Effect of different asthma treatments on risk of cold-related exacerbations.

Common colds often trigger asthma exacerbations. The present study compared cold-related severe exacerbations during budesonide/formoterol maintenance and reliever therapy, and different regimens of maintenance inhaled corticosteroids (ICS), with or without long-acting ß(2)-agonists (LABA), and with as-needed short-acting ß(2)-agonists (SABA) or LABA. Reported colds and severe exacerbations (defined by oral corticosteroid use and/or hospitalisation/emergency room visit) were assessed for 12,507 patients during 6-12 months of double-blind treatment. Exacerbations occurring ≤14 days after onset of reported colds were analysed by a Poisson model. The incidence of colds was similar across treatments. Asthma symptoms and reliever use increased during colds. Budesonide/formoterol maintenance and reliever therapy reduced severe cold-related exacerbations by 36% versus pooled comparators plus SABA (rate ratio (RR) 0.64; p=0.002), and for individual treatment comparisons, by 52% versus the same maintenance dose of ICS/LABA (RR 0.48; p<0.001); there were nonsignificant reductions versus higher maintenance doses of ICS or ICS/LABA (RR 0.83 and 0.72, respectively). As-needed LABA did not reduce cold-related exacerbations versus as-needed SABA (RR 0.96). Severe cold-related exacerbations were reduced by budesonide/formoterol maintenance and reliever therapy compared with ICS with or without LABA and with as-needed SABA. Subanalyses suggested the importance of the ICS component in reducing cold-related exacerbations. Future studies should document the cause of exacerbations, in order to allow identification of different treatment effects.

Human complement regulators: a major target for pathogenic microorganisms.

The C3 convertases of the human complement system are controlled by fluid-phase and membrane proteins in the RCA (regulators of complement activation) family. Accumulated data show that many pathogenic microorganisms interact with these complement regulators. Recent advances in this field include determination of the crystal structure of the binding domains in the measles virus receptor CD46 and identification of a CD46 transgenic mouse line that is sensitive to measles virus. Moreover, recent findings support the hypothesis that pathogenic bacteria binding fluid-phase RCA proteins exploit these proteins to escape complement attack. These studies provide novel insight into the interplay between pathogens and the innate immune system and may have implications for the plans to use animals expressing an RCA protein for xenotransplantation.

Mapping of the immunoglobulin light chain-binding site of protein L.

Protein L is a cell surface protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. The molecule binds specifically and with high affinity to immunoglobulins (Ig) of a wide range of animal species. The Ig-binding activity is mediated through five highly homologous domains, each 72 to 76 amino acid residues long, which interact with framework regions in the variable domain of Ig light chains. The interaction does not interfere with the antigen binding capacity of the antibody. The fold of the Ig light chain-binding domains of Protein L is comprised of an alpha-helix packed against a four stranded beta-sheet and is similar to the fold of the IgG heavy chain-binding domains of streptococcal protein G, despite the fact that the two proteins show no significant sequence homology. In the present work, heteronuclear NMR spectroscopy has been utilized to define the interaction between the N-terminal Ig-binding domain of Protein L and the variable domain of a human Ig kappa light chain. The Ig-binding region of the Protein L domain involves most of the residues in the second beta-strand, the C-terminal residues of the alpha-helix and the loop connecting the alpha-helix with the third beta-strand. The Ig light chain-binding surface of Protein L thus resembles the surface of Protein G which binds to the C gamma 1 domain of IgG, but is different from the portion of Protein G involved in the contact with the C gamma 2-C gamma 3 interface region. The data suggest that the global fold shared by the Ig-binding domains of Proteins L and G provide bacteria with a flexible template for the evolution of surface structures capable of interacting with different conserved parts of Ig molecules of the infected host.

NMR analysis of the interaction between protein L and Ig light chains.

