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A novel numerical methodology is used to investigate the behavior of an all-composite wing-box under a compressive load taking into account the presence of concurrent inter-laminar damages such as delaminations and skin-stringer debonds and their propagation. The proposed numerical methodology, based on linear structural analyses, is computationally inexpensive if compared to the VCCT and CZM based numerical techniques, hence, it appears to be particularly suitable for the preliminary design phase of complex structure. The presented linear approach is an evolution of a previous technique developed specifically for delaminations. Indeed, in this paper the linear approach has been enhanced to predict the propagation of inter-laminar damages with open crack surfaces such as skin-stringer debonds and it has been implemented in a commercial Finite Element platform. The effectiveness (in terms of computational cost and accuracy of results) of the suggested linear numerical methodology, in predicting the compressive behavior of complex composite structures with inter-laminar damages, has been confirmed by cross-comparisons with the standard non-linear VCCT technique. Finally, comparisons in terms of critical propagation load and Energy Release Rate distribution along the damage tip predictions, for several examined structural configurations with different damage extension and depth, have allowed to assess the field of applicability of the propose novel numerical methodology.

The investigation of the mechanical response of fibre-reinforced composite laminates under impact loads can be very difficult due to the occurrence of simultaneous failure phenomena. Indeed, as a consequence of low velocity impacts, intra-laminar damages, like fibre and matrix cracking, and inter-laminar damages, such as delaminations, can take place simultaneously. These damage mechanisms can lead to significant reductions in strength and stability of the composite structure. In this paper a joint numerical-experimental study is proposed which, by means of non-destructive testing techniques (Ultra-sound and thermography) and non-linear explicit FEM analyses, aims to completely characterise the impact induced damage in composite laminates under low velocity impacts. Indeed the proposed numerical tool has been used to improve the understanding of the experimental data obtained by Non-Destructive Techniques. Applications on samples tested according to the AECMA (European Association of Aerospace Manufacturers) prEn6038 standard at three different impact energies are presented. The interaction between numerical and experimental investigation allowed to obtain an exhaustive insight on the different phases of the impact event considering the inter-laminar damage formation and evolution.

To cite this article: Schiavi E, Barletta B, Butteroni C, Corinti S, Boirivant M, Di Felice G. Oral therapeutic administration of a probiotic mixture suppresses established Th2 responses and systemic anaphylaxis in a murine model of food allergy. Allergy 2011; 66: 499–508. Abstract Background: No effective treatment is available for food allergy and its primary management still consists of avoiding relevant allergens. Probiotics are claimed to beneficially affect the immune system. We sought to investigate the therapeutic potential of VSL#3 probiotic mixture on specific immune responses and anaphylactic reaction induced in mice by the major food allergen shrimp tropomyosin (ST). Methods: The cytokine production by spleen cell from ST-sensitized mice upon allergen re-stimulation in the presence of VSL#3 was analysed. Next, the effects of oral administration of VSL#3 on allergen-induced anaphylaxis and Th2 response in the murine model of food allergy to ST was investigated by evaluating symptom score and histamine content in the faeces after allergen challenge, antibody response in serum and faeces, and cytokine and transcription factor expression in the jejunum. Results: The in vitro studies on mouse spleen cells indicates that the VSL#3 preparation has the capacity to shift a polarized Th2 response to a Th1/T regulatory-type profile. Oral therapeutic administration of VSL#3 to ST-sensitized mice significantly reduces symptom score and histamine release in the faeces following allergen challenge, as well as specific IgE response. In the jejunum, IL-4, IL-5 and IL-13 tissue content was significantly reduced, whereas FOXP3 and IL-27 mRNA expression, IL-10, TGF-β and IFN-γ tissue content were up-regulated. Conclusions: Oral therapeutic treatment with the probiotic mixture VSL#3 is effective in redirecting allergen-specific Th2-polarized immune responses towards Th1-T regulatory responses and in the protection against anaphylactic reactions induced by the allergen in a murine model of food allergy.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Delamination and other damage mechanisms, such as matrix cracks, fibre-matrix debonding and fiber failure can appear as a consequence of impact events with foreign objects under in service conditions and maintenance operations. These phenomena are seldom analyzed together without discussing how the interferences between the different damage mechanisms can influence their evolution under different loading conditions. The present work is focused on the development of a specific numerical procedure, able to take into account the failure modes interaction in composite laminated structures subject to a low velocity impact. As a matter of fact, a very fine mesh refinement is required to correctly evaluate the stress state where the impact induced damage onsets. Hence, in order to reduce the computational cost without compromising the accuracy of results, a global/local approach, characterized by a very refined mesh in the critical impact region interacting with a coarser mesh in the rest of the geometrical domain, has been implemented in the FE model. In the present work, Multi-Point-Constraints (MPC) has been used to link the refined local domain to the coarse global domain without using transition meshes. The implementation and the analyses have been performed in the ABAQUSA (R) FE environment.

Purified recombinant Parj1 and Parj2 allergens bind an IgE repertoire common to the Parietaria species, allowing their use as marker molecules for diagnosis and therapy of allergic disease induced by the Urticaceae family. Preclinical studies on the in vivo immunogenicity of recombinant Parj1, Parj2 and their isoforms indicated differential capacity to induce IgG1 antibody responses, as indication of potential clinical use. A recombinant hetero-dimeric hybrid derivative (PjED), encompassing the shorter Parj1 isoform (Parj1.0201) and Parj2 allergen, was characterised. In vivo immunisation with PjED induces IgG1 antibodies capable of binding all the isoforms of Parietaria major allergens, overcoming the poor immunogenicity of single monomeric allergens. This feature makes PjED a promising candidate molecule to be further characterised for clinical applications in the treatment of Parietaria allergy.

