- Email: [email protected]
P259
334
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
genic properties of PDC may be related to differential expression of surface coregulatory molecules, such as programmed death ligand -1 (PD-L1). Here we present preliminary evidence of a correlation between circulating pre-DC expressing comparatively high levels of PD-L1 and Treg in operationally tolerant liver transplant recipients. Methods: We compared the expression of blood cell phenotype by multi-color flow cytometric analysis between operationally tolerant pediatric liver transplant patients (TOL; n⫽6), patients undergoing prospective, uninterrupted drug withdrawal (PW; n⫽7), patients requiring maintenance immunosuppression (MI; n⫽4) and normal healthy individuals (N; n⫽6). Results: The percentage of total T cells (CD3⫹), T cells subsets (CD4⫹, CD8 ⫹, CD4 ⫹CD8 ⫹, and CD4 -CD8 -) and NK cells (CD3CD56 ⫹) did not differ among the four groups. There was also no difference in the percentage of CD45RO⫹memory CD4⫹ or CD8 ⫹ T cells between the groups. The incidence of CD4⫹CD25 high Treg in TOL patients did not differ from that in PW patients or N. However, TOL patients had significantly higher CD4⫹CD25 high Treg (p⬍0.05) and a higher PD-L1/CD86 ratio (p⬍0.01) on pre-PDC compared with MI patients. Conclusion: Although the relevance of those findings to the tolerant state remains to be established, the results suggest a possible functional inter-relationship between PDC and Treg in operational liver transplant tolerance.
P257. FACTORS CORRELATED WITH SUCCESSFUL HUMAN ISLET ISLATION. X. Zhao 1, M. R. Garfinkel 2; 1Metrohealth Medical Center in Cleveland, Cleveland, OH, 2Univeristy of Chicago, Chicago, IL Introduction: Since the publication of the Edmonton protocol in 2000, human islet transplantation has become a promising alternative to whole pancreas transplantation in the treatment of type I diabetes. Human islet transplantation is a complex procedure where many factors affect the final islet yield. As a brief overview, a donor pancreas is procured and preserved before it is enzymatically digested and purified using continuous density gradients in a processing facility. After purification, if the final yield of islets is greater than 5,000 islet equivalents (IEQ) per kilogram of the intended recipient’s body weight, islets can be infused through hepatic portal vein injection. Even with the ever increasing number of islet transplantations reported worldwide, still less than half of all processed pancreata provided transplantable islet yields, which raises an important question, “What is (or are) the decisive factor(s) for human islet isolation?”. In this study, we retrospectively reviewed data from the 35 most recent human islet isolations processed in our facility. The descriptive statistics and correlative study were used to compare differences between the those isolations that produced a transplantable yield versus those that did not. Methods: From April 2005 through February 2006, the human islet transplantation team at U of C processed 35 human pancreata for islet transplantation, under the direct guidance of FDA-approved islet isolation Standard Operating Procedures (SOP). 7 of 35 isolations were able to generate transplantable quality islets and were subsequently infused into four different recipients. In this retrospective study, single variable such as: donor’ age, Body Mass Index (BMI), Cold Ischemia Time (CIT) and Pancreas Weight, or combination of age, BMI, CIT and pancreas weight (XYZ score) were evaluated as a predictor for transplantable yields. In XYZ score, age, BMI, CIT and pancreas weight were converted into number ranging from 0 to 5 based on pre-defined score system and XYZ score was the sum of the converted numbers. These results are expressed as mean ⫾ STDEV Also, a two-tailed Student’s t Test was used to compare the means among the two groups (yields leading to transplantation vs. yields not leading to transplantation). The correlation coefficient (r) was computed by graphpad insat., P⬍ 0.05 ⫽ not significant (ns). Results: In the 35 pancreata, 18 were from female donors and 17 from male. Five of seven isolations resulting in transplantations were from male donors. The average age was 46.8⫾14.5. The average BMI was 29.9⫾5.5. the average pancreas weight was 102.2⫾24.2. The average pre-purification and postpurification islet yields were 462557⫾245451 and 197871⫾117940. The