Protein L is a cell wall protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. It binds to immunoglobulin (Ig) light chains predominantly of the kappa subtype from a wide range of animal species. This binding is mediated by five highly homologous repeats designated as B1-B5, each of which comprises 72 to 76 amino acid residues. The fold of the Ig light chain-binding B1 domain of protein L has previously been shown to comprise an alpha-helix packed against a four-stranded beta-sheet. The Ig-binding region of the protein L domain involves most of the residues in the second beta-strand, the C-terminal residues of the alpha-helix, and residues in the loop connecting the alpha-helix with the third beta-strand. In the present study, we have identified the protein L-binding site of an Ig light chain by use of stable isotope-assisted NMR spectroscopy. The light chain of a murine monoclonal anti-17alpha-hydroxy-progesterone Fab fragment (IgG2b, kappa) was selectively labeled with 13C at carbonyl groups of Ala, Arg, Cys, Ile, Lys, Met, Phe, Trp, or Tyr. The residues in which the carbonyl 13C chemical shift was significantly perturbed upon binding of the protein L B1 domain were preferentially found in the second beta-strand of the variable kappa domain and parts of its flanking beta-strands. None of these residues were affected by the addition of the antigen against which the monoclonal Fab fragment is directed. Therefore, we conclude that protein L binds to the outer surface of the framework region of the V(L) domain, primarily involving the V(L) second strand, and that this binding is independent of antigen-binding. The present NMR data, in combination with sequence comparisons between kappa light chains with and without protein L affinity, suggest that the amino acid substitutions at positions 9, 20, and/or 74 of the kappa light chains could crucially affect the interaction between protein L and the V(L) domain.

Streptococcal protein G has affinity for both Fab- and Fc-fragments of human IgG.

We have analyzed the binding of IgG and fragments of IgG [Fc, F(ab')2, and Fab] to group C and G streptococci and to protein G, the IgG binding cell wall protein of these bacteria. A direct correlation (r = 0.87, P less than 0.0001) was observed when the binding of radiolabelled, polyclonal IgG F(ab')2- and Fc-fragments to 23 group C and G streptococcal strains was compared. One strain (G-148) was treated with increasing amounts of pepsin, trypsin or papain and the Fab-binding structure was found to be much more sensitive to the enzymes as compared to the Fc-binding. A 35 K fragment of protein G was coupled to Sepharose, and both radiolabelled IgG F(ab')2- and Fc-fragments bound to the Sepharose beads. Binding of IgG fragments was inhibited by intact IgG or by the homologous IgG fragment, whereas Fc-fragments did not inhibit Fab binding or vice versa. Two radiolabelled protein G-fragments (28 and 35 K) showed different binding to polyclonal IgG, IgG F(ab')2-, IgG Fab- and IgG Fc-fragments. Thus, in a dot binding assay the 35 K fragment bound all IgG fragments tested, whereas the 28 K protein G fragment bound only intact IgG and IgG Fc-fragments. These results indicate two independent and separate binding sites for Fab- and Fc-fragments on protein G. Different binding sites on protein G were also indicated by Western blot analysis of four different protein G-fragments (28, 35, 42 and 65 K). In these experiments the 28 K fragment showed affinity only for Fc-fragments, while the higher mol. wt protein G preparations bound both IgG Fab- and Fc-fragments.

Non-immune Fab- and Fc- mediated interactions of avian Ig with S. aureus and group C and G streptococci.

Serum samples from 19 avian species representing 8 orders were tested for their capacity to inhibit the Fab- and Fc-mediated immunoglobulin binding to protein A-carrying S. aureus and protein G-carrying group C and G streptococci. Four species (mallard, dunlin, starling and blackbird) belonging to three different orders showed a high degree of Fc-mediated protein A- and protein G-reactivity. Five species demonstrated a high level and nine species exhibited a low level of Fab-mediated protein A-reactivity. The four species identified as Fc-reactive were capable of Fab-mediated immunoglobulin binding with streptococcal surface proteins but incapable of Fab-mediated protein A binding. SDS-PAGE analysis confirmed that the protein A-Sepharose affinity purified material contained proteins corresponding to immunoglobulin chains. Inhibition results by avian sera were confirmed by direct binding of protein A-reactive proteins to bacteria, by precipitation in gel and by Western blot analysis of binding to protein A and protein G, respectively.

Isolation and molecular characterization of a novel albumin-binding protein from group G streptococci.

