Cholesterol-dependent human complement activation resulting in damage to liposomal model membranes

Human (but not guinea pig) complement-mediated damage. It was concluded that human complement was activated spontaneously by liposomes containing a high concentration (71 mol %) of cholesterol. This occurred in the absence of any recognizable antigen or antibody, and did not occur at a low concentration (43 mol %) of cholesterol. Activation of complement resulted in membrane damage and release of trapped liposomal glucose. The complement activity was inhibited by preheating (56 degrees C, 30 min), 10 mM Mg2EDTA3 or EGTA, and by prior adsorption with insoluble immune complexes. Almost all human sera had some reactivity, but it ranged from very low levels (less than 7% liposomal glucose release) to very high levels (greater than 50% glucose release). Complement activation appeared to be mediated by a serum factor which could be removed by adsorption and which was partially heat labile. The factor was transferred by adding heated high reacting human serum to unheated low reacting human serum, or to guinea pig serum. The serum factor, although quantitatively diminished in potency due to heat lability, caused equal activation of each of these two latter complement sources in the presence of high cholesterol liposomes. It did not cause activation of C4-deficient guinea pig complement. These data suggested that the classical complement pathway was activated. The liposomal membrane composition had an influence on this phenomenon. Activities of about half of the human sera were enhanced when galactosyl ceramide, or ceramide alone, was present in the liposomes. Activity was enhanced by longer fatty acyl chain lengths of lecithin when dimyristoyl-, dipalmitoyl-, or distearoyllecithin was employed in the liposomes. Liposomes containing sphingomyelin as the only phospholipid were not sensitive to cholesterol-dependent complement-mediated damage. It was concluded that human complement was activated in the presence of high concentrations of membrane cholesterol and that this was caused by an uncharacterized serum factor and was influenced by the lipid composition of the membrane.     

In vitro blood compatibility of surface-modified polyurethanes

Polyurethanes have proven durable materials for the manufacture of flexible trileaflet heart valves, during in vitro tests. The response of two polyurethanes of differing primary structure to parameters of blood compatibility has now been investigated, using an in vitro test cell. Platelet (beta-thromboglobulin) release, complement (C3a) activation, the activation of free plasma and surface-bound factor XII were studied using fresh, human blood (no anticoagulant) or citrated plasma in control and surface-modified polyurethane. Surface modifications were designed to affect material thrombogenicity and included covalent attachment of heparin, taurine, a platelet membrane glycoprotein fragment, polyethylene oxide (PEO), 3-aminopropyltriethoxysilane, and glucose or glucosamine. Unmodified control polyurethanes caused platelet release and complement activation. High molecular weight (2000 D) polyethylene oxide reduced platelet release slightly but only glucose attachment to the surface produced a significant reduction in platelet activation. All modifications reduced C3 activation compared with controls, but the greatest reduction was achieved with polyethylene oxide attachment or glycosylation. Most surface modifications were more activating of factor XII, both in plasma and on the material surfaces, than the control polyurethanes. Heparin and high molecular weight PEO produced the greatest activation of factor XII in the free plasma form, but low molecular weight PEO and glucosamine produced the greatest activation of surface-bound factor XIIa. The least activating surfaces, affecting both free plasma and surface-bound factor XIIa, were those treated with platelet membrane glycoprotein fragment and glucose. PEO surfaces performed relatively well, compared with controls and most surface modifications. The best overall surface, however, was the glucose-modified surface which was least activating considering all parameters of blood compatibility.     

A novel serum protein similar to C1q, produced exclusively in adipocytes

We describe a novel 30-kDa secretory protein, Acrp30 (adipocyte complement-related protein of 30 kDa), that is made exclusively in adipocytes and whose mRNA is induced over 100-fold during adipocyte differentiation. Acrp30 is structurally similar to complement factor C1q and to a hibernation-specific protein isolated from the plasma of Siberian chipmunks; it forms large homo-oligomers that undergo a series of post-translational modifications. Like adipsin, secretion of Acrp30 is enhanced by insulin, and Acrp30 is an abundant serum protein. Acrp30 may be a factor that participates in the delicately balanced system of energy homeostasis involving food intake and carbohydrate and lipid catabolism. Our experiments also further corroborate the existence of an insulin-regulated secretory pathway in adipocytes.     

