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.

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 (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).

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.

L-ficolin in children with recurrent respiratory infections.

The lectin pathway of complement activation is used by a collectin, mannan-binding lectin (MBL), and two ficolins, L-ficolin and H-ficolin, to opsonize microorganisms for phagocytosis. We published evidence recently that MBL insufficiency is associated with recurrent respiratory infections in childhood. We have now measured serum L-ficolin in 313 respiratory infection patients and 74 healthy control children. L-ficolin concentrations below the lower limit of the control group were found in 6% of the patients (P <0.02) and were associated most strongly with children having co-existing atopic disorders (11%; P=0.002). We suggest that L-ficolin may have a role in protection from microorganisms complicating allergic disease.

Mannan-binding lectin insufficiency in children with recurrent infections of the respiratory system.

Blood samples were collected over a 4-year period from 335 children (aged 1-16 years) suffering from recurrent respiratory infections and 78 controls. The patients were subdivided into four groups: I, children with no immune system defects detected (n = 101); II, children with allergies (n = 94); III, children with humoral response defects (n = 93); and IV, children with disturbances of cellular immunity (n = 66). Nineteen patients had both humoral and cellular abnormalities. All patients and controls were investigated to determine the exon 1 and promoter region variants of the mbl-2 gene. MBL serum concentrations were also determined in samples from 291 patients and 75 controls. The proportion of O (B, D or C) alleles was significantly higher in the patient group compared to controls, and this association was strongest for subgroup III. The promoter LX variant frequency was also commoner in the patients as a whole, and significantly so in subgroups II and IV. Genotypes markedly influenced MBL concentrations in all groups, and correlated with ability to activate the lectin pathway of complement activation. The strongest and most significant inverse correlations between serum MBL and respiratory disease were found in patient group III and in 17 patients with multiple humoral and/or cellular abnormalities. Among nine patients with unexpectedly low LP activity in view of their MBL concentrations, one person was found to be MASP-2 deficient. Our results indicate that mannan-binding lectin insufficiency, with or without a coexisting immune defect, is associated with the occurrence of recurrent respiratory infections in childhood, and this relationship is particularly strong and statistically significant in children with concomitant impairments of humoral immunity.

Nitric oxide generation from hydroxylamine in the presence of neutrophils and in the cell-free system.

Conversion of hydroxylamine (HA) to nitric oxide (NO) has been studied in the presence or absence of human neutrophils with or without myristate acetate phorbol (PMA), catalase (CAT), hydrogen peroxide (H2O2), and superoxide dismutase (SOD) and nitric oxide synthase (NOS) inhibitors. The generation of NO from HA in the presence of neutrophils was higher than in the cell-free system. We found that catalase did not influence the nitrite generation from HA in the cell-free system and in the presence of neutrophils. The H2O2 enhanced the NO generation from HA in the presence of neutrophils only. When catalase and H2O2 were added together, a high increase of NO generation from HA in both systems was observed. The addition of SOD decreased whereas addition of PMA enhanced the NO generation from HA in the presence of neutrophils. The presented data show the possible role of oxygen radicals in the decomposition of HA to NO. The addition of NOS inhibitors to the culture of neutrophils decreased the generation of nitrite from HA. Our results suggest that NO generation from HA, which is an intermediate in NO production from L-arginine, may be supported by an enzymatic pathway in which cellular NO synthase is involved.

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 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.

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.

Serological studies of an acid-labile O-polysaccharide of Proteus vulgaris OX19 lipopolysaccharide using human and rabbit antibodies.

In a Weil-Felix test, sera from patients infected with Rickettsia sp. agglutinate Proteus OX types of bacteria and Proteus lipopolysaccharide (LPS) are responsible for the cross-reaction. Data on the character of LPS of one of the OX group strains, Proteus vulgaris OX19, are contradictory, and it remained unclear whether it has an O-polysaccharide (OPS) and is thus LPS of the smooth type (S) or not (rough-type LPS). Our studies showed that P. vulgaris OX19 (strain PZH-24) produces a smooth-type LPS that contains a long-chain OPS, but it undergoes depolymerization during mild acid hydrolysis conventionally used for LPS delipidation and loses the serological activity. An elucidation of the complete structure of OPS demonstrated the presence of a glycosyl phosphate linkage responsible for the acid-lability of the polysaccharide chain. In ELISA, both IgM type antibodies in a Weil-Felix test with human anti-Rickettsia typhi sera and rabbit anti-P. vulgaris OX19 antibodies reacted with OPS. Rabbit antibodies did not inhibit the cross-reaction with human antibodies and thus bind to different epitopes.

The comparison of some biological activities of lipopolysaccharides obtained from smooth and rough Proteus mirabilis strains.

The biological activity of lipopolysaccharide (LPS) obtained from Proteus mirabilis smooth and rough strains was investigated. The tested endotoxins (differing in polysaccharide chain lenght) were isolated from wild S1959 strain as well as from its rough mutants Ra and Re. Induction of tumor necrosis factor-alpha (TNF-alpha), and nitric oxide production as well as activation of complement system by lipopolysaccharide are the pathophysiological reaction in a host response to gram-negative bacteria. In this study, it was found that S (S1959), Ra (R110) and Re (R45) chemotypes of LPS similarly induced the human neutrophils to release TNF-alpha. In contrast none of the LPS stimulated the neutrophils to synthesis of nitric oxide regardless of doses used and culture time. Te Re form of LPS showed the strongest anticomplementary activity.

