Complement activation in patients with immune thrombocytopenic purpura according to phases of disease course.

Immune thrombocytopenic purpura (ITP) is an autoimmune thrombocytopenia with shortened platelet survival and relative bone marrow failure. The pathogenesis involves antibody production, cytokine release, T cell impairment, complement activation and clearance of platelets. We measured plasma levels of C3, C4, C1q and sC5b-9 in 80 ITP patients in acute phase, 50 ITP patients in complete (CR) or partial (PR) remission and 50 age- and sex-matched healthy volunteers. Statistical analyses showed that acute ITP patients had higher plasma levels of sC5b-9 and C1q than CR or PR patients (median = sC5b-9: 200 versus 98 mg/dl, P-value < 0·001) (median C1q = 2·11 versus 1·00 mg/dl, P-value < 0·001). CR and PR ITP patients had sC5b-9 and C1q plasma levels comparable to those observed in healthy volunteers. There was a significant correlation between sC5b-9 and C1q plasma levels (Spearman's rho correlation index on 130 ITP patients equal to 0·58, P-value < 0·001). We also found that sC5b-9 plasma level is inversely correlated with the number of platelets. Furthermore, we divided acute ITP patients into subjects with detectable (24 of 80, 30%) or undetectable (56 of 80, 70%) anti-platelet antibodies; patients with detectable anti-platelet antibodies have significantly higher plasma levels of C1q and sC5b-9. This research will potentially offer novel therapeutic strategies in light of new drugs affecting complement activation for monitoring therapy response.

Pin1 contribution to Alzheimer's disease: transcriptional and epigenetic mechanisms in patients with late-onset Alzheimer's disease.

BACKGROUND: Neurofibrillary tangles and senile plaques are hallmarks of Alzheimer's disease (AD) although the molecular basis of their coexistence remains elusive. The peptidyl-prolyl cis/trans isomerase Pin1 acts on both tau and amyloid precursor protein to regulate their functions by influencing tau phosphorylation and amyloid precursor protein processing. OBJECTIVE: In order to identify potential biomarkers for AD in easily accessible cells and to gain insight into the relationship between the brain and peripheral compartments in AD pathology, we investigated Pin1 expression and activity in the peripheral blood mononuclear cells of subjects with late-onset AD (LOAD) and age-matched controls (CT). METHODS: Gene and protein expression, promoter methylation, Ser(16) phosphorylation and activity of Pin1 were evaluated in 32 samples from subjects with LOAD and in 28 samples from CT. RESULTS: In LOAD subjects, there was a statistically significant reduction in Ser(16) phosphorylation (-30%; p = 0.041) and promoter methylation (-8%; p = 0.001), whereas Pin1 expression was significantly increased (+74%; p = 0.018). CONCLUSION: The modifications of Pin1 found in LOAD subjects support its involvement in the pathogenesis of the disease with an important role being played by epigenetic mechanisms.

Neuroprotection by complement (C1) inhibitor in mouse transient brain ischemia.

The authors investigated the effect of the C1 inhibitor (C1-INH), the only known inhibitor of complement C1, in a murine model of transient focal ischemia. Ischemia was induced by intraluminal occlusion of the middle cerebral artery. After 2 hours, reperfusion was produced by removing the nylon monofilament occluding the artery. The effect of 15 U C1-INH (intravenously) was evaluated in terms of general and focal neurologic deficits, ischemic volume, neutral red staining (to identify the brain areas subject to ischemic damage), and glial fibrillary acidic protein immunoreactivity (to show astrocytic response). Forty-eight hours after ischemia, C1-INH significantly improved general and focal deficits by 36% and 54%, respectively, and significantly reduced infarct volume (CI-INH, 6.69% +/- 2.93%; saline, 24.24% +/- 8.24%) of total brain. Neutral red staining further showed the strong protective effect of C1-INH in cortex, hippocampus, and striatum. Astrocyte activation induced by ischemia was not affected by C1-INH. These findings show that C1-INH displayed a potent neuroprotective action by effectively reducing ischemia-reperfusion injury.

