4.18 THROMBELASTOGRAPHY PERFOMRED WITHOUT AN ACTIVATOR ENHANCES DETECTION OF FIBRINOLYSIS

Posted on December 22, 2014

B. A. Quinn1,2, E. Gonzalez1, H. B. Moore1, M. P. Chapman1, A. Sauaia1, A. Banerjee1, C. C. Silliman1,3, E. E. Moore1,2  1University Of Colorado Denver,Department Of Surgery,Aurora, CO, USA 2Denver Health Medical Center,Department Of Surgery,Denver, CO, USA 3Bonfils Blood Center,Research Department,Denver, CO, USA

Introduction: The fibrinolytic response to trauma can be physiologic (preventing systemic clot propagation), pathologic (favoring bleeding), or shut-down (favoring un-regulated clotting). Thrombelastography (TEG) is used to quantify fibrinolysis and facilitate appropriate use of fibrinolysis inhibitors such as tranexamic acid in bleeding patients with hyperfibrinolysis. TEG in the trauma bay is typically performed with tissue factor as an activator (rapid-TEG). However, TEG can also be performed with kaolin as an activator or as a “native assay” with no activator. Which TEG modality is optimal for detecting fibrinolysis remains to be elucidated. We hypothesized that the use of kaolin or tissue factor as activators for TEG decreases the assay’s capability to detect tissue plasminogen activator (tPA)-induced fibrinolysis.

 

Methods: Citrated and non-citrated blood samples were collected from 10 healthy adults. Fibrinolysis was achieved by addition of tPA to whole blood samples prior to running the assay in five concentrations: 0, 50, 75, 150, and 300 (ng/mL). tPA-induced fibrinolysis was quantified by three different TEG methods—citrated native (CN) (no activator), citrated kaolin (CK), and non-citrated rapid (RT) (tissue factor). The TEG variable of fibrinolysis, LY30 (percent clot lysis at 30 minutes after reaching maximum clot strength), was compared at each tPA dose amongst CN, CK, and RT. The significance of differences between groups was tested by the Friedman’s test (p<0.05). Groups with significant differences were subjected to a post-hoc pairwise comparison with Bonferroni adjustment.




Results: At baseline (0 tPA), RT vs. CN detected a greater LY30 (2.8 vs. 1.7%, p=0.03). At 50 tPA the differences in LY30 among RT, CK, and CN were not statistically significant (p=0.050). At 75 tPA, CN vs. RT detected a greater LY30 (15.8 vs. 3.6%, p<0.001). At 150 tPA, CN vs. RT detected a greater LY30 (56.1 vs. 36.7%, p=0.005). At 300 tPA there were no significant differences.

Conclusion: Using a coagulation activator decreases the threshold for detecting tPA-induced fibrinolysis by TEG. When fibrinolysis was induced to levels of tPA previously reported in trauma patients (75 and 150 ng/mL), CN detected more fibrinolysis. Early detection of hyper-fibrinolysis in injured patients is imperative for triggering treatment with anti-fibrinolytics in order to control bleeding and decrease mortality. Our data demonstrates that TEG can be used with no activator (CN) for adequate quantification of fibrinolysis.

4.19 Cytokine Gene Expression in the Gastrocnemius of Patients with Peripheral Arterial Disease

Posted on December 22, 2014

L. A. Carpenter1, J. R. Thompson1, D. M. Ha1, S. A. Swanson1, J. M. Johanning1,2, E. A. Papoutsi1, P. Koutakis1, D. A. Miserlis1, I. I. Pipinos1,2, G. P. Casale1  1University Of Nebraska Medical Center,Department Of Surgery,Omaha, NE, USA 2VA Nebraska-Western Iowa Health Care System,Omaha, NE, USA

Introduction:
Peripheral arterial disease (PAD) is characterized by limb dysfunction in association with cycles of ischemia/reperfusion that cause oxidative damage to muscles of the lower leg.  Studies evaluating the contribution of inflammation to the pathology of PAD identified increased serum concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin 6 and monocyte chemoattractant protein-1.  Recently, we demonstrated increased concentrations of cytokines including TNF-alpha, transforming growth factor beta1 (TGF-beta1) and chemokine (c-c motif) ligand 5 (CCL5) in the gastrocnemius of PAD patients.  In this study, we tested the hypothesis that these increases were due to increased gene expression in the gastrocnemius.

Methods:
Gastrocnemius biopsies were collected from PAD patients (N=24) at Fontaine Stage II and controls (N=18) with no leg impairment.  Muscle homogenates were analyzed by qPCR for TNF-alpha, TGF-beta1 and CCL5 gene transcripts expressed as fold change in relation to myosin heavy chain transcripts.

Results:
TNF-alpha, TGF-beta1 and CCL5 transcripts were increased in PAD patients (mean +/- s.e.; 1.07 +/- 0.31, 12.87 +/- 3.38 and 3.01 +/- 0.99, respectively) compared to controls (0.02 +/- 0.01, 0.56 +/- 0.25 and 0.15 +/- 0.10) at p values of 0.003, 0.001 and 0.009.  Relative changes in these transcripts are in close agreement with changes in protein expression determined in our previous study.

Conclusion:
The data support increased, local expression of the genes for TNF-alpha, TGF-beta1 and CCL5 in the gastrocnemius of PAD patients and identify a local cytokine milieu that suppresses myoblast differentiation and promotes fibrotic replacement of oxidatively damaged and necrotic myofibers.

4.20 Acute Hind Limb Ischemia in the Recombinant Polygenic Type 2 Diabetic Mouse

Posted on December 22, 2014

H. Albadawi1,3, R. Oklu2,3, T. P. Uong1, J. D. Milner1, H. Yoo1, M. T. Watkins1,3  1Massachusetts General Hospital,Department Of Surgery, Division Of Vascular And Endovascular Surgery,Boston, MA, USA 2Massachusetts General Hospital,Department Of Imaging, Division Of Interventional Radiology,Boston, MA, USA 3Harvard School Of Medicine,Brookline, MA, USA

Introduction: The polygenic mouse model of diabetes is believed to better simulate the human adult type-2 diabetes compared to the monogenic models (i.e. db/db or Ob/Ob). This model exhibits a maturity-onset transition from impaired glucose tolerance to a stable non-fasting hyperglycemia when fed a 10% high fat diet after 16 weeks. Wound healing experiments using these mice show substantial impairment in wound healing processes involving the skin. The aim of this study was to assess acute skeletal muscle injury in the polygenic mouse model of diabetes following hind limb ischemia reperfusion (IR).
 

