Jean Francois Nicolas

Effector and regulatory mechanisms in allergic contact dermatitis

Allergic contact dermatitis (ACD), one of the commonest occupational diseases, is a T‐cell‐mediated skin inflammation caused by repeated skin exposure to contact allergens, i.e. nonprotein chemicals called haptens. Allergic contact dermatitis, also referred to as contact hypersensitivity, is mediated by CD8+ T cells, which are primed in lymphoid organs during the sensitization phase and are recruited in the skin upon re‐exposure to the hapten. Subsets of CD4+ T cells endowed with suppressive activity are responsible for both the down‐regulation of eczema in allergic patients and the prevention of priming to haptens in nonallergic individuals. Therefore, ACD should be considered as a breakdown of the skin immune tolerance to haptens. Recent advances in the pathophysiology of ACD have demonstrated the important role of skin innate immunity in the sensitization process and have revisited the dogma that Langerhans cells are mandatory for CD8+ T‐cell priming. They have also introduced mast cells as a pivotal actor in the magnitude of the inflammatory reaction. Finally, the most recent studies address the nature, the mode and the site of action of the regulatory T cells that control the skin inflammation with the aim of developing new strategies of tolerance induction in allergic patients.

T-cell recognition of chemicals, protein allergens and drugs: towards the development of in vitro assays

Chemicals can elicit T-cell-mediated diseases such as allergic contact dermatitis and adverse drug reactions. Therefore, testing of chemicals, drugs and protein allergens for hazard identification and risk assessment is essential in regulatory toxicology. The seventh amendment of the EU Cosmetics Directive now prohibits the testing of cosmetic ingredients in mice, guinea pigs and other animal species to assess their sensitizing potential. In addition, the EU Chemicals Directive REACh requires the retesting of more than 30,000 chemicals for different toxicological endpoints, including sensitization, requiring vast numbers of animals. Therefore, alternative methods are urgently needed to eventually replace animal testing. Here, we summarize the outcome of an expert meeting in Rome on 7 November 2009 on the development of T-cell-based in vitro assays as tools in immunotoxicology to identify hazardous chemicals and drugs. In addition, we provide an overview of the development of the field over the last two decades.

Corticosteroid cross‐reactivity: clinical and molecular modelling tools

To cite this article: Baeck M., Chemelle J.A., Goossens A., Nicolas J.F., Terreux R. Corticosteroid cross‐reactivity: clinical and molecular modelling tools. Allergy 2011; 66: 1367–1374.

Esomeprazole-induced DRESS syndrome. Studies of cross-reactivity among proton-pump inhibitor drugs

intervals of 1, 2 and 4 weeks between the sessions. The persistence of negative results of intradermal testing was confirmed before each desensitization procedure. Loss of skin sensitivity appeared to be a good predictor of tolerance of the drug. Both patients continued receiving cyanocobalamin injections monthly with good tolerance. Drug desensitization is time dependant and re-sensitization recurs after awhile.We have little information about the length of time the drug tolerance persists. It is thought to be in the range of weeks. According to our experience, with the above patients, it persists for at least 4 weeks. We did not asses a longer time interval and therefore would advise four weekly treatmentwith the therapeuticdose. Our patients showed cross sensitivity between cyanocobalamin and hydroxicobalamin therefore we had no substitute drug available. Cross sensitivity has previously been described although it is not always found (2, 3). In a patient with hydroxicobalamin allergy and a negative result to cutaneous testing with cyanocobalamin, this latter preparation may be tolerated. We have not been able to measure specific IgE in patients serum, though the mechanism is likely to be IgE dependant. We suspect this to be true as both sensitizations occurred after a period of tolerance with increasing severity of the reaction after each injection. We were able to confirm positive skin test results in our patients as compared with negative test results in control subjects. The preparation of cyanocobalamin we used did not contain benzyl alcohol preservative which has been involved in patient reacting to vitamin B12 injection (4). The allergen involved in vitamin B12 reactions is likely to be a hapten. We are indebted to Lucy Riddington, allergy specialist nurse, for technical support.

