A Novel Agent for Improved Cryopreservation of Adipose Tissue

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 46.2. The Pathogenesis of Small-for-Size Syndrome after Living Donor Liver Transplantation. M. Ninomiya, H. Kayashima, H. Uchiyama, Y. Soejima, A. Taketomi, K. Shirabe, Y. Maehara; Kyushu University, Fukuoka, Japan Introduction: Too small liver volume after massive hepatectomy or partial liver transplantation is a major cause of subsequent liver dysfunction. Our hypothesis is that the abrupt regenerative response of small remnant liver is responsible for subsequent deleterious outcome. We have tested the hypothesis by controlling the regenerative speed pharmacologically after partial hepatectomy in a rat model. Methods: In order to slow down the regenerative speed, NS-398 (ERK1/2 inhibitor) or PD98059 (selective MEK inhibitor) was administered after 70% or 90% partial hepatectomy. The effect of regenerative speed on liver morphology, portal pressure and survival were assessed. Results: In the 70% partial hepatectomy model, NS-398 treatment suppressed the abrupt replicative response of hepatocytes during the early phase of regeneration, although liver volumes at day 7 were not different from the control group. Immunohistochemical analysis of CD31 (for sinusoids) and AGp110 (for bile canaliculi) revealed that lobular architectural disturbance was alleviated by NS-398 treatment, forming fewer hepatic islands. The elevation of portal pressure at day 3 was also suppressed by NS-398 treatment. In the 90% partial hepatectomy model, administration of NS-398 or PD98059 significantly enhanced the survival (Figure). Furthermore, acceleration of regenerative response by deletion variant hepatocyte growth factor, a potent inducer of hepatocyte replication, did not improve the survival. Conclusions: These observations suggest that the abrupt regenerative response of small remnant liver is responsible for intensive lobular derangement and subsequent liver dysfunction. The suppression of MEK/ERK signaling pathway in the early phase after hepatectomy makes the regenerative response linear and ameliorates the prognosis of small remnant liver.

46.3. NOD1 Ligands, but Not NOD2 Ligands, Potentiate Cytokines to Stimulate Nitric Oxide Production and Chemokine Production in Hepatocytes. C. Chen, Q. Sun, T. R. Billiar, M. J. Scott; University of Pittsburgh, Pittsburgh, PA Introduction: Hepatocytes (HC) express high levels of NOD1 and NOD2, which are cytoplasmic pattern recognition receptors. Both NOD1 and NOD2 are stimulated by bacterial cell wall components. Little is known, however, about hepatocyte (HC) responses to NOD ligands and the role of NOD receptors in the liver. In this study, we investigated the role of NOD1 and NOD2 in HC by assessing responses to specific NOD1 and NOD2 agonists. Methods: Isolated primary mouse HC were treated for up to 24 h with 100ng/mL LPS (TLR4-ligand), 100ng/mL iE-DAP (NOD1 ligand), or 10ug/mL MDP (NOD2 ligand). Other cells were given cytokines IFNg

