Ragweed-sensitive Patients Research Articles

Physicochemical properties of reaginic antibody: III. Further studies on the reaginic antibody in γA-globulin preparations

Three sera from ragweed-sensitive patients were fractionated by chromatography on DEAE cellulose columns and gel filtration. It was confirmed that the reaginic activity in the chromatographic fractions did not parallel γA-globulin concentration. The γA-globulin fractions from the same sera contained reaginic activity. When the γA-globulin preparations were further fractionated by chromatography on DEAE cellulosc columns, the reaginic activity was concentrated into one of the three subfractions. The precipitation of γA-globulin in the subfraction and the original γA-globulin preparation with rabbit anti-γA-globulin antibody was not accompanied by any detectable decrease of reaginic activity. The results indicated that the reaginic activity in the fractions was not associated with γA-globulin, but with impurities which were not detected by immunodiffusion methods. The subfractions of the γA-globulin preparation from a ragweed-sensitive patient blocked passive sensitization of normal human skin with reaginic antibody of a heterologous system. The blocking activity of the fractions paralleled the sensitizing activity against ragweed-pollen extract. It was also found that a fraction containing reagin but no detectable γA-globulin had a definite blocking activity, whereas γA-myeloma proteins did not. These findings suggest that both the blocking activity and reaginic activity are associated with the same protein other than γA-globulin.

Physico-Chemical Properties of Human Reaginic Antibody

Summary The reaginic activity in sera from ragweed-sensitive patients was inactivated after the precipitation of serum proteins with the rabbit antihuman globulin serum which did not contain any of the antibodies specific for γG-, γA-, γM-globulins and for light chains of immunoglobulins. The rabbit antiserum gave a γ1-globulin precipitin band with the reagin-containing fraction which contained γA but none of the γG-, γM- or γD-globulins. The specificity of the precipitin band was different from the γA-anti-γA band. By radioimmunoelectrophoresis and by radioimmunodiffusion, the antibody against ragweed pollen-extract (antigen E) was detected in the γ1-globulin, indicating the presence of a unique γ1-immunoglobulin. The protein was tentatively designated γE-globulin. Precipitation of γE-globulin anti-ragweed antibody in the reagincontaining fraction was accompanied by the loss of reaginic activity, whereas the precipitation of the γA-globulin antibody in the fraction did not result in any decrease of reaginic activity. The results highly suggest that the reaginic activity is associated with γE-globulin.

The effect of 6-mercaptopurine on antibody production in atopic individuals

Recent interest in the use of the purine antimetabolites in the treatment of diseases of proved or postulated immune etiology prompted a study of the effect of 6-mercaptopurine (6MP) on antibody formation in atopic individuals. Six prison inmate volunteers with ragweed hay fever and/or asthma (treatment group) were matched according to age, sex, and race with six ragweed sensitive patients who served as untreated controls. Subjects in the treatment group were given 6MP, 1.5 mg. per kilogram per day by mouth for 21 to 42 days. As compared with the control group, subjects in the treatment group demonstrated the following: diminished antibody response following immunization with E. coli Vi antigen; no change in pre-existing titers of ragweed reagin as measured by direct skin test and passive transfer studies; no change in pre-existing hemagglutinin titers to diphtheria toxoid; and no change in pre-existing serum concentration of gamma-1A, gamma-1M, or gamma-2 globulin. Two subjects in the treatment group developed toxicity which necessitated discontinuance of the drug. Bone marrow examinations at the end of the study revealed macronormoblasts in four treated patients. The lack of suppression of pre-existing antibody titers, including reagin, suggests that when used under the conditions studied, and aside from possible nonimmunologic effects, 6MP would be of little value in treatment of the atopic diseases.

Changes in conjunctival sensitivity following grass and ragweed emulsion treatment

There has been concern regarding the possible development of hypersensitivity following injections with emulsions. It was therefore decided to review the conjunctival threshold levels in clinically sensitive patients treated with pollen emulsions. There were 131 grass- and ragweed pollen-sensitive patients who were given conjunctival threshold tests one month before repository injections and retested one year later. The testing levels were unchanged in 52 per cent, conjunctival sensitivity was slightly increased in 28 per cent, and slightly decreased in 30 per cent. There were 44 grass- and ragweed-sensitive patients who received repository injections for three years. Conjunctival threshold tests were recorded before the repository injection was administered each year. In 46 per cent of these patients, after two years, the conjunctival threshold levels were unchanged; 17 per cent showed slight increase in conjunctival sensitivity, and 37 per cent slight decrease. Many of the changes which were noted after the first year were reversed when patients were studied for a second year. Analysis of conjunctival changes in ragweed-sensitive patients showed changes in the opposite direction from the grass-sensitive patients; since these changes were in opposite directions, they canceled each other out when both grass-sensitive and ragweed-sensitive patients were combined. The problem of the development of delayed hypersensitivity following repository therapy has not been resolved by these findings. The conjunctival threshold test, in a combined group of ragweed-sensitive and grass-sensitive patients, did not show marked increase or decrease one and two years after emulsion therapy.

