Gabriella Di Felice

Lamina propria T cells in Crohn's disease and other gastrointestinal inflammation show defective CD2 pathway-induced apoptosis

BACKGROUND & AIMS Normal human lamina propria lymphocytes manifest increased unstimulated apoptosis compared with peripheral lymphocytes, which are enhanced after stimulation via the CD2 activation pathway. This activation-induced apoptosis down-regulates cell expansion and cytokine production. In previous studies, it was shown that lamina propria T cells from patients with Crohns disease and ulcerative colitis manifest abnormal proliferation and cytokine production. It was therefore of interest to determine if such cells also showed abnormal patterns of apoptosis. METHODS Apoptosis was evaluated by propidium iodide staining of cells followed by flow cytometric analysis. Fas expression and Bcl-2 levels in cells were evaluated by immunofluorescence. RESULTS Lamina propria lymphocytes from patients with Crohns disease and ulcerative colitis as well as from 2 patients with diverticulitis showed defective CD2 pathway-induced apoptosis. Studies of the mechanisms of this defect focusing on cells from patients with Crohns disease showed that Crohns disease lamina propria lymphocytes from inflamed tissues express the same amount of cell surface Fas but are less sensitive to Fas-mediated apoptosis than control cells. In addition, lamina propria lymphocytes from inflamed Crohns disease tissues manifest increased expression of Bcl-2 after CD2 pathway stimulation and elevated Bcl-2 levels in cultures of unstimulated T cells. CONCLUSIONS T cells isolated from areas of inflammation in Crohns disease, ulcerative colitis, and other inflammatory states manifest decreased CD2 pathway-induced apoptosis. Studies of cells from inflamed Crohns disease tissue indicate that this defect is accompanied by elevated Bcl-2 levels. These changes are probably caused by the chronic inflammation and may aggravate the underlying disease processes that are present.

Specific IgE to cross-reactive carbohydrate determinants strongly affect the in vitro diagnosis of allergic diseases

BACKGROUND Cross-reacting carbohydrate determinants (CCDs) are antigenic structures shared by allergenic components from taxonomically distant sources. The case history of a patient with a great discrepancy between skin test and specific IgE results led us to investigate the role of these determinants in his specific case and in an allergic population. OBJECTIVE We sought to determine the role of CCDs in causing false-positive and clinically irrelevant results in in vitro tests. METHODS The involvement of CCDs was studied by specific IgE inhibition by using glycoproteins with a known carbohydrate structure. Direct and inhibition assays were performed by commercially available systems, in-house ELISA, and the immunoblotting technique. The binding to the periodate-oxidated carbohydrate structure of glycoproteins and allergenic extracts was also evaluated. A comparative study between skin test and specific IgE responses to the antigens studied was carried out in 428 consecutive allergic subjects. RESULTS All the tests performed suggested that cross-reacting carbohydrate epitopes were the cause of false-positive specific IgE results in one of the commercial systems and the high reactivity in all the solid-phase in vitro tests. None of the cross-reacting carbohydrate allergens yielded a positive skin test response. Periodate treatment caused variable degrees of reduction of IgE binding to the different antigens studied, indicating that CCDs played a different role in each of them. About 41% of patients allergic to pollen had specific IgE for a glycoprotein, without a positive skin test response to the same molecule. CONCLUSIONS CCDs must be taken into account when evaluating the clinical relevance of positive results in in vitro specific IgE assays, at least in the diagnosis of patients with pollen allergy. Commercial systems should be carefully assessed for the ability to detect specific IgE for carbohydrate determinants to avoid false-positive or clinically irrelevant results.

Cupressaceae Pollinosis: Identification, Purification and Cloning of Relevant Allergens

Allergy to Cupressaceae pollen is a worldwide pollinosis caused by several species. Pollen extracts prepared from allergenic species belonging to this family are characterised by low protein and high carbohydrate content. The allergenic components represented in the pollen extracts from different species of the Cupressaceae family show high levels of cross-reactivity when probed with human IgE from allergic subjects and share a number of common epitopes also identified by polyclonal rabbit antisera and monoclonal antibodies. A close relationship has also been described with the Taxodiaceae and Podocarpaceae families. Although both proteic and carbohydrate epitopes appear to be involved in IgE recognition and allergenic cross-reactivity, a large portion of the IgE reactivity of Cupressaceae-allergic patients seems to be associated with sugar moieties present on the relevant allergenic molecules. From this point of view, Cupressaceae/Taxodiaceae allergens constitute a particularly useful model to study IgE cross-reactivity, as they have been shown to display different levels of homology. Moreover, the availability of the purified allergens, together with their recombinant counterparts, may shed light on the actual role played by carbohydrate in allergic sensitisation, IgE recognition and allergenic cross-reactivity.

