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The effects of an extract of the saprophytic mold, Alternaria tenuis (AT-CE) on the humoral response to a ragweed allergen extract (DWSR), ovalbumin and sheep red blood cells (SRBC) was investigated in female Wistar rats. Animals pretreated with 100 micrograms or 2 mg AT-CE showed enhancement (p less than 0.05) in the reaginic response (IgE antibody) to DWSR at 25 and 18 days postimmunization. On the other hand, animals posttreated with AT-CE showed substantial reduction in anti-DWSR IgE antibody response. Contrasting results were obtained when ovalbumin was used as an immunizing antigen. There was a remarkable enhancement in the reaginic response to ovalbumin in rats pre- or posttreated with 10 micrograms of AT-CE. Pretreatment with AT-CE did not affect the hemagglutination titers to ovalbumin, while posttreatment with 100 micrograms or 1 mg AT-CE increased the hemagglutination titers of IgM antibody. There was a significant reduction in hemagglutinin, and hemolysin titers to SRBC in animals pretreated with all concentrations of AT-CE; at day 21, suppression was noted in animals pre- or posttreated with all concentrations of AT-CE. On the other hand, greatly increased hemagglutination titers were found in animals posttreated with 100 micrograms or 1 mg AT-CE. Hence, enhancement and suppression can both occur depending on the dose and time of administration of AT-CE together with the nature of the immunizing antigen.

Comparison of intracutaneous skin tests and RAST in 2 groups of patients, one consisting of 16 individuals having multiple allergies to pollen, mold and animal dander and the other of 10 patients allergic to mold only, revealed that skin tests were more sensitive than RAST. Skin reactions of 3+ to 4+ were frequently associated with negative RAST results. This was observed more so in mold-allergic patients. In a few cases (6%) RAST showed 2+ reactions while the skin tests were negative. Leukocytes from allergic individuals who had positive skin tests and negative RAST released a significant amount of histamine upon challenge with specific allergen as well as antihuman IgG4, suggesting that this immunoglobulin is a functional component of some immediate hypersensitivity reactions.

Effects of staphylococcal enterotoxin A (SEA) on the mouse homocytotropic antibody (HCA) system were studied. Groups of BDF mice received 10 micrograms SEA either orally or intraperitoneally at 0, 24, 48 h before or after immunization with 100 micrograms ovalbumin in 1 mg A1(OH)3 gel. Primary and secondary HCA responses were determined by 48-hour passive cutaneous anaphylactic reactions in genetically hairless mice. It was found that effects of SEA on HCA responses were dependent on the time and route of SEA administration. In general, early administration (48 h before immunization) of SEA showed suppression, while later administration (either 24 h before or after immunization) of SEA demonstrated enhancement. A further delay of SEA administration (48 h after immunization) exerted suppressive effects except when it was given intraperitoneally in the anamnestic HCA experiments. The mouse HCA system proved to be a suitable in vivo correlate of in vitro plaque-forming cell responses modulated by SEA.

The reaginic antibody response to alum-precipitated ovalbumin (OA) and the dialyzed water-soluble extracts of ragweed (DWSR) and Alternaria tenuis (DWST) in several strains of rats appeared to be wholly an IgE response. There was no evidence of a heat-stable (IgGa) antibody to OA, DWSR and DWST in the sera of the rats immunized with these antigens suspended in alum. Wistar-Furth and Lew inbred and hooded outbred rats produced comparable amounts of reaginic antibody after immunization with DWST, but BN inbred rats failed to generate a reaginic response to this antigen. The amount of antigen-induced histamine release from rat peritoneal mast cells did not always correlate with the level of circulating IgE-specific antibody.

The allergenic potencies of samples of two commercial Alternaria tenuis extracts stored under various conditions were compared at 3 months intervals over a period of 1 year. Aliquots of the extracts at 5.12 mg/ml were maintained in: saline; 50% glycerol-saline mixture; 0.4% phenol-saline; 0.4% phenol-saline containing 0.03% human serum albumin, and samples were also kept in lyophilized form. The samples were stored at -20, 4, 24, 37 and 56 degrees C. Their potency was measured by radioallergosorbent inhibition assay and by mouse IgE passive cutaneous anaphylaxis (PCA) tests. The lyophilized extracts fully maintained their activity at all temperatures for 12 months. Aqueous extracts with or without preservatives also maintained their potency if stored at -20, 4 and 24 degrees C, but showed a significant loss of PCA activity at 56 degrees C, after 3 months. A deleterious effect of phenol was observed in one of the extracts stored at -20 degrees C for 3 months, by PCA tests. The use of preservatives to retain potency over a period of 12 months is therefore unnecessary and may be deleterious. The results indicate that, while storage of A. tenuis in a lyophilized form is best at all temperatures, aqueous extracts are also remarkedly stable when stored at -20, 4 and 24 degrees C, over a 12-month period.

