Preliminary criteria for the definition of allergic rhinitis: a systematic evaluation of clinical parameters in a disease cohort (II)

BACKGROUND: This study is a development of the work done in 'Preliminary criteria for the definition of allergic rhinitis: a systematic evaluation of clinical parameters in a disease cohort (I)'. OBJECTIVE: We sought to develop criteria which could be routinely used to define allergic rhinitis. METHODS: A total of 47 allergic rhinitis and 23 normal subjects were evaluated with a detailed questionnaire and history, physical examination, serum total immunoglobulin (Ig) E, skin prick tests and serum EAST's. RESULTS: Based on this data, we developed a preliminary scoring system by which a reliable diagnosis of allergic rhinitis can be made, which is based on the prevalence adjusted strength of association delta ( partial differential) rank values described in this report. Cumulative frequency histograms were constructed for allergic rhinitis and normal subjects in a variety of configurations. CONCLUSION: A simple scoring system which can be used to diagnose allergic rhinitis on a routine basis was developed in this study.

Preliminary criteria for the definition of allergic rhinitis: a systematic evaluation of clinical parameters in a disease cohort (I).

BACKGROUND: The aim of this study is to formulate criteria for the definition of allergic rhinitis. Other studies have sought to develop scoring systems to categorize the severity of allergic rhinitis symptoms but it was never used for the formulation of diagnostic criteria. These other scoring systems were arbitrarily chosen and were not derived by any statistical analysis. To date, a study of this kind has not been performed. OBJECTIVE: The hypothesis of this study is that it is possible to formulate criteria for the definition of allergic rhinitis. This is the first study to systematically examine and evaluate the relative importance of symptoms, signs and investigative tests in allergic rhinitis. We sought to statistically rank, from the most to the least important, the multiplicity of symptoms, signs and test results. METHODS: Forty-seven allergic rhinitis and 23 normal subjects were evaluated with a detailed questionnaire and history, physical examination, serum total immunoglobulin E, skin prick tests and serum enzyme allergosorbent tests (EAST). RESULTS: Statistical ranking of variables indicated rhinitis symptoms (nasal, ocular and oronasal) were the most commonly occurring, followed by a history of allergen provocation, then serum total IgE, positive skin prick tests and positive EAST's to house dust mite, perennial rye and bermuda/couch grass. Throat symptoms ranked even lower whilst EAST's to cat epithelia, plantain and cockroach were the least important. Not all symptoms, signs and tests evaluated proved to be statistically significant when compared to a control group; this included symtoms and signs which had been considered historically to be traditionally associated with allergic rhinitis, e.g. sore throat and bleeding nose. CONCLUSION: In performing statistical analyses, we were able to rank from most to least important, the multiplicity of symptoms signs and test results. The most important symptoms and signs were identified for the first time, even though some of these were not included in our original selection criteria for defining the disease cohort i.e. sniffing, postnasal drip, oedematous nasal mucosa, impaired sense of smell, mouth breathing, itchy nose and many of the specific provocation factors.

Immobiline-based two-dimensional gel electrophoresis: an optimised protocol for resolution of human colonic mucosal proteins.

An optimised protocol for the production of two-dimensional protein patterns of human colonic mucosal cells using immobilised pH gradients in the first dimension is presented. We tested a wide variety of solubilising agents and electrophoretic parameters, separately and in combination. Protein solubilisation was found to be best using a lysis solution containing 9 M urea, 2% Triton X-100, 2% 2-mercaptoethanol, 0.8% Pharmalyte pH 3-10 and 8 mM phenylmethylsulfonyl fluoride (PMSF). Horizontal streaking of basic proteins in the first dimension was virtually eliminated by a combination of washing Immobiline strips in 100 mM asorbic acid, pH 4.5, for 24 h before use and isoelectric focusing samples at 40 degrees C. The presence of 2% glycerol in the first dimension resulted in tighter resolution throughout the entire pH range. The use of these conditions may prove to have broad applicability to the generation of optimally resolved Immobiline-based two-dimensional protein patterns of many other tissues.

Purification and characterization of a human bradykinin binding protein from inflammatory cells.