Evaluation of allergenicity of genetically modified soybean protein extract in a murine model of oral allergen-specific sensitization

Background With the development of genetically modified crop plants there has been a growing interest in the approaches available to assess the potential allergenicity of novel gene products. For additional assessment of the potential allergenicity of expressed proteins, informative data can be generated using animal models. Soybean is one of the major source of protein in human and animal nutrition, and has also been well characterized as a major allergenic source. Advances in biotechnology have resulted in an increasing number of genetically engineered foods, and among these soybean is one of the most widespread.

Hypoallergenic variants of the Parietaria judaica major allergen Par j 1: a member of the non-specific lipid transfer protein plant family.

Background: Par j 1 represents a major allergenic component of Parietaria judaica (Pj) pollen, since it is able to induce an immunoglobulin E (IgE) response in 95% of Pj-allergic patients. It belongs to the non-specific lipid transfer protein family, sharing with them a common three-dimensional structure. Methods: Disulphide bond variants of the recombinant Par j 1 (rPar j 1) allergen were generated by site-directed mutagenesis, and the immunological activity of rPar j 1 and its conformational mutants was compared with the use of the skin prick test (SPT). The ability to bind IgE antibodies was evaluated by Western blot, ELISA and ELISA inhibition. T cell reactivity was measured by peripheral blood mononuclear cell proliferation assay. Results: The disruption of Cys14–Cys29 and Cys30–Cys75 bridging (PjA mutant) caused the loss of the majority of specific IgE-binding activity. Additional disruption of the Cys4–Cys52 bridge (PjC mutant) and the latter Cys50–Cys91 bridge (PjD mutant) led to the abolition of IgE-binding activity. On the SPT, PjB (lacking the Cys4–Cys52 and Cys50–Cys91 bridges) was still capable of triggering a type I hypersensitive reaction in 9 out of 10 patients, and PjA in 3 out of 10 patients, while PjC and PjD did not show any SPT reactivity. All the mutants preserved their T cell reactivity. Conclusion: Recombinant hypoallergenic variants of the rPar j 1 allergen described herein may represent a useful tool for improved immunotherapy.

Cross-reactivity between Cupressus arizonica and Cupressus sempervirens pollen extracts

BACKGROUND Cupressus arizonica and C. sempervirens, two species belonging to the Cupressaceae family, are recognized as an important cause of respiratory allergies in countries with a Mediterranean climate. OBJECTIVE The relationship between pollen extracts from these two species was studied by evaluating the reactivity with polyclonal rabbit antisera and human IgE. METHODS The two extracts were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Cross-reactivity was evaluated by ELISA and immunoblotting inhibition experiments. RESULTS The electrophoretic patterns of the two extracts are quite different, although some components display identical molecular weights. The immunoblotting developed with human IgE from subjects allergic to members of the Cupressaceae family indicated that two major IgE-reactive components, displaying molecular weights of about 43,000 and 36,000 d, were similarly detected in both extracts. Inhibition experiments showed a high degree of crossreactivity between the two extracts when tested with rabbit polyclonal antibodies against C. arizonica and C. sempervirens. When tested with human IgE inhibition methods, both extracts were able to reciprocally inhibit all of the IgE-reactive bands, although C. arizonica extract was always a better inhibitor. CONCLUSIONS C. arizonica and C. sempervirens extracts are highly cross-reactive at the IgE level and share a number of common epitopes also identified by polyclonal rabbit antisera.

Purification and partial characterization of the major antigen of Echinococcus granulosus (antigen 5) with monoclonal antibodies

A monoclonal antibody specific for antigen 5 of Echinococcus granulosus was isolated and partially characterized. Purification of antigen 5 was accomplished by affinity chromatography using an immunoabsorbent prepared with this monoclonal antibody. Pure antigen 5 was identified by immunoelectrophoresis, double diffusion in agar gel, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting. The pure antigen displayed the electrophoretic mobility typical of antigen 5 and gave a single precipitin band in double diffusion with both monoclonal antibody and rabbit anti-pH5PPT hydatid fraction serum. Two bands of 66 and 56 kDa could be detected in the pure antigen 5 after sodium dodecyl sulphate-polyacrylamide gel electrophoresis when performed in non-reducing conditions: both bands were reactive with the monoclonal antibody in immunoblotting. After reduction with 2-mercaptoethanol, antigen 5 displayed one band only, of 39 kDa. Antigen 5 purified by this procedure was found to retain reactivity with human sera from hydatid patients in a DD5 test.

