In vitro tests of T cell-mediated drug hypersensitivity

Allergic side-effects of drugs are a major problem of drug therapy. Diagnosis of drug-hypersensitivity reactions in patient is still very difficult as a broad spectrum of different drugs can elicit various immune-mediated diseases with distinct pathomechanisms. In this review, we summarize current in vitro techniques with an emphasis on diagnosis of T cell- mediated drug allergies and discuss recent research finding that may be applied to develop tests allowing for a better diagnosis of drug hypersensitivity in the future.     

Clinical presentation and time course in hypersensitivity reactions to beta-lactams

Background:  β-lactam hypersensitivity reactions are classified as immediate or nonimmediate. Diagnosis is usually based upon skin tests and provocation challenges.
Objective:  The time course of the reactions in proven β-lactam hypersensitivities was studied and then correlated with the symptoms to determine the relationship between the clinical presentations and the time course.
Method:  All of the patients who consulted between 1996 and 2004 for a suspected β-lactam hypersensitivity reaction were studied. Two hundred and ten patients with a proven hypersensitivity reaction diagnosed according to the European Network on Drug Allergy were included in the present study.
Results:  Of the patients, 36.7% had urticaria as a single symptom, 19.1% anaphylaxis without shock, 17.6% anaphylactic shock and 19.1% maculopapular exanthema. Anaphylactic shock and anaphylaxis mostly occurred within 1 h after drug administration. Exanthema mainly occurred after 24 h. Urticaria as a single symptom occurred at any time. A firm diagnosis was determined using immediate-reading skin prick (10.0%) and intradermal tests (38.1%), late-reading skin tests (19.1%) or provocation tests (32.9%).
Conclusion and clinical implication:  Depending on the time course of the reaction, three clinical groups were identified: anaphylaxis and anaphylactic shock (immediate reaction); maculopapular exanthema (late reaction) as well as urticaria (immediate and late reaction).     

In vitro detection and characterization of drug hypersensitivity using flow cytometry

Background:  The lymphocyte transformation test (LTT) is the only in vitro test for detecting drug sensitization at the cellular level irrespective of the reaction's phenotype. However, the LTT includes working with radioactive substances and is considered impracticable for routine laboratory investigation.
Objective:  The aim of this study was to assess drug-specific cytokine production by means of flow cytometry as an alternative nonradioactive approach which may be more appropriate for routine testing and may provide in addition more information about the pathophysiology of the reaction than proliferation-based assays, like the LTT.
Method:  Peripheral blood mononuclear cells of 19 patients were incubated with culprit drugs ( n  = 28) or irrelevant antigens ( n  = 10). Ten healthy persons served as controls for all different drugs ( n  = 15). Intracellular interleukin (IL)-5, interferon (IFN)-γ and IL-10 production was investigated using flow cytometry. Accuracy of the flow cytometry test system was confirmed using different statistical tests, i.e. receiver operating characteristic curve and Mann–Whitney rank test. In addition, drug-specific secretion of IL-5, IL-2 and IFN-γ were analysed using enzyme-linked immunosorbent assay (ELISA).
Results:  Drug-specific cytokine production could be demonstrated in 75% of the patients using flow cytometry and in 79% using ELISA respectively. Combining ELISA and flow cytometry increased the sensitivity to 100%. Analysis of involved T-cell subsets [e.g. CD4⁺ or CD8⁺; T helper (TH) 1 or TH 2] allowed characterization of the in vitro lymphocyte reactivity pattern.
Conclusions:  Analysis of drug-specific cytokine production by means of flow cytometry proved a useful and reliable approach for the in vitro detection and characterization of drug hypersensitivities.     

Diagnosis and management of the drug hypersensitivity reactions in Coronavirus disease 19: An EAACI Position Paper

Coronavirus disease 2019 (COVID-19), a respiratory tract infection caused by a novel human coronavirus, the severe acute respiratory syndrome coronavirus 2, leads to a wide spectrum of clinical manifestations ranging from asymptomatic cases to patients with mild and severe symptoms, with or without pneumonia. Given the huge influence caused by the overwhelming COVID-19 pandemic affecting over three million people worldwide, a wide spectrum of drugs is considered for the treatment in the concept of repurposing and off-label use. There is no knowledge about the diagnosis and clinical management of the drug hypersensitivity reactions that can potentially occur during the disease. This review brings together all the published information about the diagnosis and management of drug hypersensitivity reactions due to current and candidate off-label drugs and highlights relevant recommendations. Furthermore, it gathers all the dermatologic manifestations reported during the disease for guiding the clinicians to establish a better differential diagnosis of drug hypersensitivity reactions in the course of the disease.     