Methods: The recombinant polygenic diabetic mice (NONcNZO10/LtJ, n=6) and its non-diabetic control strain (NON/ShiLtJ, n=5) were subjected to unilateral moderate hind limb tourniquet ischemia for 1.5 hours followed by 24 hours of reperfusion. To confirm their diabetic state, fasting blood glucose levels were measured prior to ischemia. After 24 hours of reperfusion, mice were sacrificed and muscle samples were processed for histological quantitative assessment of muscle fiber injury and inflammatory cell infiltration (Ly6G, marker of neutrophils, or Mac-3, marker of monocytes lineage) using immunohistochemistry. The protein levels of pro-inflammatory chemokine KC (CXCL1) in the serum and solubilized muscle protein extracts were measured using ELISA. Data were expressed as mean±SEM and statistical analysis was performed using student’s t-test.

Results: The fasting blood glucose levels in the diabetic mice were significantly greater than in the non-diabetic mice (472±32 vs. 165±28 mg/dL, p<0.000001). There was no significant difference in the degree of muscle fiber injury between the diabetic vs. non-diabetic mice (15±2 vs. 16±2 average injured fiber per high power field, p=0.6). The accumulation of Ly6G+ (41±10 vs. 48±15 average positive cells per field, p=0.7) and Mac3+ (42±6 vs. 33±5 average positive cells per field, p=0.7) cells in skeletal muscle following IR was similar in the diabetic vs. non-diabetic mice. Furthermore, levels of muscle KC (17±2 vs. 14±2 pg/mg protein, p=0.2) and serum KC (103±6 vs. 73±15 pg/ml, p=0.09) were also not statistically different between the two groups.

Conclusion: While excessive IR injury and increased inflammation is believed to play a major role in defective wound healing models, the pattern of acute skeletal muscle IR in the polygenic diabetic mouse does not appear to be worse than that of the non-diabetic mouse following 1.5 hours of ischemia. Further studies in these polygenic diabetic mice subjected to severe periods of ischemia (i.e. ≥3 hours) and characterization of the regenerative phase (i.e. healing) in the limb muscle is warranted.

4.01 Valproic Acid Alters Inflammatory Gene Expression after Traumatic Brain Injury and Hemorrhagic Shock

Posted on December 22, 2014

T. Bambakidis1, S. E. Dekker1, M. Sillesen1, B. Liu1, C. N. Johnson1, I. Halaweish1, Y. Li1, H. B. Alam1  1University Of Michigan,Surgery,Ann Arbor, MI, USA

Introduction: We have reported that valproic acid (VPA) can create a pro-survival gene expression profile in various models of lethal insults, and its administration can significantly decrease brain lesion size and surrounding inflammation, in a swine model of combined traumatic brain injury (TBI) + hemorrhagic shock (HS). It, however, remains unknown whether this neuroprotective effect is driven by alterations in the expression of cerebral inflammatory genes.

Methods: Computer-controlled TBI (cortical impact) and HS (40% blood volume) were induced in 10 Yorkshire swine. After two hours of shock, animals were randomly treated with either 6% hextend (HEX; 1x shed blood) or HEX+VPA (300mg/kg) (n=5/group). Six hours after resuscitation, brains were harvested, RNA isolated, and gene expression profiles measured using a Porcine Gene ST 1.1 microarray (Affymetrix, CA). Ingenuity Pathway Analysis® (IPA), Gene Ontology (GO), and Parametric Gene Set Enrichment Analysis (PGSEA) were used for pathway analysis. Key microarray findings were verified using real-time polymerase chain reaction (PCR).

Results: Of the 1753 genes modulated by VPA, significant alterations were noted in genes related to the inflammatory response. IPA analysis revealed that VPA significantly down-regulated the complement system (P<0.001), natural killer cell communication (P<0.001), and dendritic cell maturation (P<0.001) (Figure). Real-time PCR data confirmed that VPA significantly decreased the expression of genes associated with inflammation, such as CCR1 (P=0.01), IL-1β (P=0.003), TREM2 (P=0.02), and TYROBP (P=0.05).

Conclusion: This is the first high-throughput analysis of cerebral gene expression profile following TBI+HS which reveals that VPA treatment significantly attenuates inflammatory pathways.

 

4.02 The Role of Erythropoietin and Hepcidin in the Regulation of Persistent Injury-Associated Anemia

Posted on December 22, 2014

I. G. Alamo1, K. B. Kannan1, M. A. Smith1, P. A. Efron1, A. M. Mohr1  1University Of Florida,Surgery,Gainesville, FL, USA

Introduction:  The cause of persistent injury-associated anemia is multifactorial and includes blood loss, impaired proliferation of erythroid progenitor cells, altered erythropoietin (EPO) response, dysregulation of iron homeostasis, and chronic inflammation/stress. Hepcidin plays a key role in iron homeostasis and has been shown to be regulated by anemia as well as inflammation and EPO is a main regulator of erythropoiesis induced by hypoxia. The relationship between these two factors in persistent injury-associated anemia has yet to be fully elucidated. Using a combined lung injury (LC)/hemorrhagic shock (HS)/chronic restraint stress (CS) model to produce persistent injury-associated anemia, the aim of this study was to investigate the regulation of hepcidin and EPO.

 

Methods: Male Sprague-Dawley rats (N=6-9 per group) were randomly assigned into one of the four groups of rodent models: naïve, CS alone, combined LCHS, or LCHS/CS.  CS was performed using restraining cylinders every day for two hours following either LC or LCHS. During CS, rodents were exposed to 80-85 decibel alarms for two minutes every 30 minutes and rotated to prevent habituation. At day seven, blood, urine, bone marrow and lung tissue was harvested. Hemoglobin (Hgb), EPO, and hepcidin levels were assessed.  Data presented as mean±SD in each group. *p<0.05 vs naive

 

Results: Compared to naïve rodents, plasma hepcidin levels were significantly decreased in CS, LCHS, and LCHS/CS groups (Figure).  Similarly, urine hepcidin levels were significantly lower in CS, LCHS and LCHS/CS as compared to naïve (12±4*, 10±3*, 10±2* vs. 52±26 pg/mg protein). There was no change in bone marrow hepcidin mRNA levels in any group.  In the LCHS/CS group, there was a significant 70% decrease in lung hepcidin mRNA level as compared to naive. Only LCHS/CS was associated with persistent anemia despite significant elevation of EPO (Figure).  There was a strong inverse correlation between EPO and plasma hepcidin (Pearson R= -0.362, p<0.05).

 

Conclusion: Chronic stress, LCHS and LCHS/CS all significantly decrease plasma and urine hepcidin. Yet only LCHS/CS is associated with persistent anemia despite elevation of EPO. Although there is an inverse correlation between hepcidin and EPO in this model, anemia alone does not regulate hepcidin. The addition of chronic stress did not counteract hepcidin suppression.  Further study of the mechanisms involved in injury-associated persistent anemia is warranted.

