Detection of Mycoplasma antigens (Mycoplasma arthritidis and Mycoplasma fermentans) in the tissues of experimentally infected animals by using indirect immunofluorescence and aggregate-hemagglutination reactions

The analysis of the results obtained in the detection of mycoplasmic antigens in tissues of infected rabbits by means of the immunofluorescence test and the aggregate hemagglutination test, carried out in parallel, indicates that both these tests are highly specific, while the immunofluorescence test is more sensitive.     

Bronchial Reactions to Western Red Cedar (Thuja Plicata)

The clinical features and the results of investigations (including immunological tests) of three patients with asthma due to western red cedar are described. Bronchial provocation tests with extract of this wood produced immediate asthmatic reaction in one patient, late asthmatic and peripheral reactions in another and late asthmatic reaction alone in the third. While mild immediate skin reactions were detected in two patients, no late skin reactions were observed. Serum precipitins to this extract were not detected. An attempt was made to identify the responsible allergen in the red cedar extract.     

NADP-dependent mannitol dehydrogenase, a major allergen of Cladosporium herbarum

Cladosporium herbarum is an important allergenic fungal species that has been reported to cause allergic diseases in nearly all climatic zones. 5-30% of the allergic population displays IgE antibodies against molds. Sensitization to Cladosporium has often been associated with severe asthma and less frequently with chronic urticaria and atopic eczema. However, no dominant major allergen of this species has been found so far. We present cloning, production, and characterization of NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) and show that this protein is a major allergen that is recognized by IgE antibodies of approximately 57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Cla h 8 was purified to homogeneity by standard chromatographic methods, and both N-terminal and internal amino acid sequences of protein fragments were determined. Enzymatic analysis of the purified natural protein revealed that this allergen represents a NADP-dependent mannitol dehydrogenase that interconverts mannitol and d-fructose. It is a soluble, non-glycosylated cytoplasmic protein. Two-dimensional protein analysis indicated that mannitol dehydrogenase is present as a single isoform. The cDNA encoding Cla h 8 was cloned from a lambda-ZAP library constructed from hyphae and spores. The recombinant non-fusion protein was expressed in Escherichia coli and purified to homogeneity. Its immunological and biochemical identity with the natural protein was shown by enzyme activity tests, CD spectroscopy, IgE immunoblots with sera of patients, and by skin prick testing of Cladosporium allergic patients. This protein therefore is a new major allergen of C. herbarum.     

Use of the indirect hemagglutination inhibition reaction for evaluating the specific activity of allergens

To evaluate the specific activity of house-dust allergen, the passive hemagglutination inhibition test was used. Nine commercial batches of house-dust allergen were studied by means of this test. The results of the determination of the activity of house-dust allergen in the passive hemagglutination inhibition test, the skin tests and the indirect mast-cell degranulation test were in good correlation.     

Rapid production of the major birch pollen allergen Bet v 1 in Nicotiana benthamiana plants and its immunological in vitro and in vivo characterization.

Type I allergies are immunological disorders that afflict a quarter of the world's population. Improved diagnosis of allergic diseases and the formulation of new therapeutic approaches are based on the use of recombinant allergens. We describe here for the first time the application of a rapid plant-based expression system for a plant-derived allergen and its immunological characterization. We expressed our model allergen Bet v 1, the major birch pollen allergen, in the tobacco-related species Nicotiana benthamiana using a tobacco mosaic virus vector. Two weeks postinoculation, plants infected with recombinant viral RNA containing the Bet v 1 coding sequence accumulated the allergen to levels of 200 microg/g leaf material. Total nonpurified protein extracts from plants were used for immunological characterizations. IgE immunoblots and ELISA (enzyme-linked immunoassay) inhibition assays showed comparable IgE binding properties for tobacco recombinant (r) Bet v 1 and natural (n) Bet v 1, suggesting that the B cell epitopes were preserved when the allergen was expressed in N. benthamiana plants. Using a murine model of type I allergy, mice immunized with crude leaf extracts containing Bet v 1 with purified rBet v 1 produced in E. coli or with birch pollen extract generated comparable allergen-specific IgE and IgG1 antibody responses and positive type I skin test reactions. These results demonstrate that nonpurified Bet v 1 overexpressed in N. benthamina has the same immunogenicity as purified Bet v 1 produced in E. coli or nBet v 1. We therefore conclude that this plant expression system offers a viable alternative to fermentation-based production of allergens in bacteria or yeasts. In addition, there may be a broad utility of this system for the development of new and low-cost vaccination strategies against allergy.     

