This study presents the binding of ovine factor H (fH) by various serotypes of Borrelia and simultaneously correlates their complement resistance to sheep serum. Affinity ligand binding assay was employed to study the binding of borrelial proteins to ovine recombinant fH and its truncated forms (short consensus repeat, SCR 7 and SCRs 19-20). From a repertoire of 17 borrelial strains, only two strains showed affinity to sheep fH. A ~28-kDa protein of Borrelia burgdorferi sensu stricto (B. burgdorferi s.s., strain SKT-2) bound full-length fH as well as SCRs 19-20. This fH-binding protein was further identified as complement regulator-acquiring surface protein of B. burgdorferi (BbCRASP-1) by MALDI-TOF analysis. Surprisingly, a ~26-kDa protein of Borrelia bissettii (DN127) showed affinity to full-length fH but not to SCR 7 and SCRs19-20. In complement sensitivity assay, both strains-SKT-2 and DN127-were resistant to normal sheep serum. Significant complement resistance of two Borrelia garinii strains (G117 and T25) was also observed; however, none of those strains was able to bind sheep fH. Our study underscores the need of further exploration of fH-mediated evasion of complement system by Borrelia in domestic animals.

• MeSH
• Bacterial Proteins chemistry   genetics   immunology   MeSH
• Borrelia classification   genetics   immunology   isolation & purification   MeSH
• Kinetics MeSH
• Complement Factor H chemistry   immunology   MeSH
• Lyme Disease immunology   microbiology   veterinary   MeSH
• Molecular Sequence Data MeSH
• Sheep Diseases immunology   microbiology   MeSH
• Sheep MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

The whooping cough agent Bordetella pertussis secretes an adenylate cyclase toxin (CyaA) that through its large carboxy-proximal Repeat-in-ToXin (RTX) domain binds the complement receptor 3 (CR3). The RTX domain consists of five blocks (I-V) of characteristic glycine and aspartate-rich nonapeptides that fold into five Ca2+-loaded parallel β-rolls. Previous work indicated that the CR3-binding structure comprises the interface of β-rolls II and III. To test if further portions of the RTX domain contribute to CR3 binding, we generated a construct with the RTX block II/III interface (CyaA residues 1132-1294) linked directly to the C-terminal block V fragment bearing the folding scaffold (CyaA residues 1562-1681). Despite deletion of 267 internal residues of the RTX domain, the Ca2+-driven folding of the hybrid block III/V β-roll still supported formation of the CR3-binding structure at the interface of β-rolls II and III. Moreover, upon stabilization by N- and C-terminal flanking segments, the block III/V hybrid-comprising constructs competed with CyaA for CR3 binding and induced formation of CyaA toxin-neutralizing antibodies in mice. Finally, a truncated CyaAΔ1295-1561 toxin bound and penetrated erythrocytes and CR3-expressing cells, showing that the deleted portions of RTX blocks III, IV, and V (residues 1295-1561) were dispensable for CR3 binding and for toxin translocation across the target cell membrane. This suggests that almost a half of the RTX domain of CyaA is not involved in target cell interaction and rather serves the purpose of toxin secretion.

• MeSH
• Acylation MeSH
• Adenylate Cyclase Toxin metabolism   MeSH
• Bordetella pertussis pathogenicity   MeSH
• CHO Cells MeSH
• Cricetulus MeSH
• Epitopes metabolism   MeSH
• Humans MeSH
• Macrophage-1 Antigen chemistry   metabolism   MeSH
• Antibodies, Neutralizing metabolism   MeSH
• Protein Domains MeSH
• Protein Folding MeSH
• Amino Acid Sequence MeSH
• THP-1 Cells MeSH
• Calcium metabolism   MeSH
• Protein Binding MeSH
• Structure-Activity Relationship MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The interaction of Bordetella pertussis adenylate cyclase toxin (CyaA) with complement receptor 3 (CR3, CD11b/CD18) involves N-linked oligosaccharide chains. To investigate the relative importance of the individual N-glycans of CR3 for toxin activity, the asparagine residues of the consensus N-glycosylation sites of CR3 were substituted with glutamine residues that cannot be glycosylated. Examination of CR3 mutant variants and mass spectrometry analysis of the N-glycosylation pattern of CR3 revealed that N-glycans located in the C-terminal part of the CD11b subunit are involved in binding and cytotoxic activity of CyaA. We suggest that these N-glycans form a defined clustered saccharide patch that enables multivalent contact of CR3 with CyaA, enhancing both affinity and specificity of the integrin-toxin interaction.