Many streptococcal strains are known to bind the two most abundant plasma proteins, namely, immunoglobulin G and albumin. Protein G isolated from group C and G streptococci has been demonstrated to have separate binding regions for each of these proteins. However, some group G streptococcal strains bind only serum albumin. This report describes the isolation of a 48-kDa albumin-binding protein from such a strain (DG12). The affinity constant of this protein for human serum albumin was determined to be 5 x 10(9) M-1, and the protein interacted strongly also with serum albumin from several other mammalian species. The gene encoding the albumin-binding protein was cloned and expressed in Escherichia coli. DNA sequence analysis of this gene revealed a unique NH2-terminal sequence and three types of repeats in the encoded protein. One of these repeated sequences has significant homology with the albumin-binding domains of protein G, and it was demonstrated that the albumin binding of the DG12 protein was localized within these domains. Another type of repeat is localized in the putative wall-spanning region of the molecule. This repeat sequence, which has the length of only 4 amino acids (LysProGluVal), is repeated 14 times. The relationship of the albumin-binding protein to other cell-wall-associated proteins of pathogenic streptococci is discussed.

PAM, a novel plasminogen-binding protein from Streptococcus pyogenes.

The ability of group A streptococci to bind human plasminogen and plasmin has attracted interest, because it could provide the bacteria with a mechanism for invasion. M or M-like proteins account for the binding of several plasma proteins to group A streptococci. To investigate whether M or M-like proteins were responsible for the binding of plasminogen to group A streptococci, acid-extracted material from a type M53 streptococcal isolate was tested for its ability to bind plasminogen. Indeed, a 42-kDa plasminogen-binding protein was solubilized. Two oligonucleotides homologous with conserved sequences in known M protein genes were used as primers in the polymerase chain reaction, with chromosomal DNA from the M53 isolate. When cloned and expressed in Escherichia coli, a resulting fragment encoded a 43-kDa plasminogen-binding protein. Nucleotide sequence determination of the gene fragment revealed an open reading frame encoding a polypeptide of 43,580 Da, which matched the amino-terminal amino acid sequence of the plasminogen-binding protein extracted from M53 streptococci. The DNA sequence data also proved the relationship of the encoded protein, named PAM, to the M proteins. The plasminogen-binding domain was mapped to the amino-terminal third of PAM. Plasminogen absorbed by M53 streptococci or by immobilized PAM could be activated by streptokinase. The results provide further evidence of the diversity of the M protein family and suggest a new mechanism whereby these proteins contribute to the virulence of group A streptococci.

Analysis of plasminogen-binding M proteins of Streptococcus pyogenes.

Group A streptococci are common human pathogens that cause a variety of infections. They express M proteins which are important cell wall-bound type-specific virulence factors. We have found that a set of strains, associated primarily with skin infections, express M proteins that bind plasminogen and plasmin with high affinity. The binding is mediated by a 13-amino-acid internal repeated sequence located in the N-terminal surface-exposed portion of these M proteins. This sequence binds to kringle 2 in plasminogen, a domain that is not involved in the interaction with streptokinase, a potent group A streptococcal activator of plasminogen. It could be demonstrated that plasminogen, absorbed from plasma by growing group A streptococci expressing the plasminogen-binding M proteins, could be activated by exogenous and endogenous streptokinase, thereby providing the bacteria with a surface-associated enzyme that could act on the tissue barriers in the infected host.

Protein G genes: structure and distribution of IgG-binding and albumin-binding domains.

Protein G (also designated Fc receptor type III) is the IgG-binding protein of group C and G streptococci. Protein G has also been shown to bind human serum albumin but at a site that is structurally separated from the IgG-binding region. From the known gene sequence of protein G, two synthetic oligonucleotides were constructed for use as probes in DNA-hybridization experiments to study the structure and distribution of the albumin- and IgG-binding regions in bacterial strains belonging to different species. Thus, one of the probes corresponded to repeats within the IgG-binding region (I) and the other corresponded to repeats in the albumin-binding encoding region (II). Probe I showed strong hybridization to DNA isolated from 31 human group C and G strains, whereas hybridization to probe II was variable. With the three restriction endonucleases used, three restriction patterns were found in Southern blot experiments. No fundamental difference could be detected in hybridization experiments, either between strains of group C and G streptococci, or between isolates of different clinical origin. No hybridization to DNA from other bacterial species was found.

