Antigenic proteins involved in occupational rhinitis and asthma caused by obeche wood (Triplochiton scleroxylon).

BACKGROUND Obeche wood dust is a known cause of occupational asthma where an IgE-mediated mechanism has been demonstrated. OBJECTIVE To characterize the allergenic profile of obeche wood dust and evaluate the reactivity of the proteins by in vitro, ex vivo and in vivo assays in carpenters with confirmed rhinitis and/or asthma MATERIALS AND METHODS An in-house obeche extract was obtained, and two IgE binding bands were purified (24 and 12 kDa) and sequenced by N-terminal identity. Specific IgE and IgG, basophil activation tests and skin prick tests (SPTs) were performed with whole extract and purified proteins. CCD binding was analyzed by ELISA inhibition studies. RESULTS Sixty-two subjects participated: 12 with confirmed occupational asthma/rhinitis (ORA+), 40 asymptomatic exposed (ORA-), and 10 controls. Of the confirmed subjects, 83% had a positive SPT to obeche. There was a 100% recognition by ELISA in symptomatic subjects vs. 30% and 10% in asymptomatic exposed subjects and controls respectively (p<0.05). Two new proteins were purified, a 24 kDa protein identified as a putative thaumatin-like protein and a 12 kDa gamma-expansin. Both showed allergenic activity in vitro, with the putative thaumatin being the most active, with 92% recognition by ELISA and 100% by basophil activation test in ORA+ subjects. Cross-reactivity due to CCD was ruled out in 82% of cases. CONCLUSIONS Two proteins of obeche wood were identified and were recognized by a high percentage of symptomatic subjects and by a small proportion of asymptomatic exposed subjects. Further studies are required to evaluate cross reactivity with other plant allergens.

Enhanced diagnosis of pollen allergy using specific immunoglobulin E determination to detect major allergens and panallergens.

BACKGROUND Pollen is one of the main causes of allergic sensitization. It is not easy to make an etiological diagnosis of pollen-allergic patients because of the wide variety of sensitizing pollens, association with food allergy, and increasing incidence of polysensitization, which may result from the presence of allergens that are common to different species, as is the case of panallergens. OBJECTIVE To compare the results of skin prick tests (SPT) using whole pollen extract with specific immunoglobulin (Ig) E determination for several allergens (purified panallergens included) in the diagnosis of polysensitized pollen-allergic patients. METHODS The study sample comprised 179 pollen-sensitized patients who underwent SPT with pollen extract and allergen-specific IgE determination against different allergens. RESULTS The level of concordance between the traditional diagnostic test (SPT) and IgE determination was low, especially in patients sensitized to the panallergens profilin and polcalcin. In the case of SPT, the results demonstrated that patients who are sensitized to either of these panallergens present a significantly higher number of positive results than patients who are not. However, IgE determination revealed that while patients sensitized to polcalcins are sensitized to allergens from a higher number of pollens than the rest of the sample, this is not the case in patients sensitized to profilins. On the other hand, sensitization to profilin or lipid transfer proteins was clearly associated with food allergy. CONCLUSIONS Sensitization to panallergens could be a confounding factor in the diagnosis of polysensitized pollen-allergic patients as well as a marker for food allergy. However, more studies are required to further investigate the role of these molecules.

International survey on skin patch test procedures, attitudes and interpretation.

BACKGROUND Skin patch test is the gold standard method in diagnosing contact allergy. Although used for more than 100 years, the patch test procedure is performed with variability around the world. A number of factors can influence the test results, namely the quality of reagents used, the timing of the application, the patch test series (allergens/haptens) that have been used for testing, the appropriate interpretation of the skin reactions or the evaluation of the patient's benefit. METHODS We performed an Internet -based survey with 38 questions covering the educational background of respondents, patch test methods and interpretation. The questionnaire was distributed among all representatives of national member societies of the World Allergy Organization (WAO), and the WAO Junior Members Group. RESULTS One hundred sixty-nine completed surveys were received from 47 countries. The majority of participants had more than 5 years of clinical practice (61 %) and routinely carried out patch tests (70 %). Both allergists and dermatologists were responsible for carrying out the patch tests. We could observe the use of many different guidelines regardless the geographical distribution. The use of home-made preparations was indicated by 47 % of participants and 73 % of the respondents performed 2 or 3 readings. Most of the responders indicated having patients with adverse reactions, including erythroderma (12 %); however, only 30 % of members completed a consent form before conducting the patch test. DISCUSSION The heterogeneity of patch test practices may be influenced by the level of awareness of clinical guidelines, different training backgrounds, accessibility to various types of devices, the patch test series (allergens/haptens) used for testing, type of clinical practice (public or private practice, clinical or research-based institution), infrastructure availability, financial/commercial implications and regulations among others. CONCLUSION There is a lack of a worldwide homogeneity of patch test procedures, and this raises concerns about the need for standardization and harmonization of this important diagnostic procedure.

