Allergenic components of isolates of cladosporium herbarum

Six isolates of Cladosporium herbarum, IMI 49630, IMI 46932, IMI 96220, CBS 121.47, CBS 180.50 and ATCC 38810 were grown on synthetic revised tobacco medium for 28 days. Extracts of the mycelia were prepared and their biochemical and immunological properties were examined. Two isolates, IMI 49632 and CBS 180.50 did not give a significant yield of mycelial mat, and the mycelial extract of ATCC 38810 was only weakly immunoreactive. In IgE immunoblots of sodium dodecylsulphate polyacrylamide electrophoresis gels, the remaining three extracts showed strongly reactive bands at 14, 17, 25, 35, 41,47 and 97 kDa MW and IMI 96220 had two additional bands at 53 and 71 kDa. These extracts had 8 to 10 bands in isoelectric focusing with pl values between 3.75 to 5.2, the two predominant bands being at 4.5 and 5.2. In crossed — radioimmunoelectrophoresis using human atopic sera, IMI 96220 showed one dominant and three minor allergens; IMI 49630 and CBS 121.47 had one dominant and one minor allergen. In direct radioallergosorbent tests with human atopic sera and passive cutaneous anaphylaxis tests with mouse IgE, IMI 49630 and 96220 were more potent than CBS 121.47. These results indicate that mycelial extracts of IMI 49630 and 96220 contain a potent but complex mixture of the major allergens of C. herbarum and are preferred sources for a reference standard for this organism.     

Allergy to insect stings. IV. Diagnosis by radioallergosorbent test (R.A.S.T.)

Radioallergosorbent tests (RAST(s)) have been developed and assessed for the diagnosis of insect hypersensitivity by using a purified allergen from honeybee venom, phospholipase A, and crude yellow jacket venom. Sera from 193 patients positive both by history and skin test to one of these insects were compared with various groups of control sera. Eighty percent of sera from skin test-positive patients were RAST positive; positive RAST were found in 16% of sera tested from skin test-negative patients. A highly positive RAST correlates well with a positive skin test and clinical sensitivity, but serum IgE is not measurable in many patients with mast cell or basophil bound antibody. Since biologically important reactions of antigen with IgE require that the antibody be cell bound, skin testing would be preferred to RAST if one were limited to a single test for the diagnosis of insect allergy.     

Skin testing versus radioallergosorbent testing for indoor allergens

BACKGROUND: Skin testing (ST) is the most common screening method for allergy evaluation. Measurement of serum specific IgE is also commonly used, but less so by allergists than by other practitioners. The sensitivity and specificity of these testing methods may vary by type of causative allergen and type of allergic manifestation. We compared ST reactivity with serum specific IgE antibodies to common indoor allergens in patients with respiratory allergies. METHODS: 118 patients (3 mo-58 yr, mean 12 yr) with allergic rhinitis and/or bronchial asthma had percutaneous skin testing (PST) supplemented by intradermal testing (ID) with those allergens suspected by history but showed negative PST. The sera were tested blindly for specific IgE antibodies by the radioallergosorbent test (Phadebas RAST). The allergens were D. farinae (118), cockroach (60), cat epithelium (90), and dog epidermal (90). Test results were scored 0-4; ST >/= 2 + and RAST >/= 1 + were considered positive. RESULTS: The two tests were in agreement (i.e., either both positive or both negative) in 52.2% (dog epidermal) to 62.2% (cat epithelium). When RAST was positive, ST was positive in 80% (dog epidermal) to 100% (cockroach mix). When ST was positive, RAST was positive in 16.3% (dog epidermal) to 50.0% (D. farinae). When RAST was negative, ST was positive in 48.5% (cat epithelium) to 69.6% (D. farinae). When ST was negative, RAST was positive in 0% (cockroach) to 5.6% (cat epithelium). The scores of ST and RAST showed weak to moderate correlation (r = 0.24 to 0.54). Regardless of history of symptoms on exposure, ST was superior to RAST in detecting sensitization to cat epithelium and dog epidermal. CONCLUSION: For all four indoor allergens tested, ST was more sensitive than RAST. When both tests were positive, their scores showed poor correlation. Sensitizations to cat epithelium and dog epidermal are common, even in subjects who claimed no direct exposure.     