Severe community-acquired pneumonia: etiology]

A complex microbiological (sputum, protected brush biopsy of the bronchial mucosa) and immunological examination of 40 male patients (the average age of 55.4 +/- 8.8 years) with severe community-acquired pneumonia (risk classes III-V according to Fine M.J. et al., 1997) revealed the disease etiology in 52.5 per cent of the cases. The leading pathogen was Streptococcus pneumoniae. It was detected in 61.6 per cent of the cases of the etiologically verified pneumonia. Staphylococcus aureus and Klebsiella pneumoniae were also among the actual pathogens (14.3 and 14.3 per cent respectively). The Legionnaires infection was not confirmed in any of the patients (enzyme-linked immunological analysis of urine for the serotype 1-6 Legionella pneumophila antigen). In the absolute majority of the patients the isolated pneumococci were susceptible (E-test) to benzylpenicillin. Only in 1 patients with severe pneumonia and secondary bacteriemia the pneumococcal isolates were moderately resistant to benzylpenicillin (the MIC of 0.125 mg/ml). Still, they were susceptible to ceftriaxone (the MIC of 0.023 mg/ml). The data are useful in the development of a national (regional) programme for empirical antibacterial therapy of severe community-acquired pneumonia.     

Latex agglutination test for diagnosing pneumococcal pneumonia in children in developing countries.

OBJECTIVE--To prepare and assess the sensitivity and specificity of a latex agglutination test specific for the serotype of antigen in diagnosing pneumococcal pneumonia in Gambian children. DESIGN--Comparison of agglutination test specific for serotype with culture of blood and lung aspirates, countercurrent immunoelectrophoresis, and commercial latex agglutination tests in diagnosing pneumococcal pneumonia. Cross reaction studies and investigation of 102 control children to determine specificity of agglutination test specific for serotype. SETTING--General medical ward of Medical Research Council laboratories, The Gambia. PATIENTS--101 Gambian children aged between 2 months and 10 years admitted with severe pneumonia. INTERVENTIONS--Serum samples were boiled and treated with edetic acid, and urine samples were boiled and concentrated 25 times before testing. END POINT--A latex agglutination test specific for the serotype of pneumococcal antigen that is sensitive and highly specific for detecting pneumococcus in the urine of patients with pneumococcal pneumonia. MEASUREMENTS AND MAIN RESULTS--Concentrated urine samples from 16 of the 21 children (76%) with pneumococcal pneumonia established by results of culture of blood or lung aspirates gave a positive result with the agglutination test specific for serotype, whereas only four of the 102 urine samples obtained from control children without pneumonia gave positive results. The serotypes of antigens detected in the urine of children with pneumococcal pneumonia and the serotypes of pneumococci isolated from cultures of blood or lung aspirates were the same in all cases. CONCLUSIONS--When performed on urine samples the agglutination test specific for serotype has a high specificity and is more sensitive than culture of blood or lung aspirates, commercial agglutination tests, or countercurrent immunoelectrophoresis in identifying pneumococcal pneumonia. It is easy to use and should be especially useful in communities with limited laboratory facilities.     

Crystal structure and some properties of a major house dust mite allergen, Derf 2

Pyroglyphid house dust mites are a major source of allergens in house dust. Mite allergens sensitize and induce asthma, rhinitis, and eczema in a large portion of patients with allergic diseases. Here, the crystal structure of a major mite allergen, Derf 2, derived from Dermatophagoides farinae was solved by single isomorphous replacement method with anomalous scattering (SIRAS) at 2.1A resolution. The present study also demonstrated that the conformation of the allergen was critical in the determination of Th1/Th2 shift based on physicochemical and immunological analyses. This indicates that rigidly folded and singly dispersed structure is essentially required for the generation of Th2 type cells by the allergen, while conformational variant protein leads to Th1 skewing, irrespective of the same amino acid sequence. This structure/function relationship may allow us to develop a novel strategy for hyposensitization therapy in patients with allergic diseases triggered by house dust mite allergens.     

