Apoptosis of human intervertebral discs after trauma compares to degenerated discs involving both receptor-mediated and mitochondrial-dependent pathways.

Post-traumatic disc degeneration with consecutive loss of reduction and kyphosis remains a debatable issue within both the operative and nonoperative treatment regimen of thoracolumbar spine fractures. Intervertebral disc (IVD) cell apoptosis has been suggested to play a vital role in promoting the degeneration process. To evaluate and compare apoptosis-regulating signaling mechanisms, IVDs were obtained from patients with thoracolumbar spine fractures (n = 21), patients suffering from symptomatic IVD degeneration (n = 6), and from patients undergoing surgical resection of a primary vertebral tumor (n = 3 used as control samples). All tissues were prospectively analyzed in regards to caspase-3/7, -8, and -9 activity, apoptosis-receptor expression levels, and gene expression of the mitochondria-bound apoptosis-regulating proteins Bax and Bcl-2. Morphologic changes characteristic for apoptotic cell death were confirmed by H&E staining. Statistical significance was designated at p < 0.05 using the Student's t-test. Both traumatic and degenerative IVD demonstrated a significant increase of caspase-3/7 activity with evident apoptosis. Although caspase-3/7 activation was significantly greater in degenerated discs, both showed equally significant activation of the initiator caspases 8 and 9. Traumatic IVD alone demonstrated a significant increase of the Fas receptor (FasR), whereas the TNF receptor I (TNFR I) was equally up-regulated in both morbid IVD groups. Only traumatic IVD showed distinct changes in up-regulated TNF expression, in addition to significantly down-regulated antiapoptotic Bcl-2 protein. Our results suggest that post-traumatic disc changes may be promoted and amplified by both the intrinsic mitochondria-mediated and extrinsic receptor-mediated apoptosis signaling pathways, which could be, in part, one possible explanation for developing subsequent disc degeneration.

Biphasic onset of splenic apoptosis following hemorrhagic shock: critical implications for Bax, Bcl-2, and Mcl-1 proteins.

INTRODUCTION: The innate immune response to trauma hemorrhage involves inflammatory mediators, thus promoting cellular dysfunction as well as cell death in diverse tissues. These effects ultimately bear the risk of post-traumatic complications such as organ dysfunction, multiple organ failure, or adult respiratory distress syndrome. In this study, a murine model of resuscitated hemorrhagic shock (HS) was used to determine the apoptosis in spleen as a marker of cellular injury and reduced immune functions. METHODS: Male C57BL-6 mice were subjected to sham operation or resuscitated HS. At t = 0 hours, t = 24 hours, and t = 72 hours, mice were euthanized and the spleens were removed and evaluated for apoptotic changes via DNA fragmentation, caspase activities, and activation of both extrinsic and intrinsic apoptotic pathways. Spleens from untreated mice were used as control samples. RESULTS: HS was associated with distinct lymphocytopenia as early as t = 0 hours after hemorrhage without regaining baseline levels within the consecutive 72 hours when compared with sham and control groups. A rapid activation of splenic apoptosis in HS mice was observed at t = 0 hours and t = 72 hours after hemorrhage and predominantly confirmed by increased DNA fragmentation, elevated caspase-3/7, caspase-8, and caspase-9 activities, and enhanced expression of intrinsic mitochondrial proteins. Accordingly, mitochondrial pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins were inversely expressed within the 72-hour observation period, thereby supporting significant pro-apoptotic changes. Solely at t = 24 hours, expression of the anti-apoptotic Mcl-1 protein shows a significant increase when compared with sham-operated and control animals. Furthermore, expression of extrinsic death receptors were only slightly increased. CONCLUSION: Our data suggest that HS induces apoptotic changes in spleen through a biphasic caspase-dependent mechanism and imply a detrimental imbalance of pro- and anti-apoptotic mitochondrial proteins Bax, Bcl-2, and Mcl-1, thereby promoting post-traumatic immunosuppression.

The early second hit in trauma management augments the proinflammatory immune response to multiple injuries.

