Influence of a co-stimulation of human leucocytes with an Escherichia coli preparation and fixed immunoglobulins on cytokine release in the presence of hydrocortisone.

Pharmaceuticals of biological origin consisting of bacterial culture suspensions (BCS) as active ingredients have long been used for the treatment of hemorrhoidal diseases and chronic anal pruritogenic eczemas. However, some of these pharmaceuticals often contain glucocorticoids such as hydrocortisone as an anti-inflammatory supplement. Therefore, the question arises whether the claimed immunostimulatory capacity of the bacterial culture suspension might be altered by the steroid. Up to now, numerous reports support the evidence that the stimulation of the different Fc-receptor subtypes on leucocytes result in profound immunoregulatory activities influencing phagocytosis and antigen processing, antibody-dependent cytotoxicity or secretory functions thereby enhancing the overall activities of the immune system towards foreign antigens/pathogens. With these findings in mind it was investigated whether the immunomodulatory capacity(s) of the BCS in the presence of hydrocortisone will be modified by solid-phase bound immunoglobulins (Igs). For this purpose freshly prepared human peripheral blood leucocytes (PBLs) were incubated with different concentrations of the BCS (0.1, 1, 10 micrograms/ml), either with or without fixed human immunoglobulins in the presence of increasing concentrations of hydrocortisone. As a parameter of PBL activation the secretion of different cytokines was measured, e.g. tumor necrosis factor alpha (TNF-alpha), interleukin-10 (IL-10) and granulocyte-macrophage colony stimulating factor (GM-CSF). Cytokines were determined with specific sandwich ELISAs. With this modified cell culture system it was demonstrated that the immunosuppressive activities, normally caused by hydrocortisone, were partially antagonized by the combination of BCS plus fixed Igs. TNF-alpha and GM-CSF were significantly more produced, even in the presence of hydrocortisone, whereas the synthesis of IL-10 was diminished by fixed Igs. However, this effect could be reversed with increasing concentrations of hydrocortisone. These results raise the possibility that in the natural environment, e.g. the rectal mucosa, antigens derived from the BCS are bound by specific Igs, thereby modifying secretory and effector functions of locally present leucocytes in another way as free antigens. The biological relevance of these in vitro data with respect to the therapeutic benefit of the BCS preparations with hydrocortisone will be discussed considering recent findings in the literature.

Escherichia coli as a biologically active ingredient of suppositories. Solid phase extraction and quantification in a double antibody ELISA.

Techniques for isolation and quantification of an active ingredient of biological origin from a pharmaceutical product (Posterisan suppositories) were developed. By means of ELISA (enzyme-linked immunosorbent assay) in connection with a computerized evaluation, the antigenic material, a bacterial culture suspension (BCS) of Escherichia coli as a raw material was shown to be specifically and reproducibly detectable and quantifiable. As a limit of detection, a bacteria concentration of 6 x 10(4) cells/ml was determined, corresponding to 0.02% of the concentration in the product. After treating of the suppositories with organic solvents, the E. coli antigens were extracted with silica columns. The complete validations of both methods, the ELISA itself and the extraction procedure of the antigens from the matrix, in accordance with pharmaceutically accepted principles are presented. The eventual application of the new technique to the analysis of other pharmaceuticals with similar ingredients as well as the possibility of substituting conventional methods like total cell counting is discussed.

Respiratory allergies (bronchial asthma and rhinitis) due to sensitization of type I allergy to red spider mite (Panonychus ulmi KOCH).

The inhabitants of a fruit growing area often report spontaneously of sensitization to the red spider mite (RSM) (Panonychus ulmi KOCH). These are for the most part sensitizations with low clinical symptoms (rhinitis, conjunctivitis and erythema). Severe clinical developments with bronchial asthma have been observed. We investigated six patients working in a fruit growing area sensitized by RSM. The sensitizations corresponded to a Type I allergy. Skin tests and provocation tests (nasal as well as bronchial) with RSM showed immediate reactions and RAST positive results were obtained using RSM allergen disks. RAST measurements of sera from nine house-dust mite Dermatophagoides pteronyssinus allergic patients using RSM allergen disks showed RAST-class > 1 for eight patients. RAST inhibition and immunoprint suggest a possible cross-reaction between RSM and D. pteronyssinus.

Occupational wood-dust sensitivity from Euonymus europaeus (spindle tree) and investigation of cross reactivity between E.e. wood and Artemisia vulgaris pollen (mugwort).

