Multiple reaction monitoring cubed for protein quantification at the low nanogram/milliliter level in nondepleted human serum.

Mass spectrometry-based strategies for the quantification of low-abundance putative protein biomarkers in human blood currently require extensive sample fractionation steps which hamper their implementation in a routine and robust way across clinical laboratories. We demonstrate that a technique using MS(3) reconstructed chromatograms on a signature of secondary ions issued from a trapped primary product ion, termed multiple reaction monitoring cubed (MRM(3)), enables targeting protein biomarkers in the low nanogram/milliliter range in nondepleted human serum. The simple two-step workflow is based on a trypsin proteolysis of whole serum (100 microL) followed by enrichment of targeted proteotypic peptides on a solid phase extraction column using mixed-cation exchange resin. MRM(3)'s fidelity of peak detection extends the dynamic range and limit of quantitation (LOQ) of protein biomarkers to the low nanogram/milliliter range, corresponding to a concentration that is 10(6)-fold lower than the concentration of the most abundant proteins in serum. The power of the MRM(3) method is illustrated by the assay of prostate specific antigen in nondepleted human sera of patients. The results correlate well with the established method for determining PSA levels in serum, i.e., enzyme-linked immunosorbent assay (ELISA) tests.

Tritiated thymidine incorporation does not enhance sensitivity of the popliteal lymph node assay.

The popliteal lymph node (PLN) assay has been proposed as a tool to predict drugs and chemicals with the potential to induce systemic autoimmune reactions in man. In this assay, weight and cellularity indices typically are the measured endpoints. The present study was conducted to test whether incorporation of tritiated thymidine could improve sensitivity of the PLN assay. Male and female Balb/c mice were injected with 20 microCi of [3H]-methyl-thymidine intravenously 7 days after receiving 0.5, 1 or 2 mg of diphenylhydantoin, streptozotocin, sulfamethoxazole, ofloxacin, phenobarbital, or metformin intradermally. Results obtained with incorporation of tritiated thymidine were compared to weight indices. No consistent or marked differences in these endpoints were noted whatever the compound used. This study shows that incorporation of tritiated thymidine does not improve sensitivity of the PLN assay.

[Anaphylactic shock associated with ceftriaxone therapy in a newborn]. / Choc anaphylactoïde à la suite d'un traitement par ceftriaxone chez un nouveau-né.

CASE REPORT: During an hospitalization, a ten-day-old newborn infant was treated with ceftriaxone (Rocephine i.v., 390 mg/day) for an infection secondary to the presence of an umbilical catheter. A few minutes after the end of the fifth injection, the infant presented with cyanosis, initially localized at the perfusion site, then generalized, a tachycardia followed by acute circulatory failure with arterial hypotension and finally a multiple organe failure with coagulation, kidney and liver dysfunction. The infant received classical resuscitation treatment and recovered without short term sequelae. The time of onset was in favour of drug-induced accident. A postnatal sensitization during previous injections might have occurred, although the latency of immediate hypersensitivity reactions after a first sensitizing contact is usually longer. A sensitization in utero or via breast feeding was ruled out due to the absence of maternal exposure to ceftriaxone. The absence of urticaria and bronchospasm, and the initial localization of cyanosis were not in favour of a classic allergic disease. An other cause, toxic or infectious cannot be ruled out.

Adverse effects of immunotherapeutics involving the immune system.

Immunotherapeutics are pharmaceutical products intended to modify immune functions either directly or indirectly by influencing physiological systems that affect immunological functions. They include conventional immunosuppressants, monoclonal antibodies, recombinant cytokines, gene therapy products or therapeutic vaccines. A variety of adverse effects involving the immune system have been described in laboratory animals as well as in the clinic. Some of these adverse effects can be predicted, at least to some extent, from the immunopharmacological profile of these drugs, but a number of unpredicted adverse effects have been described. Immunosuppression, allergy, autoimmunity, immunoactivation are the major adverse consequences to be expected as illustrated by this overview of the leading immunotherapeutics already used in the clinic. These untoward and potentially severe consequences support the need for a careful preclinical and clinical evaluation of the immunotoxicity of these products.

