Activation of the lectin pathway of complement by cardiopulmonary bypass contributes to the development of systemic inflammatory response syndrome after paediatric cardiac surgery.

The systemic inflammatory response is a challenge in the management of paediatric patients undergoing cardiac surgery. Although multi-factorial, a contribution by the lectin pathway of complement activation has been postulated. We therefore investigated the changes in serum levels of mannose binding lectin (MBL) and activities of MBL-MBL-associated serine protease (MASP)-1 and MBL-MASP-2 complexes immediately before and during surgery, throughout the first postoperative day and at discharge from the hospital. These changes were analysed in relation to postoperative complications. Blood samples were obtained from 185 children with congenital heart disease undergoing surgical correction with the use of cardiopulmonary bypass: preoperatively (MBL-1), 15 min after initiation of cardiopulmonary bypass (CPB) (MBL-E), 30 min (MBL-2), 4 h (MBL-3), 12 h (MBL-4) and 24 h (MBL-5) post-CPB and at discharge from hospital (MBL-K). Alterations in serum MBL levels were calculated as a ratio of its serum level at subsequent time-points (MBL-2, -3, -4, -5) to the preoperative (MBL-1) value. Decreases in MBL and MBL-MASP complexes were observed in all samples, correlating with a decrease in C4 and increase in C4a, confirming activation of the lectin pathway. Changes in MBL levels between children with an uncomplicated postoperative course and those suffering from infection or low cardiac output syndrome did not differ significantly, but significant differences were observed between the SIRS and non-SIRS groups. Paediatric cardiac surgery with the use of cardiopulmonary bypass activates the complement system via the lectin pathway and the latter contributes to the development of the post-bypass systemic inflammatory response.

Can ficolin-2 (L-ficolin) insufficiency be established by a single serum protein measurement?

Serum ficolin-2 was measured in multiple (2-27) samples from 68 paediatric sepsis patients. Fourteen individuals (21%) gave values that included a change in status from 'normal' to 'insufficient' or vice versa. Therefore, if possible, ficolin-2 concentration should be determined in samples obtained when a disease is inactive.

Interaction of lectin pathway of complement-activating pattern recognition molecules with mycobacteria.

We have demonstrated that mannose-binding lectin (MBL) recognizes various slow-growing, pathogenic mycobacteria [Mycobacterium tuberculosis (MTB), M. bovis, M. kansasii, M. gordonae] as well as non-pathogenic M. smegmatis. Recognition resulted in activation of the lectin pathway (LP) of complement and an enhancement of phagocytosis (shown for M. tuberculosis). Although MBL may be considered the main factor activating the LP upon recognition of mycobacteria, involvement of ficolins has also to be considered. Interaction of ficolin-3 with M. tuberculosis, M. bovis and M. kansasii, and ficolin-1 with M. tuberculosis and M. bovis was shown for the first time. Binding of recombinant MBL or ficolin-3 to MTB H37 Rv led to the agglutination of bacteria and promoted their phagocytosis, but little effect was apparent with ficolin-1 or ficolin-2. Data from Western blots suggest mannosylated lipoarabinomannan (ManLAM) to be one of the main cell components of slow-growing mycobacteria, involved in LP activation. However, the LP was also activated by other cell fractions. Results presented here supplement considerably the data concerning the ability of complement-activating lectins to interact with mycobacteria. Ficolins (especially ficolin-3) might influence host response to infection and thus have clinical significance, at least as disease modifiers.

Structural and serological studies of the O-specific polysaccharide of the bacterium Proteus mirabilis O10 containing L-altruronic acid, a new component of O-antigens.

An acidic O-specific polysaccharide from the lipopolysaccharide of Proteus mirabilis O10 contains 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, D-galacturonic acid, and L-altruronic acid, the last-named sugar having not been found hitherto in O-antigens. Structure of a branched tetrasaccharide repeating unit of the polysaccharide was established by 1H and 13C NMR spectroscopy, including two-dimensional COSY and rotating-frame NOE spectroscopy. The lateral L-altruronic acid residue plays the immunodominant role in manifestation of the O10 specificity of Proteus, whereas a disaccharide fragment of the main chain in common with the O-specific polysaccharide of P. mirabilis O43 provides the one-way serological cross-reactivity between anti-O10 serum and O43-antigen.

Structures of the O-antigens of Proteus bacilli belonging to OX group (serogroups O1-O3) used in Weil-Felix test.

