Mario Perl

Leukocyte apoptosis and its significance in sepsis and shock

Sepsis and multiple organ failure continue to be significant problems among trauma, burn, and the critically ill patient population. Thus, a number of laboratories have focused on understanding the role of altered apoptotic cell death in contributing to immune and organ dysfunction seen in sepsis and shock. Immune cells that undergo altered apoptotic changes include neutrophils, macrophages, dendritic cells, as well as various lymphocyte populations. Evidence of epithelial as well as endothelial cell apoptotic changes has also been reported. Although mediators such as steroids, tumor necrosis factor, nitric oxide, C5a, and Fas ligand (FasL) appear to contribute to the apoptotic changes, their effects are tissue‐ and cell population‐selective. As inhibiting Fas‐FasL signaling (e.g., gene deficiency, Fas fusion protein, or Fas short interfering RNA administration), caspase inhibition (caspase mimetic peptides), and/or the overexpression of downstream antiapoptotic molecules (e.g., Bcl‐2, Akt) improve survival of septic mice, it not only demonstrates the pathological significance of this process but points to novel targets for the treatment of sepsis.

Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis.

Apoptosis and inflammation play an important role in the pathogenesis of direct/pulmonary acute lung injury (ALI). However, the role of the Fas receptor-driven apoptotic pathway in indirect/nonpulmonary ALI is virtually unstudied. We hypothesized that if Fas or caspase-8 plays a role in the induction of indirect ALI, their local silencing using small interfering RNA (siRNA) should be protective in hemorrhage-induced septic ALI. Initially, as a proof of principle, green fluorescent protein-siRNA was administered intratracheally into transgenic mice overexpressing green fluorescent protein. Twenty-four hours after siRNA delivery, lung sections revealed a significant decrease in green fluorescence. Intratracheally administered Cy-5-labeled Fas-siRNA localized primarily in pulmonary epithelial cells. Intratracheal instillation of siRNA did not induce lung inflammation via toll-like receptor or protein kinase PKR pathways as assessed by lung tissue interferon-alpha, tumor necrosis factor-alpha, and interleukin (IL)-6 levels. Mice subjected to hemorrhagic shock and sepsis received either Fas-, caspase-8-, or control-siRNA intratracheally 4 hours after hemorrhage. Fas- or caspase-8-siRNA significantly reduced lung tissue Fas or caspase-8 mRNA, respectively. Only Fas-siRNA markedly diminished lung tissue tumor necrosis factor-alpha, IL-6, IL-10, interferon-gamma, IL-12, and caspase-3 activity. Fas-siRNA also preserved alveolar architecture and reduced lung neutrophil infiltration and pulmonary epithelial apoptosis. These data indicate the pathophysiological significance of Fas activation in nonpulmonary/shock-induced ALI and the feasibility of intrapulmonary administration of anti-apoptotic siRNA in vivo.

In vivo gene silencing (with siRNA) of pulmonary expression of MIP‐2 versus KC results in divergent effects on hemorrhage‐induced, neutrophil‐mediated septic acute lung injury

Lung injury in trauma patients exposed to a secondary infectious/septic challenge contributes to the high morbidity/mortality observed in this population. Associated pathology involves a dys‐regulation of immune function, specifically, sequestration of activated polymorphonuclear neutrophils (PMN) in the lungs. The targeting of PMN is thought to involve the release of chemokines from cells within the local environment, creating a concentration gradient along which PMN migrate to the focus of inflammation. Keratinocyte‐derived chemokine (KC) and macrophage‐inflammatory protein‐2 (MIP‐2) are murine neutrophil chemokines identified as playing significant but potentially divergent roles in the pathogenesis of acute lung injury (ALI). In the current study, we examined the contribution of local pulmonary cells to the production of KC and MIP‐2 and the pathogenesis of ALI. We hypothesized that local silencing of KC or MIP‐2, via the local administration of small interference RNA (siRNA) against KC or MIP‐2, following traumatic shock/hemorrhage (Hem), would suppress signaling for PMN influx to the lung, thereby reducing ALI associated with a secondary septic challenge (cecal ligation and puncture). Assessment of siRNA local gene silencing was done in green fluorescent protein (GFP)‐transgenic, overexpressing mice. A marked suppression of GFP expression was observed in the lung 24 h following intratracheal (i.t.) instillation of GFP siRNA, which was not observed in the liver. To test our hypothesis, siRNA against KC or MIP‐2 (75 ug/C3H/Hen mouse) was instilled (i.t.) 2 h post‐Hem (35 mm Hg for 90 min, 4× LRS Rx.). Twenty‐four hours after, mice were subjected to septic challenge and then killed 24 h later. i.t. MIP‐2 siRNA significantly (P<0.05, ANOVA‐Tukey’s test, n=5–6/group) reduced tissue and plasma interleukin (IL)‐6, tissue MIP‐2 (enzyme‐linked immunosorbent assay), as well as neutrophil influx [myeloperoxidase (MPO) activity]. In contrast, KC siRNA treatment reduced plasma KC, tissue KC, and IL‐6 but produced no significant reduction in plasma IL‐6 or MPO. Neither treatment reduced tissue or plasma levels of tumor necrosis factor α compared with vehicle. These data support not only our hypothesis that local pulmonary chemokine production of MIP‐2, to a greater extent than KC, contributes to the pathogenesis of PMN‐associated ALI following Hem but also the use of siRNA as a potential therapeutic.

