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Del1 protects chondrocytes from apoptosis
ORTHOPAEDIC SURGERY with a myeloperoxidase (MPO) assay. Changes in liver permeability were assessed by measuring the wet-dry-ratio.
Del1 protects chondrocytes from apoptosis Zhen Wang, MD,* Xingju Nie, MD, Michael T Longaker, MD, MBA, FACS, George P Yang, MD, PhD, FACS Stanford University, Stanford, CA
RESULTS: The Fx-STI-group showed significantly increased serum cytokine levels as compared to the Fx- and Sham-group. The homogenized liver tissue of the Fx-STI-group showed significantly increased IL-6 levels as compared to the Sham-group. The MPO activity in lung and liver in the Fx-STI-group was significantly increased in comparison to the Fx- and Sham-group and in the Fxgroup in comparison to the Sham-group. The wet-dry-ratio of the liver was significantly increased in the Fx-STI-group as compared to the Sham-group. Table 1 Serum-Cytokine Levels
INTRODUCTION: Del1 is a secreted integrin-binding protein that can protect endothelial cells from apoptosis. Del1 is strongly expressed during development in cartilaginous tissues and we hypothesized that Del1 has anti-apoptotic affects on chondrocytes. METHODS: Chondrocytes were attached to plates coated with purified Del1 or BSA and apoptosis was induced by etoposide, doxorubicin, or TNFalpha/actinomycin D for 16-24 hr. Anoikis was performed by culture on poly-HEMA coated plates for 48 hr. Cell viability was demonstrated by WST-8 assay. Apoptosis was measured by TUNEL staining, and caspase 3/7 activity. MAPK and Akt activation was detected by immunoblotting, and blocked with the addition of specific inhibitors of MAPK and PI3K. The role of integrin binding was tested with the addition of RGD or RAD peptides.
IL-2
IL-10 31.53 ⫾ 20.75
138.02 ⫾ 40.47 ⫾ 19.37 ⫾ 33.89 ⫾ 14.32 0.96 14.08 0.71
IL-12
KC
MCP-1
129.66 ⫾ 43.18 ⫾ 301.66 ⫾ 38.86 ⫾ 14.15 1.54 93.20 1.69
Sham
58.99 ⫾ 15.57 ⫾ 37.43 ⫾ 6.38 3.41 11.72
38.78 ⫾ 33.60
Fx
62.92 ⫾ 17.60 ⫾ 56.90 ⫾ 3.68 3.05 13.45
122.51 ⫾ 123.48 ⫾ 41.52 ⫾ 395.86 ⫾ 39.62 ⫾ 44.97 ⴱ 15.22 1.77 58.64 2.17
Fx-STI 72.03 ⫾ 20.90 ⫾ 111.29 ⫾ 278.29 ⫾ 142.51 ⫾ 43.59 ⫾ 622.71 ⫾ 44.08 ⫾ 3.54 2.92 13.02ⴱ# 41.17ⴱ# 7.36 0.79 86.06ⴱ 1.84
RESULTS: Del1 protects chondrocytes from apoptosis under all of the conditions tested. Del1 protects chondrocytes exposed to etoposide, doxorubicin or TNFalpha/actinomycin D through activation of both MAPK and Akt. Del1 protects chondrocytes subjected to anoikis through activation of only Akt. MAPK and PI3K inhibitors and RGD peptides abolished Del1’s anti-apoptotic affect.
Mean ⫾ SEM; p⬍0.05 vs. Sham; #p⬍0.05 vs. Fx.
CONCLUSIONS: The degree of fracture associated soft-tissue injury appears to modify systemic inflammation following bilateral femur fracture and is able to induce remote organ dysfunction. These results may have implications that have been previously underestimated, thus warranting clinical follow-up studies.
CONCLUSIONS: Del1 has anti-apoptotic affects on chondrocytes, mediated by integrin binding. Under different apoptotic stimuli, MAPK and PI3K/Akt are variably used to prevent apoptosis. We believe Del1 plays an important role in chondrocyte biology and are exploring the role of Del1 in fracture healing are maintenance of articular cartilage.
Functional and proteomic analysis of cerebrospinal fluid derived from patients with and without traumatic brain injury Dieter Cadosch, MD, Matthew Thyer, PhD, P Frey Soenke, MD, Gu¨nter Lochnit, PhD, Luis Filgueria, MD, Rene´ Zellweger, MD, FACS Royal Perth Hospital, The University of Western Australia, Perth,
Degree of fracture-associated soft-tissue injury modifies systemic inflammation and remote organ dysfunction after bilateral femur fracture
INTRODUCTION: Patients with traumatic brain injury (TBI) and bone fractures are predisposed to accelerated fracture healing. The mechanisms for this phenomenon remain unknown. But it is hypothesized that osteoinductive factors are released from the injured brain. The aim of this study was to identify osteoinductive factors in the human cerebrospinal fluid (CSF) of TBI patients using functional in vitro tests and a proteomic approach.
