Feline immunoglobulin E: induction of antigen-specific antibody in normal cats and levels in spontaneously allergic cats.

Sera from 10 cats with symptoms consistent with atopy, from 15 normal household cats and from 11 laboratory maintained cats were assessed for allergen-specific IgE and IgG to Dermatophagoides farinae (DF) by enzyme-linked immunosorbent assay (ELISA). In addition, 10 normal cats were immunised with DF and intradermal skin tests (IDST) were performed weekly. Sera from the latter were also assessed for DF-specific IgE by ELISA and using Prausnitz-Küstner (PK) tests. Although DF-specific IgE was detectable in all the atopic cats, there was no significant difference between the levels in this group and in the clinically normal household cats. However levels in both these groups were significantly higher than those in the laboratory maintained cats. Detectable DF-specific IgE was induced in all of the 10 cats, but the levels were not correlated with the development of positive IDSTs, nor with the level of IgE as assessed by PK tests. These findings are consistent with a possible heterogeneity of IgE antibody in cats.

Production and characterisation of polyclonal antisera against feline IgE.

Cats, naturally or experimentally infected with Toxocara cati were immunised with dinitrophenylated ascaris antigen (DNP-Asc). All cats developed immediate skin reactivity to DNP coupled to bovine serum albumin (DNP-BSA) and the sera of the nine cats had a heat labile homocytotropic antibody detectable by homologous Prausnitz-Küstner (PK) tests. Reagin-rich fractions were prepared from these sera and used for the preparation of polyclonal antisera in rabbits. Resultant antisera were passed through a immunoabsorbent column of Sepharose 4B coupled to heated normal cat serum. An immunoabsorbent column prepared with the resultant antisera removed the PK reactivity from the cat sera, and the activity was recovered following acid elution. The antiserum failed to detect any recognised immunoglobulin in cat sera, but precipitated with a heat labile protein with gamma-1 electrophoretic mobility in the sera of parasited cats. These findings support the contention that the antisera are specific for feline IgE.

Assessment [correction of Asessment] of the brown Norway rat as a suitable model for the investigation of food allergy.

We have investigated the potential of the inbred Brown Norway (BN) rat as a model for food allergy using two different antigens, ovalbumin (OA) and semi-skimmed milk (SSM). The use of milk-free diet prior to and during exposure to SSM was a key factor in the induction of sensitisation to milk proteins. Investigation of dose received and timing of administration identified a sensitisation regimen using 500 micrograms SSM injected i.p. together with 1 mg CGN (adjuvant) on days 0 and 7 as the optimum conditions for induction of reaginic antibody production. In this model milk proteins were less allergenic than OA as the amount of SSM required to induce sensitivity was 20-fold greater. Examination of antigen-specificity of the IgG and reaginic antibody responses to a range of proteins, present in SSM, showed that the BN rats were capable of recognising a similar profile of allergens as those recognised by milk sensitive humans. Lactoferrin which is present in low concentrations in milk proved as allergenic as the major proteins in milk, the caseins and beta-lactoglobulin. These studies have identified conditions for induction of sensitisation to milk proteins, and have shown the antibody specificity of the response to be similar to that in man. This suggests that the BN rat could provide the basis of a model for the investigation of allergic reactions to food.

Differential suppressive influence of intranasal application of rye grass pollen extract on IgE antibody production in the mouse.

Serum antibody responses to a ubiquitous aero-allergen, rye grass pollen extract, have been monitored in BALB/c and BD1 mice following intranasal and parenteral administration. Parenteral treatments were shown to trigger antibody production while topical administration of low doses of allergen extract preferentially suppressed reaginic antibody production. However, higher doses of extract administered to the respiratory tract were found to induce the formation of IgE antibody. Thus in mice antigen administered intranasally can influence the immune response in a qualitatively different manner to parenteral exposure.

Iota-carrageenan induced reaginic antibody production in the rat--I. Characterisation and kinetics of the response.