Bradykinin (BK) is a potent mediator with a broad spectrum of pharmacological and inflammatory actions which are exerted through cell surface receptors. We report here the affinity chromatographic purification of a novel 14 kDa BK binding protein from human blood neutrophils and also peripheral blood mononuclear cells (PBMC), 80% of which are lymphocytes. Radioreceptor crosslinking experiments using bifunctional crosslinkers and radiolabelled BK identified a 14 kDa protein in these cell types both on the cell surface, in glycerol purified plasma membranes and in detergent solubilized cell extracts. Purification by BK affinity chromatography from a variety of BK responsive human cell types i.e. CCD-16Lu lung fibroblasts, HL60 promyelocytes, U937 myelomonocytes and Jurkat T lymphocytes also demonstrated a 14 kDa protein. Purified material obtained from three different BK affinity columns all demonstrated three major proteins at 190, 50 and 14 kDa when eluted with either excess BK or mild acid. Neutrophil fractions from detergent solubilized cell extracts contained an additional 150 kDa protein when eluted with mild acid. Neutrophil and PBMC crude plasma membrane BK affinity column purifications yielded only a single 14 kDa protein. Radioreceptor dot assays of the purified neutrophil eluates containing the 14 kDa protein revealed specific binding to [125I]-BK with a 160 fold excess signal ratio over the original membrane extract. Our data indicates that we have successfully isolated a 14 kDa novel human BK specific binding protein expressed on the surface of inflammatory cells.

High affinity bradykinin binding to human inflammatory cells.

Specific direct bradykinin (BK) binding and competitive inhibition was detected in human neutrophil and peripheral blood mononuclear cell (PBMC) detergent solubilized extracts and purified plasma membranes using in vitro radioreceptor ligand binding. Scatchard analyses of [125I]-BK binding revealed an equilibrium dissociation constant (Kd) of 2.9 x 10(-11) M for neutrophils and 5.6 x 10(-11) M for PBMC using [des-arg9]-BK a B1 agonist; 2.6 x 10(-11) M for neutrophils, 6.2 x 10(-11) M for PBMC with BK a B2 agonist; 5.4 x 10(-11) M for PBMC using Lys-BK a B2 agonist. The number of binding sites (Bmax) was calculated to be 0.113 fM/microgram protein (720 receptors per cell) for neutrophils and 0.200 fM/microgram protein (1289 receptors per cell) for PBMC with the B1 agonist while with the B2 agonists the values were 0.128 fM/microgram protein (818 receptors per cell) for neutrophils and 0.157 fM/microgram protein (1005 receptors per cell) for PBMC with BK, and 0.293 fM/microgram protein (1870 receptors per cell) with Lys-BK for PBMC. In a competitive binding inhibition assay using neutrophil and PBMC glycerol purified plasma membranes, high affinity binding in the nanomolar range was detected to Lys-BK and BK but with [des-arg9]-BK a 10-100 fold lower order affinity was observed this being indicative of pharmacologically defined B2 characteristics.

High resolution autoantibody detection by optimized protein G-horseradish peroxidase immunostaining in a western blotting assay.

We report a procedure for optimisation of Western blotting using protein G-horseradish peroxidase (protein G-HRP) which avoids the false positive reactions often caused by second antibodies and increases the detection of autoantibodies by protein G conjugate. A number of modifications were investigated. Higher concentrations of serum and protein G-HRP at 1:5 to 1:10 and 1:100, respectively, increased the detection to the same order as that obtained with second antibody systems and gold staining with silver enhancement. The role of various detergents in the procedure was established. 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) in incubation with protein G-HRP increased the binding between protein G and immunoglobulin G. Addition of Tween-20 for blocking produced little background so that protein blockers could be avoided. Prolonged incubation with serum increased markedly the sensitivity of the procedure when compared with the recommended 2 h incubation period. Polyvinylidene difluoride membrane provided better transfer effect, lower background and higher mechanical strength than nitrocellulose membrane. The utilization of only one antibody-specific ligand increased the simplicity, reliability, economy, efficiency and specificity of the method. These modifications make this method significantly better for detection and screening for autoantibodies.

Polymorphism of IgE gene in chronic urticaria.