The severity of atopic dermatitis and analysis of the food hypersensitivity reactions

Aim of this study is to evaluate the dependence between the occurrence of food hypersensitivity reactions and the severity of atopic dermatitis. The detailed personal history about the food hypersensitivity reactions was recorded and the severity of atopic dermatitis was evaluated with SCORAD index. The statistical evaluation of the dependence between the occurrence of food hypersensitivity reactions and the severity of atopic dermatitis was performed. Two hundred and eighty-five patients were examined – 90 men and 195 women, average age 26.2 (s.d. = 9.5). The significant dependence between the severity of atopic dermatitis and the occurrence of food hypersensitivity reactions was confirmed; 96% of patients with severe form of atopic dermatitis suffer from food reactions. In evaluating the single foods, the significant dependence was found between the severity of atopic dermatitis and the reactions to nuts, apples, and fishes.     

Immune pathomechanism and classification of drug hypersensitivity

Drug hypersensitivity reactions (DHR) are based on distinct mechanisms and are clinically heterogeneous. Taking into account that also off‐target activities of drugs may lead to stimulations of immune or inflammatory cells, three forms of DHR were discriminated: the allergic‐immune mechanism relies on the covalent binding of drugs/chemicals to proteins, which thereby form new antigens, to which a humoural and/or cellular immune response can develop. In IgE‐mediated drug allergies, a possible tolerance mechanism to the drug during sensitization and the need of a covalent hapten‐carrier link for initiation, but not for elicitation of IgE‐mediated reactions is discussed. The p‐i (“pharmacological interaction with immune receptor”) concept represents an off‐target activity of drugs with immune receptors (HLA or TCR), which can result in unorthodox, alloimmune‐like stimulations of T cells. Some of these p‐i stimulations occur only in carriers of certain HLA alleles and can result in clinically severe reactions. The third form of DHR (“pseudo‐allergy”) is represented by drug interactions with receptors or enzymes of inflammatory cells, which may lead to their direct activation or enhanced levels of inflammatory products. Specific IgE or T cells are not involved. This classification is based on the action of drugs and is clinically useful, as it can explain differences in sensitizations, unusual clinical symptoms, dependence on drug concentrations, predictability and immunological and pharmacological cross‐reactivities in DHR.     

In vitro tests for drug hypersensitivity reactions: an ENDA/EAACI Drug Allergy Interest Group position paper

Drug hypersensitivity reactions (DHRs) are a matter of great concern, both for outpatient and in hospital care. The evaluation of these patients is complex, because in vivo tests have a suboptimal sensitivity and can be time‐consuming, expensive and potentially risky, especially drug provocation tests. There are several currently available in vitro methods that can be classified into two main groups: those that help to characterize the active phase of the reaction and those that help to identify the culprit drug. The utility of these in vitro methods depends on the mechanisms involved, meaning that they cannot be used for the evaluation of all types of DHRs. Moreover, their effectiveness has not been defined by a consensus agreement between experts in the field. Thus, the European Network on Drug Allergy and Drug Allergy Interest Group of the European Academy of Allergy and Clinical Immunology has organized a task force to provide data and recommendations regarding the available in vitro methods for DHR diagnosis. We have found that although there are many in vitro tests, few of them can be given a recommendation of grade B or above mainly because there is a lack of well‐controlled studies, most information comes from small studies with few subjects and results are not always confirmed in later studies. Therefore, it is necessary to validate the currently available in vitro tests in a large series of well‐characterized patients with DHR and to develop new tests for diagnosis.     

Food hypersensitivity reactions and peripheral blood eosinophilia in patients suffering from atopic dermatitis

The aim of our study is to evaluate the count of eosinophils in peripheral blood in atopic dermatitis (AD) patients over 14 years of age and to compare it with the occurrence of food hypersensitivity (FH) reactions. Complete allergological and dermatological examination was performed in 212 patients included in the study (90 men, 122 women, average age 26.7 years, average SCORAD 32.9). According to our results, in AD patients the difference in count of eosinophils in patients with and without FH reactions is not statistically significant. When evaluating the occurrence of FH reactions to single foods, the count of eosinophils is significantly higher only in patients suffering from reactions to carrot.     