BACKGROUND: Today's management of patients with multiple injuries remains controversial with regard to damage control and the appropriate timing of operative treatment ("second hit"). Among the multitude of physiologic parameters critical to the immune defense and clinical course of recovery, recent research has proven the regulation of distinct pro- and anti-inflammatory mediators to be closely associated with posttraumatic outcome and complications, including systemic inflammatory response syndrome (SIRS) and sepsis. This study sought to investigate the significance of multiple injuries and consecutive operative treatment ("second hit") with regard to the early inflammatory profile and its importance within the host's immune function. METHODS: Peripheral whole blood was obtained from 32 patients with multiple injuries (injury severity score [ISS] >20) and 14 healthy control subjects on the day of injury (day 0) and 24 hours thereafter (day 1). Trauma patients were divided into two groups (trauma versus trauma + immediate operation ["second hit"]). Whole blood was centrifuged at 400 g at room temperature for subsequent plasma collection and analyses of Interleukin-6 (IL-6), IL-10 and soluble triggering receptor expressed on myeloid cells (sTREM)-1 plasma concentrations by enzyme-linked immunosorbent assay, respectively. RESULTS: IL-6 plasma levels from second hit trauma patients (n = 18, ISS 35.5 +/- 12.2) significantly exceeded values determined in both trauma patients without a second hit (n = 14, ISS 30.5 +/- 5.3) and healthy control subjects (n = 14) by posttrauma day 1 (p < 0.05). IL-10 plasma concentrations on day 1 were equally and significantly elevated in both trauma patient populations, when compared with control samples (p < 0.05). In contrast, sTREM-1 was exclusively increased in trauma patients with a second hit, suggesting a strong proinflammatory response in patients with multiple injuries challenged with immediate surgical care (p < 0.05). CONCLUSION: Immediate surgical treatment of patients with multiple injuries augments the proinflammatory immune response in the early phase of recovery as determined by increased IL-6 and sTREM-1 plasma levels. If not required solely for damage control, the early second hit from additional surgical stress might promote posttraumatic complications by surcharging the innate immune response to injury.

Traumatic proximal tibiofibular joint dislocation treated by open reduction and temporary fixation: a case report.

Isolated dislocations of the proximal tibiofibular joint are a rare condition. Missed diagnosis can lead to chronic knee pain and disability. Early recognition should be followed by immediate closed reduction or open reduction and joint transfixation. We present a young athlete with this injury which was treated successfully by open reduction.

Erythropoiesis in multiply injured patients.

Posttraumatic anemia in multiply injured patients is caused by hemorrhage, reduced red blood cell survival, and impaired erythropoiesis. Trauma-induced hyperinflammation causes impaired bone-marrow function by means of blunted erythropoietin (EPO) response, reduced iron availability, suppression and egress of erythroid progenitor cells. To treat posttraumatic anemia in severely injured patients, symptomatic therapy by blood transfusion is not sufficient. Furthermore, EPO, iron, and the use of red cell substitutes should be considered. The posttraumatic systemic inflammatory response syndrome (SIRS) induces posttraumatic anemia. Thus, a worsening of SIRS by a "second-hit" through blood transfusion ought to be avoided.

Concepts of transtibial amputation: Burgess technique versus modified Brückner procedure.

The technique of a long posterior myocutaneous flap described by Ernest M. Burgess in the late 1960s is one of the most frequent procedures for below-knee amputations worldwide. To account for some potential problems associated with this procedure in patients with occlusive arterial disease, Lutz Brückner developed a modified amputation technique for transtibial amputation in the 1980s. Although this new standardized procedure has been widely used in Germany, it is not well known outside central Europe, secondary to its lack of description in English published work. In this review article, we describe a comparison of the technical aspects of the Burgess procedure with the modified Brückner technique for transtibial amputations. In addition, the charts of 69 consecutive patients with end-stage occlusive arterial disease undergoing below-knee amputation by either of the two standardized procedures (Burgess, n = 29; Brückner, n = 40) were reviewed. The clinical results of the two procedures are reported and compared. Review of the German published work suggests that the two techniques for transtibial amputation in patients with occlusive arterial disease appear to have similar results. This is further supported by data from our own 10-year experience with 69 patients undergoing below-knee amputation by either of the two standardized procedures. This suggests that the Brückner technique is at least equivalent to the Burgess procedure with regard to the clinical outcome in patients with occlusive arterial disease. The potential advantages of the standardized modified Brückner procedure compared with the 'classical' Burgess technique for transtibial amputation remain to be assessed in prospective multicentre trials.

Trauma induces apoptosis in human thoracolumbar intervertebral discs.

