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Biomedical subjects

A Magnan

Publications and source records attributed to A Magnan.

At least 37 records · Page 2Linked to original sources
PubMed2003

Study of cytokine gene expression in small cell samples: use in induced sputum.

Sputum examination is being increasingly used as a non-invasive method for the study of airway inflammation. However, the technical applications of sputum are still limited because of the small number of cells recovered. In attempt to extend applications of sputum examinations, we developed and standardised, the reverse transcriptase-polymerase chain reaction (RT-PCR), a sensitive and specific technique of detection of mRNA, in induced sputum samples. Total RNA were extracted from samples containing as few as 50 to 80,000 cells, using a phenol-chloroform extraction method. RT-PCR was successfully tested on beta-actin, interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and tumour necrosis factor-beta (TGF-beta) genes. This protocol provides a simple technique to extract total RNA from a few number of induced sputum cells. It permits the semi-quantitatively study of cytokine gene expression in airways with simple means.

Actins↗
PubMed2003

T cell activation, from atopy to asthma: more a paradox than a paradigm.

During the last 15 years, it was largely shown that allergic inflammation was orchestrated by activated Th2 lymphocytes, leading to IgE production and eosinophil activation. Indeed, Th2 activation was shown to be necessary to induce allergic sensitization in animal models. In humans, a Th2 skewing was shown in atopic children soon after birth. In asthma, descriptive studies showed that Th2 cells were more numerous in patients than in controls. In addition, during specific allergen stimulation, an increase of Th2 cells was described in most cases. According to this Th2 paradigm, it was proposed that early avoidance of microbial exposure could explain the increase of atopic diseases seen in the last 20 years in developed countries, as the "hygiene hypothesis". Recently, it was proposed that early exposure to lipopolysaccharide (LPS) could be protective against atopic diseases. However, it is well established that exposure to LPS can induce asthma symptoms, both in animals and humans, although it induces a Th1 inflammatory response. In addition, most infections induce asthma exacerbations and Th1 responses. Recently, some studies have showed that some Th1 cells were present in asthmatic patients, which could be related to bronchial hyperreactivity. There is therefore an "infectious paradox" in asthma, which contributes to show that the Th2 paradigm is insufficient to explain the whole inflammatory reaction of this disease. We propose that the Th2paradigm is relevant to atopy and inception of asthma albeit a Th1 activation would account at least in part for bronchial hyperreactivity and asthma symptoms.

Animals↗
PubMed2003

Hymenoptera ultra-rush venom immunotherapy (210 min): a safety study and risk factors.

BACKGROUND: In this study, which summarizes our last 5 years of experience, we evaluated the side-effects of ultra-rush venom immunotherapy and the possibility to define some risk factors for side-effects as age, Hymenoptera venom used for treatment, treatment phase, severity of prior insect sting reaction, concentration of skin test positivity, and level of specific IgE. METHODS: In our protocol on day 1, an initial venom dose of 0.1 microg was followed by 1, 10, 20 microg at 30-min intervals and then 30 and 40 microg at 60-min intervals. Patients who completed this protocol received two booster injections of 50 microg on day 15 and one of 100 microg on day 45. Subsequently, monthly 100 microg boosters were given. RESULTS: Fifty-one children (9.20 +/- 3.41 years) and 207 adults (40.62 +/- 14.00 years) underwent an ultra-rapid venom immunotherapy (ultra-RVIT). Single ultra-RVIT was administered to 195 patients: 69 with honeybee, 123 with yellow jacket and three with wasp venoms. Two venoms were injected into 59 patients: 42 with yellow jacket and wasp, 17 with yellow jacket and honeybee. Four patients received the three venoms. The frequency of immediate systemic reactions (SR) was not significantly different between adults and children: 11.2% vs. 10.8%. SR were experienced more frequently on day 1 (n= 33). They were uncommon on day 15 (n= 2) and on day 45 (n= 1). No late reactions have been observed. Honeybee venom induced significantly more SR (30%) vs. yellow jacket (3.2%) and wasp venom (6.1%). Among the 33 patients who experienced a SR on day 1, 24 had a reaction grade 1 or 2 and nine had a reaction grade 3 or 4. There is a significant risk for patients with a prior reaction grade 3 or 4 to experience a SR during venom immunotherapy (VIT). The strength of positive skin tests and the level of specific IgE were not related to an increased risk of SR (P= NS). CONCLUSION: Treatment with honeybee extract induced more SR than the treatment with yellow jacket and wasp venom. Doses increase phase on day 1 is risk factors for SR of ultra-RVIT, as well as the severity of the prior reaction. Age, degree of positive skin tests, and specific IgE are not risk factors.

