[The range and potential contribution of irreversibly adsorbed proteins on the surface of artificial implants]. / Spektr i potentsial'naia rol' neobratimo adsorbirovannykh belkov na poverkhnosti iskusstvennykh implantiruemykh materialov.

Protein adsorption is the first stage of the interaction between prosthetic materials with tissues of the body. They undergo conformational changes depending on the chemical composition and the nanotopography surface. Adsorbed proteins induce adhesion and alter the functional state of migrating cells. Plasma samples from patients were incubated with such matrices as titanium, polypropylene or polyester with fluoropolymer coating meshes. Bound peptides were analyzed by electrophoresis. Qualitative analysis of the peptides extracted from the gel was performed by chromatography-mass spectrometry. Quantitative analysis was performed by the MRM method. More than 60 proteins were identified on the analyzed surfaces. Quantitative analysis showed preferential adsorption of vitronectin, albumin, fibrinogen a-chain, C1ѕ component of the complement system. Vitronectin had the maximum relative protein content. Since biocompatibility of the analyzed materials varies considerably this variability may be attributed to conformational changes occurring with vitronectin during its irreversible adsorption.

Elevated C1rC1sC1inh levels independently predict atherosclerotic coronary heart disease.

UNLABELLED: Clinical studies as well as animal models emphasized the importance of the complement system in the pathogenesis of coronary atherosclerosis and cardiovascular diseases. Our aim was to examine the extent and clinical implication of complement system activation in patients with stable atherosclerotic coronary heart disease (ACHD). Seventy-six patients with stable angina pectoris (SAP) scheduled for elective coronary angiography were enrolled into the study. Percutaneous coronary intervention (PCI) was performed in 24 patients, in 27 patients (NOPCI group) the coronary angiography showed significant stenosis and bypass surgery (CABG) or optimal medical therapy (OMT) were advised, whereas in 25 patients the coronary angiography was negative (NC group). 115 volunteers served as healthy controls (HC). In all individuals, the plasma level of several complement activation products - C1rC1sC1inh, C3bBbP and SC5b-9 - were determined on admission, strictly before the coronary angiography. In patients with angiographically proven ACHD (PCI and NOPCI groups), the baseline C1rC1sC1inh levels were significantly higher compared to NC group and HC (p<0.0001, for both comparisons). According to the multiple logistic regression analysis, high C1rC1sC1inh level proved to be an independent biomarker of coronary heart disease (p<0.026, OR: 65.3, CI: 1.628-2616.284). CONCLUSION: Activation of the classical complement pathway can be observed in angiographically proven coronary atherosclerosis. Elevated C1rC1sC1inh levels might represent an useful biomarker for coronary artery disease.

HLA antibody specification using single-antigen beads--a technical solution for the prozone effect.

BACKGROUND: Substantial progress in human leukocyte antigen antibody specification has been made by the introduction of Luminex single-antigen bead (SAB) assays. This progress was impaired when it turned out that this method is prone to a prozone effect leading to false-negative results in the case of high antibody titers. Testing serum and ethylenediaminetetraacetic acid (EDTA) plasma of one patient in parallel, we observed the prozone effect with the serum sample only. This led us to investigate complement component 1 (C1) as the cause of the prozone in SAB testing. We also found an easy way to overcome the prozone effect. METHODS: Sera with a prozone effect were tested in the SAB assay, applying different methods of serum pretreatment to explore the parameters leading to the prozone. RESULTS: The prozone was not observed when EDTA plasma or serum with EDTA added were tested. Further, addition of dithiothreitol, addition of C1 inhibitor, or heat inactivation of the sera abolished the prozone effect. Adding fresh nonimmune serum to heat-inactivated sera restored the prozone effect. Only beads showing a prozone were found to be covered with C1q. CONCLUSION: Our observations are consistent with the hypothesis that dissociation or destruction of complement C1 eliminates the prozone effect. Addition of EDTA to serum of highly immunized patients is the easiest way to avoid false-negative results in SAB testing caused by a prozone effect.

Hereditary angioedema in a family presenting as transient periarthritis.

Hereditary angioedema (HAE) is a rare condition known to cause episodic, self-limiting, nonpruritic, nonpitting edema that involves skin and visceral organs. It may affect any external body surface including face, extremities, and genitalia. Most commonly involved viscera are gastrointestinal and respiratory systems. Patients may have severe abdominal pain because of edema of the bowel wall. This disease can cause life threatening laryngeal edema if it involves the airway.We describe a patient with HAE who was initially diagnosed with arthritis after she had recurrent edema around her peripheral joints. Diagnosis of HAE in her led to the same diagnosis in her sister and her father. HAE should be considered in the differential diagnosis of recurrent attacks of periarticular swelling.

