderson Cancer Center,Urology,Houston, TX, USA 3University Of Texas Southwestern Medical Center,Urology,Dallas, TX, USA

Introduction: In approximately 10% of renal cell carcinoma (RCC) patients, tumor extends beyond the kidney into the venous system, increasing the risk of postoperative recurrence.  Anemia is known to predict worse survival in patients with metastatic RCC, but the prognostic ability of preoperative anemia has not been studied in non-metastatic high risk RCC patients. The purpose of this study was to evaluate whether patients with preoperative anemia had worse postoperative cancer outcomes using a multi-institutional contemporary series of non-metastatic RCC patients with venous involvement.

Methods: A comprehensive review of clinical and pathological risk factors was performed for consecutive RCC patients with thrombus treated between 2000 and 2012 at three separate institutions. Univariate and multivariate Cox proportional hazards analysis was used to evaluate association of anemia or other common risk factors for cancer specific survival (CSS) and recurrence free survival (RFS).

Results:  A total of 470 non-metastatic patients were treated surgically for RCC with venous thrombus invasion from 2000-2012 at participating centers. Thrombus extended into the renal vein in 259 (55.1%) patients, into the IVC <2cm in 65 (13.8%) patients, into the IVC >2cm but below hepatic veins in 81 (17.2%) patients, and within the IVC above the hepatic veins in 65 (13.8%) patients.

Median follow-up was 28.4 months (IQR 12.2-54.9) and 188 (40.0%) patients developed recurrent disease within the follow-up period. Of patients that developed recurrent disease, initial site of recurrent disease was solitary for 128 (68.1%) patients while 60 (31.9%) patients presented initially with multiple sites of metastatic disease.  Preoperative hemoglobin was independently predictive of recurrence with a hazard ratio of 1.727 (95% CI: 1.251-2.385, p=0.0009). Other independent predictors of recurrence included BMI ≤20, perinephric fat invasion by tumor, non-clear cell histology and tumor width.

Median CSS was 136.6 (IQR 43.8-NR) months and 112 (23.8%) patients died of RCC within the follow-up period. BMI, systemic symptoms, IVC thrombus level above the hepatic veins, and estimated blood loss were associated with CSS on univariate analysis. On multivariate analysis, only preoperative hemoglobin less than lower limit of normal (HR 2.051, p=0.02) and tumor width per cm (HR 1.078, p=0.02) were independently predictive of CSS.

Conclusion: In non-metastatic RCC patients with venous invasion, preoperative anemia and tumor diameter were independent predictors of recurrence and cancer mortality.

46.07 Right vs. Left Colectomy Outcomes in Colon Cancer Patients

Posted on December 22, 2014

H. Aziz1, M. R. Torres1, V. Nfonsam1  1University Of Arizona,Tucson, AZ, USA

Introduction:

Optimization of surgical outcomes after colectomy continues to be actively studied, but most studies group right-sided and left-sided colectomies together. The aim of our study was determine whether the complication rate differs between right-sided and left-sided colectomies for cancer.

Methods:

We identified patients who underwent laparoscopic colectomy for colon cancer in the 2005-2010 American College of Surgeons National Surgical Quality Improvement Program database and stratified cases by right and left side. The two groups were matched using propensity score matching for demographics, previous abdominal surgery, pre-operative chemotherapy and radiotherapy, and pre-operative laboratory data. Outcome measures were: 30-day mortality and morbidity.

Results:

We identified 2,512 patients who underwent elective laparoscopic colectomy for right-sided or left-sided colon cancer in the database. The two groups were similar in demographics, and pre-operative characteristics. There was no difference in overall morbidity (15% vs 15.7%; p-0.8) or 30 day mortality (1.5% vs 1.5%; p-0.9) between the two groups. Sub-analysis revealed higher surgical site infection rates (9% vs 6%; p-0.04), higher incidence of ureteral injury(0.6% vs 0.49%; p-0.04), higher conversion rate to open colectomy (51% vs. 30%; p-0.01) and a longer hospital length of stay (10.5+/-4 vs. 7.1+/-1.3 days; p-0.02) in patients undergoing laparoscopic left colectomy.

Conclusion:

Our study highlights the difference in complications between right-sided and left-sided colectomies for cancer. Further research on outcomes after colectomy should incorporate right vs left side colon resection as a potential preoperative risk factor.

46.08 Impact of laterality on perioperative morbidity and mortality following major hepatic resection.

Posted on December 22, 2014

S. C. Pawar1, A. Robinson1, R. S. Chamberlain1  1Saint Barnabas Medical Center,Surgery,Livingston, NJ, USA

Introduction: Anatomic variations and technical nuances involved in performing a left sided hepatic resection versus a right-sided hepatic resection make the procedures distinctly different. This study examined the relationship between laterality and perioperative outcomes in hepatic hemi-resection and aimed to determine whether specific complications are associated with the types of hepatic resection, and if so whether unique complication profiles exist for left and/or right hemiresections.

Methods: American College of Surgeons National Surgical Quality Improvement Program (ACS-NSQIP) was used to identify patients who underwent elective open hepatectomies between 2002 and 2012. Demographics, 60 perioperative risk factors, and 32 postoperative complications were analysed using Multivariate analyses. 

Results: Among 5,355 patients, 2,614 underwent right hepatic resection, 1,439 left hepatic resection, and 1,302 extended hepatic resection. The median age was 60 years; with a male to female ratio of 1:1 and 65 % of patients had an ASA score of 3 to 4. Among patients undergoing open elective hepatectomies 44 % had disseminated cancer and 22 % had received chemotherapy prior to surgery. The most prevalent co-morbidity was hypertension (46%).The three groups were similar with regards to preoperative comorbidities.Overall 30-day mortality rate was 3.3% (173/5,243) and morbidity was 50.5%. The 30 day mortality rate was significantly higher among trisegmentectomy and right hepatectomy patients relative to left hepatectomies (4.8% and 3.6 % Vs 1.5%).  Patients with right sided hepatic resections had a higher rate of morbidity in terms of number of cases with 5 or more complications (4.6% vs. 2%; p < 0.001), and overall morbidity (51% vs. 41%, p < 0.001). Though superficial site infection (SSI) rates were similar for both procedures (5% vs. 4.6 %, p < 0.04), right hepatic resections had a higher rate of organ/space SSI (7.3% vs. 6%, p < 0.001). Left sided hepatic resection had lower rates of blood transfusions (28.6% vs. 36%, p < 0.0001); lower respiratory complications (3.5% vs 5%, p < 0.001), lower renal complications (0.8% vs. 1.6%, p < 0.001) and a shorter hospital stay (6 vs 7, p < 0.05).

 

Conclusion: Analysis of the NSQIP perioperative outcomes data confirms that extended hepatectomies (trisegmentectomy) are associated with the highest risk for mortality and morbidity.  Right hepatic lobectomy is associated with a significantly higher incidence of post-operative complications than left hepatic lobectomy, most notably with regards to intraoperative/postoperative blood transfusions, biliary leaks, cardiac complications, sepsis and 30 day operative mortality.