Background: Allergy is an immunological disorder affecting about 25% of the population living in the industrialized countries. Specific immunotherapy is the only treatment with a long-lasting relief of allergic symptoms and able to reduce the risk of developing new allergic sensitizations and inhibiting the development of clinical asthma in children treated for allergic rhinitis. Methods: By means of DNA recombinant technology, we were able to design a head to tail dimer expressing disulphide bond variants of the major allergen of the Parietaria pollen. IgE binding activity was studied by Western blot, ELISA inhibition assays and the skin prick test. T cell recognition was studied by peripheral blood mononuclear cell proliferation. The immunogenicity of the hybrid was studied in a mouse model of sensitization. Results: In vitro and in vivo analysis showed that the disruption of specific cysteine residues in both allergens caused a strong reduction in IgE binding activity of the PjEDcys hybrid. In addition,we were able to show that a reduction in the IgE epitope content profoundly reduced the anaphylactic activity of the hybrid (from 100 to 1,000 times less than wild-type allergens) without interfering with the T cell recognition. Sera from BALB/c mice immunized with the hybrid were able to bind the natural Parietaria allergens and to inhibit the binding of human IgE to wild-type Par j 1 and Par j 2 allergens up to 90%. Conclusion: Our results demonstrate that hybrid-expressing disulphide bond variants of the major allergens of the Parietaria pollen displayed reduced allergenicity and maintained T cell reactivity for induction of protective antibodies.

Summary 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. Objective To develop and characterize a murine model of IgE-mediated soybean sensitization induced by intragastric immunization, in the presence of Cholera Toxin, with wild-type soybean extract (wt-SE) or with genetically modified soybean extract (gm-SE). Methods Balb/c mice born in our animal facilities, from females fed on soy-free food, were fed with the same soy-free food and used in all the experiments. Mice were sensitized by gavages with soybean extracts, and allergen-specific IgE and IgG responses were studied by direct ELISA and ELISA inhibition. Antigen-specific cell proliferation and cytokine production were evaluated in spleen cell cultures. Results Sensitization with both soybean extracts induced high levels of antigen-specific IgE and IgG1 and low levels of specific IgG2a. Both wt-SE and gm-SE were able to inhibit the binding of specific IgE from mice immunized with gm-SE to the same antigen used for the ELISA coating. A comparable proliferative response was obtained with the homologous as well as with the heterologous extracts. Conclusion In sensitized mice, we observed a predominantly T-helper type 2 (Th2)-type immune response, with increased soybean-specific IgE and IgG1 antibodies and a concomitant increase of IL-4 and IL-5 production. Results obtained by specific IgE ELISA inhibition and by antigen-specific T cell proliferation demonstrated that wt-SE and gm-SE shared B and T epitopes. The present murine model of soybean sensitization established by the oral route should provide valuable information about risk assessment for food allergy from new proteins of genetically modified foods.

Summary Background Histamine skin reactivity (HSR, the dimension of the skin weal elicited by histamine 10 mg/mL) is a variable that differs in children from different European countries and increases over time in the same place (Italy). Objective In this epidemiologic study, we investigated to what extent differences in HSR influence the relationship between positive allergen skin prick tests (ASPTs) and serum-specific IgE concentrations. Methods Between October 2001 and February 2002, 591 unselected 9–10-year-old schoolchildren drawn from five small towns in central Poland (Starachowice), central Italy (Ronciglione, Guardea) and Libya (Al-Azyzia, near the Mediterranean sea and Samno, 900 km south of the coast) were analysed for histamine, common ASPT and for serum total and specific IgE. Results HSR differed markedly in children from the three countries (Libya>Italy>Poland) whereas serum total IgE concentrations remained the same. The prevalence of children with measurable serum specific IgE (0.35 kU) or with a positive ASPT for five common allergens was high in Italy, lower in Poland and far lower in Libya. A 3-mm ASPT weal corresponded to a serum-specific IgE concentration that was two to threefold higher in children with low HSR compared with children with high HSR (P=0.008). Conclusion These findings suggest that HSR – a variable that differs in schoolchildren populations from the three countries studied – independently influences the results of ASPT and its influence should be considered when ASPT are assessed in international studies. The HSR differences found in the populations reported here probably reflect a complex, dynamic, environmental interaction that should be monitored in the different parts of the world.

Summary Background Par j 1 represents the major allergenic component of Parietaria judaica pollen. Its three-dimensional structure is stabilized by four disulphide bridges. A family of three-dimensional mutants of the recombinant Par j 1 (rPar j 1) allergen, showing reduced allergenicity and retained T cell recognition has been recently developed by site-directed mutagenesis. Objective To develop and characterize a murine model of IgE sensitization to rPar j 1. To evaluate similarities between the murine model and the human IgE response. To investigate in this model the recognition of a hypoallergenic mutant of Par j 1, and to study the immune responses elicited in mice by the mutant itself. Methods BALB/c mice were sensitized by two intraperitoneal immunizations with rPar j 1 in alum on days 0 and 21. Allergen-specific serum IgE and IgG responses were studied by direct ELISA and immunoblotting, ELISA inhibition and competitive ELISA. Cell proliferation was evaluated in splenocyte cultures. Results Sensitization with rPar j 1 induced high levels of IgE and IgG1 vs. low levels of IgG2a. Mouse antibodies specific to rPar j 1 were able to compete with human IgE for recognition of rPar j 1. IgE from mice immunized with rPar j 1 showed a significantly reduced binding activity towards the hypoallergenic variant rPjC, which lacks three disulphide bridges. On the contrary, rPjC was recognized by IgG1 and IgG2a antibodies as well as rPar j 1. The proliferative response to rPjC by splenocytes from mice immunized with rPar j 1 was comparable to that stimulated by rPar j 1. Immunization with rPjC induced low levels of IgE antibodies to the rPjC itself, while IgG and proliferative responses were similar to those induced by rPar j 1. Conclusion Conformational variants of allergens, displaying reduced allergenicity accompanied by retained IgG and T cell recognition, offer a safe, specific and flexible approach to immunotherapy of type I allergy. Our mouse model of IgE sensitization to a recombinant allergen, mimicking the human response to its native counterpart, could provide valuable information for pre-clinical testing of such hypoallergenic molecules.

