Effect of indoor air quality on the development of rhinitis in an urban population in Poland.

Background: Indoor air significantly impacts the incidence of rhinitis among residents of urban agglomerations. Objective: To assess the impact of indoor air pollution on rhinitis. Methods: Data were collected by using an European Community Respiratory Health Survey (ECRHS) and International Study of Asthma and Allergies in Childhood (ISAAC) standardized questionnaires (N = 18,617), and medical examinations were carried out (N = 4783) in selected Polish regions. For statistical analysis, the odds ratio (OR) was calculated with a 95% confidence interval to detect factors associated with rhinitis. The Wald test was performed to assess the significance of those factors. A value of p < 0.05 was considered statistically significant. Results: The most important factors associated with allergic rhinitis declared by adults, ages 20­44 years were the following: the age of the buildings (OR 1.34), presence of central heating system (OR 1.19), gas furnace used to heat the house (OR 1.19), solid-fuel stove (OR 1.92), and bottled-gas stove (OR 1.66). More frequent declarations of nonallergic rhinitis in the study group were associated with the use of a central heating system (children ages 6­7 years: OR 1.21; children ages 13­14 years: OR 1.22; and adults, ages 20­44 years: OR 1.27), solid-fuel stove (children ages 6­7 years: OR 2.95; children ages 13­14 years: OR 2.86; adults, ages 20­44 years: OR 2.02), and bottled-gas stove (children ages 6­7 years: OR 1.89; children ages 13­14 years: OR 1.88; adults, ages 20­44 years: OR 2.06). Diagnosed seasonal allergic rhinitis in adults, ages 20­44 years was associated with the year when the building was constructed (1970­1990) (OR 1.93) and the presence of a central heating system (OR 1.85). The year of construction of a building (1946­1969) (OR 4.84) as well as the use of central heating (OR 1.79) were causes of allergies to molds in the group of children ages 6­7 years, whereas sensitization to Dermatophagoides. pteronyssinus (OR 1.62) and Dermatophagoides farinae (OR 1.78) in children ages 6­7-years was associated with the presence of a central heating system. In children ages 13­14 years, the use of a solid-fuel stove was a cause of sensitization to D. farinae (OR 1.62). Conclusion: The age of the building, home heating systems, and pollution emitted by cooking appliances have a significant impact on the incidence of rhinitis. The highlights of the study included the following: (1) the age and condition of the building, the use of heating devices, stoves, and also mold allergens and house-dust mites contributed to a higher incidence of rhinitis, mainly among adults, ages 20­44 years; (2) gas-storage tanks and solid fuels contribute to rhinitis in the group of children ages 6­7 years and children ages 13­14 years.

Intramolecular immunological signal hypothesis revived--structural background of signalling revealed by using Congo Red as a specific tool.

Micellar structures formed by self-assembling Congo red molecules bind to proteins penetrating into function-related unstable packing areas. Here, we have used Congo red--a supramolecular protein ligand--to investigate how the intramolecular structural changes that take place in antibodies following antigen binding lead to complement activation. According to our findings, Congo red binding significantly enhances the formation of antigen-antibody complexes. As a result, even low-affinity transiently binding antibodies can participate in immune complexes in the presence of Congo red, although immune complexes formed by these antibodies fail to trigger the complement cascade. This indicates that binding of antibodies to the antigen may not, by itself, fulfill the necessary conditions to generate the signal which triggers effector activity. These findings, together with the results of molecular dynamics simulation studies, enable us to conclude that, apart from the necessary assembling of antibodies, intramolecular structural changes generated by strains which associate high- affinity bivalent antibody fitting to antigen determinants are also required to cross the complement activation threshold.

Intramolecular signaling in immunoglobulins -- new evidence emerging from the use of supramolecular protein ligands.

The postulated intramolecular signaling in immunoglobulins generated by antigen binding has been controversial for years. The high heterogeneity of immune complexes as signaling systems and the requirement of the immobilized antigen form for efficient triggering of effector activity is likely the reason for the lack of clarity. Here we present new evidence supporting the notion of intramolecular signaling, based on the use of supramolecular dyes that bind to signal-derived specific sites in immunoglobulins.