 

 

 

46.09 Aggressive Papillary Thyroid Microcarcinoma: Population based study from SEER database (1989-2011).

Posted on December 22, 2014

S. C. Pawar1, R. S. Chamberlain1  1Saint Barnabas Medical Center,Surgery,Livingston, NJ, USA

Introduction: Papillary thyroid microcarcinoma (PTMC) usually follows an indolent course and is considered a low risk tumor.  However recent studies have identified several cases of PTMC with locoregional recurrences in the neck and with distant metastases.  It is imperative that the clinicians have a better understanding of PTMC and specifically characteristics of PTMC with aggressive behavior.

Methods: The Surveillance, Epidemiology, and End Results (SEER) program was queried for patients diagnosed with PTMC between 1989 and 2011. Age, gender, tumor size, lesion type, extent of disease, stage, surgery, and mortality were analyzed for papillary thyroid micro carcinomas (≤ 1cm) and compared to papillary tumors of size (> 1 cm).  Clinical and pathological characteristics of patients with PTMC were compared to PTMC patients who died of cancer specific death using multivariate analyses. 

Results:Among 53,429 patients diagnosed with Papillary thyroid carcinoma, 36,093(67%) were diagnosed with PTC tumors of size (> 1 cm) and 17,336 (32%) were PTMC (≤ 1 cm). The median age of the entire cohort was 48 yr (PTMC (49 yr) and PTC (48 yr)), however patients with PTMC with aggressive behavior were significantly old (64 yr). Females dominated the study cohort for both PTC (> 1 cm) and PTMC (≤ 1 cm) (73% vs 81% respectively). PTMCs were more likely to present as multifocal tumors compared to PTC (> 1 cm) (14% Vs 8% respectively). 82% of PTMCs had localized disease at the time of diagnosis, 17% had regional spread, and 1% had distant metastasis. Multivariate analysis identified extrathyroidal invasion, multifocality, male gender, Asian race, and tumors with regional and distant metastasis at diagnosis as significant independent predictors of mortality in PTMC patients.  Patients with PTMC who died were more male (Odds ratio [OR], 4.34; p < 0.0001), of Asian ethnicity (OR, 1.35; p < 0.005), and elderly (OR, 9.77; p < 0.001). 

Conclusion:Although most PTMCs are considered low risk, up to 17% of patients with PTMC exhibit aggressive behavior in regards to extrathyroidal invasion, multifocality, regional spread, distant metastasis at diagnosis, and may benefit from a more radical therapeutic approach. PTMCs with aggressive behavior are more common among male gender, elderly age group, and Asian ethnicity.

 

 

46.10 Adjuvant Chemotherapy Attenuated the Impacts of Perineural Invasion in Stage III Colorectal Cancer

Posted on December 22, 2014

T. Suzuki1, K. Suwa1, K. Hanyu1, Y. Mitsuyama1, K. Eto1, M. Ogawa1, T. Okamoto1, T. Fujita1, M. Ikegami2, K. Yanaga1  1The Jikei University School Of Medicine,Department Of Surgery,Tokyo, , Japan 2The Jikei University School Of Medicine,Department Of Pathology,Tokyo, , Japan

Introduction: Perineural invasion (PNI) is associated with decreased survival in several malignancies, but no robust evidence has been documented in colorectal cancer (CRC). The aim of the present study was to evaluate the association of PNI and outcomes of patients after colorectal resection for CRC, focusing on the impact of adjuvant chemotherapy on survival rates of patients with PNI.

Methods: We retrospectively reviewed 224 consecutive patients who underwent surgery for Stage I to IV CRC between January 1999 and December 2004. The presence or absence of PNI of the tumor was determined by experienced pathologists in our institution. Overall and disease-free survival rates were estimated using the Kaplan-Meier method, and intergroup differences in survival curves were tested with the log-rank test. To evaluate the correlation between PNI and survival, PNI was entered into a Cox proportional hazards model as an independent variable.

Results: PNI was detected in 63 of the 224 patients (28%) and positively correlated with lymphatic invasion (P = 0.003), venous invasion (P = 0.006), lymph node involvement (P = 0.002), size of tumor (P = 0.019), postoperative chemotherapy (P = 0.023), and incidence of metastasis or recurrence (P < 0.0001). The 5-year disease-free survival rate was 1.6-fold in patients with PNI-negative tumors as compared to PNI-positive tumors (66% vs 44%, respectively; P < 0.001). The 5-year overall survival rate was 71% for PNI-negative tumors, which was significantly higher than 40% for PNI-positive tumors (P < 0.001). A multiple regression analysis revealed that PNI was a strong prognostic factor for overall survival. In a subset analysis of patients with stage III CRC, adverse effects of PNI on survival were attenuated by adjuvant chemotherapy, but adjuvant chemotherapy did not completely reverse the effects of PNI.

Conclusion:PNI was associated with poor prognostic markers such as lymphatic invasion and lymph node involvement, and predicted worse survival in patients with stage III CRC. Adverse effects of PNI on survival were attenuated by adjuvant chemotherapy. Intensive chemotherapy and strict surveillance seem be warranted in patients with CRC of stage III with PNI.

 

45.01 The Impact of Age and Frailty on Surgical Outcomes After Ileal Pouch-Anal Anastomosis

Posted on December 22, 2014

J. N. Cohan1,3, P. Bacchetti2, M. G. Varma1, E. Finlayson1,3  1University Of California – San Francisco,Department Of Surgery,San Francisco, CA, USA 2University Of California – San Francisco,Department Of Epidemiology And Biostatistics,San Francisco, CA, USA 3University Of California – San Francisco,Institute For Health Policy Studies,San Francisco, CA, USA

Introduction:  Historically, older patients with ulcerative colitis were not considered candidates for ileal pouch-anal anastomosis (IPAA).  However, evidence from several single-center studies suggests that IPAA can be performed in selected older patients with surgical outcomes similar to those in younger patients.  In order to evaluate whether this finding is generalizable, we used a national database including patient data from 374 US hospitals to examine outcomes after IPAA.   

Methods:  Using the American College of Surgeons National Surgery Quality Improvement Program (ACS-NSQIP) database, we identified all patients with ulcerative colitis who underwent total proctocolectomy or completion proctectomy with IPAA from 2005-2012.  We examined the associations of age and frailty trait count with length of stay and major surgical complication count using multivariate linear and negative binomial regression.  In both models, we adjusted for important procedure and patient characteristics (see table footnote).  The frailty trait count includes frailty traits present in the NSQIP database that are frequently used in frailty indices: diabetes, hypertension, congestive heart failure, functional status, and ≥10% body weight loss over six months prior to surgery.