 

4.03 Daily Propranolol Prevents Prolonged HPC mobilization in a Chronic Stress and Polytrauma Model

Posted on December 22, 2014

L. E. Bible2, L. V. Pasupuleti2, A. V. Gore2, Z. C. Sifri2, A. M. Mohr1  1University Of Florida,General Surgery,Gainesville, FL, USA 2New Jersey Medical School,Newark, NJ, USA

Introduction:  Following injury, hematopoietic progenitor cells (HPC) mobilize to the peripheral blood from the bone marrow (BM) and then home to the injured tissue. We have previously shown that propranolol decreases HPC mobilization and improves BM function following acute injury in rodent models. These acute injury models do not reflect the prolonged period of critical illness following severe traumatic injury. Using our unique lung injury (LC)/hemorrhagic shock (HS)/chronic restraint stress (CRS) model, we hypothesize that daily propranolol administration following LC/CRS and LCHS/CRS will reduce prolonged HPC mobilization without worsening lung healing.

Methods:  Male Sprague-Dawley rats (n=5-9/group) underwent six days of CRS after undergoing LC or LCHS. CRS consists of a daily two hour period of restraint within a cylinder that is interrupted every 30 minutes by alarms and repositioning. Each day following their intervention, the rats received intraperitoneal propranolol (10mg/kg). On day seven the peripheral blood was analyzed for granulocyte-colony stimulating factor (G-CSF) via ELISA and for HPC mobilization using c-kit and CD71 flow cytometry, and the lungs were examined histologically to grade injury.

Results: As previously shown, seven days following LC and LCHS, the addition of CRS significantly increased HPC mobilization which is associated with persistently elevated G-CSF and worsened lung injury scores (Table). The addition of propranolol to LC/CRS and LCHS/CRS models significantly reduces HPC mobilization in peripheral blood (Table). In addition, the administration of propranolol following LC/CRS and LCHS/CRS restores G-CSF levels to that of naïve animals without worsening lung injury scores. 

Conclusion: Daily propranolol administration following both LC/CRS and LCHS/CRS reduces prolonged HPC mobilization from the bone marrow and decreases plasma G-CSF levels. Despite the reduction of HPC mobilization, the lung healing did not worsen. Alleviating chronic stress with propranolol may be a future therapeutic target to improve healing following severe injury.

 

4.04 Interleukin-6 is Essential for Endogenous Fibrinogen Release in the Acute Phase Response to Trauma

Posted on December 22, 2014

R. A. Jacobson1,2, J. G. Schoenecker1,3  1Vanderbilt University Medical Center,Pharmacology,Nashville, TN, USA 2Rush University Medical Center,Rush Medical College,Chicago, IL, USA 3Vanderbilt University Medical Center,Orthopedics,Nashville, TN, USA

Introduction: Fibrinogen (FBG) is an acute phase reactant secreted from the liver in response to injury and consumed during hemostasis at sites of tissue damage. Recent studies show that patients deficient in FBG due to traumatic consumption or dilution secondary to fluid resuscitation are coagulopathic, with an elevated risk of further bleeding. As such, repletion of FBG is a crucial step in the endogenous and therapeutic responses to injury. However, the mechanism of induction of FBG secretion after injury is incompletely understood. This study investigated the role of the inflammatory mediator interleukin-6 (IL-6) in endogenous FBG secretion following blunt trauma. Our hypothesis is that trauma-induced circulating IL-6 is essential for upregulation of circulating FBG.

Methods: 8-week-old male wild type C57BL/6 (WT) and IL-6 deficient (IL-6-/-) mice were injured, then sacrificed at the time points indicated below (n=3 mice per time point) by CO2 inhalation with cardiac puncture for blood collection. Citrated blood was processed into plasma, and ELISA for FBG and IL-6 were performed.  Injury was induced using a modified version of the method described by Pape et al (J Surg Res 2011). Open muscle trauma was induced by clamping a large needle driver around the body of the gastrocnemius for 30s.

Results: Figure 1A shows plasma levels of IL-6 before and after injury in WT mice. IL-6 peaks above 60pg/mL 4-8 hours post-injury from a baseline (t=0) below 5pg/mL.  Levels return to baseline at 48 hours post-injury. Plasma IL-6 was undetectable in IL-6-/- mice.

Figure 1B shows plasma fibrinogen levels in WT and IL-6-/- mice following injury. In WT mice, FBG levels began to rise at the earliest time point – 4 hours, peak at 24 hours and then decline. In IL-6-/- mice, there is no immediate rise in FBG in response to trauma. A blunted, delayed increase in circulating FBG does occur between 24 and 48 hours post-injury.

Conclusion: The results presented in this study indicate that circulating IL-6 is essential for physiologic upregulation of FBG in response to trauma. This finding is in line with past work showing that IL-6 is regulates hepatic secretion of additional coagulation proteins.  In this sense, IL-6 can be viewed as an “SOS” signal released from damaged tissue, inducing the production of essential hemostatic proteins consumed at the site of injury. This schema is illustrated in Figure 1C. Future studies are needed to determine the mechanism of IL-6 release from damaged tissue, and its induction of FBG secretion from the liver. These studies could serve as proof of principle for therapeutic trials designed to treat the pathophysiologic conditions of diminished (hypocoagulable) or excessive (hypercoagulable) circulating FBG.

4.06 An Accurate Method For Predicting Death From Sepsis

Posted on December 22, 2014

J. W. Kuethe1, E. F. Midura1, K. R. Kasten2, C. M. Freeman1, T. C. Rice1, C. C. Caldwell1  1University Of Cincinnati,Division Of Research,Cincinnati, OH, USA 2East Carolina University Brody School Of Medicine,Department Of Surgery,Greenville, NC, USA

Introduction: The successful early immune response to sepsis strikes a balance between microbial eradication and host tissue injury. Unsuccessful clearance often results in persistent inflammatory / immunosuppressive catabolic syndrome (PICS).  Due to a fluctuating inflammatory state, a measure of immune status and an accurate model of risk stratification are critical to the effective use of immune modulating therapies. Determination of leukocyte numbers, their activation state, and cytokine levels has been proposed to stratify such patients. In mice, circulating IL-6 levels allow for risk stratification following cecal ligation and puncture (CLP). Although CLP is the gold standard for inducing sepsis in experimental murine models, lack of source control is a severe limitation when extrapolating to sepsis management in humans. Therefore, we utilized a CLP-Excision (CLP-E) model in which cecal excision, peritoneal wash and antibiotic treatment were performed following CLP. Using this more clinically relevant model, we hypothesized that leukocyte characterization and cytokine measurements, isolated at the time of source control, would allow us to predict survival.

Methods: Outbred mice were subjected to CLP (50% ligation / 20 gauge puncture). After CLP, the their abdomens were re-explored, the necrotic cecums debrided, the abdomens washed and a single intra-peritoneal dose of antibiotics administered. Survival was then monitored. The peritoneal wash was analyzed for IL-6 concentration by ELISA, and neutrophil numbers and activation by flow cytometry.