Liposuction combined with controlled compression therapy reduces arm lymphedema more effectively than controlled compression therapy alone

Arm lymphedema after breast cancer therapy has been treated with various forms of conservative and surgical treatment during recent years. The clinical results usually have been modest or, in some instances, even disappointing. In a previous series of patients treated with the new liposuction technique combined with controlled compression therapy, we found, however, an overall edema reduction of 106 percent after 1 year. The purpose of this study was both to investigate how much the surgical procedure contributes to the outcome and to clarify the importance of controlled compression therapy. Twenty-eight patients were, therefore, prospectively matched into two groups. One group received liposuction combined with controlled compression therapy, and one group received the therapy alone. Additionally, the therapy group was compared with our complete group of patients treated thus far with liposuction combined with therapy (n = 30). The prospective study using matched pairs (n = 14) showed that liposuction combined with controlled compression therapy is significantly more effective than the therapy alone (p < 0.0001), with a mean difference of about 1000 ml during the entire 1-year observation period. The beneficial effect of liposuction was confirmed by the comparison between the controlled compression therapy group and our complete group of patients treated with liposuction combined with the therapy, as the edema reduction figures after 1 year were 47 percent and 104 percent, respectively (p < 0.0001). In six patients who had surgery and a complete reduction of the edema, the compression garments were removed for 1 week, 1 year postoperatively. A marked increase in the arm volume was observed, which was immediately remedied by reapplying the garments. We conclude that liposuction combined with controlled compression therapy reduces arm lymphedema more efficiently than the therapy alone. Continued use of compression garments is, however, important to maintain the primary surgical outcome.     

Hereditary porcine membranoproliferative glomerulonephritis type II is caused by factor H deficiency

We have recently described hereditary membranoproliferative glomerulonephritis type II in the pig. All affected animals had excessive complement activation, revealed as low plasma C3, elevated plasma terminal complement complex, and massive deposits of complement in the renal glomeruli, and eventually died of renal failure within 11 wk of birth. The aim of the present study was to investigate the cause of complement activation in this disease. Transfusion of normal porcine plasma to affected piglets inhibited complement activation and increased survival. Plasma was successively fractionated and the complement inhibitory effect of each fraction tested in vivo. A single chain 150-kD protein which showed the same complement inhibitory effect as whole plasma was finally isolated. Immunologic cross-reactivity, functional properties, and NH2-terminal sequence identified the protein as factor H. By Western blotting and enzyme immunoassay, membranoproliferative glomerulonephritis-affected piglets were demonstrated to be subtotally deficient in factor H. At 1 wk of age, median (range) factor H concentration was 1.6 mg/liter (1.1-2.3) in deficient animals (n = 13) and 51 mg/liter (26-98) in healthy littermates (n = 52). Our data show that hereditary porcine membrano-proliferative glomerulonephritis type II is caused by factor H deficiency.     

Removal of activated complement from shed blood: comparison of high- and low-dilutional haemofiltration

BACKGROUND: Perioperative blood salvage is associated with release of inflammatory mediators. Depending on type of processing, the complement system is activated to some extent in the final blood product. The aim of the present study was to evaluate a haemofiltration technique concerning complement system activation and whether the volume of added saline will have an influence on the elimination of activated complement during processing. METHODS: Sixteen patients undergoing total hip arthroplasty received wound blood salvaged intraoperatively with a haemofiltration technique. Saline was added to the reservoir for washing in a ratio of 1:1 or 5:1 of estimated blood volume. Samples for determination of the anaphylatoxins C3a and C5a, and the terminal SC5b-9 complement complex (TCC) were drawn from the patients, the collected blood, the ultrafiltrate and the processed blood. RESULTS: Increased concentrations of C3a, C5a and TCC were found in aspirated and processed blood. Haemofiltration did not reduce the concentrations of these factors, except that of C3a in the group where saline was added in a ratio of 5:1. There were no increased concentrations of C3a, C5a or TCC in the patient plasma after reinfusion. No differences in blood pressure, heart rate, pH, arterial oxygen tension, arterial carbon dioxide tension, or base excess were found in association with reinfusion of the blood. CONCLUSION: Collected shed blood washed through haemofiltration contained moderately elevated concentrations of C3a, C5a and TCC. Reinfusion of the blood neither led to increased systemic concentrations of complement activation products, nor to disturbances in haemodynamic or biochemical parameters.     