average XYZ score is 9.2⫾2.4. Individually, Donor’s age, BMI, pancreas’ CIT and weight did not correlate with post-purified islet yields (p values are 0.12, 0.09, 0.16 and 0.18 respectively). The combination of variables or XYZ score correlated with the post-purified islet yields ( p⫽0.02). Conclusion: Human islet isolation is a complex procedure with many variables. Through the review of 35 cases, we conclude that single variable is not able to predict final islet yields. XYZ score which combines variables is able to predict final islet yields. P258. INTERMEDIATE-TERM OUTCOMES WITH EARLY STEROID WITHDRAWAL (ESW) IN AFRICANAMERICAN (AA) RENAL ALLOGRAFT RECIPIENTS (RARS). S. A. Gruber, K. Morawski, M. S. West, J. E. Losanoff, E. Cincotta, M. D. Doshi, J. M. El-Amm; Wayne State University School of Medicine, Detroit, MI Introduction: Multiple investigators have demonstrated excellent results with ESW in low-risk RARs (low panel reactive antibody [PRA] Caucasians undergoing first transplant with immediate graft function [IGF]). However, AAs are considered a high-risk subgroup, with higher reported rates of acute and chronic rejection and graft loss. As a result, there has been a greater reluctance to use ESW in this population and very few reports with at least one-year follow-up in all patients (pts). Methods: We examined the outcomes of 57 adult AA RARs transplanted from 6/22/03 to 6/29/05 receiving ESW, with minimum and mean follow-up 12 and 23 ⫾ 8 months (mo), respectively. Only pts who were on prednisone at the time of transplant or had current PRA ⱖ 50% were excluded from ESW. Our initial protocol consisted of 4 doses of Thymoglobulin (ATG), 1.5 mg/kg, for induction, and mycophenolate mofetil (MMF) 1 g BID started day 1 ⫹ tacrolimus (TCL), started day 1-2 with target trough levels 10-12 ng/ml, for maintenance. After 1/20/04, pts with IGF were randomized to receive either TCL or sirolimus (SRL; troughs 10-12 ng/ml). Pt demographics were as follows: age 47 ⫾ 13; 4.8 ⫾ 3.9 years ESRD; 7% retransplant; 19% hepatitis C⫹; 19% live donor; 15 ⫾ 7 hour cold ischemic time; 7% with current PRA ⱖ 10%; 42% with peak PRA ⱖ 10%; 4.4 ⫾ 1.4 HLA mismatches; and 40% AA donor. Results: 48 pts were initiated on TCL, 9 on SRL. Conversions between maintenance agents were performed as clinically indicated for untoward side effects. Only 10 pts received ⬎ 4 (5-7) doses of ATG. Outcome data: delayed graft function (DGF): 39%; pt survival 98% at 1 yr, 95% overall; graft survival 96% at 1 yr, 89% overall; acute rejection (AR) 18%; CMV infection 7%; creatinine at 6 and 12 mo: 1.6 ⫾ 0.5 and 1.7 ⫾ 0.9 mg/dl, respectively; mean/median initial length of stay: 4.7/4 days. 6 graft losses were due to 3 deaths (2 malignancy, 1 infection), 1 noncompliance, and 2 FSGS. There were 2 cases of BK virus nephropathy. Of 10 pts developing AR, 2 were equivocal, 5 grade 1, 1 grade 2A, and 2 antibody-mediated. 46 pts (81%) remained steroid free at 1 yr, of which 11 (24%) were also calcineurin inhibitor free. % weight gain at 6 and 12 mo (3.6 ⫾ 8.9 & 7.4 ⫾ 8.9) and incidence of posttransplant diabetes (16%) in pts at risk were numerically less, but not significantly different from, values obtained in a group of 41 historical and concomitantly-treated AA RARs receiving ATG/MMF ⫹ TCL or SRL ⫹ maintenance steroids (4.4 ⫾ 10.6 & 9.1 ⫾ 14.5; 18%, respectively). Conclusions: Our findings suggest that ESW in AA RARs with multiple risk factors including DGF can produce excellent intermediate-term antirejection and graft functional outcomes. Our results will need to be verified in larger numbers of AA RARs with longer follow-up. P259. NO COMPROMISED SURVIVAL IN DONOR HEARTS WITH ECHOCARDIOGRAPHIC ABNORMALITIES. N. Sopko, K. Shea, III, K. Ludrosky, N. Smedira, K. Hoercher, D. Taylor, R. Starling, G. Gonzalez-Stawinski; Cleveland Clinic, Cleveland, OH Objective: To report on pre-discharge outcomes and long-term survival in recipients with donor heart echocardiographic abnormalities.
