Chemical derivatization of therapeutic proteins.

Despite major advances in redesigning and producing proteins through recombinant DNA technology, many therapeutic proteins are still produced by extraction from biological tissues or fluids, or from nonrecombinant microorganisms. Modification of such proteins, to improve potency and bioavailability and reduce immunogenicity, can only be carried out post-translationally by chemical-derivatization methods. Genetic- and chemical-modification methods are not mutually exclusive, however, and may be combined to optimize protein-engineering strategies, because chemical modification can introduce structural changes that are not encoded by DNA into both recombinant, and nonrecombinant proteins.

Interference in the radioallergosorbent test by antibody light chains in rabbit urine.

Immunological techniques have been used to identify and quantitate rabbit antibody light chains in the urine of rabbits. The influence of these light chains on the radioallergosorbent test (RAST) used for the diagnosis of laboratory animal allergy to rabbit urinary protein allergens is described. The presence of IgG antibody specific for rabbit derived antigens in human serum has been confirmed.

The influence of hapten density on the assay of penicilloylated proteins in fluids.

The use of inhibition radioimmunoassays for the measurement of penicilloylated proteins in biological fluids is compromised by the dominant influence of hapten density. Precise quantitation, and therefore assessment of antigenicity and immunogenicity, cannot be achieved in the absence of knowledge of the number and distribution of haptenic groups on the protein carrier. These assays may not, therefore, be appropriate for the measurement of potential allergenic residues in food products.

Risk assessment of antibiotic residues of beta-lactams and macrolides in food products with regard to their immuno-allergic potential.

In human medicine drug allergy is a well-established side-effect of the therapeutic use of antibiotics, especially the beta-lactams. Side-effects caused by macrolides are uncommon and only a very few of these seem to be caused by allergic mechanisms. Clinically, drug allergy is characterized by a spectrum of reactions ranging from mild skin rashes to angio-oedema or life-threatening anaphylaxis. Concern has been expressed that antibiotic residues in meat and other foods might be responsible for similar hypersensitivity reactions in a small number of individuals. This review assesses the potential risk of such reactions in general, but focuses on allergy to penicillin and macrolide residues in particular. In relation to the risk of primary sensitization, it is unlikely that residues could contribute to the overall immune response in view of the very low levels that are likely to be encountered in comparison with the high levels received during therapeutic use. No evidence has been found that any individual has become sensitized by residues of either penicillins or macrolides. Furthermore, the oral route is much less sensitizing than parenteral administration and immunochemical studies with penicillin indicate that hapten-protein complexes formed in vivo are unlikely to be immunogenic because of their low dose, low epitope density and binding to autologous carrier proteins. For performed allergens, the epitope density was also too low to be immunogenic. Because of the ubiquitous nature of penicillin-producing moulds in nature and the extensive use of beta-lactam antibiotics in human medicine, it is unlikely that epidemiological studies could be undertaken that could allow quantification of the minimal risk. The risk of allergic reactions in pre-sensitized individuals can be assessed similarly and again it is concluded that factors such as dose, oral administration and low epitope density make it unlikely that a significantly antigenic derivative could be formed. However, a review of the literature on penicillin hypersensitivity revealed a very small number of previously sensitized individuals from whom there is reasonable clinical and documentary evidence that penicillin residues in milk triggered an allergic reaction, usually a rash. Although these cases are very rare (less than 10 cases reported in the last 25 years), they illustrate the continuing need to control antibiotic residues vigilantly. Animal models have not proved useful for predicting the risk of hypersensitivity reactions to drugs, since allergy in man is determined by genetic and other factors and no validated methods exist to determine a no-effect level.(ABSTRACT TRUNCATED AT 400 WORDS)

Laboratory animal allergy: the measurement of airborne urinary allergens and the effects of different environmental conditions.

Casella Simquad air samplers, with 0.5 microM cut-off filters, were employed to sample the air in a laboratory animal house environment. The extracts obtained were assayed for laboratory animal urinary protein allergens using the inhibition radioallergosorbent test (RAST inhibition). The results showed that the collection and assay methods were of value and studies were extended to the influence of air change rates and humidity on airborne allergen levels. Reducing the air changes increased allergen levels, whilst increasing the humidity from 54% to 77% caused a significant reduction in allergen levels.

Effects of beta-lactam antibiotics on eukaryotic cells.