Atopic disease and immunoglobulin E in twins reared apart and together.

Both genetic and environmental influences have been implicated in the etiology of atopic disease and in the determination of serum IgE levels. To quantify the relative contribution of these influences, we studied the prevalence of asthma and seasonal rhinitis, skin-test response, total serum IgE levels, and specific IgE, as measured by RAST, in a sample of MZ and DZ twins reared apart or together. Concordance rates for asthma, rhinitis, positive skin tests, and RAST were calculated. MZ twins, whether reared apart or together, showed a greater concordance than dizygotic twins reared apart or together. Maximum-likelihood tests of genetic and environmental components of the variation of total IgE levels revealed a substantial genetic component and a negligible contribution from common familial environmental effects.     

Identification of specific IgE binding proteins in Castor bean (Ricinus communis) pollen obtained from different source materials

Allergenic components of Ricinus communis pollen obtained from different stages of inflorescence, different time intervals, different years and places were studied by immunoblot analysis. Proteins separated by SDS-PAGE and transferred to NC were identified using pooled sera from 15 skin and RAST positive patients. The IgE binding components in M.W. range of 14 to 70 kD were identified. The protein fractions of 70, 66, 64, 60, 50, 45, 36, 22 and 14 kD are the most prominent allergenic bands. Six samples collected during same pollination season from the same place showed similar allergenic profile. Of the samples collected from different stages of inflorescence, pollen of immature buds showed only three bands as compared to 18 from mature buds and flowers. Variability was seen in the IgE binding components of pollen stored for different years and obtained from different geographic regions of India. The IgE binding pattern of fifteen sera were heterogenous. The number of bands identified by different sera varied from 3 to 18. Two protein components of 66 and 36 kD were recognised by 14 (93.3%) of the 15 sera studied. The result suggests that there exists variations in the specific IgE binding pattern in pollen samples of Castor Bean, obtained from difference source materials.     

Measurement of IgG blocking antibodies by interference in the radioallergosorbent test

We previously found that sera of patients immunized with ragweed pollen extract contained a factor that interfered with the binding of IgE antibodies to solid-phase allergens in the radioallergosorbent test (RAST). We now describe an assay, RAST interference, to measure this factor, and we present evidence that the factor is IgG blocking antibody. Sera from immunized allergic patients were heated at 56 degrees C for 4 hr to destroy heat-labile Fc determinants on IgE and were tested for their ability to prevent binding of additional IgE antibody to solid-phase allergens in the RAST. Eight of 10 sera from allergic immunized patients gave RAST interference dose-response curves that did not differ from the arbitrary standard. The factor causing interference showed specificity for the immunizing antigen, was heat-stable, eluted from Sephadex G-200 in the 7S peak, was present only in sera of immunized patients, and rose after initiation of immunization. These results indicated that RAST interference can be used to measure IgG blocking antibodies with the same reagents employed for the measurement of IgE antibodies, provided the antiserum to IgE is specific for the heat-labile FC determinants on IgE.     

Serum interleukin-1β as a marker for differentiation of asthma and chronic obstructive pulmonary disease

Asthma and chronic obstructive pulmonary disease (COPD) are diseases of airway inflammation with clinical and physiological similarities, making their differentiation difficult. Airway inflammatory changes are associated with systemic changes. However, no serum marker is known for their differentiation. Therefore, serum interleukin (IL)-1β levels were determined. Out of a total of 1023 patients screened, we included in the study ten patients each with atopic asthma, non-atopic asthma and COPD and ten healthy subjects. Skin prick tests with 14 inhalant allergens were performed on each patient. Blood was collected in the symptomatic and asymptomatic phases of the diseases and serum IL-1β and IgE levels were determined. Our results showed that in the symptomatic phase in asthmatics, serum IL-1β levels were higher (P<0.05) than in patients with COPD. Serum IgE levels were higher (P<0.05) in atopic asthmatics than in non-atopic asthmatics and in COPD patients. We conclude that serum IL-1β level determination during the symptomatic phase of the diseases may help to differentiate asthmatics from patients with COPD. Serum IgE levels may differentiate atopic asthmatics from non-atopic asthmatics and COPD patients.     

