Anti-HMGB1 monoclonal antibody ameliorates immunosuppression after peripheral tissue trauma: attenuated T-lymphocyte response and increased splenic CD11b (+) Gr-1 (+) myeloid-derived suppressor cells require HMGB1.

Although tissue-derived high mobility group box 1 (HMGB1) is involved in many aspects of inflammation and tissue injury after trauma, its role in trauma-induced immune suppression remains elusive. Using an established mouse model of peripheral tissue trauma, which includes soft tissue and fracture components, we report here that treatment with anti-HMGB1 monoclonal antibody ameliorated the trauma-induced attenuated T-cell responses and accumulation of CD11b(+)Gr-1(+) myeloid-derived suppressor cells in the spleens seen two days after injury. Our data suggest that HMGB1 released after tissue trauma contributes to signaling pathways that lead to attenuation of T-lymphocyte responses and enhancement of myeloid-derived suppressor cell expansion.

Selective roles for toll-like receptors 2, 4, and 9 in systemic inflammation and immune dysfunction following peripheral tissue injury.

BACKGROUND: Toll-like receptors (TLRs) detect endogenous ligands released after trauma and contribute to the proinflammatory response to injury. Posttraumatic mortality correlates with the extent of the immunoinflammatory response to injury that is composed of a complex regulation of innate and adaptive immune responses. Although TLRs are known to modulate innate immune responses, their role in the suppression of lymphocyte responses following traumatic tissue injury is unclear. METHODS: This study used a murine model of severe peripheral tissue injury, involving muscle crush injury and injection of fracture components, to evaluate the roles of TLR2, TLR4, and TLR9 in the early and delayed immunoinflammatory phenotype. Posttraumatic immune dysfunction was measured in our trauma model using the following parameters: ex vivo splenocyte proliferation, TH1 cytokine release, and iNOS (inducible nitric oxide synthase) induction within splenic myeloid-derived suppressor cells. Systemic inflammation and liver damage were determined by circulating interleukin 6 levels and hepatocellular injury. RESULTS: Suppression of splenocyte responses after injury was dependent on TLR4 and TLR9 signaling as was posttraumatic iNOS upregulation in splenic myeloid-derived suppressor cells. TLR2 was found to have only a partial role through contribution to inhibition of splenocyte proliferation. This study also reveals the involvement of TLR2 and TLR4 in the initial systemic inflammatory response to traumatic tissue injury; however, this response was found to be TLR9 independent. CONCLUSION: These findings demonstrate the previously unidentified role of TLR2, TLR4, and TLR9 in the T cell-associated immune dysfunction following traumatic tissue injury. Importantly, this study also illustrates that TLRs play differing and selective roles in both the initial proinflammatory response and adaptive immune response after trauma. Furthermore, results in TLR9-deficient mice establish that the upregulation of early proinflammatory markers do not always correlate with the extent of sustained immune dysfunction. This suggests potential for targeted therapies that could limit immune dysfunction through selective inhibition of receptor function following injury.

Inducible nitric oxide synthase contributes to immune dysfunction following trauma.

Trauma results in a persistent depression in adaptive immunity, which contributes to patient morbidity and mortality. This state of immune paralysis following trauma is characterized by a change in cell-mediated immunity, specifically a depression in T-cell function and a shift toward TH2 T-cell phenotype. Upregulation of inducible nitric oxide synthase (iNOS) is well recognized after injury and contributes to the inflammatory response and organ damage early after trauma. However, it is unknown whether iNOS plays a role in adaptive immune dysfunction after trauma. This study utilized a murine model of severe peripheral tissue injury to show that iNOS is rapidly upregulated in macrophages and a (Gr-1-CD11b) myeloid-derived suppressor cell subpopulation in the spleen. Through the use of iNOS knockout mice, a specific iNOS inhibitor, and a nitric oxide (NO) scavenger, this study demonstrates that iNOS-derived NO is required for the depression in T-lymphocyte proliferation, interferon γ, and interleukin 2 production within the spleen at 48 h after trauma. These findings support the hypothesis that iNOS regulates immune suppression following trauma and suggest that targeting the sustained production of NO by iNOS may attenuate posttraumatic immune depression.