• MeSH
• Adenylate Cyclase Toxin genetics   metabolism   MeSH
• CD11b Antigen chemistry   metabolism   MeSH
• CD18 Antigens chemistry   metabolism   MeSH
• Asparagine genetics   MeSH
• Bordetella pertussis metabolism   pathogenicity   MeSH
• Glutamine genetics   MeSH
• Glycosylation MeSH
• Humans MeSH
• Macrophage-1 Antigen genetics   metabolism   MeSH
• Polysaccharides metabolism   MeSH
• Amino Acid Substitution MeSH
• Protein Structure, Tertiary MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The goal of the project is to study the role of selected components of the complement system in the pathogenesis of autoimmune thyroid disorders (AITD), i.e. Graves´ disease and Hashimoto´s thyroiditis, and their relationship to the course of disease. The aims of the first part of the project include: i) to determine the prevalence of mannan-binding lectin (MBL) deficiency in patients with AITD; ii) to assess the impact of thyroid hormones on MBL serum concentrations; ii) to evaluate the relationship between MBL deficiency/serum levels of MBL and the clinical course of disease. The aim of the second part of the project is to follow the serum levels of antibodies against complement component C1q in time and to determine their relationship to the clinical course of AITD. Methods include genetic analysis of the MBL-2 gene and repeated clinical and laboratory examinations of patients with AITD, including measurements of serum MBL and anti-C1q levels.

Předmětem projektu je studium vybraných složek komplementu v etiopatogenezi autoimunitních tyreopatií (AITD), t.j. Gravesovy-Basedowovy tyreotoxikózy a chronické lymfocytární tyreoiditidy, a jejich vztahu ke klinickému průběhu onemocnění. Cílem první části projektu je: 1) zjistit prevalenci deficience mannan-vázajího lektinu (MBL) u nemocných s AITD; 2) ozřejmit vliv tyreoidálních hormonů na sérové koncentrace MBL; 3) definovat vztah MBL deficience a klinického průběhu AITD. Cílem druhé části projektu je sledovat sérové koncentrace protilátek proti C1q složce komplementu u nemocných s AITD v čase a popsat jejich vztah k průběhu onemocnění. Metodika bude zahrnovat genetickou analýzu genu pro MBL a opakovaná klinická a laboratorní vyšetření nemocných s AITD, zahrnující m.j. stanovení sérových koncentrací MBL a anti-C1q protilátek.

• MeSH
• Autoimmune Diseases MeSH
• Thyroiditis, Autoimmune MeSH
• Graves Disease MeSH
• Complement C1q analysis   antagonists & inhibitors   MeSH
• Mannose-Binding Lectin analysis   deficiency   MeSH
• Complement Pathway, Mannose-Binding Lectin MeSH
• Thyroid Diseases MeSH
• Postpartum Thyroiditis MeSH

• Conspectus
• Fyziologie člověka a srovnávací fyziologie
• NML Fields
• endokrinologie
• NML Publication type
• závěrečné zprávy o řešení grantu IGA MZ ČR

Mannose-binding lectin (MBL) is an important component of the innate immunity, and it is responsible not only for opsonization of micro-organisms, but also for efferocytosis. The aim of this study was to investigate whether MBL concentrations and lectin complement pathway activity are altered in non-pregnant women with previous adverse pregnancy outcomes. Patients were divided into four groups on the basis of their history of pregnancy complications, including control patients who had uncomplicated pregnancies and term deliveries (control, n = 33), and three groups of patients with a history of pregnancy complications, including preterm labour (n = 29), recurrent miscarriage (n = 19) or unexplained intrauterine foetal death (IUFD; n = 17). All women enrolled in the study had an interval of three to six months following their previous pregnancy, and they agreed to have a blood sample taken. We found significantly higher MBL concentrations and functional activity of the lectin complement pathway in healthy controls who had previous uneventful term pregnancies (1341 ng/mL; activity 100% (IQR: 62%-100%)), compared to women with the history of IUFD (684 ng/mL, P = .008; activity 8.5% (IQR: 0%-97.8%), P = .011), recurrent miscarriage (524 ng/mL, P = .022; activity 44% (IQR: 4%-83%), P = .011) or preterm labour (799 ng/mL, P = .022; activity 62.5% (IQR: 0%-83%), P = .003). Our results suggest that inadequate function of the complement lectin pathway is associated with a higher risk of preterm labour, recurrent miscarriage and unexplained intrauterine foetal death.

