Soledad Gallardo

Inhibition of Chronic Airway Inflammation and Remodeling by Galectin-3 Gene Therapy in a Murine Model

We previously demonstrated that treatment of acute asthmatic rats with gene therapy using plasmid-encoding Galectin-3 (Gal-3) resulted in an improvement of cellular and functional respiratory parameters. The next question that we wanted to clarify was if in a chronic situation where the treated animal continues to inhale the Ag, does this procedure prevent the chronicity and the remodeling? Chronic inflammation was induced by intranasal administration of OVA over a period of 12 wk. In the treated group, the Gal-3 gene was introduced by intranasal instillation in 50 μl of plasmid-encoding Gal-3. Noninvasive airway responsiveness to methacholine was tested at different times. Cells were obtained by bronchoalveolar lavage and used for RNA extraction and cytometric studies. Eosinophils were counted in blood and bronchoalveolar lavage fluid. Real-time PCR was used to measure Gal-3 and cytokine mRNA expression in lung. Lungs were paraffined and histologic analyses were performed (H&E, periodic acid-Schiff, and Masson Trichrome stain). Our results showed that 12 wk after the first intranasal Ag instillation in chronically asthmatic mice, treatment with the Gal-3 gene led to an improvement in the eosinophil count and the normalization of hyperresponsiveness to methacholine. Concomitantly, this treatment resulted in an improvement in mucus secretion and subepithelial fibrosis in the chronically asthmatic mice, with a quantitatively measured reduction in lung collagen, a prominent feature of airway remodeling. Plasmid-encoding Gal-3 acts as a novel treatment for chronic asthma in mice producing nearly complete blockade of Ag responses with respect to eosinophil airway accumulation, airway hyperresponsiveness, and remodeling.

Ole e I: Epitope Mapping, Cross-Reactivity with Other Oleaceae Pollens and Ultrastructural Localization

Ole e I is the major allergen derived from olive tree pollen (Olea europaea) and it is composed of two polypeptides with molecular weights (MWs) of 18 and 20 kD. A panel of six monoclonal antibodies (mAbs) has been prepared and used to map antigenic determinants on this molecule. Four epitope determinants have been identified on Ole e I. Using the purified mAbs produced against Ole e I, we have analyzed the common epitope determinants in olive (O. europaea) and different Oleaceae pollens: ash (Fraxinus excelsior); privet (Ligustrum vulgare); lilac (Syringa vulgaris), and forsythia (Forsythia suspensa). ELISA showed three reactivity groups depending on the recognition of monoclonal antibodies: (1) olive and ash; (2) olive, ash, privet and lilac; and (3) olive, ash, privet, lilac and forsythia. Immunoblotting studies on Oleaceae pollen extracts with these mAbs showed a very similar cross-reactivity pattern. The 18- and 20-kD MW proteins were present in each pollen, except in the case of forsythia. In this case the reactivity pattern was associated with 50- to 55-kD protein bands. This band was recognized by a pool of sera from olive-allergic patients. Finally, ultrastructural localization of Ole e I antigen was performed on the mature olive pollen grain. Ole e I was located in association with dilated endoplasmic reticulum cisternae. Pollen grain walls, nuclei and cytoplasmic organelles were totally devoid of the allergen.

Molecular cloning of major allergen from Cupressus arizonica pollen: Cup a 1

The family Cupressaceae is a relevant source of allergens that causes winter respiratory allergies. Cloning and sequencing the major antigen of Cupressus arizonica is important for a better diagnosis and treatment of sensitized patients.

