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.

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)

Stimulatory effects of FK156 in a panel of tests designed to detect changes in immune function.

There is a need to evaluate the utility of experimental models in immune function assessment if these are to be accepted in preclinical safety studies. We have evaluated a panel of tests measuring cellularity and functions of the lymphoid system in the Fischer rat in order to determine whether they would detect immunostimulation, rather than suppression. Injection of the peptide immunostimulant FK156 (D-lactyl-L-alanyl-y-D-glutamyl-(L)-meso-diaminopimelyl- (L)-glycine) increased the numbers of macrophages recovered from the peritoneal cavity, and stimulated their activity, as measured by chemiluminescence, adherence, and secretion of interleukin 1. In vitro, T lymphocytes had an increased background incorporation of tritiated thymidine, increased response to sub-optimal concentrations of concanavalin A, and an increase in secretion of interleukin 2 at optimal concentrations of concanavalin A. There was no change in the proliferative responses of B lymphocytes in vitro. Antibody responses to tetanus toxoid in vivo were increased. These changes were not reflected in consistent, statistically significant alterations in the numbers of lymphocytes bearing either lineage markers or the interleukin 2 receptor as a marker of activation.

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.

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.

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 specific IgG antibody levels in serum of patients on regimes comprising high total dose beta-lactam therapy.

Sera from 125 patients receiving mean total doses of beta-lactam therapy of 215 g over a mean of 14 days were assayed by radioimmunoassay. Titres of anti-penicilloyl antibodies, expressed in arbitrary units of specific IgG per microliter of serum (u/microliter), ranged from undetectable (less than 3 u/microliter) to 1,650 u/microliter. There was a higher prevalence of elevated IgG levels in patients who developed haemolytic anaemia or neutropenia compared with patients with no adverse reactions but only in those patients who developed haemolytic anaemia were the antibody titres significantly higher. A subsidiary finding was that a correlation was established between mean total dose and prevalence of positive IgG titres, on a patient group basis (r = 0.87).

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.

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.

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.

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 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.

Development and use of radioimmunoassays for the detection of human IgG antibodies to laboratory animal urinary proteins.

A radioimmunoassay method for the detection of human IgG antibodies to laboratory animal urinary proteins has been developed using partially purified protein fractions prepared from crude urine extracts by gel filtration. The proteins from the four species, mouse, rat, guinea pig and rabbit, were labelled with 125I and used in an antigen binding assay employing a sepharose-linked staphylococcal protein A immunosorbent. Results, expressed as arbitrary units of specific IgG per unit serum, show antibodies to one or more species present in 57% of current animal workers compared with 6% of non-animal workers.

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.