Summary Background Cypress pollinosis is an important cause of respiratory allergies. Recently, the Cupressus arizonica major allergen, Cup a1, has been cloned and expressed. The native counterpart of this allergen has been purified and characterized by our group. It has been suggested that sugar moieties play a role in the in vitro IgE binding on Cupressus arizonica pollen extract. Objective To characterize the immunoreactivity of the recombinant major allergen in comparison with its native counterpart. To evaluate the role of carbohydrate moieties in the IgE-mediated in vitro histamine release from basophils by using the native glycosylated Cup a1 as compared with the recombinant one. Methods Recombinant Cup a1 was expressed in E. coli. IgE reactivity of Cupressaceae-allergic patients on the native as well as the recombinant molecule was investigated by immunoblotting, ELISA experiments and histamine release test from passively sensitized basophils. Results Fourteen out of 17 Cup a1-positive sera had IgE antibodies reactive with the native molecule only and lost their reactivity after periodate deglycosylation of the allergen. Moreover, only native molecule was capable of inducing histamine release by this group of sera. Both the recombinant and the native molecules were recognized by three out of the 17 sera and were equally capable of triggering degranulation. Conclusion A large number of sera reactive with the major allergen recognize carbohydrate epitopes only. IgE from these sera are able to induce histamine release from basophils and they might play a functional role in the clinical symptoms of allergy.

Background: Airborne insect antigens represent important aeroallergens which have been widely investigated. Although it has been demonstrated that house dust contains significant silverfish (Lepisma saccharina) levels, none of the extracts obtained so far has been extensively characterized. Thus, we have prepared and characterized a silverfish extract and investigated its IgE-reactive components by testing the reactivity of sera from patients allergic to inhalant insect allergens. Methods: The extract from silverfish insect bodies was prepared by homogenizing frozen silverfish in Tris-HCl buffer. The soluble material (Sup) was filtered and the insoluble material (Ppt) was resuspended in 100 mM Tris pH 10.6. The two fractions were characterized by biochemical and immunochemical methods. IgE reactivity was investigated on both fractions before and after periodate treatment. Results: Protein content and total carbohydrates was 2 and 3% w/w for Sup and 1 and 0.3% w/w for Ppt. The SDS-PAGE profile of the two fractions showed a different pattern in the MW range of 5–175 kD. Sup and Ppt, probed with allergic sera, showed a complex pattern of IgE reactivity. When periodate-treated fractions were tested, IgE reactivity was either completely abrogated, reduced or not affected, depending on the allergic serum employed. Conclusions: The results obtained indicate that the classic aqueous-extraction procedures that have been used up to now for other insects might not be completely satisfactory, since several allergenic components are not soluble at the normally used pH. We developed a dedicated extraction procedure allowing the detection of a certain degree of reactivity in sera negative to allergens extracted following classic procedures.

Composite structures are increasingly finding more applications in the aeronautical field as well as the automotive one, thanks to their low weight – performance ratios, in terms of strength and stiffness. However, composite materials, as well known, are characterized by a critical behavior in terms of detectability of damage and performances of damaged components. A critical aspect related to damaged composite structures is, for sure, the repair aimed to restore the original stiffness and strength characteristics of the component depending on the damage typology and location. In this paper, a preliminary repair design tool is presented. The tool is aimed to help the designer by suggesting different repair typologies and proper repair size. This tool, by means of optimization analyses can provide the best repair solution with minimal adhesive shear stress and size of the repair patch. The tool has been tested against a literature case study on multistep composite-metal joints.

In this paper, a numerical study on the skin–stringer debonding growth in composite panels under compressive load is presented. A novel numerical procedure, for the selection of proper material parameters governing the traction–separation law in Cohesive Zone Model (CZM) based elements, is introduced and demonstrated. Indeed, the proposed procedure uses Virtual Crack Closure Technique (VCCT) based FEM analyses on Double Cantilever Beam (DCB) and End Notched Flexure (ENF) specimen to characterize the traction–separation law, respectively, for fracture mode I and mode II. The established traction–separation laws are then applied to composite structures containing inter-laminar damages modeled by cohesive elements. To validate the proposed approach, a single stringer panel under compression with an artificial debonding between skin and stringer, has been considered. The numerical results, in terms of displacements and debonding size as a function of applied compressive load, have been compared to experimental data available in literature providing a good numerical–experimental correlation.

Carbohydrate epitopes are capable of binding human IgE from allergic subjects and these epitopes play a role in the cross-reactivity between allergens from unrelated sources. A monoclonal antibody (5E6), specific for a carbohydrate epitope detectable on components of Cupressus arizonica pollen extract, has been produced and characterized. To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects. To investigate the presence of such carbohydrate IgE determinant in extracts from 21 pollen species belonging to 16 taxonomically related and unrelated families, by means of the monoclonal antibody. IgG-depleted fraction from protein G-purified human allergic serum was obtained. The monoclonal antibody and the IgE from the purified fraction were tested on two glycoproteins, polyamine oxidase and ascorbate oxidase, adsorbed on the ELISA plates. The relationship between the monoclonal- and the IgE-recognized epitopes was investigated by ELISA-competition experiments. Analysis of the distribution of this carbohydrate epitope was performed by direct binding of the monoclonal antibody onto the various extracts. The monoclonal antibody and the IgE were able to bind carbohydrate epitopes on the two plant glycoproteins, ascorbate oxidase and polyamine oxidase. Polyamine oxidase shows only one N-glycosilation site whose carbohydrate moiety seems to be composed of a branched chain of seven ordered sugars, i.e. two N-acetyl-D-glucosamine-, three mannose-, one fucose- and one xylose-residues. This structure bears the epitope recognized by mAb 5E6. Human IgE from the IgG-depleted fraction were found capable of inhibiting the monoclonal antibody binding. The allergenic epitope identified was shared by a large number of extracts with different levels of reactivity (OD490 ranging from 0.110 to 2.060). Our data support the finding that a monoclonal antibody specific for a carbohydrate epitope of Cupressus arizonica pollen extract detects an epitope which is also recognized by IgE from allergic subjects. This characterized reagent could be a useful tool for studying distribution of cross-reactive carbohydrate determinants in allergenic pollen extracts and their components.