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS Methods: Retrospective chart review of heart donors utilized over 6 years. Donor charts provided demographic information, cause of death, and echo results. Recipient charts were queried for pre-discharge echo and survival results. Results: From Jan 1999 to Dec 2005, 485 heart transplants were performed. Of these, 50 donors had 70 echo abnormalities. Mean donor age was 32yrs (range 4-51 yrs). Echo abnormalities included 30 (60%) wall motion abnormalities, 20 (40%) LVH, 16 (32%) mitral regurgitations, 2 (4%) aortic regurgitations, and 2 (4%) LV dilations. Mean EF was 54.0% (range 40-70%). Echo abnormalities post-transplant decreased significantly to 18 (64% decrease) pts with 21 (70% decrease) echo abnormalities. Wall motion abnormalities resolved in 27 (90% decrease) pts (p⬍0.01), LVH resolved in 14 (70% decrease) pts (p⬍0.01), and mitral regurgitation resolved in 12 (75% decrease) pts (p⬍0.01). Aortic regurgitation and LV dilation resolved in all 2 pts. However, RV dilation significantly increased in 8 pts (p⬍0.01). Mean EF at discharge was 56.9% (range 45-89%). Overall survival was 96% at a mean follow-up of 4 years (range 2-6 yrs). Conclusion: Majority of echocardiographic abnormalities in donor hearts resolve prior to discharge. 4 year survival with these hearts is as good as donor hearts without echocardiographic abnormalities. The clinical significance of RV dilation is unclear.
TRAUMA/CRITICAL CARE VII: HEMORRHAGIC SHOCK P260. ETHANOL EFFECTS PROINFLAMMATORY STATE OF NEUTROPHILS IN SHOCK. P. B. Amin, L. N. Diebel, D. M. Liberati; Wayne State University/Detroit Medical Center, Detroit, MI Introduction: Ethanol (EtOH) intoxication plays an important role in the etiology of traumatic events and has often been described as having immunosuppressive effects. EtOH has been shown to effect intestinal barrier function in prior studies. The ability of gut derived factors on neutrophil function in this setting is unknown. This study looks at the role of ethanol in modulating proinflammatory states in the neutrophil in vitro. Methods: Confluent Caco2 monolayers were exposed to 0.1% EtOH and/or Escherichia Coli C-25 (EC) under normoxia (21%O 2) or hypoxia (5%O 2) followed by normoxic conditions (H/R). Neutrophils were then incubated with the supernatants from the treated cells. Chemotaxis, elastase and superoxide anion release, and CD11b measurements were undertaken in these neutrophils and compared to controls. Results: Group PMN Alone Caco2 Caco2⫹ EtOH Caco2⫹EtoH⫹EC Caco2⫹EtOH⫹H/R Caco2⫹EtOH⫹EC⫹ H/R
%Elastase
%0 2-
%CD11b Chemotaxis
19.8⫾1.5 5.5⫾0.5 27.5⫾1.7 20.7⫾1.4 6.3⫾0.3 44.7⫾5.2* 32.3⫾1.5* 10⫾0.2* 48.2⫾4.5* 44.5⫾1.5* 12.9⫾1.3* 59.8⫾3.1* 44.4⫾0.8* 16.4⫾2.1* 55.2⫾3.3*
575⫾25 1125⫾125 2583⫾1018 2750⫾1000 4062⫾1187*
48.7⫾1.6* 17.1⫾0.2* 67.5⫾3.5* 4312⫾1687*
*p⬍0.001 vs control Conclusion: EtOH has a proinflammatory role in the activation of neutrophils at the intestinal epithelial cell barrier in shock states. EtOH may play an important role in worsening septic complication in severely traumatized patients via dysregulation of neutrophils.