Endothelial targeting with C1-inhibitor reduces complement activation in vitro and during ex vivo reperfusion of pig liver.

Tissue damage during cold storage and reperfusion remains a major obstacle to wider use of transplantation. Vascular endothelial cells and complement activation are thought to be involved in the inflammatory reactions following reperfusion, so endothelial targeting of complement inhibitors is of great interest. Using an in vitro model of human umbilical vein endothelial cells (HUVEC) cold storage and an animal model of ex vivo liver reperfusion after cold ischaemia, we assessed the effect of C1-INH on cell functions and liver damage. We found that in vitro C1-INH bound to HUVEC in a manner depending on the duration of cold storage. Cell-bound C1-INH was functionally active since retained the ability to inhibit exogenous C1s. To assess the ability of cell-bound C1-INH to prevent complement activation during organ reperfusion, we added C1-INH to the preservation solution in an animal model of extracorporeal liver reperfusion. Ex vivo liver reperfusion after 8 h of cold ischaemia resulted in plasma C3 activation and reduction of total serum haemolytic activity, and at tissue level deposition of C3 associated with variable level of inflammatory cell infiltration and tissue damage. These findings were reduced when livers were stored in preservation solution containing C1-INH. Immunohistochemical analysis of C1-INH-treated livers showed immunoreactivity localized on the sinusoidal pole of the liver trabeculae, linked to sinusoidal endothelium, so it is likely that the protective effect was due to C1-INH retained by the livers. These results suggest that adding C1-INH to the preservation solution may be useful to reduce complement activation and tissue injury during the reperfusion of an ischaemic liver.

Activation of complement and contact system in Alzheimer's disease.

beta-Amyloid protein (betaA) has been implicated in the pathogenesis of Alzheimer's disease (AD) because of its neurotoxicity and ability to trigger a local inflammatory response. Although assembly of betaA in particular aggregates seems to be crucial event in AD pathogenesis, soluble, non-fibrillar betaA may also be involved. Non-fibrillar betaA1-42, and truncated peptide 1-28, induced dose-dependent activation of C4 sparing C3. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar betaA can still activate C4 in plasma genetically deficient in C1q. A C1q independent mechanism of complement classical pathway activation could be via the activation of contact/kinin system. The possible involvement of contact system in AD is suggested by the finding that this system is massively activated in CSF of AD patients. The mechanism of activation of contact system could be the result of an anionic interaction of residues within the region 1-11 of betaA1-42 with factor XII, and of kallikrein generation. Concomitant incubation of a small cationic peptide (lysine4) with betaA abrogated its ability to trigger the cleavage of high molecular weight kininogen. In vivo, prevention of contact system activation beside the reduction of kallikrein generation, can also decrease the activation of complement system and the release of interleukin-6, both factors being considered to play an important role in the inflammatory reactions in AD brain.

The region 1-11 of Alzheimer amyloid-beta is critical for activation of contact-kinin system.

Amyloid-beta protein (Abeta) has been implicated in the pathogenesis of Alzheimer's disease (AD) because of its neurotoxicity and its ability to trigger a local inflammatory response. In the present study using truncated Abeta peptides, we identified the region between residues 1 and 11 as critical for the activation of the contact system in vitro through an ionic interaction of Abeta with factor XII and/or kallikrein. Concomitant incubation of a small cationic peptide (lysine(4)) with Abeta abrogated its ability to trigger the cleavage of high molecular weight kininogen, indicating that Abeta's activity can be blocked by an inhibitory peptide. These findings could be clinically important, since there is evidence that the contact system is activated in AD brain. Thus, prevention of contact system activation, beside diminishing the recruitment of glial cells and microvascular permeability, can also decrease the activation of complement system and the release of IL6, both factors being considered to play an important role in the inflammatory reactions in AD brain.

Idiopathic nonhistaminergic angioedema.