Measurement of absolute amounts of antigen-specific human IgE by a radioallergosorbent test (RAST) elution technique.

A technique for the absolute quantification of antigen-specific human IgE is described. It employs elution of a calculable amount of antigen-specific IgE from an allergosorbent-antibody complex by means of alkaline pH treatment, followed by measurement of the IgE content of the eluate with a modified radioimmunosorbent test (RIST). With this method IgE antibody directed against the benzylpenicilloyl determinant of penicillin (BPO) was measured quantitatively in sera from seven penicillin allergic patients. IgE specific for ragweed antigen E was measured in sera from 33 ragweed allergic patients. Values obtained for IgE anti-BPO ranged from 19 to 1806 ng/ml and comprised from 1.3 to 27.5% of total serum IgE. Values of IgE anti-antigen E ranged from 9 to 1807 ng/ml, comprising from 3 to 84% of total serum IgE. Excellent correlation (r = 0.99; p less than 0.001) was obtained for both antigen systems between values determined by the RAST elution technique and by simple RAST assay with interpolation from a reference serum of known specific IgE content as determined by the elution technique may be needed only for primary standardization of reference sera.     

Identification and partial characterization of the allergenic proteins of Ricinus communis L. pollen - a new approach

The pollen of Ricinus communis L., a potentially allergenic plant, was extracted to identify the allergenic determinants responsible for causing respiratory disorders. The soluble proteins were extracted and subjected to ammonium sulphate precipitation at 80% saturation and the total protein separated on 12% SDS-Polyacrylamide gel. In order to avoid the time consuming and expensive biochemical methods of column chromatography, each band was directly recovered from the gel by electroelution and the allergenic proteins identified directly by skin tests, without the necessity of Phadezym RAST or ELISA inhibition by reaction with serum IgE, the general procedure to identify the allergens. The fourth and the fifth band in the protein profile of R. communis pollen, RC4 (77 kD) and RC5 (66 kD) were the two major allergenic components. RC3 (91 kD) also induced a considerable amount of reactivity in sensitive patients. Contrary to the earlier reports of protein bands of R. communis ranging from 14 kD to 70 kD, 4 bands above 70 kD i.e. RC1 (123 kD), RC2 (97 kD), RC3 (91 kD) and RC4 (77 kD) are reported here for the first time. Immunodiffusion analysis with pooled sera of patients sensitive to the total extract also revealed similar results.     

Antigens of Ambrosia elatior (short ragweed) pollen. III. Crossed radioimmunoelectrophoresis of ragweed-allergic patients' sera with special attention to quantification of IgE responses

Short ragweed allergenic extract has been studied by means of crossed radioimmunoelectrophoresis (CRIE) with the use of sera from 37 allergic patients and the relevant control sera. In this study 22 of 52 antigens, detectable in crossed immunoelectrophoresis (CIE) against polyspecific rabbit anti-ragweed IgG, were able to bind specific human IgE to their corresponding immunoprecipitates. This binding was semiquantified by comparison with the binding of a standard serum pool. Nine antigens were identified as important allergens, including the previously isolated components, AgE, AgK, and Ra6. Certain allergens (e.g., AgE, AgK, and Ag 31) bound IgE in almost all patients' sera, whereas others showed a bimodal distribution for sera of responder and nonresponder patients. The total CRIE score was found to correlate significantly both with ragweed-specific serum IgE antibody determined by RAST (rs = 0.88; p less than 0.001) and with total IgE level (rs = 0.55; p less than 0.01). Patient's CRIE scores to AgE also correlated significantly with their specific IgE antibody to AgE measured by RIA (r = 0.47; p less than 0.01) and with skin-test sensitivity to AgE (r = 0.44; p less than 0.05). It was concluded that CRIE is well suited for identification of important ragweed allergens without the previous need for laborious isolation procedures.     