A symposium on effects of beta-lactam antibiotics on eukaryotic cells was held as part of the 9th International Congress of Infectious and Parasitic Diseases (Munich, Germany, July 1986). This symposium provided an opportunity to review recent work on the effect of beta-lactam structures on mammalian cells in culture and to speculate on possible clinical implications. This paper is a comment on the subject matter covered by the symposium papers which follow.

Measurement of IgG subclasses in the sera of laboratory workers exposed to rats.

Human IgG antibody subclasses have been measured in the sera of workers exposed to rats, using a crude extract of rat urinary protein antigens, in an ELISA system. The antibody titres in individuals either with or without specific IgE were similar, with the exception of IgG4 where the mean level of this subclass was lower in those individuals with measurable titres of IgE (p less than 0.01). Symptomatic individuals, with specific IgE, also had lower titres of IgG4 than the corresponding asymptomatic, IgE-positive subjects (p less than 0.05). The frequency of positives in each subclass assay was similar in both groups. These findings suggest that higher levels of IgG4 may have a protective rôle.

Development and use of three new radioallergosorbent tests in the diagnosis of penicillin allergy.

The synthesis and characterisation of three novel reagents for use in the radioallergosorbent test (RAST) for the diagnosis of penicillin allergy are described. The antigenic determinants involved are the benzyl penicillanyl, thiol-linked benzyl penicillenate and thiol-linked penicillamine. These reagents, and also one specific for the benzyl penicilloyl group, have been used to evaluate the sea of subjects suspected of suffering from penicillin allergy and to explore the aetiology of the respiratory dyspnoea experienced by some workers exposed to penicillin-containing dusts. The use of these reagents, while confirming the importance of the penicilloyl or major determinant of penicillin allergy, has shown that there is heterogeneity in the IgE response of penicillin-allergic patients and some patients have IgE antibody specific for one or more of the new determinants only. These reagents will, therefore, increase diagnostic capabilities. Their use has also confirmed that the disorder induced by occupational exposure to penicillins is not primarily mediated by IgE antibody specific for allergenic determinants represented by any of the available reagents.

Reduction in incidence of ampicillin rash by purification of ampicillin.

Purification of ampicillin (Penbritin) with respect to protein impurities has been found significantly to reduce the incidence of rashes in treated patients. This may be related to findings in animals that injections of the isolated protein impurity can induce the formation of circulating IgG antibodies and skin-sensitizing antibodies.

Immunogenicity and allergenicity studies on two beta-lactam structures, a clavam, clavulanic acid, and a carbapenem: structure-activity relationships.

Two newer beta-lactam-containing structures, a clavam, clavulanic acid, and a carbapenem, MM22383, have been studied for their intrinsic immunogenicity and allergenicity. Clavulanic acid has a very low immunogenic and allergenic potential, in contrast to MM22383 which is a contact sensitiser in guinea pigs and an immunogen in rabbits. Evidence for the allergenic potential of MM22383 in man through occupational exposure is also presented. Consideration of the chemistry of these two compounds with respect to their reactivity with protein provides a rationale for the marked difference in their behaviour. The importance of stable hapten-protein conjugates and epitope density is discussed in relation to immunogenicity.

Immunological studies on paroxetine, a novel anti-depressant drug.

Paroxetine is a novel and selective neuronal 5-hydroxy-tryptamine uptake inhibitor with anti-depressant activity. Paroxetine was examined for its ability to induce adverse immunological reactions, either as a consequence of a specific immune response or by a direct or indirect effect on the immune system. Paroxetine did not react in vitro with protein amino or thiol groups, suggesting that it lacks the capacity to form potentially immunogenic hapten protein conjugates. No anti-paroxetine antibody was detected in plasma or serum samples from patients and rats following oral administration over prolonged periods, or from epicutaneously exposed guinea pigs, or from rabbits given paroxetine in Freund's adjuvant, suggesting that paroxetine does not have the capacity to elicit humoral immune responses. Guinea pigs epicutaneously exposed to paroxetine did not develop contact sensitivity, suggesting that it does not have the capacity to elicit cell-mediated immune responses. These results suggest that paroxetine lacks intrinsic immunogenicity. Anti-SRBC antibody plaque-forming cell responses in mice were unaffected by oral administration of paroxetine, and paroxetine had no significant effect on ex vivo and in vitro murine macrophage phagocytosis of opsonized SRBC or on ex vivo murine splenocyte mitogen responses, suggesting that paroxetine does not exert modulatory effects on the immune system or on macrophage function. These findings, together with the results of pre-clinical safety evaluation studies, suggest that paroxetine is unlikely to have immunotoxic effects.