PURPOSE: We sought to describe the characteristics of a group of patients with idiopathic nonhistaminergic angioedema and their response to prophylactic treatment with tranexamic acid. METHODS: We identified 25 patients (15 men and 10 women; age at diagnosis 16 to 77 years) who had idiopathic nonurticarial angioedema that was not prevented by histamine-1 (H1) blockers. Known causes of angioedema were excluded by clinical history, physical examination, and diagnostic tests. RESULTS: The median age at the onset of symptoms was 35 years (range 8 to 66). The frequency of attacks was > 12 per year for 16 patients, six to 11 per year for 6 patients, and one to five per year for 3 patients. All patients had cutaneous attacks, 13 (52%) reported swellings of the pharynx or larynx, and 5 (20%) had symptoms consistent with bowel angioedema. Because of the similarities between these patients and patients who are deficient in C1 inhibitor, the 15 patients with severe and frequent attacks were started on prophylactic treatment with the antifibrinolytic agent tranexamic acid, 1 g three times a day orally for 3 months, tapered according to its effectiveness. The symptoms of 11 patients decreased to less than one attack per year, and the remaining 4 patients had partial remissions (less than 4 attacks per year). Fourteen patients are still being treated with tranexamic acid. CONCLUSION: Patients with idiopathic nonhistaminergic angioedema appear to have similar clinical features and response to treatment with tranexamic acid as those who are deficient in C1 inhibitor. This suggests that those two forms of angioedema might have, at least in part, a similar pathogenesis.

Consumption of C4b-binding protein (C4BP) during in vivo activation of the classical complement pathway.

C4BP has a central role in regulating the classical complement (C') pathway, but it is still uncertain whether or not it is consumed during in vivo complement activation. Attempts to demonstrate changes in C4BP plasma levels in systemic lupus erythematosus and essential mixed cryoglobulinaemia have failed, probably due to up-regulation of this protein during the inflammatory reaction. We have studied one patient with severe post-transfusion complement-mediated anaphylaxis (CMA), and 67 patients with hereditary C1 inhibitor deficiency (hereditary angioedema (HAE)). The first of these two conditions is characterized by the absence of systemic inflammatory reaction and the second by acute and chronic activation of the C' classical pathway. C4BP, C4BP-C4b complex, and soluble terminal C' complex (sC5b-9) were measured in the patients' plasmas by ELISA techniques and C3a and C4a by radioimmunoassays. In CMA, 15 min after the transfusion, there was a massive C' activation, with increases in C4a, C3a, sC5b-9, C4BP-C4b complexes and decreases in C4, C3 and C4BP. All parameters reverted to preinfusion values within 24 h. Depletion of C4 was correlated with that of C4BP. In patients with HAE, the median value of C4BP (83% range 54-165) was significantly lower (P < 0.0001) than in normal controls (99% range 70-159), with no difference between patients in remission or during acute attacks. C4BP-C4b complexes could not be detected in HAE patients. The results of this study indicate that C4BP is consumed in vivo during acute, and possibly during chronic activation of the C' classical pathway, and that this protein, after interaction with C4b, not longer circulates in plasma.

Alzheimer's beta-amyloid peptides can activate the early components of complement classical pathway in a C1q-independent manner.

beta-Amyloid (beta-A) accumulates in the brain of patients with Alzheimer's disease (AD) and is presumably involved in the pathogenesis of this disease, on account of its neurotoxicity and complement-activating ability. Although assembly of beta-A in particular aggregates seems to be crucial, soluble non-fibrillar beta-A may also be involved. Non-fibrillar beta-A does not bind C1q, so we investigated alternative mechanisms of beta-A-dependent complement activation in vitro. On incubation with normal human plasma, non-fibrillar beta-A 1-42, and truncated peptide 1-28, induced dose-dependent activation of C1s and C4, sparing C3, as assessed by densitometric analysis of immunostained membrane after SDS-PAGE and Western blotting. The mechanism of C4 activation was not dependent on C1q, because non-fibrillar beta-A can still activate C1s and C4 in plasma genetically deficient in C1q (C1qd). In Factor XII-deficient plasma (F.XIId) the amount of cleaved C4 was about 5-10% less that in C1qd and in normal EDTA plasma; the reconstitution of F.XIId plasma with physiologic concentrations of F.XII resulted in an increased (8-15%) beta-A-dependent cleavage of C4. Thus our results indicate that the C1q-independent activation of C1 and C4 can be partially mediated by the activation products of contact system. Since the activation of contact system and of C4 leads to generation of several humoral inflammatory peptides, non-fibrillar beta-A might play a role in initiating the early inflammatory reactions leading to a multistep cascade contributing to neuronal and clinical dysfunction of AD brain.

