Mary C. Johnson

Studies on correlation between symptoms of ragweed hay fever and titer of thermostable antibody.

Abstract 1.1. There was a general lack of correlation between thermostable antibody titer as determined by the methods used and degree of clinical protection. 2.2. The mechanism by which clinical improvement occurs following specific pollen therapy remains unknown.

Inhibitory Effect of Nitrogen Mustard (Bis Beta-Chloroethyl Amine) on Lesions of Experimental Serum Hypersensitiveness.

Summary Periodic intravenous injections of the nitrogen mustard, bis beta-chloroethyl amine, suppressed both antibody formation and the development of vascular lesions in rabbits injected intravenously with a massive dose of horse serum. The incidence of vascular lesions was directly correlated with the amount of antibody produced, since all rabbits with terminal amounts of antibody in excess of 0.06 mg nitrogen per ml exhibited vascular lesions. Cutaneous hypersensitiveness failed to develop in rabbits which did not produce detectable amounts of anitbody.

Detection of the “thermostable” antibody by means of the precipitin reaction: Preliminary report

Abstract 1.A precipitin method for the detection of thermostable antibody has been devised. 2.This method compares favorably with that of passive transfer and is simple to perform. 3.This report is preliminary.

Fractionation of pollen extracts by chromatography: I. Preliminary studies with ragweed extract

Abstract A method has been presented whereby a minimum of 6 different active components were obtained from ragweed pollen extract. Five of these components readily diffuse through Visking membrane and are therefore of relatively small molecular size. It has been demonstrated that 4 of the diffusible components are peptides and the major amino acid composition of 3 of these peptides has been defined. The fifth diffusible active component is a carbohydrate. In addition to the diffusible active components there is a highly active fraction of ragweed extract which is retained by Visking membrane after 16 days of dialysis against running water. It has been demonstrated that this dialyzed residue contains at least one large polypeptide or protein. The observation that this material is not readily demonstrable by the ninhydrin reaction on paper suggests that the polypeptide or protein contains relatively few free amino groups. The major amino acid composition of the protein or polypeptide components of the dialyzed residue has been determined. The activity of each of the fractions described has been demonstrated by direct intradermal test in ragweed-sensitive subjects and by passive transfer and neutralization techniques. Each ragweed-sensitive subject reacted to the dialyzed residue and a positive passive transfer reaction was obtained with each reaginic serum challenged with this material. Fifteen of 17 ragweed subjects and 22 of 25 reaginic sera reacted to one or more of the diffusible components (CHO, A, B, C, and D). The observations presented (Tables I and II) suggest that each ragweed sensitive subject has an individual pattern of reactivity to the smaller molecularsize components: the data suggesting that most subjects are reactive to certain of these components and not to others, some are reactive to all, and a few to none; however, because of a scarcity of material, observations were made with only a single concentration of the dialysate fractions and, therefore, quantitative variations in reactivity have not been fully evaluated. Regarding the origin of the fractions of dialysate, it is possible that the process of dialysis or the action of lutidin on the extract results in a separation of active fragments from a larger comples antigen. Since the active diffusible components have been identified on chromatograms of whole ragweed extract, the process of dialysis does not seem to be responsible for the breakdown of a large comples. Since electrophoretic and ultracentrifuge studies have indicated the presence of a number of active components of small molecular weight 15 it seems further probable that the active diffusible fractions obtained from chromatograms are not the result of any solvent action on a complex but rather that they are native to ragweed extracts. This study demonstrates that paper chromatography is a valuable adjunct in the investigation of a crude antigenic material and the methods employed might profitably be applied to the study of other crude allergenic extracts. Ragweed extract has been fractionated heretofore by electrophoretic techniques and by more classical chemical procedures. The fractions obtained in those studies have been difficult to evaluate and correlate since their chemical definition has in most studies been based on general qualitative chemical reactions. The methods employed in this study provide not only for a separation of the active components of ragweed extract but also provide extensive information concerning the composition of these components. A continuation of the present study of ragweed extract by paper chromatography, immunologic, and classical chemical methods is in progress.

Quantitative immunologic studies with allergens II. Deterioration of Ragweed pollen extracts as measured by precipitation, neutralization, and protein nitrogen analysis the prevention of deterioration by glycerin

Abstract 1.1. Plain unglycerinated low ragweed extracts, upon heating to 56° C. for thirty minutes and upon storage at room temperature, 6° C., −25° C., and −70° C. for periods up to one year, showed loss of activity as measured by their ability to precipitate antiragweed rabbit serum and to neutralize skin sensitizing antibodies of human ragweed sensitive serum. 2.2. Low ragweed extracts containing 50 per cent glycerin showed no loss or less loss of activity than their corresponding plain extracts after heating or storage at the four different temperatures.

