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Grass pollen allergens are shown to remain associated with protein material and a yellow pigment during paper chromatography and during dialyses and ultrafiltrations of various types. Dialysable* allergens comprise only a fraction of I per cent of the total activity and the amount of activity extractable by diethylene glycol (DEG) and similar solvents is of the same order. Besides the allergens, the DEG and aqueous extracts contain large amounts of inositol, glucose and fructose, also some yellow pigments and phosphates. Larger amounts of free and combined amino acids are found in the aqueous than in the DEG extracts, but the reverse is true for sucrose. In addition the DEG extracts contain a yellow glucoside different from the dactylen of the aqueous extracts, a glucosan and an arabinose-galactose-pigment complex, only the latter being associated with any activity. The spontaneous release of the crystalline dactylen from originally clear aqueous pollen extracts is found not to be caused by enzymes. The washed crystals are found to be chromatographically and electrophoretically homogeneous and devoid of allergenic activity. [ABSTRACT FROM AUTHOR]

To examine the validity of the old idea of the possible significance of mites for housed use atopy it was necessary to study the mite fauna of house dust. The most important findings in this investigation are that mites occur regularly, rather than occasionally, in house dust, and that the mite genus Dermatophagoides is represented by much higher numbers than any of the other genera found in dust. Furthermore, it appears that there is a seasonal periodicity in the numbers of mites in dust with a maximum from August to October, and also that the numbers of mites in the dust from various houses differ considerably. House dust from high- mountain areas contains very few mites. These observations fit very well the knowledge of the seasonal and topographical Variation in the allergen content of various dust samples. Based on laboratory observations on the temperature and humidity requirements of Dermatophagoides, a mode! is constructed which makes the relation between the degree of dampness of houses and the numbers of mites in the dust understandable. In this paper only very short mention is made of the many experiments showing the identity between the house dust allergen and the allergen produced by Dermtophagoides. From the comparison of skin reaction to equivalent extracts of different species of mites it appears that the allergenic products of mites of one family lend to be identical. [ABSTRACT FROM AUTHOR]

Heat and pH stability studies and experiments with organic solvents show that the A-antigens discussed in the preceding paper (Augustin, 1959c) are much more labile than the I-(‘inner ring’) antigens. Breakdown products and/or aggregates are produced which no longer precipitate with antisera to the original extracts, but act as inhibitors. Solutions of pollen allergens, on the other hand, are found to withstand even autoclaving for 15 min. at 20 atm. and vigorous boiling over the naked flame of a bunsen burner. None of the carbohydrates tested has a demonstrable effect on skin reactivity which is, however, destroyed by crystalline pepsin, crystalline trypsin, a crystalline mould protease and a tissue protease (a partially purified extract from rabbit spleen). It follows that the bulk of the allergens—if not all—are proteins. The relation of skin reactivity, immuno-electrophoretic patterns, carbohydrate and protein reactions to the selective destruction of the pollen antigens is investigated. Pollen components prove to have a somewhat wider range of electrophoretic mobilities than serum proteins and are probably as complicated a mixture. The most and least highly negatively charged components are without skin reactivity in allergic subjects. The skin reactive allergens appear to have the mobilities of α- and β-globulins. Not all the hay fever subjects react equally to all the components, and Cocksfoot and Timothy activity patterns vary in different subjects. [ABSTRACT FROM AUTHOR]

When lymphoid cells from hypersensitive rabbits are incubated with antigen, biologically active substances are formed and released which are capable of inhibiting the migration of normal non-sensitized mesenchymal cells. In the present paper some basic parameters of their production were determined. These substances were regularly obtained after 6 and 18 hours incubation, but not after 2 hours. Under more favourable cultivation conditions (lower density of lymphocyte suspension) an increased activity in the cell extracts as compared with the supernatants was observed. Another critical factor in the production of these substances is the quantity of antigen added. Ten micrograms of PPD leads to the production and liberation of a highly effective substance. A lower dose of antigen results in the liberation of a substance into the supernatant which by itself is almost inactive, but becomes more active when more antigen is added. The efficiency of the released substances was determined by serial dilution. The inhibiting activity was maintained at 1:5 and 1:20 and sometimes at 1:100 dilutions. [ABSTRACT FROM AUTHOR]