Pathogenetic and clinical aspects of C1 inhibitor deficiency.

People deficient in C1-INH present recurrent angioedema localized to subcutaneous or mucous tissues. The defect can be caused by impaired synthesis, due to a genetic defect (hereditary angioedema), or by increased catabolism (acquired angioedema). In our experience the majority of patients with acquired angioedema (16 of 18) have autoantibodies to C1-INH in their serum. These autoantibodies bind to C1-INH with different and generally low affinity. The vasopermeability mediator responsible for attacks is still undefined: bradykinin (derived from cleavage of high molecular weight kininogen) and a kinin-like peptide (derived from the second component of complement) still remain the two primary candidates. We examined the systems controlled by C1-INH (complement, contact system, fibrinolysis and coagulation) and found that all of them are activated during angioedema attacks. Activation of the coagulation leads to generation of thrombin whose vasoactive effect can thus influence edema formation. Treatment of severe angioedema attacks is satisfactorily performed with C1-INH plasma concentrate although patients with an acquired defect frequently need very high doses. Attenuated androgens effectively prevent attacks in hereditary angioedema, but their safety, on the very long-term, needs to be further assessed. Acquired angioedema generally fail to respond to these drugs, but can be treated prophylactically with antifibrinolytic agents.

Activation of the contact system in cerebrospinal fluid of patients with Alzheimer disease.

Several converging lines of evidence suggest that beta-amyloid and inflammation may be linked in the pathogenesis of Alzheimer disease (AD), but the mechanism of beta-amyloid neurotoxicity is unclear. In this study, by demonstrating that high molecular weight kininogen may be massively cleaved in the cerebrospinal fluid (CSF) of patients with AD, we provide evidence of the potential involvement of the contact system in the inflammatory processes taking place in this disease. In the CSF of patients with neuroimmune inflammatory disease (multiple sclerosis, chronic inflammatory demyelinating polyneuropathy), there was no evidence of increased cleavage of high molecular weight kininogen, suggesting that this finding may be characteristic of the Alzheimer brain. The data obtained from in vitro experiments seem to indicate that the cleavage of high molecular weight kininogen in vivo may be the result of the interaction of beta-amyloid with factor XII and of kallikrein generation. The actual relevance of such a phenomenon remains to be established in vivo. However, the demonstration that the contact system may be activated in the brains of Alzheimer patients points to the potential involvement of the kallikrein-kinin system in the inflammatory process of this disease.

Study of complement-mediated anaphylaxis in humans. The role of IgG subclasses (IgG1 and/or IgG4) in the complement-activating capacity of immune complexes.

