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Donor antigen-pulsed host dendritic cells combined with transient immunosuppression prolong hind-limb survival
Vol. 203, No. 3S, September 2006
Plastic Surgery II
Lymphangiogenesis and angiogenesis can occur in a guided manner. These approaches may have a significant impact on the clinical treatment of lymphedema.
Endothelial progenitor cells participate in neovascularization and engraftment of cultured skin substitutes Sachin S Vaikunth MD, Marwan Marwan MD, Jignesh Parvadia MD, Maria Ripberger BS, Barbara Kalinowska BS, Eva Uzvolgy MD, PhD, Andrew Supp BS, Datis Alaee MS, Steve Boyce PhD, Timothy Crombleholme MD, Dorothy Supp PhD Cincinnati Childrens’ Hospital Medical Center, Cincinnati, OH INTRODUCTION: Bone marrow derived Endothelial Progenitor Cells (EPCs) have been demonstrated to be involved in wound healing and neovascularization. Neovascularization is critical to the engraftment of Cultured Skin Substitutes (CSS), which are comprised of keratinocytes and fibroblasts in a biopolymer matrix and used to treat extensive burns. We hypothesize that EPCs are involved in the neovascularization of CSS. METHODS: Lethally irradiated Balb-C athymic mice were transplanted with bone marrow from FVBN Tie-2 LacZ mice. Four weeks after engraftment, human CSS was transplanted onto full thickness excisional wounds on these mice. CSS were harvested at day 7 (n⫽4) and day 14 (n⫽4)and were analyzed for vessel density using CD31 immunostaining, beta-galactosidase (B-gal) expression, and CD133 in situ hybridization for EPC quantification. RESULTS: Vessel density analysis revealed that the capillaries were located predominantly along the edges of CSS but CD133⫹ EPCs were distributed throughout the CSS, specifically 39.1 ⫾ 2.8% at day 7 and 34.1 ⫾ 2.5% at day 14 were located near the epidermaldermal junction of CSS. % CD133ⴙ cells at CSS interface
B-gal ⴙ cells/ HPF
Vessel Density (caps/ HPF)
Day 7
21.7 ⫾ 3.8
12.2 ⫾ 1.3
9.8 ⫾1.2
39.1⫾ 2.8ⴱ
22.1 ⫾ 3.1
38.8 ⫾ 2.7
Day 14
21.0 ⫾ 1.3
8.7 ⫾ 1.3
9.3 ⫾ 1.5
34.1 ⫾ 2.5#
24.6 ⫾ 1.0
41.2 ⫾ 3.4
CSS
CD133ⴙ cells/ HPF
% CD133ⴙ cells in superficial dermis
%CD133ⴙ cells in deep dermis
ⴱ p ⬍ 0.006 compared to deep # p ⫽ 0.061 compared to deep
CONCLUSIONS: These results show that B-gal⫹ EPCs are recruited from the bone marrow to sites of neovascularization in CSS. CD133⫹ cells are more superficial in the dermis compared to the CD31⫹ cells suggesting a possible role of EPCs in orchestrating neovascularization as vessels form from the deep to superficial dermis. This novel demonstration of EPCs in CSS could lead to better understanding of the EPCs’ contribution in the neovascularization and engraftment of CSS.