Results:  IPAA was performed in 2493 patients with ulcerative colitis.  Thirty-day mortality was 0.002% (N=7).  Patients older than age 50 were more likely to have diabetes (20.4% vs 2.6%), hypertension (32.9% vs 8.0%), blood transfusion (6.8% vs 3.6%), ≥1 frailty trait (39.7% vs 14.2%), and American Society of Anesthesiologists class 3-4 (35.4% vs 17.6%) than younger patients (all p≤0.001).  The majority of patients had no serious postoperative complications (age <50: 79.5%, age 50-59: 80.4%, age ≥60: 79.1%).  After adjusting for important procedure and patient characteristics, age ≥60 was associated with a minor (15%) increase in number of complications (p=0.40) and with a 0.8 day increase in length of stay (p=0.036).  The average adjusted length of stay was 7.4 days in patients age <50, 7.7 days in patients age 50-59, and 8.2 days in patients age ≥ 60.  Frailty trait count ≥1 was associated with small increases that did not reach statistical significance (Table).  

Conclusion:  Among patients with ulcerative colitis undergoing IPAA in the NSQIP database, advanced age and frailty were associated with only slightly increased postoperative complications, but the effect of age ≥60 on length of stay was close to a full day.  These findings suggest that IPAA may be a safe surgical option for older patients with ulcerative colitis.  Future work should evaluate functional outcomes in older patients across a spectrum of hospitals.

45.02 Post-Operative Troponin Testing and Cardiac Interventions among Patients with Coronary Stents

Posted on December 22, 2014

C. N. Holcomb1, L. A. Graham2, T. M. Maddox4, K. M. Itani3, M. T. Hawn1,2  1University Of Alabama At Birmingham,Department Of Gastrointestinal Surgery,Birmingham, AL, USA 2Birmingham VA Medical Center,The Center For Surgical, Medical Acute Care Research And Transitions (C-SMART,Birmingham, AL, USA 3Boston University And Harvard Medical School,Department Of Surgery,Boston, MA, USA 4University Of Colorado School Of Medicine,Cardiology,Denver, COLORADO, USA

Introduction: Patients with coronary stents undergoing non-cardiac surgery are at an increased risk of adverse cardiac events.  Knowledge of this risk may increase the number of troponin tests and influence interventions performed in the early post-operative period. 
 

Methods: The VA Patient Treatment File was used to identify coronary stents implanted in the VA system from October 1, 1999 through September 30, 2010. Patients undergoing non-cardiac surgery within 24 months of coronary stent were identified using VA Surgical Quality Improvement Program data. Each stented patient was matched to two non-stented patients undergoing non-cardiac surgery. Matching variables included age, race, surgical specialty, fiscal year of the operation, work RVU, and components of the revised cardiac risk index.  Cardiac troponin (cTroponin) levels drawn in the first 72 hours following surgery were assessed and a cTroponin >0.04ng/ml was considered a positive test.  The outcomes of interest were MI, cardiac catheterization, and death.  Bivariate frequencies were compared using chi-square test. 

Results: Over the 11 year study period, 5,855 stented patients and 11,371 non-stented patients with similar cardiac risk underwent an inpatient surgery.  Within the first 72 hours of surgery, patients with coronary stents had higher rates of cTroponin testing (25.5% vs 18.7%, p<0.001) with more positive tests (56.4% vs. 52.9%, p=0.04) compared to non-stented patients.  Among those tested, stented patients experienced higher rates of MI (9.8% vs 6.3%, p<0.001).  Myocardial infarctions were diagnosed at lower maximum cTroponin values in stented patients versus non-stented patients (Figure).   Following a diagnosis of MI, patients with stents were more likely to undergo cardiac catheterization (31.2% vs 19.9%, p<0.01) with no difference in cardiac mortality (17.2% vs 20.9%, p=0.34) between the two cohorts. 

Conclusions: Post-operative cTroponin testing among patients with coronary stents was associated with higher observed MI rates and cardiac catheterizations at lower maximum troponin levels but no observed difference in mortality following MI compared to non-stented patients.  Further studies should be done to determine factors predicting improved outcomes with cardiac interventions following surgery. 

 

 

 

 

45.03 Independent Predictors of Postoperative Ileus Development

Posted on December 22, 2014

M. Murphy4, S. Tevis4, G. Kennedy4  4University Of Wisconsin,School of Medicine And Public Health, Department Of Surgery,Madison, WI, USA

Introduction:  Postoperative ileus has a significant impact on patient wellbeing, and with a 15% incidence in colectomy patients, costs US hospitals more than 750 million dollars a year. While some causative mechanisms have been identified, little is known about what places patients at risk for ileus. We sought to characterize preoperative and intraoperative predictors of ileus in colectomy patients.

Methods:  Patients who underwent elective surgery between 2011-2012 were identified from the colectomy-specific ACS-NSQIP database. We performed descriptive statistics and evaluated demographics, comorbidities, health indicators, preoperative treatments, and operative characteristics as independent risk factors for ileus using multivariate analyses.  All analyses were performed with SPSS version 22.  A p-value < 0.05 was considered significant for the purposes of this study.

Results: We included 9734 patients in this analysis. Of the patients in this study, 1364 (14%) were found to have a postoperative ileus.  Patients who developed an ileus were more likely to develop any postoperative complication (26.4% v 9.9%, p < 0.001). In addition, patients who suffer post-operative ileus are significantly more likely to be readmitted (20% vs 13%, p < 0.001) and more likely to require reoperation (37% vs 13%, p < 0.001) compared with patients without ileus. Independent risk factors for ileus included demographic and preoperative factors including older patient age (OR 1.95, 95% CI 1.58-2.42), male gender (OR 1.41, 95% CI 1.24-1.60), obesity (OR 1.32, 95% CI 1.12-1.56), preoperative chemotherapy (OR 1.53, 95% CI 1.21-1.94), preoperative ascites (OR 1.86, 95% CI 1.07-3.22), preoperative sepsis (OR 1.74, 95% CI 1.32-2.29), and smoking (OR 1.20, 95% CI 1.02-1.41).  Intraoperative and perioperative factors included lack of oral antibiotics (OR 1.29, 95% CI 1.10-1.50), lack of mechanical bowel preparation (OR 1.15, 95% CI 1.01-1.32), open approach (OR 2.04, 95% CI 1.77-2.36), and long operation times (OR 1.63, 95% CI 1.36-1.96).

Conclusion: We identified a number of modifiable risk factors for development of ileus including smoking, weight loss, preoperative oral antibiotics, mechanical bowel preparation, and surgical approach.  Preoperative modification of risk factors for ileus may not only improve patients’ quality of life, but may also influence outcomes such as postoperative complications, readmission, and reoperation.

 

44.10 Combined Treatment with Hypothermia and Valproate Upregulates Survival Pathway in Hemorrhagic Shock

Posted on December 22, 2014

T. Bambakidis1, S. E. Dekker1, B. Liu1, J. Maxwell1, K. T. Chtraklin1, D. Linzel1, W. He1, Z. Chang1, Y. Li1, H. B. Alam1  1University Of Michigan,Surgery,Ann Arbor, MI, USA

Introduction: Therapeutic hypothermia and valproic acid (VPA, a histone deacetylase inhibitor) have independently been shown to be protective in models of trauma and hemorrhagic shock, but require logistically challenging doses to be effective. Theoretically, combined treatment may further enhance effectiveness, allowing us to use lower doses of each modality. We previously confirmed the beneficial effects of combined therapy in an in vitro model of hypoxic neuronal cells. The aim of this study was to determine whether combined Hypothermia+VPA treatment offers better cytoprotection compared to individual treatments in an in vivo hemorrhage model.