P261. STORED BLOOD CONTAINS HIGH CONCENTRATIONS OF ACTIN. T. L. Nydam 1, M. R. Kelher 2, E. E. Moore 3, R. C. McIntyre, Jr. 1, S. S. Damle 1, C. C. Silliman 2; 1 University of Colorado Health Science Center, Denver, CO, 2 Bonfils Blood Center, Denver, CO, 3Denver Health Medical Center, Denver, CO
335
The release of intracellular actin following severe injury contributes to multiple organ failure (MOF). In the circulation, actin polymerizes, causing microthrombi, endothelial injury, activation of platelets, and end-organ ischemia. Since both the amount and age of transfused blood are an independent risk factors for postinjury MOF, we hypothesized that actin accumulates in the plasma fraction of stored blood products. Methods: Serial samples were taken from day 1 and 42 stored packed red blood cells (PRBCs) and day 1 and 7 apheresis platelets, both washed (w) and unwashed (uw). Control samples of fresh blood were obtained from a healthy volunteer. Samples were diluted in 3% SDS buffer and equal volumes were analyzed by separation of the proteins by SDS gel electrophoresis, transfer to nitrocellulose and the immunoreactivity determined via western blot. The relative actin amounts were measured using densitometry (Image J, NIH). Statistical analysis was done using paired ANOVA, followed by a Bonferroni post hoc analysis for multiple comparisons and statistical significance was determined at the p⬍0.05 level. Results: Fresh plasma had small but detectable amounts of actin. The average amount of actin measured in the day 1 PRBC plasma was 5 times that in the fresh plasma. The average amount of actin measured in the day 42 PRBC plasma was 8 times that in the fresh plasma, and nearly 2 times that in the day 1. The large concentration of actin found in platelet plasma was completely removed by washing. Conclusion: The plasma in stored transfusion products contains actin concentrations much higher than that of fresh plasma. The largest increase is associated with the initial processing but continues with age. Washing completely removes the actin. These results implicate another means by which blood transfusion contributes to MOF, and suggest that pre-transfusion washing be considered for the injured patient.
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
genic properties of PDC may be related to differential expression of surface coregulatory molecules, such as programmed death ligand -1 (PD-L1). Here we present preliminary evidence of a correlation between circulating pre-DC expressing comparatively high levels of PD-L1 and Treg in operationally tolerant liver transplant recipients. Methods: We compared the expression of blood cell phenotype by multi-color flow cytometric analysis between operationally tolerant pediatric liver transplant patients (TOL; n⫽6), patients undergoing prospective, uninterrupted drug withdrawal (PW; n⫽7), patients requiring maintenance immunosuppression (MI; n⫽4) and normal healthy individuals (N; n⫽6). Results: The percentage of total T cells (CD3⫹), T cells subsets (CD4⫹, CD8 ⫹, CD4 ⫹CD8 ⫹, and CD4 -CD8 -) and NK cells (CD3CD56 ⫹) did not differ among the four groups. There was also no difference in the percentage of CD45RO⫹memory CD4⫹ or CD8 ⫹ T cells between the groups. The incidence of CD4⫹CD25 high Treg in TOL patients did not differ from that in PW patients or N. However, TOL patients had significantly higher CD4⫹CD25 high Treg (p⬍0.05) and a higher PD-L1/CD86 ratio (p⬍0.01) on pre-PDC compared with MI patients. Conclusion: Although the relevance of those findings to the tolerant state remains to be established, the results suggest a possible functional inter-relationship between PDC and Treg in operational liver transplant tolerance.
