Aqueous Extract Of Ragweed Pollen Research Articles

Pollen-derived adenosine is a necessary cofactor for ragweed allergy.

Ragweed (Ambrosia artemisiifolia) is a strong elicitor of allergic airway inflammation with worldwide increasing prevalence. Various components of ragweed pollen are thought to play a role in the development of allergic responses. The aim of this study was to identify critical factors for allergenicity of ragweed pollen in a physiological model of allergic airway inflammation. Aqueous ragweed pollen extract, the low molecular weight fraction or the major allergen Amb a 1 was instilled intranasally on 1-11 consecutive days, and allergic airway inflammation was evaluated by bronchoalveolar lavage, lung histology, serology, gene expression in lung tissue, and measurement of lung function. Pollen-derived adenosine was removed from the extract enzymatically to analyze its role in ragweed-induced allergy. Migration of human neutrophils and eosinophils toward supernatants of ragweed-stimulated bronchial epithelial cells was analyzed. Instillation of ragweed pollen extract, but not of the major allergen or the low molecular weight fraction, induced specific IgG1 , pulmonary infiltration with inflammatory cells, a Th2-associated cytokine signature in pulmonary tissue, and impaired lung function. Adenosine aggravated ragweed-induced allergic lung inflammation. In vitro, human neutrophils and eosinophils migrated toward supernatants of bronchial epithelial cells stimulated with ragweed extract only if adenosine was present. Pollen-derived adenosine is a critical factor in ragweed-pollen-induced allergic airway inflammation. Future studies aim at therapeutic strategies to control these allergen-independent pathways.

Purification and immunochemical characterization of recombinant and native ragweed allergen Amb a II

The complete sequence of a cDNA encoding Amb a II and its relationship to the Amb a I family of allergens has recently been described [Rogers et al. (1991) J. Immun. 147, 2547–2552; Griffith et al. (1991 a), Int. Archs Allergy appl. Immun. 96, 296–304]. In this study, we present results generated with rabbit antipeptide antisera that recognize Amb a II or Amb a I, but not both. The specificity of two anti- Amb a II antipeptide sera, anti-RAE-50.K and anti-RAE-51.K, was verified on Western blots of recombinant Amb a II and Amb a I.1. These two sera, directed against separate regions of the Amb a II molecule, detected three individual 38-kDa Amb a II isoforms on 2D Western blots of aqueous ragweed pollen extract. These Amb a II isoforms have pi in the 5.5–5.85 range and can be easily distinguished from Amb a I isoforms with pI in the 4.5–5.2 range detected by an anti- Amb a I specific peptide antiserum. The Amb a II isoforms have also been individually purified from pollen, positively identified as Amb a II by amino acid sequencing, and visualized as separate bands on IEF gels. An analysis of Amb a II cDNA sequences generated by PCR led to the prediction of three Amb a II isoforms with pI of 5.74, 5.86 and 5.97 that are very similar to the pI deduced from 2D Western blot analysis. Recombinant Amb a I.I and Amb a II have been expressed in E. coli, purified in their denatured form, and examined by ELISA for their capacity to bind pooled allergic human IgE. Purified native Amb a and Amb a II from pollen were shown to have very similar IgE-binding properties. In contrast, Amb a II had a markedly reduced IgE-binding capacity as compared to Amb a I.I. These data suggest that recombinant Amb a I.I and Amb a II, isolated in a denatured form, diner significantly in their IgE-binding properties whereas the native molecules isolated from pollen do not.

The clinical and immunologic specificity of immunotherapy

In order to study the specificity of immunotherapy for respiratory allergy, a group of patients sensitive to both ragweed and grass pollens were selected. From 87 volunteers with a history of both spring and fall hay fever, 42 patients with evidence of strong sensitivity by basophil histamine release to both ragweed pollen and mixed grass pollen extracts were selected for study. On the basis of the histamine release data, the patients were divided into two groups matched for sensitivity to both grass and ragweed pollens. In 1970, May and June symptom diaries showed the two groups to suffer quite similar severity of symptoms during the grass pollination season. One group of patients was started on a preseasonal course of immunotherapy with alum-precipitated aqueous extract of ragweed pollen while the other group received placebos containing histamine. By fall there had been a considerable rise in IgG-blocking antibodies to ragweed in the treated group. Symptom diaries in August and September showed that the treated group showed significantly less severe symptoms than the placebo group. Both groups received booster injections at 2-wk intervals from the fall of 1970 to the fall of 1971. Doses in the treated group were raised to attempt to administer the largest possible dose. Again there was no difference in the symptoms reported by the two groups during the grass pollination season, but an even greater difference emerged between the two groups during the ragweed season. The following year 1972 the same results were obtained. These data demonstrate that treatment with ragweed pollen extracts has little or no effect on grass pollen symptoms and confirm that immunotherapy is clinically as well as immunologically specific. Antibody responses to the second year of high-dosage booster injections was not greater than responses to a comparatively short preseasonal course given the first year.