Radioimmunoelectrophoretic studies of ragweed-binding antibodies in allergic sera

With the radioimmunoelectrophoresis technique, ragweed-binding antibodies may be demonstrated in all the immunoglobulins of human sera. One or several different binding antibodies may be present in an individual serum. Beta-2A ragweed-binding activity was present in about two thirds of sera obtained from both untreated and treated ragweed-sensitive patients. Gamma globulin binding antibodies were also present in about two thirds of the untreated sera and in almost all of the treated sera. Beta-2M binding activity was noted in 10 per cent and 29 per cent of the untreated and treated groups, respectively. No correlation could be found between most clinical parameters of ragweed sensitivity and the type of binding antibody. Beta-2M antibodies were associated with younger patients, particularly in the untreated group. Almost all patients developed gamma globulin antibodies following specific therapy. The clinical significance of binding is still unclear. There was some correlation noted between reagin titers and beta-2A binding activity. A more striking correlation between hemagglutinating antibody titers and gamma globulin binding was found.

IMMUNOLOGIC ASPECTS OF HUMAN REAGINIC ALLERGY: AN IN VITRO METHOD AND SOME APPLICATIONS.

Suspensions of human leukocytes have been used for in vitro studies of reaginic allergy. These cells when isolated from ragweed sensitive patients, release histamine on reaction with antigen at threshold levels of 10−6 μg. The release process is characterized by a lag phase and a requirement for both calcium and magnesium. The divalent metal requirement has been observed throughout the reaction course. Physiologically competent cells are required for the release process, since cells subjected to 40°C, or treated with di-isopropylfluorophosphate show an impaired response. On these grounds, it has been concluded that the release process is a multi-step phenomenon and that the passage of histamine to the extracellular environment cannot be accounted for in terms of diffusion following membrane injury. Human leukocytes carry γ-globulins which can react with specific antibody in complement fixation, or in histamine release experiments.

Demonstration of human reagin in the monkey: I. Passive sensitization of monkey skin with sera of untreated atopic patients

The technique of passive sensitization of the skin of monkeys is a convenient and reproducible method for the demonstration of skin-sensitizing antibodies. Intravenous challenge with a mixture of antigen and Evans blue dye proved to be technically easier and as reliable as administration of dye followed by antigen. The intravenous administration of antigen generally proved to be superior to the direct intradermal injection with antigen. Heating of the sera from untreated ragweed-sensitive patients at 56° C. for 2 hours completely destroyed the reaginic activity as demonstrated by passive transfer in the monkey and human skin, while the hemagglutination titers were not changed by heating. Separation of reaginic sera by ultracentrifugation with the sucrose gradient method suggests that the skin-sensitizing antibody is not a macroglobulin. This technique for demonstrating skin-sensitizing antibodies in the monkey skin avoids many problems inherent in the classical Prausnitz-Ku¨stner method, including the hazard of transmitting serum hepatitis. It has potential value in evaluating human skin-sensitizing antibody and in the standardization of allergens.

Studies of purified fractions of ragweed pollen

The allergenic and antigenic properties of three “purified” fractions of ragweed pollen, pool C, fraction IV 2, and Delta, prepared in separate laboratories by different techniques were investigated. Gel diffusion studies indicated that none of the fractions contain only one antigen. However, it appears that a number of antigens in whole ragweed extract have been reduced or eliminated in the preparation of these materials. Positive indirect hemagglutination reactions were produced in higher titers and with a greater number of sera from untreated ragweed-sensitive patients with each of the three fractions than with whole ragweed extract. The Delta fraction was most potent in eliciting hemagglutination. Pool C reacted in highest titer with most sera. Hemagglutination inhibition data suggest that all of the necessary hemagglutinating antigens are present in each fraction, but there are some quantitative differences. Each of the fractions was more skin reactive than whole ragweed extract. In vitro neutralization of reagin indicated that each of these fractions probably contained, in variable amounts, all of the allergens in whole ragweed extract. However, the in vivo neutralization studies suggested that only Delta contains most, if not all, of the allergens present in whole ragweed extract and that some of these allergens have been climinated or markedly diminished in the process of preparing pool C and fraction IV 2.