Structure of peptostreptococcal protein L and identification of a repeated immunoglobulin light chain-binding domain.

The gene for protein L, an immunoglobulin (Ig) light chain-binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus, was cloned and sequenced. The gene translates into a protein of 719 amino acid residues. Following a signal sequence of 18 amino acids and a NH2-terminal region ("A") of 79 residues, the molecule contains five homologous "B" repeats of 72-76 amino acids each. Further, toward the COOH terminus, two additional repeats ("C") were found. These are not related to the "B" repeats, but are highly homologous to each other. After the C repeats (52 amino acids each), a hydrophilic, proline-rich putative cell wall-spanning region ("W") was found, followed at the COOH-terminal end by a hydrophobic membrane anchor ("M"). Fragments of the gene were expressed, and the corresponding peptides were analyzed for Ig-binding activity. The B repeats were found to be responsible for the interaction with Ig light chains. An Escherichia coli high level expression system was adapted for the production of large amounts of two Ig-binding protein L fragments comprising one and four B repeats, respectively.

Streptococcal protein G. Gene structure and protein binding properties.

Protein G was solubilized from 31 human group C and G streptococcal strains with the muralytic enzyme mutanolysin. As judged by the mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the binding patterns of the solubilized protein G molecules in Western blot experiments, the strains could be divided into three groups, represented by the group G streptococcal strains G148 and G43 and the group C streptococcal strain C40. The 65-kDa G148 protein G and the 58-kDa C40 protein G showed affinity for both immunoglobulin G (IgG) and human serum albumin (HSA), whereas the 40-kDa G43 protein G bound only IgG. Despite the different molecular patterns, the three protein G species had identical NH2-terminal amino acid sequences. Apart from the 65-kDa peptide, digestion of G148 streptococci with mutanolysin also produced a 52-kDa IgG- and HSA-binding peptide and a 14-kDa HSA-binding peptide. It was demonstrated that these peptides resulted from cleavage of 65-kDa protein G by proteolytic components in the mutanolysin preparation. The protein G genes of the C40 and G43 strains were cloned and sequenced, and their structure was compared to the previously published sequence of the G148 protein G gene. As compared to G148, both the C40 and G43 genes lacked a 210-base pair fragment in the IgG-binding region, accounting for the 10-fold lower affinity of these proteins for IgG. The G43 gene also lacked a 450-base pair fragment in the 5'-end of the gene, explaining why the G43 protein G did not bind HSA. The differences in protein G structure did not correlate with the clinical origin of the strains used in this study. The IgG-binding region of protein G was further mapped. Thus, a peptide corresponding to a single IgG-binding unit was obtained by the cloning and expression of a 303-base pair polymerase chain reaction-generated DNA fragment. The affinity of this 11.5-kDa peptide for human IgG was 8.0 x 10(7) M-1, as determined by Scatchard plots. Finally, a 55-amino acid-long synthetic peptide, corresponding to one of the three repeated domains in the COOH-terminal half of strain G148 protein G, effectively blocked binding of protein G to IgG.

Plasminogen, absorbed by Escherichia coli expressing curli or by Salmonella enteritidis expressing thin aggregative fimbriae, can be activated by simultaneously captured tissue-type plasminogen activator (t-PA).

Curli are fimbrial structures expressed by Escherichia coli that specifically interact with matrix proteins such as fibronectin and laminin. Similar structures are also expressed by Salmonella enteritidis and have been denoted thin aggregative fimbriae. Bacteria expressing curli and thin aggregative fimbriae were found to bind radiolabelled plasminogen as well as the tissue-type plasminogen activator (t-PA). By contrast, E. coli carrying a gene locus with an insertionally inactivated chromosomal curlin subunit were unable to bind the two human proteins. The purified subunit polypeptides of curli and thin aggregative fimbriae bound plasminogen and t-PA with high affinity (1 x 10(8) to 2 x 10(8) M-1). The binding of plasminogen and t-PA to curli-expressing E. coli was only partially inhibited by fibronectin and laminin. Plasminogen absorbed from human plasma by curli-expressing E. coli was readily converted to plasmin by t-PA; both plasmin and t-PA were functionally active when bound to the bacteria. A simultaneous binding of fibrinolytic proteins and matrix proteins to fimbriae of E. coli and S. enteritidis could provide these pathogens with both adhesive and invasive properties.