P257. FACTORS CORRELATED WITH SUCCESSFUL HUMAN ISLET ISLATION. X. Zhao 1, M. R. Garfinkel 2; 1Metrohealth Medical Center in Cleveland, Cleveland, OH, 2Univeristy of Chicago, Chicago, IL Introduction: Since the publication of the Edmonton protocol in 2000, human islet transplantation has become a promising alternative to whole pancreas transplantation in the treatment of type I diabetes. Human islet transplantation is a complex procedure where many factors affect the final islet yield. As a brief overview, a donor pancreas is procured and preserved before it is enzymatically digested and purified using continuous density gradients in a processing facility. After purification, if the final yield of islets is greater than 5,000 islet equivalents (IEQ) per kilogram of the intended recipient’s body weight, islets can be infused through hepatic portal vein injection. Even with the ever increasing number of islet transplantations reported worldwide, still less than half of all processed pancreata provided transplantable islet yields, which raises an important question, “What is (or are) the decisive factor(s) for human islet isolation?”. In this study, we retrospectively reviewed data from the 35 most recent human islet isolations processed in our facility. The descriptive statistics and correlative study were used to compare differences between the those isolations that produced a transplantable yield versus those that did not. Methods: From April 2005 through February 2006, the human islet transplantation team at U of C processed 35 human pancreata for islet transplantation, under the direct guidance of FDA-approved islet isolation Standard Operating Procedures (SOP). 7 of 35 isolations were able to generate transplantable quality islets and were subsequently infused into four different recipients. In this retrospective study, single variable such as: donor’ age, Body Mass Index (BMI), Cold Ischemia Time (CIT) and Pancreas Weight, or combination of age, BMI, CIT and pancreas weight (XYZ score) were evaluated as a predictor for transplantable yields. In XYZ score, age, BMI, CIT and pancreas weight were converted into number ranging from 0 to 5 based on pre-defined score system and XYZ score was the sum of the converted numbers. These results are expressed as mean ⫾ STDEV Also, a two-tailed Student’s t Test was used to compare the means among the two groups (yields leading to transplantation vs. yields not leading to transplantation). The correlation coefficient (r) was computed by graphpad insat., P⬍ 0.05 ⫽ not significant (ns). Results: In the 35 pancreata, 18 were from female donors and 17 from male. Five of seven isolations resulting in transplantations were from male donors. The average age was 46.8⫾14.5. The average BMI was 29.9⫾5.5. the average pancreas weight was 102.2⫾24.2. The average pre-purification and postpurification islet yields were 462557⫾245451 and 197871⫾117940. The
average XYZ score is 9.2⫾2.4. Individually, Donor’s age, BMI, pancreas’ CIT and weight did not correlate with post-purified islet yields (p values are 0.12, 0.09, 0.16 and 0.18 respectively). The combination of variables or XYZ score correlated with the post-purified islet yields ( p⫽0.02). Conclusion: Human islet isolation is a complex procedure with many variables. Through the review of 35 cases, we conclude that single variable is not able to predict final islet yields. XYZ score which combines variables is able to predict final islet yields. P258. INTERMEDIATE-TERM OUTCOMES WITH EARLY STEROID WITHDRAWAL (ESW) IN AFRICANAMERICAN (AA) RENAL ALLOGRAFT RECIPIENTS (RARS). S. A. Gruber, K. Morawski, M. S. West, J. E. Losanoff, E. Cincotta, M. D. Doshi, J. M. El-Amm; Wayne State University School of Medicine, Detroit, MI Introduction: Multiple investigators have demonstrated excellent results with ESW in low-risk RARs (low panel reactive antibody [PRA] Caucasians undergoing first transplant with immediate graft function [IGF]). However, AAs are considered a high-risk subgroup, with higher reported rates of acute and chronic rejection and graft loss. As a result, there has been a greater reluctance to use ESW in this population and very few reports