The inflammatory response following treatment of abdominal aortic aneurysms: a comparison between open surgery and endovascular repair.

OBJECTIVES: to compare the inflammatory response following endovascular and conventional AAA repair. DESIGN: prospective study. PATIENTS AND METHODS: ten patients were selected for open surgery (OPEN) and ten for endovascular (ENDO) AAA repair. Leukocytes, platelets, myeloperoxidase, lactoferrin, beta-thromboglobulin, C-reactive protein (CRP), interleukin 6 (IL-6), tumour necrosis factor alpha (TNF-alpha) and complement activation products were measured before, during and after surgery. RESULTS: in the OPEN group the median hospital stay was longer (6 vs. 12 days, p=0.001) and more patients required transfusion (p=0.02). IL-6 and CRP increased postoperatively, most in OPEN (p<0.01). Platelet counts decreased after the first angiography in ENDO (p<0.01) and before aortic cross-clamping in OPEN (p<0.05). The decrease was larger in OPEN (p=0.02). Leukocyte counts decreased after the first angiography in ENDO, and thereafter increased (p=0.001). An equivalent increase was observed in OPEN after declamping (p=0.001). Leukocyte and platelet degranulation products increased after the first angiography in ENDO and after declamping in OPEN. Changes in complement activation products were small. TNF-alpha did not change significantly. CONCLUSION: endovascular AAA repair caused significant leukocyte and platelet activation. Based on the timing of activation this could be caused by radiographic contrast media.

[The interconnection between the severity of meningococcal infection and the endotoxicity levels and patient's blood complement]. / Sviaz' tiazhesti techeniia meningokokkovoi infektsii s urovniami éndotoksina i komplementa v krovi bol'nykh.

Seventy-eight patients with severe systemic meningococcal disease admitted to the Intensive Care Unit of the Second Moscow Hospital for Infectious Diseases were divided into four groups by complications of their disease: patients with refractory septic shock (RSS)--group 1; patients with early septic shock (ESS)--group 2; patients without shock but with severe mental disorders--group 3; patients without any of these complications--group 4. The LPS concentration in plasma was assessed by chromogenic method. Initial LPS levels in plasma of group 3 or group 4 patients (170 ng/l, median value and 360 ng/l, respectively) were greater than those of healthy donors (LPS < 15 ng/l). LPS concentration was significantly greater in group 2 (920 ng/l) or group 1 (12,400 ng/l). LPS levels declined exponentially in all the patients. The half-life was calculated to be 1.4 (+) -0.3 h. In group 2 and 1, respectively, the classical pathway complement activity in patients' serum was 50 and 10% of normal control values. To estimate significant prognostic factors for fatality in our patients, specificity and factor fatality difference of various clinical and laboratory factors were calculated. The cut-off LPS value for development of ESS was 600 ng/l and that for development of RSS and death was 8000 ng/l. For the prediction of fatality using the former cut-off value of LPS, sensitivity was 84% and specificity 100%. Using plasma complement activity (cut-off--15% of normal value) for prediction, sensitivity was 75% and specificity was 100%. Other factors (platelet and WBC count, blood pH, BP, etc.) had lower predictive power. Thus to date plasma endotoxin level and complement activity are the best prognostic factors in meningococcal disease.

Binding of complement components C1q, C3, C4 and C5 to a model immune complex in ELISA.

When normal human serum is added to microELISA plates coated with monomeric or aggregated IgG various complement components become bound and can be detected with specific chicken anti-C1q, anti-C3, anti-C4 and anti-C5 antibodies. Using such assays we found increased C1q- and decreased C3- and C4-binding in sera from patients with SLE. In contrast, sera from patients with rheumatoid arthritis showed decreased C3 binding but normal C1q binding. The decreases in C3 and C4 binding observed in the sera from patients with SLE were larger than the corresponding decreases determined by radial immunodiffusion. Comparing these results with those of the CH50 assay, the correlation coefficient between CH50 and the C3-binding assay was 0.48. There was no correlation between the results of the CH50 and those of the C1q-, C4- or C5-binding assays.

Plasma concentrations of complement-activation complexes correlate with disease activity in patients diagnosed with isolated central nervous system vasculitis.