Immune dysregulation increases the incidence of delayed-type drug hypersensitivity reactions

Delayed-type, T cell–mediated, drug hypersensitivity reactions are a serious unwanted manifestation of drug exposure that develops in a small percentage of the human population. Drugs and drug metabolites are known to interact directly and indirectly (through irreversible protein binding and processing to the derived adducts) with HLA proteins that present the drug-peptide complex to T cells. Multiple forms of drug hypersensitivity are strongly linked to expression of a single HLA allele, and there is increasing evidence that drugs and peptides interact selectively with the protein encoded by the HLA allele. Despite this, many individuals expressing HLA risk alleles do not develop hypersensitivity when exposed to culprit drugs suggesting a nonlinear, multifactorial relationship in which HLA risk alleles are one factor. This has prompted a search for additional susceptibility factors. Herein, we argue that immune regulatory pathways are one key determinant of susceptibility. As expression and activity of these pathways are influenced by disease, environmental and patient factors, it is currently impossible to predict whether drug exposure will result in a health benefit, hypersensitivity or both. Thus, a concerted effort is required to investigate how immune dysregulation influences susceptibility towards drug hypersensitivity.     

In vitro cytotoxicity as a marker of hypersensitivity to sulphamethoxazole in patients with HIV.

Hypersensitivity to trimethoprim-sulphamethoxazole (TMP-SMX) in patients with HIV infection may be a result of either immune dysregulation, a direct cytotoxicity of the SMX-hydroxylamine metabolite (SMX-HA) (rather than SMX per se), or glutathione deficiency. We evaluated the in vitro cytotoxicity of SMX and SMX-HA to peripheral blood mononuclear cells (PBMC) of HIV-infected subjects to determine if the degree of in vitro cytotoxicity is associated with hypersensitivity, whether glutathione inhibits cytotoxicity, and whether in vitro cytotoxicity is predictive for the development of hypersensitivity. Given that fever is often a prominent feature of hypersensitivity, we also assessed whether SMX or SMX-HA could induce the in vitro production of IL-1 beta, IL-6 or tumour necrosis factor-alpha (TNF-alpha) by PBMC. The cytotoxicities of SMX and SMX-HA to PBMC were assessed in 45 HIV-infected patients with prior TMP-SMX therapy, and in eight HIV- controls. Twelve HIV-infected subjects were studied prospectively before primary Pneumocystis carinii pneumonia (PCP) therapy or rechallenge with TMP-SMX in previously hypersensitive subjects. Cytokine production was measured in four hypersensitive and two non-hypersensitive HIV-infected subjects, and three HIV-uninfected controls. The cytotoxicity of SMX-HA to PBMC was significantly greater in the 22 HIV-infected patients with prior hypersensitivity than both the 23 HIV-infected patients without hypersensitivity and the control group. Cytotoxicity was significantly reduced by glutathione only in the hypersensitive group. SMX did not induce cytotoxicity in any group. In 12 subjects studied prospectively, SMX-HA cytotoxicity was also significantly greater in those with subsequent hypersensitivity. Exposure of PBMC to SMX-HA resulted in a modest increase in the production of IL-6, IL-1 beta and TNF-alpha, although no major difference was detected between subjects with or without hypersensitivity. These data suggest that SMX-HA and glutathione deficiency are involved in the pathogenesis of hypersensitivity to TMP-SMX in HIV-infected patients, and that in vitro cytotoxicity could be useful in the diagnosis of hypersensitivity and predicting its likelihood.     

Human leukocyte antigens (HLA) associated drug hypersensitivity: consequences of drug binding to HLA

Recent publications have shown that certain human leukocyte antigen (HLA) alleles are strongly associated with hypersensitivity to particular drugs. As HLA molecules are a critical element in T‐cell stimulation, it is no surprise that particular HLA alleles have a direct functional role in the pathogenesis of drug hypersensitivity. In this context, a direct interaction of the relevant drug with HLA molecules as described by the p‐i concept appears to be more relevant than presentation of hapten‐modified peptides. In some HLA‐associated drug hypersensitivity reactions, the presence of a risk allele is a necessary but incomplete factor for disease development. In carbamazepine and HLA‐B*15:02, certain T‐cell receptor (TCR) repertoires are required for immune activation. This additional requirement may be one of the ‘missing links’ in explaining why most individuals carrying this allele can tolerate the drug. In contrast, abacavir generates polyclonal T‐cell response by interacting specifically with HLA‐B*57:01 molecules. T cell stimulation may be due to presentation of abacavir or of altered peptides. While the presence of HLA‐B*58:01 allele substantially increases the risk of allopurinol hypersensitivity, it is not an absolute requirement, suggesting that other factors also play an important role. In summary, drug hypersensitivity is the end result of a drug interaction with certain HLA molecules and TCRs, the sum of which determines whether the ensuing immune response is going to be harmful or not.     