Background: Cow's milk allergy is a common disease of infancy and early childhood. If the baby is not breast-fed, a substitute for cow's milk formula is necessary. Objective: The aim of this study was to investigate, in vitro and in vivo, the allergenicity of mare's milk in a population of selected children with severe IgE-mediated cow's milk allergy. Methods: Twenty-five children (17 male and 8 female) aged 19 to 72 months (median age 34 months) with IgE-mediated cow's milk allergy were selected for this study. All the children underwent skin prick tests with cow's milk and mare's milk and double-blind placebo-controlled oral food challenge (DBPCOFC) with fresh cow's milk, fresh mare's milk, and, as placebo, a soy formula (Isomil, Abbott, Campoverde, Italy). We performed immunoblotting of cow's and mare's milk developed with IgE from allergic children. Results: All the children showed strong positive skin test responses to cow's milk (4+); 2 children had positive skin test responses to mare's milk (2+). All children had positive DBPCOFCs to cow's milk; one child had a positive DBPCOFC to mare's milk. No children reacted to the placebo (Isomil). In the cow's milk, some proteins are able to strongly react with human IgE; when the sera are tested with mare's milk, the bands corresponding to the same proteins are recognized by a lower percentage of sera. Conclusion: These data suggest that mare's milk can be regarded as a good substitute of cow's milk in most children with severe IgE-mediated cow's milk allergy. It would be prudent, however, to confirm its tolerability by a supervised titrated oral challenge test. (J Allergy Clin Immunol 2000;105:1031-4.)

Lymphocytosis is a marker of subcutaneous interleukin (IL)-2 therapy efficacy, whereas baseline elevated inflammatory indices were noticed in IL-2-resistant disease. The aim of this study was to analyse the relationship between pretreatment circulating values of IL-6, neopterin, sIL-2R, ESR and the changes in lymphocyte number in response to IL-2 administration. Twenty metastatic renal cell cancer patients were treated with subcutaneous IL-2 immunotherapy (6 000 000 IU day−1 for 6 days per week for 4 weeks); tumour response consisted of partial response (PR) in four patients, stable disease (SD) in eight patients and progressive disease (PD) in eight patients. Abnormally high pretreatment values of each marker were found as follows: IL-6 in seven patients, neopterin in nine patients, sIL-2R in 13 patients. In response to IL-2 immunotherapy, a significantly higher mean increase in lymphocyte number and a higher percentage of patients with tumour response or stable disease were observed when pretreatment values of IL-6, neopterin and sIL-2R were within the normal range, in comparison to patients with high values for these markers. The pretreatment excess of these serum inflammatory markers seems to negatively influence both the host and tumour response to IL-2 administration, by preventing the IL-2-induced lymphocytosis and resulting in tumour progression. Further studies are requested to verify if overall survival and quality of life may depend on pretreatment host immune status and/or lymphocyte response after IL-2 administration. © 1999 Cancer Research Campaign

Background The major allergens in Parietaria pollen, Par j 1 and Par j 2, have been identified as lipid transfer proteins. The family of the Par j 1 allergens is composed of two isoforms, which differ by the presence of a 37 amino acid peptide (Par37) exclusive to the Par j 1.0101 isoform. The goal of this study was to elucidate the biological properties of the Par37 peptide. Methods In silico analysis, spectrofluorimetric experiments and in vitro cell culture assays were used to identify the biological properties of Par37. In addition, a mouse model of sensitization was used to study the influence of Par37 in the murine immune response. Results In silico analysis predicted that Par37 displays characteristics of a host defence peptide. Spectrofluorimetric analysis, real-time PCR and ELISA assays demonstrated that Par37 possesses an LPS-binding activity influencing cell signalling in vitro. In RAW264.7 cells, LPS-induced IL-6 and TNF-α transcription and translation were inhibited after preincubation with Par37. Consistent with these data, inhibition of IFN-γ secretion was observed in murine spleen cells and in human PBMC. Finally, mice immunized with the two Par j 1 isoforms differing in the presence or absence of the Par37 peptide showed different immunological behaviours in vivo. Conclusions This study demonstrates that the Par j 1.0101 allergen displays LPS-binding activity due to the presence of a 37 amino acid COOH-terminal region and that this region is capable of influencing cytokine and antibody responses in vitro and in vivo.

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 cross-reactivity 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. (J ALLERGY CLIN IMMUNOL 1996;98:797-804.)

Two monoclonal antibodies (MoAb) specific for Parietaria judaica allergenic components were selected on the basis of their capability to recognize either the Parietaria judaica major allergen (MoAb 1A6/1D1) or several other allergenic components (MoAb 1A4/2F8) except the major allergen. These two antibodies, either individually or combined, were used to develop an ELISA-inhibition system using a reference Parietaria judaica extract (in-house Reference preparation, IHR). The assays performed with these reagents were firstly standardized by testing the IHR several times. A good reproducibility, evaluated both at the level of 50% inhibition values, and in terms of analysis of the variance of the slopes of the regression curves, was obtained. Subsequently, the potency of several Parietaria judaica extracts, either obtained by manufacturing companies or produced in other laboratories, was evaluated by these tests. Data obtained by interpolation with the IHR values and expressed in terms of arbitrary units (AU) were compared with those obtained by classical human IgE inhibition, performed with sera from allergic patients. Results indicate that the monoclonal antibodies produced in our laboratory can be successfully employed, either individually or combined, in the standardization of allergenic preparations in addition to, and possibly replacing, the classical IgE-based standardization procedures which require human specimens often available in limited amounts only.