with at least one-year follow-up in all patients (pts). Methods: We examined the outcomes of 57 adult AA RARs transplanted from 6/22/03 to 6/29/05 receiving ESW, with minimum and mean follow-up 12 and 23 ⫾ 8 months (mo), respectively. Only pts who were on prednisone at the time of transplant or had current PRA ⱖ 50% were excluded from ESW. Our initial protocol consisted of 4 doses of Thymoglobulin (ATG), 1.5 mg/kg, for induction, and mycophenolate mofetil (MMF) 1 g BID started day 1 ⫹ tacrolimus (TCL), started day 1-2 with target trough levels 10-12 ng/ml, for maintenance. After 1/20/04, pts with IGF were randomized to receive either TCL or sirolimus (SRL; troughs 10-12 ng/ml). Pt demographics were as follows: age 47 ⫾ 13; 4.8 ⫾ 3.9 years ESRD; 7% retransplant; 19% hepatitis C⫹; 19% live donor; 15 ⫾ 7 hour cold ischemic time; 7% with current PRA ⱖ 10%; 42% with peak PRA ⱖ 10%; 4.4 ⫾ 1.4 HLA mismatches; and 40% AA donor. Results: 48 pts were initiated on TCL, 9 on SRL. Conversions between maintenance agents were performed as clinically indicated for untoward side effects. Only 10 pts received ⬎ 4 (5-7) doses of ATG. Outcome data: delayed graft function (DGF): 39%; pt survival 98% at 1 yr, 95% overall; graft survival 96% at 1 yr, 89% overall; acute rejection (AR) 18%; CMV infection 7%; creatinine at 6 and 12 mo: 1.6 ⫾ 0.5 and 1.7 ⫾ 0.9 mg/dl, respectively; mean/median initial length of stay: 4.7/4 days. 6 graft losses were due to 3 deaths (2 malignancy, 1 infection), 1 noncompliance, and 2 FSGS. There were 2 cases of BK virus nephropathy. Of 10 pts developing AR, 2 were equivocal, 5 grade 1, 1 grade 2A, and 2 antibody-mediated. 46 pts (81%) remained steroid free at 1 yr, of which 11 (24%) were also calcineurin inhibitor free. % weight gain at 6 and 12 mo (3.6 ⫾ 8.9 & 7.4 ⫾ 8.9) and incidence of posttransplant diabetes (16%) in pts at risk were numerically less, but not significantly different from, values obtained in a group of 41 historical and concomitantly-treated AA RARs receiving ATG/MMF ⫹ TCL or SRL ⫹ maintenance steroids (4.4 ⫾ 10.6 & 9.1 ⫾ 14.5; 18%, respectively). Conclusions: Our findings suggest that ESW in AA RARs with multiple risk factors including DGF can produce excellent intermediate-term antirejection and graft functional outcomes. Our results will need to be verified in larger numbers of AA RARs with longer follow-up. P259. NO COMPROMISED SURVIVAL IN DONOR HEARTS WITH ECHOCARDIOGRAPHIC ABNORMALITIES. N. Sopko, K. Shea, III, K. Ludrosky, N. Smedira, K. Hoercher, D. Taylor, R. Starling, G. Gonzalez-Stawinski; Cleveland Clinic, Cleveland, OH Objective: To report on pre-discharge outcomes and long-term survival in recipients with donor heart echocardiographic abnormalities.
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS Methods: Retrospective chart review of heart donors utilized over 6 years. Donor charts provided demographic information, cause of death, and echo results. Recipient charts were queried for pre-discharge echo and survival results. Results: From Jan 1999 to Dec 2005, 485 heart transplants were performed. Of these, 50 donors had 70 echo abnormalities. Mean donor age was 32yrs (range 4-51 yrs). Echo abnormalities included 30 (60%) wall motion abnormalities, 20 (40%) LVH, 16 (32%) mitral regurgitations, 2 (4%) aortic regurgitations, and 2 (4%) LV dilations. Mean EF was 54.0% (range 40-70%). Echo abnormalities post-transplant decreased significantly to 18 (64% decrease) pts with 21 (70% decrease) echo abnormalities. Wall motion abnormalities resolved in 27 (90% decrease) pts (p⬍0.01), LVH resolved in 14 (70% decrease) pts (p⬍0.01), and mitral regurgitation resolved in 12 (75% decrease) pts (p⬍0.01). Aortic regurgitation and LV dilation resolved in all 2 pts. However, RV dilation significantly increased in 8 pts (p⬍0.01). Mean EF at discharge was 56.9% (range 45-89%). Overall survival was 96% at a mean follow-up of 4 years (range 2-6 yrs). Conclusion: Majority of echocardiographic abnormalities in donor hearts resolve prior to discharge. 4 year survival with these hearts is as good as donor hearts without echocardiographic abnormalities. The clinical significance of RV dilation is unclear.