Classical complement pathway activation on nucleated cells. Role of factor H in the control of deposited C3b

The restriction of alternative complement pathway activation in fluid phase or on nonactivator surfaces has been described as the major physiologic function of the complement regulatory protein factor H. In this study, we provide evidence that factor H is also a restriction factor of classical pathway activation on the surface of nucleated cells. We found that C3b was rapidly converted to inactivated C3b (iC3b) on human SK-MEL-93-2 melanoma cells after classical pathway activation with the murine monoclonal IgG3 Ab R24 directed against the disialoganglioside surface Ag GD3. The SK-MEL-93-2 cells are nonactivators of the alternative pathway and express neither CR1 (CD35) nor the C3b-cleaving protease p65. The cells are further characterized by the expression of only moderate amounts of DAF (CD55) and approximately 5 x 10(3) MCP (CD46) molecules/cell. FACS analysis and direct quantitation using [125I]factor H revealed high level binding of factor H to the melanoma cells (5.6 x 10(6) molecules/cell) during classical pathway activation. The binding of factor H could be inhibited under conditions that inactivate the classical complement pathway (EGTA and heat treatment), but not by factor B depletion of the serum, demonstrating that classical pathway activation was responsible for factor H binding. Treatment of factor B-depleted serum with neutralizing concentrations of polyclonal anti-factor H resulted in the prolonged presence of intact C3b on the cells and a significantly reduced generation of iC3b. The increased amount of C3b on these cells correlated with a 2.65-fold greater rate of cell death. In contrast, the increase in cell death effected by neutralizing concentrations of anti-CD46 or anti-CD55 Ab was only 0.13- or 0.35-fold, respectively. In addition, the supplementation of serum with purified factor H decreased the extent of lysis of the cells. Collectively, these data provide experimental evidence that factor H, through its cofactor activity for C3b degradation, is involved in the restriction of the classical pathway of complement on the surface of nucleated cells, a function that to date has been exclusively attributed to the membrane regulatory proteins CD35 and CD46.     

Metabolism of the fifth component of complement, and its relation to metabolism of the third component, in patients with complement activation

The metabolism of the fifth component of complement (C5), and its relatonship to metabolism of the third component of complement (C3), has been studied in normal subjects and patients by simultaneous administration of radioiodine labeled C5 and C3. In seven normal subjects the fractional catabolic rate of C5 ranged from 1.5 to 2.1% of the plasma pool/h and extravascular/intravascular distribution ratio from 0.22 to 0.78, these values being similar to those obtained for C3, and synthesis rate from 71 to 134 mug/kg per h, In patients with complement activation the increase in fractional catabolic rate of C5 was nearly always less than that of C3. The data also showed that there was increased extravascular distribution of C3 and C5 in most patients and considerable extravascular catabolism of both proteins in some. However, there were differences in metabolic parameters between patients with different types of complement activation. In patients with systemic lupus erythematosus, fractional catabolism and extravascular distribution of C3 and C5 were both increased, and there was marked extravascular catabolism of both proteins. There was increased fractional catabolism and extravascular distribution of C3 in patients with mesangiocapillary nephritis and (or) partial lipodystrophy, and fractional catabolism of C5 was also increased in three of six studies although distribution of C5 was always within the normal range; however, in two patients with nephritic factor in their serum fractional catabolism of C5 was normal despite markedly increased C3 turnover, suggesting that in patients with alternative pathway activation by nephritic factor little or no C5 convertase is generated.     