Complement and c4 null alleles in severe chronic adult periodontitis.

Severe forms of chronic periodontitis affect up to 10% of adults. Tumour necrosis factor and lymphotoxin-alpha genes in the major histocompatibility complex are associated with severe periodontitis. Complement factor C4 is a nearby, polymorphic, functionally relevant gene region. Although associated with chronic mucosal infections, C4 deficiencies have not been assessed in adult periodontitis patients. We tested whether complement levels are systemically altered and C4 deficiencies associated with severe chronic periodontitis. In a case-control study, we analysed levels of plasma C3, and C4, serum classical pathway haemolytic activity, C4 allotypes and C4 gene numbers in 37 patients with severe chronic periodontitis and in 150 voluntary controls. Plasma levels of C3 were higher, and classical pathway haemolytic activity was lower in patients than in controls. Partial C4 gene deficiencies were more frequent in patients than in controls (odds ratio 2.4, 95% confidence interval 1.1-5.5, P = 0.032). Changes in complement levels may reflect chronic, recurring inflammation. C4 gene deficiencies are associated with predisposition to chronic periodontitis.

Predictive value of serum anti-C1q antibody levels in experimental autoimmune myasthenia gravis.

Components of the complement cascade and circulating immune complexes play important roles in both experimental autoimmune myasthenia gravis and myasthenia gravis in humans. Thus far, no serological factor has been identified to predict the clinical severity of either myasthenia gravis. Upon immunization with acetylcholine receptor, levels of complement factors C1q, C3 and CIC increase with time in sera from C57BL/6 (B6) mice. Both these and plasma samples from myasthenia gravis patients also contain anti-C1q antibodies. The serum levels of anti-C1q antibodies but not C1q, C3 and CIC are significantly correlated with the clinical severity in the experimental myasthenia mice. However, this correlation is not observed in myasthenia gravis patients.

Hereditary and acquired angioedema: experience with patients in Puerto Rico.

Hereditary (HAE) and acquired (AAE) angioedema are vascular reactions involving the sub mucosal tissues, representing localized edema caused by dilatation and increased permeability of the capillaries. HAE and AAE are clinical disorders characterized by angioedema that require prompt differentiation from other causes of angioedema in order to receive the most pertinent and effective therapeutic interventions. The aim of this report is to describe the clinical characteristics of patients with both HAE and AAE identified and followed at the Immunology Clinic of the University Hospital at the Puerto Rico Medical Center, their response and side effects to danazol therapy and their comparison with other series of similar patients reported in the literature. Overall, the patients in this sample presented a similar clinical profile compared to other reported series in the literature.

Immunoglobulin gamma3-heavy-chain deposition disease: report of a case and relationship with hypocomplementemia.

The authors describe a 54-year-old woman presenting with proteinuria, hematuria, and hypocomplementemia whose renal biopsy results showed diffuse increase in mesangial matrix and nodular formations in several glomeruli with the deposition of immunoglobulin gamma3-heavy-chain and complement components C1q and C3 in the glomeruli and on the tubular basement membranes, without associated light-chain deposits. Staining for the constant domains of gamma-heavy-chain showed a deletion of the first constant domain (CH1). These findings were consistent with those of gamma-heavy-chain deposition disease (gamma-HCDD). The patient was treated monthly with melphalan and prednisolone although a bone marrow aspirate did not show findings suggestive of plasmacytoma. Six courses of melphalan and prednisolone therapy resulted in a marked reduction of urinary protein excretion and marked rise of complement levels. The current case is the fourth HCDD patient reported featuring gamma3-heavy-chain deposition who showed severe hypocomplementemia and responded to chemotherapy with improved renal parameters and complement levels. A review of previously reported cases of HCDD showed that some but not all HCDD cases were associated with hypocomplementemia. The authors also discuss here the relationship of HCDD to hypocomplementemia.

Effect of C1-esterase-inhibitor on capillary leak and inflammatory response syndrome during arterial switch operations in neonates.