Ani s 10, a new Anisakis simplex allergen: cloning and heterologous expression

Anisakiasis is a human disease caused by accidental ingestion of larval nematodes belonging to the Anisakidae family. Anisakiasis is often associated with a strong allergic response. Diagnosis of A. simplex allergy is currently carried out by test based on the IgE reactivity to a complete extract of L3 Anisakis larvae although the specificity of these diagnostic tests is poor. Improving the specificity of the diagnostic test is possible using purified recombinant allergens. A new Anisakis allergen, named Ani s 10, was detected by immunoscreening an expression cDNA library constructed from L3 Anisakis simplex larvae. The new allergen was overproduced in Escherichia coli; it is a protein of 212 amino acids and it was localized as a 22 kDa protein band in an ethanol fractionated extract from the parasite. Ani s 10 has no homology with any other described protein, and its sequence is composed by seven almost identical repetitions of 29 amino acids each. A total of 30 of 77 Anisakis allergic patients (39%) were positive both to rAni s 10 and natural Ani s 10 by immunoblotting. The new allergen could be useful in a component-resolved diagnosis system for Anisakis allergy.     

Circulating immune complexes in the diagnosis of Mycoplasma pneumoniae infection

The possibility of detecting M. pneumoniae antigen and antibodies to it, incorporated into immune complexes, in the sera of patients with acute pneumonia by means of erythrocyte diagnosticums was studied, and the immunological characterization of these complexes was made. In patients with mycoplasmal pneumonia M. pneumoniae antigen and specific antibodies, both free and incorporated into immune complexes, were found to circulate in the blood. In children, antigenemia was detected twice as frequently as in adults. Dissociated M. pneumoniae antigens had different molecular weight, their location on the gel chromatogram of the serum being in fractions 7S and 19S. The dissociation of immune complexes permits the detection of M. pneumoniae antigen and antibodies to it in a bound state by means of the passive hemagglutination test, thus increasing the frequency of positive results in the diagnosis of M. pneumoniae infection.     

Impact of Dermatophagoides pteronyssinus mite body raw material on house dust mite allergy diagnosis in a Serbian population

House dust mite (HDM) allergy has different clinical and immunological patterns in different geographic regions. The impact of raw material of commercial Dermatophadoides pteronyssinus (Acari: Pyroglyphidae) mite bodies on the quality of allergen extracts for allergy diagnosis in the Serbian population has not been previously evaluated. House dust mite bodies obtained from manufacturers in Europe, South America and Australia were used in the preparation of allergen extracts for in vivo diagnosis and serological analysis in a group of 14 HDM‐allergic adults. In the group of mite‐allergic patients, there was no statistically significant difference in skin test reactivity (Wilcoxon matched pairs test) among the three HDM body extract preparations. In a CAP inhibition assay, two extracts (A and C) achieved maximum inhibition of >90%, whereas extract B demonstrated a different inhibition slope and lower inhibition potential (80%). However, a remarkable difference in immunoglobulin E reactivity using Western blot analysis with individual patients' sera was observed in one of the preparations (extract B). These findings emphasize the need for the careful selection of starting material for the preparation of HDM diagnostic reagents intended for use in patients from geographically distinct regions as these preparations can have implications on the selection criteria for patient‐tailored immunotherapy of HDM allergy.     

The determination of allergen-specific IgE and IgG antibodies in patients sensitized to the house dust mite Dermatophagoides pteronyssinus

45 patients, hypersensitive to house-dust mites, were examined by the method of skin tests to D. pteronyssinus allergen. Besides, in their blood sera the levels of allergen-specific IgE antibodies were determined in the radioallergosorbent test and allergen-specific IgG antibodies, in the enzyme immunoassay. These tests revealed that in 91% of the patients the results of skin tests were positive, in 68% an elevated level of specific IgE antibodies and in 93% of the patients an elevated level of specific IgG antibodies were detected. All patients showed the positive result in one of the above-mentioned tests. The largest group of the patients (55%) included persons showing the positive result of the skin test and having elevated levels of allergen-specific IgG and IgE antibodies. Thus, in cases of hypersensitivity to house-dust mites the levels of allergen-specific IgG and IgE antibodies in the patients' blood sera should be determined.     