Methods: Male Sprague-Dawley rats were subjected to 40% volume-controlled hemorrhage, kept in shock for 30 minutes, and assigned to one of the following treatment groups: normothermia (36-37°C), Hypothermia (30±2°C), normothermia+VPA (300mg/kg), and Hypothermia+VPA (n=5/group). After three hours of observation, the animals were sacrificed, liver tissue was harvested and subjected to whole cell lysis, and levels of key proteins in the pro-survival Akt pathway were measured using Western Blot.

Results: Levels of pro-apoptotic protein (cleaved caspase-3) were significantly lower, and pro-survival proteins (Bcl-2 and β-catenin) significantly higher in the Hypothermia+VPA group compared to the individual treatments (P<0.05) (Figure). The level of the downstream protein Phospho-GSK-3β was significantly higher in the hypothermia and combined treatment groups (P<0.001).

Conclusion: This is the first in-vivo study to demonstrate that combined treatment with VPA and hypothermia offers better cytoprotection than these treatments given independently.

 

42.02 Identification of Novel Class II-Promiscuous HER3-Derived CD4+ Peptides for Cancer Immunotherapy

Posted on December 22, 2014

J. Datta1, S. Xu1, J. H. Terhune1, L. Lowenfeld1, C. Rosemblit1, E. Berk1, E. Fitzpatrick1, R. E. Roses1, B. J. Czerniecki1  1University Of Pennsylvania,Surgery,Philadelphia, PA, USA

Introduction: HER3 overexpression is associated with resistance to targeted therapies (e.g. BRAF inhibitor in melanoma, trastuzumab in breast cancer) and a poor oncologic prognosis. Since existing anti-HER3 strategies – antibodies inhibiting HER2-HER3 dimerization – are static but not lytic, cellular therapies targeting HER3 are needed. CD4 T-helper type 1 (Th1) cells secrete IFN-γ/TNF-α, inducing tumor apoptosis. Using the extracellular domain (ECD) of HER3 as a candidate tumor antigen, we sought to identify immunogenic Class II-promiscuous HER3 CD4 peptides that generate anti-HER3 Th1 immunity for inclusion in cancer vaccines.

Method: A library comprising 123 overlapping 15 aa-long peptides was generated from HER3-ECD. Autologous monocyte-derived DCs from patients were matured to a type 1-polarized phenotype using IFN-γ/LPS (IL-12 secreting DC1), pulsed with HER3-ECD, and co-cultured with purified CD4 T-cells. After 10 days, sensitized CD4 T-cells were restimulated against immature DCs (iDC) pulsed with HER3 peptide clusters or irrelevant CD4 control. Th1 responses, measured by IFN-γ ELISA, were considered antigen-specific if IFN-γ production was at least twice that of control groups.

Results: Th1 sensitization was performed in 6 breast cancer patients with known anti-HER3 reactivity in order to identify single immunogenic HER3 CD4 epitopes. HER3 ECD-specific CD4 Th1 were sequentially restimulated against 10-peptide clusters, narrowed to 3-peptide clusters, and subsequently to single immunogenic HER3 peptides. Four immunogenic peptides (HER351-75, HER3402-417, HER3417-432, HER3451-465) were reproducibly identified based on two-fold increase in IFN-γ production (Fig). These peptides were promiscuous across HLA-DR, DP, and DQ subtypes. When Th1 cells from 4 non-HER3 reactive donors were sensitized using DC1s pulsed with the four identified HER3 peptides, and subsequently challenged to recognize HER3 ECD-pulsed iDCs, all donors demonstrated successful sensitization not only to individual immunogenic HER3 peptides, but also recognized native HER3-ECD.

Conclusion: DC1 pulsed with an overlapping HER3-derived peptide library identified promiscuous class II peptides for CD4 Th1 vaccine development. Immunogenic HER3 CD4 peptides abrogated anti-HER3 immune self-tolerance. Utilizing these peptides in vaccine construction warrants investigation in patients harboring HER3-overexpressing cancers, and would represent the first cellular therapy targeting IFN-γ-secreting CD4 Th1 cells against HER3-ECD. These data also reveal a novel strategy to rapidly and reproducibly identify class II-promiscuous immunogenic CD4 epitopes from any self-tumor antigen for immunotherapy via a DC1-Th1 platform.

42.03 LIGHT Over-Expression in Colorectal Cancer Stimulates Lymphocyte Proliferation and In-vivo Tumor Rejection

Posted on December 22, 2014

K. H. Pardiwala1, G. Qiao1, B. Prabhakar1, A. V. Maker1  1University Of Illinois At Chicago,Chicago, IL, USA

Introduction:
T-cell infiltration in primary and metastatic colorectal cancer tumors is associated with improved patient survival. LIGHT (TNFSF14) is an immunomodulator that has been shown to increase T-cell trafficking to tumors that we have previously shown to be associated with improved patient survival when expressed at high levels in metastatic colon cancer deposits. However, the mechanism of LIGHT-associated survival benefit is not completely understood. 

Methods:
Murine colorectal cancer cells (CT26) were designed to stabily over-express LIGHT by lentiviral transduction.   Cell morphology was assessed by light microscopy, survival was evaluated with Annexin V/propridium idodide staining, proliferation was determined using the MTS colorimetric assay, and 3D cell migration assays were performed in collagen gel matrix and analyzed on Image J software.  Natural Killer (NK) and T-cells were obtained from the spleens of Balb/C mice and cultured with LIGHT+CT26 syngeneic colon cancer cells in various E:T ratios. Cell proliferation was determined with CFSE staining.  In vivo models were created by tumor cell flank injections of LIGHT+CT26 or wtCT26 cells into syngenic, immunocompetent Balb/C mice. 

Results:

LIGHT+CT26 colon cancer cells demonstrated similar morphology, survival, apoptosis, and migration compared to wtCT26 cells. T-cell proliferation was increased in the presence of LIGHT+CT26 compared to control CT26 cells(67.3%vs. 50.3% ). Similarly, when compared to wtCT26, LIGHT over-expressing CT26 cells stimulated natural-killer (NK) cell proliferation nearly three-fold (24% vs. 8 %).  Eight of eight  mice inoculated with wtCT26 formed palpable tumors with an average volume of 2458 ± 572 mm3  while 0 of 8 mice inoculated with CT26LIGHT developed tumors (p= 0.0026). (figure)

Conclusion:
Over-expression of the immunostimulatory cytokine LIGHT in colorectal cancer cells stimulates lymphocyte proliferation and inhibits in-vivo tumor growth. Further studies to determine the mechanism of LIGHT-induced anti-tumor responses and to determine its potential as an immunotherapeutic agent in colorectal cancer are warranted.