Structural and immunochemical studies of two cross-reactive Proteus mirabilis O-antigens, O6 and O23, containing beta1-->3-linked 2-acetamido-2-deoxy-D-glucopyranose residues.

A marked serological cross-reactivity was observed by ELISA and a precipitation test between anti-Proteus mirabilis O23 serum and the lipopolysaccharide as well as the O-specific polysaccharide from the Proteus mirabilis strain belonging to serogroup O6. The structures of the O-specific polysaccharides were elucidated using chemical and NMR spectroscopic analyses, and the only common component, 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-D-GlcNAc), was revealed, which was suggested to be responsible for the cross-reactivity observed. Both anti-O23 and anti-O6 sera were shown to react with 1, 3-Linked beta-D-GlcNAc-containing O-antigen from Salmonella enterica ssp. arizonae O59 also. The lack of reactivity of Smith-degraded P. mirabilis O6 O-specific polysaccharide with homologous antiserum indicated the crucial role of alpha-D-glucuronic acid in specific antibody binding.

[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.

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.

Structure of the acid-labile galactosyl phosphate-containing O-antigen of the bacterium Proteus vulgaris OX19 (serogroup O1) used in the Weil-Felix test.

The structure of the O-specific polysaccharide chain of Proteus vulgaris OX19 lipopolysaccharide which determines the O1 specificity of Proteus and is used in the Weil-Felix test for diagnostics of rickettsiosis was established. On the basis of 1H- and 13 C-NMR spectroscopy, including two-dimensional correlation spectroscopy (COSY), H-detected 1H, 13C heteronuclear multiple-quantum coherence (HMQC), and rotating-frame nuclear Overhauser effect spectroscopy (ROESY), it was found that the polysaccharide consists of branched pentasaccharide repeating units containing D-galactose, 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2-deoxy-D-galactose, and 2-acetamido-2,6-dideoxy-D-glucose (QuiNAc, two residues), which are connected to each other via a phosphate group (P): [formula: see text]. The polysaccharide is acid-labile, the glycosyl phosphate linkage being cleaved at pH 4.5 (70 degrees C) to give a phosphorylated pentasaccharide with a galactose residue at the reducing end. Structural analysis of the oligosaccharide and a product of its dephosphorylation with 48% hydrofluoric acid using 1H- and 13C-NMR spectroscopy and electrospray ionization mass spectrometry confirmed the structure of the polysaccharide.

Structural and serological studies of the O-antigen of the bacterium Proteus vulgaris OX2 (serogroup O2) used in the Weil-Felix test.

Based on monosaccharide analysis and 1H- and 13C-NMR spectroscopy, the following structure of the O-specific polysaccharide chain of Proteus vulgaris OX2 lipopolysaccharide (LPS), which defines the O2 specificity of Proteus, was established: [formula: see text] where L-QuiNAc is N-acetyl-L-quinovosamine (2-acetamido-2,6-dideoxy-L-glucose). Various strains of P. vulgaris OX2 used in the Weil-Felix test for serodiagnosis of rickettsiosis (spotted fevers, except for Rocky Mountain spotted fever) were shown to produce LPS with the same O-specific polysaccharide, which differs structurally and serologically from LPS of P. vulgaris OX19 used as antigen for serodiagnosis of typhus and Rocky Mountain spotted fever. O-Acetyl groups present in the polysaccharide are not important for manifesting the immunospecificity. ELISA confirmed that the epitope responsible for the cross-reactivity between sera from patients with Japanese spotted fever and P. vulgaris OX2 cells is located on the P. vulgaris LPS. At the same time, no cross-reaction was observed between rabbit anti-P. vulgaris OX2 antibodies and the spotted fever group (SFG) rickettsial cells. Therefore, human anti-SFG rickettsial antibodies and rabbit anti-P. vulgaris OX2 antibodies may bind to distinct epitopes on P. vulgaris OX2 LPS, and no epitope recognized by rabbit anti-P. vulgaris OX2 antibodies is present on the LPS or any other surface antigen of SFG rickettsiae.

Structural and serological studies of the O-antigen of the bacterium Proteus mirabilis OXK (serogroup O3) used in the Weil-Felix test.

Sugar analysis and 1H- and 13C-NMR spectroscopic studies showed that various strains of Proteus mirabilis OXK used as antigens in the Weil-Felix test for serodiagnosis of rickettsiosis (scrub typhus) produce lipopolysaccharides (LPSs) with the same O-specific polysaccharide chain having the following structure: [formula: see text] where GlcA and GalA are glucuronic and galacturonic acids, respectively. This polysaccharide which defines the O3 specificity of Proteus and has been found earlier in an unclassified P. mirabilis strain S1959, contains an amide of D-galacturonic acid with L-lysine which plays an important role in manifesting the immunospecificity. A cross-reaction was observed in ELISA between sera from patients with scrub typhus, caused by the bacterium Orientia (Rickettsia) tsutsugamushi, and purified LPS of P. mirabilis OXK, thus suggesting that the common epitope involved in the Weil-Felix test is located on P. mirabilis OXK LPS. Rabbit anti-P. mirabilis OXK antibodies did not cross-react with LPS-lacking O. tsutsugamushi strain Gilliam in dot-blotting and Western blotting, and the nature of the rickettsial antigen responsible for the Weil-Felix reaction remains unknown.