The intraperitoneal injection of graded amounts of ovalbumin into inbred PVG strain rats was found to produce a dose dependent anti-ovalbumin antibody response without eliciting the production of ovalbumin-specific reaginic antibodies. The injection of ovalbumin admixed with iota-carrageenan enhanced the anti-ovalbumin response and simultaneously elicited the de novo production of reaginic anti-ovalbumin antibodies. Isotype analysis of the anti-sera revealed the presence of both IgG and IgE class anti-ovalbumin antibodies and demonstrated that non-reaginic IgE anti-ovalbumin antibodies were also a feature of this reaction. The reaginic antibody phase of the response, although transient in nature, was re-elicited by secondary antigen challenge. The administration of ovalbumin prior to the ovalbumin-carrageenan preparation was shown to abrogate the reaginic antibody response without significantly influencing the non-reaginic component of the response. Conversely the injection of iota-carrageenan prior to antigen, significantly suppressed the normal agglutinating anti-ovalbumin response but did not prevent the induction of reaginic antibodies. These observations demonstrate that iota-carrageenan can, under specific conditions, function as an efficient adjuvant, and is capable of by-passing the normal mechanisms responsible for controlling antigen specific reaginic antibody production.

Grass Conjuvac. II. Pro-inflammatory activity and potential for inducing hypersensitivity.

Treatment of rats with Conjuvac induced no anti-pollen extract (PE) IgE and no sensitization, whereas alum-adsorbed pollen extract induced IgE antibody and marked sensitivity. Conjuvac induced anti-PE IgE in rats treated with Bordetella pertussis organisms but the antibody concentrations were less than those induced by PE with B. pertussis. There was no indication, either in rats injected with B. pertussis and Conjuvac or alginate (ALG), of sensitization to ALG. In guinea pigs, Conjuvac induced immediate hypersensitivity to PE but there was no delayed hypersensitivity. Furthermore, no immediate hypersensitivity and little or no delayed hypersensitivity to ALG was detected in guinea pigs injected with conjuvac or ALG. Histological studies at Conjuvac injection sites in rabbits revealed inflammatory reactions less intense than those produced by aluminium hydroxide.

Allergens of Schistosoma mansoni. II. Fractionation and characterization of S. mansoni egg allergens.

The interaction of Schistosoma mansoni crude soluble egg antigen (SEA) with IgE antibodies in sera from S. mansoni-infected mice, rats and humans has been studied by the radioallergosorbent test (RAST) and the Prausnitz-Küstner (PK) technique. IgE antibodies recognizing egg antigens were present as early as day 21 after the infection in the mouse sera and day 28 in rat sera. IgE in sera of infected humans reacted with antigenic components in the Mr range 70,000-150,000 and focusing as a broad peak in the pH range 4.5-6.5 as measured by RAST. SDS-PAGE followed by western blotting showed the presence of major components at molecular weights of 117,000 and 35,000-43,000. In the PK test, using mouse sera, components focusing in the alkaline pH range also gave a positive reaction. Most of the allergenic activity was bound by concanavalin A-Sepharose and by wheat germ agglutinin-Ultrogel. IgE in serum from an infected non-permissive host (the Fischer rat) apparently recognized egg-stage-specific allergen as indicated by differences in the time course of the IgE response to egg allergens compared to the adult material. When analyzed by SDS-PAGE and western blotting with day 45-infected rat serum, SEA showed some qualitative and quantitative differences to adult worm antigen. Molecules at molecular weights between 25,000 and 30,000 and at about 43,000 in SEA reacted with rat serum IgE and were absent from adult worm antigen. The allergenic similarities between egg and adult worm are discussed.

The relationship between specific IgE antibody and non-specific IgE in anaphylactic sensitization of mice and rats.

The mice from inbred strains BALB/c, 129, C3H/A, C57BL/6J and outbred Wistar rats were tested for their capacity for reaginic sensitization on the response to antigenic stimuli. The total serum IgE level measured before immunization was highest in 129 mice and increased (2.5-3-fold) after immunization. BALB/c mice also showed an increase of the total serum IgE after immunization, whereas in two other strains such effect was not observed. All mouse strains tested produced IgE antibodies after immunization and the results suggest, that these IgE antibodies form only minor part of the total IgE content during immunization; in 129 and BALB/c mice beside IgE antibody response the production of non-specific IgE may be potentiated and these IgE may interfere in passive sensitization of mast cells with IgE antibodies. The examination of rats also showed, that mast cells with lower concentration of non-specific IgE are better acceptors for sensitization with IgE antibodies.

Potentiated reagin response to ovalbumin in Schistosoma mansoni infected rats and in rats treated with a soluble metabolic product from adult worms.

Infection by Schistosoma mansoni in the rat was found able to maintain high levels of reaginic antibody to an unrelated antigen, ovalbumin, mainly when infection was carried out 14 days after immunization. In addition, administration of a soluble metabolic product of living worms, the immunosuppressive activity of which was removed by dialysis, was also effective in inducing a potentiated response to ovalbumin.