Allergenic components of ganoderma applanatum

Spores of Ganoderma applanatum were collected by gravity. The spore extract was prepared and its biochemical and immunological properties were examined. In isoelectric focusing, the extract had 10–12 bands with pl values between 3.5 to 6.0, the strongest being at 3.5, 3.75, 4.55 and 5.2. In crossedradioimmunoelectrophoresis using human atopic sera, the extract had two dominant and two minor allergens. In IgE immunoblots of sodium dodecylsulphate polyacrylamide gel electrophoresis, the extract showed reactivity at 82, 45, 33, 26, 16, 11 kDa MW, the strongest being at 45 kDa MW. These results indicate that G. applanatum contains a complex mixture of allergens.     

IgE responses in human filariasis. II. Qualitative characterization of filaria-specific IgE

Crossed radioimmunoelectrophoresis (CRIE) was used to characterize human IgE antibody responses to filarial parasites by using antigens derived from Brugia malayi (Bm) adult worms. A reference pool of patient sera was initially used to determine the sensitivity and specificity of CRIE. Because IgG-blocking antibodies interfered with IgE binding in certain sera, all sera were preabsorbed with protein A-Sepharose. As little as 50 ng of specific IgE antibody (determined by quantitative radioallergosorbent test [RAST]) in the reference pool bound to 20 of the 35 antigen precipitates in crossed immunoelectrophoresis. Increasing IgE antibody concentration did not increase the number of IgE-binding precipitates. Six patients from each of the three major clinical groups in lymphatic filariasis (i.e., tropical pulmonary eosinophilia [TE], chronic lymphatic pathology [CP], or circulating microfilaremia [MF]) were studied by CRIE with the use of a constant amount of IgE antibody (50 ng IgE anti-BmA). Distinct patterns of allergen recognition were observed among the groups. Individuals with TE recognized both anodic and cathodic antigens as allergens, whereas the other two groups recognized predominantly anodic antigens. The greatest number of allergens was recognized by patients with TE; this number ranged from nine to 18, whereas patients with CP or circulating MF recognized from six to 11 allergens. Although potentiated IgE responses at a quantitative level in parasitic helminth infections is a well-established phenomenon, our studies showing the diversity of antigens recognized as allergens indicate for the first time potentiated IgE responses at a qualitative level as well.     

Detection of IgG and IgE antibodies to Aspergillus fumigatus in human sera by immunogold assay

An immunogold assay (IGA) was developed to detect IgG and IgE antibodies to Aspergillus fumigatus. Sixteen sera from patients with allergic bronchopulmonary aspergillosis (ABPA), aspergilloma, and normal controls were studied. All sera were also evaluated for antibodies against A. fumigatus by biotin-avidin linked enzyme immunosorbent assay (BALISA) and by agar gel double diffusion method. A. fumigatus specific IgG and IgE antibodies could be detected by IGA in all the patients' sera but not in the sera of normal controls. Both IgG and IgE antibodies to A. fumigatus could be demonstrated in all the sera by BALISA and normal controls showed only low levels of these antibodies. There was a positive correlation between the degree of reactivity detected by IGA, the BALISA titer and the precipitins by agar gel diffusion. It can be concluded that IGA is a reliable, sensitive and simple method capable of detecting both IgG and IgE antibodies against A. fumigatus in patient serum.     

Antigen Der f I from the dust mite Dermatophagoides farinae: structural comparison with Der p I from Dermatophagoides pteronyssinus and epitope specificity of murine IgG and human IgE antibodies

The physicochemical and antigenic properties of an allergen purified from Dermatophagoides farinae, Der f I, were compared with Der p I from Dermatophagoides pteronyssinus. On SDS-PAGE, Der f I migrated as a single polypeptide chain with the same m.w. as Der p I (24,000). Two isoallergenic peaks of Der f I were identified on preparative isoelectric focusing (pI 5.7 to 6.3 and pI 6.6 to 6.95). Fractions from each peak were shown to have an identical amino acid composition (which was similar but not identical to Der p I) and the same N-terminal amino acid sequence. There was a good correlation between quantitative intradermal skin tests to both purified allergens and to D. farinae extract in mite-allergic patients, with positive results when using as little as 10(-5) micrograms/ml of Der f I. The majority of sera with detectable IgE antibody to D. farinae also had IgE antibody to Der f I both among children (29/42 = 69%) and adults (55/63 = 87%). By RAST, there was an excellent correlation between IgE antibody to Der f I and Der p I in sera from 42 mite-allergic children (n = 0.94, p less than 0.001). Polyclonal IgG antibodies from six mice immunized with Der f I showed preferential binding to that allergen, and most monoclonal antibodies (16 of 18) raised against Der f I did not bind Der p I. However, two monoclonal antibodies from this fusion showed cross-reactive binding to both allergens. Immunoabsorption experiments, using D. pteronyssinus and D. farinae extracts coupled to Sepharose, showed that a large proportion of murine antibodies (74% to Der p I and 60 to 93% to Der f I) could not be absorbed by the heterologous extract on the immunosorbent. In contrast, in sera from seven mite-allergic patients, most of the specific IgE and IgG antibody (i.e., greater than or equal to 82%) was removed by either immunosorbent. Thus, Der f I and Der p I represent a homologous pair of major allergens which possess both cross-reacting and species-specific epitopes. The antibody response in mice immunized with either allergen in complete Freund's adjuvant was largely directed against species-specific epitopes, whereas in allergic humans, IgE- and IgG-specific antibodies bound predominately to cross-reacting epitopes.     