Comparative study of the basic immunological reactions for assessing tularemia immunity in inoculated persons or those who have had the disease

In the comparative study of immunological tests used for the determination of immunity to tularemia in vaccinees the greatest number of persons with the positive reaction was detected by means of the tularine skin test. Former tularemia patients retained allergic skin reaction to this test irrespective of the time of the disease.     

Evaluation of clarithromycin efficacy in the treatment of community-acquired pneumonia on the basis of the Binax NOW test results (a prospective open trial)]

The results of the treatment of community-acquired pneumonia with clarithromycin (500 mg bid for 6-8 days) at 172 patients (military recruits aged 18-25) are presented. Diagnosis, infection performance, treatment efficacy were evaluated by complex of data (X-ray, sputum analysis by bacteriological and cultural tests and immunochromatography test Binax NOW for pneumococcal antigen identification). High efficacy of clarithromycin for the treatment of moderate and mild pneumonia (including pneumococcal pneumonia) was demonstrated. Side effects were registered at 6.2 per cent of patients (gastro-intestinal disorders at 5 patients) and 1 general urticaria at 1 patient whose treatment had to be changed).     

Alpha-Actinin Is a New Type of House Dust Mite Allergen

Main indoor allergens for humans are from house dust mites. There are more than 30 allergens in Dermatophagoides farinae but only fourteen allergens have been identified from this mite including Der f 1–3, 6, 7, 10, 11, 13–18, and 22. A native allergen protein (Der f 24, 90 kDa) was purified from D. farinae by gel filtration and anionic exchange liquid chromatography combined with IgE immunodetection. Its primary structure was determined by Edman degradation, mass spectrometry analysis and cDNA cloning. Enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, basophil activation test (BAT) and skin prick test (SPT) were performed to evaluate the allergenicity. It was identified as an alpha (α)-actinin containing a CaM-like domain with EF-hand motifs. Der f 24 reacted to sera from 85.4% (35/41) of patients on western blot analysis. It reduced ∼20% sera IgE reactivity to D. farinae extracts on a competitive ELISA. Eighty percent (8/10) of patients with D. farinae allergy showed positive reactions to Der f 24 in skin prick test. The expression of CD63 on basophils from patients was up-regulated by Der f 24 by ∼5.4-fold. Alpha-actinin was identified as a new type of house dust mite allergen. To the best of our knowledge, this is the first report of α-actinin as an allergen.     

Production and detailed characterization of biologically active olive pollen allergen Ole e 1 secreted by the yeast Pichia pastoris.

The glycoprotein Ole e 1 is a significant aeroallergen from the olive tree (Olea europaea) pollen, with great clinical relevance in the Mediterranean area. To produce a biologically active form of recombinant Ole e 1, heterologous expression in the methylotrophic yeast Pichia pastoris was carried out. A cDNA encoding Ole e 1, fused to a Saccharomyces cerevisiae alpha-mating factor prepropeptide using the pPIC9 vector, was inserted into the yeast genome under the control of the AOX1 promoter. After induction with methanol, the protein secreted into the extracellular medium was purified by ion-exchange and size-exclusion chromatography. The structure of the isolated recombinant Ole e 1 was determined by chemical and spectroscopic techniques, and its immunological properties analysed by blotting and ELISA inhibition with Ole e 1-specific monoclonal antibodies and IgE from sera of allergic patients. The allergen was produced at a yield of 60 mg per litre of culture as a homogeneous glycosylated protein of around 18.5 kDa. Recombinant Ole e 1 appears to be properly folded, as it displays spectroscopic properties (CD and fluorescence) and immunological reactivities (IgG binding to monoclonal antibodies sensitive to denaturation and IgE from sera of allergic patients) indistinguishable from those of the natural protein. This approach gives high-yield production of homogeneous and biologically active allergen, which should be useful for scientific and clinical purposes.     