The fate of (14)C-labeled allergens injected intradermally into guinea pigs, namely picryl chloride (PCl*) and 2:4 dinitrochlorobenzene (DNCB*), was followed during the induction period of delayed hypersensitivity. Both chemicals were applied in a single injection into one ear in amounts that approached their minimal sensitizing doses (PCl, 0.25 microg; DNCB, 5.0 microg). Radioactivity in the various tissues was determined by liquid scintillation counting after combustion of tissues to CO(2) and H(2)O. The injected allergens seemed to leave the injection site in three phases. A large proportion of allergen escaped rapidly from the ear, about 50% within 3 hr in the case of PCl, within 15 min for DNCB, the difference probably reflecting their unequal reaction constants. Initially there was a "half-life" escape in 2.5 hr with injected dosage of 0.25 microg PCl and in 18 hr for 5.0 microg DNCB. This escape occurred via the regional veins and not via the lymphatics. Radioactive decomposition products of the allergens were already present in the urine within 3-4 hr. After 6-8 hr, the half-life time of escape lengthened to approximately 28 hr for both allergens used in their respective initial dosages, holding up to 2 days after which there occurred still further slowing; between 2 and 4 days the time was about 43 hr for PCl, much longer (72-88 hr?) for DNCB, apparently reflecting different physicochemical properties of this second fraction. Sensitization seemed to be connected with the portion that was present between 12 hr and 4 days of the induction period. It is not known how far the escape of radioactivity during this period may represent gradual hydrolysis of attached picryl and dinitrophenyl groupings, respectively, to form picric acid and dinitrophenol. Gradual accumulation of the second fraction in the regional lymph nodes could definitely be excluded. It was noted that no hypersensitivity arose and essentially no depot of radioactivity existed between 12 hr and 4 days when DNCB was injected in a dose of 0.25 microg, owing to its ready escape from the ear; but 20 times as much DNCB caused sensitization and provided about the same fixed depot as 0.25 microg of picryl chloride. After delayed hypersensitivity had been established, traces of radioactivity were still measurable at the site. This third fraction, probably representing a different coupling product, escaped at a very low rate and was traceable up through several weeks. No demonstrable radioactivity could be detected in thymus, spleen, and mesenteric nodes when examined at short intervals between (1/2) min and 17 days. In analogy with findings on transplantation "immunity" and with studies reported in the following paper, the induction of delayed hypersensitivity can be explained by encounters between lymphoid cells and the hapten complex which is found present in the local site for 4 days, in agreement with Medawar's concept of peripheral sensitization.

Although many studies have been made on the lyophilization of BCG vaccine, few reports on its stability have appeared. The amount of living bacilli present in the vaccine and its allergenic potency can be considered to be an indication of the stability of the vaccine. The authors have already reported on the change in viability of vaccines preserved for 17 months; in this paper two experiments on lots of vaccines preserved for three years are discussed. The authors determined changes in viability after storage and examined tuberculin reactions one to eighteen months after vaccination. It was observed that the vaccines which had a relatively high potency at the time of lyophilization maintained their ability to induce allergy for at least two years.

Various aspects of the immune status were examined in patients with cystic fibrosis (CF) and the following observations were made. (1) Ten per cent of the CF patients had elevated or reduced serum IgG concentrations and there seems to be a transient low serum IgA concentration in the same number. Serum IgM concentrations were normal in CF patients. Normal levels of the three serum immunoglobulins (IgG, IgA and IgM) were detected in the siblings and heterozygotes. (2) Serum IgE concentrations were elevated in 32% of patients with CF but normal concentrations were detected in heterozygotes and siblings. (3) Precipitating antibodies to various antigens and allergens were detected in the sputum of most patients with CF, but their sera contained lower or undetectable titres of these antibodies. (4) The precipitating antibodies in CF sputum to bacteria, to agar, to seminal fluid, to urine and to human serum may be related to a common antigenic determinant of the glycoproteins present in all these fluids. (5) The total protein concentration in the sputum of CF patients seemed to reflect the extent of lung damage. (6) The third component (C3) of serum complement concentration was normal, or moderately elevated in all CF patients tested. (7) Serum transferrin concentrations were normal in CF patients but their prealbumin levels were depressed, suggesting marginal undernutrition. (8) Peripheral blood lymphocyte transformations with PHA and PWM appear to be normal in CF patients. (9) Precipitins to the food antigens and the high percentage of positive sera with reticulin antibodies may be the result of malabsorption from the gut as well as a defect of the SIgA system. (10) The presence of free J-chain in the sputum of patients with CF may be further evidence of SIgA structural abnormality. [ABSTRACT FROM AUTHOR]