OBJECTIVE: To determine if prophylactic administration of C1-esterase-inhibitor would have a beneficial effect on postoperative weight gain and the inflammatory response in neonates undergoing cardiac surgery with cardiopulmonary bypass (CPB). DESIGN: Randomized, double-blinded study. SETTING: University-affiliated heart center. PARTICIPANTS: Twenty-four neonates with transposition of the great arteries. INTERVENTIONS: In group inhibitor (INH) patients (n = 12), 100 IU/kg of C1-esterase-inhibitor (Berinert) was given 30 minutes before CPB. In group placebo (P) patients (n = 12), placebo was administered instead. Interleukin (IL)-6, C3a anaphylatoxin, C1 activity, prekallikrein, Hageman factor, D-dimers, and clinical parameters were measured 6 times perioperatively. MEASUREMENTS AND MAIN RESULTS: All 24 patients had an uneventful clinical course. Mean arterial pressure and pulmonary oxygenation after CPB were superior in group INH patients. The weight gain on postoperative days 1 to 4 was significantly less in group INH patients compared with group P (55 +/- 59 g vs. 340 +/- 121 g, day 1). The concentration of IL-6 (76 +/- 17 pg/mL vs. 262 +/- 95 pg/mL during CPB) was significantly lower in group INH patients compared with group P patients. In contrast, no influence on C3a anaphylatoxin and coagulation factors was found. CONCLUSION: Prophylactic application of C1-esterase-inhibitor in neonates undergoing arterial switch operations produces less inflammatory response compared with placebo. This difference may have contributed to improved clinical parameters, including less weight gain postoperatively.

Complement C1s activation in degenerating articular cartilage of rheumatoid arthritis patients: immunohistochemical studies with an active form specific antibody.

OBJECTIVE: The first complement component C1s was reported to have novel functions to degrade matrix components, besides its activities in the classic complement pathway. This study explores participation of C1s in articular cartilage degradation in rheumatoid arthritis (RA). METHODS: Normal articular cartilage (n = 6) and cartilage obtained from joints with RA (n = 15) and osteoarthritis (OA, n = 10) were immunostained using anti-C1s monoclonal antibodies PG11, which recognises both active and inactive C1s, and M241, which is specifically reactive to activated C1s. The effects of inflammatory cytokines on C1s production by human articular chondrocytes were also examined by sandwich ELISA. RESULTS: In normal articular cartilage, C1s was negative in staining with both PG11 and M241. In contrast, degenerating cartilage of RA was stained with PG11 (14 of 15 cases), and in most of the cases (13 of 15 cases) C1s was activated as revealed by M241 staining. In OA, C1s staining was restricted in severely degrading part of cartilage (5 of 10 cases), and even in that part C1s was not activated. In addition, C1s production by chondrocytes in vitro was increased by an inflammatory cytokine, tumour necrosis factor alpha. CONCLUSION: These results suggest that C1s activated in degenerative cartilage matrix of RA but not in that of OA. C1s is thought to participate in the pathogenesis of RA through its collagenolytic activity in addition to the role in the classic cascade.

Complement activation and CD59 expression in the motor facial nucleus following intracranial transection of the facial nerve in the adult rat.

Intracranial transection of the facial nerve has been shown to cause a massive neuronal cell death in the motor facial nucleus. Complement activation has been proposed to contribute to neuronal degeneration following axotomy. Using immunocytochemistry and in situ hybridization we show in the present study that there is complement activation in the facial nucleus after intracranial facial nerve transection as well as increase of the complement regulators CD59 and clusterin. We propose a neuroprotective role for the complement regulators CD59 and clusterin against homologous attack of complement to facial motor neurons.

Complement components and their activation products in pleural fluid.

STUDY OBJECTIVES: The aim of this study was to determine the role of complement components in pleural effusion measured with novel markers of complement activation, to assess which pathway of activation is predominant in different diseases, and to find out whether the analysis of complement components and their activation products could help in diagnostic procedure differentiating the etiologies of pleural effusion. PATIENTS: The study population consisted of 71 patients who had pleural effusion secondary to tuberculosis (n=23), rheumatic disease (n=10), or malignancy (n=38). MEASUREMENTS: Complement components and their activation products, including the soluble terminal complex SC5b-9, were measured in plasma and pleural fluid. RESULTS: In all patients with rheumatic pleurisy, pleural fluid SC5b-9 was higher than 2 AU/mL and in all patients with malignant pleural fluid it was lower than 2 AU/mL. The mean level of SC5b-9 in rheumatic pleural effusion was also significantly higher than in tuberculosis. In addition, the concentrations of pleural fluid C3 and C4 were significantly lower and the ratio C4d/C4 was significantly higher in rheumatic compared with tuberculous or malignant pleurisy. In plasma, both SC5b-9 and C1s-C1r-C1INH-complexes were significantly higher in rheumatic subjects than in other patients. In stepwise multinominal logistic regression analyses, the most significant predictors for rheumatic pleural fluid were high pleural fluid SC5b-9 and low C4. CONCLUSIONS: These observations indicate that the complement cascade is activated through both the classic and the alternative pathways in rheumatic pleurisy. Determinations of SC5b-9 and C4d/C4 in pleural fluid were the best variables differentiating rheumatic, tuberculous, and malignant effusions.

