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nhibition of IL-4 function decreases fibrosis andymphatic dysfunction in a mouse model ofymphedemaomer Avraham MD, Jamie C Zampell MD, Alan Yan MD,anjay V Daluvoy MD, Essie Kueberuwa MD,abak J Mehrara MD, FACSemorial Sloan-Kettering Cancer Center, New York, NY

NTRODUCTION: Lymphedema is characterized by chronic in-lammation and fibrosis. Previous studies have shown that fibrosis inther organs is dependent on T-helper2 (Th-2) inflammation. Theurpose of these experiments was therefore to evaluate the role ofh-2 cells in the regulation of fibrosis and lymphatic dysfunction.

ETHODS: We used a mouse tail model of lymphedema in wild-ype or nude mice to evaluate the role of T-cells on the developmentf chronic inflammation and fibrosis. In addition, in order to evalu-te the role of Th2 cells in this process, we inhibited Th2 differenti-tion using a neutralizing antibody to IL4 (IL4mab) as compared tosotype control antibody.

ESULTS: Lymphedema in wild-type mice resulted in a mixedh1-Th2 chronic inflammation response. The absence of T-cells inude mice resulted in 3-fold less tail edema and 2-fold decreasedibrosis (p�0.01). Inhibition of Th2 differentiation with IL4mabnhibited initiation of lymphedema and decreased tail volume (3-old), improved lymphatic function, decreased fibrosis (2x), de-reased the number of Th2 cells (3x), and decreased circulating/localL4, IL13, TGF-b1 expression (2-3x; p�0.01). Blockade of IL-4 inice with established lymphedema resulted in resolution of

ymphedema with decreased tail volume (4x), fibrosis (2x), and res-oration of lymphatic flow (p�0.01).

ONCLUSIONS: Chronic T-cell inflammation in general and Th2ifferentiation in particular are necessary for fibrosis and lymphaticysfunction in lymphedema. Th2 differentiation promotes fibrosisy increasing the expression of pro-fibrotic cytokines IL4, IL13, andGF-B1 locally and systemically. Inhibition of Th2 differentiationan reverse the pathologic changes associated with lymphedema.

D4� T-cells promote fibrosis and lymphaticysfunction in lymphedemalan Yan MD, Tomer Avraham MD, Jamie C Zampell MD,abak J Mehrara MD, FACSemorial Sloan-Kettering Cancer Center, New York, NY

NTRODUCTION: Chronic lymphedema results in soft tissue fibro-is and lymphatic dysfunction. However, the cellular mechanismshat regulate this response remain unknown. T-cells play an impor-ant role in fibrosis in a number of other fibroproliferative disorders.he purpose of these experiments was therefore to determine the rolef various types of T-cells in the regulation of fibrosis and lymphaticysfunction resulting from lymphatic fluid stasis.

ETHODS: Lymphedema was produced by circumferential skinxcision and microsurgical ligation of the deep lymphatic system ofdult female mice tails. Two weeks after surgery the mice were treated

ith monoclonal antibodies to deplete CD4�, CD8�, or CD25� b

S812010 by the American College of Surgeons

ublished by Elsevier Inc.

-cells for 4 weeks. Control mice were treated with isotype controlntibodies. In addition, we compared CD4 knockout (KO) miceith wild-type mice and evaluated lymphatic function.

ESULTS: Depletion of CD4� T-cells resulted in improved lym-hatic function and a two-fold decrease in tail lymphedema. Theseindings were confirmed histologically demonstrating decreasedhronic inflammation, fibrosis, and dermal thickness in CD4-epleted mice. In contrast, depletion of CD8� or CD25� T-cellsad little effect on these measures. When compared with wild-typeice, CD4 KO mice had marked decrease in tail lymphedema (50%)

nd improved lymphatic function as evidenced by microlym-hangiography 6 weeks after surgery (p�0.05).

ONCLUSIONS: We have shown that CD4� but not CD8� orD25� T-cells play an important role in fibrosis and lymphaticysfunction resulting from lymphedema. This finding is importantince identification of the mechanisms regulating these pathologicesponses may identify targeted treatment or preventative measures.

igration of systemically injected adipose-derivedtromal cells to sites of cranial and appendicularkeletal injuryaron W James MD, Benjamin Levi MD, Jason Glotzbach MD,ichelle Peng BS, Emily Nelson BS,ichael T Longaker MD, MBA, FACS

tanford University, Stanford, CA

NTRODUCTION: Adipose-derived stromal cells (ASCs) represent aultipotent cell stromal cell type with proven capacity to differenti-

te along various mesodermal lineages. In the following study, weought to examine whether ASCs would migrate to surgically createdefects of the cranial and/or appendicular skeleton in the mouse.

ETHODS: Mouse ASCs were harvested from luciferase� trans-enic mice. Next, surgical defects were created in wildtype, adultice: a 4mm defect in the parietal bone and/or a 1mm defect in the

roximal tibia. For a control, skin incisions were made without dril-ing. After recovery, either mouse or human ASCs were injectedystemically (200,000 ASCs in 2 mL NS). Scanning by luciferaseetection (IVIS) and microCT was performed weekly thereafter. An-

mals were sacrificed for routine histology 4 weeks thereafter.

ESULTS: As soon as 1 week post-injury, luciferase activity wasbserved at either cranial or tibial defect sites. In comparison, littlectivity was observed on the side contralateral to the defect, or alter-atively among control groups with a skin incision only. Luciferasectivity was observed to persist up to 4 weeks postoperatively. Inter-stingly, if simultaneous injuries were performed in the calvaria andibia, the predominance of luciferase activity was observed at theibial defect site. Interestingly, microCT and histological analysisonfirmed increased bone formation within ASC treated mice.

ONCLUSIONS: Systemically administered ASCs migrate to sitesf skeletal injury. Moreover, preferential migration to the appendic-lar skeleton was noted when two defects were created simulta-eously. Analysis suggests that these cells differentiate toward osteo-

lasts once in the defect site and contribute to repair.

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