The immunological cross-reaction between penicillins and a cephalosporin derivative (cephaloridinc CEPR) has been investigated. Cross-allergenicity was studied in twenty-four patients with established penicillin allergy using a variety of tests. Skin tests, quantitative leucocyte challenge (estimating histamine release by allergen) and lymphocyte stimulation (transformation) tests were performed, using CEPR, conjugates of CEPR with human serum albumin (HSA) and bovine gamma-globulin (BGG), benzylpenicillin and benzylpenicilloyl (BPO) conjugates with HSA and BGG. A cross-reaction was clearly established in the majority of patients. The highest percentage of positive results (cross-allergenicity) was obtained in the leucocyte challenge test (80 per cent with CEPR.BGG), followed by the lymphocyte stimulation test (50 per cent with CEPR.BGG), and then the skin test (46 per cent with CEPR.HSA). None often non-allergic controls gave a positive result in these tests. Three of the penicillin allergic patients had received cephaloridine, and all three developed allergic reactions. Cross-antigenicity has also been shown by haemagglutination and haemagglutination-inhibition tests on serum from larger groups of penicillin-allergic patients (including the previously mentioned twenty-four patients), and of non-allergic controls, who had anti-BPO antibodies. [ABSTRACT FROM AUTHOR]

Long persisting antigenic stimulation at immunogenic levels leads to a profound inhibition of antibody formation. With Brucellus abortus, there is first a brief and high IgM response. IgG antibody titres remain at a low level for some days, and then begin a slow and progressive increase, leading to a rather persistent maximum, and finally after about 300 days, to the state of inhibition. When the same total dose is given with monthly intervals, the effect is quite different, with similar IgM and IgG peaks being observed after each dose. The inhibited animals respond moderately to a ten-fold higher antigen dose, and only with IgG. Six months after interruption of the persistent antigenic stimulus, a strong response can be obtained after a new antigenic stimulation, with a substantial proportion of IgM. It is concluded that persistent antigenic stimulation plays a major role in the change from IgM to IgG synthesis. [ABSTRACT FROM AUTHOR]

This article presents a structural study of house dust allergens. Several research teams have developed methods for purification of the allergen, so at the present time there exists a consistent frame of analytical data permitting a few general conclusions on the chemical nature of the allergens. The central method favoured by most investigators for rapidly concentrating the allergen involves the treatment of clarified aqueous dust extracts with benzoic acid, thereby adsorbing allergenic glycoproteins and extraneous compounds.

Ten antigen fractions were prepared from adult Schistosoma mansoni by extraction into borate-buffered saline, precipitation at pH 4.6 and separation on Sephadex G-100. The allergic activity of these antigens was assayed by a modified Prausnitz-Kustner type reaction in rats; this test system was found to be sensitive and consistent, allowing differences in allergenicity between antigens to be accurately assessed. Skin-reactivity was detected in both acid-soluble and acid-insoluble fractions. Specific allergenicity was located in peak 3 of a G-100 separation of the acid-soluble fraction and in peaks 1 and 2 of a G-100 separation of the acid-insoluble fraction suggesting that the allergens of S. mansoni were of at least two types: (1) a protein of mol. wt above 150,000 precipitated at pH 4.6, and (2) a protein of mol. wt 20-30,000 remaining in solution at this pH. It is suggested that both these allergens are glycoproteins. Non-specific histamine-releasing agents were found in peak 1 of the G-100 separation of the acid-soluble material. [ABSTRACT FROM AUTHOR]

Saline extracts of adult Metastrongylus spp. were fractionated by both Sephadex gel filtration and anion exchange chromatography, and the fractions tested for allergenicity both by wheal and erythema tests in infected pigs, and by P-K tests in passively sensitized pigs. It is suggested that cross reactions, so commonly found when using nematode antigens in wheal and erythema reactions, can be eliminated by suitable dilution of the allergen. It was shown that the allergenic components had a molecular weight range of 5000 to 50,000, and were eluted from DEAE cellulose with a series of buffers of differing molarity. [ABSTRACT FROM AUTHOR]

The highly purified major allergenic component of rye grass pollen (Group I) was used to investigate the possibility of destroying selectively the allergenic properties of an antigen, while largely retaining its original immunizing capacities. The allergen was treated under mild conditions with formalin alone or formalin plus a reactive low molecular weight additive. Certain derivatives (allergoids) showed well over 99 per cent reduction in allergenicity, determined by the histamine released from allergic human leucocytes in vitro, but were still able to combine with rabbit antibody against native antigen. Furthermore, the allergoids stimulated production (in guinea-pigs) of appreciable amounts of antibody able to inhibit native allergen-mediated human allergic histamine release in vitro and to cross-react with native antigen by PCA tests in normal guinea-pigs. Residual allergenicity and cross-immunogenicity (by the inhibition assay) of the different formalinized derivatives varied appreciably according to the additive used in formalinization, but the cross-reactivities of the different preparations in quantitative precipitin analysis against rabbit anti-native antigen serum were similar. The residual allergenicities of individual derivatives varied by up to 1000-fold in different cell preparations, suggesting a heterogeneity of allergenic determinants. Allergoid derivatives showed no hapten-like activity in that they were unable to inhibit allergen-mediated histamine release from leucocytes. The theoretical and practical application of allergoids is discussed, including their potential usefulness in improving the immunotheraphy of atopic humans. [ABSTRACT FROM AUTHOR]