Extending the role of liposuction in body contouring with ultrasound-assisted liposuction

The initial experience with ultrasound-assisted liposuction in treating difficult fibrous areas, such as gynecomastia, hitherto not uniformly responsive to traditional suction-assisted lipoplasty, has led to the evolution and improvement of ultrasound-assisted liposuction techniques. This prospective study examined 114 consecutive patients treated with ultrasound-assisted liposuction over a 13-month period, from September of 1996 to September of 1997. The means by which this procedure helps achieve fat contouring differs from that of suction-assisted lipoplasty. Ultrasound-assisted liposuction removes fat through a fat emulsification process termed "cavitation," whereas suction-assisted lipoplasty achieves contouring through the mechanical avulsion of fat. The technique for the use of ultrasound-assisted liposuction has changed significantly from our initial series of patients to our current technique. To optimize the benefits of both ultrasound-assisted and traditional suction-assisted lipoplasty, we use a three-stage technique consisting of infiltration, ultrasound-assisted sculpturing, and suction-assisted lipoplasty for evacuation and final contouring. This has decreased our operative time, minimized complications, and optimized our body contouring results. Data were collected intraoperatively, including treatment times, treatment volumes, and treatment areas for both suction-assisted and ultrasound-assisted lipoplasty. A total of 114 patients were treated with ultrasound-assisted liposuction between September of 1996 and September of 1997. There were 23 male patients and 91 female patients. In general, the average total volume removed with this procedure decreased by about 50 percent throughout the series, whereas the suction-assisted lipoplasty volume increased correspondingly by 50 percent. Overall, suction-assisted lipoplasty volume was approximately two times ultrasound-assisted liposuction volume in the same area. Exceptions to this include the dense fibrous areas such as the back and male breast, where aspiration volumes were approximately equal. The total ultrasound-assisted liposuction treatment times were reduced after our initial 30 patients, and suction-assisted lipoplasty times increased. Total aspiration rates in our later patients averaged 36.2 cc/per minute for ultrasound-assisted and 58.4 cc/per minute for suction-assisted lipoplasty, whose rates were approximately 1.5 to 2 times faster than for ultrasound-assisted liposuction in most areas. After using this technology in our initial series of 30 patients, it became apparent that ultrasound was not a substitute for suction-assisted lipoplasty but rather a natural complement. We have found that the marriage of the techniques enhances results and minimizes complications, such as seromas, which have been reported to be 11.4 percent with ultrasound-assisted liposuction alone and are 2.6 percent in our series.     

Neutrophil and macrophage activation and anaphylatoxin formation in orthotopic liver transplantation without the use of veno-venous bypass

BACKGROUND: Activation of neutrophils and activation of complement may be an aetiologic factor behind circulatory insufficiency in association with reperfusion of the grafted liver. METHODS: Neutrophil and macrophage activation (determined as PMN elastase and neopterin release) and complement activation were evaluated in 15 consecutive patients undergoing orthotopic liver transplantation without the use of veno-venous bypass. RESULTS: The PMN elastase concentrations were increased at the end of the anhepatic phase, 2, 5 and 30 min after start of reperfusion and 6 and 24 h postoperatively. There were significantly higher PMN elastase concentrations in patients with circulatory instability (postreperfusion syndrome) compared with those without postreperfusion syndrome. The neopterin concentration was increased 2 min after the start of reperfusion and remained elevated until 6 h postoperatively. The plasma complement C3a concentrations were increased at the end of the anhepatic phase and 2, 5 and 30 min after the start of reperfusion. The plasma C3a levels were higher in patients with postreperfusion syndrome compared to those without. CONCLUSIONS: Activation of neutrophils and macrophages and of the complement cascade with the formation of biologically active substances may be one explanation for the circulatory instability often seen in patients undergoing orthotopic liver transplantation.     