The human complement C1 complex has a picomolar dissociation constant at room temperature.

Periodic sampling of serum or reconstituted C1 initially diluted 1/2000 and 1/4000 (that is, to 0.1 and 0.05 nM) into a recombinant C1s-containing solution showed a gradual decline of hemolytic activity until equilibrium was approached, consistent with a simple dissociation, reassociation equilibrium, presumably C1 <--> C1q + C1r2C1s2. The presence of excess (5 nM) recombinant C1s minimized further dissociation of the C1r2C1s2, allowing the first step to be studied independently of the dissociation of C1r2C1s2 <--> C1r2 + 2 C1s. Reassociation experiments were also performed, starting with the dissociated C1 diluted to the same concentrations and following the regain of hemolytic activity to approximately the same values, showing that the same equilibrium had been achieved from both directions. Analysis of the kinetic data yielded forward and reverse rate constants and the equilibrium constant, for which values of approximately 72 and 3 pM were estimated at 0 and 23 degrees C, respectively. The effects of temperature, ionic strength, Ca2+ ion concentration, and activation of the zymogen on the equilibrium constants were explored; extreme sensitivity to temperature, ionic strength, and activation were found. At 23 and 30 degrees C, slow activation of C1 was also evident. Highly purified, reconstituted C1 yielded approximately the same values for the kinetic and equilibrium parameters as serum C1, suggesting that the structure of the reconstituted complex was similar to or identical with that of the serum C1 complex.

[Rapid method of measuring the alternative activity of the complement system]. / Ekspress-metod opredeleniia al'ternativnoi aktivnosti sistemy komplementa.

The method is based on the ability of inulin to activate the alternative route of the complement system. The alternative activity is assessed from the consumption of the third complement component as a result of interaction with inulin. C3 is measured in experimental and control samples by kinetic titration, which permits assessing the activation of the alternative route in patients sera in less than 2 hours.

[A method of preparing a reagent for assessing the activity of the second component and factor B of the complement system]. / Sposob prigotovleniia reagenta dlia opredeleniia aktivnosti vtorogo komponenta i faktora B sistemy komplementa.

The method is intended for preparing a reagent for the detection of the second component and factor B of the complement system. It makes use of an unsophisticated device for even time-controlled heating of the serum by letting it pass through a coil pipe submerged in the bath. The resultant reagent is used to measure the activity of the second component and B factor of the complement system and may be used for assessing humoral immunity in patients.

Evidence for activation of the terminal pathway of complement and upregulation of sulfated glycoprotein (SGP)-2 in the hypoglossal nucleus following peripheral nerve injury.

In a previous study, we found immunoreactivity for complement factors C3, C3d, and C4d, as well as endogenous IgG in the hypoglossal nucleus following hypoglossal nerve transection, suggesting that activation of the complement cascade had taken place in the vicinity of the axotomized motorneurons. In the present study, we found increased immunoreactivity for complement factor C1 and C1q in reactive microglia, indicating an increased potential for initiation of the classical pathway by binding of IgG to C1q. Furthermore, we found immunoreactivity for C9, which contributes to the formation of C5b-9, the final lytic product of the complement cascade close to the axotomized neurons and perineuronal glia. In addition, immunoreactivity and mRNA labeling of sulfated glycoprotein (SGP-2), a putative complement inhibitor, was increased in a subpopulation of the axotomized motorneurons. SGP-2 immunoreactivity was also increased in astroglial cells ipsilateral to the nerve injury. The results lend further support to the hypothesis that the complement cascade is activated in the vicinity of axotomized neurons, which in turn may be protected by complement inhibitors. The balance between activation of complement and complement inhibitors might have an impact on the degenerative components of the axon reaction and, in particular, the events leading to nerve cell death.

