Angela Bonura

Institute of Immunology, Santiago de Cali, Valle del Cauca, Colombia

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Publications (27)68.15 Total impact

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    ABSTRACT: Allergen specific immunotherapy has been introduced in the clinic more than 100 years showing effectiveness and, so far, it represents the only curative approach to treat allergic disorders ameliorating the symptoms, reducing the medication costs and blocking the onset of new sensitizations. However, some questions are still open regarding to the safety of the treatment and the need to reduce the dose and time of administration to improve the compliance of the patients. All preparations that are currently available may trigger side effects. For these reasons, new formulations and route of administration have been exploited demonstrating that such products presented improved efficacy and safety. Nanotechnology for biomedical applications offers many advantages, such as improved stability and bioavailability, favourable biodistribution profiles and targeting to specific cell populations whose impact on the immune system has been evaluated in animal systems. Nanoparticles interact with the immune system, and the final outcome of this interaction depends on their physico-chemical characteristics. Concerns can be raised when immunotoxic effect are induced, resulting in inflammatory dangerous responses or in the reduction of the normal defensive activity of the immune system. In this paper, we will review the most relevant data about the synthesis of allergen/nanoparticles systems and will discuss their impact on the immune system in terms of immunomodulatory activity and immunotoxicity risk assessment.
    Current pharmaceutical design 08/2015; · 3.45 Impact Factor
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    ABSTRACT: ML superfamily represents a group of proteins playing important roles in lipid metabolism and innate immune response. In this study, we report the identification of the first component of the ML superfamily in the invertebrate Ciona intestinalis by means of a subtractive hybridization strategy. Sequence homology and phylogenetic analysis showed that this protein forms a specific clade with vertebrate components of the Niemann-Pick type C2 protein and, for this reason, it has been named Ci-NPC2. The putative Ci-NPC2 is a 150 amino acids long protein with a short signal peptide, seven cysteine residues, three putative lipid binding site and a three-dimensional model showing a characteristic β-strand structure. Gene expression analysis demonstrated that the Ci-NPC2 protein is positively upregulated after LPS inoculum with a peak of expression 1 hour after challenge. Finally, in-situ hybridization demonstrated that the Ci-NPC2 protein is preferentially expressed in hemocytes inside the vessel lumen. Copyright © 2015. Published by Elsevier Ltd.
    Developmental and comparative immunology 07/2015; 53(1). DOI:10.1016/j.dci.2015.06.018 · 2.82 Impact Factor
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    ABSTRACT: Esperimento pilota mirato all'analisi dell'effetto della stimolazione immunitaria su A. viridis, finalizzato alla produzione di composti biologicamente attivi.
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    ABSTRACT: Applicazione di un protocollo di immuno-stimolazione su individui di Pracambarus clarkii mirato alla produzione di peptidi con attività biologica.
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    ABSTRACT: 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 Author(s) 2015.
    International journal of immunopathology and pharmacology 03/2015; 28(1):142-5. DOI:10.1177/0394632015573920 · 1.62 Impact Factor
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    ABSTRACT: Leptin is involved in the lung epithelial homeostasis. Its role in the nasal tract is largely unknown. Allergic rhinitis (AR) is induced by the allergen exposure leading to consequential structural abnormalities in the nasal epithelium. Topical corticosteroids are recommended as first-line therapy in AR. Parietaria pollen is one of the most important allergenic sources in the southern Europe. In vitro, in human nasal epithelial cell line RPMI 2650, we aimed to determine whether allergen stimulation acts on leptin/leptin receptor pathway and how fluticasone furoate (FF) influences this pathway. The effects of the major allergen recombinant Par j 1 (rPar j 1), of FF, of leptin, and of TGF-β1 on cell proliferation, on leptin/leptin receptor expression and modulation (by clonogenic test, by RT-q-RT-PCR, by immunocytochemistry and by flow-cytometry), and on STAT-3 activation (assessing nuclear translocation by western blot analysis) were assessed. We found that rPar j 1 and TGF-β1 significantly decreased cell proliferation and down-regulated the leptin/leptin receptor pathway, whereas FF and leptin reverted them, both alone and in combination. Furthermore, rPar j 1 reduced, while leptin and FF increased STAT-3 activation. In conclusion, FF and leptin itself are able to preserve nasal epithelial homeostasis restoring the leptin/leptin receptor pathway altered by rPar j 1 exposure.