Our aim was to determine whether heterogeneity of the IgE (C epsilon) gene could be demonstrated in patients with chronic urticaria (CU). We performed Southern blots on DNA extracted from peripheral blood leucocytes of 20 patients with CU and 20 normal controls. Using a human C epsilon gene probe containing four exons of the constant segment of the IgE heavy chain, we showed the presence of a restriction fragment length polymorphism of the C epsilon gene segment in four of 20 patients with CU, but in none of 20 normal subjects. Family studies of two propositi revealed the presence of this C epsilon gene polymorphism in other family members. Our data show that a proportion of patients with CU have a polymorphism of the constant segment of the C epsilon gene. Further studies of this polymorphic gene fragment indicated that it was derived from duplication of the 3rd and 4th exons of functional C epsilon gene and was very likely to be located close to this gene. It raises the possibility that polymorphism of the functional C epsilon gene may affect expression of this gene. This could possibly lead to dysfunctional IgE-receptor interaction with consequent alteration in mediator release.

A preparative method for sequencing proteins and peptides: in situ gel staining with subsequent passive elution onto polyvinylidine difluoride membranes.

A preparative method for obtaining both N-terminal and internal peptide amino acid sequences from purified proteins is reported. The methodology reliably yields high fidelity signal from between 14 to 30 residues per purified protein or peptide, with low backgrounds on amino acid analysis. The procedure relies on the use of in situ staining of proteins during preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the utilisation of microconcentrators to repeatedly concentrate small amounts of proteins onto a small polyvinylidene difluoride (PVDF) disc until sufficient amounts have been adsorbed so as to give a strong sequencing signal. The protein elution and subsequent adsorption can be monitored visually with a dye and the final product, a PVDF disc with the adsorbed protein or peptide, can be directly inserted into the automated amino acid sequencer.

Purification of histamine receptor proteins from detergent-solubilized human peripheral blood mononuclear cells.

Histamine is released from mast cells and basophils by either immunological or nonimmunological mechanisms. Histamine, which is the most potent short acting mediator released from these cells, exerts its diverse biological actions by binding to cell surface histamine receptors. We report the affinity purification of histamine receptor proteins from Triton X-100 solubilized peripheral human blood mononuclear cells which include lymphocytes and monocytes. Three different designs of histamine affinity columns were constructed; all three resulted in the same material being eluted. This consisted of bands which on SDS-PAGE after boiling and reduction had the following molecular weights: 193K, 84K, 58K, 48K, 37K, and 16K. The most abundant bands were of molecular weights 193K, 48K, and 16K, and these were disulfide bonded together to form a high molecular weight complex. (The 58K band was present in lower amounts than the others, and in only a few fractions. It had the same molecular weight as the dimeric form of histamine methyltransferase which is present in small amounts in mononuclear cells and may therefore have copurified.) The histamine binding proteins described in this report were purified by conventional affinity chromatography, rather than by an expression cloning approach which obviates the use of any protein chemistry. Consequently, we had the advantage of being able to verify the histamine binding specificity of our purified proteins directly and with several independent assays as follows. The histamine binding specificity of all three columns was established by specific elution with histamine, by preabsorption of crude cell extract with excess free histamine prior to column application, and by comparison with control columns. Independent determination of the binding specificity, using a radioreceptor dot blot assay, of the eluate containing only the 193K, 48K, and 16K disulfide-linked subunits confirmed that the purified material bound specifically to [3H]histamine and that a 300-500-fold degree of purification from tissue extract had been obtained. Following cell surface radioreceptor cross-linking of radiolabeled histamine to intact mononuclear cells, the 16K band was detected, indicating it to be the ligand-binding subunit for histamine. These same three proteins were purified from T lymphocyte and monocytoid cell lines, indicating that both lymphocyte and monocyte subsets of mononuclear cells express these proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Improved detection of the histamine receptor on human peripheral blood mononuclear cells by crosslinking using tritiated as compared with radioiodinated histamine.

In order to detect histamine receptors on the surface of human peripheral blood mononuclear cells, the cells were incubated in the presence of radiolabelled histamine and then the bifunctional crosslinker disuccimidyl suberate was added in various concentrations. They were then solubilized with sodium dodecyl sulphate, boiled, reduced and the lysate separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both 3H and 125I-radiolabelled ligands bound to a 16 kDa band, to be defined although a much clearer and obviously unequivocal signal was obtained with 3H-labelled histamine. This molecule migrated with the same mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 16 kDa subunit which had been purified on a histamine affinity column from Triton X-100 solubilized mononuclear cells, indicating it to be the ligand-binding subunit for the histamine receptor on these cells. For 3H, fluorography with Entensify was required to obtain an autoradiographic signal. Although 3H took much longer to give a signal than 125I, the considerable background, artefacts and heavy lane trailing seen with [125I] histamine were completely abrogated when [3H]histamine was used. In addition, the distinction between specific and nonspecific binding was more clearly seen using [3H]histamine. The modifications reported here which improve signal detection for 3H should encourage the use of tritiated ligands in radioreceptor crosslinking, particularly those of low molecular weight which might otherwise undergo steric modification due to iodination, this having the potential for interfering with receptor ligand binding.

Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of coomassie brilliant blue and silver staining.

Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol-acetic acid-formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.

A nondenaturing vertical isoelectric focusing polyacrylamide slab gel system suitable for silver staining and electrophoretic blotting.

We have devised a nondenaturing vertical isoelectric focusing (IEF)-polyacrylamide gel electrophoresis (PAGE) system which is amenable to silver staining and electroblotting. Apart from being accessible, inexpensive, and simple to use, this new methodology overcomes problems inherent in current IEF methods, for example, pH gradient drift, nonuniform cooling, restricted sample volume, and inability to perform electroblotting. Two photopolymerization gel formulas were derived: a 5% acrylamide formula using bisacrylamide (Bis) as the crosslinker and a 6% acrylamide formula using diallyltartdiamide (DATD) as the crosslinker. The 5% acrylamide Bis gel gave excellent resolution and separation of proteins whereas the 6% acrylamide DATD gel expanded slightly during silver staining, resulting in mild band distortions. At least 80 ng of protein per band could be detected by the silver staining protocol devised. Both the DATD and the Bis gels were suitable for electroblot transfer. Parameters to ensure the optimum conditions for reproducible, high resolution vertical IEF-PAGE are described. IEF-PAGE silver staining and electroblotting procedures and silver staining of the nitrocellulose electroblot procedures are also described. The advantages of this methodology over previously published methods are discussed.

HLA, C4, Bf, Gm and autoantibodies in rheumatoid arthritis.

Sera from 48 subjects with rheumatoid arthritis (RA), more than 80% of which were seropositive, were examined for the presence of antibodies to intact nuclei (ANA) by immunofluorescence, extractable nuclear antigens (ENA), double-stranded DNA by immunoassay and immunoglobulins, i.e. rheumatoid factors (RF) by haemagglutination. The genetic markers of HLA, the fourth complement component (C4) and properdin factor (Bf), which are all coded for within the major histocompatibility complex and Gm immunoglobulin allotypic markers were all analysed in relation to various parameters of these autoantibodies. The only significant association to emerge was between low ENA antibody affinities and the Bf Fs phenotype (p less than 0.02). Although weak associations between individual autoantibody parameters and the other immunogenetic markers were detected, there were no significant HLA, C4 or Gm association with either ANA subsets or RF, a finding which agrees with, and also extends previous HLA studies of predominantly seropositive RA cohorts.

Solubilization and characterization of moderate and high affinity histamine binding sites on human blood mononuclear cells.

The Triton X-100 solubilized extract of human peripheral blood mononuclear cells, in direct binding studies with 10(-9)-10(-6) M [3H]histamine contained both high and moderate affinity sites whose dissociation constants (Kd 4.4 X 10(-9) and 6.7 X 10(-7) M) were commensurate with basal plasma histamine levels and plasma levels obtained following physiological or mild pathological stimuli, respectively. Binding was enhanced by mM concns of calcium cations and by the protease inhibitor Pepstatin A. It was inhibited by bacitracin, agents interfering with thiol groups, Triton X-100 concns greater than 0.2% and EDTA. Binding was optimal between the pH range of 7.0 and 8.5 and was enriched for in a plasma membrane preparation. Thus the histamine binding sites identified maintained their specific ligand binding properties after solubilization from the cell surface and displayed properties fulfilling the criteria for receptors.

Solubilization and characterization of a low-affinity histamine-binding site on human blood mononuclear cells.