Results: Following excision, neutrophil characteristics and wash IL-6 levels were analyzed. After assessing for survival, the measurements associated with the mice that lived (LIVE) and those that died (DIE) were used to generate an ROC curve. Two ROC curves were significant in predicting survival (Table).  A marker of neutrophil activation, CD11b was noted to be 67% more elevated in the LIVE group compared to the DIE group (p<0.0001). IL-6 concentration, a marker of inflammation, was observed to be increased 2.2 fold in the DIE group compared to the LIVE group (p<0.001). In a subsequent cohort, neutrophil CD11b and IL-6 accurately predicted risk of death using the appropriate ROC curve.

Conclusion: This technique predicts survival by analyzing surgical waste in a clinically relevant model.  We observed that neutrophil activation was blunted in the DIE cohort, but elevated in the LIVE cohort. Based on this observation, we speculate that treatments to increase neutrophil activation in the DIE cohort would improve survival, but would only exacerbate host tissue injury in the LIVE cohort, thus demonstrating a need to determine immune status prior to considering immune modulating therapies.

 

4.07 The Effects of Exercise on Soleus Function in Severe Burn with Muscle Disuse Atrophy

Posted on December 22, 2014

M. R. Saeman1, K. DeSpain1, M. Liu1, B. Carlson1, L. A. Baer2, J. Song1, C. E. Wade2, S. E. Wolf1  1University Of Texas Southwestern Medical Center,Surgery,Dallas, TEXAS, USA 2University Of Texas Health Science Center At Houston,Surgery,Houston, TEXAS, USA

Introduction:
Muscle loss is a known sequela of severe burn and critical illness that increases the risk of complications such as sepsis and prolonged recovery time. A prior study in a rat model of hindlimb unloading after burn supports that bedrest contributes significantly to muscle atrophy. The aim of our study was to evaluate if exercise mitigates the loss of muscle in this animal model.

Methods:
Two groups of 24 Sprague-Dawley rats were randomly assigned to burn ambulatory (B/A), burn hindlimb unloading (B/H), sham ambulatory (S/A), or sham hindlimb unloading (S/H). One group was trained to perform twice daily weighted resistance climbing of 1 meter with 5 repetitions; the other group had no exercise. Rats received a full thickness scald burn of 40% total body surface area or sham and were allowed to ambulate or were placed in a tail traction system for hindlimb unloading. On day 14 in situ isometric forces were measured on the left soleus muscle. Statistical analysis was performed with Sigma Plot using Student’s t-test, Mann Whitney, or ANOVA with Holm-Sidak method where appropriate. 

Results:
The soleus wet weight was lower in the hindlimb (144 mg) and the exercise (136 mg) versus the ambulatory (190 mg, p<0.001), and no exercise (180 mg, p=0.01) groups. There was no difference in weights between burn and sham. Twitch was significantly lower in the hindlimb group: 31 vs 12 g (p<0.001). Compared to no exercise, the B/H exercise group had a significantly higher twitch force 14 vs. 8 g (p=0.04). Across all other factors there was no significant difference in the twitch between exercise and no exercise. There was a significantly lower tetanic force in the hindlimb group: 55 vs 148 g (p<0.001). B/A had a lower tetanic force in the exercise group versus no exercise: 118 vs 165g (p=0.02). In B/H no difference in tetanic force was seen with or without exercise. All hindlimb groups had significantly lower specific tetanic force than ambulatory: 12 vs. 22 N/cm2 (p<0.001). The specific tetanic force in B/H was significantly higher in exercise versus no exercise: 14 vs. 7 N/cm2 (p=0.008). Fatigue index was significantly lower in the ambulatory (55%) and exercise (52%) groups versus hindlimb (69%) and no exercise (73%) groups (p=0.03, p=0.002 respectively). Muscle function of all groups included in table.

Conclusion:
Hindlimb unloading is a significant factor in muscle atrophy with or without burn. Exercise increased soleus twitch and specific force in this model. However, there was a surprising decrease in muscle mass with exercise in all groups and a decrease in the fatigue index. These findings suggest that exercise contributes to a functional muscle change in a model of disuse and critical illness. 
 

4.08 Stress Conditions Modulate Adipose Tissue Inflammatory Response and Subsequent Organ Injury

Posted on December 22, 2014

D. A. Edelman1, D. M. Liberati1, L. N. Diebel1  1Wayne State University,Surgery/School Of Medicine,Detroit, MI, USA

Introduction: Obesity is an independent risk factor for ARDS and organ failure after severe trauma.  Adipose tissue (AT) composed of adipocytes, macrophages, and other immune cells is a source of pro-inflammatory mediators that are associated with a chronic low grade inflammatory state in the obese patient.  Obesity is also associated with activation of the sympathetic nervous system, which has been linked to shock induced gut and lung injury in the trauma setting.  We hypothesized that sympathomimetic stimulation of adipose tissue would augment inflammatory signaling from adipose tissue and contribute to lung injury and ARDS after trauma.

Methods: Cell co cultures of mature adipocytes and macrophages (RAW264.7) were established and then incubated with either low or high concentrations of epinephrine (10-6 or 10-3  μM respectively). Cell culture supernatants (sup) were obtained at 12 hrs, and AT derived TNF α and IL-6 determined. In separate experiments, human microvascular endothelial cell (HMVEC) monolayers were incubated with adipocyte macrophage sup and HMVEC apoptosis (%apo), ICAM expression (MFI) and FITC-dextran permeability determined.

Results: No difference was seen in co culture data between the low dose epinephrine groups and the no dose epinephrine groups (p<0.001).

Conclusion: Both adipocytes and macrophages contribute to the "obesity related" proinflammatory state. Augmentation of this response after stress related sympathetic activation could contribute to lung injury and other remote organ failure in the injured obese patient. This response seems primarily due to stimulation of the adipocyte component of adipose tissue. The clinical impact likely depends on the magnitude of injury and the distribution/percent total body fat of the patient. Modification of the stress response following trauma may be a therapeutic target in this population. 

 

4.09 Inhibition of Histone Deacetylase 6 Protects the Intestinal Tight Junctions in Hemorrhagic Shock

Posted on December 22, 2014

Z. Chang1, W. He1, B. Liu1, I. Halaweish1, T. Bambakidis1, Y. Li1, H. B. Alam1  1University Of Michigan,Surgery,Ann Arbor, MI, USA

Introduction: We have previously shown that treatment with histone deacetylase (HDAC) 6 inhibitor Tubasatin A (Tub A) can improve outcome in a rat model of septic shock. Also, we have demonstrated that circulating levels of claudin 3, a tight junction (TJ) protein, increase after shock and return to normal after treatment with VPA (pan-HDAC inhibitor). This study investigated whether specific inhibition of HDAC 6 could protect intestinal cellular TJ and improve survival after HS.