SummaryBackground Parietaria pollen is one of the major cause of pollinosis in the southern Europe. Specific immunotherapy is the only treatment able to modify the natural outcome of the disease restoring a normal immunity against allergens. Methods We designed a recombinant molecule (PjEDloop1) comprised of genetic-engineered variants of the major allergens of the Parietaria pollen (Par j 2/Par j 1). Purity and chemical–physical properties of the derivative were analysed by RP-HPLC chromatography and Photon Correlation Spectroscopy. Immunological activity was evaluated by means of Western blotting, ELISA inhibition and PBMC proliferation assay in 10 Parietaria allergic patients. Basophil activation was studied in six subjects. The immunogenicity of the hybrid was studied looking at the immune responses induced in a mouse model of sensitization. Results The PjEDloop1 hybrid was produced as a purified recombinant protein with high stability in solution. Western blot, ELISA inhibition and basophil activation test showed that the PjEDloop1 displays a remarkable reduced IgE binding and anaphylactic activity. CD3 reactivity was conserved in all patients. Mice immunization with the rPjEDloop1 induced antibodies and T cell responses comparable to that obtained by the wild type allergens. Such antibodies shared the specificities to rPar j 1 and rPar j 2 with human IgE antibodies. Conclusion Our results demonstrated that a mutant hybrid expressing genetically engineered forms of the major P. judaica allergens displayed reduced allergenicity and retained T cell reactivity for the induction of protective antibodies in vaccination approaches for the treatment of Parietaria pollinosis.

The diagnosis and therapy of allergic disorders are usually performed with crude extracts which are a heterogeneous mixture of proteins with different allergenic potency. The knowledge of the allergenic composition is a key step for diagnostic and therapeutic options. Parietaria judaica pollen represents one of the main sources of allergens in the Mediterranean area and its major allergens have already been identified (Par j 1 and Par j 2). In addition, inhibition studies performed using a calcium-binding protein (CBP) from grass pollen (Phl p 7) showed the presence of a homologue of this cross-reactive allergen in the Parietaria extract. Screening of a cDNA library allowed us to isolate a 480 bp cDNA containing the information for an 87 AA long protein with high level of homology to calcium-binding proteins from other allergenic sources. It was expressed as a recombinant allergen in Escherichia coli and purified by affinity chromatography. Its expression allowed us to study the prevalence of this allergen in a population of allergic patients in southern Europe. Immunoblotting and inhibition studies showed that this allergen shares a pattern of IgE epitopes in common with other 2-EF-hand calcium-binding proteins from botanically non-related species. The immunological properties of the Pj CBP were investigated by CD63 activation assay and CFDA-SE staining. In conclusion, DNA recombinant technology allowed the isolation, expression and immunological characterization of a cross-reactive calcium-binding protein allergen from Parietaria judaica pollen.

Allergy is an immunological disorder affecting about 25% of the population living in the industrialized countries. Specific immunotherapy is the only treatment with a long-lasting relief of allergic symptoms and able to reduce the risk of developing new allergic sensitizations and inhibiting the development of clinical asthma in children treated for allergic rhinitis.By means of DNA recombinant technology, we were able to design a head to tail dimer expressing disulphide bond variants of the major allergen of the Parietaria pollen. IgE binding activity was studied by Western blot, ELISA inhibition assays and the skin prick test. T cell recognition was studied by peripheral blood mononuclear cell proliferation. The immunogenicity of the hybrid was studied in a mouse model of sensitization.In vitro and in vivo analysis showed that the disruption of specific cysteine residues in both allergens caused a strong reduction in IgE binding activity of the PjEDcys hybrid. In addition,we were able to show that a reduction in the IgE epitope content profoundly reduced the anaphylactic activity of the hybrid (from 100 to 1,000 times less than wild-type allergens) without interfering with the T cell recognition. Sera from BALB/c mice immunized with the hybrid were able to bind the natural Parietaria allergens and to inhibit the binding of human IgE to wild-type Par j 1 and Par j 2 allergens up to 90%.Our results demonstrate that hybrid-expressing disulphide bond variants of the major allergens of the Parietaria pollen displayed reduced allergenicity and maintained T cell reactivity for induction of protective antibodies.

Summary Background We aimed to evaluate the safety and effectiveness of a conservative approach with short-term chemotherapy with or without consolidation radiotherapy in primary resectable large-cell gastric lymphoma in patients not requiring emergency surgery at presentation. Patients and Methods Seventeen consecutive patients presenting with resectable primary large-cell lymphoma of the stomach not requiring immediate surgery were initially treated with chemotherapy with or without consolidation radiotherapy. Subtotal or total resection of the stomach was planned only as salvage treatment for those patients who failed locally, or as emergency surgery in instances of acute iatrogenic complications of treatment. Chemotherapy included four to six cycles of an anthracycline-containing regimen, and consolidation radiotherapy was planned on the entire stomach and surrounding lymph node areas for complete responders readily capable of compliance with a daily treatment schedule at our Institution. Results None of the patients in the present series experienced acute iatrogenic morbidity or mortality from local complications. After a median follow-up of almost six years, two patients failing first-line chemotherapy have died of progressivelymphoma, while 15 patients are well and currently disease-free. Conclusions Up-front chemotherapy as initial treatment for primary gastric large-cell lymphoma appears to be a safe and effective treatment by which most patients can probably be spared surgical gastrectomy. Consolidation radiation therapy on the stomach can probably improve on the effectiveness of chemotherapy alone. More experience is needed to elucidate the prognostic factors, treatment-related long-term toxic effects and the feasibility of such a treatment administered outside of highly specialized institutions.