    Molecular and Cellular Biochemistry 07/2014; 396(1-2). DOI:10.1007/s11010-014-2142-z · 2.39 Impact Factor
  • A Bonura · A Trapani · L Gulino · V Longo · R Valenta · R Asero · P Colombo
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    ABSTRACT: Parietaria judaica pollen is one of the main sources of allergens in the Mediterranean area. Its allergenic composition has been studied in detail showing the presence of two major allergens (Par j 1 and Par j 2) and two minor allergens belonging to the profilin and calcium binding protein families of allergens (Par j 3 and Par j 4, respectively). Clinical reports support the hypothesis of a limited cross-reactivity between profilin from Parietaria and unrelated sources. We screened a P. judaica cDNA library to identify novel forms of profilins with allergenic activity. This strategy allowed us to isolate a 767bp cDNA containing the information for a 131 amino acids protein with homology to profilins from unrelated sources greater than that observed with the already published Parietaria profilins. This profilin was expressed in Escherichia coli as a recombinant protein and its immunological prevalence was studied in a population of Parietaria allergic patients from Southern Europe. Immunoblotting analysis showed that the Parietaria profilin was recognized by IgE from 6.5% of the allergic population. Finally, a selected population of profilin allergic patients was enrolled to demonstrate the cross-reactivity of this novel variant with other profilins from grass and date palm. In conclusion, molecular cloning and immunological studies have allowed the isolation, expression and immunological characterization of a novel cross-reactive profilin allergen from P. judaica pollen named Par j 3.0201.
    Molecular Immunology 10/2013; 57(2):220-225. DOI:10.1016/j.molimm.2013.09.004 · 2.97 Impact Factor
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    ABSTRACT: A subtractive hybridization strategy for the identification of differentially expressed genes was performed between LPS-challenged and naive Ciona intestinalis. This strategy allowed the characterization of two transcripts (Ci8short and Ci8long) generated by the use of two Alternative Polyadenylation sites. The Ci8long transcript contains a protein domain with relevant homology to several components of the Receptor Transporting Protein (RTP) family not present in the Ci8short mRNA. By means of Real Time PCR and Northern Blot, the Ci8short and Ci8long transcripts showed a different pattern of gene expression with the Ci8short mRNA being strongly activated after LPS injection in the pharynx. In situ hybridization analysis demonstrated that the activation of the APA site also influenced the tissue localization of the Ci8short transcript. This analysis showed that the Ci8long mRNA was expressed in hemocytes meanwhile the Ci8short mRNA was highly transcribed also in vessel endothelial cells and in the epithelium of pharynx. These findings demonstrated that regulation of gene expression based on different polyadenylation sites is an ancestral powerful strategy influencing both the level of expression and tissue distribution of alternative transcripts.
    PLoS ONE 04/2013; 8(4):e63235. DOI:10.1371/journal.pone.0063235 · 3.23 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Allergy 03/2013; 68(3):297-303. DOI:10.1111/all.12086 · 6.03 Impact Factor
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    ABSTRACT: In this study we wanted to analyse the pattern of the immune response to the Parietaria major allergen Par j 1 in freshly purified peripheral blood mononuclear cell (PBMC) from healthy subjects. We observed that Par j 1 was capable of inducing IFN-γ production by CD3(-) and CD16(+)/CD56(+) cells exclusively in healthy individuals. Furthermore, a multiparametric analysis allowed us a better definition of two IFN-γ-Par j 1 specific populations (IFN-γ(dim) and IFN-γ(high)) characterized by the presence of different proportions of NKT and NK cells. We also identified the concomitant presence of a subset of IL-10(+) NK cells. Moreover, CFSE staining showed that the Par j 1 preferentially induced the proliferation of CD3(-)/CD56(+)/CD335(+) cells. Finally, a subset of CD4(+)/CD25(+)/FoxP3(+)/IL-10(-) T cells was identified. The result of this pilot study suggest that during a tolerogenic response, the major allergen of the Parietaria pollen works as an activator of both the innate and the adaptive human immune system.