S61
Donor antigen-pulsed host dendritic cells combined with transient immunosuppression prolong hindlimb survival Justin M Sacks MD, Ryosuke Ikeguchi MD, Ali E Aksu MD, Elaine Horibe MD, Jignesh Unadkat MBBS, MRCS, Aurele Taieb MD, Jeremy Breitinger BS, Angus W Thomson PhD, Maryam Feili-Hariri PhD, WP Andrew Lee MD University of Pittsburgh Medical Center, Pittsburgh, PA INTRODUCTION: Risks of chronic immunosuppression hinder composite tissue allograft (CTA) transplantation. A novel modality was investigated using transient immunosuppression and immature host dendritic cells (DC) pulsed with donor antigens (Ag) following CTA transplantation across a full MHC barrier. METHODS: Orthotopic hind-limb transplants between WistarFurth and Lewis rats were performed. Bone marrow derived DC (BM-DC) were propagated with GM-CSF and pulsed with donor splenic cell lysate. Recipient groups included: I, control; II, Cyclosporine A (CsA; 10mg/kg/day, day 0-20,i.p.); III, antilymphocyte serum (ALS; day - 4, ⫹ 1,i.p.) ⫹ CsA; and IV, CsA⫹ALS, combined with donor cell lysate-pulsed BM-DC (day 7, 14, 21,i.v.)(n⫽6-9/group). Epidermolysis defined rejection. Allograft recipients of ⬎100 days received donor and third-party skin grafts. Biopsies were obtained at 21 days and at rejection. T cells were stimulated via T-cell receptor or with donor antigens to determine T-cell function. RESULTS: Group IV demonstrated mean CTA survival (57.8 days) compared to group II (32.8 days,p⬍0.05) and group I (9.8 days,p⬍0.05). Long-term graft survival (⬎100 days) in group IV was observed in 3/9 rats. Muscle component of donor graft in group IV showed reduction in mononuclear cell infiltration relative to groups I, II and III (p⬍ 0.05). Donor skin graft was accepted, whereas third-party skin was rejected. Analysis of T cell responses in long-term surviving grafts revealed no donor cell reactivity. CONCLUSIONS: Donor alloAg-pulsed host BM-DC combined with transient immunosuppression prolonged CTA survival. This may represent the basis for a clinically applicable strategy promoting CTA survival with reduced systemic immunosuppression.
Obesity lowers tolerance to ischemia-reperfusion in the rat abdominal flap: A novel animal model for free flap breast reconstruction Mark E Feldmann MD, Ron Reyna MD, Seung-Jun O MD, Kenneth Chavin MD, PhD Medical University of South Carolina, Charleston, SC INTRODUCTION: Obesity presents a risk-factor for flap-related complications in autologous tissue breast reconstruction. A rat model for pedicled flaps in the setting of obesity has previously been described. In this study, we add pedicle cross-clamping to simulate free flap reconstruction. We hypothesize that obesity reduces the tolerance of a rat abdominal flap to warm ischemia.
Plastic Surgery II
Lymphangiogenesis and angiogenesis can occur in a guided manner. These approaches may have a significant impact on the clinical treatment of lymphedema.
Endothelial progenitor cells participate in neovascularization and engraftment of cultured skin substitutes Sachin S Vaikunth MD, Marwan Marwan MD, Jignesh Parvadia MD, Maria Ripberger BS, Barbara Kalinowska BS, Eva Uzvolgy MD, PhD, Andrew Supp BS, Datis Alaee MS, Steve Boyce PhD, Timothy Crombleholme MD, Dorothy Supp PhD Cincinnati Childrens’ Hospital Medical Center, Cincinnati, OH INTRODUCTION: Bone marrow derived Endothelial Progenitor Cells (EPCs) have been demonstrated to be involved in wound healing and neovascularization. Neovascularization is critical to the engraftment of Cultured Skin Substitutes (CSS), which are comprised of keratinocytes and fibroblasts in a biopolymer matrix and used to treat extensive burns. We hypothesize that EPCs are involved in the neovascularization of CSS. METHODS: Lethally irradiated Balb-C athymic mice were transplanted with bone marrow from FVBN Tie-2 LacZ mice. Four weeks after engraftment, human CSS was transplanted onto full thickness excisional wounds on these mice. CSS were harvested at day 7 (n⫽4) and day 14 (n⫽4)and were analyzed for vessel density using CD31 immunostaining, beta-galactosidase (B-gal) expression, and CD133 in situ hybridization for EPC quantification. RESULTS: Vessel density analysis revealed that the capillaries were located predominantly along the edges of CSS but CD133⫹ EPCs were distributed throughout the CSS, specifically 39.1 ⫾ 2.8% at day 7 and 34.1 ⫾ 2.5% at day 14 were located near the epidermaldermal junction of CSS. % CD133ⴙ cells at CSS interface
B-gal ⴙ cells/ HPF
Vessel Density (caps/ HPF)
Day 7
21.7 ⫾ 3.8
12.2 ⫾ 1.3
9.8 ⫾1.2
39.1⫾ 2.8ⴱ
22.1 ⫾ 3.1
38.8 ⫾ 2.7
Day 14
21.0 ⫾ 1.3
8.7 ⫾ 1.3
9.3 ⫾ 1.5
34.1 ⫾ 2.5#
24.6 ⫾ 1.0
41.2 ⫾ 3.4
CSS
CD133ⴙ cells/ HPF
% CD133ⴙ cells in superficial dermis
%CD133ⴙ cells in deep dermis
ⴱ p ⬍ 0.006 compared to deep # p ⫽ 0.061 compared to deep
CONCLUSIONS: These results show that B-gal⫹ EPCs are recruited from the bone marrow to sites of neovascularization in CSS. CD133⫹ cells are more superficial in the dermis compared to the CD31⫹ cells suggesting a possible role of EPCs in orchestrating neovascularization as vessels form from the deep to superficial dermis. This novel demonstration of EPCs in CSS could lead to better understanding of the EPCs’ contribution in the neovascularization and engraftment of CSS.