Induction of intracellular cytokine production in human monocytes/macrophages stimulated with ligands of pattern recognition receptors.

OBJECTIVE: This study addressed the role of the pattern recognition receptors (PRR), which recognize different molecular structures present on microorganisms, apoptotic, senescent and tumor cells, in the stimulation of human monocyte and monocyte-derived macrophages (MDM) for the production of intracellular cytokines. MATERIALS AND METHODS: Monocytes and MDM were stimulated with different ligands of scavenger receptors (SR) and mannose receptor (MR). Production of intracellular cytokines: tumor necrosis factor alpha (TNF alpha), interleukin 10 and 12 (IL-10, IL-12) was determined by flow cytometry following staining with anti-cytokine monoclonal antibodies (mAbs). RESULTS: The ligands of SR type A: fucoidan, polyguanylic acid (polyG), chemically modified low density lipoproteins (LDL), ligands of SR-B: native and chemically modified LDL, and ligand of mannose receptor (MR)-mannan induced strong expression of intracellular TNF alpha and weaker IL-10 in monocytes, while phosphatidylserine (PdS) was without effect. IL-12 was stimulated only by fucoidan and polyG. The induction of cytokine m-RNA generally followed the pattern and the magnitude of intracellular cytokine production. In MDM, intracellular TNF alpha and IL-12 expression was induced by mannan, native and modified LDL, but not other ligands. Expression of IL-10 was less pronounced and occurred following stimulation with fucoidan, polyG and modified, but not native, LDL. CONCLUSIONS: These results suggest that some PRR ligands may be involved in activation of monocytes/MDM for the production of mainly proinflammatory cytokines (TNF alpha, IL-12) implicating their role in the response to microbial and tumor invasion.

Egg yolk platelet proteins from Xenopus laevis are amyloidogenic.

The association of amphibian (Xenopus laevis) egg yolk platelet proteins, represented predominantly by lipovitellin, was studied as a model of the formation of amyloid deposits. Two kinds of molecular organization formed by this protein material - native and heat-denatured - were found to exhibit amyloid properties although they differ significantly in structural organization. The first consisted in protein molecules arranged in the natural, physiological, net-like platelet organization, with a tendency to orient uni-directionally. The second was obtained by the gradual removal of Congo red from lipovitellin denatured by heating in an excess of dye. This procedure produced the twisted fibrillar organization of molecules typical for amyloids, represented predominantly by end-to-end associated major polypeptide chains of lipovitellin. Both native and denatured structural forms bind Congo red and produce a green birefringence effect, confirming the near parallel alignment of the complexed Congo red molecules. However, a dye(1,4-bis(1-amino-4-sulfonaphtyl-2-azo)phenylene) closely related to Congo red but with a very weak self-assembling tendency appeared inactive when the spectral shift was studied in a cross-polarization system, indicating in this way that dye supramolecularity is an extra factor which may determine binding to amyloid proteins and specific spectral effects.

Heat-induced formation of a specific binding site for self-assembled Congo Red in the V domain of immunoglobulin L chain lambda.

Moderate heating (40-50 degrees C) of immunoglobulins makes them accessible for binding with Congo Red and some related highly associated dyes. The binding is specific and involves supramolecular dye ligands presenting ribbon-like micellar bodies. The L chain lambda dimer, which upon heating disclosed the same binding requirement with respect to supramolecular dye ligands, was used in this work to identify the site of their attachment. Two clearly defined dye-protein (L lambda chain) complexes arise upon heating, here called complex I and complex II. The first is formed at low temperatures (up to 40-45 degrees C) and hence by a still native protein, while the formation of the second one is associated with domain melting above 55 degrees C. They contain 4 and 8 dye molecules bound per L chain monomer, respectively. Complex I also forms efficiently at high dye concentration even at ambient temperature. Complex I and its formation was the object of the present studies. Three structural events that could make the protein accessible to penetration by the large dye ligand were considered to occur in L chains upon heating: local polypeptide chain destabilization, VL-VL domain incoherence, and protein melting. Of these three possibilities, local low-energy structural alteration was found to correlate best with the formation of complex I. It was identified as decreased packing stability of the N-terminal polypeptide chain fragment, which as a result made the V domain accessible for dye penetration. The 19-amino acid N-terminal fragment becomes susceptible to proteolytic cleavage after being replaced by the dye at its packing locus. Its splitting from the dye-protein complex was proved by amino acid sequence analysis. The emptied packing locus, which becomes the site that holds the dye, is bordered by strands of amino acids numbered 74-80 and 105-110, as shown by model analysis. The character of the temperature-induced local polypeptide chain destabilization and its possible role in intramolecular antibody signaling is discussed.