Adolescent↗
PubMed2003

Assessment of T lymphocyte cytokine production in induced sputum from asthmatics: a flow cytometry study.

BACKGROUND: Asthma results from a bronchial inflammation in which Th2 lymphocytes play a pivotal role, as shown in invasive bronchial biopsies and broncho-alveolar lavages. Induced sputum (IS) is a non-invasive method of recovery of bronchial cells, which can be repeated in the same patients. However, lymphocyte activation has not been studied in IS to date, because of the low number of T cells recovered. Herein we took advantage of flow cytometry, a method suitable for the study of small cell populations, to assess T cell cytokine production in IS. OBJECTIVES: (1) To assess induced sputum T cell cytokine production by flow cytometry in asthmatic subjects and controls. (2) To compare the T cell cytokine production between symptomatic and non-symptomatic asthmatics. METHODS: Thirteen asthmatics and 19 controls were included. Sputum was induced by a hypertonic saline. Sputum cells were stimulated and intracellular IL-13 and IFN-gamma were detected in T cells by flow cytometry. RESULTS: Stimulation induced an increase of IL-13 and IFN-gamma production by T cells. This increase was higher in asthmatics. IL-13-producing T cells were increased in asthmatics after stimulation. In symptomatic asthma, IFN-gamma-producing T cells were in higher proportion than in controlled asthma. CONCLUSION: IS T cell cytokine production indicates a basic Th2 bias in asthma, accompanied during symptoms by a Th1-like activation. These results open the field for longitudinal studies of the variation of T cell activation in asthma.

Adult↗
PubMed2003

[Pathophysiology of the IgE-dependent reaction in respiratory allergy].

The IgE-dependent reaction is a specific type of inflammatory reaction occurring in allergic persons responding to an allergen. When the allergen penetrates the airways, it is captured by the antigen presenting cells and presented to CD4+ T cells. The most important conditions of antigenic presentation are the presence of IL-4 and not IL-12 at the presentation site and the implication of certain coreceptors determining T-cell differentiation to Th2 cells, which produce IL-4, IL-3, and IL-5. Il-4 and IL-3 induce IgE production by plasmocytes and IL-5 activates eosinophils. The specific IgEs of the allergens then bid to their high-affinity receptor on the surface of mast cells (sensitivization phase). If the allergens are still present or if there is an new exposure, coaggregation of high-affinity receptors via specific IgEs induces mast cell degranulation and release of vaso- and broncho-active mediators which lead to the acute signs of rhinitis or asthma (early phase of the immediate reaction). At the same time, activated eosinophils, attracted by IL-5 as well as chemokines, infiltrate the mucosa, producing basic proteins toxic for the epithelium. These cells are responsible for the late phase of the immediate allergic reaction, and during repeated or continuous exposure, are the cause of the chronic allergic disease.

Allergens↗
PubMed2002

Allergic vs nonallergic asthma: what makes the difference?