The use of the intra-lymph node injection route for a range of doses of ovalbumin (antigen) and mycobacterial peptidoglycolipid (adjuvant) in water-in- oil emulsion failed to show any superiority of this method over the usual footpad injection, in terms of induction of cell-mediated hypersensitivity. The same result applied to anti-ovalbumin antibody levels in the serum at 3 weeks after injection, the findings with lower doses of ovalbumin were that lower levels of anti-ovalbumin resulted when the intra-lymph node injection route was used. For the induction of allergic encephalomyelitis the intra-lymph node injection route failed to prove superior to the footpad route at several dose-levels of mycobacterial peptidoglycolipid in water-in-oil emulsion with homologous brain tissue. [ABSTRACT FROM AUTHOR]

Cocksfoot and Timothy pollen extracts are each found to contain at least fifteen components antigenic in rabbits. Most of these can also be allergens for man, but only a few are regularly so. These 'principal' allergens have now been isolated in highly purified form. Procedures are given a simple method of preparing extracts for clinical purposes and for the partial separation, concentration and purifications of the allergens by means of differential extractions of the pollens and by means of ultrafiltration, isoelectric precipitation and salt fractionations (at acid and neutral pH) of the extracts. Isoelectric precipitations gave highly pigmented acid complexes, two of which moved at single-sharp peaks at pH 7.4 in free electrophoresis, but proved to be hardly active by skin tests. Acid NaCl fractionation of the remainder resulted for Cocksfoot and Timothy in the isolation of a nearly white powder (T21.111121112 = T21B) which was weight 1000-10,000 times as active as the pollen from which it had been derived. The powders have retained their activity for 7 years. By gel diffusion rests, they were found to contain two antigens (one in each preparation) which were immunologically partially related, but the Timothy preparation contained in addition the 'intermost' 'twin' antigens specific for Timothy that we had discovered previously in the crude extracts by gel diffusions methods. Skin reactions could be elicited in hay-fever subjects by tests with concentrations of 10-9-10-8 g./ml, which is equivalent to intradermal injections of 10-11-1010-10 mg and represents a 300-fold purification with respect to the concentrates of crude pollen extracts prepared by ultrafiltration and dialysis. Fractionation on DEAE-cellulose of one of the highly purified Timothy preparations (T21.11112112 = T21A) and other, crude Timothy and Cocksfoot extracts resulted in considerable and reproducible separation of the various antigens, with no indication of the continuous dissociation into smaller fragments reported in recent publications. The immunologically related antigens of Cocksfoot and Timothy were among a number not retained by the DEAE-cellulose at pH 7.4-8.5. These proved to be skin reactive in all the patients tested. When this fraction was prepared from T21.11112112(=T21A) only one main line (plus a faint subsidiary line) was found in the gel diffusion tests. At pH 6.4 the important dissimilar Cocksfoot and Timothy allergens were eluted; the fraction derived from T21A contained only the special 'intermost' twin antigens in roughly a further ten-fold purification over and above that achieved in the acid NaCl fractionation. [ABSTRACT FROM AUTHOR]

A practical immunological method for the assay of clinical biological solutions, such as allergen extracts, in agar gel plates is presented. The method permits assay of individual antigens in antigenic mixtures. Application of the method to the assay of three ‘unknown’ dilutions of each of four grass pollen extracts gave results which were within—9 per cent and +6.7 per cent of the actual values. It is postulated that an immunological assay, since it indicates the antigenic state of the solution, is a more valid index to the probable clinical potency of an allergen extract than the Noon, total nitrogen, or protein nitrogen units currently in use. The quantitative gel plate assay method is quick and simple in application and suited to routine use. [ABSTRACT FROM AUTHOR]

The mixed agglutination reaction has been used for investigating the presence of blood group antigens on the surface of human cervical carcinoma cells (HeLa) cultured for eight years in vitro. The H antigen was demonstrated in the absence of A and B. The MN-type antigen has been found as well as Tja. Treatment of HeLa cells with ficin greatly enhanced the reaction of anti-H and anti-Tja with the corresponding antigens on HeLa cells. The authors failed to show the following antigens: Rh(D) and Rh(c), S, P, Lea, Leb, Lua and Lub. [ABSTRACT FROM AUTHOR]

Gel-filtration on Sephadex, of Ascaris, Nippostrongylus, Toxocara and Toxascaris antigens revealed that the allergenic components had a molecular weight range of 10,000–50,000. Allergenicity was assessed by homologous passive cutaneous anaphylaxis, to detect reagins in man and reagin-like antibodies in the rat and rabbit. It is suggested that nematodes can provoke a Type I allergic reaction in man, rat and the rabbit. [ABSTRACT FROM AUTHOR]