Occupational asthma caused by IgE-mediated sensitization to multiple woods

Wood is a natural material that is able to trigger rhinitis and asthma in exposed subjects in occupational settings. This has been described with both soft and hard woods.1,2 Involvement of both low- and high-molecular-weight allergens has been reported, and the relevance of these is related with the wood type.1 There are cases where protein may be the responsible allergen. Crossreactivity between obeche and ramin woods3 and between obeche and latex4 has been shown. However, to the best of our knowledge, this is the first report of a multiple IgE-mediated sensitization to different woods that caused occupational respiratory symptoms in the same worker.

Antigenic Proteins Involved in Occupational Rhinitis 1 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.

Sensitization profiles and cross-reactivity among plant allergens. Molecular mechanisms of food allergy development and the role of enhancers

La prevalencia de las alergias est�� aumentando desde mediados del siglo XX, y se estima que actualmente afectan a alrededor del 2-8 % de la poblaci��n, pero las causas de este aumento a��n no est��n claras. Encontrar el origen del mecanismo por el cual una prote��na inofensiva se convierte en capaz de inducir una respuesta al��rgica es de vital importancia para prevenir y tratar estas enfermedades. Aunque la caracterizaci��n de al��rgenos relevantes ha ayudado a mejorar el manejo cl��nico y a aclarar los mecanismos b��sicos de las reacciones al��rgicas, todav��a queda un largo camino para establecer el origen de la alergenicidad y reactividad cruzada. El objetivo de esta tesis ha sido caracterizar las bases moleculares de la alergenicidad tomando como modelo dos familias de panalergenos (prote��nas de transferencia de l��pidos ���LTPs- y taumatinas ���TLPs-) y estudiando los mecanismos que median la sensibilizaci��n y la reactividad cruzada para mejorar tanto el diagn��stico como el tratamiento de la alergia. Para ello, se llevaron a cabo dos estrategias: estudiar la reactividad cruzada de miembros de familias de panal��rgenos; y estudiar mol��culas-co-adyuvantes que pudieran favorecer la capacidad alerg��nica de dichas prote��nas. Para estudiar la reactividad cruzada entre miembros de la misma familia de prote��nas, se seleccionaron LTPs y TLPs, descritas como alergenos, tomando como modelo la alergia a frutas. Por otra parte, se estudiaron los perfiles de sensibilizaci��n a al��rgenos de trigo relacionados con el asma del panadero, la enfermedad ocupacional m��s relevante de origen al��rgico. Estos estudios se llevaron a cabo estandarizando ensayos tipo microarrays con al��rgenos y analizando los resultados por la teor��a de grafos. En relaci��n al estudiar mol��culas-co-adyuvantes que pudieran favorecer la capacidad alerg��nica de dichas prote��nas, se llevaron a cabo estudios sobre la interacci��n de los al��rgenos alimentarios con c��lulas del sistema inmune humano y murino y el epitelio de las mucosas, analizando la importancia de mol��culas co-transportadas con los al��rgenos en el desarrollo de una respuesta Th2. Para ello, Pru p 3(LTP y al��rgeno principal del melocot��n) se selecci��n como modelo para llevarlo a cabo. Por otra parte, se analiz�� el papel de mol��culas activadoras del sistema inmune producidas por pat��genos en la inducci��n de alergias alimentarias seleccionando el modelo kiwi-alternaria, y el papel de Alt a 1, al��rgeno mayor de dicho hongo, en la sensibilizaci��n a Act d 2, al��rgeno mayor de kiwi. En resumen, el presente trabajo presenta una investigaci��n innovadora aportando resultados de gran utilidad tanto para la mejora del diagn��stico como para nuevas investigaciones sobre la alergia y el esclarecimiento final de los mecanismos que caracterizan esta enfermedad. ABSTRACT Allergies are increasing their prevalence from mid twentieth century, and they are currently estimated to affect around 2-8% of the population but the underlying causes of this increase remain still elusive. The understanding of the mechanism by which a harmless protein becomes capable of inducing an allergic response provides us the basis to prevent and treat these diseases. Although the characterization of relevant allergens has led to improved clinical management and has helped to clarify the basic mechanisms of allergic reactions, it seems justified in aspiring to molecularly dissecting these allergens to establish the structural basis of their allergenicity and cross-reactivity. The aim of this thesis was to characterize the molecular basis of the allergenicity of model proteins belonging to different families (Lipid Transfer Proteins ���LTPs-, and Thaumatin-like Proteins ���TLPs-) in order to identify mechanisms that mediate sensitization and cross reactivity for developing new strategies in the management of allergy, both diagnosis and treatment, in the near future. With this purpose, two strategies have been conducted: studies of cross-reactivity among panallergen families and molecular studies of the contribution of cofactors in the induction of the allergic response by these panallergens. Following the first strategy, we studied the cross-reactivity among members of two plant panallergens (LTPs , Lipid Transfer Proteins , and TLPs , Thaumatin-like Proteins) using the peach allergy as a model. Similarly, we characterized the sensitization profiles to wheat allergens in baker's asthma development, the most relevant occupational disease. These studies were performed using allergen microarrays and the graph theory for analyzing the results. Regarding the second approach, we analyzed the interaction of plant allergens with immune and epithelial cells. To perform these studies , we examined the importance of ligands and co-transported molecules of plant allergens in the development of Th2 responses. To this end, Pru p 3, nsLTP (non-specific Lipid Transfer Protein) and peach major allergen, was selected as a model to investigate its interaction with cells of the human and murine immune systems as well as with the intestinal epithelium and the contribution of its ligand in inducing an allergic response was studied. Moreover, we analyzed the role of pathogen associated molecules in the induction of food allergy. For that, we selected the kiwi- alternaria system as a model and the role of Alt a 1 , major allergen of the fungus, in the development of Act d 2-sensitization was studied. In summary, this work presents an innovative research providing useful results for improving diagnosis and leading to further research on allergy and the final clarification of the mechanisms that characterize this disease.