A 44-year-old goldsmith suffered from rhinitis and conjunctivitis after having worked with wood dust from Euonymus europaeus (E.e.) for 15 years. The material was used for drying pieces of jewelry. Very strong reactions could be seen after friction test, scratch test and nasal challenge using wood dust of E.e. RAST-class 3 could be measured with the serum of this patient using E.e. wood and Artemisia vulgaris (A.v. pollen) allergen disks. RAST-inhibition, western-blot (WB) and immunoprint (IP) indicated common allergens in extracts of E.e. wood and A.v. pollen of different degree. In addition this study indicated that subjects suffering from A.v. pollen allergy also show sensitization to E.e. wood since in 22 of 37 A.v. pollen allergies A.v. (RAST class 2-4) IgE-antibodies could be seen. The present case probably demonstrates for the first time an IgE-mediated type I allergy to E.e. wood.

Determination of IgE-specificities in nasal secretions and sera of allergic subjects by crossed radio-immunoelectrophoresis.

Allergen-specific IgE antibodies have not only been demonstrated in the serum, but also in the nasal secretions of allergic patients by means of the radio-allergosorbent test (RAST). In this preliminary study we adapted the technique of crossed radio-immunoelectrophoresis (CRIE) to nasal secretions in order to obtain further insight into the specificity of locally collected antibodies. Based on RAST results we compared CRIE patterns in serum, nasal secretions in 13 out of 23 grass pollen allergic subjects and two out of three house dust mite allergic subjects. In the nasal secretions the same IgE specificities were found as in the corresponding serum of the individual subject and as in a reference serum pool. These data indicate that the same specificity of IgE antibody can be found in the serum and the target organ. From these data there is no evidence for a local mucosal immune response that differs in specificity from the serum response.

Allergens of horse epithelium. I. Physicochemical and immunochemical characterization of five different horse epithelium raw materials used for allergen extract preparation.

We investigated five horse epithelial allergen extracts prepared from different qualities of raw material by several biochemical and immunochemical methods. Horse serum albumin and horse serum were used to identify serum-related antigens. We found high similarities as well as marked differences between the extracts. There were strong differences in the protein contents, the protein patterns obtained by isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis, the total allergenic activities obtained by radioallergosorbent test inhibition assays and the amounts and numbers of serum-related proteins. The patterns of dander-related allergens in crossed radioimmunoelectrophoresis were similar but showed partly strong differences in allergen concentrations. Allergen compositions determined by blotting methods showed only minor differences between the investigated epithelial extracts. It appears that some of the differences, e.g. the content of serum-related proteins, depend on the way the different raw materials are prepared. Because serum-related extract components are not considered as major allergens, their content should be low in horse epithelial allergen extracts. For this reason whole skin cannot be recommended as starting material for horse epithelium allergen extract preparation.

IgE-mediated allergic reactions to potatoes.

We investigated sera of 12 patients with IgE-mediated hypersensitivity reactions to potato, using different in vitro methods. Radioallergosorbent test classes 2-4 (7.6-46.5% binding) were measured with potato allergen disks. Immunoblot detected allergen bands with an isoelectric point range of approximately 4.5-5.2 and mainly in a molecular weight range between 16.00 and 30.00 kD. But also at molecular weights of 43.00 and 65.00 kD allergen bands (IgE specificities) could be detected. Histamine release assay performed with isolated fractions of the potato extract showed a great individual variation and positive results of fractions of molecular weights between 10.00 and 80.00 kD. From our data we conclude that raw potato is a 'multiallergenic' vegetable.

Comparison of Alternaria tenuis extracts prepared from different raw materials.

Alternaria extracts, prepared from three different sources under the same conditions, were compared by several biochemical/immunochemical methods. Two raw materials (A,G) contained mostly mycelia. The third raw material (C) contained mycelia (35%) and spores (65%). Extracts A and G were different in total allergenic potency, antigen/allergen pattern, and in the Alt 1 content. Extract C was of similar total allergenic potency as extract A but showed a somewhat different antigen/allergen pattern and a different Alt 1 content. Although there were some compositional differences among the extracts, all extracts regardless of source materials (spores and mycelia) demonstrated strong RAST inhibition activity and parallelism of the inhibition curves.

Double-blind, placebo-controlled immunotherapy with mixed grass-pollen allergoids. III. Efficacy and safety of unfractionated and high-molecular-weight preparations in rhinoconjunctivitis and asthma.