Isolated central nervous system (CNS) vasculitis is rare medium- sized vessel disease limited to intracerebral vessels. The two most common symptoms of this inflammatory disorder observed at entry to a hospital are headaches and mild memory deficits. Further progression of this disease may result in focal neurologic alterations and seizures. Currently, the most common laboratory abnormality noted is an elevated erythrocyte sedimentation rate. The complement (C) system is known to play a role in many inflammatory processes; it may also be involved in CNS vasculitis. In this longitudinal study of patients with CNS vasculitis, we detected C activation by highly sensitive and specific assays that are capable of identifying breakdown products formed after C activation: C3a des arg, C4a des arg, C5a des arg, C1rC1s-C1-inhibitor complex, and terminal C complex (C5b-9). We present two cases of documented CNS vasculitis in which serial measurements of these C-activation products correlate with disease activity. Our results indicate that a temporal association exists between C activation and the clinical presentation of CNS vasculitis. We conclude that physicians should monitor C-activation by-products in plasma when they attempt to follow the clinical course of CNS vasculitis.

A mechanism for the spontaneous activation of the first component of complement, C1, and its regulation by C1-inhibitor.

We have developed a method to initiate spontaneous activation of the first component of complement in serum, by the removal of C1-inhibitor through complexation with added C1s. Preliminary experiments to test this method using C1 reconstituted from its purified subcomponents led to an unexpected result: pre-incubation of the reassembled subcomponents with C1-inhibitor, followed by its removal with C1s, altered the subsequent pattern of spontaneous activation. Thus, pre-incubation with C1-inhibitor at 37 degrees C for 1 h resulted in sigmoidal activation of C1 with a prolonged lag phase. In contrast, pre-incubation with C1-inhibitor on ice for the same time resulted in subsequent rapid, pseudo first order activation of C1 with a half-life of about 5 min. We have examined the activation kinetics under a variety of conditions, and our data are consistent with a model proposed by Lepow and coworkers in 1965, involving both spontaneous activation and C1 catalyzed activation: (1) C1----k1 C1 (2) C1----k2C1 C1 According to this model, the role of C1-inhibitor is to eliminate the second step by rapidly forming a tight complex with C1 which becomes irreversible at 37 degrees C. When C1s was added to normal human serum, activation at 37 degrees C was also sigmoidal, similar to that of reconstituted C1.

Assembly of the membrane attack complex promotes decay of the alternative pathway C3 convertase on Neisseria gonorrhoeae.

C3, C4, factor B, properdin, and C2 binding to serum-sensitive and serum-resistant gonococci was quantitated in C8-deficient and normal human serum by using fluorescein-conjugated antibodies and 3H-labeled components. Organism and serum-specific differences were noted, the most striking of which involved factor B and properdin binding to the serum-sensitive strains in the different sera. C3 binding to these organisms was quantitatively and kinetically equivalent in C8-deficient and normal human serum. In contrast, factor B and properdin binding reached a plateau after 5 min in C8-deficient serum but peaked and fell to control values in normal human serum. Identical results were obtained with normal human serum immunochemically depleted of C8. Between 7 and 15% of the bound C3 participated in formation of the alternative pathway convertase C3bBb/P. Reconstitution of the C cascade by adding purified C8 to C8-deficient serum led to the loss of factor B previously bound to the organisms. Factor B loss occurred coincident with bacterial killing and membrane disruption as observed by electron microscopy. Prevention of membrane disruption by depleting normal human serum of lysozyme had no effect on killing and failed to prevent factor B loss. Stabilization of the C3bBb complex with Ni2+ prevented factor B loss as well as gross membrane disruption but not bacterial killing. C2 (the classical pathway analog of factor B) binding to gonococci was equivalent in C8-deficient and normal human serum peaking within 2.5 min and falling to control values in both sera thereafter. We conclude that the assembly of the membrane attack complex promotes decay of C3bBb/P with release of factor B and properdin but not C3 from the organism surface. Membrane disruption does not appear to be required for this effect. This activity may represent a mechanism to limit continued C consumption.

Eosinophil granule major basic protein regulates generation of classical and alternative-amplification pathway C3 convertases in vitro.

