C3a Concentrations Research Articles

Reduced neuronal cell death after experimental brain injury in mice lacking a functional alternative pathway of complement activation

BackgroundNeuroprotective strategies for prevention of the neuropathological sequelae of traumatic brain injury (TBI) have largely failed in translation to clinical treatment. Thus, there is a substantial need for further understanding the molecular mechanisms and pathways which lead to secondary neuronal cell death in the injured brain. The intracerebral activation of the complement cascade was shown to mediate inflammation and tissue destruction after TBI. However, the exact pathways of complement activation involved in the induction of posttraumatic neurodegeneration have not yet been assessed. In the present study, we investigated the role of the alternative complement activation pathway in contributing to neuronal cell death, based on a standardized TBI model in mice with targeted deletion of the factor B gene (fB-/-), a "key" component required for activation of the alternative complement pathway.ResultsAfter experimental TBI in wild-type (fB+/+) mice, there was a massive time-dependent systemic complement activation, as determined by enhanced C5a serum levels for up to 7 days. In contrast, the extent of systemic complement activation was significantly attenuated in fB-/- mice (P < 0.05,fB-/- vs. fB+/+; t = 4 h, 24 h, and 7 days after TBI). TUNEL histochemistry experiments revealed that posttraumatic neuronal cell death was clearly reduced for up to 7 days in the injured brain hemispheres of fB-/- mice, compared to fB+/+ littermates. Furthermore, a strong upregulation of the anti-apoptotic mediator Bcl-2 and downregulation of the pro-apoptotic Fas receptor was detected in brain homogenates of head-injured fB-/- vs. fB+/+ mice by Western blot analysis.ConclusionThe alternative pathway of complement activation appears to play a more crucial role in the pathophysiology of TBI than previously appreciated. This notion is based on the findings of (a) the significant attenuation of overall complement activation in head-injured fB-/- mice, as determined by a reduction of serum C5a concentrations to constitutive levels in normal mice, and (b) by a dramatic reduction of TUNEL-positive neurons in conjunction with an upregulation of Bcl-2 and downregulation of the Fas receptor in head-injured fB-/- mice, compared to fB+/+ littermates. Pharmacological targeting of the alternative complement pathway during the "time-window of opportunity" after TBI may represent a promising new strategy to be pursued in future studies.

Alterations in Plasma Complement Levels Following Human Ischemic Stroke

Stroke is a leading cause of morbidity and mortality in the United States. Recent animal studies have implicated the complement system in cerebral ischemia/reperfusion injury and suggest that complement inhibition may improve stroke outcomes. To assess the applicability of these findings to humans, we evaluated the characteristics and time course of human complement activation after stroke. We compared peripheral blood levels of complement factor 3a (C3a), 5a (C5a), and sC5b-9 drawn from 15 patients on poststroke Days 1, 2, 3, 7, 14, 21, and 28 to age-, race/ethnicity-, and sex-matched controls from the same population. Statistical analysis was performed using unpaired Mann-Whitney nonparametric tests with Bonferroni correction. All data is presented as the mean +/- standard deviation. Mean C3a concentrations showed significant early elevations in stroke patients relative to matched controls (controls: 1080 +/- 189 ng/ml; Day 1: 1609 +/- 422 ng/ml, P = 0.0008; Day 3: 1520 +/- 317 ng/ml, P = 0.0005; Day 7: 1526 +/- 386 ng/ml, P = 0.001). C3a was also significantly elevated on Day 28 (1448 +/- 386 ng/ml, P = 0.004). Before poststroke Day 7, mean C5a levels did not differ significantly from controls. However, beginning on Day 7 and continuing through Day 14, there were significant elevations in C5a (controls: 3.33 +/- 2.1 ng/ml; day 7: 6.86 +/- 3.5 ng/ml, P = 0.005; Day 14: 7.65 +/- 4.6 ng/ml, P = 0.004). Mean sC5b-9 concentrations showed early depressions that reached significance on Days 1 and 2 (controls: 275.6 +/- 107 ng/ml; Day 1: 167.0 +/- 108 ng/ml, P = 0.006; Day 2: 156.3 +/- 80.0 ng/ml, P = 0.005) and did not differ significantly from controls at any other time point. C3a is acutely elevated after human ischemic stroke, C5a shows delayed elevations 7 to 14 days after cerebral ischemia, and sC5b-9 is acutely depressed after stroke. Together, these data confirm complement activation after stroke and suggest that this activation is a heterogeneous process, with varying responses for different components.