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(100U/ml), TNFa (500U/ml), IL-1b (100U/ml), alone and in combination with NOD ligands. Whole cell lysates were collected and immunoblotted for iNOS. Cell supernatants were analyzed by ELISA for chemokines (RANTES, MCP-1, KC, MIG) and acute phase proteins (SAA, LBP) and nitrite (NO) concentration determined by Greiss reaction. Western blots of whole cell lysates assessed iNOS expression All samples were run in duplicate, and experiments repeated at least 4 times using hepatocytes isolated from 4 separate mice. Results: iNOS was strongly upregulated in HC by cytokine mix (TNFa, IFNg, and IL-1b). LPS, iEDAP or MDP alone did not induce NO production or upregulate iNOS. However, iEDAP (NOD1 ligand) significantly potentiated iNOS and NO production induced by cytokine mix (7.47mMþ/-0.03 vs 34.72mMþ/-0.2; p < 0.0001). iNOS and NO was also significantly upregulated by iEDAP with IFNg. Treatment of HC with iEDAP for 24 h also significantly increased RANTES and KC levels compared with LPS (RANTES: 1189þ/12pmol vs 508þ/-10pmol; p < 0.05). MDP did not increase chemokine production in HC. Acute phase proteins (SAA and LBP) were also not significantly increased after treatment with MDP or iEDAP. Conclusion: Our data show that HC respond to NOD1 ligand, iEDAP, to produce chemokines (RANTES and KC), but do not respond to MDP alone. iEDAP also potentiates cytokine effects, especially IFNg, to upregulate iNOS and release NO. These data suggest that HC are programmed to respond to NOD stimulation by attracting and activating leukocytes to a site of infection. Our findings may help determine new treatments to regulate inflammatory responses in the liver during sepsis. 46.4. A Novel Agent for Improved Cryopreservation of Adipose Tissue. M. A. Medina, III, J. Kirkham, J. Lee, J. T. Nguyen, M. C. McCormack, M. A. Randolph, W. G. Austen, Jr; Massachusetts General Hospital, Boston, MA Background: Improved cryopreservation of fat would significantly enhance reconstruction with fat grafts by providing for multiple treatments without additional harvesting. Currently, cryopreservation is limited by the toxicity of standard agents such as DMSO. In our study of adipocyte resuscitation using a tri-block copolymer (P188) we have been able to demonstrate a significant improvement in graft preservation. We hypothesized that a similar strategy may be utilized to protect cryopreserved fat as well. In this study adipose tissue was treated with various agents and cryo-banked for 6 weeks followed by analysis in a nude mouse model. Methods: Fat was obtained via human liposuction aspirates, washed with saline and centrifuged. Aliquots of fat were treated with one of four agents: polymer (P188), PARPi (anti-apoptosis control), DMSO þ Trehalose (gold standard), or saline as a negative control. Groups were snap frozen and stored at -80C for six weeks or slow cooled at -20C (24hrs) then stored at -80C for six weeks. Thawed samples were then implanted into nude mice (1.0 cc, 0.97gm). Samples were serially harvested at 3, 6, and 9 days and at 6 weeks. The explanted fat nodules were weighed and analyzed for ATP activity, DNA content, and apoptotic activity. Results: During the first 9 days there was neither a statistical difference between any of the groups in implant weight nor apoptotic activity. However at 6 weeks the gold standard DMSO þ Trehalose controls exhibited up to 60% re-absorption. PARPi demonstrated a similar 53% resorption (p ¼ 0.004). Significantly, grafts treated with P188 and snap frozen demonstrated only 25% resorption (p ¼ 0.012) at 6 weeks (see Chart). The ATP levels at 6 weeks were higher in P188 treated grafts when compared to saline controls. Histological examination demonstrated superior adipose tissue structure in the P188 treated samples versus the other groups. Interestingly, the DMSO þ Trehalose samples histologically contained large amounts of fibrotic tissue and large vacuolated spaces. Conclusions: Treatment of cells with a membrane stabilizing agent P188 may be a new strategy for cryopreservation of fat without the potentially toxic effects of DMSO. Although more study is needed to optimize the effect of P188 on frozen grafts, this polymer may provide a clinically viable agent for the banking of adipose tissue. Additionally future directions of this technology

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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS

are wide reaching, with the possibility of cryoperservation beta-islet cells for transplant, or even as a whole organ preservative.

skin recipients (102.7 vs. 36.9 x10^5) were markedly greater than controls, p < 0.05. Plasma cells in the spleen were significantly greater after rejection of cardiac (209.2 vs 24.6 x10^5, p < 0.05) and skin allografts (12.8 vs. 1.0x10^5, p < 0.05) than non-transplanted controls. Conclusion: Coincident with a large surge of circulating DSA, immunoglobulin rich plasma cells and plasmablasts markedly increase within the bone marrow and spleen after rejection of murine cardiac and skin allografts.

