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PubMed2000

Purification of human ceruloplasmin as a by-product of C1-inhibitor.

Human ceruloplasmin (Cp) has been purified from cryoprecipitate-poor plasma as a by-product of the C1-inhibitor production chain. Highly purified Cp was obtained by subsequent ion-exchange chromatography on sulfate-Fractogel EMD and TMAE-Fractogel EMD. Treatments for viral safety included application of the solvent-detergent method and two nanofiltration steps using 35- and 15-nm pore size filters at the end of the process. Overall antigen yield was 95 (+/-5) %. Purified human ceruloplasmin was studied by electron spin resonance (ESR) to characterize its different types of copper complexes and to check its antioxidant properties. We distinguished three types of complexes: one type-2 Cu(II) with g// = 2.25 and A// = 180 G and two type-I Cu(II) exhibiting different narrow hyperfine splitting (A// = 72 G and A// = 90 G) with close g// (2.20 and 2.21). Purified Cp has a specific activity of 24.5+/-0.2 mU/mg of proteins. This process provides a method for Cp purification that could be easily integrated into modern plasma fractionation.

Antioxidants↗
PubMed2006

Functional analysis of splicing mutations and of an exon 2 polymorphic variant of SERPING1/C1NH.

Several sequence changes have been reported in hereditary angioedema patients in intron 2 of the SERPING1/C1NH gene, but their consequences on splicing have not been determined. We examined in cell transfection assays the consequences at the mRNA level of splicing mutations affecting either the +3 or the +5 donor site positions, or the conserved canonical splicing signals of exon 2, using mutant C1 inhibitor minigene constructs. Both intron 2 mutations, c.51+3A>G and c.51+5G>A, resulted in marked exon 2 skipping in these assays, but also yielded a large fraction of normal transcripts. We show that the c.51+3A>G mutation cosegregates with low C1 inhibitor protein levels in one family. Moreover, the second base of exon 2 of the SERPING1/C1NH gene is the site of a polymorphic variant, which has been proposed as a modifier of disease severity. We found that the c.-21C allele at this position yields low but significant levels of exon 2 skipping in transfected Hep G2 or Hep 3B cells, suggesting that this allele may contribute, at the RNA level, to more severe forms of angioedema. Furthermore, we describe a previously not detected alternative splicing of exon 3, found in peripheral blood cell mRNA but not in the liver or in hepatoma cell lines and we show that, in cultured monocytes of a patient carrying the c.51+3A>G mutation, this alternative splicing is shifted from exon 3 exclusion to skipping of both exons 2 and 3. The latter finding suggests that mutations affecting splicing of exon 2 of the SERPING1/C1NH gene may have different consequences in monocytes versus other cell types.

Adolescent↗
PubMed2002

Detection of C1 inhibitor (SERPING1/C1NH) mutations in exon 8 in patients with hereditary angioedema: evidence for 10 novel mutations.

Hereditary angioedema (HAE) is caused by mutations in the C1 inhibitor gene (SERPING1, C1NH) and the result is C1 inhibitor deficiency, either in levels or function. We have searched exon 8 for mutations by direct sequencing and analyzed the rest of the exons by SSCP in 87 Spanish families affected by HAE. Out of 87 screened families, we have detected exon 8 mutations in 26. Among these, 17 different mutations were identified: 14 point mutations and 3 frameshift. Seven of the point mutations and the three frameshift were not previously reported. Mutations were: S438P; R444P; V451G; W460X; V468D; G471E; X479R; S417fsX427; I440fsX450; E429fsX450. The rest of the families presented previously reported mutations, 5 missense and two nonsense. In none of the 26 families was an additional change identified in the rest of the exons by SSCP, and, in 20 out of the 22 families with point mutation, we verified that the mutation did not affect a healthy relative. Seven of these families had no history of the disease, and in five of them we were able to verify that the progenitors did not have the mutation. Therefore, they were de novo mutations.

Angioedema↗
PubMed2003

Laryngeal edema and death from asphyxiation after tooth extraction in four patients with hereditary angioedema.