Comparison of In Vitro and In Vivo Complement Activation by Metal and Bioabsorbable Screws Used in Anterior Cruciate Ligament Reconstruction

The purpose of the study was to evaluate the biocompatibility of polylactide (PLLA) screws in comparison with standard metal screws for fixation of the patellar tendon graft in human anterior cruciate ligament (ACL) reconstruction. A total of 41 patients (22 women and 19 men) were prospectively randomized for the use of metal interference screws (20 patients) or biologically resorbable PLLA screws from Linvatec, Largo, FL (21 patients). Average age at the time of surgery was 26 years (15 to 51 y). Synovial fluid and plasma were collected preoperatively and after 6 weeks in both groups. Plasma was analyzed for C5a and synovial fluid, as well as for terminal SC5b-9 complement complex (TCC) and interleukin (IL)-8. At 1 year after surgery, serum was incubated with metal, PLLA, and no screws; this was followed by analysis of C5a after 1 and 6 hours of incubation. Inflammatory mediators were measured through enzyme-linked immunosorbent assay (ELISA). In the BioScrew group, 4 patient samples showed high C5a concentration in synovial fluid after 6 weeks, but no statistically significant difference was observed between the 2 groups (P = .11). One patient in the BioScrew group had a high TCC value after 6 weeks, but no statistically significant difference was seen between the 2 groups (P = .20). In the in vitro study, no increased C5a generation was observed in sera incubated with a BioScrew or a metal screw compared with controls. No statistically significant difference was observed between the BioScrew and metal screw groups concerning C5a, TCC, and IL-8 formation. However, some patients in the BioScrew group showed elevated values. Level II, prospective randomized trial.

Role of C5a in Acute Lung Injury

The complement system plays an important role in the pathogenesis of various infectious and non-infectious diseases with the complement activation product C5a acting as a key mediator of the inflammatory response. In the present study the role of C5a in acute lung injury (ALI) models in rodents was evaluated. ALI was induced in mice by either immune complexes (IC) or by intratracheal (i.t.) deposition of bacterial lipopolysaccharide (LPS). Bronchoalveolar lavage (BAL) fluids were then obtained at various time points and C5a levels were measured by ELISA. The C5a concentrations in BAL fluids from IC injury lungs increased with time (peaking between 4 and 6h). In accordance, the permeability indices were elevated as well, indicating a capillary leak due to the injury. The blockade of C5a with anti-C5a IgG deposition (i.t.) at the time of IC injury induction prevented the increase in C5a levels and also led to a lower level of injury. In contrast, after LPS induced injury no elevated C5a levels in BAL fluids were detectable. Surprisingly, when mice were neutrophil (PMN) depleted (with anti- Ly-6G (Gr-1) found on PMN surfaces) 18 h prior to the onset of immune complex formation, the C5a levels in the BAL fluids increased little, if at all. In summary, these findings indicate a crucial role of C5a in the development of ALI induced by immune complexes, whereas the injury in the LPS model does not seem to be mediated via complement activation. The presence of PMNs at the site of inflammation may contribute to formation of C5a and lung injury. This work was supported by NIH Grants No. GM-61656, GM-29507 and HL-31963.

Dynamics of protein kinase C mediated phosphorylation of the C5a receptor on Serine‐334

Phosphorylation of Ser-334, one out of three key serine phosphorylation sites within the C5aR carboxyl terminus, was shown to be required for the C5a-stimulated internalization of this chemotactic leukocyte receptor (Braun et al., J Biol Chem 278:4277, 2003). Because of the functional significance of this phosphorylation site for C5aR endocytosis we generated phosphosite-specific monoclonal antibodies which specifically react with phosphoserine-334 in ligand-activated C5aR. Treatment of db-cAMP stimulated U937 cells, which endogenously express C5aR, with activators (PMA) or inhibitors (bisindolylmaleimide) of protein kinase C (PKC) revealed that both homologous, C5a-induced and heterologous, C3a-induced phosphorylation of Ser-334 was mediated by PKC. A C5aR synthetic peptide was found to be a substrate for recombinant conventional (α,β I, β II, γ) and novel (δ) PKC isozymes in an in vitro phosphorylation assay. However, in intact myeloid cells only a PKCβ I/β II-selective inhibitor (LY333531), but not a PKCδ inhibitor efficiently blocked Ser-334 phosphorylation. Consistent with our previous observation in the related CCR5 receptor system, PKC phosphorylated C5aR at low C5a concentrations in a very rapid (t½ ~ 20 sec), albeit transient manner. Surprisingly, at high concentrations (>10nM) of C5a the PKC-mediated phosphorylation of serine-334 was essentially blocked. This could be attributed to the even faster (t½ < 5 sec) GRK-mediated phosphorylation of C5aR and consecutive firm binding of ß-arrestin. Our findings indicate that β-arrestin modulates PKC-mediated receptor phosphorylation and thus define a new regulatory role for this important adaptor protein.