Diagnosis and Management of Grain-Induced Asthma

Grain-induced asthma is a frequent occupational allergic disease mainly caused by inhalation of cereal flour or powder. The main professions affected are bakers, confectioners, pastry factory workers, millers, farmers, and cereal handlers. This disorder is usually due to an IgE-mediated allergic response to inhalation of cereal flour proteins. The major causative allergens of grain-related asthma are proteins derived from wheat, rye and barley flour, although baking additives, such as fungal ��-amylase are also important. This review deals with the current diagnosis and treatment of grain-induced asthma, emphasizing the role of cereal allergens as molecular tools to enhance diagnosis and management of this disorder. Asthma-like symptoms caused by endotoxin exposure among grain workers are beyond the scope of this review. Progress is being made in the characterization of grain and bakery allergens, particularly cereal-derived allergens, as well as in the standardization of allergy tests. Salt-soluble proteins (albumins plus globulins), particularly members of the ��-amylase/trypsin inhibitor family, thioredoxins, peroxidase, lipid transfer protein and other soluble enzymes show the strongest IgE reactivities in wheat flour. In addition, prolamins (not extractable by salt solutions) have also been claimed as potential allergens. However, the large variability of IgE-binding patterns of cereal proteins among patients with grain-induced asthma, together with the great differences in the concentrations of potential allergens observed in commercial cereal extracts used for diagnosis, highlight the necessity to standardize and improve the diagnostic tools. Removal from exposure to the offending agents is the cornerstone of the management of grain-induced asthma. The availability of purified allergens should be very helpful for a more refined diagnosis, and new immunomodulatory treatments, including allergen immunotherapy and biological drugs, should aid in the management of patients with this disorder.

Component-resolved diagnosis of wheat flour allergy in baker's asthma.

Baker's asthma is one of the most common types of occupational asthma and its prevalence is increasing in the last years. Diagnosis of occupational asthma is complex. The poor specificity of current diagnostic approaches may be associated with insufficient purity of wheat extracts or lack of inclusion of major allergens in them. In this work, we use microarray technology to characterize the allergenic profiles of baker's asthma patients from three regions in Spain and to analyze the influence of other environmental allergens on the sensitization pattern.