Why Congo red binding is specific for amyloid proteins - model studies and a computer analysis approach.

BACKGROUND: The complexing of Congo red in two different ligand forms - unimolecular and supramolecular (seven molecules in a micelle) - with eight deca-peptides organized in a b-sheet was tested by computational analysis to identify its dye-binding preferences. Polyphenylananine and polylysine peptides were selected to represent the specific side chain interactions expected to ensure particularly the stabilization of the dye-protein complex. Polyalanine was used to verify the participation of non-specific backbone-derived interactions. MATERIAL AND METHODS: The initial complexes for calculation were constructed by intercalating the dye between the peptides in the middle of the beta-sheet. The long axis of the dye molecule (in the case of unimolecular systems) or the long axis of the ribbon-like micelle (in the case of the supramolecular dye form) was oriented parallel to the peptide backbone. This positioning maximally reduced the exposure of the hydrophobic diphenyl (central dye fragment) to water. In general the complexes of supramolecular Congo red ligands appeared more stable than those formed by individual dye molecules. Specific interactions (electrostatic and/or ring stacking) dominated as binding forces in the case of the single molecule, while non-specific surface adsorption seemed decisive in complexing with the supramolecular ligand. RESULTS: Both the unimolecular and supramolecular versions of the dye ligand were found to be likely to form complexes of sufficient stability with peptides. The low stability of the protein and the gap accessible to penetration in the peptide sheet seem sufficient for supramolecular ligand binding, but the presence of positively charged or hydrophobic amino acids may strengthen binding significantly. CONCLUSIONS: The need for specific interaction makes single-molecule Congo red binding rather unusual as a general amyloid protein ligand. The structural feature of Congo red, which enables specific and common interaction with amyloid proteins, probably derives from the ribbon-like self-assembled form of the dye.

Evidence that supramolecular Congo red is the sole ligation form of this dye for L chain lambda derived amyloid proteins.

The mechanism of Congo red binding to amyloid protein was studied in order to establish which of two structural dye versions present in water solutions--unimolecular and supramolecular--represent its actual ligation form. Immunoglobulin L chain lambda of amyloidogenic nature, expressed by Congo red binding and easy gel formation, was used as the model amyloid protein. Congo red was coassembled with rhodamine B, designed to be a marker of the Congo red micellar organisation in complexation with protein. The particular suitability of rhodamine B for this role results from significant difference in its binding affinity to Congo red and to protein. It associates readily with Congo red, becoming incorporated into its micellar organisation, but as homogenous dye it shows an almost complete inability to bind to protein. In view of these properties, Congo red was used as a vehicle to draw rhodamine B into complexation with protein, at the same time supplying evidence of its supramolecular ligation form. The results show that both soluble amyloid precursor L chain and the derived gel material attach rhodamine B coassembled with Congo red but not the homogenous rhodamine B. Despite its dynamic, supramolecular character, Congo red participates in complexation with amyloid proteins as an integral ligand unit.

The conformational characteristics of Congo red, Evans blue and Trypan blue.

The structures of the closely related bis-azo dyes Evans blue, Trypan blue and Congo red, which appeared to have different self-assembly properties and correspondingly different abilities to form complexes with amyloids and some other proteins, were compared in this work. Ab initio and semi-empirical methods were used to find the optimal structures and partial charge distributions of the dyes. The optimal structures were searched using different widely used programs. The structures of Congo red and evans blue were found to be planar, except for the torsion on the central diphenyl bond connecting the two halves of the dye. Both symmetrical parts of the molecules appeared very close to planarity. However, Trypan blue exhibits non planarity on the di-azo bonds, as well as on the central bond between the symmetrical parts of the dye. In a consequence, the non planarity of this molecule is higher than in the case of its isomer, Evans blue and Congo red as well. The extra rotation around the azo bonds extorted by the close proximity of the sulfonic groups may be the direct cause of its poor self-assembling and complexation properties versus Evans blue.