Effect of Regional Citrate Anticoagulation on Thrombogenicity and Biocompatibility During CVVHDF

This study was designed to assess the principal markers of thrombogenicity and biocompatibility during continuous venovenous hemodiafiltration (CVVHDF) using regional citrate anticoagulation (RCA). In a prospective study, 11 procedures with a polysulfone membrane were performed in nine critically ill patients with acute renal failure and impaired hemostasis. Blood samples were taken before and during CVVHDF at diafilter outlet—before calcium-induced reversal of the effect of citrate—at 15, 60, 360, and 1440 minutes. In four patients, 10 CVVHDF sessions were performed with systemic heparin anticoagulation (HA) using a polyacrylonitrile membrane. During RCA, blood thrombocyte count, plasma thrombin-antithrombin III complexes, beta-thromboglobulin, and von Willebrand factor levels did not differ significantly from baseline. Plasma D dimer levels rose significantly at 360 minutes; however, the difference between diafilter inlet and outlet levels was nonsignificant. There was a significant increase in plasma C5a concentrations and a decline in blood leukocyte count in the early phase of CVVHDF. Just as in RCA, no increase in plasma thrombogenicity indices was observed during HA. However, clotting times in blood entering patients' circulation were significantly prolonged. Plasma C5a concentrations increased significantly at the beginning of CVVHDF. RCA can effectively inhibit the thrombogenic effect of the extracorporeal circuit in CVVHDF. The effect of HA may be similar, however, at the expense of systemic anticoagulation and risk of bleeding. RCA, performed in a way that overcomes thrombogenicity, does not completely eliminate complement activation and/or transient leukopenia during CVVHDF.

Isolated hyperthermic liver perfusion with cytostatic-containing perfusate activates the complement cascade.

Eight patients with advanced liver malignancy undergoing isolated hyperthermic liver perfusion with melphalan and cisplatin were studied with regard to complement activation and formation of anaphylatoxins (C3a and C5a) and terminal C5b-9 complement complexes (TCCs). Blood samples for complement variables (C1-INH, C3, C4, C5, C3a, C5a and TCCs) were taken before surgery, 1 min before the start of perfusion, 1, 2 and 3 h after the start of perfusion, and 24 h after operation. Samples were drawn from the perfusate 1 h after the start of perfusion. Activation of complement was observed during perfusion. Raised plasma concentrations of C3a and TCCs were recorded and high levels of C3a and TCCs were found in the perfusate. In vitro tests indicated that melphalan and cisplatin may activate complement. This activation occurred at 37 and 42 degrees C but was more pronounced at 42 degrees C.

Complement C3a and C4a Increased in Plasma of Patients with Aspirin-induced Asthma

Aspirin-induced asthma (AIA) is a distinct clinical syndrome that affects up to 10% of adults with asthma. Although eicosanoid metabolites appear to play an important role in AIA, the exact pathogenic mechanism for the syndrome remains obscure. In addition, the proposed mechanism fails to explain why aspirin does not cause bronchoconstriction in all individuals. We aimed to identify proteins that were differentially expressed in between AIA and aspirin-tolerant asthma (ATA) plasma. By using a proteomics approach, six proteins were found to be differentially expressed in plasma between patients with AIA and patients with ATA at baseline, and eight proteins were significantly up- or down-regulated after aspirin challenge in patients with AIA. These proteins, which were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, can be classified into four groups: complement components, apolipoproteins, modified albumin, and unknown proteins. Among them, the complement component levels in plasma were validated by using ELISA. Plasma concentrations of C3a and C4a were higher in patients with AIA (n = 30) than in patients with ATA (n = 24). After the aspirin challenge, C3 decreased in both patients with AIA and those with ATA, but the C3a concentration increased in the AIA patient group (p = 0.019). Moreover, C3a and C4a levels and the ratios of C3a/C3 and C4a/C4 were correlated with the changes of FEV(1) values after aspirin challenge. Aspirin intolerance may be related to alterations in the levels of complements, as well as those of lipoprotein and other proteins.