Specific immunotherapy with unmodified formalinized allergoids is effective in grass-pollen allergy, but systemic reactions have been observed. A high-molecular-weight formalinized allergoid (HMW-GOID) was fractionated by gel filtration, retaining molecules of greater than 85,000 daltons in the expectation of improving safety without sacrificing efficacy. HMW-GOID and unfractionated allergoid (GOID) had a similar allergenic activity assessed by RAST inhibition, but the HMW-GOID preparation was 65 times less reactive when it was tested by skin prick test than the GOID preparation. A double-blind, placebo-controlled study was carried out in grass pollen-allergic patients with placebo (14 patients), GOID (15 patients), and HMW-GOID (13 patients). An additional group of 18 patients was treated by a rush schedule with a standardized orchard grass-pollen extract. A similar mean cumulative dose was administered with both allergoids. The fractionated allergoid only elicited minor systemic reactions similar to reactions elicited by placebo, whereas 20% of patients treated by GOID and 5.5% of patients receiving the standardized extract had a severe systemic reaction. For rhinitis, conjunctivitis, and asthma, the HMW-GOID and the standardized extract had a similar efficacy, significantly greater than placebo. GOID was less effective than the other two active treatments but was significantly more effective than placebo treatment for asthma and conjunctivitis.

Investigation of the allergenic activity of isolated fractions of a standardized partly purified whole mite body (Dermatophagoides pteronyssinus) extract.

Twelve fractions of a molecular weight range of 1.35-670.00 kilodaltons (kD) were isolated from a biologically standardized partly purified whole mite body extract (Dermatophagoides pteronyssinus) by preparative size exclusion high-performance liquid chromatography. The allergenic activity and the antigen and allergen patterns of the isolated fractions were investigated by RAST, RAST inhibition and crossed (radio)immunoelectrophoresis (CIE/CRIE). By CRIE, each of the fractions showed allergen patterns, mostly of different compositions. Each fraction showed allergenic activity of different degrees by RAST inhibition. The highest allergenic activity could be measured by RAST inhibition with fractions which contained predominantly the major allergens Der pI and PY as detected by CRIE. Also proteins of higher molecular weights (greater than 158 kD) showed IgE-binding capacities. Nearly all antigens detected by CIE could be identified as allergens using CRIE.

The international collaborative study on the first international standard of birch (Betula verrucosa)-pollen extract.

A selected candidate international standard preparation of birch (Betula verrucosa)-pollen extract was studied together with other birch-pollen extracts in a multinational study involving 20 laboratories in 11 countries. The biologic activity of the extract had previously been demonstrated in quantitative skin prick testing. The study methods comprised RAST inhibition, histamine release, quantitative immunoelectrophoresis, isoelectric focusing, and other methods. The results from RAST inhibition were calculated as parallel-line assays with statistical tests for linearity and parallelism. Analysis of variance was applied to test the significance of differences between potency estimates. In all assay methods, the candidate standard could be used to assign relative potencies to other birch-pollen extracts. The candidate standard was adequately stable during 36 months of storage at or below 5 degrees C. On the basis of this study, the World Health Organization has established the preparation as the International Standard for birch-pollen extract with assigned units of 100,000 IU per ampule.

Investigations of patients allergic to the house dust mite Dermatophagoides pteronyssinus by crossed radioimmunoelectrophoresis using purified mite bodies and whole mite culture extracts.

Sera of 20 patients allergic to the house dust mite Dermatophagoides pteronyssinus were investigated by crossed radioimmunoelectrophoresis (CRIE) using extracts prepared from purified mite bodies (PMB) and whole mite culture (WMC). By CRIE, different allergen patterns and different numbers of allergens were detected in the two extracts. By summarizing the values of the CRIE patterns of the 20 patients' sera into allergograms, 6 out of 16 allergens in PMB and 5 out of 10 allergens in WMC extracts could be identified as major allergens. These diverging results, together with the published literature, emphasize the necessity to reevaluate the International Standard for D. pteronyssinus.

Double-blind, placebo-controlled immunotherapy with mixed grass-pollen allergoids. I. Rush immunotherapy with allergoids and standardized orchard grass-pollen extract.

Forty-five grass pollen-allergic patients were randomly assigned to three groups according to their skin test and RAST sensitivities and the severity of seasonal rhinitis. Eleven patients were treated with placebo (group 1), 19 patients (group 2) were treated with a six-mixed grass-pollen allergoid prepared by mild formalinization with a two-step procedure, and 15 other patients were treated with a standardized orchard grass-pollen extract (group 3). Because of a different immunotherapy schedule, only patients placed in groups 1 and 2 received the extracts in a double-blind fashion. Rush immunotherapy was performed in 3 to 6 days, and the maintenance dose was subsequently administered weekly for 4 weeks and every 2 weeks until the end of the grass-pollen season. During the season, a coseasonal treatment was administered. Systemic reactions occurred during the rush protocol in 36.8% of patients treated with allergoid and 20% of patients who received the standardized extract. Only patients treated with allergoid had systemic reactions during maintenance dose. The reactions observed with the standardized extract were more severe. Total doses of allergoid ranged from 2350 to 13,500 protein nitrogen units. Symptoms and medication scores during the peak of the season were analyzed. Patients treated with the standardized allergen had a significant reduction of the number of days of symptoms during the month of June (9.5 +/- 6.7 days; p less than 0.005) and of medication scores (1.3 +/- 1.4; p less than 0.01) compared to patients receiving placebo (19.4 +/- 8.1 days; medication score, 2.8 +/- 2.1).(ABSTRACT TRUNCATED AT 250 WORDS)

