The human leukocyte antigen (HLA) system is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. HLA genes are highly polymorphic, which means that they have many different alleles, allowing them to fine-tune the adaptive immune system. HLA genotyping is based on reverse sequence-specific oligonucleotide-polymerase chain reaction using DNA from peripheral blood.

Pharmacogenetic testing is not widely used in routine clinical practice to optimize drug choice or clinical management (Philipps, 2006). This gap between scientific knowledge and clinical application may be explained by the fact that the successful incorporation of a pharmacogenetic test into routine practice requires a combination of high-level evidence that can be generalized to diverse clinical setting, wide-spread availability of cost-effective and reliable laboratory tests, and effective strategies incorporate testing into routine clinical practice (Mallal et al., 2008).

Several clinical studies, as reported in the review by Mayorga et al. (2016), correlate hypersensitivity reaction caused by pharmaceuticals with the presence of different HLA allele. In particular HLA-B^*57:01 has been found to be associated with abacavir hypersensitivity; for carbamazepine, the most important association has been established with HLA-B^*15:02 and HLA-A31:01, while HLA-B^*58:01 allele has been associated with allopurinol hypersensitivity.

Prospective HLA-allele screening may be widely useful, but there are some controversial points like the cost-effectiveness of the test, that depend on several estimates that vary among populations, the health care setting, and also the availability of appropriate laboratory assays (Martin et al., 2006; Hammond et al., 2007).

The LTT measures the in vitro proliferative response of T cells that emerge from the clonal expansion of naïve T cell, after drug exposure (Nyfeler and Pichler, 1997; Lanzavecchia and Sallusto, 2000). The proliferative response of lymphocytes is measured by the incorporation of ³H-thymidine during DNA synthesis or by carboxyfluorescein diacetate succimidyl ester (CFSE). The most widely studied drugs are betalactams (Mayorga et al., 2016). The LTT limits are represented by the use of radioactivity, which limits the application of the method to specific research laboratories, it is a quite long procedure, the sensitivity is quite low (60–70%), and it depends upon the conditions employed (Nyfeler and Pichler, 1997; Romano et al., 1997; Beeler and Pichler, 2007).

ELISpot is a technique used to determine the number of cells able to produce cytokines and cytotoxic markers after their activation by the drug or its metabolites (Sullivan et al., 2015). Clinical studies report the use of IFN-γ ELISpot to diagnose non-immediate reactions to betalactames; granzyme B and granulysin ELISpot for evaluating severe cutaneous reactions induced by amoxicillin, ciprofloxacin, carbamazepine, sulphonamides, allopurinol, mefenamic acid, oxipurinol, and lamotrigine (Zawodniak et al., 2010; Porebski et al., 2013). One advantage of this in vitro test is that drug-reactive T cells remain detectable for long time after the reaction and could be appropriate for high throughput screening but to improve the accuracy of the test, two or more cytokines determination could be necessary.

After drug stimulation, T-cells express or up-regulate a number of surface molecules and produce different inflammatory mediators. Cytokine expression and secretion can be evaluated by several methods such as reverse transcription polymerase chain reaction (RT-PCR), flow cytometric analysis, and enzyme linked immunosorbent assay (ELISA). RT-PCR is used to measure cytokine at the transcriptional levels, while flow cytometric analysis are used to study intracellular cytokines and cell surface markers. Finally, ELISA is used to measure the amount of secreted cytokines in cell culture supernatants (Khalil et al., 2008).

Sachs et al. demonstrated that accumulation of eosinophils following IL-5 secretion, and to a lesser extent also with IL-10 and IFN-γ, from drug-specific stimulated T cells is a characteristic histological feature of drug-induced skin eruption. In vitro determination of drug-specific IL-5 secretion by peripheral blood mononuclear cells may be relevant for the detection of in drug-induced maculopapular exanthems (Sachs et al., 2002). CD69 is up-regulated after 48–72 h, and its determination by flow cytometry correlates with LTT for betalactames, sulphamethoxazole and carbamazepine HDR (Beeler et al., 2008). CD69 may be used for the evaluation of non-immediate reactions.

These in vitro tests represent important tools for diagnosis. They are, however, used mainly as research methods rather than as routine procedures. It must be also taken into consideration the timing of sample collection, which is critical as mediators can be secreted in transitory peaks with variants in the maintenance of detectable levels (Mayorga et al., 2006), and chemokines and cytokines can be degraded by protease (Niwa et al., 2000).

Other in vitro tests have been proposed for the study of drug allergy reactions. Among these the cellular allergen stimulation test (CAST-ELISA®) for the measurement of leukotrienes after peripheral blood leukocyte stimulation, basophil histamine release tests and a basophil activation test (BASO-Test®) can be mentioned. CAST-ELISA® is commercially available but it has not been sufficiently evaluated to recommend as a standard investigation outside the context of prospective studies. Basophil activation markers using fluorescence activated cell sorter analysis are currently being evaluated for certain type of drug allergic reactions but there seems to be no evidence currently of any advantage of these tests over skin testing (Mirakian et al., 2009).