Immunodeficient Mice Are Poor Mobilizers -Novel Evidence That Demonstrates a Pivotal Role of Complement in Triggering Mobilization of HSPC.

Complement (C) is activated by immunoglobulin-dependent classical and immunoglobulin-independent alternative pathways, and we recently reported that the activation of C enhances the responsiveness of hematopoietic stem/progenitor cells (HSPC) to an SDF-1 gradient cascade by releasing C3a and desArgC3a cleavage fragments (Blood 2003; 101:3784; Blood 2004; 103: 2071). We also found that bone marrow (BM) concentration of C3a and desArgC3a increases during mobilization with G-CSF or cyclophosphamide (CY). To explain this phenomenon we envisioned that these mobilizing agents expose a neo-antigen in BM tissue by turning BM into a highly proteolytic microenvironment. As a consequence of this, the newly exposed neo-epitope becomes bound by natural IgM antibodies leading to the activation of the classical C cascade. In our studies to elucidate the role of C activation in triggering the mobilization of HSPC, we found that C is effectively activated in the BM of G-CSF- or CY- mobilized wild-type (wt) but not IgM-deficient (RAG2null) mice. More importantly we found that several immunodeficient murine strains (RAG2null, SCID and Xid) displayed severely reduced G-CSF-induced mobilization of HSPC which we believe is a result of lack of B lymphocytes and complement-activating immunoglobulins. Supporting this, we found T cell depletion in wt mice did not affect mobilization. Moreover, G-CSF-induced mobilization in RAG2null, SCID and Xid animals was restored after infusion of murine inmmunoglobulins. Furthermore, since mobilization by zymosan or sulfated glycans activates C via the alternative immunoglobulin-independent pathway, we focused on the role of C activation during zymosan-induced mobilization. As expected, zymosan-induced immunoglobulin-independent C activation and mobilization of HSPC were unaffected in immunodeficient (RAG2null, SCID and Xid) mice which have a normal serum level of C3. However, these processes were severely reduced in C3-deficient animals. Thus our data strongly support the notion that C and innate immunity is an important trigger of mobilization of HSPC. While G-CSF and cyclophosphamide activate C by a classical IgM-dependent pathway, zymosan and sulfated polyglycans activate it by employing an alternative pathway. In conclusion, mobilization of HSPC could be envisioned as part of a more global immune response that is triggered/mediated by C3 activation/cleavage.

Impact of Endograft Material on the Inflammatory Response After Elective Endovascular Abdominal Aortic Aneurysm Repair

The purpose of this paper is to examine the impact of endograft material on the inflammatory response after elective endovascular abdominal aortic aneurysm repair. Consecutive patients (n = 22, all men, 53 to 82 years old) were divided into 2 groups according to the graft material used: In group A (n = 12) the endovascular device was made of polyester and in group B (n = 10) the device was made of expanded polytetrafluoroethylene (ePTFE). All patients received antiinflammatory drugs in the perioperative period. Fever, white blood cells and platelet count, serum concentrations of cytokines (interleukin 6 [IL-6], tumor necrosis factor alpha [TNF-alpha], interleukin 8 [IL-8], acute-phase proteins high-sensitivity C-reactive protein [hsCRP] and alpha1-antitrypsin [alpha1-antitrypsin]), and complement protein (C3a) were measured preoperatively and 1, 3, 6, 24, 48, and 72 hours after aneurysm exclusion. One patient in each group had a systemic inflammatory response syndrome with 2 of the systemic inflammatory response syndrome (SIRS) criteria. No other complication associated with inflammation were present in any patient. Fever was more frequent in group A patients. Increases of white blood cells and serum concentrations of IL-6, TNF-alpha, hsCRP, alpha1-antitrypsin, and C3a and decrease of platelet count were recorded in both groups, but no statistically significant difference between them was recorded. However, serum concentrations of IL-8 were significantly higher in group A patients 24 hours postoperatively (p = 0.01). No significant difference was apparent in the biological response between patients receiving a polyester or an ePTFE stent graft, except for fever and serum concentrations of IL-8.