Physiopathologie de l’urticaire et approches thérapeutiques

L’urticaire (nom féminin dérivé d’urtica ou ortie) est un synrome cutanéomuqueux inflammatoire très fréquent, puisque 12 à 0 % de la population présente au moins un épisode d’urticaire au ours de son existence. Il s’agit d’une éruption papuleuse, érythéateuse, prurigineuse et fugace (moins de 24 heures d’évolution our chaque lésion). L’urticaire peut être superficielle (œdème ermique) ou profonde (angio-œdème ou œdème du tissu sousutané) et disparaît sans laisser de cicatrice ni de pigmentation ésiduelle. Toute éruption qui ne rentre pas dans cette définition st dite « urticariforme » mais ce n’est pas a priori de l’urticaire. La cellule clé de l’urticaire est le mastocyte ; son rôle physioloique est d’établir une première ligne de défense sous-épithéliale ontre les microorganismes pathogènes et les parasites pénétrant ar cette voie. L’activation des mastocytes cutanés a schématiqueent trois conséquences (Fig. 1) : la dégranulation brutale avec

IgE‐mediated allergy to pristinamycin: the value of skin tests and basophil activation tests

Allergy to pristinamycin (PMC), an antibiotic related to the macrolide family, is rare and the published cases exclusively concern delayed (T cell-mediated) allergic reactions where the skin tests [patch and intradermal tests (IDT)] are of good diagnostic value (1). We report three cases of IgE-mediated anaphylaxis to PMC where the diagnosis, highly suspected on the clinical symptoms, was confirmed by the positivity of both skin and immunobiological tests. A 60-year-old woman developed an anaphylactic reaction (hypotension, malaise, urticaria and angioedema) 15 min after first taking two PMC tablets for prevention of infectious endocarditis during dental treatment. The symptoms improved in a few hours following an adrenaline injection. A 50-year-old patient developed an anaphylactic reaction (malaise and hypotension associated with angioedema) 30 min after first taking two tablets of PMC for prevention of infectious endocarditis during dental treatment. The symptoms rapidly resolved after the injection of corticosteroids. A 45-year-old woman developed an anaphylactic reaction (malaise without hypotension associated with urticaria, angioedema and nausea) 15 min after first taking one tablet of PMC for cutaneous infection. The symptoms rapidly resolved after the injection of antihistaminics. Skin tests and immunological tests were performed 1–3 months after the reaction according to standard protocols (2, 3). Sterile solutions of PMC and related macrolides were prepared by the hospital pharmacy by dissolving the drugs in isotonic saline at a concentration of 50 mg/ml. Two series of tests were performed. The first one tested PMC alone and the relevant controls. Prick tests (50 mg/ml) and IDT (10 dilution of the prick solution, i.e. 50 lg/ml) to PMC were positive in the three patients but negative in two healthy nonPMC allergic individuals. Prick tests and IDT to the solvent were negative in the three patients, excluding a nonspecific positivity of skin tests to PMC. The second series of tests (prick and IDT) concerned five antibiotics of the macrolide family (spiramycin, roxithromycin, clarithromycin, telithromycin and erythromycin) to check for potential cross-reactivities. Skin tests with all the macrolides tested were negative in the three patients. The basophil activation test (BAT), analyzing the expression of CD203c on patients basophils by FACS (2), was positive for PMC in the three patients. The results are expressed as % of basophils expressing CD203c after in vitro PMC re-stimulation compared to % of basophils expressing CD203c without re-stimulation. The BAT was positive at 31% (control 2%), 69% (control 2%) and 70% (control 2%) for patients 1–3, respectively. The BAT was negative for the five antibiotics of the macrolide family. To our knowledge, this is the first report of immediate allergy to PMC. We demonstrate that both skin tests and BAT are of value for the diagnosis of immediate allergy to PMC.

Characterization of Flucloxacillin-specific CD8+ T-cells in a mouse model

Drug-induced liver injury (DILI) involving the adaptive immune system is a serious complication in both patient treatment and drug development. Flucloxacillin, a -lactam antibiotic effective against -lactamase-producing bacteria, is a common cause of immunological DILI through cholestasis. We have recently isolated CD8+ flucloxacillin-specific T-cells from patients with DILI, which indicates that T-cells participate in the pathogenesis of the disease. There are currently no animal models of DILI where the adaptive immune system has shown to damage liver, and therefore, it is difficult to explore the mechanistic basis of the tissue injury. Thus, the aim of this study was to use C57/Bl6 CD4 deficient mice with a mutation in the gene encoding for MHC class II molecules to explore the immunogenicity of flucloxacillin and whether flucloxacillin-responsive CD8+ T-cells damage hepatocytes. In initial experiments sensitization was achieved through epicutaneous application (1g/ml; in 70% DMSO; 50L). Proliferation, IFN- and granzyme-B secretion from draining lymph node cells was measured ex vivo following treatment with flucloxacillin. Flucloxacillin-specific CD8+ T-cell mediated killing of hepatocytes was measured using the ApoTox-Glo kit (Promega, UK). In separate experiments, flucloxacillin-specific T-cells were forced to migrate to the mesenteric lymph nodes using retinoic acid, prior to administration of oral flucloxacillin for 10 days, followed by analysis of plasma biomarkers of liver injury. CD8+ T-cells from draining lymph nodes of flucloxacillin-treated mice proliferated in a concentration-dependent manner following ex vivo secondary stimulation. The proliferative response was associated with IFN- and granzyme-B secretion. In contrast, proliferative responses and cytokine secretion was not detected with cells from vehicle control mice. Flucloxacillin-specific hepatocyte toxicity and apoptosis was observed when CD8+ T-cells were cultured with dendritic cells and flucloxacillin for 24h, washed and transferred to the hepatocyte cultures. Flucloxacillin sensitization followed by 10 day oral exposure resulted in marked gall bladder swelling and mild elevations in ALT. Other plasma biomarkers of liver damage were negative. In conclusion, these data show successful sensitization of mice against flucloxacillin. Thus, the C57/Bl6 CD4 deficient mouse represents a promising model to study the role of the adaptive immune system in DILI.

