Patrick S. Wolf

Epithelioid sarcoma: the University of Washington experience.

BACKGROUND Epithelioid sarcoma is a rare sarcoma with a high local recurrence rate that frequently metastasizes to lymph nodes. We reviewed our experience with adjuvant therapy in patients with this disease. METHODS Between 1990 and 2003, we treated 11 patients with epithelioid sarcoma. Patient, tumor, and treatment characteristics were analyzed, and effect of treatment on survival was evaluated by the Kaplan-Meier method. RESULTS Nine men and 2 women were treated. Tumors presented on the trunk, the upper extremities, and the lower extremities. Five patients developed nodal disease. All patients underwent surgery for the primary tumor, and 7 patients had nodal evaluation. Ten patients underwent adjuvant chemotherapy, and 9 underwent radiotherapy. Recurrence developed in 9 patients. Five-year disease-free and overall survival rates were 46% and 65%, respectively. Chemotherapy and radiation therapy did not impact disease-free survival. CONCLUSIONS Although surgery remains the primary treatment modality, multi-institutional trials are needed to develop more effective adjuvant therapy for patients with epithelioid sarcoma.

Stress-activated protein kinase inhibition to ameliorate lung ischemia reperfusion injury

OBJECTIVE Inhibition of cytokines offers modest protection from injury in animal models of lung ischemia-reperfusion. Improved strategies would selectively inhibit the transcriptional activation response to oxidative stress. Mitogen-activated protein kinases (p38, c-jun N-terminal kinase, extracellular signal-regulated kinase) have been shown to be activated after oxidative stress and in animal models of acute inflammatory lung injury. We hypothesized that mitogen-activated protein kinase inhibition would block downstream transcriptional activation, providing robust protection from lung ischemia-reperfusion injury. METHODS Experimental rats received inhibitors of p38, c-jun kinase, or extracellular signal-regulated kinase before in situ left lung ischemia-reperfusion. Immunohistochemistry localized cellular sites of mitogen-activated protein kinase activation. Several markers of lung injury were assessed. Enzyme-linked immunosorbent assay measured soluble cytokine and chemokine contents. Western blotting assessed mitogen-activated protein kinase phosphorylation. Electromobility shift assays measured transcription factor nuclear translocation. RESULTS Immunohistochemistry localized p38 and c-jun kinase activations in positive controls to alveolar macrophages. Extracellular signal-regulated kinase was activated in endothelial and epithelial cells. Animals treated with p38 or c-jun kinase inhibitor demonstrated significant reductions in transcription factor activation and markers of lung injury. Extracellular signal-regulated kinase inhibition was not protective. Western blotting confirmed inhibitor specificity. CONCLUSION Inhibition of p38 and c-jun kinase provided significant protection from injury. The alveolar macrophage appears to be the key coordinator of injury in response to oxidative stress. Therapeutically targeting specific cell population (macrophage) responses to oxidative stress has the potential benefit of reducing lung reperfusion injury severity while leaving host immune responses intact.

Lipopolysaccharide pre-conditioning is protective in lung ischemia-reperfusion injury

BACKGROUND The availability of suitable lung donors has remained a significant barrier to lung transplantation. The clinical relevance of an isolated positive Gram stain in potential donor lungs, which occurs in >80%, is unclear. Low doses of lipopolysaccharide (LPS) have been protective in several models of ischemia-reperfusion injury through a pre-conditioning response. We sought to demonstrate that low-dose LPS is protective against subsequent lung ischemia-reperfusion injury. METHODS Pathogen-free Long-Evans rats were pre-treated with vehicle or LPS 24 hours before 90 minutes of ischemia and up to 4 hours of reperfusion. Lungs were assessed for vascular permeability, myeloperoxidase content, bronchoalveolar lavage inflammatory cell and cytokine/chemokine content, as well as nuclear translocation of nuclear factor kappaB (NFkappaB) and activator protein-1 (AP-1), and interleukin-1 receptor-associated kinase-1 (IRAK-1) and stress-activated protein kinase (SAPK) activation. RESULTS Compared with positive controls, LPS pre-treatment resulted in reductions in vascular permeability (70%, p < 0.001), myeloperoxidase content (93%, p < 0.001), bronchoalveolar lavage inflammatory cells (91%, p < 0.001), and inflammatory cytokine/chemokine content (cytokine-induced neutrophil chemoattractant, 99%, p = 0.003; interleukin-1beta, 72%, p < 0.0001; tumor necrosis factor-alpha, 76%, p < 0.0001), NFkappaB (86%, p < 0.001) and AP-1 (97%, p < 0.001) nuclear translocation, and IRAK-1 (87%, p < 0.001) and SAPK (80%, p < 0.001) phosphorylation. CONCLUSIONS Lipopolysaccharide pre-treatment reduced lung injury and inflammatory mediator production after subsequent exposure to ischemia-reperfusion. Understanding the clinical significance of lipopolysaccharide in donor lungs has the potential to expand and clarify donor inclusion criteria.