Molecular and Biological Analysis of Potato virus M (PVM) Isolates from the Czech Republic

The sequences of the 3′‐terminal region of four Czech Potato virus M isolates VIRUBRA 4/007, VIRUBRA 4/009, VIRUBRA 4/016 and VIRUBRA 4/035 were determined and compared with sequences of PVM isolates available in GenBank. Among the Czech isolates, VIRUBRA 4/007 and 4/016 as well as VIRUBRA 4/016 and 4/035 showed the highest nucleotide identity (93%). Isolates VIRUBRA 4/007, 4/016 and 4/035 were most similar to the PV0273 isolate from Germany and to the wild isolate from Russia. Interestingly, isolate VIRUBRA 4/009 significantly differed from the other three Czech isolates and was the only European isolate that showed the highest nucleotide identity with American isolates. Moreover, the PVM isolates from the Czech Republic and Germany differed in their host range. Phylogenetic analysis based on ORF5 coding for coat protein showed that the Czech isolates could be classified in two of the three groupings of the phylogenetic tree obtained. This is the first report on molecular and biological analysis of the genome sequences of PVM isolates from the Czech Republic.     

Paracoccidioides brasiliensis antigen batches from the same isolate show immunological and biochemical differences

We investigated the occurrence of antigenic and biochemical variability among Paracoccidioides brasiliensis antigen batches prepared according to the same protocol. Initially (experiment #1), we analyzed two antigen lots of two human isolates (Bt1 & Bt2), cultured in two media (PYG: bactopeptone, yeast extract, glucose; MMM: McVeigh & Morton medium) in SDS-PAGE and in two immunological tests (imunodiffusion-ID and footpad swelling test-FPT). Afterwards (experiment #2), we compared the antigenic profile of three antigen batches from three human isolates (Bt1, Bt2 & Bt3) by two-dimensional immunoelectrophoresis (2 D-IEP) against a reference system for P. brasiliensis antigens. In experiment #1, there were important intra- and inter-strain antigenic differences between batches of the fungal isolates cultured on both media. The block titration of the antigen batches for the immunological tests revealed correlation between protein concentration and biological activity in ID and no correlation in FPT. In experiment #2, the reference system for P. brasiliensis showed 26 antigen peaks. There were important differences between batches prepared from the same isolate and between batches from different isolates. Our data suggested the occurrence of instability in the synthesis of antigenic components by a same P. brasiliensis isolate, under controlled incubation conditions.     

Leukotoxic Activity of Actinobacillus actinomycetemcomitans Isolated from Brazilian Periodontal Patients

