Mugwort-fennel-allergy-syndrome associated with sensitization to an allergen homologous to Api g 5.

BACKGROUND: The cross-reactive allergen responsible for the so called "mugwort-celery-spice-syndrome", a pollen-food allergy that occurs in a minority of mugwort pollen-allergic patients, is still undefined. OBJECTIVE: To identify the allergen responsible for the cross-reactivity between mugwort pollen and plant-derived foods. METHODS: The serum from one index patient with both fennel and mugwort pollen allergy was used to identify IgE-reactive allergens by direct ELISA and Immunoblot analysis. Cross-reactivity between mugwort pollen and fennel was checked by cross-inhibition experiments. Fennel and mugwort allergens selected on the basis of IgE reactivity and inhibition tests were excised from SDS-PAGE gels and microsequenced. The amino acid sequences obtained were used to screen the NCBI database using the protein BLAST software. RESULTS: On ELISA inhibition experiments, serum absorption with fennel extract completely inhibited the IgE response to mugwort. On immmunoblot analysis periodate treatment caused the disappearance of all bands of IgE reactivity except one at about 60 kDa. The 60 kDa bands from both mugwort and fennel PAGE-SDS gels revealed the presence of distinct proteins. The N-terminal amino acid sequencing gave the same major amino acid sequence corresponding to an Api g 5-like allergen. The MS/MS spectra were analyzed and a provided evidence of a fennel-specific protein with sequence similarity to phosphoglyceromutase from Apium graveolens. CONCLUSION: A 60 kDa allergen, highly homologous to Api g 5, was recognized in fennel by patient's IgE. Inhibition experiments showed a high degree of cross-reactivity between this fennel allergen and the homologous mugwort pollen allergen. This allergen might be responsible for the mugwort-celery-spice syndrome.

Cellular and molecular immune responses of the sea bass (Dicentrarchus labrax) experimentally infected with betanodavirus.

Naïve sea bass juveniles (38.4 + or - 4.5 g) were intramuscularly infected with a sublethal dose of betanodavirus isolate 378/I03, followed after 43 days by a similar boosting. This infection resulted in an overall mortality of 7.6%. At various intervals, sampling of fish tissues was performed to investigate: i) B and T lymphocyte content in organs and tissues; ii), proliferation of leucocytes re-stimulated in vitro with inactivated virus; iii) presence of serum antibody specific for betanodavirus; iv) expression of genes coding for the following immunoregulatory molecules involved in innate and acquired responses: type I IFN, Mx, IL-1, Cox-2; IL-10, TGF-beta, TCRbeta, CD4, CD8alpha, IgM, by using a quantitative PCR array system developed for sea bass. The obtained results showed a detectable increase of T cells and B cells in PBL during betanodavirus infection. Furthermore, leucocytes obtained from blood, head kidney, and gills showed a detectable "in vitro" increase in viability upon addition of inactivated viral particles, as determined by measuring intracellular ATP concentration. ELISA analysis of sera showed that exposure to nodavirus induced a low, but specific antibody titer measured 43 days after infection, despite the presence of measurable levels of natural antibody. Finally, a strong upregulation of genes coding for type I IFN, Mx, and IgM was identified after both infection and boosting. Interestingly, an upregulation of Cox-2 until boosting, and of TGF-beta and IL-10 after boosting was also observed, while the other tested genes did not show any significant variations with respect to mock-treated fish. Overall, our work represents a first comprehensive analysis of cellular and molecular immune parameters in a fish species exposed to a pathogenic virus.

Use of new "barrier socks" in contact allergic dermatitis.

We present the case of a fifty-year-old construction worker with contact allergic dermatitis in his feet. Given the limited results obtained with a costly topical therapy, we tried, for first time, using completely breathable "barrier socks", which solved the persistent problem in a matter of days. In addition to the improvement in the patient's quality of life and the renewed possibility of his wearing protective shoes, a net reduction in the costs incurred with topical therapy was also obtained.

Tear and serum soluble leukocyte activation markers in conjunctival allergic diseases.

