Publications by authors named "Anna Carla Goldberg"

37 Publications

Comparison of senescence progression in mesenchymal cells from human umbilical cord walls measured by immunofluorescence and flow cytometry of p16 and p21.

Einstein (Sao Paulo) 2020 16;18:eAO5236. Epub 2020 Oct 16.

Hospital Israelita Albert Einstein, São Paulo, SP, Brazil.

Objective: To follow the expansion of mesenchymal stem cells from umbilical cords by two classic senescence markers, p16 (INK4A) and p21 (CDKN1A), using practical, fast, and less expensive methods than the gold standard Western blotting technique, to evaluate its applicability in the laboratory.

Methods: Mesenchymal stem cells from umbilical cords were isolated from Wharton's jelly and, after quality control, morphological and immunophenotypic characterization by flow cytometry, were expanded in culture until coming close to cell cycle arrest (replicative senescence).

Results: A comparison was made between young cells, at passage 5, and pre-senescent cells, at passage 10, evaluating the protein expression of the classic cell senescence markers p16 and p21, comparing the results obtained by Western blotting with those obtained by flow cytometry and indirect immunofluorescence.

Conclusion: Follow-up of cell cultures, through indirect p16 immunofluorescence, allows the identification of mesenchymal stem cells from umbilical cord cultures at risk of reaching replicative senescence.
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http://dx.doi.org/10.31744/einstein_journal/2020AO5236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546682PMC
October 2020

Optimizing the Decellularized Porcine Liver Scaffold Protocol.

Cells Tissues Organs 2020 Oct 9:1-10. Epub 2020 Oct 9.

Cellular and Molecular Cardiology Laboratory, Carlos Chagas Filho Biophysics Institute, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil,

There are few existing methods for shortening the decellularization period for a human-sized whole-liver scaffold. Here, we describe a protocol that enables effective decellularization of the liver obtained from pigs weigh 120 ± 4.2 kg within 72 h. Porcine livers (approx. 1.5 kg) were decellularized for 3 days using a combination of chemical and enzymatic decellularization agents. After trypsin, sodium deoxycholate, and Triton X-100 perfusion, the porcine livers were completely translucent. Our protocol was efficient to promote cell removal, the preservation of extracellular matrix (ECM) components, and vascular tree integrity. In conclusion, our protocol is efficient to promote human-sized whole-liver scaffold decellularization and thus useful to generate bioengineered livers to overcome the shortage of organs.
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http://dx.doi.org/10.1159/000510297DOI Listing
October 2020

The Immunomodulatory Potential of Wharton's Jelly Mesenchymal Stem/Stromal Cells.

Stem Cells Int 2019 11;2019:3548917. Epub 2019 Jun 11.

Hospital Israelita Albert Einstein, São Paulo, Brazil.

The benefits attributed to mesenchymal stem/stromal cells (MSC) in cell therapy applications are mainly attributed to the secretion of factors, which exhibit immunomodulatory and anti-inflammatory effects and stimulate angiogenesis. Despite the desirable features such as high proliferation levels, multipotency, and immune response regulation, there are important variables that must be considered. Although presenting similar morphological aspects, MSC collected from different tissues can form heterogeneous cellular populations and, therefore, manifest functional differences. Thus, the source of MSC should be a factor to be considered in the development of novel therapies. The following text presents an updated review of recent research outcomes related to Wharton's jelly mesenchymal stem/stromal cells (WJ-MSC), harvested from umbilical cords and considered novel and potential candidates for the development of cell-based approaches. This text highlights information on how WJ-MSC affect immune responses in comparison with other sources of MSC.
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http://dx.doi.org/10.1155/2019/3548917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594275PMC
June 2019

FAM3B/PANDER inhibits cell death and increases prostate tumor growth by modulating the expression of Bcl-2 and Bcl-X cell survival genes.

BMC Cancer 2018 01 22;18(1):90. Epub 2018 Jan 22.

School of Arts, Sciences and Humanities, University of São Paulo, São Paulo, SP, Brazil.

Background: FAM3B/PANDER is a novel cytokine-like protein that induces apoptosis in insulin-secreting beta-cells. Since in silico data revealed that FAM3B can be expressed in prostate tumors, we evaluated the putative role of this cytokine in prostate tumor progression.

Methods: FAM3B expression was analyzed by quantitative PCR in tumor tissue clinical samples and prostate tumor cell lines. Culture growth and viability of DU145 cell line were evaluated after treatment with either exogenous FAM3B protein obtained from conditioned media (CM) of 293 T cells overexpressing FAM3B or a recombinant FAM3B protein produced in a bacterial host. DU145 cells overexpressing FAM3B protein were produced by lentiviral-mediated transduction of full-length FAM3B cDNA. Cell viability and apoptosis were analyzed in DU145/FAM3B cells after treatment with several cell death inducers, such as TNF-alpha, staurosporine, etoposide, camptothecin, and serum starvation conditions. Anchorage-independent growth in soft agarose assay was used to evaluate in vitro tumorigenicity. In vivo tumorigenicity and invasiveness were evaluated by tumor xenograft growth in nude mice.

