Inhibition of complement C5a prevents breakdown of the blood-brain barrier and pituitary dysfunction in experimental sepsis

INTRODUCTION: Septic encephalopathy secondary to a breakdown of the blood-brain barrier (BBB) is a known complication of sepsis. However, its pathophysiology remains unclear. The present study investigated the effect of complement C5a blockade in preventing BBB damage and pituitary dysfunction during experimental sepsis. METHODS: Using the standardised caecal ligation and puncture (CLP) model, Sprague-Dawley rats were treated with either neutralising anti-C5a antibody or pre-immune immunoglobulin (Ig) G as a placebo. Sham-operated animals served as internal controls. RESULTS: Placebo-treated septic rats showed severe BBB dysfunction within 24 hours, accompanied by a significant upregulation of pituitary C5a receptor and pro-inflammatory cytokine expression, although gene levels of growth hormone were significantly attenuated. The pathophysiological changes in placebo-treated septic rats were restored by administration of neutralising anti-C5a antibody to the normal levels of BBB and pituitary function seen in the sham-operated group. CONCLUSIONS: Collectively, the neutralisation of C5a greatly ameliorated pathophysiological changes associated with septic encephalopathy, implying a further rationale for the concept of pharmacological C5a inhibition in sepsis

An essential role for complement C5a in the pathogenesis of septic cardiac dysfunction

Defective cardiac function during sepsis has been referred to as “cardiomyopathy of sepsis.” It is known that sepsis leads to intensive activation of the complement system. In the current study, cardiac function and cardiomyocyte contractility have been evaluated in rats after cecal ligation and puncture (CLP). Significant reductions in left ventricular pressures occurred in vivo and in cardiomyocyte contractility in vitro. These defects were prevented in CLP rats given blocking antibody to C5a. Both mRNA and protein for the C5a receptor (C5aR) were constitutively expressed on cardiomyocytes; both increased as a function of time after CLP. In vitro addition of recombinant rat C5a induced dramatic contractile dysfunction in both sham and CLP cardiomyocytes, but to a consistently greater degree in cells from CLP animals. These data suggest that CLP induces C5aR on cardiomyocytes and that in vivo generation of C5a causes C5a–C5aR interaction, causing dysfunction of cardiomyocytes, resulting in compromise of cardiac performance

A novel human skin chamber model to study wound infection ex vivo

Wound infections with multi-drug resistant bacteria increase morbidity and mortality and have considerable socioeconomic impact. They can lead to impaired wound healing, resulting in rising treatment costs. The aim of this study was to investigate an ex vivo human wound infection model. Human full-thickness skin from the operating room (OR) was placed into the Bo-Drum® and cultivated for 7 days in an air–liquid interphase. On day 8, the skin was inoculated with either (1) Pseudomonas aeruginosa, (2) Staphylococcus aureus (105 CFU, n = 3) or (3) carrier control. 1, 3 and 7 days after inoculation colony forming units in the tissue/media were determined and cytokine expression was quantified. A reliable and reproducible wound infection could be established for 7 days. At this timepoint, 1.8 × 108 CFU/g tissue of P. aeruginosa and 2 × 107 CFU/g tissue of S. aureus were detected. Immunohistochemical analysis demonstrated bacterial infection and epidermolysis in infected skin. RT-PCR analysis exhibited a significant induction of proinflammatory cytokines after infection. The BO-drum® is a robust, easy-to-use, sterilizable and reusable ex vivo full-skin culture system. For investigation of wound infection, treatment and healing, the BO-drum® presents a convenient model and may help to standardize wound research

The Complement Anaphylatoxin C5a Induces Apoptosis in Adrenomedullary Cells during Experimental Sepsis