Diagnosis of tuberculous meningitis: a comparative analysis of 3 immunoassays, an immune complex assay and the polymerase chain reaction.

OBJECTIVE: To compare 3 immunoassays, an immune complex assay, and an application of the polymerase chain reaction (PCR) for the diagnosis of tuberculous meningitis (TBM). MATERIAL: Cerebrospinal fluid (CSF) from 33 patients with TBM and from 34 control patients with infectious and non-infectious CNS diseases was analysed. RESULTS: The antibody immunoassays were either nonspecific or insensitive. However, detection of mycobacterial IgG immune complexes correlated strongly with infection, as they were detected in the CSF from 64% of the patients with TBM compared to only 3 (9%) of the control samples. PCR analysis, using Mycobacterium tuberculosis-specific oligonucleotide primers, also strongly correlated with infection, as DNA was amplified from 54% of the samples from patients with TBM, but from only 2 (6%) of the control samples. Both 'false positive' samples were also positive in the immune complex assay and came from 2 patients with otogenic brain abscesses. It is conceivable that these patients suffered from otogenic tuberculosis with secondary non-mycobacterial meningitis. When combining the immune complex assay with DNA-detection by PCR, 100% of the culture positive and 74% of culture negative samples were found to be positive, while maintaining a high specificity. CONCLUSION: Parallel analysis to detect mycobacterial immune complexes and M. tuberculosis-specific DNA by PCR from the CSF of patients may offer a sensitive and specific tool for the diagnosis of TBM.

Protein LG: a hybrid molecule with unique immunoglobulin binding properties.

Immunoglobulin (Ig)-binding bacterial proteins have attracted theoretical interest for their role in molecular host-parasite interactions, and they are widely used as tools in immunology, biochemistry, medicine, and biotechnology. Protein L of the anaerobic bacterial species Peptostreptococcus magnus binds Ig light chains, whereas streptococcal protein G has affinity for the constant (Fc) region of IgG. In this report, Ig binding parts of protein L and protein G were combined to form a hybrid molecule, protein LG, which was found to bind a large majority of intact human Igs as well as Fc and Fab fragments, and Ig light chains. Binding to Ig was specific, and the affinity constants of the reactions between protein LG and human IgG, IgGFc fragments, and kappa light chains, determined by Scatchard plots, were 5.9 x 10(9), 2.2 x 10(9), and 2.0 x 10(9) M-1, respectively. The binding properties of protein LG were more complete as compared with previously described Ig-binding proteins when also tested against mouse and rat Igs. This hybrid protein thus represents a powerful tool for the binding, detection, and purification of antibodies and antibody fragments.

Protein LA, a novel hybrid protein with unique single-chain Fv antibody- and Fab-binding properties.

Existing Ig-binding proteins all suffer from limitations in their binding spectrum. In the pursuit of the ultimate, non-restricted, Ig-binding protein, we have constructed the hybrid protein LA, by fusing four of the Ig kappa light-chain-binding domains of peptostreptococcal protein L with four of the IgGFc- and Fab-binding regions of staphylococcal protein A. Ligand-blot experiments demonstrated that the L and the A components were both functional in the hybrid, as the protein was shown to bind purified kappa light chains and IgGFc. Protein LA bound human Ig of different classes and IgG from a wide range of mammalian species. IgG, IgM and IgA were purified from human serum and saliva by affinity chromatography on protein LA agarose. Similarly, single-chain Fv (scFv) antibodies carrying the kappa light-chain variable domain or expressing the V(H)III (variable domain of the heavy chain of Ig) determinant, were efficiently purified on immobilized protein LA. As judged by surface plasmon resonance (SPR), protein LA showed enhanced affinity for all tested ligands, including several scFv antibodies, compared with proteins L and A alone. SPR analysis also demonstrated that binding of a ligand to one of the components in protein LA did not affect the ability of the hybrid protein to interact simultaneously with a ligand for the other component. The antigen-binding capacity of a kappa-expressing scFv antibody was unaffected by the interaction with protein LA, whereas the binding of a V(H)III-expressing scFv antibody to its antigen was, unexpectedly, blocked by protein A and protein LA. Together, these data demonstrate that protein LA represents a highly versatile Ig-binding molecule.