DENSITY-GRADIENT SEDIMENTATION OF SKIN-SENSITIZING ANTIBODY AND BETA-2A-GLOBULIN IN SERUM OF ALLERGIC INDIVIDUALS.

The molecular size of the skin-sensitizing antibody in the serum of 4 ragweed-sensitive patients was studied, using the method of sucrose density-gradient sedimentation. The skin-sensitizing activity was present in a protein fraction of serum which sedi-mented somewhat faster than the 7S globulins but definitely slower than the 19S globulins. The skin-sensitizing antibody was associated with β2A-globulin, although the latter extended into the lighter 7S region.

Hyposensitization therapy including repository: A double-blind study

1. 1. Ragweed-sensitive patients who had never received previous hyposensitization therapy were divided randomly into 3 groups. Either repository, placebo, or aqueous therapy was administered to each group. Following aqueous therapy, 16 of 21 patients responded satisfactorily. In both the repository and placebo groups, 11 of 19 patients had a satisfactory clinical response. The study was conducted in a double-blind manner. 2. 2. There was no correlation between the clinical response and the change in hemagglutination titer. 3. 3. There was a slight suggestion that patients with an elevated reagin titer or a rising reagin titer have a poorer therapeutic response to specific therapy.

The association of skin-sensitizing antibody with the beta 2A-globulins in sera from ragweed-sensitive patients.

1. The removal of the beta(2)A-globulins from three sera from treated ragweed-sensitive individuals by immune absorption was associated with the loss of all detectable skin-sensitizing antibody activity as demonstrated by Prausnitz-Küstner testing. 2. Gel filtration studies, with sephadex G-200, indicated that the fractions containing only macroglobulins were devoid of all detectable skin-sensitizing antibody activity. 3. The immune absorption of the gamma-globulins from a serum fraction containing beta(2)A-globulins, gamma-globulins, and a trace of beta(2)M-globulins had no detectable influence on the skin-sensitizing antibody activity. 4. The removal of a portion of the albumin from the allergic sera by immune absorption, with retention of the skin-sensitizing activity, indicated that the loss of skin-sensitizing antibody was not due to non-specific absorption on an antigen-antibody precipitate. 5. No inhibition of the Prausnitz-Küstner reaction was observed when sheep serum, normal human serum, or normal human gamma-globulins were tested in concentrations described. 6. We conclude that the skin-sensitizing antibody activities in the three sera from treated ragweed-sensitive individuals studied were associated with the beta(2)A-globulins.

The Allergens in Ragweed Pollen

Abstract It has been shown that when the dialysable fraction (dialysate) of the water soluble extract of ragweed pollen (WSR), which constitutes approximately 95% of the total dry weight of WSR (1), is injected into the skin of ragweed sensitive patients, it will give rise to a positive reaction in all treated cases but induce no reaction in over 80% of those who have had no prior treatment (2). It is therefore of considerable interest to determine the composition of the component(s) which is capable of inducing a skin sensitizing antibody in man. This paper deals with the purification of the allergen(s) in dialysate and its physicochemical properties. Methods and Materials. I. Precipitation of the allergen. Aliquots of varied concentrations of phosphotungstic acid (PTA), picric acid and trichloracetic acid were mixed with 2-ml aliquots of dialysate (20 mg/ml) to determine which of these reagents could precipitate the allergen in as pure a state as possible.

Immunologic studies of ragweed-sensitive patients treated by a single repository antigen injection

T HE treatment of allergic rhinitis and asthma due to pollens by the injection of allergens emulsified in oil has been attempted in the past on several occasions. However, the results were not very encouraging.‘, 2 It is only recently33 4, 5 that practical and useful methods of treatment, based on the emulsion principle, have been devised. Although this mode of treatment is now widely used, several pertinent questions remain unanswered, such as the utilization of objective methods which might demonstrate the efficiency or superiority of such treatment over the conventional meth0d.O This paper is primarily concerned with immunologic studies ( SSAt titers, blocking antibody titers, hemagglutinin titers) on the sera of 35 ragweed-sensitive patients who were all treated by a single repository ragweed injection in June, 1960, at the Royal Victoria Hospital Allergy Clinic.