Contribution of Anaphylatoxins to Allergic Inflammation in Human Lungs

The contribution of complement activation to allergic asthma remains controversial. In order to elucidate the role played by the complement split products, anaphylatoxins C3a and C5a, we evaluated their effects on production of cysteinyl-leukotrienes (cysLTs) by human lung fragments following an anaphylactic reaction. The lung tissues obtained from two patients with lung cancer showed C5aR-, C5L2R-, and C3aR-mRNA expression. When the chopped lung fragments passively sensitized with human IgE were incubated with anti-human IgE antibody, a significant amount of cysLTs was generated in comparison with the control (without anti-IgE antibody). The co-addition of human C5a at doses of 0.1 to 10 ng/ml to the anti-IgE antibody potentiated cysLT production. The response was bell-shaped in distribution, significant, and peaked at a C5a concentration of 1 ng/ml. The co-addition of human C3a up to 1,000 ng/ml seemed to increase cysLT production, but not to any significant extent. A novel C5a receptor complementary peptide, acetylated peptide A, dose-dependently inhibited cysLT production by the human lung fragments following the anaphylactic reaction in the presence of 1 ng/ml C5a. However, this peptide did not inhibit cysLT production in the presence of 100 ng/ml C3a. It is suggested that the anaphylatoxin C5a potentiates cysLT production in human lung tissues and contributes to allergic inflammation in disorders such as asthma, thus acetylated peptide A may be useful for suppressing allergic inflammation in the lungs.

Relationship between granulocyte elastase and C3a under protamine dosing in on-pump cardiac surgery

The complement cascade and granulocytes are activated in on-pump cardiac surgery. If activation of complement directly regulates granulocytes, granulocyte elastase (GEL) should increase significantly after protamine administration. We examined the effect of protamine on granulocytes by protamine administration and observation of the effect on GEL and C3a. Thirty patients who underwent coronary artery bypass grafting were randomly assigned to two groups. In 15 patients, protamine was administered 5 min after the termination of cardiopulmonary bypass, and was administered 35 min after cardiopulmonary bypass in the other 15 patients. All patients were perfused with heparin-coated circuits and received 300 IU/kg heparin and 3 mg/kg protamine. GEL and C3a concentrations were measured at 7 time points. GEL concentrations increased significantly just before aortic declamping and did not increase significantly after protamine administration. C3a concentrations, however, did not increase during cardiopulmonary bypass and did increase significantly after protamine administration. This study indicates that GEL does not increase after protamine administration and that complement concentration does not directly affect GEL release.

Selective inhibition of the C5a chemotactic cofactor function of the Vitamin D binding protein by 1,25(OH) 2 Vitamin D 3

The Vitamin D binding protein (DBP) is a multifunctional plasma protein that can significantly enhance the chemotactic response to complement fragment C5a. The chemotactic cofactor function of DBP requires cell surface binding in order to mediate this process. The goal of this study was to investigate the effect of ligating DBP with its two primary physiological ligands, Vitamin D and G-actin, on both binding to neutrophils and the ability to enhance chemotaxis to C5a. There was no difference in neutrophil binding between of the holo (bound) forms versus the apo (unbound) form of radioiodinated DBP, indicating that the cell binding region of DBP is likely distinct from the Vitamin D sterol and G-actin binding sites. Likewise, G-actin, 25(OH)D 3, and G-actin plus 25(OH)D 3 bound to DBP did not alter its capacity to enhance chemotaxis toward C5a. However, the active form of Vitamin D (1,25(OH) 2D 3) completely eliminated the chemotactic cofactor function of DBP. Dose–response curves demonstrated that as little as 1 pM 1,25(OH) 2D 3 significantly inhibited chemotaxis enhancement. Moreover, at physiological concentrations 1,25(OH) 2D 3 needs to be bound to DBP to mediate the inhibitory effect. Neutrophil chemotaxis to optimal concentrations of C5a, formyl peptide, CXCL8 or leukotriene B 4 was not altered by 1,25(OH) 2D 3, indicating that the active vitamin does not have a global inhibitory effect on neutrophil chemotaxis. Finally, inhibition of cell surface alkaline phosphatase (AP) with sodium orthovanadate completely reversed the inhibitory effect of 1,25(OH) 2D 3. These results indicate that the cell binding and co-chemotactic functions of DBP are not altered when the protein binds G-actin and/or Vitamin D. Furthermore, the co-chemotactic signal from DBP can be eliminated or counteracted by 1,25(OH) 2D 3.

