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.

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.

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.

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.

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.

Effect of treatment with 17 alpha-alkylated androgens on C4 conversion products in hereditary angioedema studied by crossed immunoelectrophoresis.

During agarose electrophoresis C4 in the normal human serum is converted into cleavage products of Beta 1 and Beta 2 mobility. By contrast in the serum of untreated patients with hereditary angiodema C4 gives only one Beta 2 peak on crossed immunoelectrophoresis. The normal C4 electrophoretic pattern is restored in serum of patients treated with stanazolol but not with danazol despite the same C1-esterase inhibitor (C1 INH) activities and C4 serum concentration. We suggest that stanazolol besides having specific effect on C1 INH activity can interfere with other protease inhibitors affecting C1 activation.

Hereditary angioedema: an appraisal of 104 cases.

One hundred and four patients affected by hereditary angioedema belonging to 31 families have been studied. Twenty-two percent had the variant form related to the deficiency of the functional activity of serum C1 esterase inhibitor. The remaining 78% of patients had the predominant form, characterized by low antigenic levels and low functional activity of serum C1 esterase inhibitor. Attacks of swelling affected the subcutaneous tissue in 86% of patients; the upper airways in 76% of patients, and the bowel mucose in 75% of patients. Before treatment was available the mortality rate was 56%. One or more attacks a month were present in 46% of cases. The infusion of C1 inhibitor concentrate promptly reversed 14 severe attacks without any side effect. Twenty-nine patients were given long term prophylactic treatment with androgen derivatives with full success. Tranexamic acid reduced the frequency of swelling of 70% of the patients.

Studies of complement activation in ARDS patients treated by long-term extracorporeal CO2 removal.

To investigate the role of complement activation in the adult respiratory distress syndrome (ARDS) and in the complications of extracorporeal circulation (ECC), several parameters (CH50, C3 split products, C3a, C5a, PMN aggregating activity, carboxypeptidase activity) of the complement profiles of 23 ARDS patients were measured. Twenty patients were treated by long-term extracorporeal support. Before connection to ECC, marked leukocytosis (18,250 +/- 5,950) and significantly high plasma C3a levels (p less than 0.005) were observed. After connection, C3a levels increased further, up to values eight times higher than the basal ones. The WBC count transiently decreased to 41% of prebypass levels after 15 minutes of ECC. At the same time C3 split products appeared and PMN aggregating activity was shown in 52% of the patients. C5a levels remained normal during bypass, even in two samples in which PMN aggregating activity was detected. Later decreases in CH50 titers (p less than 0.001) and carboxypeptidase activity (p less than 0.005) were observed. Complement activation was no longer evident after the 24th hour of bypass. We conclude that there is a low-degree complement activation in ARDS, and ECC is a further strong stimulus for complement activation. This phenomenon appears, however, to be self-limited.

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.

[Pathological effects of recently isolated complement derived peptides]. / Effetti patologici dei peptidi di derivazione complementare recenti acquisizioni.

The C3a and C5a polypeptides release, under conditions of complement activation, causes a change in PMN shape and adhesiveness with formation of leukoemboli in microvascular districts. Those modified granulocytes produce oxygen radicals with toxic activity. This mechanism seems to play a role in different pathological situations like pulmonary distress in hemodialysis, leukapheresis, cardiopulmonary by-pass and extent of necrosis in acute myocardial infarction. The same mechanism has been largely investigated in Adult Respiratory Distress Syndrome (A.R.D.S.). In this condition the leukoembolization toxicity seems to be the most important pathogenetic factor of the alveolo-capillary membrane damage which is the base of the disease.

Pityriasis rubra pilaris and and retinol-binding protein.

Serum levels of retinol-binding protein (the specific carrier of vitamin A) were measured in eleven patients with pityriasis rubra pilaris and in some of their close relatives. The level of retinol-binding protein was markedly reduced in every patient, and in some of the relatives. It is postulated that defective synthesis of retinol-binding protein is a biochemical marker for pityriasis rubra pilaris, probably transmitted as a Mendelian dominant.

Acquired C1 inhibitor deficiency with angioedema symptoms in a patient infected with Echinococcus granulosus.

A patient with echinococcosis, acquired deficiency of the inhibitor of the activated first component of complement and angioedema symptoms has been studied. These symptoms started 7 months after the surgical removal of an echinococcus liver cyst. Eight years later, when the complement was investigated, a marked deficiency of the C1 inhibitor, C1, C4 and CH50 was present. The patient was therefore successfully treated with tranexamic acid. After 4 years, the woman needed another operation because of a relapse of echinococcosis; afterwards she was symptom-free without medications, while the complement profile remained unchanged. Circulating immune complexes were detected by the conglutinin method. The patient's serum was demonstrated to possess an anticomplementary activity without affecting the C1 inhibitor when incubated with normal human serum at 37 degrees C. At present, 16 years after the onset of the symptoms, there are no signs of lymphoproliferative disease.

Substitution of a single amino acid (aspartic acid for histidine) converts the functional activity of human complement C4B to C4A.

The C4B isotype of the fourth component of human complement (C4) displays 3- to 4-fold greater hemolytic activity than does its other isotype C4A. This correlates with differences in their covalent binding efficiencies to erythrocytes coated with antibody and complement C1. C4A binds to a greater extent when C1 is on IgG immune aggregates. The differences in covalent binding properties correlate only with amino acid changes between residues 1101 and 1106 (pro-C4 numbering)--namely, Pro-1101, Cys-1102, Leu-1105, and Asp-1106 in C4A and Leu-1101, Ser-1102, Ile-1105, and His-1106 in C4B, which are located in the C4d region of the alpha chain. To more precisely identify the residues that are important for the functional differences, C4A-C4B hybrid proteins were constructed by using recombinant DNA techniques. Comparison of these by hemolytic assay and binding to IgG aggregates showed that the single substitution of aspartic acid for histidine at position 1106 largely accounted for the change in functional activity and nature of the chemical bond formed (ester vs. amide). Surprisingly, substitution of a neutral residue, alanine, for histidine at position 1106 resulted in an increase in binding to immune aggregates without subsequent reduction in the hemolytic activity. This result strongly suggests that position 1106 is not "catalytic" as previously proposed but interacts sterically/electrostatically with potential acceptor sites and serves to "select" binding sites on potential acceptor molecules.

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)