Structures of the O-specific polysaccharide chains of lipopolysaccharides from Proteus group OX strains (serogroups O1-O3) used as antigens in Weil-Felix test for diagnosis of rickettsiosis, were established. From them, the acid-labile polysaccharide of Proteus vulgaris OX19 (O1) is built up of the following branched pentasaccharide repeating units connected via a phosphate group: [structure in text] where QuiNAc stands for 2-acetamido-2,6-dideoxyglucose (N-acetylquinovosamine). The basis of serospecificity of the Proteus group OX antigens and their cross-reactivity with human anti-rickettsial antibodies is discussed.

Comparison of serological reactions of Rickettsiae-infected patients and rabbit anti-Proteus OX antibodies with Proteus OX2, OX19 and OXK lipopolysaccharides.

The reactivity of anti-Rickettsiae human antibodies with Proteus OX cells is used as a presumptive rickettsial diseases diagnostic test Weil-Felix reaction. In presented studies we compare the reactivity of human anti-Rickettsiae and rabbit anti-Proteus antibodies with series of Proteus OX2, OX19 and OXK lipopolysaccharides (LPS). Polyclonal rabbit anti-OX2, anti-OX19 and anti-OXK sera reacted only with the homologous LPS--OX2, OX19 and OXK, respectively. The antibodies of Japanese spotted fever patients were less specific and reacted with OX2 as well as OX19 LPS. The antibodies of scrub typhus patients recognized Proteus OXK LPS, only. The serological reactions of O-antigen of P. mirabilis S1959 indicated that this, previously serologically not classified, strain may belong to the OXK group. Bacteria, used in the studies, came from the American, Japanese and European strain collections. The series of OX2, OX19 and OXK LPS, isolated from these Proteus strains, presented pattern of electrophoretic mobility and serological reactivities specific for each of OX-group types.

Serum IgG antibodies in children and adults reacting with Helicobacter pylori lipopolysaccharides.

The presence of IgG antibodies reacting with Helicobacter pylori lipopolysaccharides (LPSs) in sera from children and adults diagnosed as H. pylori-infected, as well as healthy persons, was tested. There was no correlation between the production of antibodies reacting with H. pylori surface proteins and LPSs. Also no correlation between reactivity of tested sera with H. pylori antigens and deep rough mutant (Re types) enterobacterial LPSs was revealed. The prevalence of anti-LPS IgG in randomly selected children was relatively high.

Structure and serological characterization of an Nepsilon-[(R)-1-carboxyethyl]-L-lysine-containing O-chain of the lipopolysaccharide of Proteus mirabilis O13.

In this paper we present the structure and describe serological properties of the O-specific polysaccharide of Proteus mirabilis O13 lipopolysaccharide, which contains a unique component: an amide of D-galacturonic acid (D-GalA) with an unusual amino acid, Nepsilon-[(R)-1-carboxyethyl]-L-lysine (alaninolysine, AlaLys). Selective chemical degradations of either GalA or AlaLys resulted in the loss of the serological reactivity of the polysaccharide with anti-O serum against P. mirabilis O13. Neither synthetic stereoisomers of AlaLys nor the isolated amide of GalA with AlaLys inhibited the reaction of the O-antiserum with the homologous lipopolysaccharide. The O-antiserum did not cross-react with the lipopolysaccharide of Providencia alcalifaciens O23 containing an amide of D-glucuronic acid with AlaLys. These data showed that both uronic acid and amino acid components of the amide play an important role in manifesting the P. mirabilis O13-specificity, but the full specific epitope also includes another O-specific polysaccharide component(s). A cross-reactivity of anti-O13 serum with some other P. mirabilis strains was observed and attributed to a common heat-stable antigen(s) different from the lipopolysaccharide.

The structure of the carbohydrate backbone of the core-lipid A region of the lipopolysaccharide from Proteus vulgaris serotype O25.

The following structure of the lipid A-core region of the lipopolysaccharide (LPS) from Proteus vulgaris serotype O25 was determined by using NMR and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of LPS, of the products of alkaline deacylation of the LPS, and of the products of LPS deamination: [structure: see text] Terminal residues of beta-GlcNAc and beta-Kdo (indicated by bold italics) are present alternatively in approximately 3:2 amount, leaving no unsubstituted beta-Gal. All sugars are in the pyranose form, alpha-Hep is the residue of L-glycero-alpha-D-manno-Hep, alpha-DDHep is the residue of D-glycero-alpha-D-manno-Hep.

Structure of an acidic O-specific polysaccharide of Proteus mirabilis O5.

The following structure of the O-specific polysaccharide of Proteus mirabilis O5 was established by 1H and 13C NMR spectroscopy at 500 MHz, including two-dimensional COSY, TOCSY, NOESY, and H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC) experiments: [formula: see text] where O-acetylation of alpha-D-GlcNAc at both positions is nonstoichiometric.