Sepsis remains a poorly understood, enigmatic disease. One of the cascades crucially involved in its pathogenesis is the complement system. Especially the anaphylatoxin C5a has been shown to have numerous harmful effects during sepsis. We have investigated the impact of high levels of C5a on the adrenal medulla following cecal ligation and puncture (CLP)-induced sepsis in rats as well as the role of C5a on catecholamine production from pheochromocytoma-derived PC12 cells. There was significant apoptosis of adrenal medulla cells in rats 24 hrs after CLP, as assessed by the TUNEL technique. These effects could be reversed by dual-blockade of the C5a receptors, C5aR and C5L2. When rats were subjected to CLP, levels of C5a and norepinephrine were found to be antipodal as a function of time. PC12 cell production of norepinephrine and dopamine was significantly blunted following exposure to recombinant rat C5a in a time-dependent and dose-dependent manner. This impaired production could be related to C5a-induced initiation of apoptosis as defined by binding of Annexin V and Propidium Iodine to PC12 cells. Collectively, we describe a C5a-dependent induction of apoptotic events in cells of adrenal medulla in vivo and pheochromocytoma PC12 cells in vitro. These data suggest that experimental sepsis induces apoptosis of adrenomedullary cells, which are responsible for the bulk of endogenous catecholamines. Septic shock may be linked to these events. Since blockade of both C5a receptors virtually abolished adrenomedullary apoptosis in vivo, C5aR and C5L2 become promising targets with implications on future complement-blocking strategies in the clinical setting of sepsis

C5a Enhances Dysregulated Inflammatory and Angiogenic Responses to Malaria In Vitro: Potential Implications for Placental Malaria

Placental malaria (PM) is a leading cause of maternal and infant mortality. Although the accumulation of parasitized erythrocytes (PEs) and monocytes within the placenta is thought to contribute to the pathophysiology of PM, the molecular mechanisms underlying PM remain unclear. Based on the hypothesis that excessive complement activation may contribute to PM, in particular generation of the potent inflammatory peptide C5a, we investigated the role of C5a in the pathogenesis of PM in vitro and in vivo.Using primary human monocytes, the interaction between C5a and malaria in vitro was assessed. CSA- and CD36-binding PEs induced activation of C5 in the presence of human serum. Plasmodium falciparum GPI (pfGPI) enhanced C5a receptor expression (CD88) on monocytes, and the co-incubation of monocytes with C5a and pfGPI resulted in the synergistic induction of cytokines (IL-6, TNF, IL-1beta, and IL-10), chemokines (IL-8, MCP-1, MIP1alpha, MIP1beta) and the anti-angiogenic factor sFlt-1 in a time and dose-dependent manner. This dysregulated response was abrogated by C5a receptor blockade. To assess the potential role of C5a in PM, C5a plasma levels were measured in malaria-exposed primigravid women in western Kenya. Compared to pregnant women without malaria, C5a levels were significantly elevated in women with PM.These results suggest that C5a may contribute to the pathogenesis of PM by inducing dysregulated inflammatory and angiogenic responses that impair placental function

Insight in microcirculation and histomorphology during burn shock treatment using in vivo confocal-laser-scanning microscopy

PURPOSE: Microcirculatory disturbances are well known during shock; however, the accompanied histomorphological alterations are widely unknown. We used high resolution confocal-laser-scanning microscopy for the evaluation of microcirculation and histomorphology during Burn Shock treatment. METHODS: Confocal-laser-scanning microscopy was performed in 10 burn shock patients (4 women, 6 men; aged 40.6 +/- 11.4 years, burn extent >20% body surface area) initially and 24 hours after shock resuscitation. Ten matched hemodynamic stable burn intensive care unit patients served as controls. The following parameters were evaluated: quantitative blood cell flow, cell size of the granular layer, basal layer thickness, and epidermal thickness. RESULTS: Quantitative blood cell flow in controls was 62.45 +/- 3.39 cells per minute. Burn shock significantly reduced blood cell flow to 37.27 +/- 3.64 cells per minute; fluid resuscitation effectively restored baseline blood flow (65.18 +/- 3.76 cells per minute) after 24 hours. Granular cell size was 793.61 +/- 41.58 mum(2) in controls vs 644.27 +/- 42.96 mum(2) during burn shock. Post resuscitation granular cell size measured 932.74 +/- 38.83 mum(2). Basal layer thickness was 14.84 +/- 0.59 mum in controls, 13.26 +/- 0.54 mum in burn patients at admission and before resuscitation, and 17.50 +/- 0.46 mum after resuscitation. Epidermal thickness in control patients was 49.60 +/- 2.36 mum, 37.83 +/- 2.47 mum in burn patients at admission and 69.50 +/- 3.18 mum after resuscitation. CONCLUSIONS: Confocal-laser-scanning microscopy provides a noninvasive tool for simultaneous evaluation of microcirculation and tissue histomorphology. It may help to assess the adequacy of and response to resuscitation of burn patients early after trauma