Direct or C5a-induced activation of heterotrimeric Gi2 proteins in human neutrophils is associated with interaction between formyl peptide receptors and the cytoskeleton

The binding of ligands to N-formyl peptide chemoattractant receptors in human neutrophils results in a rapid association of these receptors with a cytoskeletal fraction and a specific activation and release of Gi2 alpha-subunits from this fraction. In the present study we could show that pretreating neutrophils with GDPbetaS prevented the fMet-Leu-Phe-induced association of its receptor with a cytoskeletal fraction and also blocked the release of Gi2 alpha-subunits from the same cytoskeletal fraction. In contrast, direct activation of Gi2 proteins by addition of GTPgammaS or AlF4- not only caused a release of Gi2 alpha-subunits from the cytoskeleton but also an association of formyl peptide receptors with the cytoskeleton. The receptor for complement fragment 5a, which transduces its signaling through the same Gi2 protein, triggers both a release of Gi2 alpha-subunits from the cytoskeleton fraction and, of even greater interest, an association between formyl peptide receptors and the cytoskeleton. The close relationship between the activation and release of Gi2 alpha-subunits from the cytoskeleton and the association of formyl peptide receptors with the cytoskeleton might, however, not be a matter of protein-protein exchange, since the increased binding of formyl peptide receptors to the cytoskeleton occurs more rapidly than the release of Gi2 alpha-subunits from the cytoskeleton. The present findings suggest a possible mechanism for the initiation of formyl peptide receptor desensitization during neutrophil locomotion.     

Monocyte tissue factor expression and ongoing complement generation in ventricular assist device patients

BACKGROUND: Ongoing complement activation in patients with a ventricular assist device may contribute to observed hemostatic abnormalities and cellular aggregation by mediating leukocyte and platelet activation, formation of leukocyte-platelet conjugates, and the tissue factor pathway of coagulation. METHODS: Blood from 30 patients was collected before ventricular assist device implantation and during the implantation period. Plasma levels of thrombin-antithrombin III complexes, C3a, and SC5b-9 were measured by commercial enzyme-linked immunosorbent assay. Flow cytometry was used to measure circulating monocyte tissue factor expression and circulating monocyteplatelet and granulocyte-platelet conjugates. RESULTS: Thrombin-antithrombin III complex level and monocyte tissue factor expression peaked in the early postoperative period, with maxima occurring on postoperative days 5 and 3, respectively. Levels of C3a and SC5b-9 remained dramatically elevated over normal values for the duration of the study (6 and 5 times upper normal, respectively). Levels of monocyte-platelet conjugates were normal before implantation, decreased during the first 4 postoperative days, and then increased and remained elevated. Levels of granulocyte-platelet conjugates were elevated over the normal range before implantation and remained elevated from postoperative days 3 to 21. A positive correlation was found between levels of SC5b-9 and granulocyte-platelet conjugates (Spearman R=0.66; p < 0.001), and between levels of C3a and thrombin-antithrombin III complex (Spearman R=0.13; p=0.021). CONCLUSIONS: The data suggest a model in which complement mediates formation of leukocyte-platelet aggregates and may indirectly contribute to thrombin generation through monocyte tissue factor expression.     

CRP-mediated activation of complement in vivo: assessment by measuring circulating complement-C-reactive protein complexes

The in vivo function of C-reactive protein (CRP) is unknown. Among the in vitro functions assigned to CRP is the ability to activate complement via the classical pathway. To date, there is no evidence supporting that CRP exerts this function in vivo. We here show a novel approach to assess CRP-mediated complement activation in vivo, which is based on the property that activated complement factors C3 and C4 fix to CRP during complement activation induced by this acute phase protein. We developed specific ELISAs for complexes between CRP and C4b, C4d, C3b, or C3d. We established that in vitro complement-CRP complexes were formed only during CRP-dependent activation, and not during activation by other activators, even in the presence of high CRP levels. Circulating levels of complement-CRP complexes were undetectable in normal donors, but significantly increased in nine patients following implantation of a renal allograft. Importantly, levels of complement-CRP complexes did not change in these patients upon a bolus infusion of mAb OKT3, which induces activation of the classical complement pathway, demonstrating in vivo that complement-CRP complexes are not formed during CRP-independent activation of complement, even when CRP is elevated. We conclude that measurement of complement-CRP complexes provides a suitable tool to study CRP-mediated activation of complement in vivo. Furthermore, increased levels of these complexes occur in clinical samples, indicating that CRP may induce activation of complement in vivo.     