Enriched mannose glycosylation contributes to Act d 2 allergenicity.

Allergens are responsible for the Th2 response in patients as part of complex mixtures of proteins, fatty acids and other molecules. Plant allergens have hitherto been included in several protein families that share no common biochemical features. Their physical, biochemical and immunological characteristics have been widely studied, but no definite conclusion has been reached about what makes a protein an allergen. N-glycosylation is characteristic of plant allergen sources but is not present in mammals.

Papel de la interleuquina 23 en la diferenciaci��n hacia Th17

The incidence of inflammatory and autoimmune diseases has increased among developed countries in the past 30 years, creating a demand for the development of effective and economic therapies for these diseases. Interleukin 23 (IL-23) is a pro-inflammatory cytokine whose increased production has been shown to play a key role in the establishment and maintenance of inflammatory and autoimmune diseases in different murine models such as inflammatory bowel disease, psoriasis and experimental autoimmune encephalomyelitis. More importantly, increased levels of IL-23 have been found in biopsies from patients with Crohn���s disease and ulcerative colitis, and psoriasis. The pathological consequences of excessive IL-23 signalling have been linked to its ability to promote the production of interleukin 17 (IL-17), particularly in the subpopulation of CD4 T cells Th17. However, the precise molecular mechanisms by which IL-23 sustains the Th17 response and induces pathogenic effector functions in these cells remain largely unknown. The global objective of the experiments carried out in this work was to determine the effect of IL-23 on the proliferation, survival and IL-17 and interferon gamma (IFN-��) production in Th17 cells. These experiments have shown that IL-23 does not promote proliferation or survival of in vitro generated Th17 cells, and that there is no difference in the production of IL -17 in the absence or presence of IL -23. The IL-23 receptor, like other cytokine receptors, lacks intrinsic enzymatic activity. Instead, IL-23 receptor associates with members of the Janus tyrosine kinase family (Jaks). Cytokine binding to a Jak-associated receptor triggers the activation of the Signal Transducers and Activators of Transcription (STAT) family of transcription factors. Previous work indicated that the IL-23 receptor complex is associated with the tyrosine kinases Jak2 and Tyk2 that promote STAT3 phosphorylation. Subsequent studies showed that IL23 activation of STAT3 induces the expression of the transcription factor ROR��t, which is crucial for IL-17 production. This work has explored the IL-23 signalling cascade, determining the optimal conditions for STAT3 activation and demonstrating the activation of other transcription factors such as STAT4, STAT5 and STAT1 that contribute to IL-23-mediated signalling pathways.

Immunomodulation of experimental autoimmune encephalomyelitis by oral administration of copolymer 1

The activity of copolymer 1 (Cop 1, Copaxone, glatiramer acetate) in suppressing experimental autoimmune encephalomyelitis (EAE) and in the treatment of multiple sclerosis patients when injected parenterally has been extensively demonstrated. In the present study we addressed the question of whether Cop 1 can induce oral tolerance to EAE similar to myelin basic protein (MBP). We now have demonstrated that oral Cop 1 inhibited EAE induction in both rats and mice. Furthermore, oral Cop 1 was more effective than oral MBP in suppressing EAE in rats. The beneficial effect of oral Cop 1 was found to be associated with specific inhibition of the proliferative and Th1 cytokine secretion responses to MBP of spleen cells from Cop 1-fed mice and rats. In all of these assays, oral Cop 1 was more effective than oral MBP. The tolerance induced by Cop 1 could be adoptively transferred with spleen cells from Cop 1-fed animals. Furthermore, Cop 1-specific T cell lines, which inhibit EAE induction in vivo, could be isolated from the above spleen cells. These T cell lines secrete the anti-inflammatory cytokines IL-10 and transforming growth factor type ��, but not IL-4, in response to both Cop 1 and MBP. In conclusion, oral Cop 1 has a beneficial effect on the development of EAE that is associated with down-regulation of T cell immune responses to MBP and is mediated by Th2/3 type regulatory cells. These results suggest that oral administration of Cop 1 may modulate multiple sclerosis as well.