S61
Donor antigen-pulsed host dendritic cells combined with transient immunosuppression prolong hindlimb survival Justin M Sacks MD, Ryosuke Ikeguchi MD, Ali E Aksu MD, Elaine Horibe MD, Jignesh Unadkat MBBS, MRCS, Aurele Taieb MD, Jeremy Breitinger BS, Angus W Thomson PhD, Maryam Feili-Hariri PhD, WP Andrew Lee MD University of Pittsburgh Medical Center, Pittsburgh, PA INTRODUCTION: Risks of chronic immunosuppression hinder composite tissue allograft (CTA) transplantation. A novel modality was investigated using transient immunosuppression and immature host dendritic cells (DC) pulsed with donor antigens (Ag) following CTA transplantation across a full MHC barrier. METHODS: Orthotopic hind-limb transplants between WistarFurth and Lewis rats were performed. Bone marrow derived DC (BM-DC) were propagated with GM-CSF and pulsed with donor splenic cell lysate. Recipient groups included: I, control; II, Cyclosporine A (CsA; 10mg/kg/day, day 0-20,i.p.); III, antilymphocyte serum (ALS; day - 4, ⫹ 1,i.p.) ⫹ CsA; and IV, CsA⫹ALS, combined with donor cell lysate-pulsed BM-DC (day 7, 14, 21,i.v.)(n⫽6-9/group). Epidermolysis defined rejection. Allograft recipients of ⬎100 days received donor and third-party skin grafts. Biopsies were obtained at 21 days and at rejection. T cells were stimulated via T-cell receptor or with donor antigens to determine T-cell function. RESULTS: Group IV demonstrated mean CTA survival (57.8 days) compared to group II (32.8 days,p⬍0.05) and group I (9.8 days,p⬍0.05). Long-term graft survival (⬎100 days) in group IV was observed in 3/9 rats. Muscle component of donor graft in group IV showed reduction in mononuclear cell infiltration relative to groups I, II and III (p⬍ 0.05). Donor skin graft was accepted, whereas third-party skin was rejected. Analysis of T cell responses in long-term surviving grafts revealed no donor cell reactivity. CONCLUSIONS: Donor alloAg-pulsed host BM-DC combined with transient immunosuppression prolonged CTA survival. This may represent the basis for a clinically applicable strategy promoting CTA survival with reduced systemic immunosuppression.
Obesity lowers tolerance to ischemia-reperfusion in the rat abdominal flap: A novel animal model for free flap breast reconstruction Mark E Feldmann MD, Ron Reyna MD, Seung-Jun O MD, Kenneth Chavin MD, PhD Medical University of South Carolina, Charleston, SC INTRODUCTION: Obesity presents a risk-factor for flap-related complications in autologous tissue breast reconstruction. A rat model for pedicled flaps in the setting of obesity has previously been described. In this study, we add pedicle cross-clamping to simulate free flap reconstruction. We hypothesize that obesity reduces the tolerance of a rat abdominal flap to warm ischemia.