Chemicals can elicit T-cell-mediated diseases, including adverse drug reactions. The T cell priming assay (TCPA) was developed primarily for the identification of contact allergens within the integrated EU project SENS-IT-IV. This assay allows the detection of chemical-specific T cells in naive human peripheral T-cell population by measurement of proliferation and, at the single cell level, of IFN-γ production in CD4+ and CD8+ T cells. This assay may be a valuable, highly specific element in an integrated testing strategy for the predictive in vitro identification of contact allergens and possibly drugs that cause T cell-mediated adverse drug reactions (Dietz et al., 2010; Martin et al., 2010; Richter et al., 2013).

The MUSST is an in vitro method proposed to assess skin sensitization. Dendritic cell activation following exposure to sensitizers was modeled in the U937 human myeloid cell line by measuring the induction of the expression of CD86 by flow cytometry after 48 h of chemical treatment. A test substance is predicted to have a dendritic cell activating potential indicative of being a sensitizer when CD86 induction exceeds the threshold of 1.5-fold with respect to vehicle treated cells at any tested concentration showing a cell viability ≥70 % in at least two independent experiments (Urbisch et al., 2015).

In the modified version of the MUSST (mMUSST), a test substance is predicted to have a dendritic cell activating potential when CD86 induction exceeds a threshold of 1.2-fold (Bauch et al., 2012). Among more than 145 substances available from these two in vitro assays, also some drug sensitizers, namely benzocaine, hydroquinone, p-benzoquinone and diphenylclopropenone were tested and resulted correctly classified with the MUSST (Bauch et al., 2012; Natsch et al., 2013; Urbisch et al., 2015).

The h-CLAT quantifies changes in CD86 and CD54 expression in the human THP-1 cell line following 24 h exposure to the test chemical. The changes in surface marker expression are measured by flow cytometry. Cytotoxicity measurement is conducted concurrently to assess whether up-regulation of surface maker expression occurs at sub-cytotoxic concentrations. The prediction model use the relative fluorescence intensity of surface markers compared to solvent control to discriminate between sensitizers and non-sensitizers. Among the 166 substances tested in this in vitro assay, the allergenic drugs benzocaine, clofibrate, pyridine, hydroquinone, p-benzoquinone and diphenylclopropenone resulted correctly classified (Nukada et al., 2012; Takenouchi et al., 2013; Urbisch et al., 2015).

Overall, even if the number of drugs tested is very limited, data suggests that pharmaceuticals may share with chemical allergens a common mechanism of action that activates dendritic cells, and support the possibility to use these in vitro methods also for the identification of drugs potentially associated with hypersensitivity reactions.

As mentioned above, most drugs are small molecules and are by themselves, not immunogenic. During the haptenization process, these small molecules bind to carrier proteins to form a complete immunogenic complex (Chang and Gershwin, 2010). The hypersensitivity reaction then requires the activation and maturation of dendritic cells (DCs), which will then drive the activation of specific T cells (Martin, 2012). DCs are antigen-presenting cells (APC) that play a central role in the initiation and regulation of adaptive immune responses. Following the contact with antigens, DCs undergo a process of maturation associated with the expression of several co-stimulatory molecules on the membrane such as CD80, CD86 and CD40, various adhesion molecules (CD2, CD11a, CD54, CD58), and secrete different cytokines, including IL-1β and IL-8 (Quah and O'Neill, 2005). Once activated, DCs migrate into the regional lymph node or in the spleen, where they present antigen to specific T lymphocytes, through MHC class II molecules (Ryan et al., 2007) and co-stimulatory adhesion molecules expressed on both DC (i.e., CD86) and T cell (i.e., CD28) to ensure the necessary contact to achieve full T-cell activation. Following stimulation, a clone of T cells is produce with the ability to react to the antigen, resulting in the clinical manifestation of HDR.

Within the European project SENS-IT-IV, we have previously established an in vitro method able to identify contact and respiratory allergens based on the use of the human THP-1 cell line (the same cell line used in the h-CLAT) and IL-8 release: the THP-1 activation assay (Mitjans et al., 2008, 2010). IL-8 is a potent chemotactic peptide for neutrophils as well as for T lymphocytes, basophils, and NK cells. In parallel to IL-8 production, several of the proposed in vitro methods, including the ones mentioned above, are based on DC and CD86 alone or in combination with CD54 expression for the identification of chemical sensitizers, due to their roles in antigen presentation and T cell activation.