Eosinophil major basic protein (MBP), a highly charged polycation, forms the core of the eosinophil granule and mediates tissue damage in allergic disease. Purified MBP was studied for capacity to regulate the generation of classical and alternative-amplification pathway C3 convertases because previous studies have shown that other polycations (protamine, poly-L-lysine) and polyanions (heparin) may play important roles in regulating C activation. MBP inhibited the generation of EAC1,4b,2a and EAC4b,3b,Bb,P but appeared to inhibit the generation of classical pathway convertase more than the alternative amplification pathway convertase at a given dose. Dose-response curves with MBP were steeper than curves seen with polyanion (heparin). MBP did not lyse cellular intermediates at concentrations that caused almost total inhibition of convertase generation. One mechanism of inhibition of convertase generation may have been through an action on C3b, because preincubation of MBP with an EAC4b,3b cellular intermediate interfered with the ability of this cellular intermediate to be lysed. Furthermore, MBP prevented consumption of B in a reaction mixture that contained factors B, D, and C3b, also suggesting an action on C3b. Reduced and alkylated MBP (A-MBP) was compared with native MBP, which possesses two reactive sulfhydryl groups, to determine whether charge alone is responsible for blocking convertase generation; native MBP rapidly associates and is relatively insoluble at neutral and alkaline pH whereas A-MBP remains soluble. A-MBP impaired convertase generation, did not appear to remain bound to cellular intermediates and did not suppress B consumption in the fluid phase assay. This suggests that the ability of MBP to regulate C activation is complex and not entirely through its net charge. Finally, although heparin or MBP alone may prevent C activation, when these substances were present at the same time there was no effect on C activation suggesting that charge neutralization may abrogate the effects of these charged substances on C activation. Taken together, these studies suggest that MBP at physiologic concentrations may regulate in vivo C activation at the tissue level.

Persistently circulating C3 nephritic factor (C3 NeF)-stabilized alternative pathway C3 convertase (C3 CoF) in serum of an 11-year-old girl with meningococcal septicemia--simultaneous occurrence with free C3 NeF.

Hemolytic complement was found to be absent in the serum of an 11-yr-old girl (R.N.) with meningococcal septicemia. C1, C4, and C2 were slightly decreased, C3 was absent, C5-C9 within the normal range. B levels immunochemically and electrophoretic mobility of B were normal. C3d was greater than 1000% of a pooled EDTA-plasma standard indicating hypercatabolism of C3. On incubation of the patient's serum with normal human serum activation of C3 occurred even in the presence of 0.04 M EDTA. The amount of C3b generated was, however, greater without any chelating agent or in Mg-EGTA. On gel filtration of the serum two protein containing peaks were found to be responsible for activation of C3: the IgG containing peak was able to activate C3 in normal human serum without chelating agents and in Mg-EGTA but not in the presence of EDTA. The IgM-containing peak activated the third component of complement even in the presence of EDTA. The factor responsible for this phenomenon was termed C3 converting factor (C3 CoF). The IgG fraction of the patients serum caused activation of C3 in Mg-EGTA. However, in the presence of EDTA no activation of C3 could be induced even if physiological concentrations of the patients IgG were added to normal human EDTA-plasma. Thus the activity of the patient's IgG did not differ from typical C3 nephritic factor. The decay of C2 in EAC42 intermediates in the presence of the patient's IgG was uninfluenced indicating that it did not carry autoantibody activity against the classical pathway convertase C4b,2a, an activity recently termed NFc.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of the activity of platelet-bound C3 convertase of the alternative pathway of complement by platelet factor H.

The alternative pathway of complement is regulated on the surface of homologous blood cells at the C3 amplification step by the membrane protein decay-accelerating factor, as well as by the plasma protein factor H. We have reported elsewhere that platelets from patients with paroxysmal nocturnal hemoglobinuria regulate the activity of the C3 convertase C3bBb, even though they lack decay-accelerating factor. We now report that normal human platelets contain factor H, which was released from the platelet in response to complement deposition or thrombin stimulation. Factor H was localized to the platelet alpha granules by immunocytochemical techniques. As determined by a solid-phase radioimmunoassay, thrombin-stimulated platelets released approximately equal to 54 ng of factor H per 10(8) platelets. The release of factor H in response to complement or thrombin was inhibited by treating the platelets with metabolic inhibitors. Such inhibition resulted in a 3-fold increase in the activity of C3bBb. Platelets that released factor H bound only half as many molecules of radiolabeled factor B to platelet-bound C3b than platelets that could not release factor H. Treatment of platelets with anti-decay-accelerating factor antibody had no effect on the activity of C3bBb unless the release of factor H was blocked. Therefore, so far as we know, human platelets have a unique mechanism for the regulation of the alternative pathway of complement.