Update on the evaluation of hypersensitivity reactions to betalactams

Hypersensitivity reactions to betalactams (BLs) are classified as immediate or nonimmediate. The former usually appear within 1 h of drug-intake and are mediated by specific IgE-antibodies. Nonimmediate reactions are those occurring more than 1 h after drug-intake, and they can be T-cell mediated. The diagnostic evaluation of allergic reactions to BLs has changed over the last 5 years, for several reasons. Major and minor determinants are no longer commercially available for skin testing in many countries. In immediate allergic reactions, the sensitivity of skin testing and immunoassays is decreasing and new in vitro methods, such as the basophil activation test, are gaining importance for diagnosis. For nonimmediate reactions, skin testing appears to be less sensitive than previous results, although more studies need to be carried out in this direction. Nevertheless, the drug provocation test is still necessary for diagnosis.     

Risk factors for oxaliplatin-induced hypersensitivity reactions in Japanese patients with advanced colorectal cancer

Objective: Previously, we suggested that oxaliplatin (L-OHP)-related grade 3/4 hypersensitivity reactions occurred immediately after the initiation, but grade 1/2 reactions did not. This study was conducted to clarify the risk factors for L-OHP-related hypersensitivity reactions.Methods: Clinical data from 108 Japanese patients with colorectal cancer were analyzed, who were treated with L-OHP-containing regimens, FOLFOX4 and/or mFOLFOX6. The risk factors examined included demographic data, preexisting allergies, laboratory test data, treatment regimen, treatment line of therapy, pretreatment with steroids, total number of cycles and cumulative amount of L-OHP.Results: The incidence of grade 1/2 and grade 3/4 hypersensitivity reactions were found at 13.0% (14/108) and 9.3% (10/108), respectively. Female (P=0.037), preexisting allergies (P=0.004) and lower level of lactate dehydrogenase (P=0.003) were risk factors for grade 1/2 hypersensitivity reactions, and higher neutrophil count (P=0.043) and lower monocyte count (P=0.007) were for grade 3/4 reactions. Total number of cycles were larger in the patients with grade 3/4 reactions than those without reactions (P=0.049).Conclusions: Further extensive examination with a large number of patients is needed to establish a patient management strategy.     

Hypersensitivity reactions to biologicals: An EAACI position paper

Biologicals are crucial targeted therapeutic agents in oncological, immunological, and inflammatory diseases, and their use in clinical practice is broadening. In recent years, the spread of Personalized Precision Medicine has facilitated a proliferation of new treatment options, especially biologicals. Consequently, biologicals are now among the drugs that most frequently cause hypersensitivity reactions (HSRs). Patients can develop HSRs to these agents during the first-lifetime exposure or after repeated exposure, and these HSRs can be potentially life-threatening or limit therapeutic options. Despite the relatively high prevalence, the underlying mechanisms of these HSRs remain obscure, and the optimal management pathways are still a matter of discussion. In this Position Paper, the authors will provide evidence-based recommendations for diagnosing and managing HSRs to biologicals. Additionally, the document defines unmet needs as an opportunity to shape future research.     

Molecular basis of polymorphic drug metabolism

Genetic polymorphisms with functional effects occur in many of the genes encoding drug metabolizing enzymes and are an important cause of adverse drug reations. Recent advances in the understanding of the molecular genetics of drug-metabolizing enzymes, particularly the cytochromes P450, has enabled the molecular basis of several polymorphisms to be elucidated and genotyping assays using the polymerase chain reaction to be developed. Polymorphisms in this category include those in the cytochrome P450 genes CYP2D6, CYP2C19, CYP2A6, CYP2C9 and CYP2E1, the glutathione S-transferase genes GSTM1 and GSTT1 and the N-acetyltransferase gene NAT2. The molecular basis and impotance to drug metabolism of the various polymorphisms as well as evidence for the existence of polymorphisms in other genes encoding drug-metabolizing enzymes such as the UDP-glucuronosyltransferases, the sulphotransferases and the methyltransferases are discussed.Abbreviations DHEA ST Dehydroepiandrosterone sulphotransferase - EST Oestrogen sulphotransferase - TL ST Thermolabile sulphotransferase - TS ST Thermostable sulphotransferase