BACKGROUND: Recurrent angioedema is the hallmark of various inherited or acquired angioedema diseases. Hereditary angioedema, or HAE, due to C1 inhibitor, or C1NH, deficiency has considerable implications for dental health care providers because dental surgery may trigger distressing and even life-threatening episodes. CASE DESCRIPTION: The authors reviewed the literature, focusing on the pathogenesis, clinical signs and treatment of HAE. They also provided case reports of four patients who died from laryngeal edema induced by tooth extraction. In patients with HAE, dental surgery--including tooth extraction--may be followed by self-limiting edema episodes, including lip swelling, facial swelling, tongue edema and laryngeal edema with upper airway obstruction. Preoperative prophylaxis has been performed with attenuated androgens, fresh frozen plasma, C1NH concentrate and antifibrinolytics. The four patients described underwent tooth extraction, which, after a symptom-free latency of four to 30 hours, provoked laryngeal edema. Three of the patients died of asphyxiation the night after surgery, and the fourth died on the second night. In three of the patients, laryngeal edema had not occurred previously. CLINICAL IMPLICATIONS: Before undergoing dental surgery, patients with a history of recurrent angioedema should be evaluated for C1NH deficiency. If it is present, they are at risk of developing life-threatening laryngeal edema.

Adult↗
PubMed2006

Hereditary angioedema: new findings concerning symptoms, affected organs, and course.

PURPOSE: Hereditary angioedema (HAE) due to C1 inhibitor deficiency is clinically characterized by relapsing skin swellings, abdominal pain attacks, and life-threatening upper airway obstruction. Our aim was to examine a temporal and spatial pattern of the edema episodes by evaluating the long-term course of hereditary angioedema in order to establish a specific swelling pattern. SUBJECTS AND METHODS: Data were generated from 221 patients with C1 inhibitor deficiency by asking them about symptoms they experienced during their edema episodes. Documentation was accomplished through the use of standardized questionnaires. RESULTS: A total of 131110 edema episodes were observed. Clinical symptoms started at a mean age of 11.2 (SD 7.7) years. During the following cumulative 5736 years, only 370 (6.5%) symptom-free years occurred. Skin swellings, including extremity, facial, genital, and trunk swellings, and abdominal attacks occurred in 97.4% of all edema episodes of the disease. The other episodes were laryngeal edema (0.9%); edema of the soft palate (0.6%); tongue swellings (0.3%); headache episodes (0.7%); episodes affecting urinary bladder (0.3%), chest (0.2%), muscles (0.4%), joints (0.1%), kidneys (0.1%), and esophagus (0.05%), and were partly combined with other edema episodes. The per-patient analysis and the per-episode analysis revealed markedly discrepant results. On average, women had a more severe course of the disease than men. Patients with early onset of clinical symptoms were affected more severely than those with late onset. CONCLUSION: The described swelling pattern is specific for HAE and allows a tentative diagnosis based on clinical symptoms and the course of the disease.

Adolescent↗
PubMed2001

Rapid detection by fluorescent multiplex PCR of exon deletions and duplications in the C1 inhibitor gene of hereditary angioedema patients.

Hereditary angioedema (HAE) is due to a variety of defects in the C1 inhibitor gene (C1NH gene), including approximately 20% of partial deletions/duplications whose boundaries are usually within Alu repeats. To ensure complete molecular characterization of C1 inhibitor deficiencies a fluorescent multiplex assay was constructed to amplify simultaneously five exons of C1NH and an exon of the BRCA1 gene. PCR protocols were optimized for these amplicons (size range between 300 and 700 bp). Forward and reverse chimeric primers that carry strand-specific 5' tags of 16 nucleotides were used to ensure similar levels of PCR products for each amplicon in the multiplex. Data were analyzed by superposing fluorescent profiles of test and control DNA and by visually comparing the normalized peak levels of corresponding amplicons, rather than by calculating the ratios of peak areas. Tests on a collection of known defects, including five different Alu-mediated deletions and a partial duplication have validated this approach. In a study of 19 sporadic cases of HAE, of which four had failed to reveal mutations upon screening all exons by fluorescent chemical cleavage, three de novo deletions were diagnosed by using this multiplex PCR approach: a deletion of exon 4, a deletion of exons 5 and 6, and an apparently complete gene deletion. Besides being suitable for the initial DNA screening of the C1NH gene in HAE patients prior to screening for point mutations, this method can be easily adapted to complex genes for the screening of rearrangements.

Angioedema↗
PubMed2003

Mutation screening of the C1 inhibitor gene among Hungarian patients with hereditary angioedema.