12 of 93 patients with untreated seasonal ragweed rhinitis, strong skin sensitivity and 2+ to 4+ RAST gave insignificant (less than or equal to 14%) histamine release from their leukocytes on challenge with ragweed allergen.

Fraction A, a concentrate derived from short ragweed extract by (NH4)2SO4 precipitation, contains all major allergens including antigen E. Therapeutic efficacy and safety of this alum-adsorbed product was evaluated in a multicenter double-blind clinical trial by comparing it to placebo in 131 randomly matched ragweed hay fever patients who were not treated for at least 1 year. Therapeutic response to fraction A injections was more significant than placebo (p less than 0.05) by investigators' evaluation, by average daily symptom score (1.097 vs. 1.378, p less than 0.05) during the peak ragweed season, or by average proportion of days in which medication was required (0.411 vs. 0.584, p = 0.01). Fraction A patients exhibited a significantly higher (p less than 0.0005) increase in hemagglutinating titer after treatment than placebo controls. Radioallergosorbent binding, evaluated in a subgroup of 31 patients before and after the ragweed season, was unchanged or decreased in 12 of 15 patients treated with fraction A. In contrast, 9 of 16 placebo-treated patients showed an increase in this index. Systemic effects were noted after 42 (1.5%) of 2.641 injections, while local reactions occurred at the rate of 2.3%. It is concluded that a single course of preseasonal immunotherapy wih alum-adsorbed fraction A is effective and relatively safe.

The biochemical and immunological characteristics of a series of commercial samples of Alternaria tenuis were examined, including samples from two different companies. There was a considerable variation in the biochemical parameters such as the gel filtration profile on Sephadex G-100, isoelectric focussing pattern and immunodiffusion precipitin band pattern. The content and proportion of two high molecular weight polysaccharides, one a galactomannan and the other a glucan, also varied greatly. In contrast, the allergenic properties of the samples were more consistent. In rat IgE passive cutaneous anaphylaxis tests, extensive cross-reactivity was observed among batches from the same company, but this cross-reactivity was weaker when batches from the two companies were compared. In direct radioallergosorbent tests (RAST) the samples showed similar reactivities, but RAST inhibition tests showed a 40-fold range in activity between samples from one company and 55-fold between batches from the two sources. It was found that the proportions of the major A. tenuis allergen and a related hypoallergenic antigen in the samples were relatively constant, hence RAST inhibition can be used as a measure of allergenic potency despite the presence of this cross-reacting antigen. The lack of correlation between allergenic and biochemical properties of the various samples suggest that a simple biochemical test is unlikely to be a satisfactory replacement for the RAST inhibition assay in standardizing A. tenuis extracts.

Based upon previous clinical data suggesting that the potential therapeutic benefits of hyperimmune specific IgG could involve neutralization and regulatory effects, this cooperative study evaluated the efficacy and safety of combined active and passive immunotherapy in previously untreated ragweed-sensitive patients. Symptom scores and indices of patients receiving ragweed-specific globulin plus rush desensitization with ragweed extract were significantly reduced (p less than 0.05), as compared to control patients treated with albumin and active rush immunotherapy. Postseasonal RAST levels were either unchanged or decreased in 28 of 33 patients receiving hyperimmune gamma-globulin. These data were significantly different from those in albumin-treated patients (p less than 0.002). The usual anamnestic rise of ragweed-specific IgE was modified without affecting the ongoing synthesis of ragweed specific IgG. Mild constitutional symptoms were observed in patients undergoing rush immunization, but the overall procedure was relatively well tolerated and no serious or treatment-refractory constitutional problems were encountered. This clinical trial suggests that ragweed immune gamma-globulin might be a useful therapeutic adjunct when administered in close association with optimal amounts of allergen.