PURPOSE: To measure markers of leukocyte activation in patients with an exclusively ocular inflammatory or bacterial disease. METHODS: Neutrophil myeloperoxidase, eosinophil cationic protein, eosinophil neurotoxin, and soluble interleukin-2 receptor were measured in serum and tears of 17 patients with allergic vernal keratoconjunctivitis, seven with atopic keratoconjunctivitis, 11 with seasonal allergic conjunctivitis, seven with giant papillary conjunctivitis, 13 with rosacea blepharokeratoconjunctivitis, seven with bacterial conjunctivitis, and 13 normal subjects as controls. RESULTS: In serum of patients with vernal and atopic keratoconjunctivitis, levels of eosinophil cationic protein, eosinophil neurotoxin, and interleukin-2 receptor were significantly increased compared with control subjects but were not correlated with the severity of ocular symptoms. In tears of patients with vernal and atopic keratoconjunctivitis and seasonal allergic conjunctivitis, as well as in the nonallergic diseases, rosacea blepharokeratoconjunctivitis and bacterial conjunctivitis, levels of eosinophil cationic protein, neurotoxin, and interleukin-2 receptor were significantly increased compared with control subjects. The highest values of these markers were found in vernal keratoconjunctivitis samples. Neutrophil myeloperoxidase was significantly increased in vernal and atopic keratoconjunctivitis, rosacea blepharokeratoconjunctivitis, and bacterial conjunctivitis. In vernal keratoconjunctivitis, tear markers were correlated to the clinical score of the disease, but not with cytology. CONCLUSIONS: Tear histamine was measured in 10 allergic patients after allergen challenge. Although none of the above markers can be considered specific to a single disease, their measurement may still be useful for the quantification of local cell activation in ocular inflammatory diseases.

Viral encephalopathy and retinopathy of farmed marine fish species in Italy.

Viral encephalopathy and retinopathy, otherwise known as fish encephalitis or viral nervous necrosis (VNN), is an emerging problem in several farmed marine fish species in various geographic areas all over the world. Since summer 1995, heavy losses affecting mainly juvenile and adult sea bass (Dicentrarchus labrax) have been observed in several on-growing facilities in Italy. Dying fish show abnormal swimming behaviour and, at temperatures higher than 20-22 degrees C, mortality rates range between 15 and 50%. Neither significant external nor internal gross pathological signs, except frequent abnormal swim bladder hyperinflation, were detected. Histological investigations reveal vacuolations in the grey matter of the brain and spinal cord and in the granular layers of the retina. Serial tissue sections examined by an immunohistochemical method carried out with antisera against fish nodaviruses showed a positive reaction. Additionally, spherical virus-like particles 22-25 nm in diameter were detected by electron microscopy in negative stained preparations of brain tissues, and the same samples gave a positive reverse transcription-polymerase chain reaction (RT-PCR) for the T4 region of the fish nodavirus gene. These results indicate that both juvenile and adult sea bass subject to mass mortality in Italy since summer 1995 are infected with a fish nodavirus and strongly suggest that the identified virus is the cause of the observed mortality.

Measurement of specific immunoglobulin E: intermethod comparison and standardization.

Recently introduced "second-generation" techniques for specific IgE measurement have produced some analytical improvement, offering better clinical sensitivity than previous techniques. The aims of our study were to compare the analytical and clinical performances of four second-generation techniques for allergen-specific IgE measurement in serum and to ascertain whether the new system for reporting quantitative results contributes to greater clinical agreement between findings using the techniques considered. Allergen-specific IgE was measured using the CAP System, CARLA, ENEA, and AlaSTAT, and the findings were compared. A significant disagreement was found between CAP and ENEA for all allergens and between CAP and CARLA for D1 and G5. However, the clinical discrepancies were reduced by selecting method-specific thresholds using ROC analysis. Second-generation techniques enable us to obtain better standardization of results; however, the identification of a specific threshold appears to be a prerequisite for the appropriate clinical interpretation of the test findings.

Procollagens and inflammatory cytokine concentrations in tarsal and limbal vernal keratoconjunctivitis.