Hereditary angioneurotic edema (HAE) is an autosomal dominant disorder characterized by episodic local subcutaneous and submucosal edema caused by the deficiency of activated C1 esterase inhibitor protein (C1-INH, type I (C1NH): reduced serum antigen level, type II: reduced activity and normal serum antigen level). The aim of the present study was to determine the disease-causing mutations in the C1INH gene (SERPING1) among Hungarian HAE-patients. The estimated number of affected HAE-families in Hungary is 40-50, out of which 26 families (type I:23, type II:3) managed in a single center were enrolled in the current study. To detect large deletions/insertions, we used Southern-blotting analysis followed by real time PCR based gene dosage analysis. In the absence of large structural changes, we employed direct sequencing covering the whole coding region and splicing sites of the C1INH gene. Large deletions were detected in 4/23 (17.4%) type I families. We found the g.16788C>T (p.Arg444Cys) mutation in each 3, type II HAE-families. In the remaining type I families, 13 previously unreported mutations (g.638G>A, g.2238C>T, g.2534_2535delCT, g.2579_2620del42, g.2533G>A, g.2695G>A, g.2696_2697insT, g.4467C>T, g.14224A>T, g.14107delA, g.16749_;16775dup, g.16810T>A, g.16885C>G) were detected in 16 families affecting primarily exon 3 (6/13) of the C1INH gene. In the 3 remaining families, known mutations were identified affecting primarily exon 8 (2/3).

Angioedema↗
PubMed2005

Hereditary angioedema: the mutation spectrum of SERPING1/C1NH in a large Spanish cohort.

Hereditary angioedema (HAE) is a disease caused by defects in the C1 inhibitor gene (SERPING1/C1NH). We screened the entire C1NH gene for mutations in a large series of 87 Spanish families (77 with type I, and 10 with type II HAE) by SSCP, sequencing, Southern blotting, and quantitative multiplex PCR of short fluorescent fragments (QMPSF), and we characterized several defects at the mRNA level. We found large rearrangements in 13 families, and point mutations or microdeletions/insertions in 74 families. The 13 large rearrangements included nine exon deletions, of which at least eight were distinct, two were distinct exon duplications, and two were rearrangements whose precise nature could not be determined. We confirmed that exon 4 is particularly prone to rearrangements. Thirty-six mutations were unreported, and included 10 microdeletions/insertions, 10 missense, five nonsense, eight splicing, and three splicing or missense mutations. Moreover, we detected six novel uncharacterized sequence variants (USV). RT-PCR studies showed that in addition to several intronic splice site mutations tested, the exonic mutations c.882C>G and c.884T>G, located near the 3' end of exon 5, also produced exon skipping. This is the first evidence of SERPING1/C1NH mutations in coding regions that differ from the canonical splice sites that affect splicing, which suggests the presence of an exonic splicing enhancer (ESE) in exon 5.

Alternative Splicing↗
PubMed1998

C1 inhibitor gene sequence facilitates frameshift mutations.

Mutations disrupting the function or production of C1 inhibitor cause the disease hereditary angioneurotic edema. Patient mutations identified an imperfect inverted repeat sequence that was postulated to play a mechanistic role in the mutations. To test this hypothesis, the inverted repeat was cloned into the chloramphenicol acetyltransferase gene in pBR325 and its mutation rate was studied in four bacterial strains. These strains were selected to assay the effects of recombination and superhelical tension on mutation frequency. Mutations that revert bacteria to chloramphenicol resistance (Cmr) were scored. Both pairs of isogenic strains had reversion frequencies of approximately 10(-8). These rare reversion events in bacteria were most often a frameshift that involved the imperfect inverted repeat with a deletion or a tandem duplication, an event very similar to the human mutations. Increased DNA superhelical tension, which would be expected to enhance cruciform extrusion, did not accentuate mutagenesis. This finding suggests that the imperfect inverted repeat may form a stem-loop structure in the single-stranded DNA created by the duplex DNA melting prior to replication. Models explaining the slippage can be drawn using the lagging strand of the replication fork. In this model, the formation of a stem-loop structure is responsible for bringing the end of the deletion or duplication into close proximity.

Angioedema↗
PubMed1982

Dissociation of C1 and concentration dependence of its activation kinetics.

The activation of the zymogen C1s to the enzyme C1s in the human C1 complex [C1q(c1rC1s)2] was studied as a function of the concentrations of (C1rC1s)2 and C1q which were saturated with oligomers of rabbit IgG. A large concentration dependence of the sigmoidal kinetics was observed in the 2-180 nM concentration range. This was explained by association-dissociation equilibria between the antibody-saturated C1q and various forms of the (C1rC1s)2 complex (unactivated to activated). The establishment of these equilibria (binding constant 2 x 10(7) M-1) was assumed to be fast as compared to the rates of the activation steps (rate constants 10(-3) and 10(-2) sec-1 at 30 degrees C). The fast re-equilibration of the C1 complex explains the finding that small amounts of antibody-saturated C1q catalysed the activation of large amounts of C1s. The interpretation of the kinetic results was supported by a direct demonstration of the dissociation of C1 into C1q and (C1rC1s)2 by analytical and density gradient centrifugation. No difference was found between the rates of activation and the dissociation properties of reconstituted C1 and C1 isolated from serum.