Fixed volume or fixed pressure: a murine model of hemorrhagic shock.

It is common knowledge that severe blood loss and traumatic injury can lead to a cascade of detrimental signaling events often resulting in mortality. These signaling events can also lead to sepsis and/or multiple organ dysfunction (MOD). It is critical then to investigate the causes of suppressed immune function and detrimental signaling cascades in order to develop more effective ways to help patients who suffer from traumatic injuries. This fixed pressure Hemorrhagic Shock (HS) procedure, although technically challenging, is an excellent resource for investigation of these pathophysiologic conditions. Advances in the assessment of biological systems, i.e. Systems Biology have enabled the scientific community to further understand complex physiologic networks and cellular communication patterns. (14) Hemorrhagic Shock has proven to be a vital tool for unveiling these cellular communication patterns as they relate to immune function. This procedure can be mastered! This procedure can also be used as either a fixed volume or fixed pressure approach. We adapted this technique in the murine model to enhance research in innate and adaptive immune function. Due to their small size HS in mice presents unique challenges. However due to the many available mouse strains, this species represents an unparalleled resource for the study of the biologic responses. The HS model is an important model for studying cellular communication patterns and the responses of systems such as hormonal and inflammatory mediator systems, and danger signals, i.e. DAMP and PAMP upregulation as it elicits distinct responses that differ from other forms of shock. The development of transgenic murine strains and the induction of biologic agents to inhibit specific signaling have presented valuable opportunities to further elucidate our understanding of the up and down regulation of signal transduction after severe blood loss, i.e. HS and trauma. There are numerous resuscitation methods (R) in association with HS and trauma. A fixed volume resuscitation method of solely lactated ringer solution (LR), equal to three times the shed blood volume, is used in this model to study endogenous mechanisms such as remote organ injury and systemic inflammation. This method of resuscitation is proven to be effective in evaluating the effects of HS and trauma.

Pseudofracture: an acute peripheral tissue trauma model.

Following trauma there is an early hyper-reactive inflammatory response that can lead to multiple organ dysfunction and high mortality in trauma patients; this response is often accompanied by a delayed immunosuppression that adds the clinical complications of infection and can also increase mortality. Many studies have begun to assess these changes in the reactivity of the immune system following trauma. Immunologic studies are greatly supported through the wide variety of transgenic and knockout mice available for in vivo modeling; these strains aid in detailed investigations to assess the molecular pathways involved in the immunologic responses. The challenge in experimental murine trauma modeling is long term investigation, as fracture fixation techniques in mice, can be complex and not easily reproducible. This pseudofracture model, an easily reproduced trauma model, overcomes these difficulties by immunologically mimicking an extremity fracture environment, while allowing freedom of movement in the animals and long term survival without the continual, prolonged use of anaesthesia. The intent is to recreate the features of long bone fracture; injured muscle and soft tissue are exposed to damaged bone and bone marrow without breaking the native bone. The pseudofracture model consists of two parts: a bilateral muscle crush injury to the hindlimbs, followed by injection of a bone solution into these injured muscles. The bone solution is prepared by harvesting the long bones from both hindlimbs of an age- and weight-matched syngeneic donor. These bones are then crushed and resuspended in phosphate buffered saline to create the bone solution. Bilateral femur fracture is a commonly used and well-established model of extremity trauma, and was the comparative model during the development of the pseudofracture model. Among the variety of available fracture models, we chose to use a closed method of fracture with soft tissue injury as our comparison to the pseudofracture, as we wanted a sterile yet proportionally severe peripheral tissue trauma model. Hemorrhagic shock is a common finding in the setting of severe trauma, and the global hypoperfusion adds a very relevant element to a trauma model. The pseudofracture model can be easily combined with a hemorrhagic shock model for a multiple trauma model of high severity.