Cypress allergy: an underestimated pollinosis

independent. In postmarketing surveillance studies, the overall incidence of ACEI-associated angioedema approximated 0.1-0.2% and was found to be more common in Afro-Americans. As in our patient, the time of onset is usually within hours or, at most, the first days of admitiistration (1); however, reports of delayed-onset angioedema occurring months to years after initiation of the drug have been published (1). Evidence has emerged that interruptions of the ACEI treatment may represent a possible risk factor for late-onset ACEI angioedema. Though the exact pathogenesis of ACEI-mediated angioedema remains unknown, the nonapeptide bradykiniti appears to play a key role. Bradykinin, cleaved from high-molecular-weight kininogens by kallikrein, is a potent activator of the L-arginine nitric oxide system that causes vasodilation and increases capillary leakage (4).

Molecular characterization of a cross-reactive Juniperus oxycedrus pollen allergen, Jun o 2: A novel calcium-binding allergen

BACKGROUND Species belonging to the Cupressaceae family are a relevant source of allergens that are present in a wide number of countries. OBJECTIVE We sought to identify, purify, and characterize recombinant allergens from Juniperus oxycedrus, a species belonging to the Cupressaceae family. METHODS Double-stranded cDNA was synthesized from mRNA and cloned into the lambda-ZAP expression vector. IgE screening of the library was performed with a pool of sera from subjects allergic to Cupressaceae. A recombinant 6xHis-tagged Juniperus oxycedrus allergen, Jun o 2, was expressed in Escherichia coli and purified by Ni2+ affinity chromatography. It was studied further by immunoblotting inhibition with pollen extracts from other Cupressaceae, Oleaceae, Urticaceae, and Graminaceae. The role of protein-bound calcium on the allergens IgE-binding capacity was tested in a plaque assay in the presence or absence of EGTA. RESULTS A cDNA coding for a newly identified Juniperus oxycedrus pollen allergen, rJun o 2, was isolated. The deduced amino acid sequence contained four typical Ca2+ binding sites and showed a significant sequence similarity to calmodulins. Depletion of Ca2+ in the plaque assay led to a loss of IgE-binding capacity of rJun o 2. Immunoblotting inhibition revealed that J. oxycedrus, J. ashei, Cupressus arizonica, C. sempervirens, Parietaria judaica, Olea europaea, and Lolium perenne pollen extracts were able to inhibit IgE binding to blotted rJun o 2 at different concentrations. CONCLUSION rJun o 2 contains IgE-binding epitopes shared by taxonomically unrelated species, and therefore it can be regarded as a new panallergen. These findings could contribute to an explanation for the phenomenon of multiple positive test results in polysensitized patients and the potential symptom-eliciting role of allergenic sources previously not encountered.

T-cell and antibody response to Parietaria Judaica allergenic fractions in atopic and nonatopic individuals

The in vitro proliferative response to separated immunologically relevant components of Parietaria judaica pollen extract (PjE) was investigated by proliferation assay and limiting dilution analysis, in peripheral blood mononuclear cells from Parietaria‐allergic subjects and nonallergic controls. In the same subjects, the profile of the antibody response to the PjE fractions was also studied by immunoblotting to evaluate the functional significance of allergen‐induced T‐cell activation in the two groups. The estimated frequency of PjE‐reactive T cells in peripheral‐blood mononuclear cells was low in both groups. No difference was found between the Parietaria‐allergic subjects and nonallergic controls. To assess the overall contribution to the cellular response of PjE components of different molecular weights, we separated the extract by the SDS‐PAGE technique, and the fractions were blotted onto nitrocellulose and solubilized. Almost all the 14 fractions tested induced T‐cell proliferation, at different degrees of magnitude. Responses were similar in the allergic subjects and nonallergic controls. Immunoblotting demonstrated specific IgG antibodies to the 14 PjE fractions not only in the allergic subjects, but also in the healthy controls, whereas IgE antibodies were found, as expected, only in the sera from atopic subjects. These findings indicate that PjE fractions elicit similar T‐cell activation and IgG production in allergic and normal subjects.