Complement activation in amyloid plaques in Alzheimer's disease brains does not proceed further than C3.

In Alzheimer's disease (AD) patients, the complement components Clq, C4 and C3 can be detected in different types of beta/A4 plaques, one of the hallmarks of AD. Contradictory findings on the presence of late complement components in AD brains have been reported. Nevertheless, it was suggested in recent studies that in AD brain complement activation results in complement membrane attack complex (MAC) formation and that complement activation may act as an intermediate between beta/A4 deposits and the neurotoxicity observed in AD. In the present study the presence of a number of complement components and regulatory proteins in AD temporal cortex and, for comparison, in glomerulonephritis (GN) was analysed. In GN kidneys, besides Clq, Clr, Cls and C3, the late components and the C5b-9 complex are also associated with capillary basement membrane and mesangial immune complex deposits. In AD temporal cortex Clq, C4 and C3 are co-localized with beta/A4 deposits. However, in contrast to the GN kidney, the late complement components C5, C7 and C9, as well as the C5b-9 membrane attack complex cannot be detected in beta/A4 positive plaques. The absence of the cytolytic C5b-9 complex in AD brain suggests that in AD, the complement MAC does not function as the proposed inflammatory mediator between beta/A4 deposits and the neurofibrillary changes.

Charge-remote fragmentation of peptides derivatized with 4-aminonaphthalenesulphonic acid.

A series of small peptides has been studied by negative-ion fast-atom bombardment mass spectrometry with collision-induced dissociation. It has been found that by derivatizing peptides with 4-aminonaphthalenesulphonic acid in a peptide linkage at the C-terminus, negative-ion formation can be enhanced and fragmentation in collision-induced dissociation reactions controlled. The peptide-naphthalenesulphonates show charge-remote fragmentations and the resultant spectra give sequence information.

Time course of complement activation and inhibitor expression after ischemic injury of rat myocardium.

Activation of the complement (C) system has been documented in both experimental and clinical studies of myocardial infarction, but the exact time course and mechanisms leading to C activation have remained unclear. Our earlier postmortem study on human beings showed that formation of the membrane attack complex (MAC) of C was associated with loss of CD59 (protectin), an important sarcolemmal regulator of MAC, from the infarcted area. The recent discovery of a rat analogue of CD59 has now allowed the first experimental evaluation of the temporal and spatial relationship between C component deposition and loss of CD59 in acute myocardial infarction (AMI). After ligating the left coronary artery in rats the earliest sign of C activation, focal deposition of C3, was observed at 2 hours. Deposition of the early (C1, C3) and late pathway (C8, C9) components in the AMI lesions occurred at 3 hours. Glycophosphoinositol-anchored rat CD59 was expressed in the sarcolemmal membranes of normal cardiomyocytes. In Western blot analysis extracts of normal rat heart CD59 appeared as a band of 21 kd of molecular weight under nonreducing conditions. Loss of CD59 in the AMI lesions was observed in association with deposits of MAC from day one onward. Our results show that C activation universally accompanies AMI in vivo. It is initiated within 2 hours after coronary artery obstruction via deposition of C3, which may be due to generation of the alternative pathway C3 convertase in the ischemic area. Deposition of C1 and late C components also starts during the early hours (2 to 4 hours) after ischemia. Subsequent loss of the protective CD59 antigen may initiate postinjury clearance of the irreversibly damaged tissue.

C1 inhibitor functional deficiency in systemic lupus erythematosus (SLE).

C1 inhibitor (C1-inh) was assayed in eight SLE patients presenting with consistently low levels of intact C4. C1-inh antigenic levels were normal in all patients; however, the function of the C1-inh tested against C1s and C1r was variable and outside the normal functional range in seven of the eight patients. The molecular weight of patients' C1-inh protein was 105 kD, corresponding to the size of the intact molecule. The C1-inh gene was analysed in all patients. Restriction fragments generated with TaqI, PstI and HgiAI gave no indication of a major C1-inh gene rearrangement. Direct genomic sequencing of exon VIII revealed three polymorphic point mutations, but there were no changes from the normal gene in or around the reactive-centre residue of C1-inh. Furthermore, we found no evidence for a C1-inh autoantibody in patients which could affect normal C1-inh function in vitro. These results indicate that the etiology of C1-inh dysfunction in SLE is heterogeneous and distinct from that reported in either hereditary or acquired angioedema.