Based on the notion that drug sensitizers and chemical sensitizers share the same mode of action, we recently investigated the possibility to use the THP-1 activation assay developed for skin and respiratory sensitizers, for the in vitro identification of pharmaceuticals, which may be associated with in vivo drug hypersensitivity reactions (Corti et al., 2015). It is well known that allergen drugs share with chemical allergens common mechanisms of cell activation and for reason we propose the THP-1 activation assay also for the hazard identification of immune-mediated hypersensitivity reactions induced by pharmaceuticals. Drugs were selected on the basis of clear in vivo immune-adverse reactions reported in literature, post-marketing data or labeling information, and on the commercial availability as pure drugs. Clonidine, ofloxacine, procainamide, streptozotocin, sulfamethoxazole which have been associated with a relatively high incidence of immune-mediated hypersensitivity reactions (Weaver et al., 2005), methyl salicylate and probenecid, which have been reported to cause irritant or allergic contact dermatitis and anaphylactic reactions (Corti et al., 2015), have been tested. We developed a strategy based on IL-8 production, CD86 and/or CD54 expression in THP-1 cells useful for the in vitro identification of drug sensitizers (see Figure 2). There are some important differences with the previous mentioned h-CLAT. First, the method we use to calculate CD86 and CD54 expression is different from h-CLAT protocol: in h-CLAT only the MFI is considered, while we also include the percentage of positive cells. The second point is the concentration tested. In fact, we observed that quite often a CV75 couldn't be reached with drugs compared to chemicals, meaning that drugs are less cytotoxic. The test we developed allowed the correct identification of all the selected drugs tested, including sulfamethoxazole, probenecid and procainamide for which metabolism is needed. Penicillin G, another drug frequently associated with hypersensitivity reactions (Siegel and Coleman, 1957), was previously tested, and found to be able to induced a dose-related release of IL-8 following 48 h of exposure (Mitjans et al., 2008).

Exposure of THP-1 cells to sensitizing drugs results in most cases in dose related release of IL-8 and increase in CD86 expression, with some differences among drugs, markers and times of exposure. As shown in Table 1, the combination of both IL-8 and CD86 expression allows the identification of all drugs tested. The use of IL-8 mRNA expression at 3 h or CD54 expression at 24 h may offer an alternative to the 48 h exposure and increase our confidence in the negativity of a drug (see Corti et al., 2015). The expression of IL-8 mRNA at 3 h is based on previous observation we made on chemical allergens failing to induce the release of IL-8: all chemicals sensitizers tested including pro-hapten induced IL-8 mRNA at 3 h (Galbiati et al., 2012).

Using streptozotocin as reference drug to study the mechanisms of action, we could demonstrate a key role for p38 mitogen-activated protein kinase (p38 MAPK) and PKC-β activation in streptozotocin-induced IL-8 release and CD86 expression (Corti et al., 2015), confirming previous results obtained with chemical allergens (Mitjans et al., 2008; Corsini et al., 2014).

Evidence indicates that oxidative stress is involved in chemical-induced skin allergic and inflammatory diseases (Okayama, 2005; Byamba et al., 2010). Chemical-induced oxidation of the cell surface thiols appears to be one of the triggers of DC maturation, resulting in intracellular redox imbalance that generate stress-related signal (Figure 3). The Keap1/Nrf2-signaling pathway is dedicated to the detection of electrophilic stress in cells leading to the up-regulation of genes involved in protection or neutralization of chemicals reactive species (Natsch and Emter, 2007). It has been shown in human monocyte-derived dendritic cells that chemical sensitizers induced oxidative stress measured by the glutathione GSH/GSSG ratio, as a redox marker (Mizuashi et al., 2005). The reduction of the glutathione GSH/GSSG ratio was accompanied by CD86 up-regulation and p38 MAPK activation, suggesting that the electrophilic properties of chemicals sensitizers may be perceived by DCs as a danger signal leading to DC maturation (Sasaki and Aiba, 2007). Engagement of certain Toll like receptors (TLR1, 2, and 4) leads to mitochondrial translocation of the signal adaptor TRAF6. At the mitochondria, TRAF6 interacts with ECSIT, a protein implicated in the assembly of complex I, leading to its ubiquitylation, which results in increased ROS production. Proteins that are reversibly modulated by ROS are of high interest. In this context, protein kinases and phosphatases, which act co-ordinately in the regulation of signal transduction through the phosphorylation and dephosphorylation of target proteins, have been described to be key elements in ROS-mediated signaling events. In particular, PKC isoforms have been shown to contain a unique structural feature that is susceptible to oxidative modification (Cosentino-Gomes et al., 2012). The high levels of cysteine residues render the regulatory domain susceptible to redox regulation (Gopalakrishna and Jaken, 2000; Giorgi et al., 2010). Currently, evidence supports the direct activation of different PKC isoforms by ROS generation; in particular the β isoform is able to induce ROS generation through mitochondrial damage (Pinton et al., 2007).

To summarize the drugs tested in the preclinical available in vitro methods and known to be able to induce hypersensitivity reactions, a list is reported in Table 2.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer NV and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.