46.5. Plasma Cells and Circulating Donor Specific Alloantibodies Markedly Increase after Rejection of Murine Cardiac and Skin Transplantation. R. Parsons, K. Vivek, S. Rostami, A. Naji; University of Pennsylvania School of Medicine, Philadelphia, PA Introduction: Donor specific alloantibodies (DSA) are recognized as major contributors to allograft rejection. However, little is known about the ontogeny of antibody-secreting cells (i.e. plasmablasts and plasma cells) following transplantation. Plasma cells are very rare, terminally differentiated, non-dividing effector cells that are derived from the B cell lineage. The down-regulation of common B cell markers on these potent cells makes enumeration by flow cytometry challenging. Large quantities of intracellular immunoglobulin (Ig) within the rough endoplasmic reticulum are characteristic of the plasma cell phenotype. Following rejection of murine skin or cardiac allografts, the DSA response was determined and the number of plasmablasts, and plasma cells were detected with multispectral flow cytometry. We utilized an intracellular (IC) Ig kappa light chain antibody to quantify plasma cell populations. Methods: Skin and cardiac allografts were performed (BALB/cJ / C57BL6/J). Six months following transplantation, flow-crossmatch was performed after incubation of recipient serum with syngeneic control and donor splenocytes. Bone marrow, spleen, and lymph node cells were harvested for characterization of B cell populations using nine flurochromes on an LSRII (BD Biosciences) flow cytometer. After doublet and nonviable cell exclusion, cells were sorted through a single gate that included T lymphocytes, macrophages, and granulocytes (antibodies against CD4, CD8, F4/80, and GR1, respectively). B cells were defined as follows: memory B cells (CD21-/CD23-/B220þ/CD19þ), plasmablasts (IC Ig light chain kappahiþ/IgM-/B220-/CD19þ), and plasma cells (IC Ig kappahiþ/CD138þ/IgM-/B220-/CD19-). Results: Skin (n ¼ 6) and cardiac (n ¼ 5) transplants were rejected promptly (MST 12 and 9 days, respectively). The B cell crossmatch was defined as the mean fluorescence index (MFI) of Ig bound to B220þ splenocytes, while the T cell crossmatch was defined as the MFI of Ig bound to CD4þ splenocytes. DSA was identified as the fold difference in FITC-conjugated anti-IgG binding to donor splenocytes versus syngeneic control splenocytes. DSA for B cell and T cell crossmatch was markedly higher post cardiac transplant compared to pretransplant serum (20.9 vs. 1.6, p < 0.05) and (9.4 vs. 1.0, p < 0.05), respectively. The number of memory B cells of recipients was not significantly different than non-transplanted controls (4.4 vs. 3.0 vs. 3.3 x10^5). However, the number of plasma cells and plasmablasts from the bone marrow of cardiac recipients (258.2 vs. 36.9 x10^5 and 25.8 vs. 2.1 x10^5, respectively) were significantly greater than non-transplanted controls, p < 0.05. Similarly, plasma cells from the bone marrow of

46.6. Toll-Like Receptor 4 Deficient Mesenchymal Stem Cells Improve Stem Cell Mediated Myocardial Recovery after Ischemia-Reperfusion Injury. J. A. Poynter, A. M. Abarbanell, Y. Wang, J. L. Herrmann, B. R. Weil, M. C. Manukyan, J. Tan, D. R. Meldrum; Indiana University, Indianapolis, IN Introduction: Toll-like receptor 4 (TLR4) performs a critical function in the innate immune response. However, TLR4 is also known to potentiate inflammation in the setting of myocardial ischemia/reperfusion injury (I/R). Mesenchymal stem cells (MSCs) are known to attenuate I/R and improve myocardial functional recovery in part through the release of cardioprotective paracrine growth factors such as vascular endothelial growth factor (VEGF). However, it is unknown whether knockout of TLR4 on MSCs will further improve MSC mediated myocardial recovery after I/R. We hypothesized that: 1) preischemic treatment of rat hearts with TLR4-deficient (TLR4KO) MSCs would improve myocardial recovery after I/R as compared to wild-type (WT) MSCs; and 2) TLR4KO MSCs would produce more VEGF as compared to the WT. Methods: Adult male Sprague-Dawley rat hearts were perfused via the Langendorff method and subjected to 15 min of equilibration, 25 min of warm global ischemia, and 40 min of reperfusion. Immediately prior to ischemia, hearts were infused with vehicle (n ¼ 8), WT MSCs (1x10^6 cells, n ¼ 6) or TLR4KO MSCs (1x10^6 cells, n ¼ 6). To assess VEGF production, WT and TLR4KO MSCs were treated in vitro with tumor necrosis factor-alpha (TNF, 50 ng/mL) for 24 hours and the supernatants were collected for enzyme-linked immunosorbent assay. Results: Hearts treated with TLR4KO and WT MSCs had improved recovery of left ventricular developed pressure (LVDP) as compared to the vehicle (49.67 þ/- 3.14 vs. 40.12 þ/-2.81 vs. 30.46 þ/- 2.17 mm Hg) with significantly greater benefit seen with TLR4KO MSCs. TLR4KO and WT MSC treatment also significantly improved þdP/dt with respect to the vehicle (49.77 þ/- 4.18 and 42.40 þ/- 4.09 vs. 28.71 þ/- 3.66 mm Hg/s) but no difference was seen between the MSC groups. TLR4KO and WT MSC treatment improved -dP/dt versus the vehicle (-49.74 þ/- 2.08 vs. -44.63 þ/- 5.44 vs. -33.83 þ/-3.15 mm Hg/s), but this was only significant for the TLR4KO MSC treated hearts. In vitro, basal production of VEGF by TLR4KO