Summary Background The presence of specific IgE antibodies to invertebrates is common among patients with rhinitis and asthma. Tropomyosin has been described as an invertebrate cross-reactive allergen. We have recently characterized an allergenic extract from silverfish (Lepisma saccharina). Since this insect could be a new source of tropomyosin in the indoor environment, we have thought important to clone and characterize the tropomyosin from it. Methods Recombinant tropomyosin was cloned and characterized by means of immunoblotting with tropomyosin-specific monoclonal antibodies, rabbit polyclonal antibodies and IgE from allergic patients. Its allergenic activity was investigated in histamine release assays. Immunoblotting and ELISA inhibition were carried out to identify the natural tropomyosin in the silverfish extract and to study the cross-reactivity among other arthropod tropomyosins. Results Tropomyosin-specific antibodies recognized in immunoblotting the natural tropomyosin in the insoluble fraction of silverfish extract. The silverfish tropomyosin (Lep s 1) was cloned and fully expressed. It shared high homology with other arthropod tropomyosins. rLep s 1 was recognized by tropomyosin-specific monoclonal and polyclonal antibodies and by IgE of allergic patients. It was able to inhibit the IgE binding to the insoluble fraction of silverfish extract, and to induce histamine release by an arthropod-allergic serum. Inhibition experiments revealed IgE cross-reactivity between rLep s 1 and other arthropod tropomyosins. Conclusion rLep s 1 is the first allergen cloned and characterized from silverfish extract. It enabled us to identify the natural counterpart in the insoluble fraction of silverfish extract, suggesting that the tropomyosin is not readily extractable with a classic aqueous extraction procedure. rLep s 1 displayed biological activity, suggesting that it could be regarded as a useful tool to study the role of silverfish tropomyosin in the sensitization to invertebrate allergic sources.

Cypress pollinosis is an important cause of respiratory allergies. Recently, the Cupressus arizonica major allergen, Cup a1, has been cloned and expressed. The native counterpart of this allergen has been purified and characterized by our group. It has been suggested that sugar moieties play a role in the in vitro IgE binding on Cupressus arizonica pollen extract.To characterize the immunoreactivity of the recombinant major allergen in comparison with its native counterpart. To evaluate the role of carbohydrate moieties in the IgE-mediated in vitro histamine release from basophils by using the native glycosylated Cup a1 as compared with the recombinant one.Recombinant Cup a1 was expressed in E. coli. IgE reactivity of Cupressaceae-allergic patients on the native as well as the recombinant molecule was investigated by immunoblotting, ELISA experiments and histamine release test from passively sensitized basophils.Fourteen out of 17 Cup a1-positive sera had IgE antibodies reactive with the native molecule only and lost their reactivity-after periodate deglycosylation of the allergen. Moreover, only native molecule was capable of inducing histamine release by this group of sera. Both the recombinant and the native molecules were recognized by three out of the 17 sera and were equally capable of triggering degranulation.A large number of sera reactive with the major allergen recognize carbohydrate epitopes only. IgE from these sera are able to induce histamine release from basophils and they might play a functional role in the clinical symptoms of allergy.

Airborne insect antigens represent important aeroallergens which have been widely investigated. Although it has been demonstrated that house dust contains significant silverfish (Lepisma saccharina) levels, none of the extracts obtained so far has been extensively characterized. Thus, we have prepared and characterized a silverfish extract and investigated its IgE-reactive components by testing the reactivity of sera from patients allergic to inhalant insect allergens.The extract from silverfish insect bodies was prepared by homogenizing frozen silverfish in Tris-HCl buffer. The soluble material (Sup) was filtered and the insoluble material (Ppt) was resuspended in 100 mM Tris pH 10.6. The two fractions were characterized by biochemical and immunochemical methods. IgE reactivity was investigated on both fractions before and after periodate treatment.Protein content and total carbohydrates was 2 and 3% w/w for Sup and 1 and 0.3% w/w for Ppt. The SDS-PAGE profile of the two fractions showed a different pattern in the MW range of 5-175 kD. Sup and Ppt, probed with allergic sera, showed a complex pattern of IgE reactivity. When periodate-treated fractions were tested, IgE reactivity was either completely abrogated, reduced or not affected, depending on the allergic serum employed.The results obtained indicate that the classic aqueous-extraction procedures that have been used up to now for other insects might not be completely satisfactory, since several allergenic components are not soluble at the normally used pH. We developed a dedicated extraction procedure allowing the detection of a certain degree of reactivity in sera negative to allergens extracted following classic procedures.

This study aimed to evaluate the influence of environmental and sociodemographic factors and the effect of smoking, alcohol, and dietary habits on the risk of gastric intestinal metaplasia (IM) in Helicobacter pylori-infected subjects.The investigation was based on 2598 consecutive volunteer blood donors tested for the presence of antibodies against H. pylori from March 1995 to March 1997. Endoscopy with multiple biopsies was offered to all H. pylori-positive, symptomatic subjects. The presence or absence of IM was diagnosed by gastric biopsies. A serologically H. pylori-positive subject with gastric IM was defined as a case, whereas serologically H. pylori-positive subjects without IM were used as controls. All patients answered a detailed questionnaire collecting sociodemographic characteristics and smoking, alcohol drinking, and dietary habits. Odds ratios (ORs) and their 95% CIs were estimated by unconditional logistic regression, including terms for age and sex, to assess the association between the data collected and IM.Three hundred forty-four subjects with serological H. pylori infection and upper-GI symptoms underwent GI endoscopy, during which biopsies were taken for histological diagnosis. Histology revealed metaplasia in 74 subjects (21.5%). Incomplete IM was found in 37.8% of these cases. No significant associations were found between IM and anthropometric or sociodemographic factors. There was a significant association between age and IM (chi2 for trend, 6.67; p value, 0.009). Current smokers of over 20 cigarettes per day had a 4-fold risk of IM (OR, 4.75, 95% CI, 1.33-16.99). A 2-fold increased risk was found for high butter consumers (OR, 2.17; 95% CI, 1.14-4.11). No significant specific associations were found between the variables studied and complete or incomplete IM.This study found that smoking and high butter consumption may increase the risk of having gastric IM in H. pylori-positive subjects.