A sensitive specific hemolytic assay for proenzyme C1.

The traditional hemolytic assay of the functional activity of C1, the first component of the classical complement pathway, was modified to permit differentiation between proenzyme (unactivated) C1 and the activated state of the enzyme (C1). A two-step assay was developed to quantitate proenzyme C1. The C1 sample to be assayed was first preincubated with C1 inhibitor, a process that specifically inhibits the enzymatic activity of C1 without affecting the subsequent activation of proenzyme C1 by EAC4, a model immune complex. Since the rate of reaction between C1 inhibitor, a serum regulatory protein, and C1 is concentration-dependent, this step is performed at high C1 and C1 inhibitor concentrations. Subsequent dilutions of the sample prevents C1 inhibitor-mediated inactivation of the C1 that is activated during the C1 hemolytic assay. Thus, in the presence of C1 inhibitor, the level of C1 hemolytic activity specifically reflects the activity of proenzyme C1, while in the absence of C1 inhibitor, the hemolytic activity reflects the total activity of C1. Both the absolute and the relative amounts of the proenzyme (unactivated) and activated C1 can thereby be quantitated in most samples. Furthermore, a partially purified C1 inhibitor reagent, easily prepared from serum, was shown to function identically to the purified C1 inhibitor, obviating the need for a multistep isolation procedure for this protein. Using this simple yet sensitive assay to investigate the efficiency of reconstitution of C1 activity from the purified components C1q, C1r, and C1s, we also find evidence for temperature- and concentration-dependent reaction steps in the formation of functional C1.

C3 nephritic factor (C3NeF): dissociation of cell-bound and fluid phase stabilization of alternative pathway C3 convertase.

Fluid phase C3 conversion by C3 nephritic factor (C3NeF) is usually easily detectable and forms the basis of the C3NeF screening test. We have described a group of patients with membranoproliferative glomerulonephritis or partial lipodystrophy and hypocomplementaemia who have an unusual C3NeF which stabilizes cell-bound C3 convertase of the alternative pathway (C3bBb) but causes such weak fluid phase C3 conversion that a C3NeF screening test is negative. These patients have low concentrations of C5 in serum.

Complement subcomponent C1q secreted by cultured human monocytes has subunit structure identical with that of serum C1q.

An enzyme-linked immunosorbent assay (e.l.i.s.a.) that is capable of quantifying C1q concentrations as low as 2 ng/ml and a sensitive haemolytic assay were used to study the appearance of material that cross-reacts with human serum C1q as well as C1q haemolytic activity in human monocyte culture media. This material was detected in the medium after 10-14 days and continued to be secreted through to day 28 of culture, at which time the cultures were terminated. Material specifically immunoabsorbed with Sepharose-anti-C1q antibody from a culture medium of cells that was metabolically labelled with [3H] proline or [35S] methionine demonstrated a polypeptide pattern identical with that of serum C1q on SDS/polyacrylamide-gel electrophoresis. Under non-reducing conditions two protein bands were detected migrating with the same Rf values as the serum C1q A-B and C-C dimers. On reduction three bands were evident, which migrated identically with the A, B and C chains of serum C1q. The amount of radioactivity in these bands increased with time in culture, consistent with the e.l.i.s.a. and haemolytic C1q assays. These bands were reactive with monospecific anti-C1q antibody after transfer to nitrocellulose.

Erythema multiforme: a possible pathogenetic role of increased reactive oxygen species.