Cardiopulmonary, histological, and inflammatory alterations after lung contusion in a novel mouse model of blunt chest trauma

Severe blunt chest trauma remains an important injury with high morbidity and mortality. However, the associated immunological alterations are poorly understood. Existing big animal models require large-scale settings, are often too expensive, and research products for immunological studies are limited. In this study we aimed to establish a new model of blunt, isolated and bilateral chest trauma in mice and to characterize its effects on physiological and inflammatory variables. Male C3H/HeN mice (n = 9–10/group) were anesthetized and a femoral artery was catheterized. The animals were subjected to trauma or sham procedure and monitored for 180 min. Blunt chest trauma was induced by a blast wave focused on the thorax. Trauma intensity was optimized by varying the exposure distance. Blood pressure, heart rate, respiratory rate, arterial blood gases and plasma cytokine levels were measured. Macroscopic and microscopic examinations were performed. In addition, outcome was evaluated in a 10-day survival study. Chest trauma caused a drop (P < 0.05) in blood pressure and heart rate, which partly recovered. Blood gases revealed hypoxemia and hypercarbia (P < 0.05) 180 min after trauma. There was marked damage to the lungs but none to abdominal organs. Histologically, the characteristic signs of a bilateral lung contusion with alveolar and intrabronchial hemorrhage were found. Plasma interleukin-6 and tumor necrosis factor &agr; were considerably increased after 180 min. Blunt chest trauma resulted in an early mortality of 10% without subsequent death. On the basis of these findings, this novel mouse model of blunt chest trauma appears suitable for detailed studies on immunological effects of lung contusion.

Shock and hemorrhage: an overview of animal models.

Shock resulting from life-threatening blood loss (hemorrhage) remains a common complication of traumatic injury. Intensive experimental efforts are needed if we are to understand the pathological effect(s) of hemorrhagic shock, alone or in association with traumatic tissue injury, and to reverse this deleterious process in trauma patients. Here, we overview selected studies that are representative of the different hemorrhagic shock models, considering their advantages and disadvantages from a scientific and clinical perspective. Fixed-pressure versus fixed-volume versus uncontrolled hemorrhage models, with or without tissue injury, will be discussed, as well as small versus large animal models. Most of these models are nonlethal in nature, and allow the researcher to understand the changes that contribute to increased susceptibility to subsequent infection or the development of multiple organ failure. We also consider some of the confounders in these models, including anesthesia, the nature of resuscitation, and the use of anticoagulants. The selection of model must take into consideration not only the need for experimental control but must also adequately reflect the clinical pathobiology of shock if we are to develop better pharmacological interventions.

Pathogenesis of indirect (secondary) acute lung injury

At present, therapeutic interventions to treat acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) remain largely limited to lung-protective strategies, as no real molecular–pathophysiologic-driven therapeutic intervention has yet become available. This is in part the result of the heterogeneous nature of the etiological processes that contribute to the state of ALI/ARDS. This article sets out to understand the development of ALI resulting from indirect pulmonary insults, such as extrapulmonary sepsis and trauma, shock, burn injury or mass transfusion, as opposed to direct pulmonary challenges, such as pneumonia, aspiration or lung contusion. Here, we consider not only the experimental and clinical data concerning the roles of various immune (neutrophil, macrophage, lymphocyte and dendritic) as well as nonimmune (epithelial and endothelial) cells in orchestrating the development of ALI resulting from indirect pulmonary stimuli, but also how these cell populations might be targeted therapeutically.

The Pattern of Preformed Cytokines in Tissues Frequently Affected by Blunt Trauma

The aim of this prospective study was to determine the local concentrations of inflammatory mediators in various tissue types frequently affected by trauma to estimate the role of prestored cytokine release by mechanical tissue trauma in the induction of a systemic inflammatory response syndrome. The degree of tissue damage, evaluated by its systemic release of inflammatory mediators, represents an important factor concerning the outcome of trauma patients. Clinical trials indicate that the kind of traumatized tissue influences the cytokine pattern measured in patients blood afterwards. However, the tissue-specific mediator composition underlying this systemic mediator release is rarely elucidated. Upon approval of the local IRB/EC, skin, subcutaneous fat, muscle, cancellous bone, and lung tissue were obtained during standard surgical procedures. The protein-based concentrations of Interleukin (IL)-6, IL-8, IL-10, and IL-12 were determined in tissue homogenates by enzyme-linked immunoabsorbant assay (ELISA; n = 60 samples). Albumin was measured to evaluate the degree of blood contamination of tissue samples. IL-6 and IL-8 were consistently detectable in more than 95% of the tissue specimens. Lung and cancellous bone presented by far the highest concentrations of these cytokines, whereas skin, subcutaneous fat, and muscle showed significantly lower levels. IL-10 was not detectable in 88%; IL-12 could not be measured in 63% of the samples. Cytokine concentrations did not correlate with the amount of albumin measured in tissue specimens. Due to their consistent presence at the tissue level, high systemic concentrations of IL-6 and IL-8 in patients blood, seen after pulmonary trauma, long bone fractures, or soft tissue injury, may be interpreted as an overspill of local trauma mediators. This indicates their relevance in post-traumatic monitoring. Furthermore, albumin is a suitable and necessary indicator to evaluate influences of possible blood contamination in tissue samples.