To quantify the presence of inflammatory/fibrogenic cytokines and procollagens type I (PICP) and III (PIIIP) in active and non-active tarsal and limbal forms of vernal keratoconjunctivitis (VKC), tear and blood samples were collected from 27 VKC patients (20 active and 7 non-active) and 15 normal subjects. Upper tarsal conjunctival biopses were obtained from 8 controls and 8 tarsal VKC patients. From biopses of 4 tarsal VKC, fibroblasts were cultured in F12 medium with 10% FCS. TGF-beta 1, IL-1 alpha, IL-1 beta, IL-6 and TNF-alpha, PICP and PIIIP were measured in: (1) tears, (2) homogenized conjunctival tissues, (3) serum, (4) supernatants of tissue cultures at 24 hr, and fibroblast primary passage cultures. Results showed: (1) in tears, TGF-beta 1 and TNF were identified in several active VKC patients without significant differences between the tarsal and the limbal forms. IL-1 beta (27 +/- 51 pg ml-1, P = 0.03) and IL-6 (28 +/- 43 pg ml-1, P = 0.006) were significantly increased in tarsal VKC compared to controls. Both control and non-active VKC tear samples had undetectable levels of all of the above cytokines. PICP and PIIIP were significantly increased in tarsal VKC compared to both limbal VKC and controls. Non-active VKC levels were similar to controls. (2) In homogenized VKC tissues, TGF-beta 1 and IL-6 were both significantly increased compared to controls (P < 0.01) while no increases were observed in IL-1 and TNF-alpha. (3) In serum, IL-1 alpha, IL-1 beta and TNF-alpha were higher in VKC patients compared to controls. (4) In vitro fibroblasts from VKC patients showed an increased production of TGF-beta 1, IL-1 alpha, IL-1 beta, IL-6, TNF-alpha, PICP, and PIIIP over time. Increased levels of TGF-beta 1, IL-1 and IL-6 in VKC tissues and tears indicate a local production of these cytokines in active VKC. Collagen hyperproduction occurs only in active tarsal VKC and may be related to high levels of TGF-beta 1, IL-1 and IL-6. Increased serum levels of IL-1 and TNF-alpha suggests that systemic immunological changes occur in VKC. Cell culture can be used as a model to further study the pathogenesis of VKC and its characteristic local fibroblast activation.

Effect of lodoxamide and disodium cromoglycate on tear eosinophil cationic protein in vernal keratoconjunctivitis.

AIM: To validate the use of tear eosinophil cationic protein (ECP) as a marker for eosinophil activation, and its pharmacological modulation, in addition to evaluating the efficacy of lodoxamide and sodium cromoglycate in the treatment of vernal keratoconjunctivitis (VKC). METHODS: Tears were collected from 30 patients affected by active mild to moderate VKC before and after therapy with disodium cromoglycate 4% (DSCG) (n = 15) or lodoxamide 0.1% (n = 15) for 10 days. Tear cytology and ECP measurement were performed, and ocular signs and symptoms evaluated. RESULTS: While statistically significant changes did not occur after DSCG therapy, mean tear ECP increased from 343 (SD 363) micrograms/l to 571 (777) micrograms/l due to marked elevation in six eyes. The clinical score in DSCG eyes did not improve. After lodoxamide therapy, both clinical signs and symptoms, and tear ECP levels (560 (756) micrograms/l to 241 (376) micrograms/l) decreased significantly (p < 0.0001 and p < 0.01, respectively). Compared with DSCG treatment, lodoxamide was more effective in reducing signs and symptoms (p < 0.005). ECP levels were significantly correlated with signs, symptoms, corneal involvement, and number of eosinophils in tears (p < 0.0001). CONCLUSIONS: In patients with VKC, lodoxamide significantly reduced ECP tear levels, and thus, eosinophil activation, and was more effective than DSCG in reducing clinical signs and symptoms.

Clinical evaluation of a new quantitative method for specific IgE antibodies.

