A systematic analysis of the complement pathways in patients with neuromyelitis optica indicates alteration but no activation during remission.

Neuromyelitis optica (NMO) is an autoimmune demyelinating inflammatory disorder, mediated by pathogenic autoantibodies against aquaporin 4 (AQP4), the main water channel of the central nervous system (CNS). NMO is characterized by local IgG deposition and complement activation within the CNS, but the three complement pathways have not been systematically investigated. We evaluated the overall activation of the classical, alternative, and MBL-lectin pathways in the peripheral blood of 25 patients with AQP4-seropositive NMO spectrum during remission and 113 healthy controls by three ways: (1) we measured the concentrations of native complement proteins of the three pathways [C1-inhibitor (C1-inh), C1q, C4, C3, C5, factor I, factor B, properdin]; (2) the concentrations of complement products suggesting in vivo activation (C1rC1sC1-inh, C3a, C3bBbP, and SC5b-9); and (3) the total activity of the three complement pathways. Additionally we measured levels of C1rC1sC1-inh, C3a, C3bBbP in cerebrospinal fluid (CSF) of 6 patients with relapsing NMO and of 18 patients with relapsing multiple sclerosis (MS). The serological studies indicated that total complement activity of the classical [median (interquartile range) 72 (61-82) vs. 65 (56-73) CH50/mL; p=0.0122] and of the lectin pathways [73 (59-111) vs. 49 (3-92)%; p=0.0078)] were elevated compared with the controls, whereas that of the alternative pathway was not significantly different. The levels of C3 [1.1 (0.9-1.3) vs. 1.4 (1.2-1.5)g/L; p<0.0001], factor B [89 (77-115) vs. 103 (93-113)%; p=0.0397] and factor I [85 (69-95) vs. 101 (93-107)%; p=0.0007], as well as of properdin [92 (74-104) vs. 108 (97-122)%; p=0.0028] were significantly lower in the patients than in the controls. The only increase in the patients was ascertained in the relative concentration of C1rC1sC1-inh vs. the C1-inhibitor (42.3 [31.9-65.0] vs. 30.8 [13.5-43.5] AU/mg; p=0.0007). The absolute and relative levels of the other complement activation products were not elevated in the patients. On the contrary, the serum concentrations of C3a, C3bBbP, and SC5b-9 of the patients were lower than those of the controls. The absolute concentration of the complement activation products (C1rC1sC1-inh, C3bBbP, C3a) and the ratio of C3bBbP/C1rC1sC1-inh did not differ in NMO and MS CSF samples. The ratio of C3bBbP/C1rC1sC1-inh was similar in NMO plasma and CSF samples. We found a higher ratio of C3bBbP/C1rC1sC1-inh in the plasma of control subjects compared to those in any pathological samples. Our results do not indicate substantial systemic complement activation if NMO activity is adequately controlled; nevertheless, the complement system is abnormally affected even during remission. The relative ancillarity of the alternative compared to the classical pathway may also suggest that suppression of the alternative pathway by treatment may be important to achieve remission.

The role of complement activation in the pathogenesis of Fuchs' dystrophy.

PURPOSE: Inflammation can be an etiologic factor of Fuchs' dystrophy according to previous studies. Our aim was to analyse the activation of the complement system in the aqueous humor in this pathological condition. METHODS: 100 µl aqueous humor sample was taken during keratoplasty of 11 Fuchs' dystrophic patients and during phacoemulsification surgery of 18 control patients. The samples were mixed with EDTA and stored at -80 °C. Concentrations of C1rC1sC1Inh and C3bBbP complexes as markers of the activation of the classical and alternative complement pathways, respectively, were measured with ELISA method. The results of the patient group and the control group were compared with statistical analysis (non-parametric Mann Whitney test). RESULTS: Both the concentrations of C1rC1sC1Inh [4.3 (3.2-20.2)AU/ml] and of C3bBbP [15.3 (7.8-22.6)AU/ml] were significantly higher in the Fuchs' dystrophic group than in the control group [C1rC1sC1Inh: 0.0 (0.0-5.6)AU/ml, C3bBbP: 1.4 (0.0-7.8)AU/ml]. The median value is shown along with the (25% and 75% percentiles). CONCLUSIONS: Based on our results, the complement system may be activated both through the classical and alternative pathways in the aqueous humor of the patients with Fuchs' dystrophy.