Philipp Kobbe, MD, David Kaczorowski, MD, Kevin Mollen, MD, Timothy Billiar, MD, FACS, Hans-Christoph Pape, MD University of Pittsburgh, Pittsburgh, PA INTRODUCTION: Patients with bilateral femur fractures are known to be at a high risk for systemic complications. The role of fractureassociated soft-tissue injury in the induction of systemic inflammation is poorly understood. To address this gap, the systemic inflammatory response and remote organ dysfunction following bilateral femur fracture with various degrees of soft-tissue injuries were investigated.
METHODS: CSF samples were collected from seven TBI patients and nine controls. The osteoinductive effect of CSF was first verified in proliferation assays using the osteoblastic cell line hFOB1.19. The CSF-proteins were labeled with biotin prior to incubation with hFOB1.19 cells. After cell lysis, the biotin-labeled proteins that had bound to the cells were isolated through an avidin column and separated using two-dimensional gel electrophoresis.
METHODS: 6-8 weeks old male C57/BL6 mice were grouped (n ⫽ 4-8 animals) as follows: Control-group; Sham-group (anaesthesia); Fx-group (anaesthesia, bilateral femur fracture with minor soft-tissue injury); Fx-STI-group (anaesthesia, bilateral femur fracture with severe soft-tissue injury). Six hours after bilateral femur fracture serum cytokine levels were measured. Furthermore, IL-6 levels of homogenized liver tissue were assessed. Neutrophil accumulation in liver and lung was determined
© 2007 by the American College of Surgeons Published by Elsevier Inc.
IL-4
TNF-␣
IL-6
Control 56.94 ⫾ 11.45 ⫾ 6.42 ⫾ 8.75 3.58 1.14
RESULTS: All samples derived from TBI patients were found to have a significantly higher mean proliferation in the osteoinductive in vitro test. Using biotin labeling of CSF proteins and selective binding to hFOB1.19, clear differences could be detected on corresponding two-dimensional gels between the TBI and control sam-
ISSN 1072-7515/07/$32.00
S45
Del1 protects chondrocytes from apoptosis Zhen Wang, MD,* Xingju Nie, MD, Michael T Longaker, MD, MBA, FACS, George P Yang, MD, PhD, FACS Stanford University, Stanford, CA
RESULTS: The Fx-STI-group showed significantly increased serum cytokine levels as compared to the Fx- and Sham-group. The homogenized liver tissue of the Fx-STI-group showed significantly increased IL-6 levels as compared to the Sham-group. The MPO activity in lung and liver in the Fx-STI-group was significantly increased in comparison to the Fx- and Sham-group and in the Fxgroup in comparison to the Sham-group. The wet-dry-ratio of the liver was significantly increased in the Fx-STI-group as compared to the Sham-group. Table 1 Serum-Cytokine Levels
INTRODUCTION: Del1 is a secreted integrin-binding protein that can protect endothelial cells from apoptosis. Del1 is strongly expressed during development in cartilaginous tissues and we hypothesized that Del1 has anti-apoptotic affects on chondrocytes. METHODS: Chondrocytes were attached to plates coated with purified Del1 or BSA and apoptosis was induced by etoposide, doxorubicin, or TNFalpha/actinomycin D for 16-24 hr. Anoikis was performed by culture on poly-HEMA coated plates for 48 hr. Cell viability was demonstrated by WST-8 assay. Apoptosis was measured by TUNEL staining, and caspase 3/7 activity. MAPK and Akt activation was detected by immunoblotting, and blocked with the addition of specific inhibitors of MAPK and PI3K. The role of integrin binding was tested with the addition of RGD or RAD peptides.