Isolation using triflic acid solvolysis and identification of N(epsilon)-[(R)-1-carboxyethyl]-N(alpha)-(D-galacturonoyl)-L-lysine as a component of the O-specific polysaccharide of Proteus mirabilis O13.

An amino acid was released from the O-specific polysaccharide of Proteus mirabilis O13 by acid hydrolysis and identified as N(epsilon)-[(R)-1-carboxyethyl]-L-lysine by comparison with the authentic sample. An amide of this amino acid with D-galacturonic acid was isolated from the polysaccharide by solvolysis with anhydrous trifluoromethanesulfonic (triflic) acid and characterised by 1H and 13C NMR spectroscopy. These and published data enabled determination of the full structure of the repeating unit of the polysaccharide.

[Estimation of mean molecular weight of Enterobacteriaceae lipopolysaccharides using gas chromatography for determination of fatty acids]. / Okreslanie sredniej masy molowej lipopolisacharydów paleczek Enterobacteriaceae metoda oznaczania kwasów tluszczowych przy uzyciu chromatografii gazowej.

In the paper, we propose a method for estimation of the mean molecular weight of lipopolysaccharide, which is important for accuracy of endotoxin activity investigation. In our study, it was assumed that lipid A portion in Enterobacterial lipopolysaccharide is substituted by four 3-hydroxytetradecanoic acid residues. Lipopolysaccharides of S, Ra, Rc and Re chemotypes being laboratory preparations as well as purchased from Sigma were investigated. Fatty acids were determined by of gas chromatography as methyl esters according to the procedure described by Wollenweber and Rietschel. Mean molecular weight was calculated by the formula: MMW = [formula: see text]. A high agreement between the estimated and the theoretical molecular weight values was demonstrated in the case of Salmonella minnesota R595 (Re) LPS preparation. As expected, LPS heterogeneity increase together with enlargement of polysaccharide chain length which is visible in electrophoregrams also. Except for LPS mean molecular weight estimation, the method allows its detection in various preparations and samples, distinguishing of R and S LPS forms as well as the determination of mean length of O-specific chain in lipopolysaccharides which structures are known.

[Structural and immunologic studies of Proteus mirabilis 033 O-specific polysaccharide]. / Badania strukturalne i immunologiczne polisacharydu O-swoistego Proteus mirabilis 033.

O-specific polysaccharide was obtained by mild acid degradation of P. mirabilis 033 lipopolysaccharide (LPS). It was found to contain N-acetyl-D-glucosamine, D-glucuronic acid and N-acetyl-L-fucosamine in a ratio 1:1:1. On the basis of the data obtained from 13C-NMR and methylation analysis, the following structure of repeating unit was established: [formula: see text] Selective removal of the D-GlcA significantly decreased reactivity of 033 O-specific polysaccharide with homologous antiserum. This component was plays an immunodominant role. Cross reactivity between anti-033 serum and disaccharide alfa-L-FucNAc-beta-D-GlcNAc containing P. vulgaris 023 and S. arizonae 059 O-specific polysaccharides was also observed.

Levels of mannose binding lectin in early pregnancy complicated by diabetes mellitus type 1--preliminary report.

The complement system plays an important role as a product of innate and acquired immune reaction. It can be activated via three different routes: the classical pathway, the alternative pathway and the lectin pathway. MBL (mannose-binding lectin) is considered to be a pathogen recognising receptor (PRR), an important factor in recognising pathogen associated molecular pattern (PAMP). The aim of study was to evaluate MBL in early pregnancy of diabetic mothers. Higher values of MBL were observed in diabetic non-pregnant women compared to healthy non-pregnant. Subjects early pregnancy seems to decrease MBL values in both diabetic and healthy pregnant women.

Mannan-binding lectin (MBL) in women with tumours of the reproductive system.

Mannan-binding lectin (MBL) is an important factor of innate immunity contributing to the clearance of microorganisms. Recently, an antitumourigenic role of MBL has been suggested. We investigated mbl2 genotypes, MBL concentrations, and MBL-MASP-2 complex activity in patients with ovarian cancer. The expression of both mbl2 and masp-2 genes were investigated in ovarian tissue sections. Additionally, samples from patients with other malignant and benign tumours of the reproductive tract were tested. A significantly higher incidence of MBL deficiency/insufficiency-associated genotypes was found among patients with malignant disease compared to age-matched controls. Unexpectedly, no differences in median MBL level or MBL-MASP-2 complex activity were found between the groups. This was partly a reflection of higher MBL concentrations and MBL-MASP-2 activity in cancer patients compared with healthy women carrying corresponding genotypes. MBL-specific mRNA expression was detected in several normal and malignant ovarian tissues, as well as in ovarian epithelial cell lines. Intracellular staining with MBL-specific antibodies demonstrated the presence of MBL in ovarian cell lines, and in normal as well as malignant ovarian tissue sections. In contrast, MASP-2-specific mRNA expression was detected only in the ovary tissues of patients with malignant disease. No significant changes in MBL concentration during 3 months of chemotherapy were noticed. MBL was detected in ascites and in the fluid of benign ovarian cysts. Our findings may reflect anti-tumourigenic activity of MBL protein which might suggest potential therapeutic application. However, it cannot be excluded that mbl-2 mutant alleles may be in linkage disequilibrium with an unidentified tumour susceptibility gene(s).