Streptokinase activates plasminogen bound to human group C and G streptococci through M-like proteins.

An ability to interact with plasminogen or plasmin could provide micro-organisms with a mechanism for invasion. Thus, group A, C and G streptococci secrete streptokinase which binds and activates plasminogen. Some streptococci also express surface structures which bind plasminogen without causing its activation. Plasminogen-binding surface proteins were extracted from one group C and one group G streptococcal isolate. Both proteins were found to bind plasmin, fibrinogen and serum albumin in addition to plasminogen. Gene fragments encoding the streptococcal proteins were amplified by PCR and were subsequently cloned and expressed in Escherichia coli. DNA sequence determination revealed for both genes open reading frames encoding proteins which contained repetitive domains and a carboxyl-terminal unrepeated region that were typical of M and M-like proteins. Though the amino-terminal regions of the group C and G streptococcal proteins demonstrated a rather high overall similarity between themselves, they were not similar to the variable regions of other M-like proteins with one exception: there was a 46% identity between the first 22 amino acids of the group G streptococcal protein and the corresponding sequence of PAM, the plasminogen-binding M-like protein of type M53 group A streptococci. Like the proteins extracted from the streptococci, the recombinant proteins bound plasminogen, fibrinogen and albumin. The three plasma proteins bound to separate sites on the streptococcal M-like proteins. Plasminogen bound by the group C and G streptococcal proteins was readily activated by streptokinase, providing evidence for a functional link between the secreted plasminogen-activator and proteins exposed on the bacterial surface.

Selective distribution of a high-affinity plasminogen-binding site among group A streptococci associated with impetigo.

Group A streptococci can be classified according to their tendency to cause either impetigo, pharyngitis, or both types of infection. Genotypic markers for tissue site preference lie within emm genes, which encode fibrillar surface proteins that play a key role in virulence. emm gene products (M and M-like proteins) display an extensive array of binding activities for tissue and plasma proteins of the human host. In a previous study, a high-affinity binding site for human plasmin(ogen) was mapped to the emm53 gene product. In this report, a structurally similar plasminogen-binding domain is found to be widely and selectively distributed among group A streptococci harboring the emm gene marker for the skin as the preferred tissue site for infection. The findings are highly suggestive of a central role for bacterial modulation of host plasmin(ogen) during localized infection at the epidermis.

Zinc-dependent conformational changes in domain D5 of high molecular mass kininogen modulate contact activation.

Human high molecular mass kininogen (HK) participates as nonenzymatic cofactor in the contact system. Here, we show that recombinant domain D5 of HK (rD5) prolongs the clotting time of the intrinsic pathway of coagulation and attenuates the generation of bradykinin. Further studies indicate that a correct fold of domain D5 within HK is required for the activation of the contact system. The folding of rD5 seems to be modulated by the metal ions Zn2+, Ni2+, and Cu2+ as a specific antibody directed against the zinc-binding site in HK binds to HK and rD5 in a metal ion concentration dependent manner. The finding that these three metal ions specifically affect contact activation suggests that they regulate the accessibility of rD5 for negatively charged surfaces. Support for the assumption that the observed phenomena are due to conformational changes was obtained by fluorescence spectroscopy of rD5, demonstrating that its fluorescence spectrum was changed in the presence of ZnCl2. Moreover, negative staining electron microscopy experiments suggest that the zinc-induced changes in D5 also affect the conformation of the entire HK protein. The present data emphasize the role of zinc and other metal ions in the regulation of contact activation.