Methods: Experiment I: Sprague Dawley rats underwent hemorrhagic shock (40% blood loss over 10 min) followed by treatment with Tub A (70 mg/kg), without any fluid resuscitation. The experimental groups were: (1) sham (no hemorrhage, no treatment), (2) control (hemorrhage, no treatment), and (3) treatment (hemorrhage with Tub A treatment). Animals were sacrificed 6 hrs later, and intestinal tissues were used to create whole cell lysate, which were analyzed for acetyl-tubulin, total tubulin, claudin 3 and zonula occludens 1 (ZO-1) proteins by Western blot. Experiment II: human intestinal epithelial cells (Caco-2) were divided into 3 groups: (1) sham (no hypoxia), (2) control (hypoxia, no treatment), (3) treatment (hypoxia, treatment with tub A). After 12 hours in a hypoxia chamber, cells were analyzed for viability, Lactate dehydrogenase (LDH) levels measuring cellular injury, and TJ protein (claudin 3 and ZO-1) levels.

Results: Hemorrhage decreased, whereas treatment with Tub A increased, expression of claudin 3 and ZO-1. Administration of Tub A also acetylated the tubulin protein. In vitro study showed that hypoxia decreased the viability of caco- 2 cells, which was prevented by Tub A treatment. Similar results were seen when cellular cytotoxicity was determined by the LDH activity. The expression of TJ protein claudin 3 and ZO-1 was significantly decreased by hypoxia, which was significantly prevented by  Tub A treatment. Immunofluorescent study further confirmed that Tub A attenuated the hypoxia-induced claudin 3 and ZO-1 signal loss in the caco-2 cells (Figure). 
 

Conclusion: Selective inhibition of HDAC6 with Tub A preserves the expression of intestinal TJ proteins in models of hemorrhagic shock in vivo and cellular hypoxia in vitro.

4.11 Opioids Promote Anastomotic Leak Following Colo-rectal Surgery in Rats: The Role of the Microbiota

Posted on December 22, 2014

B. A. Shakhsheer1, J. R. Defazio1, J. N. Luo1, R. Klabbers2, I. D. Fleming1, N. Belogortseva1, A. Zaborin1, O. Zaborina1, J. C. Alverdy1  1Pritzker School Of Medicine, University Of Chicago,Department Of Surgery,Chicago, IL, USA 2Radboud University Nijmegen Medical Centre,Department Of Surgery,Nijmegen, , Netherlands

Introduction:  The most dreaded complication after resection of the gastrointestinal tract is anastomotic leak. The effects of opioids on outcomes after gastrointestinal surgery continue to be discovered. Use of patient-controlled analgesia pumps has been associated with increased deep surgical site infection rates by unknown mechanisms. Two findings from our lab may shed light on the mechanisms by which opioids increase anastomotic leak rates: 1. opioids (morphine) directly enhance the virulence of intestinal pathogens and 2. intestinal bacteria play a key causative role in the pathogenesis of leak. Therefore the aim of this study was to examine the effect of opioids on colonic anastomotic leak in a rat model.

Methods:  Rats undergoing one-centimeter colectomy at the peritoneal reflection and primary anastomosis were treated with slow release subcutaneous morphine pellets or placebo pellets implanted in the nape of the neck. Rats were sacrificed on post-operative day six and autopsied for gross signs of anastomotic leak. Microbial composition and phenotype (i.e. collagenase production) was investigated via culture of tissues and intraperitoneal lavage. Local anastomotic tissue was subjected to high magnification microscopy and phenotype analysis.

Results: Rats treated with high-dose (15-mg sustained release) subcutaneous morphine pellets developed a 56% leak rate compared with a 3% leak rate in the placebo treated group (n=73, p=0.0045) High powered images of the anastomotic site demonstrated mucosal ulceration in the morphine group with visible disruptions in the anastomotic integrity whereas no such findings were observed in the placebo treated group. Culture of local tissue and intraperitoneal lavage fluid identified the presence of gram-negative bacteria producing high levels of collagenase which may a play role in anastomotic disruption and non-healing. 

Conclusion: Morphine significantly increases anastomotic leak rates in rats. The role of microbial composition (i.e. gram negative pathogens) and phenotype (i.e. collagenase productions) remains to be investigated and their predictive value confirmed. Taken together these findings provide a rationale to limit opioid use following colorectal surgery and/or to block their peripheral effects with selective opioid antagonists as a countermeasure to prevent deep organ space infection and anastomotic leak.

 

4.12 Agent-based model of Non-Toxigenic Clostridium difficile as Prophylaxis for C. difficile Infection

Posted on December 22, 2014

D. Streicher2, G. An1  1University Of Chicago,Surgery,Chicago, IL, USA 2University Of Michigan,College Of Literature, Science And Arts,Ann Arbor, MI, USA

Introduction:  Non-Toxigenic Clostridium difficile (NTCD) is a generic term for several naturally occurring strains of C. difficile that lack the Pathogenicity Locus (PaLoc) Region, which contains the DNA sequences for Toxins A and B, which are responsible for the pathologic changes in Clostridium difficile Infection (CDI). Otherwise, NTCD shares behavioral properties with other strains of C. difficile, and depletion of commensal microbes by systemic antibiotics affect the suppression of NTCD spores and can trigger their shift from spore to germinated state. Pre-administration of NTCD has been suggested as a prophylaxis for CDI in high-risk population, with the initiation of early stage clinical trials for its study. However, safety concerns have been raised due to recent reports that NTCD is capable of undergoing horizontal gene transfers of the PaLoc region from toxigenic C. difficile. We investigate the feasibility and safety of NTCD prophylaxis with an Agent-based model (ABM) of the gut microbial ecology.

Methods:  A previously produced agent-based model of CDI (CDIABM) was expanded by adding NTCD with both its Germinated (NTCD-G) and Sporulated (NTCD-S) forms. Horizontal Gene Transfer of the PaLoc region was implemented as a stochastic contact effect with toxigenic C. difficile in its germinated form, resulting in the acquisition of toxin producing ability in the target NTCDs. Simulation experiments were performed to examine the prophylactic suppressive and treatment efficacy of NTCD on the development of CDI following systemic antibiotic administration. PaLoc gene transfer events were examined with respect to their impact on persistence and recurrence of CDI.

Results: NTCDs were successfully implemented into the existing CDIABM, and demonstrated qualitatively validated sporulation and germination dynamics. Simulations of different prophylactic NTCD regimens produced a dose dependent suppressive effect on the development of subsequent CDI; however, there was an inflection point identified in the dosing of NTCD and a late phase worsening of CDI. This infection point was associated with the number of horizontal gene transfer events of PaLoc, and demonstrated an exponential effect in low nutrient conditions. Post CDI administration of NTCD did not qualitatively change the trajectory of the CDI.