T4 and T3 release from the perfused canine thyroid isolated in situ

A method for once-through perfusion of the canine thyroid isolated in situ is described. The perfusion medium was a modified Krebs Ringer buffer with 4% dextran added. In 4 control experiments of the T4 and T3 concentratios in effluent were stable or slightly falling during 3 h perfusion. There were no significant alterations in the T4/T3 ratio in the effluent during these experiments. A 10-min infusion of bovine TSH (1 mU/ml) caused an increase in the release of T4 and T3 after 15-25 min. The T4/T3 ration in the effluent was significantly reduced after TSH stimulation. However, the ratio returned to pre-stimulation values while the hormone release was still very high. T4 and T3 content of the contralateral thyroid was determatio in the homogenate was twice as high as the T4(3 ratio in the effluent during control perfusion. Thus there was a preferential secretion of T3 from the perfused canine thyroid and this was increased after TSH stimulation.     

Treatment of low back pain and sciatica by intradiskal injection of chymopapain

Single injections of 50 mug estradiol-17beta (E2beta) into overiectomized sheep caused biphasic changes in plasma luteinizing hormone (LH). An initial 8-h period of LH inhibition was followed by a period (12-20 h after E2beta) of facilitated LH release. Pituitary LH responsiveness to small dosages of synthetic gonadotropin-releasing hormone (GnRH) was tested repeatedly at 2-h intervals during the time periods when plasma LH was inhibited and when it was facilitated. Reduced sensitivity to GnRH (91% decrease) characterized only the initial 4 h of the inhibitory period, suggesting that E2beta suppressed endogenous LH-releasing factor (LRF) during the latter part of the inhibitory period. Hypersensitivity (2-fold increase) to GnRH was briefly observed at the beginning of the period of E2beta-facilitated LH release. This transient and modest hypersensitivity does not completely account for the very large E2beta-induced increases in plasma LH. Therefore, E2beta probably increased endogenous LRF during the period of facilitated LH release.     

Participation of serum proteins in the inflammation-primed activation of macrophages

Inflamed lesions release degradation products of membrane lipids, lysophospholipids, and inflamed tumor tissues release alkylglycerols. Macrophages were activated by administration of lysophosphatidylcholine (lyso-Pc) or dodecylglycerol (DDG) to mice. In vitro treatment of mouse peritoneal cells (mixture of nonadherent and adherent cells) with lyso-Pc or DDG in fetal calf serum supplemented medium for 30 min, followed by 3-h cultivation of adherent cells (macrophages) alone, resulted in greatly enhanced Fc-receptor mediated phagocytic activity and superoxide generating capacity of macrophages. The tumor lipid metabolite, DDG, is far more potent (400-fold) than lyso-Pc in terms of doses required for the maximal levels of macrophage activation. The inflammation-primed macrophage activation required a serum factor, vitamin D binding protein, as a precursor for the macrophage activating factor. Treatment of mouse peritoneal cells with 1 microgram lyso-Pc/ml or 50 ng DDG/ml in a serum-free 0.1% egg albumin supplemented medium for 30 min, followed by 3-h cultivation of the treated peritoneal cells in a medium supplemented with a very small amount (0.0005-0.05%) of ammonium sulfate [20-50% saturated (NH4)2SO4] precipitable protein fraction of FCS, resulted in greatly enhanced superoxide generating capacity of macrophages. The ammonium sulfate precipitable fraction was found to contain vitamin D binding protein.     

Complement activation during CAPD

Complement activation was monitored in 20 CAPD patients and 20 normal individuals using markers of the alternative (Bb fragment), classical (C4d fragment), common (iC3b) and terminal pathways (SC5b-9, the soluble form of the membrane attack complex, MAC), together with C3, C4 and factor B. CAPD plasma SC5b-9 was higher than normal although this was not due to increased complement activation in the plasma. The calculated cleavage for C3, C4 and factor B to iC3b, C4d and Bb respectively, due to spontaneous activation, was similar in both groups. C3, C4 and factor B in dialysate were less than 1% of plasma concentration, consistent with vascular leakage, whereas iC3b, Bb and SC5b-9 were at higher concentrations, suggesting generation in the peritoneum by the alternative pathway. 2.4% C4d is consistent with leakage of this small molecule but may indicate slight classical activation. It is concluded that complement activation occurs in the peritoneum during CAPD. MAC and the anaphylatoxins which are also generated may contribute to an increased risk of infection and other inflammatory complications.     