DR7 and DQ2 are positively associated with immunoglobulin-E response to the main antigen of olive pollen (Ole e I) in allergic patients

We have studied the relationship between HLA class II haplotypes and alleles, and the IgE antibody response to a highly purified allergen, Ole e I, in allergic patients. Ole e I, is the major antigen from the pollen of olive tree that grows mainly in the Mediterranean. Genomic DNA typing was performed in 40 unrelated patients with seasonal allergic pollenosis who had specific IgE antibodies against Ole e I, detected by double-antibody radioimmunoassay. HLA-DRB and -DQB loci were analyzed by PCR-SSO and RFLP. Phenotypic frequencies were compared with those of 179 healthy unrelated individuals. Significant increases in the phenotypic frequencies of DR7 (pf = 67.5% vs 31.3% in the control population, pc = 0.0023) and DQ2 (pf = 90.0% vs 48.0%, pc = 0.0003) were found, indicating an association between DRB1*0701/2, DQB1*0201 alleles and the IgE antibody response to Ole e I. This is the first time that the HLA-DQ gene has been associated with a positive allergic response.

Immunopathogenesis of human gastrointestinal infection by Anisakis simplex

BACKGROUND Anisakis simplex is a parasite of fish, and in the case of human infestation, it should be considered as a possible cause of gastrointestinal disease, especially in countries where raw or undercooked fish is a frequent food. Clinical features of anisakiasis may simulate acute abdominal pain, such as that found in patients with gastric ulcers, appendicitis, and Crohns disease. Furthermore, many cases of anisakiasis are diagnosed as eosinophilic gastroenteritis, which is a broad term for a specific disease. OBJECTIVE The purpose of this study was to investigate the immunopathogenesis of human gastrointestinal infestation by A simplex. METHODS Thirteen intestinal biopsy specimens from patients with anisakiasis were analyzed for the presence of messenger (m)RNA for different cytokines and inflammatory mediators by RT-PCR. Specific IgE, eosinophil cationic protein, eosinophil protein X, and tryptase levels were measured in each patients serum. Also, cell cultures were set up with lymphocytes from some patients and stimulated in vitro with Anisakis and Ascaris antigens. RESULTS We performed immunologic phenotyping in 13 patients. All patients underwent biopsy after emergency surgery caused by episodes of acute abdominal pain. In all cases inflammatory infiltrate composed of eosinophils and lymphocytes was found in the intestinal wall. We demonstrated that after infestation, a T(H2)-type immune response occurred. Also, major basic protein, nitric oxide, and eotaxin were found in the tissue, and eosinophil cationic protein and eosinophil protein X levels were elevated in sera. CONCLUSION These data and in vitro lymphocyte cultures indicate that a T(H2) mechanism plays an important role in the inflammatory infiltrate produced by the anchorage of parasites in the gastrointestinal wall.

Cytokine mRNA expression in lung tissue from toxic oil syndrome patients: a TH2 immunological mechanism

In 1981, an epidemic occurred in Spain, toxic oil syndrome (TOS), in people who consumed rapeseed oil denatured with 2% aniline, and it was one of the largest intoxication epidemics ever recorded. In 1989, a similar disease, eosinophilia-myalgia syndrome (EMS) was reported in the USA and was associated with the ingestion of L-tryptophan. The pathologic findings in TOS showed primary endothelial injury, with cell proliferation and perivascular inflammatory infiltrates. Immunologic mechanisms have presumably been operative in the pathogenesis and perpetuation of TOS. Our previous findings pointed to a T-cell activation during acute phase of the disease. In order to analyze which T-cell subset is involved on TOS, we have developed an mRNA extraction procedure from paraffin-embedded lung tissues in patients with pulmonary involvement. We analyzed mRNA expression from different cytokines (IL-1, IL-2, IL-4, IL-5, IFN-gamma, GM-CSF) and CD25 (interleukin 2 receptor) and CD23 (low affinity IgE receptor), using RT-PCR technique. In lung tissues from these patients a T-cell activation was observed. We found a significant increase in Th1 (P = 0.006) and Th2 (P = 0.003) cytokine profile in TOS patients with respect to controls. The increment in TH2 response with respect to TH1 is significant (P = 0.03) in TOS lung specimens. Non-significant differences were obtained in other cytokines and receptors studied as IL-1, CD25, CD23 and GM-CSF. Data presented in this paper are the first clear evidence that an immunological mechanism is directly implicated in this illness.