Mechanism of injection pain with long and longmedium chain triglyceride emulsive propofol

t has been suggested that long-medium chain triglyceride (LCT/MCT) emulsive propofol causes less injection pain than long chain triglyceride (LCT) emulsive propofol because of the decreased propofol concentration in the aqueous phase. Alternatively, LCT propofol generates bradykinin causing the injection pain and activates complement, but these effects when using LCT/MCT propofol have not been examined. To identify the mechanism for reduced pain with LCT/MCT propofol, injection pain, bradykinin generation and complement activation with use of both propofol products were compared. Two hundred adult patients randomly allocated to two groups were given 1.5 mg x kg(-1) iv of either LCT propofol or LCT/MCT propofol at a rate of 200 mg x min(-1) in a double-blind manner and were asked to grade pain scores. In another study, bradykinin and activated complement 3 (C3a) concentrations were measured using blood obtained from 13 healthy volunteers mixed with saline, LCT propofol or LCT/MCT propofol. There was a significant difference in pain scores between groups, showing a lower incidence of injection pain in the LCT/MCT propofol group. The bradykinin concentrations in blood mixed with LCT and LCT/MCT propofol were significantly higher than in blood mixed with saline. The C3a concentrations showed similar results. LCT/MCT propofol causes less pain on injection compared with LCT propofol. Bradykinin generation and complement activation are similar with both LCT and LCT/MCT propofol. Thus, the reason for less pain on injection with LCT/MCT propofol may be attributed to a decreased concentration of propofol in the aqueous phase.

Normal pregnancy is characterized by systemic activation of the complement system

Background. The complement system, a major component of innate immunity, has recently been implicated in the mechanisms of fetal loss and placental inflammation in the anti-phospholipid antibody syndrome. Inhibition of complement has been proposed as an absolute requirement for normal pregnancy. Yet, pregnancy is characterized by a generalized activation of the innate immune system. This study was conducted to determine whether or not normal pregnancy is associated with complement activation in the maternal circulation.Methods. Anaphylatoxins (C3a, C4a and C5a) were determined in the plasma of normal pregnant (20–42 wks; n = 134) and non-pregnant women (n = 40). These complement split products (C3a, C4a and C5a) were measured using specific immunoassays. Non-parametric statistics were used for analysis.Results. 1) The median plasma concentrations of C3a, C4a and C5a were significantly higher in normal pregnant women than in non-pregnant women (all p < 0.001); 2) the concentration of C3a, C4a and C5a did not change with gestational age (p > 0.05); and 3) the median plasma concentration of C3a had a positive correlation with the plasma C4a and C5a concentrations (r = 0.36, p < 0.001 and r = 0.35, p < 0.001, respectively).Conclusion. 1) Normal human pregnancy is associated with evidence of complement activation, as determined by higher concentrations of the anaphylatoxins C3a, C4a and C5a in the maternal circulation; and 2) we propose that physiologic activation of the complement system during pregnancy is a compensatory mechanism aimed at protecting the host against infection.

Increased concentration of the complement split product C5a in acute pyelonephritis during pregnancy

Objective. Pregnant women with acute pyelonephritis develop acute respiratory distress syndrome (ARDS) more frequently than non-pregnant women. The reasons for this remain unknown. The complement system is a complex set of self-assembling proteins that have been implicated in the pathophysiology of ARDS and sepsis. The purpose of this study was to determine if activation of the complement system occurs in pregnant women with acute pyelonephritis.Methods. A cross-sectional study was conducted to determine the plasma concentrations of C3a, C4a and C5a (i.e., complement split products) in pregnant patients with acute pyelonephritis (n = 38) and normal pregnant women (n = 38). The complement split products C3a, C4a and C5a were measured using ELISA. Data were analyzed using non-parametric statistics.Results. 1) The median plasma concentration of C5a in pregnant patients with acute pyelonephritis was significantly higher than that in normal pregnant women (p < 0.001); 2) there was no statistical difference in the median plasma concentration of C3a and C4a between the two groups (p > 0.05); and 3) concentrations of C3a, C4a and C5a were not different among patients with acute pyelonephritis with and without bacteremia.Conclusions. 1) Pyelonephritis in pregnant women is associated with an increased plasma concentration of C5a, but not C3a and C4a; and 2) an excess of C5a can predispose pregnant women to develop ARDS and multi-organ failure in pyelonephritis. This finding may have clinical implications since blocking C5a improves ARDS in experimental sepsis.