Measurement of circulating ragweed antibodies and antigen: Correlation with symptoms and treatment

T HE purpose of this investigation was to study and correlate seasonal circulating ragweed antigen, which was de,scribed in a previous communication,l with thermostable antihody and the relation of each of these to reagin titer, symptoms, pollen count, and treatment with pollen extract. During this study, blood samples were obtained from both treated and untreated ragweed-sensitive patients and from normal, untreated individuals. No set schedule of treatment in respect to dosage or interval between injections was adopted. The pat,ients st,ndied received either the perennial or annual type of treatment, and varied from those extremely sensitive to those relatively insensitive. Considerable care was ta!:en in the evalntion pf symptoms and, in spite of detailed records, it was realized that appraisal of symptoms is always subjective on the part of hoth the clinician and the patient. Tndividual variations in exposure to pollen modify the improvement produced by specific pollen therapy. However, an evaluation of sympt,oms was attempted by reviewing each week a daily memorandum kept by the patient. A symptom score system was then adopted whereby symptoms for each w-eek were classified as 0, 1, 2, 3, and 4 in ratio to severity, and these were added as an accumulative score for the season for each patient (Table II). Blood samples were drawn at four-week intervals from March until August 15, after which the interval was shortened to one week. The samples were always obtained during each clinic visit prior to injection with pollen extract. Whole ragweed ext,ract was used for therapeutic injections. Circulating antigen, thermostable antihody, and reagin were determined on each blood within twent,y-four hours after it was received. This entailed the setting up and titration of several hundred tubes each week. For this rcason, the number of patients studied was limited to 29 treated cases and 11 controls. METHODS

Studies on the optimum concentration of glycerine in the preparation and preservation of ragweed pollen extract

Abstract 1.1. Extracts of low ragweed pollen, prepared in the concentration of 20 grams per 100 ml. of extracting fluid which contained 0, 5, 10, 20, 30, 40, and 50 per cent glycerine by volume, were observed after storage for five and twelve months at refrigerator (4° C.), deep-freeze (−25° C.), and room temperatures (20° C. to 40° C.), as well as heating at 56° C. for forty-five minutes. 2.2. Each extract was evaluated in terms of appearance of sediment, changes in concentration of PTA nitrogen and reducing substances, and loss of activity in neutralization of skin-sensitizing antibody. 3.3. The higher the concentration of glycerine, the greater was the protective effect against loss of reagin-neutralization activity at the various temperatures studied. 4.4. There was essentially no change in PTA nitrogen after heating, regardless of the concentration of glycerine. There was, however, an appreciable loss in PTA nitrogen of the stored extracts, most pronounced in those extracts containing smaller concentrations of glycerine. 5.5. The loss in bound carbohydrate varied under the different conditions of temperature. There was no change in any extract after heating at 56° C. for forty-five minutes. However, at room temperature, glycerine in the higher concentration showed a protective effect not evident in the lower concentrations. 6.6. An important observation was the clarity of extracts of high glycerine content in comparison with the sedimentation in extracts containing either no glycerine or glycerine of low concentration. 7.7. Whereas 30 and 40 per cent glycerine afforded significant protection against deterioration, the greatest protection was observed at the 50 per cent concentration.

The preparation of highly concentrated allergenic extracts by increasing the concentration of extraction: I. Timothy and low ragweed pollens

Abstract 1.1. Highly concentrated timothy and low ragweed pollen extracts have been prepared by direct extraction. 2.2. Extracts of timothy and low ragweed pollens, prepared in increasing weight by volume ratios, from 5 to 50 per cent, showed a distinct proportionality, indicating extraction in linear ratios, as measured by total and phosphotungstic acid-precipitable nitrogen content, concentration of reducing substances, and reagin neutralization activity. 3.3. The use of glycerinated extracting fluids was necessary in the preparation of such highly concentrated and potent extracts to avoid sedimentation and to preserve activity.

A quantitative method for measurement of precipitin reactions

Abstract 1.1. A technique is described wherein the Meloche microcentrifuge tube is used for the quantitative measurement of precipitate formed by the precipitin reaction. 2.2. The method is reliable and relatively simple and has possibilities of being applicable to the measurement of precipitates in other immunologic reactions.

Fractionation of pollen extracts: II. Chemical and paper chromatographic investigation of ragweed pollen extracts☆

Abstract 1.1. The solvent for paper chromatographic separations of skin reactive allergens must be chosen with great care. Lutidine can combine chemically with certain compounds and is in itself capable of producing skin reactions. 2.2. Fractions very similar to those previously obtained with lutidine were separated by use of a nonskin reactive solvent, methyl cellosolve. Such fractions exhibited skin reactivity. From this it has been concluded that the originally separated lutidine fractions were actually active components of dialysate. 3.3. Activity in the dialysate of ragweed extracts appears to be due to breakdown of originally larger particles, as shown by the extreme lability of the fractions isolated by paper chromatography. 4.4. Nearly all of the skin reactivity present in dialysate was precipitable by making an aqueous solution 60 per cent with respect to acetone. 5.5. Chemical fractionation of the nondialyzable portion of ragweed extract failed to produce an active fraction which was completely free of either protein or carbohydrate, respectively. Warm alkaline hydrolysis and phenol extraction came closest to producing such a fraction although the specific activity was reduced in both cases. Dialysate did, however, produce a few skin reactive carbohydrate-free fractions. 6.6. The active components appear to consist of a protein-carbohydratepigment complex, the carbohydrate component being a polysaccharide of arabinose.