TRAUMA/CRITICAL CARE VII: HEMORRHAGIC SHOCK P260. ETHANOL EFFECTS PROINFLAMMATORY STATE OF NEUTROPHILS IN SHOCK. P. B. Amin, L. N. Diebel, D. M. Liberati; Wayne State University/Detroit Medical Center, Detroit, MI Introduction: Ethanol (EtOH) intoxication plays an important role in the etiology of traumatic events and has often been described as having immunosuppressive effects. EtOH has been shown to effect intestinal barrier function in prior studies. The ability of gut derived factors on neutrophil function in this setting is unknown. This study looks at the role of ethanol in modulating proinflammatory states in the neutrophil in vitro. Methods: Confluent Caco2 monolayers were exposed to 0.1% EtOH and/or Escherichia Coli C-25 (EC) under normoxia (21%O 2) or hypoxia (5%O 2) followed by normoxic conditions (H/R). Neutrophils were then incubated with the supernatants from the treated cells. Chemotaxis, elastase and superoxide anion release, and CD11b measurements were undertaken in these neutrophils and compared to controls. Results: Group PMN Alone Caco2 Caco2⫹ EtOH Caco2⫹EtoH⫹EC Caco2⫹EtOH⫹H/R Caco2⫹EtOH⫹EC⫹ H/R
%Elastase
%0 2-
%CD11b Chemotaxis
19.8⫾1.5 5.5⫾0.5 27.5⫾1.7 20.7⫾1.4 6.3⫾0.3 44.7⫾5.2* 32.3⫾1.5* 10⫾0.2* 48.2⫾4.5* 44.5⫾1.5* 12.9⫾1.3* 59.8⫾3.1* 44.4⫾0.8* 16.4⫾2.1* 55.2⫾3.3*
575⫾25 1125⫾125 2583⫾1018 2750⫾1000 4062⫾1187*
48.7⫾1.6* 17.1⫾0.2* 67.5⫾3.5* 4312⫾1687*
*p⬍0.001 vs control Conclusion: EtOH has a proinflammatory role in the activation of neutrophils at the intestinal epithelial cell barrier in shock states. EtOH may play an important role in worsening septic complication in severely traumatized patients via dysregulation of neutrophils.
P261. STORED BLOOD CONTAINS HIGH CONCENTRATIONS OF ACTIN. T. L. Nydam 1, M. R. Kelher 2, E. E. Moore 3, R. C. McIntyre, Jr. 1, S. S. Damle 1, C. C. Silliman 2; 1 University of Colorado Health Science Center, Denver, CO, 2 Bonfils Blood Center, Denver, CO, 3Denver Health Medical Center, Denver, CO
335
The release of intracellular actin following severe injury contributes to multiple organ failure (MOF). In the circulation, actin polymerizes, causing microthrombi, endothelial injury, activation of platelets, and end-organ ischemia. Since both the amount and age of transfused blood are an independent risk factors for postinjury MOF, we hypothesized that actin accumulates in the plasma fraction of stored blood products. Methods: Serial samples were taken from day 1 and 42 stored packed red blood cells (PRBCs) and day 1 and 7 apheresis platelets, both washed (w) and unwashed (uw). Control samples of fresh blood were obtained from a healthy volunteer. Samples were diluted in 3% SDS buffer and equal volumes were analyzed by separation of the proteins by SDS gel electrophoresis, transfer to nitrocellulose and the immunoreactivity determined via western blot. The relative actin amounts were measured using densitometry (Image J, NIH). Statistical analysis was done using paired ANOVA, followed by a Bonferroni post hoc analysis for multiple comparisons and statistical significance was determined at the p⬍0.05 level. Results: Fresh plasma had small but detectable amounts of actin. The average amount of actin measured in the day 1 PRBC plasma was 5 times that in the fresh plasma. The average amount of actin measured in the day 42 PRBC plasma was 8 times that in the fresh plasma, and nearly 2 times that in the day 1. The large concentration of actin found in platelet plasma was completely removed by washing. Conclusion: The plasma in stored transfusion products contains actin concentrations much higher than that of fresh plasma. The largest increase is associated with the initial processing but continues with age. Washing completely removes the actin. These results implicate another means by which blood transfusion contributes to MOF, and suggest that pre-transfusion washing be considered for the injured patient.