An antibody-based ELISA for quantification of Ani s 1, a major allergen from Anisakis simplex.

Anisakis simplex is a nematode parasite that can infect humans who have eaten raw or undercooked seafood. Larvae invading the gastrointestinal mucosa excrete/secrete proteins that are implicated in the pathogenesis of anisakiasis and can induce IgE-mediated symptoms. Since Ani s 1 is a potent secreted allergen with important clinical relevance, its measurement could assess the quality of allergenic products used in diagnosis/immunotherapy of Anisakis allergy and track the presence of A. simplex parasites in fish foodstuffs. An antibody-based ELISA for quantification of Ani s 1 has been developed based on monoclonal antibody 4F2 as capture antibody and biotin-labelled polyclonal antibodies against Ani s 1 as detection reagent. The dose-response standard curves, obtained with natural and recombinant antigens, ranged from 4 to 2000 ng/ml and were identical and parallel to that of the A. simplex extract. The linear portion of the dose-response curve with nAni s 1 was between 15 and 250 ng/ml with inter-assay and intra-assays coefficients of variation less than 20% and 10%, respectively. The assay was specific since there was no cross-reaction with other extracts (except Ascaris extracts) and was highly sensitive (detection limit of 1·8 ng/ml), being able to detect Ani s 1 in fish extracts from codfish and monkfish.

A three-year aeropalynological study in Estepona (southern Spain).

An aeropalynological study was carried out in the atmosphere of Estepona, a very popular tourist resort situated in the "Costa del Sol", (southern Spain) based on the data obtained during a three year air-monitoring programme (March 1995 to March 1998) using a volumetric pollen trap. The 34 taxa that reached a 10-day mean air pollen concentration equal to or greater than 1 grain of pollen/m(3) of air are reflected in the calendar. The first 10 taxa, in order of abundance, were: Cupressaceae, Olea europaea, Quercus, Poaceae, Urticaceae, Plantago, Pinus, Chenopodiaceae-Amaranthaceae, Ericaceae and Castanea, the first 3 of which accounted for approximately 56 % of the annual total pollen count. The greatest diversity of pollen type occurred during spring, while the highest pollen concentrations were reached from February-June, when approximately more than 80 % of the annual total pollen was registered. The lowest concentrations were obtaining during January, August and September. The annual quantity of pollen collected, the intensity and the dates on which the maximum peaks were recorded differed for the 3 years studied, which can be explained by reference to various meteorological parameters, especially rainfall and temperature. The pollen calendar spectrum is typically Mediterranean and similar to those of nearby localities, in which many pollen types are represented and the long tails indicating long flowering periods.

Anaphylaxis caused by imported red fire ant stings in Málaga, Spain.

A 27-year-old woman suffered from anaphylaxis after being stung by Solenopsis invicta ants while she was handling wood from South America. The patient reported no previous adverse reactions to stings by other hymenopteran species. Intradermal skin tests with hymenoptera venom (Vespula vulgaris, Polistes species, Apis melifera) were negative. Serum specific immunoglobulin (Ig) E yielded positive results for S invicta (5.28 kU/L) and negative results for A melifera, Ves v 5 and Pol a 5. Immunodetection assays showed the presence of serum IgE against the Sol i 2 allergen. The patient had probably been stung previously although inadvertently by red fire ants while she handled infested wood from South America, and precautionary measures are thus advisable when this material is to be handled. To our knowledge this is the first case of anaphylaxis from red fire ant stings reported in Europe.

Neutrophil defensins: their possible role in allergic asthma.