The leukotoxic activity of 31 Actinobacillus actinomycetemcomitans isolates from Brazilian periodontal patients [nine from Localized Juvenile Periodontitis (LJP) patients, 22 from patients with AIDS-associated Necrotizing Ulcerative Periodontitis (AIDS/NUP)], and from the reference strain A. actinomycetemcomitans ATCC43718, were analysed for their cytotoxicity on human monocytes. A cytotoxicity inhibitory assay of the isolate P35 and the reference strain ATCC 43718 with sera from ten LJP patients and ten healthy subjects was also performed and leukotoxin reactivity was evaluated with serum from rabbits immune to leukotoxin from A. actinomycetemcomitans ATCC 43718. The cytotoxicity results were not statistically different among groups of A. actinomycetemcomitans isolates from LJP and AIDS/NUP patients, but the individual analysis of each isolate showed two isolates (P24 and P35) from LJP patients with high leukotoxic activity (P<0.05). Also, a high leucotoxic inhibitory effect with LJP patients' sera compared with healthy subjects with sonic extract from isolate P35 (P<0.05) and the reactivity of rabbit antiserum to leukotoxin were observed. Both leukotoxic and non-leukotoxic strains of A. actinomycetemcomitans can be isolated from LJP and AIDS/NUP patients, but A. actinomycetemcomitans with high leukotoxic activity is more frequent in PJL than AIDS/NUP patients. Even though A. actinomycetemcomitans exhibits leukotoxic activity, there is an immune response to the leucotoxin in LJP patients.     

Diagnostic utility of monoclonal antibodies raised against microfilarial excretory-secretory antigens in bancroftian filariasis

Two monoclonal antibodiesWuchereria bancrofti E 33 andWuchereria bancrofli E 34 raised againstWuchereria bancrofti microfilarial excretory-secretory antigens were studied for their diagnostic utility.Wuchereria bancrofti E 34 monoclonal antibody was found to be relatively specific and sensitive in detection of circulating filarial antigen. WhenWuchereria bancrofti E 34 monoclonal antibody was used alongwith immunoglobulin G fraction of human filarial serum immunoglobulins in double antibody sandwich enzyme linked immunosorbent assay. 68% of microfilaraemic sera (26 out of 38). 12% of clinical filarial sera (3 out of 25), 13% endemic normal sera (2 out of 15) and none of the 20 non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detected byWuchereria bancrofti E 34 monoclonal antibody in double antibody sandwich enzyme linked immunosorbent assay is possibly associated with the active stage (microfilaraemia) of infection.     

Immunofluorescence studies of the immunoglobulin receptors of B-lymphocytes]

The surface membrane Ig receptors of B lymphocytes in patients with atopic pollen asthma were studied by the direct immunofluorescence method. Use was made of labelled sera against human IgE, and IgG. It was shown that on the average 2.1 +/- 0.33% lymphocytes had IgE receptors, 8.4 +/- 0.63% IgG receptors and 18.7 +/- 1.16% human globulin receptors. A specific nature of B lymphocytes is suggested.     

IgG and IgE immune response against the surface glycoprotein gp200 of Saccharomyces cerevisiae in patients with atopic dermatitis

The heat-stable and soluble glycoprotein gp200 (molecular weight 200 kDa) is part of the cell wall of S. cerevisiae. Recently, an association was shown between IgA and IgG against gp200 and inflammation in Crohn's disease. Gp200 is able to induce a proliferation of human lymphocytes in vitro, together with a natural killer cell associated cytotoxicity. Specific IgE against Saccharomyces cerevisiae (baker's or brewer's yeast) may be detected in approximately 73 %, against Candida albicans in 68% of those patients suffering from severe atopic dermatitis. The aim of this study was to elucidate the possible role of an anti-gp200 immune response for the pathogenesis of atopic dermatitis by immunoblot analysis. Anti-gp200 IgE was found in 55% of healthy individuals, in 67% of individuals with atopic predisposition without eczema, in 63% of the patients with mild atopic dermatitis, and in 86% of patients with severe atopic dermatitis, respectively. On the contrary, anti-gp200 IgG could be shown in 55% of healthy individuals, in 89% of individuals with atopic predisposition but without eczema, in 100% of patients with mild atopic dermatitis, and in 79% with severe atopic dermatitis, respectively. No immunoreactivity was found when an extract of Arxula adeninivorans was used as antigen. These results underline the specificity of the immunoblot results with gp200 from Saccharomyces cerevisiae. It can be concluded that occurrence of specific IgE against Saccharomyces cerevisiae cannot be explained by a cross reactivity, e.g., against Candida albicansallergens. Further investigations with the recombinant gp200 will give information on the role of this glycoprotein both in atopic dermatitis and Morbus Crohn. This revised version was published online in August 2006 with corrections to the Cover Date.