Centrifugation, Density Gradient↗
PubMed2006

Symptoms, course, and complications of abdominal attacks in hereditary angioedema due to C1 inhibitor deficiency.

OBJECTIVES: Recurrent abdominal attacks belong to the cardinal and most distressing symptoms of hereditary angioedema (HAE) due to C1 inhibitor deficiency. They are characterized by crampy pain, but may include vomiting, diarrhea, and other features. Detailed clinical data about the symptoms and course of abdominal attacks have not been reported. METHODS: We retrospectively observed a total of 33,671 abdominal attacks in 153 patients with HAE including a prospectively examined subgroup of 23 patients. Symptoms, course, frequency of attacks, and complications were analyzed. RESULTS: The relation of mild, moderate, and severe attacks was 1:1.4:5.6 in the prospective part of the study. Extra-abdominal symptoms preceded the abdominal symptoms. The mean maximal pain score was 8.4 (range 1-10). Vomiting occurred in 73% (24,696) and diarrhea in 41% (13,682) of the attacks. Circulatory collapse accompanied 4.4% (1,468) of the attacks, with loss of consciousness (LOC) occurring in 2.2% (739). Nine patients could clearly distinguish two types of abdominal attacks: vomiting and diarrhea. Rare complications included tetany, hemorrhagic stools, and intussusception of the colon. In 28% (43) of the patients, recurrent abdominal attacks had started before the characteristic swelling of the skin had ever occurred. A model is proposed to classify the severity of the attacks and to describe the clinical course. CONCLUSIONS: Abdominal attacks in HAE constitute a more disabling and complex syndrome than previously assumed. Our results add to the understanding of symptoms and course of HAE and may aid in the early recognition of an impending attack and improve clinical management.

Abdominal Pain↗
PubMed2003

Approaches toward reversal of increased vascular permeability in C1 inhibitor deficient mice.

C1 inhibitor (C1INH) deficient mice have increased vascular permeability that can be demonstrated by the extravasation of Evans Blue dye. This vascular leak is reversed with protease inhibitors, such as C1INH itself, DX88 (a recombinant variant Kunitz domain plasma kallikrein inhibitor), and the bradykinin receptor type 2 antagonist, Hoe140. The studies described here were undertaken for the following reasons: (1) To provide a more quantitative analysis of the effects of these interventions; (2) to provide data to further test the hypothesis that increased vascular permeability results from contact system activation with kallikrein-mediated release of bradykinin; (3) to test the hypothesis that the amino terminal non-serpin domain of C1INH modulates access to complex proteases, such as kallikrein complexed with high molecular weight kininogen (HK); and (4) to determine whether attenuated androgens or estrogens exert a direct effect on C1INH synthesis. To characterize the differences in these reagents, the dose-response and the rate of reappearance of increased vascular permeability in C1INH(-/-) mice were determined for the following agents: human plasma-derived C1INH, a recombinant Kunitz domain plasma kallikrein inhibitor (DX88), a bradykinin receptor antagonist (Hoe140), and a recombinant C1INH with an amino terminal truncation at amino acid 98 and substitution of the P2 Ala with a Val (Cserp98,A443V). C1INH and Cserp98,A443V were equivalent in activity, which provides further support for the hypothesis that the vascular leak is mediated by bradykinin and suggests that the amino terminal domain neither enhances nor interferes with access to kallikrein within the kallikrein-HK complex. DX88 was effective at very low doses, as was Hoe140. The duration of action of Hoe140 was quite prolonged. The data indicate that, in the mouse, neither danazol nor estrogens have a significant effect on C1INH synthesis.

Animals↗
PubMed1998

Characterization of a heparin binding site on the heavy chain of factor XI.