Complement activation in the aqueous humor of pseudophakic bullous keratopathy patients.

BACKGROUND/AIMS: According to some studies, inflammation is a potential etiological factor in pseudophakic bullous keratopathy (PBK). Our aim was to obtain information on the activation of the complement system in the aqueous humor in this disorder. METHODS: Aqueous humor samples were collected during keratoplasty of 12 PBK patients, as well as during phacoemulsification surgery of 18 control patients. The concentrations of the protein-protein complexes generated during complement activation (C1rC1sC1inh and C3bBbP) through the classical and alternative pathways, respectively, as well as of the C3 cleavage product C3a, were measured with ELISA methods. The correlation among the complement factors and between the duration of the edema, the stage of the disease, and the level of the complement activation products was examined. RESULTS: The concentration of C1rC1sC1inh, C3bBbP complex and C3a was significantly higher in the PBK group (p < 0.001) compared to the control group. In PBK patients, a correlation was found between the levels of the C1rC1sC1inh complex and C3a only. CONCLUSION: Our new findings indicate that in PBK the complement system is activated - via the classical pathway - in the aqueous humor. The activated complement may play a role in increased endothelial cell loss.

Interleukin-23 receptor gene variants in Hungarian systemic lupus erythematosus patients.

OBJECTIVE: We investigated the association between systemic lupus erythematosus (SLE) and polymorphisms of interleukin-23 receptor (IL23R) gene, which was recently found to be associated with autoimmune diseases, including Crohn's disease, rheumatoid arthritis, psoriasis and ankylosing spondylitis. SUBJECTS: We analysed 383 SLE patients and 253 controls for rs11805303, rs10889677, rs1004819, rs2201841, rs11209032, 11209026, rs10489629, rs7517847 and rs7530511 variants. METHODS: The analysis was carried out using PCR-RFLP methods. Logistic regression analysis was used to compare the genotype distributions of the polymorphisms and haplotypes between the SLE patients and healthy controls. RESULTS: We observed no significant difference of the examined variants between the patient and control groups. CONCLUSIONS: Our results suggest that neither single nucleotide variants nor haplotypes of IL23R indicate susceptibility to developing SLE in the Hungarian population.

[The Danubian Biobank Initiative: synchronizing the biobanking activities of the Danube universities]. / Danubian Biobank Konzorcium: a Duna menti egyetemek "biobanking" tevékenységének összehangolása.

Aging disorders pose an increasing challenge for the public health care systems in Europe. An important approach to cope with this task is the identification of relevant novel disease genes and the control of risk factors using new technological capabilities. A key element in this process is the availability of well classified, large enough patient cohorts and the establishment of quality-controlled central banks for DNA, serum, plasma, and cells/tissues/RNA/proteins together with the development of an IT based infrastructure to provide samples and data required for biomedical studies. The Danubian Biobank initiative connects universities, associated teaching hospitals and endpoint-related rehabilitation clinics along the Danube river and in neighbouring regions. The scientific network focuses on diabetes-related endpoints, vascular disease (e.g. myocardial infarction, stroke, arterial thrombosis, kidney failure), metabolic disease (e.g. obesity, diabetes, metabolic syndrome), and neurodegenerative disorders (e.g. dementia syndromes, Parkinsonism). Task forces are set up for the relevant topics of the biobank project including patient recruitment, sample and data management, public health, epidemiology and genetics, enabling technologies, and research strategies. The project aims to select the most relevant and promising scientific targets utilizing the core competences developed in the individual partner institutions. For this purpose a series of dedicated workshops and conferences are organized as well as joint research grant proposals are submitted.

[The autoimmune features of vasculopathies]. / Vasculopathiák autoimmun vonatkozásai.