[Cutaneous adverse drug reactions: enhanced imputation score by skin testing]. / Réactions cutanées médicamenteuses: la réalisation de tests cutanés augmente le score d'imputabilité

INTRODUCTION: A careful diagnosis and the identification of the causative drug after a cutaneous adverse reaction can avoid relapses. Skin tests facilitate the identification of the causative molecule by producing a hypersensitivity reaction at the application site. Adverse drug reactions are reported to Pharmacovigilance Centres who determine the imputation score of the suspected drugs. The aim of this study was to assess to what extent skin testing after a suspected allergic drug reaction can be helpful to identify the causative drug and whether an impact on the final imputation score could be evidenced. METHOD: A 18-month prospective study was performed. All patients with a history of cutaneous adverse drug reaction of suspected immunoallergic origin were included provided skin tests could be performed within 6 to 12 weeks after the adverse drug reaction. The imputation score was determined using the French imputation method prior to and after skin testing. RESULTS: Thirthy-nine patients were included in the study. Positive skin tests were observed in 11 of 20 patients with a C2S2 (I2: plausible) initial imputation score and in 6 of 15 patients with a C2S1 (I1: doubtful) initial imputation score. One patient with a C1S1 (I1: doubtful) initial imputation score had positive skin tests. DISCUSSION: The results of skin tests helped change the imputation score of the suspected drug in 18 patients out of 39. In 55 p. 100 of cases, the imputation score was increased from C2S2 (I2) to C2S3 (I3: likely) and from C2S1 (I1) to C2S3 (I3) in 40 p. 100 of cases. The increase in imputation scores was helpful to improve warning signals after an immunoallergic reaction. Skin tests led to a more accurate diagnosis in 50 p. 100 of cases. Thus, more adequate advices for further drug treatment were possible, particularly in avoiding the irrelevant prohibition of innocent drugs.

Induction of delayed-type hypersensitivity to sulfamethoxazole in mice: role of metabolites.

Although cutaneous adverse drug reactions (ADRs) are relatively frequent and potentially severe, their mechanisms are poorly understood and no validated predictive experimental model is available. Sulfamethoxazole (SMX) is commonly used to treat infections in HIV-positive patients and severe cutaneous ADRs have been described. This study was undertaken to test whether sensitization to SMX could be achieved in mice using a combination of in vivo and in vitro endpoints. No delayed-type hypersensitivity (DTH) response could be evidenced following SMX injection in the back and subsequent challenge into the footpad or onto the ear. Pretreatment with the enzymatic inducers phenobarbitone and betanaphtoflavone, or depletion in CD4(+) T-lymphocytes were not successful either. In contrast, the injection of SMX/S9 mix in the back and challenge with SMX/S9 mix induced a significant increase in footpad thickness. A significant proliferation of spleen cells from SMX- or SMX/S9 mix-treated mice was evidenced following incubation with SMX/S9 mix, but not SMX alone. This study provides indirect evidence that SMX metabolites are involved and confirms previous in vitro results obtained with lymphocytes from patients with a history of SMX-induced ADRs cultured with murine microsomes. Further investigations using other drugs known to induce similar ADRs are warranted to establish the predictive value of this murine model.

Drug allergy diagnosis in humans: possibilities and pitfalls.

Due to the potential hazards of drug allergies, an early and reliable diagnosis is crucial. The use of in vivo tests is not recent but, because of the hazards of skin testing in patients with a history of anaphylaxis, they had been abandoned for a while. Recent reevaluations have shown that for some drugs, e.g. antibiotics-reliable skin tests can ensure the diagnosis of drug allergy in up to 70% of cases. Many in vitro tests based on well-defined mechanisms, e.g. the basophil degranulation test have been used for the diagnosis of totally unrelated allergic mechanisms. It is almost impossible to interpret their validity as diagnosis tools. Nevertheless, other tests, e.g. the lymphocyte transformation test which have been evaluated in well-conducted recent studies, seem to have a good predictive value. Their use is still restricted to clinical trials or research studies. A reliable clinical approach as well as a detailed examination of the drug intake remains obligatory to diagnose drug allergy. Available in vivo and in vitro tests are sometimes used to confirm the diagnosis. The sensitivity and specificity of these tests is evaluated in clinical studies. Research to improve the existing tests and to develop new diagnostic tools is still of paramount importance.