The extract of human peripheral blood lymphocytes and monocytes treated with Triton X-100, in direct- and competitive-binding studies, with 10(-6)-10(-2) M [14C]histamine contained a low-affinity binding site whose dissociation constant (Kd 1.8 X 10(-4) M) was commensurate with the concns of histamine (10(-6)-10(-3) M) that result from mast cell and basophil degranulation. Binding was enhanced by millimolar concns of divalent cations and by raising the incubation temp from 4 to 37 degrees C. It was inhibited by trypsin, EDTA, agents interacting with thiol groups, and by Triton X-100 concns greater than 0.2%. Thus a low-affinity histamine receptor that maintains its ligand-binding properties after solubilization from the cell surface was identified.

Extractable nuclear antigen (ENA) autoantibodies in SLE: an immunogenetic relationship with HLA, C4 and Bf alleles.

Sera from 36 subjects with systemic lupus erythematosus (SLE) were examined for extractable nuclear antigen (ENA) autoantibodies by immunoassay with the ribonucleoprotein (RNP) subset being determined by immunodiffusion. The prevalence of the genetic markers of HLA, the fourth complement component (C4) and properdin factor (Bf), which are all coded for within the major histocompatibility complex on chromosome 6 were analysed in relation to various parameters of these autoantibodies. The following associations were observed: The lowest ENA antibody titres of the RNP negative group were associated with HLA A9 (P less than 0.05), while the lowest RNA-ase sensitive ENA (RSE) subset antibody levels were associated with HLA Dr 1 (P less than 0.05). For the complement markers, C4 AQo was associated with the lowest affinity ENA antibodies (P less than 0.05), while the BF F allele and Fs phenotype had lower RSE antibody levels than did the S allele (P less than 0.05) and the SS phenotype (P less than 0.05) respectively. This study demonstrated diverse association between various MHC markers and ENA antibody parameters, indicating that there are distinctive immunogenetic influences over ENA autoantibodies in SLE.

Antibodies to extractable nuclear antigens (ENA) in rheumatoid arthritis assayed by ELISA. A clinicopathological correlation.

A clinicopathological study of autoantibodies in the sera of 53 RA patients was performed. Antibodies to extractable nuclear antigens (ENA) were assayed by ELISA and were found in 42% of the subjects, all bound to RNAase sensitive ENA, and these antibodies were significantly associated with the presence of tendon nodules (p less than 0.05). Antibodies to dsDNA were found in 16%, and rheumatoid factor (RF) was present in 81%; neither of these antibody groups were associated with any of the clinical abnormalities examined for. Comparisons between anti-ENA, anti-dsDNA and anti-immunoglobulin autoantibody parameters in RA subjects revealed ENA and dsDNA antibody levels to be significantly mutually related (p less than 0.01) but both were independent of RF levels. We concluded that in RA, ENA antibodies constitute a unique autoantibody subset, that may result from an immune response to an autoantigen directly linked with the aetiopathogenesis of RA.

Subsets of autoantibodies to extractable nuclear antigens (ENA) as assayed by ELISA correlate with specific clinical abnormalities in SLE.

A clinicopathological study of antinuclear antibodies was performed in 43 patients with SLE. Autoantibodies to double-stranded DNA, measured by RIA, were found in 56% of subjects and were associated with the presence of mesangioproliferative glomerulonephritis, Raynaud's phenomenon and cryoglobulinaemia. Antibodies to extractable nuclear antigens (ENA), when assayed by ELISA, were found in 81% of subjects and were associated with photosensitivity and thrombocytopaenia. Antibodies to RNA-ase-resistant ENA were found in 42% and were associated with cryoglobulinaemia and sclerodactyly, while antibodies to RNA-ase sensitive ENA which had a 49% prevalence were associated with haemolysis and neutropaenia. RNP antibodies (detected by immunodiffusion in 40%) were also associated with photosensitivity. RNP antibody positive and negative sera differed from each other by virtue of their relationship with other autoantibodies, and because RNP-positive sera had significantly higher ENA antibody titres and affinities than did RNP-negative sera. We therefore concluded that various combinations of antinuclear antibodies may predispose to the development of specific clinicopathological lesions in SLE.

Extractable nuclear antigen autoantibodies and their association with other autoantibodies and thymoma in myasthenia gravis.

The presence of autoantibodies to Extractable Nuclear Antigens (ENA) in myasthenia gravis (MG) was evaluated. These antibodies represent a subset of antinuclear antibodies (ANA), with ENA consisting of RNA and nucleoprotein. ENA autoantibodies had a prevalence of 44% in MG when tested by an