A Comparative Study of the Hemagglutinating and Skin Sensitizing Activities of Ragweed Sensitive Human Sera

Summary This study was undertaken to determine whether skin sensitizing antibodies are responsible for the capacity of sera from untreated ragweed sensitive patients to agglutinate red cells sensitized with ragweed extract by bisdiazotized benzidine or in tanned cell antiglobulin tests. The procedures used were a) absorption tests employing sensitized red cells; b) inhibition tests with a highly concentrated ragweed extract dialysate; and c) comparison of the hemagglutinating and skin sensitizing titers of different sera. Absorption tests showed somewhat variable results (probably due in large part to the heterogeneity of the system), but in a number of instances complete absorption of hemagglutinating activity was accomplished with no change in skin sensitizing activity. Sera neutralized with a highly concentrated ragweed extract dialysate showed no change in BDB test titers but significant losses of passive transfer activity. No significant correlation was observed between BDB or TCAG hemagglutination test titers and skin sensitizing titers of different sera. Together with recent information from other laboratories, these results indicate that the hemagglutinating activity of sera from untreated ragweed sensitive patients in BDB and TCAG tests is not due solely, if at all, to skin sensitizing antibodies.

Demonstration of Antigen-Reagin Interaction by Free Boundary Electrophoresis Using Buffer Contaminated with Specific Antigen.

Disturbances caused by interaction of reaginic sera of ragweed sensitive patients and its specific antigen are observed during free boundary migrations. Their respective changes of composition and mobilities of fractions are calculated and studied. Alteration of beta and, to a lesser extent, gamma globulin occur at optimum concentrations of ragweed contaminated buffer.

Repository antigen injections: Preparation and therapy

1. 1. Using 2 parts of a solution of 10 per cent Arlacel-A in light mineral oil to 1 part of pollen extract in a Coca-Merthiolate medium, we have prepared emulsions which can be given in amounts of 50 to 100 times the aqueous dose tolerated by the patient. 2. 2. The emulsions are examined grossly and microscopically, and the aqueous phase is determined by scratch tests on several subjects who have been tested with serial dilutions of the aqueous preparation of the antigen. 3. 3. The treatment of a small group of ragweed-sensitive patients with such emulsions in the Chicago area has produced encouraging but not superior results. It would appear that protection of ragweed patients is considerably more difficult in areas of greater pollen prevalence than in the Eastern areas. 4. 4. It is possible that with better timing, larger doses, or an improvement in the preparation, the results might be better in the future. 5. 5. The method is not fool-proof. It requires experience in the preparation and examination of the product and in the evaluation of the patient's tolerance. There is also the possibility of local tissue damage and the production of hypersensitivity of the delayed type. 6. 6. The method offers a possible approach in therapy, perhaps in selected cases. It is not yet a method to be adopted for wide, indiscriminate use.

Immunologic studies of ragweed-sensitive patients: III. Clinical aspects: The relationship of reagin and hemagglutinating antibody titers to results of hyposensitization therapy

Immunologic studies of ragweed-sensitive patients: III. Clinical aspects: The relationship of reagin and hemagglutinating antibody titers to results of hyposensitization therapy

Immunologic studies of ragweed-sensitive patients: I. Specificity and sensitivity of hemagglutination reactions

Immunologic studies of ragweed-sensitive patients: I. Specificity and sensitivity of hemagglutination reactions

Immunologic studies of ragweed-sensitive patients: II. Effect of specific hyposensitization therapy on hemagglutinating antibody titers

Immunologic studies of ragweed-sensitive patients: II. Effect of specific hyposensitization therapy on hemagglutinating antibody titers

The Lack of Absorption of Ingested Bovine Antibody in Humans

Abstract In the past few years several articles in professional journals (1, 2, 3) and numerous reports in the public press have claimed that antibodies in cows' milk are absorbed in active form via the human gastrointestinal tract. Further, it has been suggested that by using milk from appropriately immunized cows, this method of passive immunization may be employed with benefit in the treatment of such human diseases as pollen allergies. So attractive is this hypothesis that clinical trials of milk from ragweed pollen immunized cows have been conducted in ragweed sensitive patients without, however, any apparent benefit (4). In these clinical trials the lack of a relatively pure antigen makes it impossible to determine quantitatively the amount of antibody in the milk or in the subject's serum after ingestion of milk, so that all results must be based on subjective observations.