Supramolecularity creates nonstandard protein ligands.

Congo red and a group of structurally related dyes long used to stain amyloid proteins are known to associate in water solutions. The self-association of some dyes belonging to this group appears particularly strong. In water solutions their molecules are arranged in ribbon-like micellar forms with liquid crystalline properties. These compounds have recently been found to form complexes with some native proteins in a non-standard way. Gaps formed by the local distribution of beta-sheets in proteins probably represent the receptor sites for these dye ligands. They may result from higher structural instability in unfolding conditions, but also may appear as long range cooperative fluctuations generated by ligand binding. Immunoglobulins G were chosen as model binding proteins to check the mechanism of binding of these dyes. The sites of structural changes generated by antigen binding in antibodies, believed to act as a signal propagated to distant parts of the molecule, were assumed to be suitable sites for the complexation of liquid-crystalline dyes. This assumption was confirmed by proving that antibodies engaged in immune complexation really do bind these dyes; as expected, this binding affects their function by significantly enhancing antigen binding and simultaneously inhibiting C1q attachment. Binding of these supramolecular dyes by some other native proteins including serpins and their natural complexes was also shown. The strict dependence of the ligation properties on strong self-assembling and the particular arrangement of dye molecules indicate that supramolecularity is the feature that creates non-standard protein ligands, with potential uses in medicine and experimental science.

Supramolecular ligands: monomer structure and protein ligation capability.

The aim of this work was to define the chemical structure of compounds self-assembling in water solutions, which appear to interact with proteins as single ligands with their supramolecular nature preserved. For this purpose the ligation to proteins of bis azo dyes, represented by Congo red and its derivatives with designed structural alterations, were tested. The three parameters which characterize the reactivity of supramolecular material were determined in the same conditions for all studied dyes. These were: A) stability of the assembly products; B) binding to heat-denatured protein (human IgG); and C) binding to native protein (rabbit antibodies in the immune complex) measured by the enhancement of hemagglutination. The structural differences between the Congo red derivatives concerned the symmetry of the molecule and the structure of its non-polar component, which occupies the central part of the dye molecule and is thought to be crucial for self-assembly. Other dyes were also studied for the same purpose: Evans blue and Trypan blue, bis-ANS and ANS, as well as a group of compounds with a structural design unlike that of bis azo dyes. Compounds with rigid elongated symmetric molecules with a large non-polar middle fragment are expected to form a ribbon-like supramolecular organization in assembling. They appeared to have ligation properties related to their self-assembling tendency. The compounds with different structures, not corresponding to bis azo dyes, did not reveal ligation capability, at least in respect to native protein. The conditions of binding to denatured proteins seem less restrictive than the conditions of binding to native molecules. The molten hydrophobic protein interior becomes a new binding area allowing for complexation of even non-assembled molecules.

Effect of self association of bis-ANS and bis-azo dyes on protein binding.

A correlation was found between the ability of dyes (ANS, bis-ANS, Congo red, Evans blue) to form self-associated supramolecular structures in water and their tendency to form complexes with proteins. The self-association ability of dyes was measured as the resistance of a molecular sieve to their penetration. Quantitative evaluation of dye-protein interaction involved measuring the effect of dye on antibodies that agglutinate sheep red blood cells. Enhancement of agglutination by dye was assumed to represent its protein complexation ability. The results confirm that, relative to monomers, self-associated ligands also have altered protein binding properties.

The use of congo red as a lyotropic liquid crystal to carry stains in a model immunotargeting system--microscopic studies.

The lyotropic liquid crystal dye-Congo Red was used as a carrier in a model immunotargeting system constructed from sheep red blood cells (SRBC) representing the antigen target and rabbit IgG anti-SRBC as the specific driving immunoglobulin. Rhodamine B and Hemin stains were chosen as example chemicals carried to the target. The carried stains were introduced to the micellar organization of Congo Red by intercalation. Preserving its supramolecular organization, Congo Red binds spontaneously and selectively to antibodies that have altered structure extorted by interaction with the antigen in the immune complex. The functionality of the studied immunotargeting model was verified by fluorescence and electron microscopy. The results indicate that the supramolecular nature of protein ligands offers new ligation capabilities possibly useful for carrying stains or drugs in immune-oriented systems.