Conclusion: The modified CDIABM plausibly reproduced the behavior of NTCD in the gut. Prophylactic NTCD appeared to slow the rate and severity of CDI, though it does not prevent damage entirely. The risk of virulizing NTCD through horizontal gene transfer of PaLoc is low, though present with a NTCD dose dependent effect. NTCD was not demonstrated to be effective as a treatment of active CDI. As NTCD prophylaxis is currently being considered in clinical trials, computational investigations of this type can help to augment research and trial design by identifying potential negative effects, characterizing safety concerns, and establishing potential efficacy ranges.

 

4.13 The Anti-Cholinergic Pathway Protects Against Intestinal Barrier Dysfunction and DAMPs Release

Posted on December 22, 2014

M. E. Diebel1, D. M. Liberati1, L. N. Diebel1  1Wayne State University,Michael And Marian Ilitch Department Of Surgery,Detroit, MI, USA

Introduction: Intestinal barrier injury occurs following major trauma and leads to an  intestinal inflammatory response and subsequent remote organ dysfunction. This response may be modulated by either vagal nerve stimulation or pharmacologic stimulation of the alpha7-cholinergic receptor (nAChR) anti-inflammatory pathway. The downstream mediators in this pathway are relatively unknown. Gut injury is also associated with the release of endogenous damage associated molecular patterns (DAMPs) which may modulate shock induced organ dysfunction. The impact of stimulation of the intestinal cholinergic anti-inflammatory pathway on DAMPs release and resultant tissue injury was studied in vitro.

Methods: Intestinal epithelial cell (IEC-6) monolayers were subjected to hypoxia-reoxygenation (H/R) challenge. Cell subsets were treated after hypoxic challenge with nicotine or AR-R17779, a specific nAChR agonist. Nuclear factor kappa light-chain-enhancer of activated B cell (NFkB) activation was determined by ELISA and IEC monolayer integrity was indexed by permeability to an FITC-dextran probe (4,000 mw; FD-4). DAMPs production was indexed by high mobility group box 1 (HMGB-1) (western blot) and mitochondrial DNA (coxIII using RT-PCR) release. Human pulmonary microvascular endothelial cells (HMVEC) were then co cultured with IEC culture supernatants and monolayer permeability and ICAM-1 expression determined.

Results: mean ± S.D., N = 4 for each group

Conclusion: Pharmacologic stimulation of the nAChR pathway protected against H/R induced intestinal barrier derangement, NFkB activation and DAMPs release. Decreased IEC mediated DAMPs release was associated with protection against lung microvascular injury and ICAM-1 expression in this in vitro study. Modulation of this pathway may be helpful in the clinical setting.

 

3.06 Immunotherapeutic Virus GLV-1h153 Fascilitates 131I Radiotherapy and Imaging in Cholangiocarcinoma

Posted on December 22, 2014

C. Johnsen1, J. W. Ady1, K. Mojica1, A. Pugalenthi1, D. Love1, V. Longo6, P. Zanzonico6, N. G. Chen5, R. J. Aguilar5, Y. A. Yu5, A. A. Szalay5, Y. Fong2  1Memorial Sloan-Kettering Cancer Center,Surgery,New York, NY, USA 2City Of Hope National Medical Center,Surgery,Duarte, CA, USA 3University Of California – San Diego,4Department Of Radiation Medicine And Applied Sciences, Rebecca & John Moores Comprehensive Cancer Center,San Diego, CA, USA 4University Of Würzburg,5Department Of Biochemistry, Rudolph Virchow Center For Experimental Biomedicine, And Institute For Molecular Infection Biology,Würzburg, BAVARIA, Germany 5Genelux,Research And Development,San Diego, CA, USA 6Memorial Sloan-Kettering Cancer Center,Small Animal Imaging Core,New York, NY, USA

Introduction:  Cholangiocarcinoma (CC), a deadly carcinoma of the bile ducts, is clinically silent in the majority of patients until curative surgery is no longer an option. With most patients succumbing to the disease within 6 months of diagnosis, novel treatment options are needed.  Oncolytic viruses are promising cancer therapy agents because they selectively infect, replicate within, and kill cancer cells.  In this study, we assess the ability of the human sodium iodide symporter (hNIS) expressing recombinant oncolytic vaccinia virus GLV-1h153 to kill and image CC when combined with 131I radiotherapy.

Methods:  Three human CC cell lines were assayed for infectivity, cytotoxicity and viral replication in vitro.  hNIS mediated 131I radiouptake was assayed in vitro. Flank CC xenografts were treated with intratumoral GLV-1h153 alone and/or with 131I to assess tumor burden reduction. SPECT/CT was performed to visualize 131I uptake in infected tumor in vivo.

Results: All cell lines demonstrated infectivity and oncolysis in a time and concentration dependent manner. Significant viral replication was supported in all cell lines. Flank xenografts treated with GLV-1h153 combined with 131I demonstrated significant tumor reduction as compared to controls.  131I mediated SPECT/CT demonstrated significant uptake in infected tumors. 

Conclusion: GLV-1h153 efficiently kills human CC in vitro. When combined with 131I, GLV-1h153 allows for SPECT/CT imaging of CC tumors and significantly reduces tumor burden in vivo. These results indicate that GLV-1h153 is a promising novel imaging and therapeutic agent for patients with CC.

 

3.07 A Flavonoid and Midkine. Novel Targeted Therapeutic Approach for Hepatocellular Carcinoma

Posted on December 22, 2014

K. M. Sokolowski1, S. Kunnimalaiyaan1, M. Balamurugan1, S. T. Koprowski1, T. C. Gamblin1, M. Kunnimalaiyaan1  1Medical College Of Wisconsin,Surgical Oncology/Surgery/Medical College Of Wisconsin,Milwaukee, WI, USA

Introduction: Despite improvement in therapeutic strategies, median survival in advanced hepatocellular carcinoma (HCC) remains less than one year. Therefore, molecularly targeted compounds with less toxic profiles are needed. Xanthohumol (XN), a prenylated chalcone flavonoid has been shown to have anti-proliferative effects in various cancers types in vitro. XN treatment in healthy mice and humans yielded favorable pharmacokinetics and bioavailability. Although a report providing therapeutic potential of XN in prostate cancer in transgenic mice, the effects of XN on HCC proliferation is unknown. A potential molecular target, midkine (MK- heparin-binding growth factor) is negatively correlated with HCC prognosis and readily detected in serum. Therefore, investigating the effects of XN on HCC cellular proliferation and the evaluation of midkine as a possible therapeutic biomarker is integral.

Methods: The effects of XN on a panel of HCC cell lines were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colometric and colonogenic assay. Cell lysates were analyzed via Western blotting for pro-apoptotic (c-PARP and cleaved caspase-3) and anti-apoptotic proteins (Bcl2, Survivin, and Mcl-1). Mechanism of XN on HCC cellular proliferation was also examined. Midkine expression was evaluated following XN treatment of conditioned HCC media.