• MeSH
• Adult MeSH
• Pregnancy Complications blood   epidemiology   MeSH
• Mannose-Binding Lectin blood   MeSH
• Complement Pathway, Mannose-Binding Lectin immunology   MeSH
• Humans MeSH
• Immunity, Innate immunology   MeSH
• Prospective Studies MeSH
• Risk Factors MeSH
• Pregnancy MeSH
• Pregnancy Outcome MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

• MeSH
• Neoplasm Invasiveness MeSH
• Plasma virology   MeSH
• Humans MeSH
• Antibodies blood   MeSH
• Check Tag
• Humans MeSH

PURPOSE: dialysis-induced inflammatory response including leukocyte and complement activation is considered a significant cofactor of chronic morbidity in long-term hemodialysis (HD) patients. The aim of this study was to provide better insight into its molecular background. EXPERIMENTAL DESIGN: in 16 patients, basic biocompatibility markers, i.e. leukocyte counts and C5a levels, were monitored during HD on a polysulfone membrane. Proteins adsorbed to dialyzers were eluted and separated by 2-DE. Selected proteins were identified by MS; ficolin-2 plasma levels were assessed. Data are given as medians (quartile ranges). RESULTS: in total, 7.2 (34.7) mg proteins were retrieved from dialyzer eluates and were resolved into 217 protein spots. The proteins most enriched in eluates (and hence selectively adsorbed) were those involved in complement activation (C3c, ficolin-2, mannan-binding lectin serine proteases, properdin) and cell adhesion (actin, caldesmon, tropomyosin, vitronectin, vinculin). A significant decrease of plasma ficolin-2 (41% [4.7], p<0.001) was evidenced during one HD session, associated with leukopenia (r=0.73, p=0.001) and C5a production (r=-0.62, p=0.01) at 15 min. CONCLUSIONS AND CLINICAL RELEVANCE: ficolin-2 adsorption to polysulfone dialyzer initiates the lectin pathway of complement activation, mediates dialysis-induced leukopenia, and results in a significant depletion of ficolin-2, an essential component of innate immunity.

• MeSH
• Adsorption MeSH
• Renal Dialysis instrumentation   methods   MeSH
• Complement Pathway, Mannose-Binding Lectin MeSH
• Lectins chemistry   metabolism   MeSH
• Humans MeSH
• Membranes, Artificial MeSH
• Polymers chemistry   metabolism   MeSH
• Proteome analysis   MeSH
• Gene Expression Profiling MeSH
• Sulfones chemistry   metabolism   MeSH
• Inflammation immunology   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Activation of the lectin pathway of the complement system, as demonstrated by elevated levels of mannan-binding lectin proteins (MBL), contributes to vascular pathology in type 1 diabetes (T1D). Vascular complications are greatest in T1D individuals with concomitant insulin resistance (IR), however, whether IR amplifies activiation of the lectin pathway in T1D is unknown. We pooled pretreatment data from two RCTs and performed a cross-sectional analysis on 46 T1D individuals. We employed estimated glucose disposal rate (eGDR), a validated IR surrogate with cut-points of: <5.1, 5.1-8.7, and > 8.7 mg/kg/min to determine IR status, with lower eGDR values conferring higher degrees of IR. Plasma levels of MBL-associated proteases (MASP-1, MASP-2, and MASP-3) and their regulatory protein MAp44 were compared among eGDR classifications. In a subset of 14 individuals, we assessed change in MASPs and MAp44 following improvement in IR. We found that MASP-1, MASP-2, MASP-3, and MAp44 levels increased in a stepwise fashion across eGDR thresholds with elevated MASPs and MAp44 levels conferring greater degrees of IR. In a subset of 14 patients, improvement in IR was associated with significant reductions in MASPs, but not MAp44, levels. In conclusion, IR in T1D amplifies levels of MASP-1/2/3 and their regulator MAp44, and improvement of IR normalizes MASP-1/2/3 levels. Given that elevated levels of these proteins contribute to vascular pathology, amplification of the lectin pathway of the complement system may offer mechanistic insight into the relationship between IR and vascular complications in T1D.