Models of lower extremity damage in mice: time course of organ damage and immune response.

BACKGROUND: Post-traumatic inflammatory changes have been identified as major causes of altered organ function and failure. Both hemorrhage and soft tissue damage induce these inflammatory changes. Exposure to heterologous bone in animal models has recently been shown to mimic this inflammatory response in a stable and reproducible fashion. This follow-up study tests the hypothesis that inflammatory responses are comparable between a novel trauma model ("pseudofracture", PFx) and a bilateral femur fracture (BFF) model. MATERIALS AND METHODS: In C57BL/6 mice, markers for remote organ dysfunction and inflammatory responses were compared in four groups (control/sham/BFF/PFx) at the time points 2, 4, and 6 h. RESULTS: Hepatocellular damage in BFF and PFx was highly comparable in extent and evolution, as shown by similar levels of NFkappaB activation and plasma ALT. Pulmonary inflammatory responses were also comparably elevated in both trauma models as early as 2 h after trauma as measured by myeloperoxidase activity (MPO). Muscle damage was provoked in both BFF and PFx mice over the time course, although BFF induced significantly higher AST and CK levels. IL-6 levels were also similar with early and sustained increases over time in both trauma models. CONCLUSIONS: Both BFF and PFx create similar reproducible inflammatory and remote organ responses. PFx will be a useful model to study longer term inflammatory effects that cannot be studied using BFF.

Role of hemorrhage in the induction of systemic inflammation and remote organ damage: analysis of combined pseudo-fracture and hemorrhagic shock.

This study was performed to analyze the role of hemorrhage-induced hypotension in the induction of systemic inflammation and remote organ dysfunction. Male C57/BL6 mice (6- to 10-week old and 20-30 g) were used. Animals were either subjected to pseudo-fracture [PF; standardized soft-tissue injury and injection of crushed bone, PF group: n = 9], or PF combined with hemorrhagic shock (HS + PF group: n = 6). Endpoint was 6 h. Systemic inflammation was assessed by IL-6 and IL-10 levels. Myeloperoxidase (MPO) and NF-κB activity in the lung and liver tissue were obtained to assess remote organ damage. The increases of systemic cytokines are similar for animals subjected to PF and PF + HS (IL-6: 189 pg/ml ± 32.5 vs. 160 pg/ml ± 5.3; IL-10: 60.3 pg/ml ± 15.8 vs. 88 pg/ml ± 32.4). Furthermore, the features (ALT; NF-κB) of liver injury are equally elevated in mice subjected to PF (76.9 U/L ± 4.5) and HS + PF (80 U/L ± 5.5). Lung injury, addressed by MPO activity was more severe in group HS + PF (2.95 ng/ml ± 0.32) than in group PF (1.21 ng/ml ± 0.2). Both PF and additional HS cause a systemic inflammatory response. In addition, hemorrhage seems to be associated with remote affects on the lung.

Disease progression following imatinib failure in gastrointestinal stromal tumors: role of surgical therapy.

Gastrointestinal stromal tumors (GISTs) represent the most common mesenchymal neoplasms of the GI tract. The optimal management of GISTs has been evolving rapidly over the past 5 years and depends on proper histopathologic and radiologic diagnosis as well as appropriate multidisciplinary medical and surgical treatments. Complete surgical resection of primary localized GIST with negative margins remains the best therapeutic option today. In the setting of locally advanced or metastatic disease, imatinib mesylate has emerged as the initial treatment of choice, administered either as cytoreductive or as definitive treatment. Surgery or ablative modalities in this setting are becoming increasingly employed, particularly when all disease becomes amenable to gross resection or destruction, or to manage complications arising from the disease following imatinib failure. We report on the surgical management of an unusual and clinically significant complication following progression of disease secondary to imatinib resistance. The role of surgical therapy in the management of GIST complications following resistance to imatinib and the integration of surgical and molecular therapy of locally advanced or metastatic GISTs are discussed.