IL-2
IL-10 31.53 ⫾ 20.75
138.02 ⫾ 40.47 ⫾ 19.37 ⫾ 33.89 ⫾ 14.32 0.96 14.08 0.71
IL-12
KC
MCP-1
129.66 ⫾ 43.18 ⫾ 301.66 ⫾ 38.86 ⫾ 14.15 1.54 93.20 1.69
Sham
58.99 ⫾ 15.57 ⫾ 37.43 ⫾ 6.38 3.41 11.72
38.78 ⫾ 33.60
Fx
62.92 ⫾ 17.60 ⫾ 56.90 ⫾ 3.68 3.05 13.45
122.51 ⫾ 123.48 ⫾ 41.52 ⫾ 395.86 ⫾ 39.62 ⫾ 44.97 ⴱ 15.22 1.77 58.64 2.17
Fx-STI 72.03 ⫾ 20.90 ⫾ 111.29 ⫾ 278.29 ⫾ 142.51 ⫾ 43.59 ⫾ 622.71 ⫾ 44.08 ⫾ 3.54 2.92 13.02ⴱ# 41.17ⴱ# 7.36 0.79 86.06ⴱ 1.84
RESULTS: Del1 protects chondrocytes from apoptosis under all of the conditions tested. Del1 protects chondrocytes exposed to etoposide, doxorubicin or TNFalpha/actinomycin D through activation of both MAPK and Akt. Del1 protects chondrocytes subjected to anoikis through activation of only Akt. MAPK and PI3K inhibitors and RGD peptides abolished Del1’s anti-apoptotic affect.
Mean ⫾ SEM; p⬍0.05 vs. Sham; #p⬍0.05 vs. Fx.
CONCLUSIONS: The degree of fracture associated soft-tissue injury appears to modify systemic inflammation following bilateral femur fracture and is able to induce remote organ dysfunction. These results may have implications that have been previously underestimated, thus warranting clinical follow-up studies.
CONCLUSIONS: Del1 has anti-apoptotic affects on chondrocytes, mediated by integrin binding. Under different apoptotic stimuli, MAPK and PI3K/Akt are variably used to prevent apoptosis. We believe Del1 plays an important role in chondrocyte biology and are exploring the role of Del1 in fracture healing are maintenance of articular cartilage.
Functional and proteomic analysis of cerebrospinal fluid derived from patients with and without traumatic brain injury Dieter Cadosch, MD, Matthew Thyer, PhD, P Frey Soenke, MD, Gu¨nter Lochnit, PhD, Luis Filgueria, MD, Rene´ Zellweger, MD, FACS Royal Perth Hospital, The University of Western Australia, Perth,
Degree of fracture-associated soft-tissue injury modifies systemic inflammation and remote organ dysfunction after bilateral femur fracture
INTRODUCTION: Patients with traumatic brain injury (TBI) and bone fractures are predisposed to accelerated fracture healing. The mechanisms for this phenomenon remain unknown. But it is hypothesized that osteoinductive factors are released from the injured brain. The aim of this study was to identify osteoinductive factors in the human cerebrospinal fluid (CSF) of TBI patients using functional in vitro tests and a proteomic approach.
Philipp Kobbe, MD, David Kaczorowski, MD, Kevin Mollen, MD, Timothy Billiar, MD, FACS, Hans-Christoph Pape, MD University of Pittsburgh, Pittsburgh, PA INTRODUCTION: Patients with bilateral femur fractures are known to be at a high risk for systemic complications. The role of fractureassociated soft-tissue injury in the induction of systemic inflammation is poorly understood. To address this gap, the systemic inflammatory response and remote organ dysfunction following bilateral femur fracture with various degrees of soft-tissue injuries were investigated.
METHODS: CSF samples were collected from seven TBI patients and nine controls. The osteoinductive effect of CSF was first verified in proliferation assays using the osteoblastic cell line hFOB1.19. The CSF-proteins were labeled with biotin prior to incubation with hFOB1.19 cells. After cell lysis, the biotin-labeled proteins that had bound to the cells were isolated through an avidin column and separated using two-dimensional gel electrophoresis.
METHODS: 6-8 weeks old male C57/BL6 mice were grouped (n ⫽ 4-8 animals) as follows: Control-group; Sham-group (anaesthesia); Fx-group (anaesthesia, bilateral femur fracture with minor soft-tissue injury); Fx-STI-group (anaesthesia, bilateral femur fracture with severe soft-tissue injury). Six hours after bilateral femur fracture serum cytokine levels were measured. Furthermore, IL-6 levels of homogenized liver tissue were assessed. Neutrophil accumulation in liver and lung was determined
© 2007 by the American College of Surgeons Published by Elsevier Inc.
IL-4
TNF-␣
IL-6
Control 56.94 ⫾ 11.45 ⫾ 6.42 ⫾ 8.75 3.58 1.14
RESULTS: All samples derived from TBI patients were found to have a significantly higher mean proliferation in the osteoinductive in vitro test. Using biotin labeling of CSF proteins and selective binding to hFOB1.19, clear differences could be detected on corresponding two-dimensional gels between the TBI and control sam-
ISSN 1072-7515/07/$32.00
S45