Epithelial cell apoptosis and neutrophil recruitment in acute lung injury-a unifying hypothesis? What we have learned from small interfering RNAs.

In spite of protective ventilatory strategies, Acute Lung Injury (ALI) remains associated with high morbidity and mortality. One reason for the lack of therapeutic options might be that ALI is a co-morbid event associated with a diverse family of diseases and, thus, may be the result of distinct pathological processes. Among them, activated neutrophil- (PMN-) induced tissue injury and epithelial cell apoptosis mediated lung damage represent two potentially important candidate pathomechanisms that have been put forward. Several approaches have been undertaken to test these hypotheses, with substantial success in the treatment of experimental forms of ALI. With this in mind, we will summarize these two current hypotheses of ALI briefly, emphasizing the role of apoptosis in regulating PMN and/or lung epithelial cell responses. In addition, the contribution that Fas-mediated inflammation may play as a potential biological link between lung cell apoptosis and PMN recruitment will be considered, as well as the in vivo application of small interfering RNA (siRNA) as a novel approach to the inhibition of ALI and its therapeutic implications.

Early Complementopathy After Multiple Injuries in Humans.

ABSTRACT After severe tissue injury, innate immunity mounts a robust systemic inflammatory response. However, little is known about the immediate impact of multiple trauma on early complement function in humans. In the present study, we hypothesized that multiple trauma results in immediate activation, consumption, and dysfunction of the complement cascade and that the resulting severe “complementopathy” may be associated with morbidity and mortality. Therefore, a prospective multicenter study with 25 healthy volunteers and 40 polytrauma patients (mean injury severity score = 30.3 ± 2.9) was performed. After polytrauma, serum was collected as early as possible at the scene, on admission to the emergency room (ER), and 4, 12, 24, 120, and 240 h post-trauma and analyzed for the complement profile. Complement hemolytic activity (CH-50) was massively reduced within the first 24 h after injury, recovered only 5 days after trauma, and discriminated between lethal and nonlethal 28-day outcome. Serum levels of the complement activation products C3a and C5a were significantly elevated throughout the entire observation period and correlated with the severity of traumatic brain injury and survival. The soluble terminal complement complex SC5b-9 and mannose-binding lectin showed a biphasic response after trauma. Key fluid-phase inhibitors of complement, such as C4b-binding protein and factor I, were significantly diminished early after trauma. The present data indicate an almost synchronical rapid activation and dysfunction of complement, suggesting a trauma-induced complementopathy early after injury. These events may participate in the impairment of the innate immune response observed after severe trauma.

Biochemical characterization of individual injury pattern and injury severity

BACKGROUND Estimation of trauma severity currently relies on clinical diagnoses and scoring systems. However, the early estimation of the severity of chest trauma and overall soft tissue trauma (STT) remains insufficient. Traditional trauma scoring systems fail to reflect the individual trauma pattern and severity, neglecting the different outcomes after injuries in different body regions. Therefore, the aim of this prospective study was to detect laboratory markers that may reflect the pattern and extent of individual trauma in the very early phase after injury. PATIENTS AND METHODS In 107 non-selected trauma patients, blood samples were collected almost immediately and then at short intervals after the trauma. In addition to the biochemical analysis of 20 different mediators viewed as potential trauma markers, the following data were correlated with the laboratory results: injury severity score (ISS), polytrauma score (PTS), Ulmer score HTAPE (trauma pattern specific: head (H), thorax (T), abdomen (A), pelvis (P), extremities (E); 0-3 degrees each), multiple organ failure score (MOF), overall, primary and secondary lethality. RESULTS ISS and the severity of head injury were clearly higher in non-survivors (n=17) than in survivors (n=90) (median ISS: 35 versus 18; median severity of head injury (H): 3 versus 1). Whereas head injury was correlated with early death (<or=3 days: r=0.45), late death (>3 days post-trauma) was influenced by thoracic trauma (r=0.15) as well as by soft tissue trauma (STT, r=0.12). Of all investigated mediators, interleukin-6 (IL-6) displayed the highest correlations (r=0.66, P<0.00001) with the extent of chest trauma, followed by correlations with PTS, STT, fracture trauma (FT) and ISS during the first hour after trauma. There was no correlation between IL-6 and head injury. The extent of STT was correlated best to IL-8 (r=0.75), IL-6 (r=0.54), and creatine kinase (CK, r=0.49) plasma concentrations. CONCLUSION In the very early stage after an accident the severity of chest trauma is strongly correlated with the plasma concentration of IL-6, and the extent of overall soft tissue trauma (STT) to plasma concentrations of IL-8, IL-6, and CK.