Antigenic properties of polymers formed by beta-lactam antibiotics.

Purified polymers have been isolated from 6-aminopenicillanic acid and from semi-synthetic penicillins and cephalosporins. All the polymers were shown to react with rabbit antibody of penicillins and cephalosporins. All the polymers were shown to react with rabbit antibody of penicilloyl specificity, as demonstrated by passive cutaneous anaphylaxis in guinea pigs, and the reactions were shown to be penicilloyl-specific by hapten inhibition experiments. The cephalosporin-derived polymers in addition reacted with rabbit antibodies raised to the corresponding cephalosporin conjugates of bovine gamma-globulin. Using direct skin tests, passive cutaneous anaphylaxis and an in vitro assay, no evidence was obtained that the polymers induced the formation of specific antibodies in baboons, guinea pigs and rabbits, but in baboons the induction of cell-mediated immunity, demonstrable by delayed skin test reactions, was shown.

Standardization of Dermatophagoides pteronyssinus extracts and tyrosine adsorbates.

Human skin test, inhibition of RAST and the radioimmunosorbent technique of Ceska have been shown to be useful assays for the standardization of D. pteronyssinus aqueous extracts. The development of these assay systems for the standardization of D. pteronyssinus tyrosine adsorbed formulations is described. Tyrosine solubilisation procedures are detailed together with the influence of these procedures on the three assays. Good correlation has been shown between the human skin test and the radioimmunosorbent technique. The inhibition of RAST appears to be more sensitive than the other techniques to mild conformational changes in the allergen, induced in this work by acid pH. This may limit its value for the standardization of D. pteronyssinus tyrosine adsorbates but it may prove useful in monitoring manufacturing processes.

A clinical environmental study of the aeroallergens of the islands of Bermuda.

The major allergen responsible for allergic respiratory disease in the islands of Bermuda has been shown to be derived from Dermatophagoides pteronyssinus. 73% of the atopic group included in the survey gave weal and erythema reactions to extracts of this mite, whereas only 30% reacted to mixed pollen extracts and 10% to mould extracts. D. pteronyssinus was isolated from all house dust samples and it represented nature, with only a modest seasonal influence, and pollen counts were low throughout the year.

Prevalence and diagnosis of laboratory animal allergy.

A survey of the prevalence of laboratory animal allergy to rats, mice, guinea pigs and rabbits among sixty-nine animal workers and 308 other subjects on a pharmaceutical research site revealed a 22% prevalence of laboratory animal allergy among the animal workers. The overall prevalence of atopy was 67% in persons with allergy to laboratory animals. This was significantly greater than the 31% prevalence in other animal workers. Skin-prick tests and specific IgG and IgE assays to urinary protein extracts strongly correlated with the occurrence of laboratory-animal allergy and would appear to have diagnostic value. However, a number of clinically diagnosed laboratory-animal-allergy subjects gave no evidence of immunological response to the urinary allergens and wider diagnoses may have to be applied in these cases.

The development and validation of radioallergosorbent tests for the detection of specific human IgE antibody directed against laboratory animal urinary proteins.

Radioallergosorbent tests (RAST) using urinary proteins from mice, rats, guinea pigs and rabbits have been developed and used in the diagnosis of laboratory animal allergy (LAA). Of the 273 subjects tested, 15 had been previously diagnosed as laboratory animal allergic and 8 of these (53%) gave one or more positive RAST results. Of the 258 symptom-free individuals, only 9 (3.5%) had one or more positive RAST. Of these 9, 7 had previously worked with animals or had occupational exposure to the appropriate species; the remaining 2 individuals had only some pet exposure. RAST was, therefore, of value in the diagnosis of LAA. During the development of these RAST assays, several sources of potential error were identified. Modest titres of total IgE (600 IU/ml and above) were found to influence the specific RAST index observed and lead to false positive results. The presence of human IgG antibody specific for rabbit serum proteins was also identified in four sera, and was responsible for interference in the rabbit urinary protein RAST system.