Carbohydrate epitopes are capable of binding human IgE from allergic subjects and these epitopes play a role in the cross-reactivity between allergens from unrelated sources. A monoclonal antibody (5E6), specific for a carbohydrate epitope detectable on components of Cupressus arizonica pollen extract, has been produced and characterized. To study the relationship between the epitopes recognized by the monoclonal antibody and by IgE from allergic subjects. To investigate the presence of such carbohydrate IgE determinant in extracts from 21 pollen species belonging to 16 taxonomically related and unrelated families, by means of the monoclonal antibody. IgG-depleted fraction from protein G-purified human allergic serum was obtained. The monoclonal antibody and the IgE from the purified fraction were tested on two glycoproteins, polyamine oxidase and ascorbate oxidase, adsorbed on the ELISA plates. The relationship between the monoclonal- and the IgE-recognized epitopes was investigated by ELISA-competition experiments. Analysis of the distribution of this carbohydrate epitope was performed by direct binding of the monoclonal antibody onto the various extracts. The monoclonal antibody and the IgE were able to bind carbohydrate epitopes on the two plant glycoproteins, ascorbate oxidase and polyamine oxidase. Polyamine oxidase shows only one N-glycosilation site whose carbohydrate moiety seems to be composed of a branched chain of seven ordered sugars, i.e. two N-acetyl-D-glucosamine-, three mannose-, one fucose- and one xylose-residues. This structure bears the epitope recognized by mAb 5E6. Human IgE from the IgG-depleted fraction were found capable of inhibiting the monoclonal antibody binding. The allergenic epitope identified was shared by a large number of extracts with different levels of reactivity (OD490 ranging from 0.110 to 2.060). Our data support the finding that a monoclonal antibody specific for a carbohydrate epitope of Cupressus arizonica pollen extract detects an epitope which is also recognized by IgE from allergic subjects. This characterized reagent could be a useful tool for studying distribution of cross-reactive carbohydrate determinants in allergenic pollen extracts and their components.

Background: A rapid method for the purification of the major 43-kDa all_ergen of Cupressus arizonica pollen, Cup a 1, was developed. Methods: The salient feature was a wash of the pollen in acidic buffer, followed by an extraction of the proteins and their purification by chromatography. Immunoblotting, ELISA, and lectin binding were tested on both the crude extract and the purified Cup a 1. Biochemical analyses were performed to assess the Cup a 1 isoelectric point, its partial amino-acid sequence, and its glycan composition. Results: Immunochemical analysis of Cup a 1 confirmed that the all_ergenic reactivity is maintained after the purification process. Partial amino-acid sequencing indicated a high degree of homology between Cup a 1 and all_ergenic proteins from the Cupressaceae and Taxodiaceae families displaying a similar molecular mass. The purified protein shows one band with an isoelectric point of 5.2. Nineteen out of 33 sera (57%) from patients all_ergic to cypress demonstrated significant reactivity to purified Cup a 1. MALDI-TOF mass spectrometry indicated the presence of three N-linked oligosaccharide structures: GnGnXF3 (i.e., a horseradish peroxidase-type oligosaccharide substituted with two nonreducing N-acetylglucosamine residues), GGnXF3/GnGXF3 (i.e., GnGnXF with one nonreducing galactose residue), and (GF)GnXF3/Gn(GF)XF3 (with a Lewisa epitope on one arm) in the molar ratio 67:8:23. Conclusions: The rapid purification process of Cup a 1 all_owed some fine studies on its properties and structure, as well as the evaluation of its IgE reactivity in native conditions. The similarities of amino-acid sequences and some complex glycan stuctures could explain the high degree of cross-reactivity among the Cupressaceae and Taxodiaceae families.

A reduction of IgE immunoreactivity after periodate-treatment has been previously reported for various glycoprotein allergens.The aim of this study was to investigate the role of glycan moiety of a C. arizonica extract in the binding of patients' IgE and to identify the carbohydrates possibly involved.The reactivity of IgE with C. arizonica extract, before and after periodate-treatment, was evaluated by immunoblotting and ELISA inhibition. The specificity of carbohydrate-reactive IgE was evaluated by ELISA using unrelated glycoproteins with known sugar composition and structure, such as pineapple bromelain, honeybee venom phospholipase A2, and ovalbumin, before and after periodate treatment.When periodate-treated C. arizonica extract was probed after SDS-PAGE and immunoblotting with patients' IgE, no reactivity could be detected. Furthermore, a very poor inhibitory activity of the periodate-treated C. arizonica extract as compared with the untreated sample could be observed in the ELISA inhibition experiments performed using C. arizonica extract as antigen. When phospholipase A2 and bromelain were used as antigens in ELISA, they were recognized by patients' IgE, whereas ovalbumin was negative. Treatment of phospholipase A2 and bromelain with periodate completely abolishes the IgE reactivity.A large portion of the IgE reactivity of Cupressaceae-allergic subjects appears to be associated with sugar moieties of C. arizonica extract which appear to be shared by bromelain and phospholipase A2, thus suggesting that the IgE of patients reacting with such epitopes probably react with beta 1 --2 xylose, alpha 1 --3 fucose and/or alpha 1 --6 fucose.

