Angela Bonura

National Research Council, Roma, Latium, Italy

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Publications (24)54.96 Total impact

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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. · 2.65 Impact Factor
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    ABSTRACT: 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.
    Allergy 03/2013; 68(3):297-303. · 5.88 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 01/2013; 8(4):e63235. · 3.73 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; · 2.81 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. · 4.79 Impact Factor
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    Clinical and Translational Allergy. 08/2011; 1(1).
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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 01/2011; 6:2953-62. · 3.46 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. · 3.68 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. · 2.65 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.
    Inflammation & allergy drug targets. 07/2009; 8(2):104-9.
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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. · 2.25 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. · 2.65 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.
    12/2007;
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    ABSTRACT: T helper type 2 (Th2) cell differentiation requires the expression of GATA-3, a transcription factor that allows transcriptional activation of Th2 cytokine genes through chromatin remodelling. We investigated the role of the negative costimulatory receptor cytotoxic T-lymphocyte antigen 4 (CTLA-4) in the regulation of GATA-3 expression, Th2 differentiation and immunoglobulin production during the immune response to allergens. BALB/c mice were immunized with a recombinant major allergenic component of Parietaria judaica pollen, rPar j I, and treated with blocking anti-CTLA-4 or control antibodies. Results showed that in vivo CTLA-4 blockade enhanced the Par j I-specific immunoglobulin E (IgE) serum level. In contrast, Par j I-specific IgG2a serum level was reduced, suggesting that CTLA-4 blockade skewed immunoglobulin production towards interleukin-4 (IL-4) -dependent immunoglobulin isotypes. Consistently, CTLA-4 blockade increased the frequency of Par j I-specific Th2 cells but not Th1 cells, as well as IL-4 and IL-5 but not interferon-gamma production. Our data also showed that CTLA-4 blockade enhanced the GATA-3 : T-bet messenger RNA ratio. Interestingly, in vivo CTLA-4 blockade did not increase the frequency of GATA-3 protein-expressing cells. In contrast, it enhances GATA-3 protein level per cell. Further, in vitro results show that the anti-CTLA-4 monoclonal antibody, by competing with CD80 for CTLA-4 binding, induced an enhancement in the frequency of IL-4-producing cells that correlates with the increase in GATA-3 protein level per cell. In conclusion, CTLA-4, by affecting the level of GATA-3 per cell, contributes to keeping this factor under the threshold required to become a Th2 effector cell. Consequently, it affects IgE/IgG2a production and contributes to the outcome of allergen-specific immune responses.
    Immunology 06/2007; 121(1):62-70. · 3.71 Impact Factor
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    ABSTRACT: 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.
    International Archives of Allergy and Immunology 02/2007; 142(4):274-84. · 2.25 Impact Factor
  • P Colombo, A Bonura
    European annals of allergy and clinical immunology 10/2006; 38(7):224-5.
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    ABSTRACT: Parietaria pollen is one of the most important outdoor allergenic sources in all the Mediterranean countries, with a large number of subjects showing a positive skin prick test to both P. judaica and P. officinalis pollen extracts. A cross-reactivity between the two species has been already reported although few data are known at the molecular level. Twenty-five consecutive patients with Parietaria pollen allergy were selected on the basis of their clinical history. Skin prick test to P. judaica and officinalis extracts was performed. In vitro IgE measurement to both allergenic sources was performed by using a quantitative assay. ELISA inhibition experiments were made by using the rParj1 and rParj2 allergens. All the patients showed a positive skin prick test to both Parietaria species. Quantitative IgE measurement showed similar antibody concentration for P. judaica and P. officinalis extracts. ELISA inhibition experiments demonstrated that the cross-reactivity between the two species was due to the presence of the Parj1 and Parj2 allergens in the extracts with a high conserved IgE epitopes content. We conclude that P. judaica and P. officinalis pollens contain highly cross-reactive species-specific major allergens useful for the diagnosis and therapy of both allergenic sources.
    Allergy and Asthma Proceedings 08/2006; 27(5):378-82. · 2.19 Impact Factor
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    ABSTRACT: 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. 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. 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. 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. 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.
    Clinical & Experimental Allergy 04/2004; 34(3):470-7. · 4.79 Impact Factor
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    ABSTRACT: Parietaria is a genus of dicotyledonous weeds of the Urticaceae family including several species and its pollen grain is one of the most important allergenic sources in the Mediterranean area. Species belonging to this genus induce IgE responses in approximately 10 million people. Identification of allergens by means of independent strategies suggest that the allergens of the two more common species, Parietaria judaica and Parietaria Officinalis, show molecular weights ranging between 10 and 14 kD and that the allergens of the two extracts are highly cross-reactive. Biochemical analysis and molecular cloning allowed the isolation and immunological characterization of the two major allergens of the P. judaica pollen, Par j 1 and Par j 2. Sequence comparison suggests that the P j major allergens of P. Judaica belong to the nonspecific lipid transfer protein family, and three-dimensional modeling by homology has revealed that both proteins present a very conserved structural motif composed of four alpha-helices. Immunological analysis has shown that Par j 1 and Par j 2 are able to bind most of the P. Judaica-specific IgE and some of their IgE determinants have been mapped. Recombinant Par j 1 and Par j 2 allergens have been shown to possess immunological properties equivalent to their natural counterpart and their availability represents a fundamental tool for the diagnosis and therapy of Parietaria pollen allergy.
    International Archives of Allergy and Immunology 04/2003; 130(3):173-9. · 2.25 Impact Factor
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    ABSTRACT: Par j 1 represents a major allergenic component of Parietaria judaica (Pj) pollen, since it is able to induce an immunoglobulin E (IgE) response in 95% of Pj-allergic patients. It belongs to the non-specific lipid transfer protein family, sharing with them a common three-dimensional structure. Disulphide bond variants of the recombinant Par j 1 (rPar j 1) allergen were generated by site-directed mutagenesis, and the immunological activity of rPar j 1 and its conformational mutants was compared with the use of the skin prick test (SPT). The ability to bind IgE antibodies was evaluated by Western blot, ELISA and ELISA inhibition. T cell reactivity was measured by peripheral blood mononuclear cell proliferation assay. The disruption of Cys14-Cys29 and Cys30-Cys75 bridging (PjA mutant) caused the loss of the majority of specific IgE-binding activity. Additional disruption of the Cys4-Cys52 bridge (PjC mutant) and the latter Cys50-Cys91 bridge (PjD mutant) led to the abolition of IgE-binding activity. On the SPT, PjB (lacking the Cys4-Cys52 and Cys50-Cys91 bridges) was still capable of triggering a type I hypersensitive reaction in 9 out of 10 patients, and PjA in 3 out of 10 patients, while PjC and PjD did not show any SPT reactivity. All the mutants preserved their T cell reactivity. Recombinant hypoallergenic variants of the rPar j 1 allergen described herein may represent a useful tool for improved immunotherapy.
    International Archives of Allergy and Immunology 10/2001; 126(1):32-40. · 2.25 Impact Factor