Molecular cloning and characterization of an IgE-reactive protein from Anisakis simplex: Ani s 1.

Ingestion of the parasitic nematode Anisakis simplex in undercooked fish can cause severe allergic reactions in some individuals. Using pooled human sera from sensitized patients we have probed an expression library for A. simplex antigens. One positive clone was found to encode a full length 21 kDa protein with strong homology to nematode troponins. The recombinant protein was expressed as a GST-fusion protein and found by immunoblot analysis to react with sera from 20% of allergic patients. The presence of functional EF-hand Ca(2+) binding motifs was demonstrated by gel-shift analysis.

Allergy to olive pollen : T-cell response from olive allergic patients is restricted by DR7-DQ2 antigens

Backgound We have recently described the association between the IgE antibody response to Ole e I (the major antigen from olive tree pollen) and the DR7‐DQ2 haplotype in a Spanish population.

Genetic restrictions in olive pollen allergy

BACKGROUND The major antigen of olive tree pollen, Ole e 1, produces an IgE response restricted by DQ2. OBJECTIVE Our purpose was to further analyze the genetic restrictions associated with IgE and IgG antibodies against Ole e 1 and IgE against the recently described antigen Ole e 3. METHODS Twenty-two nuclear olive pollen-allergic families (n = 88) were selected. DRB1 and DQB1, TCR-Valpha 8.1, the high-affinity receptor of IgE (FcepsilonRI-beta) Rsa I exon 7 and intron 2 and TNF-beta (LTalpha-Nco I) polymorphisms were determined by PCR and analyzed for association with allergic traits by the multiallelic transmission disequilibrium test. RESULTS Significant associations were found among HLA-DQB1*0201 (n = 29) and high levels of IgG (P =.023) and IgE (P =.0136) antibodies to Ole e 1 and with IgE specific to Ole e 3 (P =.0368). DRB1*0701 was associated with high levels of total serum IgE (P =.04) and IgG against Ole e 1 (P =.025). The FcepsilonRI-beta Rsa I exon 7, allele 1 (n = 39), was associated with high levels of total serum IgE (P =. 01), IgE antibodies against Olea europaea extract (P =.004), and specific antibodies to Ole e 1, IgG (P =.04), and IgE (P =.006). The FcepsilonRI-beta Rsa I intron 2, allele 2 (n = 33), was associated with IgE antibodies to O europaea extract (P =.003) and specific antibodies to Ole e 1, IgG (P =.025), and IgE (P =.05). CONCLUSIONS We found a new association between IgE antibody response to Ole e 3 and DQB1*0201 and verified the previously reported association between Ole e 1-specific response and DQB1*0201. Also, the association between FcepsilonRI-beta and IgE antibodies against Ole e 1 was demonstrated.

Immunological basis of toxic oil syndrome (TOS)

The toxic oil syndrome (TOS), a multisystemic disease, that occurred in Spain in 1981, was caused by the ingestion of rapeseed oil denatured with 2% aniline. Due to the clinical course of the disease, immunopathological mechanisms have been suspected but a direct connection was never demonstrated. To analyse this possibility, we determined several immunological parameters in the sera of patients with TOS and without the disease, using a case-control design: total immunoglobulins, IgG and IgE antibodies against different toxic agents (oleylanilide, aniline, linoleyl-anilide, and 3-phenylaminopropane-1-2-diol), autoantibodies, cytokines (IL-4, IL-6, TNF, GM-CSF) and soluble receptors (sCD23 and sIL-2R). We detected high levels of sIL-2R in TOS patients compared to controls (P < 0.0001). A higher levels of sCD23 and IgE were also found. In addition, the response to oleyl-anilide of peripheral blood lymphocytes from TOS patients was studied and a significant proliferative response in 30% of TOS patients versus 5% controls was observed. Our data support the implication of the immune system in the acute phase of TOS, with a possible activation of T-cells and release of cytokines, that could explain some of the clinical findings in this phase of the disease.