Species of the Cupressaceae family are a worldwide cause of respiratory allergies. We used monoclonal antibodies (mAbs) to investigate the presence and the nature of cross-reacting epitopes shared by various components within Cupressus arizonica pollen extract (CaE) or by CaE and pollen extract from C. sempervirens (CsE). mAbs were produced in mice immunized with whole CaE (4A6 and 5E6) or with the major allergen components (2D5). Their reactivity was investigated by ELISA and immunoblotting before and after CaE periodate treatment. Cross-reactivity was evaluated by ELISA inhibition and immunoblotting. mAbs 2D5 and 4A6 recognized periodate-resistant epitopes, whereas the mAb 5E6 reacted with a periodate-sensitive determinant. The former mAbs recognized epitopes present on CaE major allergen and also shared by other components. mAb 5E6 showed a spread reactivity on CaE, with exclusion of the major allergen. When the three mAbs were tested with CsE, a restricted pattern of reactivity to mAbs 2D5 and 4A6 was obtained, whereas mAb 5E6 maintained a spread reactivity. The CaE major allergen is represented by two components recognized by human IgE and sharing common epitopes, as proven by mAbs reactivity. The use of these mAbs demonstrates that cross-reactivity within CaE components and between CaE and CsE is due to the presence of periodate-sensitive as well as -resistant epitopes.

Disorders of essential elements arise from inadequate intake, genetic defects, excessive exposure, or impaired elimination. Severe deficiency is rare, but aspecific symptoms and reduced protection against risk factors have been ascribed to suboptimal intake of trace elements. Inherited disturbances of the metabolism of copper, zinc, and molybdenum determine severe diseases, which may have fatal outcomes. Besides occupational and environmental exposure, metal toxicity has been reported in clinical conditions such as parenteral nutrition and hemodialysis. The prevention, diagnosis, and monitoring of pathological conditions related to trace elements, as well as pharmacokinetic studies of metallodrugs and therapy monitoring, rely mainly on the dosage of metals in appropriate compartments, such as blood, urine, and tissues (hair, nails, biopsies, and postmortem samples). However, appropriate compartments may not always be amenable for sampling, and determination of the total amount of an element may not be informative or may even be misleading if the compartment volume also undergoes changes or the different chemical forms of the element vary in opposite directions. New biochemical tests may become available for the prevention, diagnosis, and monitoring of disorders of trace elements as analytical techniques gain improved detection and resolution power. In particular, interest is focused on the dosage of biologically active elemental species and the application of stable isotopes in tracer studies.

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.The relationship between pollen extracts from these two species was studied by evaluating the reactivity with polyclonal rabbit antisera and human IgE.The two extracts were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Cross-reactivity was evaluated by ELISA and immunoblotting inhibition experiments.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.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.

It is known that advanced cancer patients may show abnormally low levels of IL-2. The immunotherapy with IL-2 can induce objective tumor regressions, but at present there are no data about the influence of a chronic exogenous IL-2 administration on endogenous secretion of IL-2. This preliminary study was performed to evaluate whether a prolonged IL-2 injection may be able to correct an eventual IL-2 endogenous deficiency in cancer patients. The study included 10 metastatic renal cancer patients, who underwent an immunotherapeutic cycle consisting of IL-2 at 6 million IU/day subcutaneously for 6 days/week for 4 weeks. Serum levels of IL-2 evaluated on venous blood samples collected before and 21 days after the end of IL-2 injection. Before the onset of treatment, abonormally low levels of IL-2 were seen in 6/10 patients. In patients with response or stable disease, mean levels of IL-2 observed 21 days after IL-2 cycle were significantly higher than those seen before therapy, whereas no difference occurred in those who progressed. This preliminary study would suggest that a prolonged subcutaneous injection of low-dose IL-2 may correct an eventual IL-2 endogenous deficiency in advanced cancer patients.

SUMMARY The role of cytokines in human hydatidosJs (Echinococcus granulosus infection) was evaluated in immunoassays determining production of IL-4, IL-10 and interferon-gamma (IFN-γ) in peripheral blood mononuclear cell (PBMC) cultures from 30 hydatid patients and 14 uninfected controls. In ceil cultures from hydatid patients parasite and non-parasite antigen stimulation significantly increased IL-4 production (P·0·005). Spontaneous and milogen-driven IL-4 production was similar in patients and controls. IL-10 and IFN-γ production did not differ statistically in the two groups, even though some hydatid patients produced these cytokines in large amounts. Notably, antigen-driven IFN-γ concentrations were invariably higher in patients than in uninfected controls. Data analysis showed a relationship between IgE and IgG4 responses and parasite-driven cytokine production. High IgE and IgG4 responders produced high IL-4 and IL-10 concentrations. High IgE responders showed decreased IFN-γ production, but high IgG4 responders had IFN-γ levels slightly higher than those of low responders. Cytokine response patterns did not relate to the clinical stage of disease. The significantly increased IL-4 and the high IL-10 concentrations found in PBMC from many hydatid patients in this study are consistent with Th2 cell activation in human hydatidosis. The presence of antigen-driven IFN-γ production in patients with E. granulosus infection implies concurrent intervention of the Th1 or Th0 cell subset.

IgG antibody response to the inhalant allergen Parietaria judaica (Pj) and IgG subclass distribution were studied in 82 normal subjects, divided into three groups according to age (0-1, 1-20, and 20-60 years) and in 32 allergic subjects aged 20-60 years. Both normal and allergic subjects showed an IgG response, and all had IgG1 antibodies specific for PjE. Serum IgG2, IgG3, and IgG4 against PjE were detectable in 36%, 46%, and 22% of normal subjects, and in 58%, 31%, and 65% of allergic subjects, respectively. A significant difference in class distribution between allergic and age-matched normal subjects was found only for IgG4 antibodies against PjE (65% and 17%; P < 0.01). The ELISA results were also analyzed quantitatively, taking into account the relative proportion of specific antibodies. Thus, in normal subjects IgG1 antibodies showed a decreasing trend as the age rose, while no differences according to the age of the subject were found for IgG2 and IgG4. When data from allergic subjects (20-60 years) and the age-matched normal group were compared, they were different for the relative percentage of IgG2 only, showing for this a significantly lower value (P < 0.001). The present data indicate that normal and allergic subjects show differences in the IgG isotype distribution depending on their sensitivity and duration of allergen exposure.