Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer\'s disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar K(D) values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.

Persistent peripheral blood eosinophilia can be associated with a variety of diseases, ranging from parasitic infection to gastrointestinal disease to vasculitis to the hypereosinophilic syndrome (HES). Mucosal ulcerations are a variant presentation of the HES that appear to be markers for a mutation that characterizes a subgroup of patients with HES responsive to treatment with imatinib mesylate. We present a patient with peripheral blood eosinophilia and severe recurrent mucosal ulcers and discuss his presentation in the context of new information about the evaluation, disease progression, and treatment of HES.

OBJECTIVE: To study the importance of sensitisation to occupational allergens for the occurrence of asthma and rhinitis in bakers.
METHODS: This is a nested clinical case-referent study of bakers based on a cohort of Swedish former bakery students. Cases were asthmatic ( n=25) or rhinitic bakers ( n=20). Randomly selected bakers ( n=44) were referents. All subjects underwent skin prick tests (SPTs) with common allergens, flours, fungal alpha-amylase and the storage mite L. destructor. Indices of airway inflammation were assessed in serum and the nose.
RESULTS: Seven of the asthmatics and eight of the rhinitics reported onset of disease during bakery work. Flour SPTs were positive in 43% of the asthmatics or rhinitics vs 16% of referents. The corresponding figures for alpha-amylase were 29%, 25%, and 7%. The odds ratio, adjusted for atopy, for an SPT positive to flour or alpha-amylase for asthmatics with onset during bakery work was 5.8 (95% confidence interval 1.1-32), and 2.6 (0.4-16) for the corresponding rhinitics. The positive predictive value of sensitisation to flour or alpha-amylase in relation to a clinical diagnosis of asthma or rhinitis was 71%. Sensitisation to L. destructor was rare. The indices of airway inflammation were similar in cases and in referents.
CONCLUSIONS: Bakers\' asthma is associated with sensitisation to flour and/or alpha-amylase, atopy taken into account. A similar association was suggested in bakers\' rhinitis. Indices of airway inflammation were of low predictive value for detecting bakers\' asthma or rhinitis in this study.

A 50-year-old man had been well until three months earlier, when he felt general fatigue, and cutaneous rash with itching. Thereafter a general muscular weakness developed and the patient could not walk for a month. Four weeks before referral to our hospital, he had high fever and could not role over in the bed. On admission, the patient was able to walk. He had no skin rash. Neurologically, he showed mild weakness in proximal muscles. Hematologic examination showed mild eosinophilia and serum creatine kinase was mildly elevated. Needle electromyogram revealed a diffuse myogenic pattern in extremities. Eosinophilic myositis was diagnosed by a biopsy of the left calf muscle showing mild infiltration of eosinophilis which was identified using antibodies against eosinophilic granule protein.

The current knowledge on atopic dermatitis comes mainly from cross-sectional studies, which are not suited to establish time-courses or causal links in complex diseases. As an alternative approach, the method of longitudinal case analysis by the autoregressive integrated moving average (ARIMA) method has been introduced to investigate the pathogenesis of atopic dermatitis. The method was applied to the investigation of 2 patients suffering from severe and moderate atopic dermatitis. Disease activity, peripheral blood parameters (differential blood count, lymphocyte subpopulations, immunoglobulin E, eosinophilic cationic protein, soluble interleukin-2 receptor), mental stress and environmental factors were determined daily for 50 days. Both patients showed a positive correlation between CD4+ and CD25+ T-cells, a negative correlation between CD16/56+ natural killer cells and CD4+ T-cells, a negative correlation between eosinophils and polymorphonuclear leukocytes, and a time-shifted positive correlation of up to 2 days between scores quantifying mental stress and disease activity. A positive correlation between T-cells and polymorphonuclear leukocytes, CD4+ T-cells and the CD45RA+ subtype, as well as a negative correlation between stress and eosinophils, sports and eosinophils, and sports and disease activity were found only in one patient with more severe atopic dermatitis. In conclusion, longitudinal time-series analysis might represent an interesting and adequate method to generate and test new hypotheses on biomedical problems which cannot be addressed by cross-sectional studies.

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Objective improvement in the diagnosis of immuno-allergology can only be obtained by application of reliable and reproducible immuno-biological methods, but until now, only measurements of total and specific IgE can be used and this has certain limitations. Presence of specific serum IgE, correlated with skin tests, favours a sensitization and implies nothing about the responsibility of the allergens. This is why we must consider if a definite improvement of diagnostic methods can be obtained by measurement of mediators. From an observation of food allergy to pork meat, we now show that it is possible to use sequential measurements of the mediators plasma histamine and urinary methylhistamine, ECP and serum tryptase to refine the diagnosis and provide proof of the responsibility of the food allergen. We report here a didactic observation which is characterised by reproducibility and specificity of the measurements. It illustrates the progress in diagnostic methods in allergy: we give a statistical diagnosis by measurement of antibodies and a dynamic diagnosis by measurement of mediators.

A 53-year-old man underwent chemotherapy (CDDP, VDS, MMC) for treatment of lung cancer. He was given 125 micrograms/m2 of GM-CSF subcutaneously every day for 8 consecutive days, in order to prevent neutropenia. Three days after starting GM-CSF therapy, marked eosinophilia in peripheral blood was observed. The maximum eosinophil count was 89% of leukocytes. Nine days after stopping the treatment with GM-CSF, the number of eosinophils had normalized spontaneously. There were no clinical symptoms except for slight fever, up to 37.5 degrees C. Moreover, there was no relationship between the number of eosinophils and the serum levels of cytokines (IL-3, IL-5, GM-CSF), although we observed minimal but significant elevation of serum ECP level. This case indicates that GM-CSF may induce marked eosinophilia rather than widely stimulating granulocytes and monocytes.

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