Earlier, it was reported that most of the allergenic activity of Alternaria tenuis seemed to reside in the last Sephadex G-100 fraction (G3, molecular weight approximately 20,000). These conclusions were based on results obtained by the in vitro techniques of radioallergosorbent tests (RAST) and RAST inhibition. In the present studies, the allergenic activity of A. tenuis fractions was measured in vivo. It was found that fraction G3 was neither capable of inducing reaginic (IgE) antibodies in rats nor eliciting a skin reaction in rats sensitized with reagins against A. tenuis non-dialyzable fraction (MW greater than 12,000). On the other hand, subfractions G2D3 and G2D4 obtained from the middle fraction of G-100 (G2, MW approximately 30,000-40,000) followed by ion-exchange chromatography on DEAE-cellulose, showed the greatest allergenic activity in vivo, giving high titered reaginic antibodies in rats. When compared by RAST inhibition, the activities of fractions G2D3 and G2D4 were slightly lower than that of G3. Fraction G3 was immunogenic in rabbits. In immunodiffusion tests, G2D3, G2D4 and G3 appeared to be identical and they were cross-inhibiting in RAST inhibition tests. It was concluded that the sizes and multivalent properties of G2 and G3 antigens exerted a strong influence on their allergenic activities in rats (IgE antibody production) but this factor was of little importance for their antigenic activity in rabbits (precipitin antibody production) and their behaviour in the in vitro tests.

Leukocyte migration inhibitory factor assay was employed as an in vitro diagnostic aid in nickel dermatitis, the second most common contact dermatitis in North America. 15 patch test-positive and 5 patch test-negative patients, all giving a past history suggestive of nickel dermatitis, were investigated. Significant inhibition of leukocyte migration in both groups of patients was obtained only with nickel sulfate-albumin conjugate and not with unconjugated nickel sulfate. Specificity of this system was tested by utilizing an unrelated metallic albumin complex, and no inhibition was found. When patch testing is equivocal or contraindicated, this in vitro technique may be a practical alternative.

Extracts of Alternaria tenuis and Alternaria solani were separated into dialyzable (molecular weight less than 10,000) and non-dialyzable forms. The latter was further fractionated by gel filtration through Sephadex G-100 followed by ion-exchange chromatography on DEAE-cellulose. The dialyzable material was fractionated by gel filtration through Sephadex G-50. The allergenic activities of the fractions obtained from the A. tenuis extract was measured in vitro by the radioallergosorbent test assay and the allergenic potency was measured by radioallergosorbent test inhibition assay. Allergenic activity was detected in most of the non-dialyzable fractions, the majority of the activity being in the last G-100 fraction (MW approximately 20,000) which was predominantly protein in nature. The same component may be responsible for the activity found in the dialyzate and its first G-50 fraction since the immunodiffusion studies indicated that the last G-100 fraction has antigenic components in common with those of the first G-50 fraction. In addition, cross-reactions between A. tenuis and A. solani extracts show that the two species share common antigenic determinants.

Six extracts were prepared from Alternaria tenuis isolated ATCC 6663, ATCC 16086 and DAOM (Agriculture Canada) 183905 grown separately on two synthetic media, revised tobacco and Czapek's, and their biochemical and immunological properties were examined. High-performance liquid chromatography revealed considerable variations in UV absorbance and carbohydrate profiles among extracts from the different isolates. These differences were less marked among samples of the same isolate cultured on different media. Enzyme screening showed that all extracts contained large amounts of phosphatases and glucosidases and moderate quantities of esterases. Only the alpha-galactosidase activity showed any correlation with allergenic activity. No significant variation was observed in isoelectric focusing patterns. Extensive antigenic cross-reactivity even between the different isolates was found in precipitin studies. In mouse IgE passive cutaneous anaphylaxis tests, all extracts gave reactions of similar intensity. In direct RAST and RAST inhibition assays, ATCC 16086 grown on revised tobacco medium was found to be the most potent and approached the activity of an extract from a commercial material (B-I). DAOM 183905 grown on either medium was next in potency while ATCC 6663 samples were the least potent. The results indicate that it is possible to obtain extracts of high allergenic potency for standardization purposes from growth of selected A. tenuis isolates on a chemically defined medium.