42.04 The Role of Transcription Factor ROR-γt in the Tumor-Driven Induction of Th17 and Treg cells

Posted on December 22, 2014

S. Downs-Canner1, N. Obermajer1, R. Ravindranathan1, K. Odunsi3, R. Edwards1, P. Kalinski1, D. Bartlett1  1University Of Pittsburgh,Surgical Oncology,Pittsburgh, PA, USA 3Roswell Park Cancer Institute,Gynecologic Oncology,Buffalo, NY, USA

Introduction:  To evaluate the role of the Th17 cells in the course of tumor development, we examined the interplay of Th17 and Treg cells.  Tumor-infiltrating Th17 cells constitute a prominent part of the cancer microenvironment and are a potent inflammatory subset of T lymphocytes characterized by their signature cytokine IL-17A and their master transcription factor RORγt.  While Th17 cells from human or murine tumors appear to favor the early growth of a variety of malignancies by promoting angiogenesis or suppressing tumor immunity, limited studies imply that at later time points, adoptively transferred Th17 cells could mediate durable antitumor responses.

Methods:   We used human ascites material to define key subsets of Th17 cells present in ovarian cancer patients. For in depth in vivo mechanistic studies, we used transplantable (ID8-MOSEC and MC-38 cells) and inducible (azoxymethane ± dextran sodium sulfate) tumor models to define the key features of Th17 cell plasticity in the setting of tumor. Further, we evaluated Th17 plasticity in vivo in IL-17aCreRosaeYFP reporter mice which enable fate mapping of cells with highly activated IL-17A, regardless of their IL-17A production at the time of analysis.  We determined the impact of Th17 cell plasticity on tumor progression in B6.129P2(Cg)-Rorctm2Litt/J (Rorγ -/-) mice (characterized by RORγ t deficient T cells).

Results:  Our data demonstrate increased infiltration of IL17+ and FoxP3+ CD4+ T cells in the human cancer microenvironment, with the presence of a distinct IL17+FoxP3+ subset, and a significant correlation between tumor-associated Th17 and Treg cells in cancer patients. The data in IL-17aCreRosaeYFP reporter mice indicate plasticity between Th17 and Treg cells. Moreover, RORγ t is required not only for Th17 development, but also for effective Treg cell induction. While FoxP3 expression was not induced in ROR-γ  -/- cells, TGF-b1 strongly induced FoxP3 expression in control cells. Further, tumor bearing ROR-γ  -/- mice showed significantly less FoxP3+ Treg cells, but higher IFNg+ T cells compared to wild type animals.

Conclusion:  Tumor development fosters an imbalance in the Th17/Treg ratio, driven by plasticity between the two cell types, both regulated in a RORγt dependent manner. The possibility to control Th17/Treg plasticity and prioritize Th1-like immune responses through manipulation of RORγt offers a novel approach to overcome tumor-associated immunosuppression and enhance clinical effectiveness of anticancer immunotherapy by improving local antitumor activities of CTLs, Th1- and NK cells and preventing dominance and persistence of Treg – governed tumor immunosuppression.

 

42.05 Myeloid Cell PD-1 Expression Drives Acute Inflammation in a Model of Obstructive Jaundice

Posted on December 22, 2014

M. Thorn1, R. A. Burga1, G. Point1, N. J. Espat1,2, S. C. Katz1,2  1Roger Williams Medical Center,Department Of Surgery,Providence, RI, USA 2Boston University,Department Of Surgery,Boston, MA, USA

Introduction: Obstructive jaundice (ObJ) is a life-threatening clinical condition that results in liver inflammation and impaired immunity.  The immunological mechanisms for liver injury due to ObJ are incompletely understood. Programmed death-1 (PD-1, CD279) is an immunoinhibitory molecule expressed by T cells and classically results in T cell exhaustion. Recent reports suggest that PD-1 may have surprising pro-inflammatory effects when activated on myeloid cells (MC) by programmed death ligand-1 (PDL-1). We hypothesized that acute inflammation due to ObJ is driven by PD-1 activation on intrahepatic polymorphonuclear cells (PMN) and Kupffer cells (KC). 

Methods: Bile duct ligation (BDL) or sham operations were performed in C57Bl/6 mice and liver leukocytes isolated at 3, 7, or 10 days. In vivo anti-PD-1 injections (200 µg/mouse) were given intraperitoneally. Liver lysate cytokines were evaluated by ELISA and cell phenotype by flow cytometry. KC were defined as CD11b+CD11c-F4/80+, PMN as CD11b+Gr1+, and MC as CD11b+.

Results: Intrahepatic PD-1 expression was not limited to liver T cells, but also expressed at significant levels by MC, including KC and PMN as early as 3 days following BDL. At baseline, 6.9% of liver PMN and 4.1% of KC expressed PD-1. Following 7 days of ObJ, PD-1 expression increased to 18.5% and 24.3% among liver PMN (p=0.014) and KC (p=0.004). PD-1 expression levels declined slightly on day 10 following BDL, but remained elevated on PMN (12.5%, p<0.001) and KC (14.3%, p=0.003) compared to baseline. Both liver PMN (14.3-32.2%+) and KC (31.2-65.3%+) expressed high levels of PDL-1 in jaundiced mice. Following BDL, we observed PD-1-dependent elevations of CXCL2, a chemokine that attracts MC. Intrahepatic CXCL2 levels were 465.9 pg/ml in jaundiced mice, but decreased to 225.9 pg/ml with anti-PD-1 treatment (p=0.045). Accordingly, anti-PD-1 treatment led to a contraction of the liver PMN population as a percentage of liver leukocytes on day 3 following BDL (19.4% vs 9.5%, p=0.03). We also examined the effects of PD-1 blockade on liver HIF1, VEGF, and IL6 levels to measure the broad anti-inflammatory effects of this treatment. While VEGF and HIF1 levels were not affected by anti-PD-1 injections, IL6 levels decreased from 376.1 pg/ml to 255.3 pg/ml in jaundiced animals treated with PD-1 blockade (p=0.009).

Conclusion: We have demonstrated increased expression of PD-1 on liver PMN and KC in response to ObJ. As these cell types also expressed PDL-1, they are likely able to activate PD-1, which we demonstrate to mediate inflammation via increased CXCL2 and IL6 production in the liver. PD-1 blockade may reduce inflammation and restore normal T cell function in the inflamed liver.

 

42.06 Transplantation of Stem Cell-Derived Hepatocytes in an Animal Model of Acute Liver Failure

Posted on December 22, 2014

R. Ramanathan1, G. Pettinato3, M. Mangino1, D. Lee4, X. Wen3, R. A. Fisher2  1Virginia Commonwealth University Medical Center,Surgery,Richmond, VA, USA 2Beth Israel Deaconess Medical Center,Transplantation,Boston, MA, USA 3Virginia Commonwealth University,Bioengineering,Richmond, VA, USA 4Mayo Clinic,Transplantation,Jacksonville, FL, USA

Introduction:
Hepatocyte cell transplantation can be life-saving in patients with acute liver failure (ALF), however it is limited by donor hepatocyte scarcity. We investigate the effect and function of stem cell-derived hepatocyte-like cells in an animal transplant model of ALF. 