The Ets transcription factor ERM is Th1-specific and induced by IL-12 through a Stat4-dependent pathway

Interleukin 12 (IL-12)-induced T helper 1 (Th1) development requires Stat4 activation. However, antigen-activated Th1 cells can produce interferon �� (IFN-��) independently of IL-12 and Stat4 activation. Thus, in differentiated Th1 cells, factors regulated by IL-12 and Stat4 may be involved in IFN-�� production. Using subtractive cloning, we identified ERM, an Ets transcription factor, to be a Th1-specific, IL-12-induced gene. IL-12-induction of ERM occurred in wild-type and Stat1-deficient, but not Stat4-deficient, T cells, suggesting ERM is Stat4-inducible. Retroviral expression of ERM did not restore IFN-�� production in Stat4-deficient T cells, but augmented IFN-�� expression in Stat4-heterozygous T cells. Ets factors frequently regulate transcription via cooperative interactions with other transcription factors, and ERM has been reported to cooperate with c-Jun. However, in the absence of other transcription factors, ERM augmented expression of an IFN-�� reporter by only 2-fold. Thus, determining the requirement for ERM in Th1 development likely will require gene targeting.

Direct visualization of antigen-specific T cells: HTLV-1 Tax11���19- specific CD8+ T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11���19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2���Ig complex to directly visualize HTLV-1 Tax11���19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8+ lymphocytes specific for the HTLV-1 Tax11���19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11���19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11���19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11���19-specific CD8+ T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-�� and ��-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11���19-specific CD8+ T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

Differential expression of major histocompatibility complex class II genes on murine macrophages associated with T cell cytokine profile and protective/suppressive effects

Protective/suppressive major histocompatibility complex (MHC) class II alleles have been identified in humans and mice where they exert a disease-protective and immunosuppressive effect. Various modes of action have been proposed, among them differential expression of MHC class II genes in different types of antigen-presenting cells impacting on the T helper type 1 (Th1)���Th2 balance. To test this possibility, the expression of H-2 molecules from the four haplotypes H-2b, H-2d, H-2k, and H-2q was determined on bone marrow-derived macrophages (BMDMs) and splenic B cells. The I-Ab and I-Ek molecules, both well characterized as protective/suppressive, are expressed at a high level on almost all CD11b+ BMDMs for 5���8 days, after which expression slowly declines. In contrast, I-Ad, I-Ak, and I-Aq expression is lower, peaks over a shorter period, and declines more rapidly. No differential expression could be detected on B cells. In addition, the differential MHC class II expression found on macrophages skews the cytokine response of T cells as shown by an in vitro restimulation assay with BMDMs as antigen-presenting cells. The results indicate that macrophages of the protective/suppressive haplotypes express MHC class II molecules at a high level and exert Th1 bias, whereas low-level expression favors a Th2 response. We suggest that the extent of expression of the class II gene gates the back signal from T cells and in this way controls the activity of macrophages. This effect mediated by polymorphic nonexon segments of MHC class II genes may play a role in determining disease susceptibility in humans and mice.

Major histocompatibility complex (MHC) class I KbDb ���/��� deficient mice possess functional CD8+ T cells and natural killer cells

We obtained mice deficient for major histocompatibility complex (MHC) molecules encoded by the H-2K and H-2D genes. H-2 KbDb ���/��� mice express no detectable classical MHC class I-region associated (Ia) heavy chains, although ��2-microglobulin and the nonclassical class Ib proteins examined are expressed normally. KbDb ���/��� mice have greatly reduced numbers of mature CD8+ T cells, indicating that selection of the vast majority (>90%) of CD8+ T cells cannot be compensated for by ��2-microglobulin-associated molecules other than classical H-2K and D locus products. In accord with the greatly reduced number of CD8+ T cells, spleen cells from KbDb ���/��� mice do not generate cytotoxic responses in primary mixed-lymphocyte cultures against MHC-disparate (allogeneic) cells. However, in vivo priming of KbDb ���/��� mice with allogeneic cells resulted in strong CD8+ MHC class Ia-specific allogeneic responses. Thus, a minor population of functionally competent peripheral CD8+ T cells capable of strong cytotoxic activity arises in the complete absence of classical MHC class Ia molecules. KbDb ���/��� animals also have natural killer cells that retain their cytotoxic potential.