BACKGROUND: The aim of this work was to describe clinical similarities and differences between allergic and nonallergic asthmatics, notably concerning the nasosinusal involvement. METHODS: A total of 165 asthmatics (122 allergics and 43 nonallergics) and 193 controls (40 allergics and 153 nonallergics), recruited in the frame of EGEA study (Epidemiological study on the Genetics and Environment of Asthma, bronchial hyperresponsiveness and atopy), were included. Asthmatics were included on the basis of positive answer to four standardized items. To establish differences and similarities between allergic and nonallergic asthmatics, general characteristics (age, sex, smoking habits, history of hay fever and allergic dermatitis), history of asthma, severity and nasosinusal involvement were examined. Clinical assessment was based on the answers to a detailed questionnaire, and spirometry. RESULTS: Greater age, female sex, sinusal polyposis, and FEV1 below 80% of the predicted value increased the risk of displaying a nonallergic type of asthma, whereas history of hay fever, seasonal exacerbation of asthma, and asthma duration lowered this risk. Unexpectedly, we found no difference in terms of rhinitic symptoms between both groups, probably resulting from distinct causes. CONCLUSION: These results give new insights into the contrasts between clinical features of allergic and nonallergic asthma. The terminology of extrinsic asthma was first introduced by Rackeman in 1947 (1) and referred to the triggering role of allergens in asthma. By symmetry, he described intrinsic asthma as a disease characterized by later onset in life, female predominance, higher degree of severity, and more frequent association to nasosinusal polyposis. As these asthmatics were not improved by conventional treatment, this author considered their disease as caused by a nonallergic, unknown phenomenon. It is now widely admitted that nonallergic asthma can be objectively distinguished from allergic asthma based on negative skin tests to usual aeroallergens. On the other hand, positive skin test shows a tendency to produce IgE antibodies in response to low doses of allergens. "Atopy" and "atopic" are the terms used to describe this clinical trait and predisposition (2). Allergic clinical manifestations of atopy are of various types, for example rhinitis and asthma. Nowadays the terminology of "extrinsic" and "intrinsic" asthma should no longer be used, and should be replaced by the terminology of "allergic" or "nonallergic" asthma (2).

Adult↗
PubMed2002

[Induced sputum as a new approach of the assessment of inflammation in asthma].

Asthma is an inflammatory condition. Traditionally bronchoalveolar lavage and bronchial biopsies obtained by bronchoscopy have been used to demonstrate inflammation. Induced-sputum is a non-invasive, reliable, reproducible and safer technique for monitoring inflammatory activity in patients with asthma. Studies have shown that induced-sputum measures aspects of inflammation distinct to that measured by bronchoalveolar lavage or bronchial biopsies. Numerous studies have suggested that induced-sputum is a potentially useful tool for early diagnosis of exacerbation, monitoring of therapy, identification of the lowest effective dose and assessing compliance in asthmatics. In this respect, we suggest that this test can be routinely used in the management of difficult asthmatics.

Asthma↗
PubMed2001

T cell development and T cell responses in mice with mutations affecting tyrosines 292 or 315 of the ZAP-70 protein tyrosine kinase.

After stimulation of the T cell receptor (TCR), the tyrosine residues 292 and 315 in interdomain B of the protein tyrosine kinase ZAP-70 become phosphorylated and plausibly function as docking sites for Cbl and Vav1, respectively. The two latter proteins have been suggested to serve as substrates for ZAP-70 and to fine-tune its function. To address the role of these residues in T cell development and in the function of primary T cells, we have generated mice that express ZAP-70 molecules with Tyr to Phe substitution at position 292 (Y292F) or 315 (Y315F). When analyzed in a sensitized TCR transgenic background, the ZAP-70 Y315F mutation reduced the rate of positive selection and delayed the occurrence of negative selection. Furthermore, this mutation unexpectedly affected the constitutive levels of the CD3-zeta p21 phosphoisoform. Conversely, the ZAP-70 Y292F mutation upregulated proximal events in TCR signaling and allowed more T cells to produce interleukin 2 and interferon gamma in response to a given dose of antigen. The observation that ZAP-70 Y292F T cells have a slower rate of ligand-induced TCR downmodulation suggests that Y292 is likely involved in regulating the duration activated TCR reside at the cell surface. Furthermore, we showed that Y292 and Y315 are dispensable for the TCR-induced tyrosine phosphorylation of Cbl and Vav1, respectively. Therefore, other molecules present in the TCR signaling cassette act as additional adaptors for Cbl and Vav1. The present in vivo analyses extend previous data based on transformed T cell lines and suggest that residue Y292 plays a role in attenuation of TCR signaling, whereas residue Y315 enhances ZAP-70 function.