We studied the analytical and clinical efficiency of a "second generation" technique for the in vitro determination of specific IgE, the so-called CARLA system (Capture Assay Radim Liquid Allergen). Reproducibility studies demonstrated satisfactory intra- and inter-assay analytical imprecision. The linearity test gave good results, and no significant interference by non-specific total IgE was found. The diagnostic efficiency of the system for the five most representative aeroallergens (D1, Dermatophagoides pteronyssinus; E1, Cat's epithelium; G5, Lolium perenne; W19, Parietaria officinalis and W5, Arthemisia absinthium) was evaluated using the Receiver Operating Characteristic (ROC) analysis in eighty-nine patients with suspected inhalant allergies. For each aeroallergen the area under the ROC curve and the most efficient threshold were calculated. Satisfactory areas under the ROC curve were found for D1, G5, E1 and W19, while a lower diagnostic efficiency was observed for W5. The most efficient thresholds differed from one allergen to another, although the differences were not great.

Receiver-operating characteristic (ROC) curves: a fundamental tool for improving the clinical usefulness of in vitro IgE tests.

In order to establish the most efficient thresholds for serum allergen-specific IgE measured by the Pharmacia CAP System, a "second-generation" in vitro method, we evaluated results from 89 subjects with suspected inhalant allergies, using receiver-operating characteristic (ROC) curve analysis. Sera samples were analyzed by the Pharmacia CAP System for specific IgE, the choice of allergens to be tested being based upon the symptoms and clinical history of each patient. Results were analyzed by ROC analysis for the five most representative allergens, cat dander (e1), Dermatophagoides pteronyssinus (d1), Lolium perenne (g5), wall pellitory (W19), and wormwood (w5). The areas under the ROC curves were found to be satisfactory, ranging from 0.931 (e1) to 0.974 (g5) when we excluded w5, which had a significantly smaller area (0.81). To establish the most efficient threshold for each allergen, we calculated the clinical sensitivity, specificity, efficiency, and negative and positive predictive values. The thresholds giving a higher diagnostic efficiency were as follows: 0.40 kUa/l for d1, 0.55 kUa/l for g5, 0.50 for e1, 0.65 kUa/l for w19, and 1.00 kUa/l for w5. It is concluded that quantitative reporting of specific IgE measurement has numerous advantages, but the choice of the positive threshold seems to be a prerequisite for obtaining the optimal clinical efficiency. It is also suggested that a specific threshold should be adopted for each allergen.

Multicentre evaluation of Capture Assay Radim Liquid Allergen for measurement of specific IgE antibodies.

A multicentre trial of Capture Assay Radim Liquid Allergen was performed to define the sensitivity, specificity and clinical reliability of the system in diagnostic allergology. The results of the evaluation were compared with clinical data and in vivo testing. Good agreement was obtained for Dermatophagoides pteronyssinus (D1), Cat's epithelium (E1), Betula verrucosa (T3) and Olea europea (T9), Artemisia vulgaris (W6) and Parietaria officinalis (W19). Some spreading of data was observed for Artemisia absinthium (W5), Cynodon dactylon (G2), and Lolium perenne (G5). We found a high number of negative cases for Alternaria alternata (M6). The advantages offered by the system are the automation, the small quantity of serum requested, the supply of quantitative results in international units of specific IgE, the user-friendly software. The data are sufficiently reliable for the diagnostic system to be introduced into the clinical laboratory allergological routine.

Eosinophil cationic protein in tears of normal subjects and patients affected by vernal keratoconjunctivitis.

The objectives of this study were to determine: 1) levels of tear eosinophil cationic protein (ECP) in patients with vernal keratoconjunctivitis (VKC); 2) the effect of pharmacologic therapy on ECP release; and 3) the correlation of this mediator with the severity of the disease. Tears were collected from 10 controls and 20 VKC patients before and after therapy for cytologic analysis and ECP measurement by radioimmunoassay. Ocular signs and symptoms were evaluated before tear collection. Mean ECP levels in controls were 7.5 +/- 0.4 microgram/l, and in VKC patients, 988.3 +/- 128 micrograms/l before therapy (P < 0.001) and 566.3 +/- 121 micrograms/l after therapy (P < 0.005). In dexamethasone (Dex) 0.1% or cyclosporin A (CsA) 2% patients (five per group), tear ECP decreased significantly after 7-14 days of treatment. Disodium cromoglycate (DSCG) 4% (five patients) for 14 days did not significantly affect ECP levels. ECP levels were significantly correlated with allergic signs (P < 0.001), symptoms (P < 0.001), and the number of eosinophils in tears (P < 0.005). The results of this study suggest that tear ECP levels accurately reflect the clinical status of VKC patients. The measurement of ECP may prove useful not only in the diagnosis and monitoring of allergic disease, but also as an objective parameter for the evaluation of new antiallergic therapies.