BACKGROUND Neutrophil defensins, originally identified as broad-spectrum antimicrobial peptides, have been implicated in the regulation of inflammatory and immunological processes. OBJECTIVES To investigate whether the in vitro challenge of neutrophils from patients with bronchial asthma with allergens stimulated the release of alpha-defensins and whether levels released were dependent on lung infections. METHOD The neutrophils were cultivated with different agonists and the concentration of alpha-defensin in cell-free supernatant was measured with enzyme-linked immunosorbent assay (ELISA). RESULTS Neutrophils from allergic patients released alpha-defensins via an allergen-dependent mechanism. Our results indicate that the in vitro activation of neutrophils is highly allergen-specific. In this context, allergens other than those which produced clinical symptoms did not elicit alpha-defensin release, and allergens had no effect on neutrophils from healthy donors. However, neutrophils from both allergic patients and healthy controls were able to release alpha-defensins upon treatment with PMA. In the allergen-stimulated neutrophils, cells from asthmatic patients stimulated with a sensitizing allergen showed a significantly higher production of alpha-defensin under respiratory tract infection than cells from the same patients without such an infection. CONCLUSION Neutrophils from allergic patients release alpha-defensins via an allergen-dependent mechanism.

Quality of life in patients with respiratory allergy is influenced by the causative allergen.

OBJECTIVE We aimed to analyze health-related quality of life (HRQOL) in adults with newly diagnosed respiratory allergy according to the sensitization profile for relevant aeroallergens in their usual area of residence. METHODS We performed a cross-sectional, epidemiological, observational, descriptive, multicenter study in allergy clinics in Spain. The sample comprised adults diagnosed with rhinitis, asthma, or both caused by significant allergens in their residential area (olive and/or grass pollen or house dust mite). Allergic rhinitis was classified according to the Allergic Rhinitis and its Impact on Asthma guidelines; asthma was classified according to the Guía Españiola para el Manejo del Asma (Spanish Guideline on the Management of Asthma). HRQOL was studied according to the ESPRINT-15 questionnaire and Mini Asthma Quality of Life Questionnaire. Control of asthma was measured using the Asthma Control Questionnaire 5. RESULTS We studied 1437 patients. Rhinitis was the most common respiratory disease. The HRQOL of rhinitis patients was lower in those sensitized to olive pollen only and in those with combined sensitization to olive and grass pollens. HRQOL associated with rhinitis was worse in patients diagnosed with both rhinitis and asthma than in patients diagnosed with rhinitis only. Asthma patients sensitized to olive pollen or olive and grass pollens had worse HRQOL. CONCLUSIONS In our study population, the HRQOL of patients with respiratory allergies varied with the allergen responsible for symptoms. In patients with rhinitis, the presence of asthma significantly worsened rhinitis-associated HRQOL.

Allergens Induce the Release of Lactoferrin by Neutrophils from Asthmatic Patients.

BACKGROUND Despite the evidence that Lactoferrin (Lf) is involved in allergic asthma processes, it is unknown whether neutrophils can be one of the main cellular sources of this key inflammatory mediator directly in response of an IgE mediated stimulus. The present study was undertaken to analyze this question. METHODS Neutrophils from healthy subjects (n = 34) and neutrophils from allergic asthmatic patients (n = 102) were challenged in vitro with specific allergens to which the patients were sensitized, PAF, or agonist mAbs against IgE-receptors, and the levels of Lf were measured in the culture supernatant. The levels of serum IgE together with the severity of symptoms were also analyzed. RESULTS Lf was released into the culture supernatant of neutrophils from allergic asthmatic patients in response to allergens and PAF. This response was highly allergen-specific, and did not happen in neutrophils from healthy donors. Allergen effect was mimicked by Abs against FcεRI and galectin-3 but not by FcεRII. The levels of released Lf correlated well with the levels of serum specific IgE and severity of asthma symptoms. These observations represent a novel view of neutrophils as an important source of Lf in allergic asthma. Importantly, the levels of released Lf by neutrophils could therefore be used to evaluate disease severity in allergic asthmatic patients.

Ki-67 MIB1 labelling index and the prognosis of primary TaT1 urothelial cell carcinoma of the bladder.