Congo red-stabilized intermediates in the lambda light chain transition from native to molten state.

Disruption of tertiary interaction makes a protein accessible to penetration by different small molecular compounds. Their interaction may stabilize the altered protein conformation. Congo red is proposed here as a stabilizer of the molten globule state and also of highly reversible intermediates in the transition from native to molten state. Human immunoglobulin lambda light chain (dimer) was used. Two protein-Congo red complexes were found after heating lambda chain in the presence of Congo red. They differed in the amount of attached dye molecules. The binding of dye was interpreted as a two-step dye penetration process involving the peripheral parts of the protein in the first step (at lower temperatures). It was concluded that the liquid crystal properties of Congo red enable it to form specific complexes with proteins, which have become accessible to penetration by ligands after global or local disruption of tertiary interaction. This dye may thus be used as a stabilizer of unfolding intermediates in the step preceding the molten globule state.

The detection of specific acute phase serum protein complexes and immune complexes by congo red binding.

The study concerns the problem of selective binding of Congo Red dye to serum proteins. The dye-binding structural motifs were assumed to appear in proteins as the result of local disruptions of tertiary interaction associated with biological activities. Functional structures of serum proteinase inhibitors and haptoglobins induced by interaction of these proteins with their natural substrates were found to bind Congo Red. Soluble immune complexes formed by bivalent hapten-bridged anti-TNP rabbit antibodies also appeared able to interact with the dye. The possible use of these effects for diagnostic analysis was suggested.

Bis azo dye liquid crystalline micelles as possible drug carriers in immunotargeting technique.

Lyotropic liquid crystal mesophases, represented in this work by Congo Red bis azo dye solution, were proposed as systems to carry drugs to immuno-selected targets. The possible use of Congo Red for this aim arises from its liquid crystalline features, enabling it to attach to immune complexes as polymolecular, ordered conglomerates and simultaneously to incorporate many organic compounds into its mesophase, largely independent of the water with a specific but adaptive molecular organization. Molecules with planar rigid structure, and/or large hydrophobic fragments, especially those with a positively charged group in the molecule, are found to be incorporated best. Rhodamine B, Rhodamine 6G and adriamycine, which have the assumed binding features, were tested as model compounds and were found to be readily engaged into Congo Red mesophase. The effect of hydrophobicity on ligand binding was evaluated following the incorporation of homologic 10, 12, 14, 16 carbon chain organic acids and the effect of charge using small mobile tandem molecules of pI differing by a few pH units (lysine-norleucine, tyrosine-tyramine). Positive charge seems to affect binding especially by influencing the organization of molecules and the shape of the micelle simultaneously. Congo Red immobilized to heat-aggregated immunoglobulins and antibodies in the immune complex was found to retain its binding ability, confirming its possible usefulness for drug transport.

The melting of native domain structure in effector activation of IgG studied by using congo red as a specific probe.

The nature of structural changes in IgG molecules associated with the binding to antigen and/or heat aggregation was studied using bis azo dye (Congo Red) as the specific probe. It was found, that protein conformation responsible for binding the dye represents an unfolding intermediate with properties corresponding to a molten globule state. The properties of the dye-protein complex reveal the signs of an unfolding of the peptide chain with simultaneously preserved relatively compact packing. Immunoglobulins which were induced by heating, or binding to antigen in order to form the complex with dye ligands, become more susceptible for digestion. The main peptide of molecular weight 30,000 D which appears in products was suggested to originate from a heavy chain after its splitting in the region of CH1 domain. The energetic evaluation of stability of IgG domains also indicates that CH1 is the least stable fragment of the heavy chain and its conformation may be destabilized first. It was concluded that destabilized tertiary packing of antibodies bound to antigen may favour the association of closely situated immunoglobulin molecules increasing the stability of the immune complex and influencing in the result its effector activity.

Heat-induced structural changes in the Fab fragment of IgG recognized by molecular dynamics stimulation--implications for signal transduction in antibodies.