    Immunobiology 11/2012; 218(7). DOI:10.1016/j.imbio.2012.11.006 · 3.04 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Clinical & Experimental Allergy 03/2012; 42(3):471-80. DOI:10.1111/j.1365-2222.2011.03938.x · 4.77 Impact Factor
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    ABSTRACT: Parietaria pollen is one of the major causes of allergic reaction in southern Europe, affecting about 30% of all allergic patients in this area. Specific immunotherapy is the only treatment able to modify the natural outcome of the disease by restoring a normal immunity against allergens. The preparation of allergen-solid lipid nanoparticles as delivery vehicles for therapeutic proteins, P. judaica major allergen Par j 2, was investigated. The Par j 2 allergen was expressed in a large amount in Escherichia coli and purified to homogeneity. Its immunological properties were studied by western blotting and enzyme-linked immunosorbent assay inhibition. Solid lipid nanoparticles were obtained by water-in-oil-in-water multiple emulsion method and characterized in terms of mean size and surface charge. These systems (approximately 250 nm diameter and negative surface charge) incorporated recombinant Par j 2 with 40% or greater efficiency. Moreover, the endotoxin level and anaphylactic activity of the empty solid lipid nanoparticles and recombinant Par j 2-loaded solid lipid nanoparticles were evaluated by looking at the overexpression of CD203c marker on human basophils. These results demonstrate that recombinant Par j 2-nanoparticles could be proposed as safe compositions for the development of new therapeutic dosage forms to cure allergic reactions.
    International Journal of Nanomedicine 11/2011; 6:2953-62. DOI:10.2147/IJN.S24264 · 4.38 Impact Factor
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    08/2011; 1(1). DOI:10.1186/2045-7022-1-S1-P5
  • A Bonura · P Colombo
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    ABSTRACT: Specific immunotherapy is a well established and clinically proved strategy to cure allergic reactions. The impressive boost of knowledge derived from DNA recombinant technology applied to this field allowed the identification, cloning and expression of several clinically relevant allergens. Recombinant allergens can be easily produced in a pure and reproducible way with immunological properties comparable to natural allergens and matching the requirements of pharmaceutical companies. Parietaria pollinosis is a major health problem in the Mediterranean basin with prolonged symptoms. In this review we will discuss the rational approaches to design hypoallergenic derivatives of the major allergens of this pollen, their immunological properties and possible clinical future implications.
    International journal of immunopathology and pharmacology 04/2011; 24(2):297-304. · 1.62 Impact Factor
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    ABSTRACT: The CAP superfamily is a group of proteins that have been linked to several biological functions such as reproduction, cancer, and immune defense. A differential screening between lipopolysaccharide (LPS)-challenged and naive Ciona intestinalis has been performed to identify LPS-induced genes. This strategy has allowed the isolation of a full-length 1471-bp cDNA encoding for a 413-amino-acid protein (CiCAP). In silico analysis has shown that this polypeptide displays a modular structure with similarities to vertebrate CAP-superfamily proteins and to a collagen-binding adhesin of Streptococcus mutans. Domain organization analysis and alignment of CiCAP to other vertebrate CAP proteins have revealed a novel structure suggesting that this protein originated from a common ancestor gene that gave rise to many subfamilies of mosaic proteins with novel functions. Quantitative mRNA expression performed by real-time polymerase chain reaction analysis has demonstrated that this gene is rapidly activated in the pharynx of C. intestinalis a few hours after LPS injection. Moreover, in situ hybridization has shown that CiCAP mRNA is highly expressed by hemocytes with large granules contained inside the pharynx vessels. Thus, CiCAP represents a protein with novel structural domains involved in ascidian immune responses.
    Cell and Tissue Research 11/2010; 342(3):411-21. DOI:10.1007/s00441-010-1072-7 · 3.57 Impact Factor
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    ABSTRACT: Collectins are a family of calcium-dependent lectins that are characterized by their collagen-like domains. Considerable interest has been focused on this class of proteins because of their ability to interact with components of the complement system activating a cascade of events responsible for the activation of the innate immune system. A differential screening between LPS-challenged and naïve Ciona intestinalis has been performed allowing the isolation of a full length cDNA encoding for a 221 AA protein. In silico analysis has shown that this polypeptide displays protein domains with similarities to mannose-binding lectins. A phylogenetic analysis suggested that C. intestinalis MBL has evolved early as a prototype of vertebrate MBL. Real-time PCR assay demonstrated that this gene is strongly activated after LPS injection in the tunica. In situ hybridization performed in LPS-induced animals has shown that this gene is expressed in granular amoebocytes and large granules hemocytes in the inflamed body wall tissue. Finally, an antimicrobial activity of the C. intestinalis MBL has been demonstrated.