• MeSH
• Diabetes Mellitus, Type 1 * MeSH
• Insulin Resistance * MeSH
• Complement System Proteins MeSH
• Mannose-Binding Lectin * MeSH
• Lectins metabolism   MeSH
• Humans MeSH
• Cross-Sectional Studies MeSH
• Mannose-Binding Protein-Associated Serine Proteases metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

• Keywords
• komplementový systém,
• MeSH
• Complement Activation * physiology   immunology   MeSH
• Anaphylatoxins physiology   MeSH
• Apoptosis physiology   MeSH
• Complement C3a physiology   MeSH
• Complement Pathway, Mannose-Binding Lectin physiology   immunology   MeSH
• Humans MeSH
• Immunity, Innate physiology   MeSH
• Hereditary Complement Deficiency Diseases diagnosis   classification   physiopathology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Review MeSH

Cílem práce je rekapitulace aktuálních znalostí o deficitu lektinové dráhy aktivace komplementu ve vztahu k možným rizikům během gravidity na příkladu názorné kazuistiky z klinické praxe. Deficit manózu vázajícího lektinu (MBL) je autozomálně recesivně dědičné onemocnění – porucha oligomerizace tripletů MBL je geneticky determinovaná. U většiny mutací nacházíme podobný fenotyp, homozygoti nebo heterozygoti produkují velice nízké, často nedetekovatelné koncentrace MBL, kdy jde především o triplety monomerů, které nejsou schopny aktivovat komplement a neumí vázat manan. Poruchy syntézy MBL a s nimi sdružený deficit lektinové dráhy aktivace komplementu mohou být sdruženy s těhotenskými komplikacemi. Jsou dávány do souvislosti s funkčními poruchami placenty typu placentární insuficience vedoucí k intrauterinní růstové retardaci plodu, dále se častěji MBL deficit vyskytuje u žen s opakovanými časnými potraty, ale i s komplikacemi pokročilé gravidity – s preeklampsií, předčasnými porody, HELLP syndromem a především s infekčními těhotenskými komplikacemi až ve formě purulentních chorioamnionitid. Popsaná případová studie popisuje tři těhotenské ztráty ve 2. trimestru těhotenství u mladé a jinak zdravé ženy. Druhotrimestrální potraty byly u pacientky sdruženy s histopatologicky verifikovanou purulentní chorioamnionitidou. Diagnostikovaná MBL deficience a deficit lektinové dráhy aktivace komplementu otevírají možnosti imunointervenční a případně protimikrobiální léčby v dalším těhotenství.

The aim of the study is to summarize the current knowledge about the lectin complement pathway disorders in relation to pregnancy complicati-ons on the example of the case report from out clinical practice. Mannose-binding lectin (MBL) deficiency is an autosomal recessive hereditary disease – MBL triplet oligomerization disorder is genetically determined. In most mutations, a similar phenotype is found – homozygotes and/or heterozygotes produce very low, often undetectable concentrations of MBL, especially triplets of monomers that are unable to activate com-plement and bind mannan. MBL deficiency and the associated lectin pathway disorder may be associated with pregnancy complications. They are associated with functional placental disorders such as placental insufficiency leading to intrauterine growth retardation of the fetus, MBL deficiency occurs more frequently in women with recurrent miscarriages and with complications of advanced pregnancy such as preeclampsia, premature birth, HELLP syndrome, and especially infectious pregnancy complications in the form of purulent chorioamnionitis. The current case report describes three pregnancy losses in the 2nd trimester of pregnancy in a young and otherwise healthy woman. Advanced pregnancy losses were associated with histopathologically verified purulent chorioamnionitis. Diagnosis of MBL deficiency and the lectin pathway disorder opens the possibilities of immune-interventional and (if necessary) antimicrobial treatment in the oncoming pregnancy.

• MeSH
• Adult MeSH
• Abortion, Habitual etiology   immunology   MeSH
• Histological Techniques MeSH
• Immunologic Techniques MeSH
• Pregnancy Complications, Infectious * prevention & control   MeSH
• Clinical Laboratory Techniques MeSH
• Mannose-Binding Lectin * genetics   deficiency   MeSH
• Complement Pathway, Mannose-Binding Lectin * physiology   immunology   MeSH
• Humans MeSH
• Abortion, Spontaneous etiology   immunology   MeSH
• Check Tag
• Adult MeSH
• Humans MeSH
• Female MeSH
• Publication type
• Case Reports MeSH