BACKGROUND: Vertebral fractures resulting from high energy trauma often comprise the risk of posttraumatic degenerative changes in the affected intervertebral discs (IVD). Particularly in conservatively treated patients, or in cases after implant removal of an exclusively posterior stabilization, consecutive disc degeneration and the associated functional losing of the spinal segment clearly represent detrimental treatment results. In this regard, apoptosis of IVD cells has been suggested to be involved in the critical changes of the extracellular matrix. METHODS: To investigate whether fractures of the vertebrae induce apoptosis in the affected IVD, disc tissue from patients (n = 17) undergoing open reduction and internal fixation of thoracolumbar spine fractures were analysed in regards to caspase activity, apoptosis-receptor expression levels and gene expression of apoptosis-regulating proteins such as Bax and Bcl-2. Healthy IVD tissue (n = 3) obtained from patients undergoing surgical resection of adjacent vertebrae were used as control samples. RESULTS: In contrast to healthy control IVD tissues, samples from traumatic thoracolumbar IVD showed positive TUNEL staining and a significant increase of caspase-3/7 activity. Interestingly, analyses of the initiator caspase-8 and -9 revealed significantly increased activation levels compared to control values, suggesting the coexistent activation of both the extrinsic (receptor-mediated) and intrinsic (mitochondria-mediated) apoptosis pathway. Accordingly, expression levels of the Fas receptor (FasR) mRNA were significantly increased. Although the TNF receptor I (TNFR I) was only slightly upregulated, corresponding TNFalpha from trauma IVD presented significantly increased mRNA expression values. Furthermore, traumatic IVD cells demonstrated significantly reduced expression of the mitochondria-bound anti-apoptotic Bcl-2, thereby maintaining baseline transcriptional levels of the pro-apoptotic Bax protein when compared to control IVD cells. CONCLUSION: Our data suggest that thoracolumbar fractures induce early caspase-dependent apoptosis in IVD cells of the affected intervertebral disc, in part, by downregulation of the anti-apoptotic protein Bcl-2 (intrinsic apoptosis pathway), as well as signalling via the death receptor complex (TNFR I and FasR).

Interleukin-18: a novel prognostic cytokine in bacteria-induced sepsis.

OBJECTIVE: Severe inflammation and sepsis remain a serious clinical challenge worldwide. Despite modern supportive medicine and an improved understanding of the underlying pathophysiology, mortality rates remain high in patients suffering from this severe inflammatory process. The often excess production of pro- and anti-inflammatory cytokines frequently found in the circulation of septic patients has stimulated the search for reliable inflammatory mediators that can be used for the diagnosis and prediction of clinical outcome. Interleukin (IL)-18, formerly termed interferon-gamma inducing factor, is a pro-inflammatory and Th1 cytokine suggested to play a significant role in the pathogenesis of this disease. This review focuses on our current understanding of the pro-inflammatory cytokine, IL-18, and its potentially unique role in sepsis. METHODS: Bibliographic search of the most recent literature (1995-2005) relating to IL-18 and its role in inflammatory diseases, with emphasis on its pathophysiological importance in sepsis. In addition, a summary of the author's own experimental data from this particular field of research set in the context of current knowledge regarding IL-18. RESULTS AND CONCLUSIONS: Several studies have shown elevated plasma IL-18 concentrations to be associated with poor clinical outcome in severe inflammatory and septic conditions. Moreover, a significant increase in IL-18 concentrations has been shown to discriminate between Gram-positive and Gram-negative related sepsis, and, thus, may potentially augment existing diagnostic tools. Biological neutralization of IL-18 via caspase-1 intervention or through the administration of IL-18-binding protein has been promulgated as a promising therapeutic approach, but additional studies are required to evaluate its full potential in acute inflammatory diseases.

Local thymic caspase-9 inhibition improves survival during polymicrobial sepsis in mice.

Caspase-9 is believed to play an essential role in sepsis-induced lymphocyte apoptosis. The aim of this study was therefore to evaluate its contribution within the caspase-dependent apoptosis pathway in a murine model of polymicrobial sepsis. Local injections of Z-LEHD-fmk, a specific caspase-9 inhibitor, into thymi of septic mice led to the complete inhibition of caspase-9, decreased apoptosis of resident tissue cells, and, in addition, reduced further downstream caspase-3 activity. In contrast to its systemic administration, only local injections improved the overall survival of septic mice. However, local injections of a pancaspase inhibitor (Z-VAD-fmk) did not improve survival, although caspase-3 activity was reduced to a similar degree as by the administration of Z-LEHD-fmk. These results indicate that local apoptosis of lymphatic tissue in polymicrobial sepsis is processed dependent of caspase-9 and suggests alternative caspase-dependent beneficial effects, which may determine a positive outcome.

IL-18: a key player in neuroinflammation and neurodegeneration?