Methods:
A model of near-lethal ALF was developed in athymic nude rats using intraperitoneal D-galactosamine. A cell xeno-transplant model in rats was established using hepatic cells isolated from mouse livers. Human induced pluripotent stem cells (iPSC) and human mesenchymal stem cells (MSC) were differentiated into hepatocyte-like cells using a novel stem cell embryoid body suspension culture protocol. A subset of stem cell-derived hepatocyte-like cells were co-cultured with endothelial cells. Stem cell-derived hepatocyte-like cells were transplanted into the spleens of athymic nude rats with ALF with monitoring of survival and in vivo function through the presence of human albumin in the rat serum.

Results:
A reliable near-lethal model of acute liver failure was achieved with 975 mg/kg of D-galactosamine in rats weighing 270-350mg with >90% death within 3 days. Hepatic cells were reliably isolated from mouse livers with >85% viability and used to establish positive controls. In vitro differentiation of iPSC and MSC into hepatocyte-like cells was confirmed using genetic and protein studies. In vitro, stem cell-derived hepatocyte-like cells metabolized ammonia. They secreted albumin and fibrinogen in the presence of primary hepatic and endothelial cells respectively. 20 rats were transplanted with iPSC-derived hepatocyte-like cells and 12 rats were transplanted with MSC-derived hepatocyte-like cells. Compared to negative controls, transplant with iPSC-derived and MSC-derived hepatocyte-like cells were associated with increased survival (70% and 67% vs. 14%, p<0.05). Human albumin was detected in rat serum at three days after transplant in over half the animals transplanted with hepatocyte-like cells. Only animals transplanted with endothelial cell co-cultured hepatocyte-like cells had prolonged rat serum human albumin production. Despite transplantation into the spleen, human albumin was detected in the rat liver only, while human endothelial cells were detected in the rat spleen only (Figure).

Conclusion:
In a reliable near-lethal model of d-galactosamine induced acute liver failure, transplantation of stem cell-derived hepatocyte-like cells improved survival with evidence of in vivo cell functionality. Despite splenic transplantation, migration to cells into the liver is noted. Sustained in vivo function was noted in cells co-cultured with endothelial cells.
 

 

42.07 Environmental Mediated Intestinal Homeostasis In Neonatal Mice

Posted on December 22, 2014

C. Culbreath1, S. Tanner1, T. Berryhill1, R. G. Lorenz2, C. A. Martin1  1University Of Alabama,Pediatric Surgery,Birmingham, Alabama, USA 2University Of Alabama,Pathology,Birmingham, Alabama, USA

Introduction:  Maintenance of intestinal homeostasis by protection against pathogenic bacteria and dietary antigens is critical for intestinal homeostasis.  Immunoglobulins, specifically IgA, play a key role in coating luminal antigens and preventing translocation of harmful bacteria.  The aryl hydrocarbon receptor (AhR) is a basic-helix-loop transcription factor, that when stimulated by exogenous pollutants, microbial products, and dietary components, activates factors important for barrier function and intestinal homeostasis. To date, the function of AhR has not been studied in the developing innate immune system.  Furthermore it is not clear how the extrauterine environmental influences the establishment of host/microbial mutualism.  We hypothesize that AhR signaling is critical for establishment of intestinal homeostasis in neonates.  

Methods:  Three groups of mice were used. C57BL/6 (B6) AhR+/+ (WT), B6.AhR-/- (KO) , and B6.AhR+/+  raised on an AhR Ligand Free diet (AhR LF).   AhR LF is defined as mice whose parents and the subsequent pups were maintained on a nutritionally balanced diet as well as cage bedding that was free of all exogenous AhR ligands.   Enzyme-linked immunosorbent assay (ELISA) was used to measure fecal IgA levels in these groups of mice at 2 and 8 weeks of age.  To determine the contribution of IgA from breast milk the gastric contents of 2 week old pups were harvested from the 3 groups of mice.  Intestinal homeostasis was measured by culturing the mesenteric lymph nodes (MLN) under aerobic conditions to determine the number of bacterial colony forming units (CFU) after 72 hours.  Results were analyzed by the Student’s unpaired T-test and expressed as the mean ± standard error of the mean.  

Results:  Two week old  KO mice had significantly less fecal IgA (37 ± 37 µg/ml) compared to WT (2519 ± 807 µg/ml ) and AhR LF (3043 ± 764 µg/ml), p value = 0.0393.  The amount of IgA from the gastric contents of 2 week old mice was not significant, WT (40 ± 14 µg/ml), KO (40 ± 18 µg/ml), AhR LF (24 ± 5 µg/ml), p value = 0.322.    At 8 weeks of age AhR LF mice (1769 ± 369 µg/ml) had significantly less fecal IgA than WT (17,574 ± 4916 µg/ml) and KO (12,553 ± 2666 µg/ml), p value = 0.0077.  At 2 weeks, KO mice had significantly higher levels of bacterial translocation (158 ± 37 CFU) compared to WT (2 ± 1 CFU) and AhR LF (9 ± 5 CFU), p value = 0 .0132.  At 8 weeks AhR LF had significantly higher levels of bacterial translocation (119 ± 57 CFU) compared to WT (15 ± 7) and KO (11 ± 10) , p value = 0.019

Conclusion:  In neonatal mice, the lack of AhR signaling is associated with loss of  intestinal homeostasis, evidenced by decreased levels of IgA and increased bacterial translocation.   In adult mice, exogenous AhR ligand and not receptor signaling is necessary for maintenance of intestinal integrity. 

 

 

42.08 Macrophages from Visceral Adipose Tissue Contribute to the Onset of Insulin Resistance

Posted on December 22, 2014

M. Rodrigues1, A. Whittam1, M. Sorkin1, M. Januszyk1, R. Kosaraju1, R. C. Rennert1, A. McArdle1, Z. Maan1, D. Duscher1, G. C. Gurtner1  1Stanford University,Department Of Surgery, Division Of Plastic Surgery,Palo Alto, CA, USA

Introduction:  Chronic low-grade inflammation that develops during obesity is postulated to contribute to insulin resistance. Although there is significant interest in understanding how inflammation contributes to diabetes, the cellular and molecular mechanisms that underlie these processes remain unclear. In this study we test the contribution of macrophages to insulin resistance. 