Measurement of the absolute levels of IgE antibodies in patients with ragweed hay fever: Effect of immunotherapy on seasonal changes and relationship to IgG antibodies

The effect of immunotherapy with aqueous ragweed pollen extract on changes in IgE antibody was analyzed in 40 untreated patients with ragweed hay fever and compared to changes in 63 treated patients. Treated patients received cumulative doses prior to and during treatment ranging from 1.4 × 10 5 to 4.8 × 10 6 protein nitrogen units (PNU). IgG antibody to ragweed antigen E (AgE) was measured by radioimmunoprecipitation, while IgE antibody to allergens in crude ragweed extract was measured by the radioallergosorbent test (RAST). The RAST procedure was calibrated using a serum whose content of IgE antibody in nanograms per milliliter had been determined by immunoabsorption, and with this method the absolute quantities of IgE antibodies to ragweed allergens could be measured. Control experiments indicated that the IgG antibodies in the sera of the treated patients did not interfere with the measurements of IgE antibodies. The levels of IgE antibody in serum varied from less than 5 ng/ml to approximately 3,000 ng/ml. IgE antibodies decreased from October, 1973, to July, 1974, and rose sharply from July to October, 1974, following the pollination season. In both untreated and treated patient groups the magnitudes of the decreases were related (p < 0.001) to the levels of IgE antibody in October, and the magnitudes of the rises were related to the levels of IgE antibody in July. In the treated patients with IgE antibody less than 71 ng/ml in October, the rate of decrease (October to July) was less than in the untreated patients. Comparison of the rises in IgE antibody from July to October revealed that these were partially suppressed in the treated group (p < 0.001), and this effect was most marked in patients with July levels less than 36 ng/ml. Levels of IgG antibody to AgE in the treated patients were approximately 70-fold greater than in untreated patients and showed little change over the study period. The levels of IgG and IgE antibody in the treated patients were positively correlated (p < 0.001) at all time periods, and patients with high levels of IgG antibody also had greater declines and rises in IgE antibodies. The results indicate that immunotherapy is associated with (1) a reduction in the seasonal decrease in IgE antibody from October to July in patients with the low levels of IgE antibody, (2) a partial suppression of the rises in IgE antibody from July to October, and (3) that both these effects are related to the level of IgE antibody before the change. In many treated patients both IgE and IgG antibodies were low in spite of parenteral administration of ragweed extract. This result is consistent with the view that overall humoral immunologic reactivity to ragweed antigens is reduced in these patients.

Stability of antigen E in commercially prepared ragweed pollen extracts

Aqueous ragweed pollen extract (20,000 or 78,000 PNU per milliliter) lose 50 per cent of their antigen E content within five months of storage at 4 °C. Glycerinated extracts and lyophilized ragweed pollen extracts retain their antigen E content for over 15 months with little or no loss. The preservative phenol in aqueous extracts is destructive to antigen E, probably by protein denaturation. The stability of ragweed extracts as measured by antigen E content is markedly enhanced with little loss after 15 month storage at 4 °C. when the extract is prepared as an alum-precipitated suspension.

Identification of human reagins isolated with immunosorbent as IgE

An immunosorbent prepared by attaching covalently the constituents of the aqueous extract of ragweed pollen to aminocellulose was used to remove reagins from a fraction of allergic serum enriched with respect to skin-sensitizing activity. The adsorbed antibodies were eluted with a solution of 2M NaI, pH 9.1, or of glycine-HCl, pH 2.5. The presence of ragweed-binding IgE antibodies in these eluates was revealed by radioimmunoelectrophoresis and radio-Ouchterlony analysis. Guinea pigs, which had been rendered tolerant to normal human cord serum proteins, were immunised with these eluates and produced anti-IgE antibodies, as demonstrated by radioimmunodiffusion and by reversed PCA in monkeys. These results confirm that human reagins elicited to the allergens of ragweed pollen belong to the IgE class of immunoglobulins.

Fine structural changes in livers of dogs in anaphylaxis

Reaginic serum was used in the passive transfer of ragweed pollen sensitivity to normal dogs. Anaphylaxis was induced by intravenous injection of a challenge antigen consisting of an aqueous extract of ragweed pollen, and pathologic changes in liver were studied in serial biopsies taken at intervals ranging from 15 seconds to 20 minutes. The results showed destruction of endothelial cells and hepatocytes as early as 15 seconds; later changes consisted of vacuolation of hepatocytes, continued cellular disruption, and filling of sinusoids by cell debris. These changes persisted throughout the period of observation.