Postinjury neutrophil priming and activation: an early vulnerable window

BACKGROUND: Generation of extracellular, cytotoxic superoxide anion (O2-) by polymorphonuclear neutrophils (PMNs) contributes to an unbridled inflammatory response that can precipitate multiple organ failure (MOF). Release of O2- is markedly enhanced when activated PMNs have been previously "primed" by inflammatory mediators, such as those expressed after trauma. We therefore hypothesized that PMN priming occurs as an integral part of the early inflammatory response to trauma. METHODS: PMNs were obtained from 17 high-risk patients with torso trauma at 3, 6, 12, 24, 48, and 72 hours after injury, as well as from 10 healthy donors, and the in vitro release of O2- was quantitated with a kinetic, superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay. PMN O2- release was measured in the presence and absence of 1 mumol/L N-formyl-methionyl-leucyl-phenylalanine (fMLP) and after priming and activation with 20 nmol/L platelet-activating factor (PAF) and 1 mumol/L fMLP, respectively. RESULTS: In vitro PMN O2- release was used to determine whether postinjury PMNs were (1) activated in vivo, (2) primed in vivo, or (3) primable in vitro. Unstimulated PMNs from trauma patients spontaneously expressed modest amounts of O2- in vitro from 6 to 48 hours after injury, suggesting endogenous activation. Also, fMLP-activated PMNs collected between 3 and 24 hours after injury expressed more O2- than controls (p < or = 0.02), indicating in vivo, trauma-related priming. Furthermore, postinjury PMNs were maximally primed in vivo (i.e., in vitro exposure to PAF before fMLP activation failed to significantly enhance O2- release) as compared to PMNs treated with fMLP. CONCLUSIONS: These data indicate that major torso trauma (first hit) primes and activates PMNs within 3 to 6 hours after injury. Consequently, we postulate that postinjury priming of PMNs may create an early vulnerable window during which a second hit (e.g., a secondary operation or delayed hemorrhage) activates exuberant PMN O2- release, rendering the injured patient at high risk for MOF.     

Infusion of ovine C5a into sheep mimics the inflammatory response of hemodialysis

Previous studies in our group have explored the inflammatory response in sheep to dialysis with a variety of different hemodialysis membranes. In the present study we investigated the potential role of C5a in mediating inflammatory responses that have been attributed to complement activation in the extracorporeal setting. Sheep C5a was infused into sheep in a manner that simulated exposure to this anaphylatoxin during dialysis. C5a infusion into sheep was shown to produce a dose-dependent neutropenia that was quantitatively and temporally identical to the response of sheep undergoing dialysis with complement-activating membranes. The two lowest doses used (0.25 and 0.50 micrograms/kg), which resulted in concentrations below the detectable limits of current assays (10 ng/ml), produced significant neutropenia (21.8% and 78.1%, respectively). The ability of the neutrophils (PMNs) to bind fluorescein isothiocyanate-C5a or initiate a respiratory burst in response to phorbol myristate acetate were also affected in a dose-dependent manner. In contrast, C5a alone was not able to produce significant release of lactoferrin, a specific granule constituent, suggesting that degranulation of PMN-specific and primary granules requires secondary stimuli. The production of thromboxane A2 and thromboxane's consequent cardiopulmonary effect of increasing mean pulmonary artery pressure were both observed in a dose-dependent fashion. However, larger amounts of C5a were required to elicit these latter responses as compared with the PMN activities. These results suggest that C5a may be a primary mediator of complement-dependent events that occur during extracorporeal therapies such as hemodialysis, and they also suggest that very little complement activation is necessary to activate leukocytes, whereas higher thresholds are required to produce cardiopulmonary responses.