Interleukin (IL)-18 is a potent inflammatory cytokine of the IL-1 family. It is synthesized as an inactive precursor (pro-IL-18), which is cleaved into its functionally active form by caspase-1. Resident cells of the CNS express IL-18 and caspase-1 constitutively, thus providing a local IL-18-dependent immune response. Recent studies have highlighted a crucial role for IL-18 in mediating neuroinflammation and neurodegeneration in the CNS under pathological conditions, such as bacterial and viral infection, autoimmune demyelinating disease, and hypoxic-ischemic, hyperoxic and traumatic brain injuries. This review provides a synopsis of the current knowledge of IL-18-dependent mechanisms of action during acute neurodegeneration in immature and adult brains.

Plasma cytokine measurements augment prognostic scores as indicators of outcome in patients with severe sepsis.

Despite recent advances in the prospective identification of the patient with sepsis who may benefit from anti-inflammatory or antithrombotic therapies, successful treatment regimens have been fairly modest. We have explored whether determination of several proinflammatory cytokine or mediator concentrations can complement physiologic scoring systems to identify patients with severe sepsis who will survive or expire within 28 days. The design of the study included an exploratory analysis performed in conjunction with a prospective, randomized, double-blind, placebo-controlled, multicenter, clinical trial and involved 33 academic institutions in the United States. One hundred twenty-four patients with severe sepsis with or without septic shock were included in this analysis. Blood samples were obtained at baseline and on days 1 through 4, and were evaluated for proinflammatory and anti-inflammatory cytokine concentrations, as well as for procalcitonin and total protein C levels. Baseline concentrations and changes in the concentrations of these mediators were evaluated in relationship to the Acute Physiology and Chronic Health Evaluation (APACHE) II and multiple organ dysfunction (MOD) scores, and 28-day all-cause mortality. Using univariate logistic regression analyses, APACHE II and MOD scores, age (but not gender), and baseline plasma interleukin (IL)-6 and soluble tumor necrosis factor receptor (sTNFR) 1 (log transformed) concentrations were all predictive of increased 28-day all-cause mortality (P < 0.01). Baseline total protein C, IL-8, IL-10, TNF-alpha, and procalcitonin concentrations, and the change in plasma cytokine concentrations from baseline over the initial 4 days were not useful in predicting outcome. Selected baseline proinflammatory cytokine concentrations and APACHE II score were correlated (P < 0.01). IL-6 concentration is a strong candidate for predicting clinical outcome in patients with severe sepsis alone, or when combined with the APACHE II or MOD scores. The potential usefulness of the combination of cytokine measurements and prognostic scores to identify patients who may benefit from treatment with anti-inflammatory or antithrombotic therapies should be further evaluated.

Functional modification of dendritic cells with recombinant adenovirus encoding interleukin 10 for the treatment of sepsis.

Control of dendritic cell (DC) function is critical for strategies to modulate innate and acquired immune responses. We examined whether transduction of murine DCs with adenoviral vectors (Adv) expressing interleukin (IL)-10 could alter their phenotype and T cell stimulatory function. Murine bone marrow-derived DCs were transduced with AdV encoding human IL-10 or green fluorescent protein (GFP). Whereas transduction of immature DCs with AdV/GFP resulted in dose-dependent maturation, DCs transduced with Adv/IL-10 maintained an immature state with low major histocompatibility complex (MHC) class II, CD86, and IL-12 expression. The Adv/IL-10 transduced DCs were phenotypically unique, characterized by suppression of IL-12 expression, failure to stimulate Th1 or Th2 cytokine responses, and retained capacity to endocytose antigen. Importantly, Adv/IL-10-transduced DCs were biologically active in vivo, in that administration of these DCs into mice before a generalized peritonitis significantly improved survival. We conclude that Adv/IL-10 transduction of DCs provides an efficient means to modulate DC function. The capacity to modify DCs by adenoviral expression of IL-10 may provide a novel ex vivo or in vivo approach to mitigate acute and chronic inflammatory diseases like sepsis.

Influence of recombinant adenovirus on liver injury in endotoxicosis and its modulation by IL-10 expression.