Aims: To evaluate whether ki-67 labelling index (LI) has independent prognostic value for survival of patients with bladder urothelial tumours graded according to the 2004 World Health Organisation classification. Methods: Ki-67 LI was evaluated in 164 cases using the grid counting method. Non-invasive (stage Ta) tumours were: papilloma (n = 5), papillary urothelial neoplasia of low malignant potential (PUNLMP; n = 26), and low (LG; n = 34) or high grade (HG; n = 15) papillary urothelial carcinoma. Early invasive (stage T1) tumours were: LG (n = 58) and HG (n = 26) carcinoma. Statistical analysis included Fisher and x2 tests, and mean comparisons by ANOVA and t test. Univariate and multivariate survival analyses were performed according to the Kaplan–Meier method with log rank test and Cox’s proportional hazard method. Results: Mean ki-67 LI increased from papilloma to PUNLMP, LG, and HG in stage Ta (p,0.0001) and from LG to HG in stage T1 (p = 0.013) tumours. High tumour proliferation (.13%) was related to greater tumour size (p = 0.036), recurrence (p = 0.036), progression (p = 0.035), survival (p = 0.054), and high p53 accumulation (p = 0.015). Ki-67 LI and tumour size were independent predictors of disease free survival (DFS), but only ki-67 LI was related to progression free survival (PFS). Cancer specific overall survival (OS) was related to ki-67 LI, tumour size, and p27kip1 downregulation. Ki-67 LI was the main independent predictor of DFS (p = 0.0005), PFS (p = 0.0162), and cancer specific OS (p = 00195). Conclusion: Tumour proliferation measured by Ki-67 LI is related to tumour recurrence, stage progression, and is an independent predictor of DFS, PFS, and cancer specific OS in TaT1 bladder urothelial cell carcinoma.

Influence of HLA DRB1 alleles in the susceptibility of rheumatoid arthritis and the regulation of antibodies against citrullinated proteins and rheumatoid factor.

INTRODUCTION The purpose of this study was to investigate the association between HLA-DRB1 alleles with susceptibility to rheumatoid arthritis (RA) and production of antibodies against citrullinated proteins (ACPA) and rheumatoid factor (RF). METHODS We studied 408 patients (235 with RA, 173 non-RA) and 269 controls. ACPA, RF and HLA-DR typing were determined. RESULTS We found an increased frequency of HLA DRB1 alleles with the shared epitope (SE) in ACPA-positive RA. Inversely, HLA DRB1 alleles encoding DERAA sequences were more frequent in controls than in ACPA-positive RA, and a similar trend was found for HLA DR3. However, these results could not be confirmed after stratification for the presence of the SE, probably due to the relatively low number of patients. These data may suggest that the presence of these alleles may confer a protective role for ACPA-positive RA. In RA patients we observed association between SE alleles and ACPA titers in a dose-dependent effect. The presence of HLA DR3 or DERAA-encoding alleles was associated with markedly reduced ACPA levels. No association between RF titers and HLA DR3 or DERAA-encoding alleles was found. CONCLUSIONS HLA DRB1 alleles with the SE are associated with production of ACPA. DERAA-encoding HLA-DR alleles and HLA DR3 may be protective for ACPA-positive RA.

CD40: Novel Association with Crohn's Disease and Replication in Multiple Sclerosis Susceptibility

Background: A functional polymorphism located at 21 from the start codon of the CD40 gene, rs1883832, was previously reported to disrupt a Kozak sequence essential for translation. It has been consistently associated with Graves’ disease risk in populations of different ethnicity and genetic proxies of this variant evaluated in genome-wide association studies have shown evidence of an effect in rheumatoid arthritis and multiple sclerosis (MS) susceptibility. However, the protective allele associated with Graves’ disease or rheumatoid arthritis has shown a risk role in MS, an effect that we aimed to replicate in the present work. We hypothesized that this functional polymorphism might also show an association with other complex autoimmune condition such as inflammatory bowel disease, given the CD40 overexpression previously observed in Crohn’s disease (CD) lesions. Methodology: Genotyping of rs1883832C.T was performed in 1564 MS, 1102 CD and 969 ulcerative colitis (UC) Spanish patients and in 2948 ethnically matched controls by TaqMan chemistry. Principal Findings: The observed effect of the minor allele rs1883832T was replicated in our independent Spanish MS cohort [p= 0.025; OR (95% CI)= 1.12 (1.01–1.23)]. The frequency of the minor allele was also significantly higher in CD patients than in controls [p= 0.002; OR (95% CI)= 1.19 (1.06–1.33)]. This increased predisposition was not detected in UC patients [p= 0.5; OR (95% CI)= 1.04 (0.93–1.17)]. Conclusion: The impact of CD40 rs1883832 on MS and CD risk points to a common signaling shared by these autoimmune conditions

Systematic survey of clonal complexity in tuberculosis at a populational level and detailed characterization of the isolates involved.

Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal variants) by applying mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) analysis to M. tuberculosis isolates from two population-based samples of respiratory (703 cases) and respiratory-extrapulmonary (R+E) tuberculosis (TB) cases (71 cases) in a context of moderate TB incidence. Clonally complex infections were found in 11 (1.6%) of the respiratory TB cases and in 10 (14.1%) of those with R+E TB. Among the 21 cases with clonally complex TB, 9 were infected by 2 independent strains and the remaining 12 showed the simultaneous presence of 2 to 3 clonal variants. For the 10 R+E TB cases with clonally complex infections, compartmentalization (different compositions of strains/clonal variants in independent infected sites) was found in 9 of them. All the strains/clonal variants were also genotyped by IS6110-based restriction fragment length polymorphism analysis, which split two MIRU-defined clonal variants, although in general, it showed a lower discriminatory power to identify the clonal heterogeneity revealed by MIRU-VNTR analysis. The comparative analysis of IS6110 insertion sites between coinfecting clonal variants showed differences in the genes coding for a cutinase, a PPE family protein, and two conserved hypothetical proteins. Diagnostic delay, existence of previous TB, risk for overexposure, and clustered/orphan status of the involved strains were analyzed to propose possible explanations for the cases with clonally complex infections. Our study characterizes in detail all the clonally complex infections by M. tuberculosis found in a systematic survey and contributes to the characterization that these phenomena can be found to an extent higher than expected, even in an unselected population-based sample lacking high infective pressure.

Structured intermittent interruption of chronic HIV infection treatment with highly active antiretroviral therapy: Effects on leptin and TNF-alpha.

The changes in nutritional parameters and adipocytokines after structured intermittent interruption of highly active antiretroviral treatment of patients with chronic HIV infection are analyzed. Twenty-seven patients with chronic HIV infection (median CD4+ T cell count/microl: nadir, 394; at the beginning of structured interruptions, 1041; HIV viral load: nadir, 41,521 copies/ml; at the beginning of structured interruptions <50 copies/ml; median time of previous treatment: 60 months) were evaluated during three cycles of intermittent interruptions of therapy (8 weeks on/4 weeks off). CD4+ T cell count, HIV viral load, anthropometric measures, and serum concentrations of triglycerides, cholesterol, leptin, and tumor necrosis factor and its soluble receptors I and II were determined. After the three cycles of intermittent interruptions of therapy, no significant differences in CD4+ T cell count/microl, viral load, or serum concentrations of cholesterol or triglycerides with reference to baseline values were found. A near-significant higher fatty mass (skinfold thicknesses, at the end, 121 mm, at the beginning, 100 mm, p = 0.100), combined with a significant increase of concentration of leptin (1.5 vs. 4.7 ng/ml, p = 0,044), as well as a decrease in serum concentrations of soluble receptors of tumor necrosis factor (TNFRI, 104 vs. 73 pg/ml, p = 0.022; TNFRII 253 vs. 195 pg/ml, p = 0.098) were detected. Structured intermittent interruption of highly active antiretroviral treatment of patients with chronic HIV infection induces a valuable positive modification in markers of lipid turnover and adipose tissue mass.

DRB1*03:01 haplotypes: differential contribution to multiple sclerosis risk and specific association with the presence of intrathecal IgM bands.

BACKGROUND Multiple sclerosis (MS) is a multifactorial disease with a genetic basis. The strongest associations with the disease lie in the Human Leukocyte Antigen (HLA) region. However, except for the DRB1*15:01 allele, the main risk factor associated to MS so far, no consistent effect has been described for any other variant. One example is HLA-DRB1*03:01, with a heterogeneous effect across populations and studies. We postulate that those discrepancies could be due to differences in the diverse haplotypes bearing that allele. Thus, we aimed at studying the association of DRB1*03:01 with MS susceptibility considering this allele globally and stratified by haplotypes. We also evaluated the association with the presence of oligoclonal IgM bands against myelin lipids (OCMB) in cerebrospinal fluid. METHODS Genotyping of HLA-B, -DRB1 and -DQA1 was performed in 1068 MS patients and 624 ethnically matched healthy controls. One hundred and thirty-nine MS patients were classified according to the presence (M+, 58 patients)/absence (M-, 81 patients) of OCMB. Comparisons between groups (MS patients vs. controls and M+ vs. M-) were performed with the chi-square test or the Fisher exact test. RESULTS Association of DRB1*03:01 with MS susceptibility was observed but with different haplotypic contribution, being the ancestral haplotype (AH) 18.2 the one causing the highest risk. Comparisons between M+, M- and controls showed that the AH 18.2 was affecting only M+ individuals, conferring a risk similar to that caused by DRB1*15:01. CONCLUSIONS The diverse DRB1*03:01-containing haplotypes contribute with different risk to MS susceptibility. The AH 18.2 causes the highest risk and affects only to individuals showing OCMB.