Reduction in skin test reactions to inhaled allergens during a 12 weeks stay in the alpine climate

Fiftyfour allergic patients with bronchial asthma or obstructive lung disease were investigated at admission and discharge after a 3 months hospitalization period in the alpine valley Davos. An intracutaneous skin test procedure was carried out with 16 common inhalant allergens. Histamine 0.01 mg/ml was used as a positive control and a phosphate buffered saline with 0.03% HSA and 0.5% phenol were used as negative controls. A liophylized control for house dust mite was used for every patient on admission and discharge, in order to control loss of allergen potency of the vial. Furthermore, an additional freshly prepared vial of house dust mite allergen was analyzed after it was opened and stored at 4°C after 3 months. The wheal and flare reactions were calculated separately for the early reaction (at 15 minutes) and the late reaction (at 6 hours). The sum of wheal/histamine index and fare/histamine index were calculated for the early phase reactions and analyzed according two-tailed paired student's t tests. Using RAST inhibition, the liophylized house dust mite showed the same allergen concentration as in the fluid form. The 3 months old vial was analyzed and showed the same allergen concentration as was expected. Results for histamine reactions on admission and discharge showed no significant difference. Patients showed no reactions on the negative controls. Differences were found between skin test reactions on admission and discharge for the sum of early wheal reactions (p = 0.0001), and to certain allergen wheal such as house dust mite and some other common allergens. We conclude that during a 12 weeks stay in the alpine climate Davos, intracutaneous early wheal reactions to certain allergens are decreasing possibly reflecting a decreased exposure. The late reaction showed no significant change.     

Identification of grass pollen allergens by two-dimensional gel electrophoresis and serological screening

Approximately 50% of allergic patients are sensitized against grass pollen allergens. The characterization of specific immunoglobulin E (IgE) reactivity to allergen components in pollen-allergic patients is fundamental for clinical diagnosis and for immunotherapy. Complex allergen extracts are commonly used in diagnostic tests as well as in immunotherapy preparations, but their composition in single allergenic molecules is only partially known. Diagnostic tests which utilize recombinant or immuno-purified allergens have been made available in clinical practice. They allow to obtain specific profiles of IgE reactivity, but the panel of available molecules is far from complete. Here, we used a proteomic approach in order to detect grass allergens from a natural protein extract. A five-grass pollen extract used for diagnosis and immunotherapy was resolved by two dimensional gel electrophoresis (2-DE), and assayed with 9 sera from pollen-allergic patients whose sensitization profile was dissected by using IgE reactivity to recombinant allergens. 2-DE immunoreactivity patterns were matched with IgE reactivity to identify protein spots as candidate allergens. Identity was confirmed by mass spectrometry analysis. We identified 6 out of 8 expected clinically relevant allergens in the natural grass extract. Moreover, we identified different molecular isoforms of single allergens, thus obtaining a more detailed profile of IgE reactivity. Some discrepancies in protein isoform profile and sera immunoreactivity between recombinant and native allergen 5 from Phleum pratense were observed and a new putative allergen was described. The proteomic approach applied to the analysis of a natural allergen allows the comprehensive evaluation of the sensitization profile of allergic patients and the identification of new allergens.     

A recombinant functional variant of the olive pollen allergen Ole e 10 expressed in baculovirus system

Pollens have been reported as important sources of antigens causing type-I allergy and, among them, olive pollen has high clinical relevance in Mediterranean countries. The most recently described olive allergen, Ole e 10, is involved in cross-reactivity phenomena and related to asthma induction in allergic patients. These immunologic features make this allergen a good candidate to be included in diagnosis and therapy of protocols of allergic diseases. Since the availability of Ole e 10 from the olive pollen is limited, the allergen has been efficiently expressed in the baculovirus/insect cell system. The Ole e 10-cDNA inserted into the transfer vector pBacPAK8 allowed the expression of the recombinant protein in cultured Sf21 cells. Recombinant Ole e 10 (rOle e 10) was purified from the culture after dialysis and three chromatographic steps. Mass spectrometry, Edman degradation, IgE- and IgG-binding analyses were employed to characterize the recombinant allergen, which showed molecular and immunological equivalence with the natural protein. Affinity gel electrophoresis in presence of laminarin (1,3-beta-glucan) revealed that rOle e 10 retains identical carbohydrate-binding capacity than the natural allergen. In conclusion, the recombinant expression of Ole e 10 in baculovirus/insect cell system produces a homogeneous and biologically active allergen that could be useful for clinical and scientific purposes.