The role of Ig classes and subclasses in complement activation has been investigated both in vitro and in experimental animals, but not in humans. This study was conducted to determine the immunologic events of post-transfusion anaphylaxis in humans, and the effects of immune complexes of different IgG subclass compositions on complement activation. The ability of immune complexes containing mixed IgG1 and IgG4 or IgG4 Ab only to activate complement was investigated in two patients with von Willebrand's disease (a congenital bleeding disorder). This disease was complicated by precipitating IgG alloantibodies to von Willebrand factor (vWF), which triggered complement-mediated anaphylaxis after infusion of vWF-containing preparations. Complement activation was greater in the presence of mixed IgG1- and IgG4-vWF complexes than with IgG4-vWF alone. In one patient, the generation of large amounts of C4a, C3a, and terminal complement complexes following the formation of mixed IgG1- and IgG4-vWF was associated with life-threatening anaphylaxis. In this patient, IgG4 appeared to have no inhibitory effect on antigen-bound IgG1-mediated complement activation. In the other patient, formation of IgG4-vWF led to a lesser degree of complement activation and was associated with moderately severe anaphylaxis. Since neither patient showed any biochemical alterations indicating the involvement of mast cells or the contact phase of coagulation at any time, it is probable that the pathogenetic mechanism of the clinical syndrome was a direct effect of complement anaphylatoxins on vascular permeability and smooth muscle contraction. In both patients, IgG-vWF bound C4 and C3 (IgG4-vWF to a lesser extent than mixed IgG1- and IgG4-vWF), and this probably prevented serum sickness as a complication.

Infusion of C1-inhibitor plasma concentrate prevents hyperamylasemia induced by endoscopic sphincterotomy.

Hyperamylasemia after endoscopic sphincterotomy is a common event, occurring in about 70% of cases. Clinical acute pancreatitis may also develop in 1% to 6% of cases. Previous attempts to prevent this reaction with inhibitors of exocrine pancreatic secretion (somatostatin and octreotide) provided conflicting and often disappointing results. Kallikrein is one of the proteases that sustain the inflammatory process in acute pancreatitis; the C1 inhibitor is the only physiologic inhibitor of the first component of the human complement cascade and is a major inactivator of kallikrein and Factor XII. Therefore, we tested the C1 inhibitor in the prevention of hyperamylasemia in 40 consecutive patients undergoing endoscopic sphincterotomy for common bile duct stones or benign papillary stenosis. They were given either C1 inhibitor (20 cases) or placebo (20 cases) before the procedure. Serum amylase levels were determined at baseline and 2, 4, 8, and 24 hours thereafter. Significant differences in serum amylase levels between groups were observed at 2 hours (p < .01), 4 hours (p < .0005), and 8 hours (p < .005) after sphincterotomy. The differences in amylase levels were also significant among the 24 subjects with pancreatic ductal filling (2 hours, p < .05; 4 hours, p < .005; 8 hours, p < .01) and the 9 patients with previous episodes of acute pancreatitis (4 hours, p < .05; 8 hours, p < .05; 24 hours, p < .05). The infusion of C1-inhibitor plasma concentrate resulted in a 50% increase in functional levels of C1 inhibitor (in the 8 cases for whom they were assayed), which persisted throughout the observation period.

Posttransfusion anaphylactic reactions in a patient with severe von Willebrand disease: role of complement and alloantibodies to von Willebrand factor.

Previous studies have suggested that activation of the complement system might be a major mechanism for posttransfusion non-immunoglobulin (Ig) E-mediated anaphylactic reactions, but its causative effect has not been clearly demonstrated in human models. Serial plasma samples were collected from a patient with severe von Willebrand disease, IgG alloantibodies against von Willebrand factor (vWF), and a history of posttransfusion anaphylaxis. During an 18-day period the patient was treated with factor VIII-vWF concentrate and with recombinant factor VIII. Complement system activation was assessed from plasma levels of C4a, C3a, cleavage products of complement factor B, soluble terminal complement complex, C1 inhibitor and C4-binding protein, and the contact phase of coagulation was assessed from plasma levels of activated factor XII and cleaved high-molecular-weight kininogen. Plasma levels of antibodies to vWF and complement-fixing IgG-vWF complexes were also evaluated. Symptoms of anaphylaxis and signs of complement activation were present only when IgG antibodies to vWF were measurable during replacement with factor VIII-vWF concentrate (days 1 and 6). IgE, IgA, and IgM antibodies to vWF were not detectable in plasma at any time. Replacement with recombinant factor VIII (days 7 to 18) secured hemostasis and did not elicit anaphylactic reactions, and complement parameters did not significantly change even when antibodies to vWF reached peak plasma levels. This prospective study of a natural clinical model indicates a cause-effect relationship between formation of IgG-vWF complexes and massive complement activation in posttransfusion non-IgE-mediated anaphylactic reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of two immunoassays for the complement protein C4b-binding protein in health and disease.