Comparison of two dosages of ragweed extract in the treatment of pollenosis

A double-blind study was carried out to determine the effect of dosage in the treatment of ragweed hay fever with injections of aqueous ragweed pollen extract. Two concentrations of extract were used, one 20-fold smaller than the other. The volumes administered were such that the dose at the higher concentration was close to the maximum that it is practical to administer. During the ragweed season, the group of patients receiving the higher concentration fared significantly better than the group receiving the lower concentration. It is concluded that large dosages of pollen extract are more effective than small dosages in the treatment of hay fever. The findings indicate that the very low dosages recommended by some would have little influence on the course of pollenosis.

Comparison of Two Dosages of Ragweed Extract in the Treatment of Pollenosis

A double-blind study was carried out to determine the effect of dosage in the treatment of ragweed hay fever with injections of aqueous ragweed pollen extract. Two concentrations of extract were used, one 20-fold smaller than the other. The volumes administered were such that the dose at the higher concentration was close to the maximum that it is practical to administer. During the ragweed season, the group of patients receiving the higher concentration fared significantly better than the group receiving the lower concentration. It is concluded that large dosages of pollen extract are more effective than small dosages in the treatment of hay fever. The findings indicate that the very low dosages recommended by some would have little influence on the course of pollenosis.

Characterization of the dialyzable constituents of the aqueous extract of ragweed pollen

Four fractions of the dialyzable components of the aqueous extract of ragweed pollen were prepared and their physicochemical and immunologic properties were examined. All these fractions were skin active with respect to sera from both treated and nontreated individuals. All the antigenic and allergenic determinant groups of the dialysate fractions were found to be present in the nondialyzable components of the whole aqueous extract. Contrary to previous conclusions, it was demonstrated by cross-neutralization studies that new reagin(s) directed exclusively against components of the dialysate is not produced by hyposensitization treatment with the whole aqueous extract. Rather, any increase in reagin concentration caused by hyposensitization is considered to be the result of a rise in the concentration of reagin (s) already being produced by the allergic individual.

A quantitative study of anaphylaxis in the guinea pig: Kabat, E. A., and Landow, H.: J. Immunol. 43: 69, 1942

An immunosorbent prepared by attaching covalently the constituents of the aqueous extract of ragweed pollen to aminocellulose was used to remove reagins from a fraction of allergic serum enriched with respect to skin-sensitizing activity. The adsorbed antibodies were eluted with a solution of 2M NaI, pH 9.1, or of glycine-HCl, pH 2.5. The presence of ragweed-binding IgE antibodies in these eluates was revealed by radioimmunoelectrophoresis and radio-Ouchterlony analysis. Guinea pigs, which had been rendered tolerant to normal human cord serum proteins, were immunised with these eluates and produced anti-IgE antibodies, as demonstrated by radioimmunodiffusion and by reversed PCA in monkeys. These results confirm that human reagins elicited to the allergens of ragweed pollen belong to the IgE class of immunoglobulins.

TREATMENT OF RAGWEED DERMATITIS

The first treatment of ragweed (contact) dermatitis was made in 1919. Hannah1reported good results in a single instance with coseasonal injections of an aqueous extract of ragweed pollen. In the same year, Sutton2reviewed 4 cases of the same condition, in 3 of which the patients were treated with a glycerin-phenol-saline solution extract of the pollen and showed decided improvement. However, none of these persons was given patch tests either with the oleoresin, which has since been proved to be the causative factor, or with ragweed leaves, which contain an abundance of the potent product. In 1930 Sulzberger and Wise3noted improvement in 1 person following intradermal injections of water-soluble ragweed pollen antigen. In 1931 Brown, Milford and Coca4showed that ragweed (contact) dermatitis is due to the fat-soluble fraction of the plant or pollen rather than to the water-soluble fraction, which causes hay fever

A sheep antibody which blocks the Prausnitz-Küstner reaction: Cohen, M. B., and Nelson, T.: J. Immunol. 34: 63, 1938

Two sheep have been repeatedly injected with an aqueous extract of ragweed-pollen. The sera of these sheep contain an antibody which inhibits the Prausnitz-Kustner reaction to an extract of ragweed-pollen.

A Sheep Antibody Which Blocks the Prausnitzküstner Reaction

Summary and Conclusions Two sheep have been repeatedly injected with an aqueous extract of ragweed-pollen. The sera of these sheep contain an antibody which inhibits the Prausnitz-Küstner reaction to an extract of ragweed-pollen.