Animals↗
PubMed2001

[Asthma and allergy].

Allergy is routinely taken into account in asthma. The allergic reaction is based on Th2 lymphocytes specific to the allergen, which induce production of E immunoglobulins (IgE) and activation of eosinophils. In atopic asthma, domestic allergens are more often responsible than pollen. They play a part early in the development of the disorder, and subsequently as factors triggering exacerbations. Work-related asthma should always be suspected. History-taking should seek the relevance of allergens in the clinical manifestations, confirmed by skin tests, and if necessary assessment of specific IgE. Provocation tests should be used only diagnosis of work-related asthma. Removal of allergens should be suggested. Specific desensitisation may be indicated in order to decrease high-dosage drug treatment. Rhinologic treatment is part of patient management. Treatments being developed include the use of anti-IgE antibodies, anti-interleukin (IL)-5, soluble IL-4 receptors and recombinant allergens.

Allergens↗
PubMed2001

Venom immunotherapy induces monocyte activation.

BACKGROUND: Venom immunotherapy (VIT) is an efficient treatment of hymenoptera venom allergy. The mechanism of VIT is based on the induction of tolerance of allergen-specific Th2 cells. The mechanisms of this T cell modulation are unknown, and could depend on cytokines produced by other cell types such as interleukin (IL)-12, tumour necrosis factor (TNF)-alpha and IL-10 by monocytes. OBJECTIVE: To assess if VIT modifies the monocyte production of IL-12, TNF-alpha and IL-10 during the 45 first days of treatment. METHODS: Fourteen patients and seven controls were included. Blood samples were taken once in controls and at day (D)1, D30 and D45 of VIT in patients. Monocytes were isolated, cultured with and without lipopolysaccharide (LPS), and the culture supernatant was harvested. IL-10, IL-12 and TNF-alpha were assayed in supernatants by ELISA. RESULTS: Baseline cytokine levels were not statistically different between patients and controls. During treatment, an increase of spontaneous monocyte production of IL-12 and TNF-alpha was observed at D15 and D45. The production of IL-10 increased at D15 and D45 but not significantly. After LPS-stimulation, IL-12, TNF-alpha and IL-10 monocyte production was not modified by VIT. CONCLUSION: VIT induces a monocyte activation characterized by a delayed overproduction of IL-12 and TNF-alpha. These cytokines could be relevant to the inhibition of Th2 cells during VIT. Therefore, VIT-induced tolerance could depend not only on the specific action of venom antigens on T cells, but also on a secondary non-specific action of monocytes.

Adult↗
PubMed2001

Flow cytometric microsphere-based immunoassay: analysis of secreted cytokines in whole-blood samples from asthmatics.

The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P = 0.003) and less IFN-gamma (P = 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-gamma than that of atopic nonasthmatic subjects (P = 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.

Adult↗
PubMed2001

Cytokines, from atopy to asthma: the Th2 dogma revisited.

Asthma is a spreading condition in Western countries, in most cases in relationship with atopy. Atopy is defined by an individual predisposition to develop allergic diseases in response to environmental allergens. The atopic immune system is characterized by a Th2 deviation determined by genetic and environmental factors. Among these factors, the role of allergen exposure, dietary behavior, air pollution and early exposure to microbes is discussed. In asthma, a Th2 cell activation is evident, but is accompanied by a Tc1 cell activation. These Tc1 cells probably down-regulate Th2 cells, but are also relevant to the bronchial hyperresponsiveness characterizing asthma. We propose that Tc1 activation in asthma could be the link between allergy and bronchial hyperresponsiveness.

Adjuvants, Immunologic↗