Molecular dynamics simulation was used to identify the conformational alterations in the Fab fragment (Kol), driven by heating at 300, 320, and 340 K. Comparison of heat-modified VH,CH1 and VL,CL domain structures with the corresponding crystal conformations revealed specific differences, most definitely expressed in the CH1 domain. These are dislocations of predominantly peripheral peptide loops exposed to the V-C interdomain interface, comprising in particular the 175-185 amino acids of the heavy chain, as well as the 112-123 amino acids of the interdomain hinge fragment. The deviations, limited to peripheral domain regions at 300 and 320 K, spread at 340 K. The resulting relaxation of the tertiary packing in the protein (including the hydrophobic core) initiates global melting of the domain. These theoretical results were supported by experimental findings concerning penetration and binding of dyes (Congo Red, Trypan Blue, ANS) to the protein. Packing relaxation of the CH1 domain is turned on after disruption of the specific peptide arrangement formed at the V-C interdomain interface basically at the hinge portion (117-122) and at fragments of adjacent peptide loops (149-154, 171-179) of CH1 origin, most probably playing the role of a switching mechanism. The dislocations also comprise the 131-141 amino acids of the loops accommodated at the CH1-CH2 interface. However, the lack of crystallographic data concerning the Fab-Fc interface limits discussion of this effect to speculation. It was concluded that the unconcerted movements of the V and C parts of the Fab fragment are an intrinsic driving mechanism, introducing structural alterations into the C domains. It is suggested that the domain relaxation, induced by heating or mechanical constraints, allows for intermolecular interactions, affecting in this way the stability of the immune complex.

Bis azo dyes--studies on the mechanism of complex formation with IgG modulated by heating or antigen binding.

The mechanism of binding of azo dyes (bis azo) to immunoglobulin G of altered conformation, induced by heating or interaction with antigen was analysed in this work. Azo dyes: Congo Red, Evans Blue and Trypan Blue were selected for these studies. The molecules of Congo Red and Evans Blue associate readily in water and exist as polymolecular micellar species of liquid crystalline organization. Such organization of molecules appeared necessary for these dyes to interact with antibodies and to affect the formation of immune complex. It was proved by studying the properties of isomeric dyes Evans Blue and Trypan Blue, whose ability to form polymolecular conglomerates in water differs, being high for Evans Blue and low for Trypan Blue. The dyes seem to influence the formation of the immune complex generally by interacting with individual immunoglobulin molecules. The rate of exchange of dye molecules in conglomerate bound to protein varies and is the lowest for the portion of molecules which are engaged directly in the complex with protein. Electron microscopic studies also confirmed the polymolecular form of the dye in the dye-protein complex. The computational simulation of dye-dye and dye-protein interaction was performed in a model system. The micelle in the system was represented by three molecules of Congo Red. The peptide loop composed of amino acids 68-88 originating from VL IgG domain, was selected to represent protein. Amino acid side chains of this fragment were reduced to C beta. The best fitness was found for peptide chains of twisted beta conformation and independently optimized conformation of the dye in a form of "twisted ribbon" micelle. It was concluded that the IgG domains become accessible for penetration of the dye after being relaxed in the result of heating or interaction with the antigen.

Bis-azo dyes interference with effector activation of antibodies.

Azo dyes (Congo Red, Evans Blue, Trypan Blue) were used to study the relation between antigen binding and effector activity in SRBC rabbit anti-SRBC IgG system. Antigen binding was tested in this study by agglutination and effector activity by complement fixation. Antibodies induced by the antigen binding appeared to become structurally susceptible for interaction with the dye. The dyes cause the enhancement of antigen binding by antibodies and inhibition of their effector activity. The intensity of both effects caused by binding the dye seems correlated. The increase of antibody concentration makes the effector activity of immune complexes more resistant to dye inhibition indicating that the influence of antigen binding may be exerted on assembling of antibody in the immune complex rather than on structural modulation of Fc. Also the analysis of dye effects on antibody derivatives deprived of effector activity (Fab)2, IgG with split interheavy chain disulphides supplied evidence that the induced by the antigen long distance structural effects may play the role of immune signal by influencing the assembling of antibodies and stability of immune complex.