Results: Up to 2 µM concentration of XN resulted in cell viabilities exceeding 90% in HCC cell lines. However, 5 µM and above of XN significantly reduced cell viability in a dose-dependent manner. Colonogenic ability also decreased with increasing concentrations of XN (Figure). Additionally, growth suppression due to apoptosis was evidenced by increased expression of pro-apoptotic and reduced anti-apoptotic proteins. Importantly, MK secretion into media was significantly reduced following XN treatment. Furthermore, XN inhibited Notch1 signaling.

Conclusion: Xanthohumol effectively inhibits HCC growth in cell culture and reduces MK secretion. This may provide XN as a novel therapeutic agent and midkine as a potential biomarker. We report for the first time that XN targets Notch1 signaling in HCC cells. The favorable cytotoxic profile based on both healthy mice and human studies along with these findings warrant further preclinical in vivo analysis. Evaluation of Notch1 reduction and its effect on HCC cells following XN treatment is innovative. In addition, confirmation of MK as a therapeutic marker for HCC to XN treatment will be novel. To our knowledge, this is the first characterization of MK and Notch1 inhibition in HCC in response to XN treatment.

 

3.08 Bidirectional TSTA of SHIP-DTA Enhances the Effect of Gene Therapy for Insulinoma

Posted on December 22, 2014

S. Liu1, J. Wu1, G. Zhou1, J. Yu1, R. Sanchez1, F. Brunicardi1  1University Of California – Los Angeles,Department Of Surgery, David Geffen School Of Medicine,Los Angeles, CA, USA

Introduction: Two limitations of gene therapy are tissue specificity and tissue expression of the transgene. We hypothesize that synthetic promoters have greatly enhanced activities over endogenous promoters to drive gene expression and can be used to enhance the effect of gene therapy without loss of target specificity. We have demonstrated that the synthetic human insulin promoter (SHIP) has far greater activity than HIP or rat insulin promoter (RIP) in driving gene expression with subsequent enhanced effect of gene therapy in neuroendocrine tumors which overexpress pancreatic and duodenal homeobox1 (PDX1) transcription factor. Bidirectional two-step transcriptional amplification (TSTA) has been shown to enhance gene expression. In this study we sought to determine that TSTA enhances the expression and therapeutic effect of SHIP-diphtheria toxin A (SHIPTSTA-DTA) in PDX1 expressing cells, such as insulinoma.

Methods: SHIP-eGFP vs SHIPTSTA-eGFP, SHIP- firefly luciferase (FLuc) vs SHIPTSTA-FLuc and SHIP-DTA vs SHIPTSTA-DTA vectors were constructed using subcloning techniques. Activity and specificity of vectors were determined by bioluminescence and eGFP assays in mouse insulinoma (βTC6) cells, human primary pancreatic (HPP) cells, pancreatic ductal epithelial (HPDE) cells and PDX1-HPDE cells, since PDX1 is the primary activator of SHIP, with or without PDX-1 shRNA co-transfection. Cytotoxicity was determined by MTS assay and glucose-stimulated insulin secretion (GSIS) was determined by ELISA. Statistical analysis was performed via paired T test; p<0.05 = significant.

Results: SHIPTSTA-FLuc activity in βTC6 cells was 8 fold higher than that of SHIP-FLuc (p<0.05)(Fig. A). SHIP-FLuc was equal to CMV-driven expression, however was specific for cells expressing PDX1. PDX-1 shRNA co-transfection resulted in decreased SHIPTSTA-FLuc activity.  Similarly, increased cell numbers of eGFP and expression intensity of eGFP were observed in SHIPTSTA-eGFP transfected βTC6 cells vs controls. No FLuc activity was detected in both SHIPTSTA-FLuc and SHIP-FLuc transfected HPP and HPDE cells. SHIPTSTA-DTA resulted in enhanced cytotoxicity and enhanced suppression of proliferation and GSIS from βTC6 cells compared to SHIP-DTA (p<0.05)(Fig. B).

Conclusions:  SHIPTSTA markedly enhanced SHIP activity without loss of tissue specificity; SHIPTSTA-DTA in PDX1+ insulinoma cells enhanced cytotoxicity and suppressed proliferation and insulin secretion. The data support the hypothesis that synthetic promoters of the target gene have greatly enhanced expression efficiency over endogenous promoters and can be used to enhance the effect of gene therapy without loss of target specificity. 
 

3.09 Mitigation of Hypertrophic Scar Contraction in vivo via a Biostable Polyurethane Scaffold

Posted on December 22, 2014

M. M. Ibrahim1, E. R. Lorden2, K. J. Miller1, L. Bashirov1, E. Hammett2, C. Quiles1, A. Rastegarpour1, A. Selim3, K. W. Leong2, H. Levinson1,3  1Duke University Medical Center,Department Of Surgery, Division Of Plastic Surgery,Durham, NC, USA 2Duke University Medical Center,Department Of Biomedical Engineering,Durham, NC, USA 3Duke University Medical Center,Department Of Pathology,Durham, NC, USA

Introduction:

Over 2.4 million Americans and tens of millions of patients worldwide suffer from hypertrophic scar (HSc) contraction following thermal injuries. HSc contraction is a debilitating condition that results in disfigurement and decreased range of motion in affected joints. The current standard of care involves skin grafting with or without the placement of a collagen based, biodegradable, bioengineered skin equivalent (BSE). Commercial BSEs assist in tissue regeneration, but do not focus on mitigating the debilitating effects of HSc and they are marginally effective. To overcome this significant unmet medical need, we have created and tested an elastomeric, polyurethane (PU) based BSE that will last throughout the remodeling phase of repair.  In unwounded skin, collagen is arranged randomly, myofibroblasts are absent, and dermal stiffness is low. Conversely, in scar contractures, collagen is arranged in linear arrays, myofibroblast density is high, and dermal stiffness increases. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that will promote random cellular orientation, decrease myofibroblast formation, and mitigate HSc contraction. 

Methods:

Electrospun PU scaffolds were fabricated, covalently coated with bovine type-1 collagen, characterized for their tensile mechanical properties, and tested in vivo. Scaffolds were surgically inserted beneath skin grafts in a validated immune competent murine burn and HSc model we have previously developed.  Wounds were excised at day 30 post-surgery. Collagen was assessed with Masson. Macrophages were detected with F4/80 immune. Vascularity was assessed with CD31 immune. Myofibroblasts were assessed with alpha-smooth muscle actin (ASMA) immune. Elastic moduli were analyzed using a microstrain analyzer. All were compared to Integra™.