Charge-dependent translocation of Bordetella pertussis adenylate cyclase toxin into eukaryotic cells: Implication for the in vivo delivery of CD8+ T cell epitopes into antigen-presenting cells

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400-aa residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver T cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted CD8+ cytotoxic T cell responses in vivo. Here, we constructed a series of recombinant toxins harboring at the same insertion site various peptide sequences of 11���25 amino acids, corresponding to defined CD8+ T cell epitopes and differing in the charge of the inserted sequence. We show that inserted peptide sequences containing net negative charges (���1 or ���2) decreased or completely blocked (charge of ���4) the internalization of the toxin into target cells in vitro and abolished the induction of cytotoxic T cell responses in vivo. The blocking of translocation due to the inserted acidic sequences can be relieved by appropriate mutations in the flanking region of CyaA that counterbalance the inserted charges. Our data indicate that (i) the electrostatic charge of the peptides inserted within the catalytic domain of CyaA is critical for its translocation into eukaryotic cells and (ii) the delivery of T cell epitopes into the cytosol of antigen-presenting cells by recombinant CyaA toxins is essential for the in vivo stimulation of specific cytotoxic T cells. These findings will help to engineer improved recombinant CyaA vectors able to stimulate more efficiently cellular immunity.

Neuropeptides, by direct interaction with T cells, induce cytokine secretion and break the commitment to a distinct T helper phenotype

Searching for nervous system candidates that could directly induce T cell cytokine secretion, I tested four neuropeptides (NPs): somatostatin, calcitonin gene-related peptide, neuropeptide Y, and substance P. Comparing neuropeptide-driven versus classical antigen-driven cytokine secretion from T helper cells Th0, Th1, and Th2 autoimmune-related T cell populations, I show that the tested NPs, in the absence of any additional factors, directly induce a marked secretion of cytokines [interleukin 2 (IL-2), interferon-��, IL-4, and IL-10) from T cells. Furthermore, NPs drive distinct Th1 and Th2 populations to a ���forbidden��� cytokine secretion: secretion of Th2 cytokines from a Th1 T cell line and vice versa. Such a phenomenon cannot be induced by classical antigenic stimulation. My study suggests that the nervous system, through NPs interacting with their specific T cell-expressed receptors, can lead to the secretion of both typical and atypical cytokines, to the breakdown of the commitment to a distinct Th phenotype, and a potentially altered function and destiny of T cells in vivo.

CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose

There are two major mechanisms reported to prevent the autoreactivity of islet-specific CD8+ T cells: ignorance and tolerance. When ignorance is operative, na��ve autoreactive CD8+ T cells ignore islet antigens and recirculate without causing damage, unless activated by an external stimulus. In the case of tolerance, CD8+ T cells are deleted. Which factor(s) contributes to each particular outcome was previously unknown. Here, we demonstrate that the concentration of self antigen determines which mechanism operates. When ovalbumin (OVA) was expressed at a relatively low concentration in the pancreatic islets of transgenic mice, there was no detectable cross-presentation, and the CD8+ T cell compartment remained ignorant of OVA. In mice expressing higher doses of OVA, cross-presentation was detectable and led to peripheral deletion of OVA-specific CD8+ T cells. When cross-presentation was prevented by reconstituting the bone marrow compartment with cells incapable of presenting OVA, deletional tolerance was converted to ignorance. Thus, the immune system uses two strategies to avoid CD8+ T cell-mediated autoimmunity: for high dose antigens, it deletes autoreactive T cells, whereas for lower dose antigens, it relies on ignorance.