Adenovirus-based gene therapy offers a unique opportunity to target gene expression to the liver by systemic delivery. However, systemic administration of a first generation adenoviral construct elicits an inflammatory response leading to TNF-alpha-dependent liver injury. The aim of this study was to evaluate whether the systemic administration of recombinant adenovirus exacerbates a subsequent TNF-alpha-dependent liver injury induced by D-galactosamine and lipopolysaccharide. Surprisingly, low-dose adenovirus administration (10(5) particles) protects, while high-dose adenovirus (10(10) particles) is associated with an exaggerated hepatic inflammatory response from a subsequent D-galactosamine and lipopolysaccharide challenge. This exacerbation is TNF-alpha dependent, since treatment with a TNF inhibitor fully protects against the liver injury. Moreover, intravenous administration of an adenoviral construct expressing the anti-inflammatory protein interleukin-10 reduces TNF-alpha appearance and attenuates the increased hepatocyte injury. Taken together, this report demonstrates potential additive effects of TNF-alpha responses induced by adenovirus and other inflammatory signals, and suggests that the response can be mitigated by relative adenovirus particle dose or by inhibitors, such as TNF-binding protein or interleukin 10.

Molecular characterization of the acute inflammatory response to infections with gram-negative versus gram-positive bacteria.

Sepsis caused by gram-negative bacteria and that caused by gram-positive bacteria often manifest similar clinical features. We investigated plasma proinflammatory cytokine profiles in patients with sepsis due to gram-positive and gram-negative bacteria and studied the cytokine production and differential gene regulation of leukocytes stimulated ex vivo with Escherichia coli lipopolysaccharide or heat-killed Staphylococcus aureus. Concentrations of tumor necrosis factor alpha, interleukin 1 receptor antagonist (IL-1Ra), IL-8, IL-10, IL-18 binding protein, procalcitonin, and protein C in plasma did not differ between patients with sepsis due to gram-negative and gram-positive bacteria. However, plasma IL-1beta, IL-6, and IL-18 concentrations were significantly higher in patients with sepsis due to gram-positive bacteria. Ex vivo stimulation of whole blood with heat-killed S. aureus markedly increased IL-1beta and IL-18 levels more than E. coli lipopolysaccharide stimulation. Microarray analysis revealed at least 359 cross-validated probe sets (genes) significant at the P < 0.001 level whose expression discriminated among gram-negative-organism-stimulated, gram-positive-organism-stimulated, and unstimulated whole-blood leukocytes. The host inflammatory responses to gram-negative and gram-positive stimuli share some common response elements but also exhibit distinct patterns of cytokine appearance and leukocyte gene expression.

Increased survival in sepsis by in vivo adenovirus-induced expression of IL-10 in dendritic cells.

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (10(5)) yields only local expression, while transduction with higher particle numbers (10(7) and 10(10)) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (10(5)) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.

Interleukin-10: A complex role in the pathogenesis of sepsis syndromes and its potential as an anti-inflammatory drug.

Interleukin (IL)-10 is a pleiotropic cytokine produced by both T cells and macrophages and possesses both anti-inflammatory and immunosuppressive properties. IL-10 circulates in the blood of patients with sepsis syndromes, and increased concentrations of IL-10 have been associated with an adverse clinical outcome. Experimental studies in rodents and primates have demonstrated that endogenously produced and exogenously administered IL-10 can reduce the magnitude of the inflammatory response and improve outcome, primarily in models of endotoxemic and bacteremic shock. However, endogenous IL-10 production and systemic administration can also exacerbate T-cell dysfunction, decrease T-cell apoptosis, reduce antimicrobial function, and increase mortality in other less acute bacterial models of sepsis or after thermal injury. Targeted delivery of IL-10 to individual tissues may obviate the adverse effects of systemic delivery. The potential anti-inflammatory properties of IL-10 will have to be carefully weighed against its immunosuppressive properties when considering its use in patients with acute inflammation and sepsis syndromes.

Interleukin-10: a complex role in the pathogenesis of sepsis syndromes and its potential as an anti-inflammatory drug.

Interleukin (IL)-10 is a pleiotropic cytokine produced by both T cells and macrophages and possesses both anti-inflammatory and immunosuppressive properties. IL-10 circulates in the blood of patients with sepsis syndromes, and increased concentrations of IL-10 have been associated with an adverse clinical outcome. Experimental studies in rodents and primates have demonstrated that endogenously produced and exogenously administered IL-10 can reduce the magnitude of the inflammatory response and improve outcome, primarily in models of endotoxemic and bacteremic shock. However, endogenous IL-10 production and systemic administration can also exacerbate T-cell dysfunction, decrease T-cell apoptosis, reduce antimicrobial function, and increase mortality in other less acute bacterial models of sepsis or after thermal injury. Targeted delivery of IL-10 to individual tissues may obviate the adverse effects of systemic delivery. The potential anti-inflammatory properties of IL-10 will have to be carefully weighed against its immunosuppressive properties when considering its use in patients with acute inflammation and sepsis syndromes.