Gell and Coombs's classification: is it still valid?

The Gell and Coombs's classification divides drug allergies into four pathophysiological types, namely anaphylaxis (type I), antibody-mediated cytotoxic reactions (type II), immune complex-mediated reactions (type III), and delayed type hypersensitivity (type IV). Although this classification was proposed more than 30 years ago, it is still widely used. As only a limited number of drug allergies fit into this classification which does not include our current understanding of the immune response, its use is not recommended, particularly in the context of the preclinical safety evaluation of new therapeutic agents. In fact, three different situations can be identified, namely pseudo-allergic reactions, primarily antibody-mediated reactions and cell-mediated reactions, which could serve as a basis for modern and more adequate classifications

Responses of the immune system to injury.

Three categories of immunotoxic effects are identified: direct immunotoxicity, hypersensitivity, and autoimmunity. Direct immunotoxicity consists of immunosuppression and immunostimulation. Total abrogation of the immune response (immunosuppression) results in more frequent, severe, and often atypical and relapsing infections and lymphomas. Immunostimulation is associated with febrile reactions, the induction/facilitation of autoimmune diseases and allergic reactions to unrelated allergens, and impaired hepatic drug biotransformation. Hypersensitivity is manifested by a variety of symptoms involving either antigen-specific or non-antigen-specific humoral and cellular adverse responses. Autoimmune reactions are divided into organ-specific and systemic reactions. Because of the involvement of many redundant mechanisms, it is difficult to predict responses of the immune system to a given immunotoxic injury. In laboratory animals, histologic but also functional changes are necessary to show evidence of and to predict such adverse responses.

Increased production of interferon-gamma, but not IL-4 mRNA, by streptozotocin in the popliteal lymph node assay.

The popliteal lymph node (PLN) assay has been proposed as a tool to predict systemic autoimmune reactions induced by medicinal products and chemicals, the mechanisms of which are poorly understood. To determine whether PLN responses involved Th1 or Th2 cell control, or both, the effects of streptozotocin (STZ), a prototypic immunotoxic compound, were analysed on the production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) mRNA by lymph node cells after injection into the hind footpad of C57 BL/6 mice. Streptozotocin induced a dramatic increase in IFN-gamma mRNA production, which correlated with PLN responses as evidenced by augmented weight and cellularity indices. No effect on IL-4 mRNA synthesis was noted. These results suggest that a Th1 response is involved in the PLN response to STZ.

The popliteal lymph node response to streptozotocin is under type 1, MHC class-I restricted, CD8(+) T-cell control.

The popliteal lymph node (PLN) assay has been proposed to predict the 'autoimmunogenic' potential of xenobiotics. A better understanding of the processes involved in PLN responses is needed to establish the value of this assay for preclinical safety evaluation. In order to determine whether PLN responses involve CD4(+) or CD8(+) T-cells, the effects of streptozotocin (STZ), a prototypic immunotoxic compound, were analyzed after injection into the hind footpad of C57 BL/6 mice and major histocompatibility complex (MHC) class I or II deficient mice. The involvement of type 1 or type 2 cell control on the production of cytokine mRNAs was analyzed in lymph node cells by quantitative RT-PCR, together with the analysis of a wide range of cytokine mRNAs after STZ injection (IL-1alpha, IL-1beta, TNF-alpha, IFN-gamma, IL-2, IL-2 receptor, IL-4, IL-5, IL-6, IL-10 and IL-12). We have found that mice depleted in CD8(+) T-cells did not respond to STZ, whereas mice depleted in CD4(+) T-cells exhibited the expected positive PLN responses, with increased weight and cellularity indices. STZ induced a low production of interleukin (IL)-2 mRNAs, a mild increase in IL-1alpha and IL-6 mRNAs production, and a dramatic increase in IFN-gamma, IL-1beta, TNF-alpha, IL-12 and IL-2 receptor mRNAs, which correlated with positive PLN responses. No effects on IL-4, IL-5 and IL-10 mRNAs synthesis were noted. In CD8(+) T-cell deficient mice, there was no production of IFN-gamma or IL-6 mRNAs. These results suggest that PLN responses to STZ are under the control of type 1, MHC class-I-restricted, CD8(+) T-cells. This is in accordance to the known physiopathology of STZ-induced diabetes. Additional studies are necessary to establish the mechanism of CD8+ T-cell activation.