Skin tests may induce DRESS relapse

DRESS is a rare but life threatening drug allergic disease. The value of skin tests, especially patch-tests (PT) for defining the culprit drug were recently reported. Nevertheless DRESS has been associated with high risk of disease flare linked to drug rechallenge (culprit drug or not) or viral reactivation . In this study we analyzed the frequency of patch test-induced DRESS flares. Method Between 01/2009 and August 2013, patients with DRESS syndrome (diagnosis score KARDAUN >5 : definite case) were hospitalized for drug allergic evaluation and received skin patch tests (Patch tests and/or IDT) with 72h reading. In case of DRESS flare blood viral load (HHV6, EBV, CMV), white blood cells count , liver enzymes and kidney function were performed. Results 68 DRESS were included. Among them, 39 patients showed positive PT (57%) and 29 were negative at the 72h reading. From the 39 PT positive patients, 8 patients (20%) experienced a DRESS flare in the form of a mild skin rash developing in the 48h following the realization of skin tests. The rash started before the 72h reading in all cases. In two cases, general signs were noted and in one case eosinophilia was found (760/mm3 N 5. Liver and kidney assays were normal. Viral blood PCR for herpes viruses were negative at the onset of the relapse and during the two following days. The culprit drugs were dominated by betalactam antibiotics (4/8 : 50%). Skin biopsies of the skin rash were compatible with a delayed hypersensitivity reaction in all cases. Interestingly 4 patients (50%) were able to induce non specific rash after introducing drugs unrelated to the culprit one with negative blood herpes viral load. Conclusion Our study demonstrates that positive skin tests may induce a mild reactivation of DRESS in a noticeable proportion of patients. The delay between skin tests and flare (in all cases <48h) confirms that DRESS is a drug specific delayed hypersensitivity reaction. The negativity of viral load does not confirm the relation of DRESS flare with viral reactivation. Many hypotheses could explain the occurrence of skin test-induced DRESS rash in these patients : T reg dysfunction after DRESS syndrome , very strong T cell sensitization in these patients, long term persistence of drug in skin, T cell chronic pre-activation state, as suggested by the frequency of non specific DRESS flare following alternative drug administration. Clinicians should be aware of the risk of skin test-induced DRESS flares in order to optimize the management of these patients during the allergological work up.

Drug Reaction with Eosinophilia and Systemic Symptoms (DRESS): clinco-histopathologic studies of 45 cases

Results A facial edema in DRESS was associated with a definite case of DRESS (p=0.005) and a liver involvement (p=0.045). A younger age (50 years vs 66 years) was associated with a liver involvement (p=0.013). A severe neutropenia was associated with severe DRESS (p=0.012). Spongiosis and keratinocyte damage are the 2 most frequent epidermal changes noticed 55% and 53%. Pustules are not rare (26%). In the dermis a vascular alterations were frequent (88%) with a true leukocytoclastic vasculitis in 28%, a mild lymphocytic vasculitis in 13% and minor alterations in 44% of cases. In the dermis, there was a constant lymphocytic infiltrate mainly moderate in a perivascular distribution. In 57% the infiltrate is polymorphous with association of eosinophils neutrophil or atypical lymphocyte. In two third of cases, the changes are polymorphous and numerous, with no specific clear pattern. In the other one third of cases with few alterations, the presence of eosinophils, keratinocyte damage and spongiosis are usually present. A spongiosis was associated with non severe form of DRESS (p=0.041) and the absence of kidney involvement (p=0.023). A a higher of alterations per slides was associated a liver involvement (p=0.018). Our study do not support a prognostic value of keratinocyte damage.