Plasma concentration of procalcitonin and systemic inflammatory response syndrome after colorectal surgery

To study whether plasma concentrations of procalcitonin (PCT), interleukin-6 (IL-6), complement 3a (C3a), C-reactive protein and white blood cell count (WBC) correlate with the presence of systemic inflammatory response syndrome (SIRS) during the early post-operative period after major colorectal surgery. Prospective, observational study during the first 24 h post-operatively. The setting for the study was the operating theatre and the recovery unit at the university hospital. Fifty consecutive patients, operated on electively with major resection of the large bowel or rectum. PCT levels increased significantly to the maximum level 18 h postoperatively. PCT levels were significantly higher in the SIRS group in comparison to the non-SIRS group of patients 6 and 12 h after surgery (P < 0.05). The IL-6 levels were increased directly after the surgery and then decreased gradually in both study groups. Twenty-four hours after the surgery, C3a levels decreased and then returned to normal levels. Twenty-four hours post-operatively, patients with SIRS had a higher plasma concentration of C3a compared with patients without SIRS (P < 0.05). CRP and WBC increased during the study period in both groups (P < 0.05). During the early post-operative period after uncomplicated major abdominal surgery, SIRS was reflected by the increase in plasma PCT and C3a concentrations. IL-6, CRP and WBC increased to the same extend in both the SIRS and the non-SIRS group of patients.

Unexplained intrauterine fetal death is accompanied by activation of complement

Activation of the complement system has recently been implicated in the mechanisms of fetal loss in the antiphospholipid syndrome. It is, however, possible that complement activation is also involved in other causes of fetal death in the second and third trimesters of pregnancy. We therefore conducted a study to determine whether fetal death is associated with changes in the maternal plasma concentrations of complement split products or anaphylatoxins (C3a, C4a and C5a). A cross-sectional study was designed to include normal pregnant women (n=60) and patients with fetal death (n=60). Patients with fetal death were classified according to the cause of fetal demise into: a) unexplained (n=44); b) associated with preeclampsia (n=8); and c) associated with chromosomal abnormalities or major congenital fetal anomalies (n=8). The plasma concentrations of C3a, C4a and C5a were measured using sensitive and specific ELISAs. Non-parametric statistics were used for analysis. A P value of <0.05 was considered significant. 1) The median plasma concentration of C5a was higher in patients with fetal death than in normal pregnant women [median 16 ng/mL (range 4.5-402.5) vs. median 11.6 ng/mL (range 1.2-87.1), respectively; P<0.001]; 2) patients with an unexplained fetal death and those associated with preeclampsia had a higher median plasma C5a concentration than normal pregnant women (P=0.002 and P<0.001, respectively); 3) no differences were observed in the maternal plasma concentrations of C3a and C4a among the study groups. Unexplained fetal death is associated with evidence of complement activation.

Participation of the 27kDa heat shock protein (hsp27) in mid-trimester amniotic fluid: Ethnic variability and association with anti-inflammatory immunity