Endocrine and neurological adverse effects of the therapeutic interferons.

There is experimental evidence that the nervous central and the neuroendocrine systems can influence the immune system, which can in turn influence the brain activity. Endogenous cytokines are known to play a critical role in the pathophysiology of many diseases. The recently acquired experience on the adverse effects of therapeutic cytokines, particularly neurological and endocrine adverse effects, are further illustrative of these interferences. Interferons-alpha have been used in thousands of patients, so that the information accumulated with this group of closely related products is essential to delineate the potential and severity for non-immunological, but largely immune-mediated adverse effects to develop in patients treated with immuno-activating agents.

Value of animal models for predicting hypersensitivity reactions to medicinal products.

Although hypersensitivity reactions induced by medicinal products and chemicals are relatively common, few predictive models are available. A major difficulty is our currently limited understanding of the mechanisms involved, and efforts should be paid to better defining drug immunogenicity, hapten formation and immune effector mechanisms. A second difficulty is the multiplicity of clinical manifestations presumably due to varying mechanisms. Available models can only predict a few of these reactions. Anaphylaxis models in guinea-pigs can be only used for the safety assessment of macromolecules which are neither humanized or of human origin, whereas guinea-pig or mouse models can detect the majority of human contact sensitizers. In addition to the extensive validation of existing models, promising avenues of research are expected to be found in the use of novel animal models, particularly those using genetically modified animals, such as transgenic and knock-out mice.

Increased levels of interleukin 5 are associated with the generation of eosinophilia in drug-induced hypersensitivity syndrome.

Hypersensitivity syndrome (HSS) usually refers to severe drug eruption associated with systemic symptoms and eosinophilia. Interleukin (IL)-5 regulates eosinophil counts with the help of IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF). Blood IL-5 levels have been reported to be increased in patients with eosinophilia secondary to parasitic infections or idiopathic eosinophilia, but have never been evaluated in drug-induced eosinophilia. The aim of our study was to determine whether IL-5, IL-3 and GM-CSF are involved in eosinophilia in patients with drug-induced HSS. Plasma levels of IL-3, IL-5 and GM-CSF were assayed by ELISA in seven patients with drug-induced HSS, in eight patients with cutaneous adverse drug reactions not associated with eosinophilia, and in five patients with eosinophilia unrelated to drug treatment. IL-5 levels were normal in all eight patients with drug eruptions without eosinophilia, and increased in five of the seven patients with HSS. In the latter patients, IL-5 levels peaked several days before highest eosinophil counts were noted, and returned to normal within a few days, even when eosinophilia persisted. In patients with eosinophilia unrelated to drug treatment, IL-5 levels, although significantly increased remained lower than in HSS patients. IL-3 and GM-CSF could not be detected in any group, at any time. Our results show that IL-5 is involved in drug-related eosinophilia. As IL-5 production was only involved in the early stages of the reaction, it is suggested that IL-5 mainly derives from activated lymphocytes rather than eosinophils. Our results support the clinical relevance of previous in vitro findings. Further studies are needed to test whether assays of IL-5 production by lymphocytes of patients stimulated by the suspected drug and/or its metabolites, are useful in establishing causality in drug-induced reactions associated with eosinophilia.