Methods:  Using a vav-mtmg murine model with hematopoietic cells labeled with GFP, and non-hematopoietic cells labeled with RFP, combined with single cell transcriptional analyses, we first developed a marker profile for macrophages in murine adipose tissue. These markers were used to fluorescently sort and analyze the distribution of macrophages in murine and human subcutaneous (SAT) or visceral adipose tissue (VAT). Next, to determine the role of macrophages during the onset of obesity, mice were either fed with normal chow, or high-fat diet, to induce obesity, and diabetes. A second well-established diabetic transgenic murine model Leprdb was used for the isolation of diabetic macrophages. Single-cell transcriptional analyses determined changes in distribution of macrophage-subtypes upon obesity, and during diabetes. Finally, macrophages isolated from adipose tissue of diabetic or non-diabetic mice were injected into the visceral adipose tissue of non-diabetic wild type mice. These mice were either fed with a high fat diet, or normal chow. Blood glucose, insulin, cholestrol and triglyceride levels were measured as an indication of diabetes and diabetic complications.

Results: Single cell analysis of vav-mtmg adipose tissue revealed that macrophages comprise 64% of hematopoietic cells in the VAT and 36% of hematopoietic cells in the SAT. These cells positively expressed cd45 cd14 cd11b emr1 cd64 csf1r cd33 fcgr3 msr1 cd36. Onset of diabetes increased the number of macrophages (CD45+ CD11b+ F4/80+) in murine VAT by 4-fold with no significant increase in the macrophages in the SAT. A similar trend was seen in human adipose samples from non-diabetic and diabetic patients. Single-cell analysis revealed that diabetes induced an increase in the number of pro-inflammatory M1 macrophages in the VAT, highly expressing il1b and il6. Transplantation of these macrophages into the perigonadal adipose tissue of non-diabetic mice in the presence of a high fat diet led to significant increases in body weight, adipose tissue and liver mass, as well as increased blood triglyceride and cholestrol levels. These mice showcased hyperglycemia within 4 weeks, significantly faster than mice injected with macrophages from non-diabetic mice, which took 8 weeks to become hyperglycemic. 

Conclusion: Pro-inflammatory M1 macrophages in the visceral adipose tissue increase with obesity, and quicken the onset of diabetes in the presence of high-fat food. Specific targeting of these cells will lead to ways of containing diabetic onset. 

 

42.09 The impact of an inadequate tolerogenic stimulus in prenatal transplantation.

Posted on December 22, 2014

A. E. Lee1, A. M. Alhajjat2, B. S. Strong1, L. E. Turner1, R. K. Wadhwani1, T. Newkold1, A. F. Shaaban1  1Cincinnati Children’s Hospital Medical Center,Center For Fetal Cellular And Molecular Therapy,Cincinnati, OH, USA 2University Of Iowa,Department Of Surgery,Iowa City, IA, USA

Introduction:

In utero hematopoietic cellular transplantation (IUHCT) has the potential to treat congenital benign cellular disease without the need for chemotherapy or radiation.  Prenatal tolerance is essential in this regard.  Previous studies have shown that allospecific prenatal natural killer (NK) tolerance requires a threshold level of circulating chimerism (> 1.8%) during the critical phases of NK cell education resulting in the elimination of alloreactive NK cell phenotypes.  However, it remains unclear if sub-threshold levels of chimerism have any impact on the education of developing NK cells.  Given the lack of sustained tolerance, we hypothesized that exposure to sub-threshold levels of hematopoietic chimerism does not lead to durable changes in NK cell selection.  

 

Methods:

To challenge this hypothesis, we compared NK cell education in stable murine prenatal chimeras (engrafters) to littermates that rejected their grafts (rejecters) in a Balb/c →  B6 murine model of IUHCT.  Following the prenatal transplantation of allogeneic fetal liver hematopoietic cells at E14, the level of chimerism and the frequency of alloreactive NK cell phenotypes were measured in the peripheral blood of either group and compared to naïve age-matched controls at serial time points during the critical phases of NK cell education.  In particular, we measured frequency of naturally occurring donor-reactive NK cells in wild-type B6 mice that express the donor-reactive Ly49D activating receptor without co-expression of donor-specific Ly49A, L49F or Ly49G inhibitory receptors (hostile NK cells).    

 

Results:

As expected, all of the engrafter mice exhibited stable long-term engraftment while the rejecter mice universally rejected their grafts several weeks after birth.  This pattern was unaffected by fostering of the recipients by naive pregnant dams suggesting no contribution of the maternal humoral immune response to these outcomes.  At weeks 3 and 4 after birth, both engrafter and rejecter mice exhibited low frequencies of phenotypically hostile NK cells (Ly49D+AFG-) when compared to age-matched controls (4.4 and 5.2% vs. 19.6%).  Both engrafter and control mice maintained a stable level of hostile NK cells through the 14 week time point (4.7% and 22.3% respectively) while rejecter mice displayed a steady rise in the frequency of hostile NK cells throughout the study period (6 weeks: 12.5%, 10 weeks: 12.9%, 14 weeks: 18%) eventually reaching levels similar to control mice.    

Conclusion:

From these findings, we conclude that: 1) durable prenatal NK cell selection requires the stable persistence of a threshold level of circulating tolerogen; 2) transient reductions in the frequency of hostile NK cells are inadequate to maintain long-term engraftment; and 3) the emergence of either tolerance or rejection is intrinsic to the education of developing lymphocytes in prenatal chimeras and is independent of the maternal humoral immune response.   Further study is needed to examine functional response of hostile NK cells in rejecter mice and the potential for donor-specific memory to develop after rejection.  
 

42.10 Virus Induced CD28 Down-Regulation as a Driver of Costimulation Resistant Allograft Rejection

Posted on December 22, 2014

D. Mou1, J. R. Espinosa1, J. Kwun1, N. N. Iwakoshi1, A. D. Kirk2  1Emory University School Of Medicine,Surgery,Atlanta, GA, USA 2Duke University Medical Center,Surgery,Durham, NC, USA

Introduction:
Belatacept, a B7-specific fusion protein that blocks CD28-B7 costimulation to prevent kidney allograft rejection, is ineffective in a sizable minority of transplant recipients. Although T cell receptor and CD28 engagement initiates T cell activation, many human antigen-experienced memory T cells lose CD28, and can be activated without CD28 signals. We posit that these cells are central drivers of belatacept resistant rejection (BRR) and propose that they may arise from antigen exposure. CD28 loss is poorly described in mice, which are typically kept in pathogen-free conditions.

Methods:
To study mice in a clinically relevant scenario of viral exposure, we characterized T cell CD28 expression after sequential infections, 3 weeks apart, with Polyomavirus (PyV, BK virus homolog), murine CMV, and mHV68 (EBV homolog). 5 mice cohorts, each containing 10 C57BL/6 mice, were defined as mock infections, single PyV infection, single mCMV infection, single mHV68 infection, or ‘all 3’ infections. Flow analysis was performed on the day of infection, at peak infection, and at the memory time point. Mixed lymphocyte reactions (MLRs) of splenocytes were conducted to assess in vitro alloreactivity. Alloreactivity was further evaluated both in the presence and absence of costimulation blockade (CTLA4-Ig and MR1). We are currently performing heart transplants in infected and uninfected mice to assess the phenotypic role of CD28 loss in BRR.