Extremes of L-ficolin concentration in children with recurrent infections are associated with single nucleotide polymorphisms in the FCN2 gene.

L-ficolin (also called ficolin-2, P35 or hucolin) is a soluble pattern recognition molecule of suspected importance in anti-microbial immunity. It activates the lectin pathway of complement and acts as an opsonin. l-ficolin, encoded by the FCN2 gene, recognizes microbial polysaccharides and glycoconjugates rich in GlcNAc or GalNAc. We report here data concerning four single nucleotide polymorphisms (SNPs) of the FCN2 gene and their relationship to l-ficolin serum concentrations. There are two pairs of SNPs in linkage disequilibrium: ss32469536 (located in promoter) with rs7851696 (in exon 8) and ss32469537 (promoter) with ss32469544 (exon 8). We selected groups possessing low or high serum l-ficolin concentrations (or= 4.5 microg/ml, respectively) from Polish children suffering from recurrent respiratory infections (n = 146). Low l-ficolin levels were associated with variant alleles for ss32469536 and rs7851696 and normal alleles for ss32469537 and ss32469544. Conversely, high l-ficolin levels were associated with variant alleles of ss32469537 and ss32469544. FCN2 genotyping should be a valuable additional tool for disease association studies.

Mannan-binding lectin in children with chronic gastritis.

The involvement of mannan-binding lectin (MBL) insufficiency in the pathogenesis of chronic gastritis (CG) in children was investigated. Blood samples were collected from 78 paediatric patients suffering from CG associated with Helicobacter pylori infection (group Hp(+)) and from 41 with the disease not associated with such an infection (group Hp(-)). Control group consisted of 77 children. The frequency of mbl-2 gene mutations and serum protein concentrations did not differ significantly in both groups as compared with controls. An expression of mbl-2 gene in gastric biopsies of CG patients was demonstrated. It was found to be stronger in H. pylori-infected children. The results presented in this paper suggest that MBL deficit/dysfunction probably does not contribute to an increased risk of CG (both associated and not associated with H. pylori infection) in children. However, MBL opsonic effect and/or the lectin pathway of complement activation may be taken into account as possible host defence mechanisms in gastric patients.

The structure of the core region of the lipopolysaccharide from Klebsiella pneumoniae O3. 3-deoxy-alpha-D-manno-octulosonic acid (alpha-Kdo) residue in the outer part of the core, a common structural element of Klebsiella pneumoniae O1, O2, O3, O4, O5, O8, and O12 lipopolysaccharides.

The structure of lipid A-core region of the lipopolysaccharide (LPS) from Klebsiella pneumoniae serotype O3 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of the LPS: [carbohydrate structure see text] where P is H or alpha-Hep; J is H or beta-GalA; R is H or P (in the deacylated oligosaccharides). Screening of the LPS from K. pneumoniae O1, O2, O4, O5, O8, and O12 using deamination showed that they also contain alpha-Hep-(1-->4)-alpha-Kdo-(2-->6)-GlcN and alpha-Kdo-(2-->6)-GlcN fragments.

[Serologic studies of cross reactions between strains of Proteus mirabilis OXK (prO 10/52) and Proteus mirabilis S1959]. / Badania serologicznych reakcji krzyzowych miedzy szczepami Proteus mirabilis OXK (PrO 10/52) I Proteus mirabilis S1959.

Strong cross-reactions are described between Proteus mirabilis strains having the same structures of repeating units of their O-specific polysaccharides. These strains are used in routine diagnosis of rickettsiae.

[Serologic cross reactions between strains of Proteus mirabilis belonging to serogroup O43 I O10]. / Serologiczne reakcje krzyzowe miedzy szczepami Proteus mirabilis nalezacymi do serogrup O43 I O10.

In this work an epitope has been determined on lipopolysaccharide of Proteus mirabilis O43. This epitope is recognized by specific antibodies.