Recently, it has been suggested that reactive oxygen species (ROS) produced by activated polymorphonuclear leukocytes (PMNs) exert auto-oxidative tissue damage at the site of inflammation. In the present study, a possible role of ROS in the pathogenesis of erythema multiforme (EM) was investigated by determining the capacity of the sera from patients for generating ROS from PMNs. Significantly increased hydroxyl radical production was observed, which is one of the most potent ROS capable of causing tissue damage. This change was not observed when PMNs were incubated with sera from patients with bullous pemphigoid (BP) or inflammatory acne, indicating that this finding was not a common feature of immunologically mediated and/or inflammatory cutaneous disorders. Elevated C1q activities and depositions of immunoreactants in the blood vessels were also noticed in some patients. These findings suggest that ROS generated by PMNs are involved in the formation of cutaneous lesions of EM and that immune complexes (ICs) may provide an important mechanism in PMN activation.

[Effect of Expafusion (HES 40/0.5) on the corpuscular elements of blood and inhibitors of blood coagulation]. / Einfluss von Expafusin (HAS 40/0.5) auf die korpuskulären Anteile des Blutes und die Inhibitoren der Blutgerinnung.

The present study reports on an investigation of 32 normal subjects who were given intravenously 500 ml of hydroxyethyl starch (HES) over 30 min. In 16 normal subjects, 500 ml of blood had been previously withdrawn over 20 min. Tolerance proved to be good. Side effects such as hyperergic reactions, influence on circulatory or cardiac function were not observed. Changes in corpuscular elements of blood did not exceed the dilution effect and mostly returned to the initial value 60 min after termination of HES infusion. The change in thrombocyte function, immunoglobulins and the inhibitors alpha 1-antitrypsin, alpha 2-macroglobulin, as well as C1-inactivator and C3-activator only slightly, but not more pronounced than it could have been expected by the dilution effect. A favorable effect could be recorded in erythrocyte filtration according to Schmidt-Schoenbein. Here, we found an improved flow property which exceeded the hemodilution effect and lasted over the 4-hour test period. In addition, thrombocyte aggregation during ADP administration in the Born chamber did not show any change which suggests that a clinically relevant effect on thrombocyte function and thus the danger of hemorrhage during HES administration can be excluded.

Effect of ascorbic acid nutriture on protein-bound hydroxyproline in guinea pig plasma.

This paper provides indirect evidence that ascorbate nutriture affects plasma concentrations of complement component C1q in the guinea pig. C1q is a protein with a hydroxyproline-rich region similar in structure to collagen. It is essential for complement-mediated lysis of pathogens and may also facilitate phagocytic activity of macrophages and neutrophils. Since C1q is the only hydroxyproline-containing protein in the euglobulin fraction of plasma, it can be quantified indirectly by precipitating this fraction, hydrolyzing it and estimating hydroxyproline colorimetrically. We investigated the effect of ascorbate nutriture on protein-bound hydroxyproline (PBH) in the euglobulin fraction of plasma of young male guinea pigs. The animals had been depleted of ascorbate for 3 wk to produce scurvy and then repleted (6 wk) as follows: 0.5, 2.0 and 10.0 mg ascorbate/100 g body weight per d or 10 g ascorbate per liter of drinking water. PBH values were significantly correlated (P less than 0.001) with dietary ascorbate (+ 0.74) and with liver ascorbate (+ 0.75). Plasma PBH was significantly higher (P less than 0.01, Scheffé's test) in guinea pigs fed ample ascorbate (10.0 mg/100 g body weight per day) or tissue-saturating levels (10 g/L of drinking water) than in those fed adequate (2.0 mg/100 g body weight) or suboptimal (0.5 mg/100 g body weight) levels. These data are consistent with the known biochemical role of ascorbic acid in hydroxyproline biosynthesis and suggest a possible link between ascorbate and the immune response via C1q.

Regulation and deregulation of the fluid-phase classical pathway C3 convertase.