The glycosaminoglycan heparin enhances several reactions involving coagulation factor XI (FXI) including activation of FXI by factor XIIa, thrombin, and autoactivation; and inactivation of activated FXI (FXIa) by serine protease inhibitors. We examined the effect of heparin on inhibition of FXIa by the inhibitors C1-inhibitor (C1-INH) and antithrombin III (ATIII). Second order rate constants for inhibition in the absence of heparin were 1.57 x 10(3) and 0.91 x 10(3) M-1 s-1 for C1-INH and ATIII, respectively. Therapeutic heparin concentrations (0.1-1.0 units/ml) enhanced inhibition by ATIII 20-55-fold compared with 0.1-7.0-fold for C1-INH. For both inhibitors, the effect of heparin over a wide range of concentrations (10(-1) to 10(5) units/ml) produced bell-shaped curves, demonstrating that inhibition occurs by a template mechanism requiring both inhibitor and protease to bind to heparin. This implies that FXI/XIa contains structural elements that interact with heparin. Human FXI contains a sequence of amino acids (R250-I-K-K-S-K) in the apple 3 domain of the heavy chain that binds heparin (Ho, D., Badellino, K., Baglia, F., and Walsh, P. (1998) J. Biol. Chem. 273, 16382-16390). To determine the importance of this sequence to heparin-mediated reactions, recombinant FXI molecules with alanine substitutions for basic amino acids were expressed in 293 fibroblasts, and tested in heparin-dependent assays. Inhibition of FXIa by ATIII in the presence of heparin was decreased 4-fold by alanine substitution at Lys253 (A253), with smaller effects noted for mutants A255 and A252. FXI undergoes autoactivation to FXIa in the presence of heparin. The rate of autoactivation was decreased substantially for A253 with modest decreases for A255 and A252. Substituting all four charged residues in the sequence resulted in a profound decrease in autoactivation, significantly greater than for any single substitution. Relative affinity for heparin was tested by determining the concentration of NaCl required to elute FXIa from heparin-Sepharose. Wild type FXIa eluted from the column at 320 mM NaCl, whereas FXIa with multiple substitutions (A252-254 or A250-255) eluted at 230 mM NaCl. All proteins with single substitutions in charged amino acids eluted at intermediate NaCl concentrations. The data indicate that FXI/XIa must bind to heparin for optimal inhibition by ATIII and for autoactivation. Lys253 is the most important amino acid involved in binding, and Lys255 and Lys252 also have roles in interactions with heparin.

Alanine↗
PubMed2006

Molecular and functional characterization of a ToxR-regulated lipoprotein from a clinical isolate of Aeromonas hydrophila.

Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.

Aeromonas hydrophila↗
PubMed1980

Immune complexes in Sjögren's syndrome.

Sera from 48 patients with Sjögren's syndrome were examined for immune complexes by the Raji cell assay. There was no correlation between levels of immune complexes and the degree of lymphocytic infiltration of the labial salivary glands. Serum complement levels were normal. Five patients were serially followed during the development of pseudolymphoma or malignant lymphoma. Immune complex levels in 4 of the 5 patients were generally unchanged throughout the illness and did not parallel disease activity, rheumatoid factor, or SS-A and SS-B concentrations. Possible roles for immune complexes in Sjögren's syndrome are discussed.

Antibody Formation↗
PubMed2001

Impact of inhibition of complement by sCR1 on hepatic microcirculation after warm ischemia.

Recent observations provide evidence that complement is implicated as an important factor in the pathophysiology of ischemia/reperfusion injury (IRI). Here, we assessed the effects of complement inhibition on hepatic microcirculation by in vivo microscopy (IVM) using a rat model of warm hepatic ischemia clamping the left pedicle for 70 min. Ten animals received the physiological complement regulator soluble complement receptor type 1 (sCR1) intravenously 1 min prior to reperfusion. Controls were given an equal amount of Ringer's solution (n = 10). Microvascular perfusion and leukocyte adhesion were studied 30 to 100 min after reperfusion by IVM. Microvascular perfusion in hepatic sinusoids was significantly improved in the sCR1 group (80.6 +/- 0.6% of all observed sinusoids were perfused [sCR1] vs 67.3 +/- 1.2% [controls]). The number of adherent leukocytes was reduced in sinusoids (49.9 +/- 3.4 [sCR1] vs 312.3 +/- 14.2 in controls [adherent leukocytes per square millimeter of liver surface]; P < 0.001) as well as in postsinusoidal venules after sCR1 treatment (230.9 +/- 21.7 [sCR1] vs 1906.5 +/- 93.5 [controls] [adherent leukocytes per square millimeter of endothelial surface]; P < 0.001). Reflecting reduced hepatocyte injury, liver transaminases were decreased significantly upon sCR1 treatment compared to controls. Our results provide further evidence that complement plays a decisive role in warm hepatic IRI. Therefore, we conclude that complement inhibition by sCR1 is effective as a therapeutical approach to reduce microcirculatory disorders after reperfusion following warm organ ischemia.

Animals↗