Results:
CD28 MFI analysis of both CD4 and CD8 cells showed significant (P<0.05) CD28 down-regulation in the effector TMs all infected cohorts. MLRs revealed that the mHV68 and triply infected cohorts exhibited significantly (P<0.05) higher alloreactivity than the naïve cohorts. Interestingly, the triple infected cohort demonstrated significantly (P<0.05) more IFNy production than the mHV68 cohort. The increased alloreactivity is resistant to costimulation blockade.

Conclusions:
Clinically relevant viruses PyV, mCMV, and mHV68 induce CD28 down-regulation in mice, suggesting that Polyoma, CMV, and EBV infections may play a role in human BRR. Additionally, mHV68 exposure confers enhanced alloreactivity in MLRs, suggesting that viruses may lead to functionally relevant changes in the immune repertoire. The MLR observation that the triply infected cohort produced more IFNy than the mHV68 cohort is suggestive of an additive alloreactivity effect that is driven by multiple viral infections. Overall, the data suggest that clinically relevant viruses may play a significant role in determining how solid organ transplant patients respond to costimulation blockade therapies.

43.01 Sirt1 Stimulation Preserves Mitochondria and Enhances Autophagy in Hepatocytes after Hypoxic Injury

Posted on December 22, 2014

A. Khader1,2, W. Yang1,2, J. M. Prince1, J. Nicastro1, G. F. Coppa1, P. Wang1,2  1Hofstra North Shore-LIJ School Of Medicine,Surgery,Manhasset, NEW YORK, USA 2Elmezzi Graduate School Of Molecular Medicine,Manhasset, NEW YORK, USA

Introduction: Liver ischemia-reperfusion (I/R) often occurs in trauma, transplantation and prolonged shock state, where it lacks specific treatment. We have recently demonstrated that pharmacologic sirtuin 1 (Sirt1) activation, with SRT1720, is protective in a murine model of liver I/R. However, the detailed mechanism of which is not yet characterized. Sirt1 is an energy-sensing enzyme with multiple roles, including the regulation of energy metabolism, mitochondrial function and autophagy. We therefore hypothesized that SRT1720 protects hepatocytes from hypoxia-induced injury through the activation of mitochondrial biogenesis and the autophagy salvage pathway.

Methods:  Rat hepatocyte epithelial H4IIE cells were subjected to 6 h of hypoxia using 5% Oxyrase, followed by reoxygenation in the presence or absence of SRT1720 (500 nM) for 4 or 24 h. Cell survival was determined by cell counts and ATP assay. Cells were stained with MitoTracker green and red FM for measuring mitochondrial mass (mtMass) and membrane potential (MMP), respectively, by flow cytometry. Acridine orange stain was used to assess the formation of autophagic vesicles under fluorescent microscopy. Protein levels were determined by Western blot. 

Results: We observed a 27.6% and 21.7% reduction in cell numbers and ATP levels, respectively, at 4 h of reoxygenation (H/R), while SRT1720-treated cells were comparable to the normoxia control. At 4 h of H/R, the mtMass and MMP per cell, as measured by mean fluorescent intensity (MFI), were decreased by 44.6% and 27.6%, respectively, compared to the normoxia control. In contrast, treatment with SRT1720 enhanced both parameters by 3.4- and 5.5-fold, respectively, compared to untreated cells at 4 h of H/R. This enhancement of mtMass and MMP by SRT1720 persisted to 24 h of reoxygenation. Treatment with SRT1720 increased the expression of PGC1α, the master regulator of mitochondrial biogenesis, by 11.0% and 36.7% at 4 h and 24 h of H/R, respectively. In addition, there was a significant enhancement in autophagy in the SRT1720 treated cells as demonstrated by an increase in LC3aII/LC3aI protein ratio to 3.3 compared to 1.7 in the untreated cells at 4 h of H/R. Further, there was an observable increase in autophagic vesicles (bright contrast) at 24 h of reoxygenation which was remarkably increased by SRT1720, as shown by the increase in vesicle numbers and intensity of acridine orange precipitation (Figure).

Conclusion: Pharmacologic activation of Sirt1 improves the survival of hepatocytes under hypoxic stress, which is associated with maintenance of mitochondrial mass and integrity as well as an increase in autophagic activity. Thus, stimulation of mitochondrial function may provide a new protective strategy against liver I/R injury. 

43.02 Cytokine-induced MicroRNA-155 Limits iNOS Synthesis in Hepatocytes During Redox Stress

Posted on December 22, 2014

P. R. Varley1, Z. Guo1, D. A. Geller1  1University Of Pittsburgh,General Surgery,PITTSBURGH, PA, USA

Introduction:  Redox stress is an important mediator of liver damage ischemia reperfusion (I/R) injury. Previously our group has described the role of inducible nitric oxide synthase (iNOS) in the liver, which exacerbates liver injury under redox stress.  MicroRNA (miR)-155 is up-regulated by a number of inflammatory stimuli, however its role in the liver and effect on iNOS expression have not been reported. We hypothesized that miR-155 is induced in hepatocytes during inflammation and regulates iNOS expression.

Methods:  Rat and human hepatocytes were isolated through collagenase dissociation and density gradient centrifugation. To determine the ability of miR-155 to regulate iNOS expression, primary human hepatocytes were isolated and treated with miR-155 mimics and then stimulated with a cytokine mix (CM) ((interferon-γ, IL-1β and TNF-α) for 24 hours. The Griess method was used to measure nitrite concentrations in the cell culture supernatant as endproduct for induced NO synthesis. To evaluate miR-155 expression, rat hepatocytes were treated with hydrogen peroxide (H2O2) and CM alone or in combination for up to 24 hours. Trizol purification was used to isolate RNA from the cells, and miR-155 levels were measured with TaqMan qRT-PCR. 

Results: Transfection of primary human hepatocytes with miR-155 mimics resulted in near complete abrogation of nitrite production in CM-treated cells (NO2–  1.1 uM in miR-155+CM vs. 18.5 uM in CM alone, p < 0.05) suggesting significant suppression of cytokine-induced NO synthesis.  H2O2 stimulation alone had a non-significant effect on miR-155 expression at 24 hrs, while CM stimulation resulted in significant up-regulation of miR-155 expression at 24hr (Table 1). Co-stimulation of hepatocytes with CM + H2O2resulted in a dramatic 21.3-fold increase in miR-155 expression at 24 hours.

Conclusion: This study is the first to describe down-regulation of iNOS by miR-155, a well-known inflammatory microRNA. Not only is miR-155 capable of iNOS regulation, but here we show that inflammatory cytokine stimuli under redox stress act in a synergistic fashion to induce miR-155. Our previous work has shown the iNOS expression peaks at 6-12 hours after introduction of inflammatory stimuli and returns to baseline by 24 hours. Taken together with the data from this study, we suggest that miR-155 blocks iNOS-mediated NO synthesis in a post-translational manner as a homeostatic control mechanism to prevent the untoward consequences of excessive iNOS expression and limit hepatocellular damage.

 

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