AMNIOTIC FLUID: ETHNIC VARIABILITY AND ASSOCIATION WITH ANTIINFLAMMATORY IMMUNITY SRIRAM C. PERNI, EMRE KARASAHIN, ROBIN B. KALISH, STEPHEN T. CHASEN, STEVEN S. WITKIN, Weill Medical College of Cornell University, Obstetrics and Gynecology, New York, New York OBJECTIVE: Modulation of pro-inflammatory immune responses during gestation is essential for pregnancy prolongation. The extracellular release of the 27 kDa heat shock protein (hsp27) induces anti-inflammatory immunity. However, the possible presence of hsp27 in amniotic fluid (AF) and its interaction with other AF components has not been determined. We investigated whether hsp27 was present in mid-trimester amniotic fluid and determined its relationship with other AF immuno-modulating components and with maternal ethnicity. STUDY DESIGN: 278 pregnant women with singleton gestations who presented for genetic amniocentesis were enrolled. Clara cell protein 16 (CCP16) and cortisol, two anti-inflammatory components of AF, as well as the cytokines interleukin (IL)-1b, IL-1 receptor antagonist (IL-1ra), IL-6, IL-13, and hsp27 were measured by ELISA. Mann-Whitney U tests and linear regression analysis were used for statistical analysis. RESULTS: Hsp27 was detected in all samples with a median (range) of 5.8 ng/ mL (0.2-43.3). Median AF hsp27 concentrations were higher in blacks (6.3 ng/ mL) than in whites (4.5 ng/mL) (P = .04). IL-1b was identified in 67.8% of the AFs with a median concentration of 13.8 pg/mL (1.0-92.8). AF concentrations of hsp27 were strongly negatively correlated with AF IL-1b concentrations (r = .4360, P ! .0001) and positively correlated with CCP16 (r = .2199, P = .0012) and cortisol (r = 0.1396, P = .04) levels. CCP16 concentrations were also negatively associated with IL-1b (r = .1753, P = .006), but not as strongly as hsp27. There was no relationship between cortisol and IL-1 b levels. There was no relationship between hsp27 levels and IL-1ra, IL-6, IL-13 concentrations or neonatal gender. CONCLUSION: Hsp27 is present in AF and contributes to the down-regulation of IL-1b-induced pro-inflammatory immunity during the mid-trimester. Whether differences in baseline hsp27 levels between blacks and whites contribute to observed ethnic variations in rates of preterm birth and neonatal morbidities remains to be determined. Supported by NIH HD41676. 568 UNEXPLAINED INTRAUTERINE FETAL DEATH IS ACCOMPANIED BY ACTIVATION OF COMPLEMENT KARINA RICHANI, ELEAZAR SOTO, JIMMY ESPINOZA, JYH KAE NIEN, TINNAKORN CHAIWORAPONGSA, JERRIE REFUERZO, SEAN BLACKWELL, SAMUEL EDWIN, MOSHE MAZOR, ROBERTO ROMERO, Wayne State University School of Medicine, Detroit, Michigan, Perinatology Research Branch, NICHD, NIH, DHHS, Bethesda, Maryland, Wayne State University School of Medicine, Department of Obstetrics and Gynecology, Detroit, Michigan, Soroka University Medical Center, Beer Sheva, Israel, Israel OBJECTIVE: The complement system has recently been implicated in the mechanisms of fetal loss in the anti-phospholipid antibody syndrome (JCI 2003; 112:1644). We propose that complement activation may also be involved in other causes of intrauterine fetal death (IUFD) in the second and third trimesters of pregnancy. This study was conducted to determine if IUFD is associated with changes in maternal plasma C5a concentration, the most biologically relevant product generated during complement activation. STUDY DESIGN: A cross-sectional study was designed to include normal pregnant women (n = 60) and patients with IUFD (n = 60). The IUFD group was classified according to the cause of fetal death into: (a) unexplained (n = 44); (b) associated with preeclampsia (n = 8); and (c) chromosomal abnormality or major malformation (n = 8). The plasma concentration of C5a was measured by ELISA. Non-parametric statistics were used for analysis. RESULTS: (1) The median plasma concentration of C5a in patients with IUFD was significantly higher than that of normal pregnant women (median 16.05 ng/mL, range 4.59–402.52 ng/mL vs. median 11.64 ng/mL, range 1.22– 87.13 ng/mL, respectively; P! .001); (2) Among patients with IUFD, those with unexplained fetal death and fetal death with preeclampsia had a median plasma C5a concentration higher than that of normal pregnant women (P = .002 and P ! .001, respectively); (3) There was no significant difference in median plasma concentration of C5a between IUFD with congenital anomalies and normal pregnancy. CONCLUSION: Unexplained IUFD is accompanied by complement activation.

Unexplained intrauterine fetal death is accompanied by activation of complement

The complement system has recently been implicated in the mechanisms of fetal loss in the anti-phospholipid antibody syndrome (JCI 2003; 112:1644). We propose that complement activation may also be involved in other causes of intrauterine fetal death (IUFD) in the second and third trimesters of pregnancy. This study was conducted to determine if IUFD is associated with changes in maternal plasma C5a concentration, the most biologically relevant product generated during complement activation.