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Background: We report on two cases of allergic contact dermatitis to chloramphenicol and azidamphenicol respectively, with in vivo and in vitro lymphocyte reactivity to both compounds. The molecular features determining lymphocyte reactivity were explored because chloramphenicol, azidamphenicol, and thiamphenicol exhibit almost identical chemical structures.
Methods: With chloramphenicol, azidamphenicol, and the chemically related thiamphenicol, we performed patch tests and lymphocyte transformation tests with both patients. Furthermore, the interleukin-5 and interferon-γ concentrations in the cultures of peripheral blood mononuclear cells of one patient were determined.
Results: Patch tests showed delayed hypersensitivity reactions to chloramphenicol and azidamphenicol, but not to thiamphenicol. These results were confirmed by lymphocyte transformation tests with peripheral blood mononuclear cells of the patients, showing a proliferative T-cell response to azidamphenicol and chloramphenicol. Moreover, lymphocytes from one patient secreted large amounts of interleukin-5, but not of interferon-γ upon coculture with azidamphenicol.
Conclusions: Since lymphocyte reactivity was observed to chloramphenicol and azidamphenicol, but not to thiamphenicol, the epitope(s) recognized by the allergen-reactive T cells may be formed by the nitro-group of the benzene ring shared by chloramphenicol and azidamphenicol.
Chloramphenicol and azidamphenicol are chemically closely related antibiotics (Fig. 1). The administration of chloramphenicol has decreased due to the occurrence of life-threatening aplastic anemia, which may also follow resorption after external application (1). Allergic contact dermatitis to topical chloramphenicol or azidamphenicol in eye-drops or antibiotic ointments, respectively, has been described (2, 3). The sensitizing potential of topically applied chloramphenicol is considered to be low according to animal studies (1). Cross-reactivities in patch tests between chloramphenicol and azidamphenicol and between chloramphenicol and thiamphenicol have been reported (2, 4). The possibility of cosensitization, however, has not definitely been excluded. Chloramphenicol is also used in veterinary medicine, possibly leading to the presence of residues in the meat of treated animals (5). Whether these residues, in addition to the promotion of resistance to antibiotics, suffice for the sensitization of man or may, on the contrary, lead to chloramphenicol tolerance considering the oral ingestion of the residues remains speculative. Since T-cell recognition of the antigen is the initial step in the immunologic cascade of allergy, sensitized T cells are present in both immediate and delayed-type hypersensitivity reactions. The lymphocyte transformation test (LTT) detects sensitized T cells and has thus proven useful as an in vitro test for the detection of drug allergy at the cellular level irrespective of the clinical phenotype (6). In the present cases, lymphocyte reactivity to chloramphenicol and azidamphenicol, but not to thiamphenicol, could be demonstrated in patch tests and with the LTT. In one case, the culture supernatants in the LTT revealed large amounts of interleukin-5 (IL-5) in the cultures incubated with azidamphenicol, corresponding to the strong proliferation observed in these wells, whereas no relevant amounts of interferon-γ (IFN-γ) could be determined.
Figure 1. Chemical structure of chloramphenicol, thiamphenicol, and azidamphenicol. Relevant molecular features are framed by rectangles. Chloramphenicol and azidamphenicol share common nitro-group. Like chloramphenicol, thiamphenicol has same p-side chain on opposite side of benzene ring containing two chloride ions, whereas azidamphenicol exhibits N3H2 group at this position.
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In the presented case reports, allergic contact dermatitis to chloramphenicol and azidamphenicol was demonstrated both in vivo by epicutaneous testing and in vitro by the LTT. The molecular features determining lym-phocyte reactivity could be explored since chloram-phenicol, azidamphenicol, and thiamphenicol share an identical chemical backbone structure differing only in distinct side chains (Fig. 1). Chloramphenicol and azidamphenicol are structurally identical except for the p-side chain opposite the nitro-group. In this position, chloramphenicol displays two chloride ions and azidamphenicol three nitrogen ions and a hydrogen ion. Thiamphenicol however, differs from chloramphenicol and azidamphenicol by having an SO2CH3 group in-stead of the nitro-group, and from azidamphenicol in addition by the chloride ions contained in the opposite side chain. Thus, the greatest structural difference can beobserved between thiamphenicol and azidamphenicol, making cross-reactivity between these two compounds more unlikely than between chloramphenicol and azidamphenicol or thiamphenicol. If we assume that only one side chain of the molecules is recognized by T cells in connection with the MHC molecule, the observed lymphocyte reactivity to chloramphenicol and azidamphenicol, and the lack of reactivity to thiamphenicol suggest
involvement of the nitro-group in the formation of the nominal T-cell epitope(s)
a true T-cell cross-reactivity on the cellular level.
Since the present investigations were carried out with PBMC, the existence of different T-cell clones circulating in the peripheral blood specific for chloramphenicol and azidamphenicol, respectively, cannot be ruled out completely. Cross-reactivity between chloramphenicol and thiamphenicol has also been described (2), suggesting a role for the –CO-CHCl2 side chain present in both compounds in the formation of the nominal T-cell epitope(s), or the existence of T-cell clones recognizing chloramphenicol and thiamphenicol, respectively. Interestingly, it has been postulated that the nitro-groupof chloramphenicolis the structural feature causing chloramphenicol-induced aplastic anemia, probably by undergoing chloramphenicol reduction to the nitroso-chloramphenicol intermediate (9). It has been reported that nitroso-chloramphenicol has a damaging effect on DNA, whereas even large concentrations of chloramphenicol and thiamphenicol lacking the nitro-group were without effect (9). Since reactive metabolites play a crucial role in carcinogenesis and immunologic sensitization, either by forming DNA-adducts or by binding to proteins (10), formation of a nitroso derivative may lead to T-cell reactivity to the nitro-group of chloramphenicol. In the present investigation, no microsomes were added to the PBMC cultures. However, oxidating and reducing capacities have also been attributed to leukocytes in vitro (11), suggesting the possibility of reduction of the nitro-group by the PBMC in the cultures.
In addition to the significant lymphocyte proliferation, PBMC from patient 1 upon coculture with azidam-phenicol secreted relevant amounts of IL-5, but not of IFN-γ. A Th2-skewed cytokine profile of human drug-specific T cells has also been reported in systemic drug allergies (12, 13). Thus, it is tempting to speculate that the molecular mechanisms leading to systemic exan-thems, as well as to allergic contact dermatitis upon challenge with distinct drugs, such as the chloramphen-icol derivatives, may be similar.