    Molecular Immunology 08/2009; 46(11-12):2389-94. DOI:10.1016/j.molimm.2009.04.035 · 2.97 Impact Factor
  • Angela Bonura · Paolo Colombo
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    ABSTRACT: Allergen-specific immunotherapy was introduced into clinical practice at the beginning of the 20(th) century and its efficacy in the treatment of seasonal allergic rhinitis has been confirmed in many clinical studies which have shown that it can prevent the onset of new sensitizations to different allergens and reduces the development of asthma in patients with allergic rhinitis. Progress in molecular cloning and characterization of allergens have made it possible to produce single recombinant allergens whose immunological properties have been tested in vitro and in vivo and have demonstrated that they retain properties resembling their natural counterpart. Several rational approaches are being developed to improve the efficacy of SIT by reducing immunoglobulin IgE-mediated adverse reactions. Some of these molecules have been tested in the clinic, demonstrating the feasibility of using biotechnology-derived products as new standardized, improved and safer therapeutic compositions.
    07/2009; 8(2):104-9. DOI:10.2174/187152809788462608
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    ABSTRACT: The immunological mechanisms responsible for the immunomodulatory and anti-allergic effects of probiotic bacteria are still poorly defined. The combined effects of mixtures of different species of probiotic bacteria have been explored only in part. The present study describes the immunomodulatory activity of the VSL#3 probiotic preparation in in vitro and in vivo systems. The activation and cytokine production by in vitro probiotic-stimulated bone-marrow dendritic cells (BM-DCs) and spleen cells isolated from naïve or Par j 1-sensitized mice were investigated. Mice were intranasally administered a sonicate preparation of VSL#3 before immunization with rPar j 1. Serum antibody levels and cytokine expression in the lung were determined. Both live and sonicated VSL#3 preparations induced maturation and cytokine production by BM-DCs. Cytokine production by spleen cells from naïve or Par j 1-sensitized mice was modulated by the probiotic preparations towards a Treg/Th0 profile, characterized by increased IL-10 and IFN-gamma production. In vivo prophylactic treatment with VSL#3 induced a significant reduction of serum specific IgG1. At lung level, VSL#3 pre-treatment remarkably reduced IL-13 and IL-4 mRNA expression and increased IL-10 expression. The VSL#3 preparations have not only the capacity to bias primary immune responses towards a Treg/Th0-type profile, but also to modify in the same way the functional characteristics of established in vitro Th2 responses. In vivo studies on a mouse model of Par j 1 sensitization indicate that the prophylactic intranasal treatment with probiotic bacteria is able to modulate the development of Th2-biased responses.
    International Archives of Allergy and Immunology 06/2009; 150(2):133-43. DOI:10.1159/000218116 · 2.67 Impact Factor
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    ABSTRACT: 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.
    Molecular Immunology 06/2008; 45(9):2465-73. DOI:10.1016/j.molimm.2008.01.009 · 2.97 Impact Factor
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    ABSTRACT: Mapping an epitope on a protein by gene fragmentation and/or point mutations is often expensive and time consuming. Analysis of a D model can be utilized to detect the amino acids residues which are exposed to the solvent surface and thus represent potential epitope residues. Parj1 and Parj2 are the two major allergens of the Parietaria judaica pollen belonging to the Lipid Transfer Protein family. Using their three-dimensional structures as a guide, a head to tail dimer expressing disulphide bond variants of the major allergens was generated by means of DNA recombinant technology. The hybrid was expressed in E.coli and its immunological activity studied in vivo and in vitro. Our results demonstrate that a hybrid polypeptide expressing disulphide bond variants of the major allergens of the Parietaria pollen displayed reduced allergenicity and enhanced T cell reactivity for induction of protective antibodies able to block human IgE induced during the natural course of sensitization against the Parietaria pollen.

Publication Stats

228 Citations
68.15 Total Impact Points


  • 2007–2015
    • Institute of Immunology
      Santiago de Cali, Valle del Cauca, Colombia
  • 2014
    • INO - Istituto Nazionale di Ottica
      Florens, Tuscany, Italy
  • 2003–2013
    • National Research Council
      • Institute of Biomedicine and Molecular Immunology "Alberto Monroy" IBIM
      Roma, Latium, Italy
  • 2011
    • Università degli Studi di Palermo
      Palermo, Sicily, Italy
  • 2006
    • Università degli Studi di Perugia
      Perugia, Umbria, Italy