A manufacturer's criteria for in-house reference preparations for RAST inhibition.

Reagents employed for RAST-inhibition need careful characterization. An allergen reference preparation was produced from carefully selected raw materials. After immunochemical and biochemical characterization, the reference preparation was used to produce allergen reference paper disks. Their quality was controlled by the amount of bound protein and serum titration, which should yield an S-shaped dose-response curve and specific binding of IgE of at least ten times more than background binding. In parallel, the biologic activity of the allergen reference preparations was established in quantitative skin prick tests in at least 20 allergic patients. From these patients, serum samples were collected and pooled after careful characterization. All three necessary reagents for RAST-inhibition (extract, disks, and serum pool) were stabilized by freeze-drying. The reproducibility of the assay procedure itself was checked by documenting the 50% inhibition value of the reference preparation, accepting only values within +/- 2 SD. By employing such reagents we could measure the activity of alum-adsorbed grass pollen extracts with sufficient precision, ie, the coefficient of variation of the mean potency of 12 batches was less than 40%.

Immunological cross-reactions between a cat hair and skin scraping extract and a cat serum and investigation of common allergenic molecules of cat 1 with cat serum.

Investigations of cross reactions between a cat hair and skin scraping extract (CHSS) and a cat serum (CS) were done by several biochemical and immunochemical methods. Furthermore, we looked for common allergenic molecules in a cat 1 reference preparation and in a CS. In crossed (radio) immunoelectrophoresis (CIE/CRIE) of CHSS vs. anti CS-serum one to eight allergen bands could be detected depending on the human serum employed. By constructing allergograms from the CRIE patterns of 17 patients' sera one out of eight allergens could be defined as a major allergen. CRIE of the cat 1 reference vs. anti CS-serum, using the sera of two cat allergic patients, displayed four to nine allergen bands, indicating that the cat 1 reference is not a pure preparation but rather an enriched fraction.

[Immunoglobulin E-mediated contact urticaria and bronchial asthma caused by household rubber gloves containing latex. 3 case reports]. / Immunglobulin E-vermittelte Kontakturtikaria bzw. Asthma bronchiale durch Latex-enthaltende Haushaltsgummihandschuhe. Drei Fallberichte.

During a period of a few months we observed 3 female patients suffering from contact urticaria by use of rubber gloves. In one case the local urticarial reaction at the fore arms was followed by asthma. In all 3 cases we could prove type I allergy against latex by radio allergo sorbent test (RAST).

Allergenic activity of short ragweed pollen (Ambrosia elatior) from different years and/or suppliers: criteria for the selection of an in-house allergen reference preparation.

We have investigated dialyzed extracts (A-F) of short ragweed pollens (Ambrosia elatior) from six consecutive production years and four different companies by protein nitrogen units, high performance liquid chromatography, isoelectric focusing, SDS-PAGE, RAST inhibition, crossed radioimmunoelectrophoresis, and antigen E contents. Extract A showed lowest yields in all quantitative assays. Isoelectric focusing, SDS-PAGE, crossed immunoelectrophoresis, and crossed radioimmunoelectrophoresis showed comparable patterns for all extracts except for extract A. Apparently, the activity of short ragweed pollens depends on the year of production and/or the skill of the producer. These findings are in contrast to similar examinations of other pollen species.

Molecular weight determination of allergen extracts and isolation of allergenic molecules by high-performance liquid chromatography.

The usefulness of size-exclusion high-performance liquid chromatography (HPLC) for the molecular weight determination of allergen extracts as a method of process control was investigated by measuring seven different batches of timothy pollen extracts. Preparative HPLC was used for the isolation of the major allergen Cat 1, from a crude cat hair and skin scraping (CHSS) extract. The chromatograms of the seven batches of the timothy pollen extracts looked very similar, suggesting the same molecular weight distribution in each extract. The major allergen, Cat 1, could be isolated from the crude CHSS extract by preparative HPLC in acceptable purity, determined by crossed radioimmunoelectrophoresis.