Three mechanisms that regulate the formation and function of the fluid-phase classical pathway C3 convertase (C4b2a) have been elucidated: a) a temperature-mediated intrinsic decay of the enzyme; b) an extrinsic accelerated decay mediated by the effect of the serum protein C4b-binding protein (C4-bp); and c) the inactivation of C4b in the C4b-C4b-p complex by the proteolytic action of C3b/C4b inactivator (I), which cleaves the alpha 1-chain of C4b yielding C4d (alpha 2-chain), and C4c (alpha 3-, alpha 4-, beta-, gamma-chains). A fourth mechanism is described based on the observation that the IgG fraction of the serum of certain patients with glomerulonephritis contains a protein that prevents the intrinsic and C4-bp-mediated decay of surface-bound C4b2a. This protein prolongs the half-life of fluid-phase C4b2a from 10 min to more than 5 hr, increasing the utilization of C3. It also inhibits the decay mediated by C4-bp by preventing the dissociation of C2a from the C4b, 2a complex. In addition, I alone or in the presence of C4-bp fails to cleave the alpha 1-chain of C4b in the stabilized C4b, 2a complex. This protective property of the stabilizing factor (NFc) requires the presence of C2a because C4b was not protected unless it was bound to C2a. Therefore, NFc provides a mechanism by which the serum regulatory proteins are bypassed.

The determination of the complement components C1q, C4 and C3 in serum and cerebrospinal fluid by radioimmunoassay.

Non-competitive 2-site radioimmunoassays (RIA) for the determination of the complement proteins C1q, C4 and C3 in cerebrospinal fluid (CSF) are described. The quantitative results of the RIAs were the same as those obtained by other assay methods: radial immunodiffusion and turbidimetry and, in the case of C4, the haemolytic assay. The concentrations of the complement proteins in paired CSF and serum samples from a group of 60 patients were measured, as well as those of albumin and IgG. The ratios (concentration in CSF)/(concentration in serum) of the complement proteins correlated poorly with that of albumin. In contrast, the ratio of IgG was significantly correlated with that of albumin. The ratios of the complement proteins were higher than might be expected on the basis of their molecular masses. This suggests that these proteins may be synthesized within the normal central nervous system.

Atypical hypocomplementemic vasculitis syndrome in a child.

We report a patient who developed recurrent urticaria and angioedema at age 2 years, severe hypocomplementemic glomerulonephritis at 11 years, and end-stage renal disease at 14 years. His disease resembled the hypocomplementemic vasculitis syndrome but was atypical in its early age of presentation, severe hypocomplementemia, and progression to end-stage renal disease. Serum C1q levels were extremely low, and C4, C2, C3, and C5 levels were significantly reduced. Serum C1 inhibitor (C1INH) levels were slightly low, presumably from consumption. Circulating C1INH-C1r-C1s complexes were evidenced by reduced ratios of functional to antigenic C1INH and antigenic C1r to C1s. Family members had normal functional and antigenic levels of all complement components studied. The patient's serum, erythrocytes, platelets, and mononuclear cells did not activate complement when mixed with normal target serum. Absence of a circulating complement activator and the low serum C3 and C5 levels suggested the presence of a solid-phase complement activator, possibly related to renal or systemic vascular endothelium. As in patients with homozygous deficiencies of classical pathway components, a severe, prolonged, acquired C1q deficiency may have predisposed this patient to the development of glomerulonephritis.

Serum C1q levels as a prognostic guide to articular erosions in patients with rheumatoid arthritis.

C1q was measured serially by single radial immunodiffusion in 54 rheumatoid arthritis (RA) patients over a period of more than 5 years, and values were correlated with laboratory, radiographic, and clinical findings. The number of joints with erosion (NJE) was determined retrospectively from radiographs of patients who had RA of greater than 7 years duration. In patients with clinically "burned out" RA, C1q levels were not statistically different from those of healthy adults. During the period of active disease, each patient's C1q level remained very constant, irrespective of erythrocyte sedimentation rate, C-reactive protein (CRP) level, or whether the RA was active or in remission. No sustained correlation was found between the C1q level and the other 2 acute phase reactants, but patients with C1q levels of at least 250 micrograms/ml showed a positive CRP over a period of years, in contrast to those with C1q levels below 250 micrograms/ml. Patients with an initial C1q above 250 micrograms/ml had more erosive RA when compared with those having C1q levels below 250 micrograms/ml. These data suggest that active RA can be classified into two subsets by C1q levels, one with persistent inflammation and a high NJE and another without persistent inflammation and with a low NJE.