In the development of atherosclerotic plaques we can identify three autoantigens, the pathological value of which has been proven by experimental and clinical data. These antigens are the 60 kDa heat-shock-protein, beta2-glycoprotein I and oxidized LDL. They have role in the antigen-specific T-cell differentiation processes, moreover, against these antigens autoantibodies are produced, which have prothrombotic activity, leading to the acceleration of atherosclerosis. In autoimmune diseases besides these factors other mechanisms are present, which lead to the development of autoimmune vasculopathies. The current review gives an overview of these vasculopathies.

Rapid quantification of human complement component C4A and C4B genes by capillary gel electrophoresis.

Complement component 4 (C4) is an important plasma protein playing a major role in the human defense mechanism against infectious diseases and inflammatory processes. The C4A and C4B genes, encoding the two isoforms of complement 4, are located in the nuclear serine/threonine protein kinase-C4A or B gene-cytochrome 21-hydroxylase-tenascin X module (RP-C4-CYP21-TNX) and manifested by variable copy numbers among individuals between zero to six in the human diploid genome. Quantification of the C4A and C4B genes has great clinical importance since unbalanced production of C4A and C4B proteins might be associated with pathological immune processes. Albeit, high-throughput analysis methods for C4 gene dosage determination are not yet available. Here we present a novel combination of allele-specific PCR and CGE separation for rapid quantification of the C4A and C4B genes where a single-step, single-tube PCR reaction generates two allele-specific (C4A and C4B) and two control amplicons, followed by CGE analysis of the four fragments. The method presented in this paper enables automated and high-throughput gene dosage analysis of large sample cohorts.

[C-reactive protein levels determined by an ultrasensitive method in a healthy Hungarian population]. / Az ultraszenzitív módszerrel meghatározott C-reaktív fehérjekoncentráció megoszlása az egészséges magyar populációban.

INTRODUCTION: C-reactive protein (CRP) is a well-known acute phase protein. The concentration of CRP in serum is increased in response to inflammatory stimuli. Increased levels serve to identify organic disease, to monitor disease activity and to assist differential diagnosis. AIM: The aim of the authors' cross-sectional study was to determine CRP distribution of the healthy Hungarian population. METHOD: 207 (79 male, 128 female; mean age: 4 +/- 68 years) healthy blood donors were enrolled for the study. The following parameters were registered: sex, age, body mass index, smoking habits, diabetes mellitus and blood pressure. Serum samples were assayed for total serum cholesterol, triglyceride, erythrocyte sedimentation rate, hemoglobin and for white blood cell count. CRP was measured by ultrasensitive, particle enhanced immunoturbidimetric assay. RESULTS: CRP levels were less than 5 mg/L in 81% of the blood donors. Mean level of CRP in the study population was 3.57 mg/L (SD +/- 5.33); the distribution was comparable to the data of already published studies. Comparing laboratory parameters and the risk status stratified according to CRP levels (less or more than 5 mg/L) significant differences were found in BMI (p = 0.0015), in total serum cholesterol (p = 0.0136), in triglyceride (p < 0.0001), in erythrocyte sedimentation rate (p < 0.0001), in white blood cell (p = 0.0007) and granulocyte count (p = 0.0014). Significant correlation was found between age and the concentration CRP (r = 0.22; p = 0.0011). CONCLUSION: The CRP measurement by ultrasensitive method is suitable for cardiovascular risk estimation in apparently healthy men and women. Risk prediction adapted for the Hungarian situation may be stimulated by these data.

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).

Complement Synthesis Influencing Factors Produced by Acute Myeloid Leukemia Blast Cells.

In a previous study, we found hypercomplementaemia in the sera of acute myeloid leukemia patients. In this study we show that the supernatants of mononuclear cells, derived from peripheral blood taken in the blastic phase, from patients with acute myeloid leukemia (CM-AML) increased the in vitro complement protein synthesis of HepG2 hepatocellular carcinoma cells. This effect of CM-AML was mediated by heat labile soluble factors and involved the synthesis of mRNA and protein. Inhibition experiments with anti-cytokine antibodies and immunoaffinity chromatography revealed that this effect of CM-AML is mostly mediated by IL-1 and IL-6.