Poly (ADP) ribose synthetase inhibition in alveolar macrophages undergoing hypoxia and reoxygenation

BACKGROUND Inhibition of the nuclear enzyme poly ribose synthetase (PARS) protects against in vivo lung ischemia reperfusion injury (LIRI). The effectiveness of intratracheal treatment suggests that PARS inhibition may primarily modulate alveolar macrophage (AM) activation. These studies attempted to characterize the effects of PARS on AM activation in response to oxidative stress. METHODS Primary cultures of AM were rendered hypoxic for 2 h and reoxygenated for up to 4 h. Cells were preincubated with INO-1001, a specific PARS inhibitor 1 h prior to hypoxia. Gel shift assays characterized nuclear factor kappa B (NFkappaB), and enzyme linked immunosorbent assay quantitated chemokine/cytokine protein secretion. RESULTS Hypoxia and reoxygenation resulted in an increase in the early nuclear translocation of NFkappaB, and an increase in the secretion of the cytokine tumor necrosis factor-alpha (TNF-alpha), chemokines macrophage inflammatory protein (MIP-1alpha), monocyte chemoattractant protein one (MCP-1) and cytokine induced neutrophil chemoattractant (CINC). Pretreatment of AM with INO-1001 decreased both the early translocation of NFkappaB and the production of TNF-alpha (p<0.05) and MIP-1alpha p=0.02, but did not affect CINC or MCP-1 production. CONCLUSIONS These findings indicate that PARS inhibition in the AM blunts their response to oxidative stress and may help explain the protective effects of intratracheal PARS inhibition in LIRI.

Synergistic Protection in Lung Ischemia-Reperfusion Injury With Calcineurin and Thrombin Inhibition

BACKGROUND Ischemia-reperfusion injury impairs lung transplant outcomes. The transcription factors, activator protein-1, and nuclear factor kappa B, are activated early in reperfusion and drive the development of injury. Thrombin inhibition with hirudin, and calcineurin inhibition with tacrolimus have independently been shown to ameliorate lung ischemia-reperfusion injury by reducing activator protein-1 and nuclear factor kappa B activation, respectively. However, high doses were required to achieve protection using individual agents, raising concerns about potential toxicities. We sought to determine if low-dose combination therapy reduced injury through synergistic inhibition of pretranscriptional signaling events. METHODS Rats were pretreated with either intravenous hirudin or tacrolimus at low doses or high doses, or both at low doses, prior to undergoing left lung ischemia and reperfusion. Lungs were assessed for markers of lung injury, including bronchoalveolar lavage cytokine-chemokine content and transcription factor transactivation of activator protein-1 and nuclear factor kappa B. RESULTS High-dose monotherapy with hirudin or tacrolimus reduced lung injury and transactivation of activator protein-1 and nuclear factor kappa B activation, respectively, whereas low-dose monotherapy with either agent did not alter transcription factor activation or lung injury compared with positive controls. Low-dose combination therapy was more protective than high-dose monotherapy with either drug, and correlated with a reduction in activation of both transcription factors and their associated cytokines. CONCLUSIONS The significant decrease in lung injury severity and transcription factor activation with combined pathway inhibition suggests pretranscriptional signaling redundancy between the calcineurin and thrombin dependent pathways in lung reperfusion injury.

Alveolar Macrophage Secretory Products Effect Type 2 Pneumocytes Undergoing Hypoxia-Reoxygenation

BACKGROUND Activation of the alveolar macrophage is centrally important to the development of lung ischemia reperfusion injury. Alveolar macrophages and type 2 pneumocytes secrete a variety of proinflammatory mediators in response to oxidative stress. The manner in which they interact and how the macrophage may influence pneumocyte responses in lung ischemia reperfusion injury is unknown. Utilizing an in vitro model of hypoxia and reoxygenation, we sought to determine if the proinflammatory response of type 2 pneumocytes to oxidative stress would be amplified by alveolar macrophage secretory products. METHODS Cultured pneumocytes were exposed to control media or media from cultured macrophages exposed to hypoxia and reoxygenation. Pneumocytes were subsequently subjected to hypoxia and reoxygenation and assessed for both nuclear translocation of nuclear factor kappa B and inflammatory cytokine and chemokine secretion. To examine for any reciprocal interactions, we reversed the experiment, exposing macrophages to conditioned pneumocyte media. RESULTS In the presence of media from stimulated macrophages, production of proinflammatory mediators by type 2 pneumocytes was dramatically enhanced. In contrast, exposure of the macrophage to conditioned pneumocyte media had an inhibitory effect on macrophage responses subsequently exposed to hypoxia and reoxygenation. CONCLUSIONS The alveolar macrophage drives the development of lung reperfusion injury in part through amplification of the inflammatory response of type 2 pneumocytes subjected to hypoxia and reoxygenation.