Dimerisation increases the immunogenicity of recombinant Parj1/Parj2 allergens.

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.

The major allergen of the Parietaria pollen contains an LPS-binding region with immuno-modulatory activity.

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.

Characterization of a Par j 1/Par j 2 mutant hybrid with reduced allergenicity for immunotherapy of Parietaria allergy.

BACKGROUND: 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.

Oral therapeutic administration of a probiotic mixture suppresses established Th2 responses and systemic anaphylaxis in a murine model of food allergy.

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-ß 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.

Isolation, expression and immunological characterization of a calcium-binding protein from Parietaria pollen.

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 480bp 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.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

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.

A highly sensitive enzyme-linked immunosorbent assay for idiotype-bearing antibodies.

A sensitive and specific immunoenzyme assay (ELISA) for quantitation of total and cross-reactive idiotype-bearing (CRI) anti-ABA antibodies is described. Total anti-ABA antibodies are directly assessed in ABA-BGG coated polyvinyl wells with enzyme-labelled rabbit anti-mouse immunoglobulins. By interpolation on a standard curve absorbance values give the concentration of anti-ABA antibodies with a sensitivity of 30 ng/ml. CRI+ antibodies are quantitated by inhibition of enzyme-labelled monoclonal CRI+ antibody binding to solid-phase coated rabbit anti-CRI immunoglobulins. The concentration of CRI+ antibodies, evaluated by interpolation on a standard inhibition curve, can be measured at the level of 10 ng/ml. This highly sensitive, rapid, specific and reproducible assay is easily used, with minor modifications, to detect specific antibodies in any idiotype system.

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.

Determinants of Helicobacter pylori seroprevalence among Italian blood donors.

BACKGROUND/AIM: Helicobacter pylori is a worldwide infection. It is estimated that approximately 50% of the general population is affected, but this percentage varies considerably between countries. To investigate the prevalence of H. pylori infection, a cross-sectional epidemiological study, based on the serological determination of the IgG antibodies against H. pylori, was carried out in healthy Italian blood donors by using a commercially available kit. METHODS: From March 1995 to March 1997, a total of 2598 consecutive volunteer blood donors were tested for the presence of antibodies against H. pylori. All patients answered a detailed questionnaire which collected sociodemographic characteristics, and smoking, alcohol drinking and dietary habits. Test-positive subjects with gastrointestinal symptoms underwent endoscopy, with biopsies taken for histological diagnosis. RESULTS: The global prevalence of H. pylori infection in our study was 1161/2598 (45%). It was directly correlated with age (67% in subjects aged > or = 50 years). The prevalence of H. pylori infection was higher in men (46.4%) than women (38.4%), and more frequent in patients with a low educational level, in the lower quintile of height and in the upper quintile of body mass index (BMI). No significant association with smoking and alcohol drinking was found. Inverse associations were found with elevated consumption of milk (chi-square for trend 5.49, P < 0.05), but not other examined food groups. Multivariate analysis selected sex, age, BMI and educational level as the variables independently related to H. pylori infection. CONCLUSION: This study confirms relatively high prevalence of H. pylori seropositivity among Italian healthy adults and points to sex, age, BMI and sociocultural class as persisting determinant features of H. pylori infection.

Reactivation of a normal endogenous secretion of interleukin-2 in metastatic cancer patients by a chronic subcutaneous injection of interleukin-2.

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.

[Functional status of intestinal t lymphocytes, regulatory mechanisms, and their variations in the course of Crohn disease and ulcerative colitis]. / Stato funzionale dei linfociti T intestinali, meccanismi di regolazione e loro variazioni nel corso di morbo di Crohn e colite ulcerosa.

We analyzed the phenotype, proliferative responsiveness, cytokine production and apoptosis susceptibility of lamina propria lymphocytes to different activation pathways. Lamina propria lymphocytes is a population enriched of activated lymphocytes showing a "memory" phenotype. As opposite to peripheral blood lymphocytes, lamina propria lymphocytes show proliferative hyporesponsiveness when stimulated via TCR/CD3 pathway while proliferative response to the CD2 activation pathway is relatively preserved. Under the latter activation pathway, cytokine production, especially IL-4 and IFN-gamma, is higher than that observed in peripheral lymphocytes. When compared to controls, lamina propria lymphocytes isolated from inflammatory bowel disease (Crohn's disease and ulcerative colitis) show distinctive variation in the cytokine production. In particular, Crohn's disease is characterized by an increased production of IFN-gamma, while in ulcerative colitis an increased production of IL-5 is observable. Among the different regulatory mechanisms contributing to maintain immunological homeostasis we analyzed the susceptibility to apoptosis of lamina propria lymphocytes. We found that CD2-activation pathway is regulated by Fas-mediated apoptosis, which regulates proliferation and cytokine production. In inflammatory bowel disease this apoptosis is defective thus contributing to the chronic inflammation and cytokine dysregulation.

Allergens and atopic diseases.

Allergens are molecules normally present in the environment, responsible of inducing the so called atopic diseases. These diseases are characterized by the high production of allergen-specific IgE antibodies, which bind to mast-cells and basophils. The cross-linking of the cell-bound IgE induces the production and/or release of various inflammatory mediators (histamine, leukotrienes, platelet-activating-factor, etc.), which trigger a cascade of events resulting in the allergic syndromes. Once the diagnosis of allergy has been made, two different therapeutic approaches can be followed: a) the pharmacologic management, aimed mainly at preventing or controlling the symptoms resulting from the release of mediators by effector cells; b) the specific immunotherapy, consisting in the administration to the patient of increasing amount of the relevant allergens, opportunely prepared and eventually chemically modified. In order to have the latter approach correctly applied it is necessary to gain a wider knowledge on both regulation of IgE synthesis and allergens characteristics.

Use of monoclonal antibodies for the characterization of allergenic extracts.

The diagnosis of allergic diseases is commonly carried out by using poorly standardized preparations containing allergenic and non-allergenic components. Recently, monoclonal antibody technology has been applied to the biochemical characterization and to the purification of allergens, in order to evaluate their potential role in the standardization of diagnostic and therapeutic extracts. Three main results have been achieved: 1) the characterization of allergenic epitopes recognized by IgE and IgG from patients' sera; 2) the purification by affinity chromatography of relevant allergenic components; 3) the development of immunoassays for the quantitation of allergens in the commercially available extracts used for diagnosis and therapy.

[Endoscopic hemostatic injection treatment of bleeding gastroduodenal ulcer]. / Le traitement injectif hémostatique de l'ulcère gastroduodénal saignant.

Out of 88 patients suffering from bleeding gastroduodenal ulcer 36 with active bleeding or stigmata of recent hemorrhage and shock underwent endoscopic hemostatic injection treatment. There were three rebleedings, two of them from the same bleeding source (chronic duodenal ulcer). These two patients underwent emergency surgery without postoperative complications. A third patient had been operated for perforating gastric ulcer and died from pulmonary embolism. Injection treatment is considered the hemostatic technique of choice in patients with bleeding peptic ulcer.