An enzyme-linked immunoassay for human C4b-binding protein (concentration range 25-400 ng/ml) was developed using two monoclonal antibodies; the intra- and interassay coefficients of variation were less than 7.2%. In 50 normal subjects, 20 patients with liver cirrhosis and 34 full-term newborns, the plasma levels of C4b-binding protein were very similar to those measured by Laurell electroimmunoassay; in 24 patients with elevated erythrocyte sedimentation rates, levels measured by enzyme-linked immunoassay were higher then those measured by the Laurell method (270 +/- 70% vs. 223 +/- 67%). In these patients crossed immunoelectrophoresis showed a pattern different from that of normal individuals, which may explain the lower values found with the Laurell method.

Antiproteasic activity of C1 inhibitor. Therapeutic perspectives.

Kallikrein is a protease involved in the inflammatory process causing acute pancreatitis. Attempts to prevent this process with antiprotease agents have been successful in experimental animal models but disappointing in humans. We studied 40 consecutive patients undergoing endoscopic papillosphincterotomy. This procedure can induce a transient, moderate pancreatic inflammatory reaction, characterized by hyperamylasemia, which in 1-6% of the patients may evolve to acute pancreatitis. To assess the capacity of C1 inhibitor, the main physiological inhibitor of kallikrein, to prevent such complications, we pretreated 20 patients with 3000 U of C1 inhibitor plasma concentrate i.v.; 20 patients served as controls. Serum levels of amylase and functional C1 inhibitor were determined before the procedure and after 2, 4, 8 and 24 hours. Serum levels of amylase in the control group (146 +/- 21 IU) and in the group treated with C1 inhibitor (158 +/- 25 IU) were similar before treatment. Four and 8 hours after the end of the procedure, amylase levels were significantly lower (p < 0.001) in the treated group (231 +/- 46 and 355 +/- 104 IU) than in the control subjects (969 +/- 229 and 923 +/- 207 IU). After 24 hours both groups had normal amylase levels. In treated patients, functional levels of C1 inhibitor increased from 104 +/- 30 to 175 +/- 30% and remained elevated throughout the observation period. These data indicate that C1 inhibitor plasma concentrate can prevent hyperamylasemia following pancreas injury, probably, by inhibiting the kallikrein-mediated inflammatory process. C1 inhibitor might benefit patients at high risk of pancreatitis who undergo endoscopic papillosphincterotomy.

Localization of the human complement component C3 binding site on the IgG heavy chain.

The location of the covalent binding site of the third component of complement (C3) on the IgG heavy chain was determined by sequence analysis of peptides generated by cyanogen bromide digestion of C3-IgG adducts. Activation of the alternative pathway by incubation of heat-aggregated human IgG1 with fresh normal human plasma formed covalent adducts of C3b-IgG. CNBr peptides of these adducts were transferred to a polyvinylidene difluoride membrane, and amino-terminal sequences were determined. A 40-kDa dipeptide containing the covalent bond was identified by labeling the free thiol group (generated during activation of the internal thioester of C3b) with iodo[1-14C]acetamide and analyzed by amino acid sequencing. The resulting double sequence suggested an adduct with NH2 termini at residue 938 (pro-C3 numbering) of C3 (75 residues NH2-terminal to the thioester) and residue 84 in the variable region of the IgG heavy chain. These results combined with results from hydroxylamine treatment (splits ester linkage between C3b and IgG) imply that this adduct peptide consists of a 22-kDa C3 fragment and an 18-kDa IgG fragment. Therefore, C3 binds covalently within the region extending from the last 20 residues of the variable region through the first 20 residues of CH2.