Full automation in allergy testing: measurement of specific IgE by the ENEA System.

UNLABELLED: We evaluated the ENEA System, a fully automated instrument for the measurement of specific IgE antibodies. The instrument dispenses sera and reagents, incubates, washes, and reads and prints results automatically. The "core" of the instrument is the reactive unit called ACE (Allergy Chamber Enzymatic), which is a new solid phase to which the allergens are linked. The system uses calibration curves specific for the major allergen families, and data are supplied qualitatively (five classes) and quantitatively. We evaluated the analytic efficiency of the system and its correlation with the in vivo test (skin prick test (SPT)) results in 60 patients with inhalant allergic diseases and in 20 controls. RESULTS: 450 results were available within 4 h. A satisfactory within-run (CVs between 1.58 and 6.2%) and between-run (CVs 6.3-11.5%) precision was found. No significant carry-over was observed. A wide linearity of the assay was demonstrated. With the concordance between the clinical history and SPT as the reference value, the clinical sensitivity of the ENEA System was 84.1%, the specificity 82.8%, and the overall efficiency 83.4%. Finally, a good agreement with the results of another technique for the in vitro measurement of specific IgE (Pharmacia CAP System) was proven.

Clinical efficiency of in vitro and in vivo tests for allergic diseases.

BACKGROUND: Specific serum IgE determination is widely used in the diagnosis of IgE-mediated allergic diseases but the relative merits of in vitro measurement of IgE antibody in comparison to in vivo skin tests are still debated. OBJECTIVE: The aim of this study was to investigate the clinical efficiency of a "second generation" technique for in vitro analysis of IgE antibody (Pharmacia CAP System). METHODS: Eighty-six patients with suspected inhalant and/or food allergies and 20 asymptomatic subjects for a total of 655 tests were evaluated. Sera with divergent results between in vitro and in vivo techniques were further analyzed by using ImmunoCAP inhibition and immunoblotting. For the calculation of sensitivity and specificity of both in vitro and in vivo tests we considered as true value (reference value) either the concordant results or, in case of discordance, the datum confirmed by ImmunoCAP inhibition or immunoblot (ie, vitro positive, vivo negative, ImmunoCAP inhibition positive; true result: positive). RESULTS: The obtained results demonstrate that the in vitro results correlate well in terms of specificity and sensitivity to this new reference standard. In particular a higher specificity for Pharmacia CAP System in comparison to in vivo skin prick test for grass pollens and a better sensitivity for mites and cat allergens were found. CONCLUSIONS: Our results suggest that the in vitro "second generation" testing provides reliable results in all clinical situations.

Eosinophil cationic protein (ECP), histamine and tryptase in peripheral blood before and during inhalation challenge with toluene diisocyanate (TDI) in sensitized subjects.

To determine whether the measurement of specific markers of inflammatory cells in peripheral blood might be used to detect the inflammatory activity in the airways in asthma induced by toluene diisocyanate (TDI), we measured the levels of eosinophil cationic protein (ECP), histamine and tryptase in peripheral blood before and during inhalation challenge with TDI or methacholine in two groups of subjects who exhibited or did not exhibit an asthmatic reaction after exposure to toluene diisocyanate in the laboratory. When the subjects developed a late asthmatic reaction after exposure to TDI, they showed an increase in their ECP serum levels. By contrast, there were no significant changes in serum ECP levels after exposure to TDI in the control group or after methacholine challenge in either group. Tryptase levels in serum were not detectable before or during inhalation challenge with TDI or methacholine. There was no significant increase in plasma histamine levels during inhalation challenge with TDI or methacholine. These results suggest that eosinophils are 'activated' in subjects who develop a late asthmatic reaction after exposure to TDI and that the measurement of ECP levels in peripheral blood may be a useful marker to monitor airway inflammation.