Results:

In vivo, at D30, collagen coated PU scaffolds integrated into the host tissue and restricted wound contraction to 70±4% of the original wound size. Meanwhile, control mice treated with skin graft alone contracted to 45±2%, while wounds treated with Integra™ beneath skin graft contracted down to 28±1.5% of the original size. Histological analysis demonstrated that PU scaffolds promote fibroblast invasion, angiogenesis, and macrophage recruitment compared to skin graft alone and Integra™. ASMA stains showed decreased numbers of myofibroblasts in PU scaffolds compared to control skin grafts and Integra™. Elastic modulus of excised PU treated wounds was significantly lower than skin-grafted control scars.  

Conclusion:

These data suggest that collagen-coated elastomeric electrospun PU scaffolds provide mechanical support to prevent wound bed contraction during healing, and decrease myofibroblast activation associated with HSc . Our long-term goal is to develop a rationally designed, translational medical therapy to mitigate HSc,

3.11 Apigenin Reduces PTHrP-Mediated Inflammation, PSC Proliferation, and ECM Synthesis in Pancreatitis

Posted on December 22, 2014

A. A. Mrazek1, V. Bhatia2, M. Falzon2, M. R. Hellmich1, C. Chao1  1University Of Texas Medical Branch,Surgery,Galveston, TX, USA 2University Of Texas Medical Branch,Pharmacology,Galveston, TX, USA

Introduction: Chronic pancreatitis (CP) is characterized by repeated bouts of acute pancreatitis, resulting in inflammation, glandular necrosis, and irreversible stromal fibrosis. Apigenin (Api) is a natural compound which we have shown to preserve acini and significantly inhibit interstitial fibrosis in a preclinical mouse model of CP. We found that Api down-regulates parathyroid hormone related protein (PTHrP), a pro-inflammatory mediator responsible for amplifying acinar and pancreatic stellate cell (PSC) response to injury. In vitro, Api reduced the viability and induced apoptosis PSCs, the cells responsible for secretion of inflammatory cytokines and the over-deposition of extracellular matrix (ECM). The aim of this study is to determine if Api acts at a transcriptional level, limiting the synthesis of inflammatory mediators (IL-6 and IL-8), markers of PSC proliferation (TGF-β and PCNA), and major components of the ECM, collagen type I and fibronectin (COL1A1 and FN).

Methods: Following an IRB-approved protocol, human PSC were isolated from discarded OR tissue by a standard outgrowth method. The PSC were pretreated with Api 50μM for 1h or vehicle (DMSO) and then stimulated with PTHrP (Bachem, Torrance, CA) 1ng/ml for 12h. Total RNA was isolated (Ambion, Austin, TX); RT-PCR was performed using SYBR Green Supermix (Applied Biosystems, Carlsbad, CA). The relative expression of transcripts was normalized to 18S expression. One-way ANOVA and post-hoc Tukey’s test were assessed with SPSS (IBM, Armonk, NY), and significance was set at p<0.05.

Results: Pretreatment with Api significantly reduced basal mRNA expression of IL-6 and IL-8, TGF-β, PCNA, and fibrillar protein COL1A1 (p<0.001) [Table 1], but not FN. When PSCs were stimulated PTHrP, Api significantly decreased the transcriptional response of all transcripts evaluated (p<0.001).

Conclusions: Api significantly lowers the threshold of basal transcriptional activity in PSC of IL-6 and IL-8 (pro-inflammatory cytokines), TGF-β and PCNA (measures of cellular proliferation), and COL1A1 (the most abundant ECM protein). Additionally, Api reduced PTHrP-stimulated increases in all factors evaluated. Pretreatment with Api did not decrease the basal mRNA levels of FN at 12h, an ECM protein which has been shown to be overabundant in CP. Changes in FN mRNA may be revealed at longer time intervals. These RT-PCR results offer mechanistic insight into the action of Api and confirm the preclinical findings in our murine model of CP, where Api reduced pancreatic fibrosis while preserving normal acinar unit architecture. This further supports apigenin’s development as a therapeutic in pancreatitis.

3.12 IAP Decreases Intestinal Permeability Prior to Developing NEC through Stabilization of Claudin-1

Posted on December 22, 2014

S. Dillman1,2, N. Heinzerling1,2, K. Fredrich1,2, J. Fawley1,2, D. M. Gourlay1,2  1Medical College Of Wisconsin,Pediatric Surgery,Milwaukee, WI, USA 2Children’s Hospital Of Wisconsin,Children’s Research Institute,Milwaukee, WI, USA

Introduction: Recent studies indicate an increase in intestinal permeability precedes necrotizing enterocolitis (NEC).  Prior research has shown supplemental enteral Intestinal Alkaline Phosphatase (IAP), an endogenous protein expressed by the intestines, decreases intestinal permeability and the severity of NEC in a rodent model. The tight junction proteins, claudins, are essential in creating a functional epithelial intestinal barrier. Increased expression and altered distribuiton of Claudin-1 (Cl-1) during intestinal inflammation was associated with increased intestinal permeability leading to a breach in mucosal barrier. We hypothesize that IAP decreases intestinal permeability prior to the development NEC through modification of CL-1. 

Methods: One day preterm Sprague Dawley rat pups were delivered via cesarean section and fed formula. Select pups received 4 units/kg of IAP in their formula. Pups were sacrificed on day of life 2-4 and the terminal ileum was harvested. Intestinal permeability was measured by filling loops of intestine with a fluorescein isothiocyanate-dextran (FITC-dextran) solution and measuring flux into the incubating media. Three day old dam-fed wild-type, heterozygotes, and IAP KO rat tissue was also harvested. To examine the effect of IAP on intestinal epithelial cell, in vitro experimentation involved FHs 74, a fetal epithelial cell culture. Cells were given either formula or formula plus 4 units of IAP. ZO-1, CL-1, 2, and 3 were measured by RT-PCR and Western Blot on all cells and ileal intestinal homogenates. Statistical analysis was performed using a paired t-test and a p<0.05 considered significant. 

Results: Intestine loops exposed to lipopolysaccharide and FITC-dextran had a greater statistically significant increase in permeability over controls.  Supplemental, enteral IAP significantly decreased intestinal permeability.  Evaluation of ileal intestinal homogenates showed a significant decrease messenger RNA levels for CL-1, 2, and 3 in IAP fed rats. For Western Blot analysis, ZO-1 and CL-1 displayed a significant increase in the IAP fed group while CL-2 showed a slight decrease in protein expression.  In the IAP gene knockout rats, CL-1 showed a significant increase mRNA expression compared to wild-type.  In the FHs 74 cell line, Cl-1 expression was significantly decreased in the IAP group while the other claudins didn’t have noticeable expression in either group. 

Conclusion: Neonatal intestinal permeability is increased by formula feeding and LPS and may increase the risk of NEC and sepsis. These data suggest IAP has a direct effect on expression and stability of CL-1 in the neonatal intestinal epithelial cell that is a novel mechanism by which IAP may function to protect newborns from NEC. .  

 

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