Relationship between tissue sensitization and IgE antibody production in rats infected with the nematode, Nippostrongylus brasiliensis.

In Nippostrongylus brasiliensis rats, tracheal sensitivity to worm allergens developed prior to intestinal sensitivity and correlated with the early local synthesis of reaginic antibody in the mediastinal (bronchial) lymph nodes. Skin and intestinal sensitivity to worm allergens more nearly correlated with serum reaginic antibody and its synthesis by mesenteric lymph nodes and other tissues. Prostaglandins appeared to modulate intestinal responsiveness to worm allergens. Thus, local reagin synthesis and other microenvironmental factors influence local tissue sensitization and responsiveness to allergens.

Hypersensitivity in rats infected with Fasciola hepatica: possible role in protection against a challenge infection.

Rats infected with Fasciola hepatica showed an increase in intestinal mast cells which reached a peak between four and six weeks and fell to control levels by week 14. Following a challenge infection sensitised rats showed evidence of a transitory mild intestinal anaphylactic response. The numbers of intestinal eosinophils, already increased as a result of the primary infection, were rapidly supplemented. In previously uninfected rats the majority of flukes penetrated the mid gut region, but in sensitised rats there was a shift towards the caecal end. Resistance to challenge and a pronounced intestinal eosinophil response were evident in previously infected rats irrespective of the presence or absence of detectable serum reaginic antibody. Systemic anaphylaxis, induced by intravenous fluke antigen administration, occurred whether serum reagins could be detected or not.

Preparation of a highly purified allergen from Dirofilaria immitis. Reaginic antibody formation in mice.

A Dirofilaria immitis protein allergen was purified; it had a molecular weight of 15,000-20,000 and a carbohydrate content of 2%. The allergenic activity of adult Dirofilaria extracts was assayed by passive cutaneous anaphylaxis (PCA) in rats using mouse sera obtained by immunization with various fractions. The mouse-Dirofilaria system was used to study the degree of purification of allergen. The purified Dirofilaria allergen appeared as one band after sodium dodecyl sulphate polyacrylamide gel (SDS-gel) electrophoresis and one precipitin arc by immunoelectrophoresis (IEP). It was inclined to aggregate in buffered solution. The results suggested that the allergen--reagin axis was a simple single antigen-antibody interaction. Immunological responses to the purified allergen were compared among four inbred strains, two hybrid strains and two outbred strains of mice. They produced relatively high titres of reaginic antibody but did not produce detectable indirect haemagglutinating test (IHA) antibody. Among the strains tested, BALB/c was a high responder and also contained to produce the reaginic antibody for longer than the other strains.

Suppression of reaginic antibodies with modified allergens. III. Preparation of tolerogenic conjugates of common allergens with monomethoxypolyethylene glycols of different molecular weights by the mixed anhydride method.

Our previous findings that antigens, such as ovalbumin (OA) and the extract of ragweed pollen (RAG), could be rendered nonantigenic, nonallergenic and tolerogenic by conjugation with polyethylene glycol (PEG) have been extended in the present study to the synthesis of conjugates of a variety of antigens with monofunctional monomethoxy-PEGs (mPEGS) of different molecular weights by the use of the mixed anhydride method. Thus, mPEGs with molecular weights of 2,000, 5,000, 10,000 and 20,000 were coupled to proteins such as dog serum albumin (DA), bovine pancreatic ribonuclease, OA and the constituents of pollen, helminth and bacterial allergens (RAG, Timothy grass pollen, Ascaris suum and Micropolyspora faeni). All these mPEG conjugates depressed markedly the ongoing IgE antibody formation in sensitized animals, in spite of additional injections of the sensitizing dose of the appropriate antigen. Moreover, the allergenicity of the proteins was either totally abolished or markedly reduced after coupling to mPEGs. Conjugates of DA and OA of varying degree of substitution (i.e. number of mPEG molecules attached per protein molecule) were prepared with mPEGs of different molecular weights and their immunological properties were assessed. It appears that, for a series of tolerogenic conjugates of the same antigen, there exists some inverse relationship between the degree of substitution and the molecular weight of mPEG, i.e. a high level of tolerogenicity with a concomitant reduction or total loss of allergenicity was achieved with a lower degree of substitution utilizing mPEGs of increasing molecular weights. On the basis of these results, it is concluded that a variety of allergens may be converted by conjugation with mPEGs to tolerogenic products with a potential for use in the therapy of patients allergic to a wide spectrum of common allergens.