Results: We observed an increase in FAM3B expression in prostate tumor samples when compared to normal tissues. DU145 cell viability and survival increased after exogenous treatment with recombinant FAM3B protein or FAM3B-secreted protein. Overexpression of FAM3B in DU145 cells promoted inhibition of DNA fragmentation and phosphatidylserine externalization in a time and dose-dependent fashion, upon apoptosis triggered by TNF-alpha. These events were accompanied by increased gene expression of anti-apoptotic Bcl-2 and Bcl-XL, decreased expression of pro-apoptotic Bax and diminished caspase-3, -8 and -9 proteolytic activities. Furthermore, inhibition of Bcl-2 anti-apoptotic family proteins with small molecules antagonists decreases protective effects of FAM3B in DU145 cells. When compared to the respective controls, cells overexpressing FAM3B displayed a decreased anchorage- independent growth in vitro and increased tumor growth in xenografted nude mice. The immunohistochemistry analysis of tumor xenografts revealed a similar anti-apoptotic phenotype displayed by FAM3B-overexpressing tumor cells.

Conclusions: Taken together, by activating pro-survival mechanisms FAM3B overexpression contributes to increased resistance to cell death and tumor growth in nude mice, highlighting a putative role for this cytokine in prostate cancer progression.
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http://dx.doi.org/10.1186/s12885-017-3950-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5778767PMC
January 2018

Intrinsic Variability Present in Wharton's Jelly Mesenchymal Stem Cells and T Cell Responses May Impact Cell Therapy.

Stem Cells Int 2017 5;2017:8492797. Epub 2017 Jul 5.

Hospital Israelita Albert Einstein, São Paulo, SP, Brazil.

Wharton's jelly mesenchymal stem cells (WJ-MSC) exhibit immunomodulatory effects on T cell response. WJ-MSC are easy to collect, process, and proliferate rapidly in culture, but information on the variability of individual cell samples impacting upon in vitro expansion, immunomodulatory potential, and aging processes is still lacking. We propose to evaluate the immunomodulatory cytokine profile and capacity to inhibit T cell proliferation of WJ-MSC progressing to replicative senescence in order to analyze if expected responses are affected. Our results show that the gene expression of immunomodulatory molecules varied among samples with no specific pattern present. In coculture, all WJ-MSC were capable of inhibiting mitogen-activated CD3 T cell proliferation, although to different degrees, and each PBMC responded with a different level of inhibition. Thus, we suggest that each WJ-MSC displays unique behavior, differing in patterns of cytokine mRNA expression and immunomodulatory capacity. We believe that variability between samples may play a role in the effectiveness of WJ-MSC employed therapeutically.
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http://dx.doi.org/10.1155/2017/8492797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5516721PMC
July 2017

Novel CD28 antagonist mPEG PV1-Fab' mitigates experimental autoimmune uveitis by suppressing CD4+ T lymphocyte activation and IFN-γ production.

PLoS One 2017 1;12(3):e0171822. Epub 2017 Mar 1.

Hospital Israelita Albert Einstein, São Paulo, Brazil.

Autoimmune Uveitis is an important chronic inflammatory disease and a leading cause of impaired vision and blindness. This ocular autoimmune disorder is mainly mediated by T CD4+ lymphocytes poising a TH1 phenotype. Costimulatory molecules are known to play an important role on T cell activation and therefore represent interesting therapeutical targets for autoimmune disorders. CD28 is the prototypical costimulatory molecule for T lymphocytes, and plays a crucial role in the initiation, and maintenance of immune responses. However, previous attempts to use this molecule in clinical practice achieved no success. Thus, we evaluated the efficacy of mPEG PV1-Fab' (PV1), a novel selective CD28 antagonist monovalent Fab fragment in the treatment of Experimental Autoimmune Uveitis (EAU). Here, we showed that PV1 treatment decreases both average disease score and incidence of EAU. A decrease in the activation profile of both T CD4+ and T CD8+ eye-infiltrating lymphocytes was evidenced. In the periphery, T CD4+ cells from PV1-treated mice also showed a decrease in their activation status, with reduced expression of CD69, CD25, and PD-1 molecules. This suppression was not dependent on Treg cells, as both their frequency and absolute number were lower in PV1-treated mice. In addition, frequency of CD4+IFN-γ+ T cells was significantly lower in PV1-treated group, but not of IL-17-producing T cells. Moreover, after specific restimulation, PV1 blockade selectively blocked IFN-γ production by CD4+ lymphocytes Taken together, our data suggest that mPEG PV1-Fab' acts mainly on IFN-γ-producing CD4+ T cells and emphasize that this specific CD28 blockade strategy is a potential specific and alternative tool for the treatment of autoimmune disorders in the eye.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0171822PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331984PMC
August 2017

Analysis of cytokine profile and growth factors in platelet-rich plasma obtained by open systems and commercial columns.

Einstein (Sao Paulo) 2016 Jul-Sep;14(3):391-397

Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.

Objective:: To evaluate growth factors and cytokines in samples of platelet-rich plasma obtained by three different centrifugation methods.

Methods:: Peripheral blood of six individuals with no hematological diseases, aged 18 to 68 years, was drawn to obtain platelet-rich plasma, using the open method and commercial columns by Medtronic and Biomet. The products obtained with the different types of centrifugation were submitted to laboratory analysis, including pro-inflammatory cytokines and chemokines by flow cytometry assays, the concentration of fibroblast growth factors-2 (FGF-2) and transforming growth factor-beta1 (TGF-β1).

Results:: The diverse separation methods generated systematically different profiles regarding number of platelets and leukocytes. The Medtronic system yielded a product with the highest concentration of platelets, and the open method, with the lowest concentration of platelets. The results of cytokine analysis showed that the different types of centrifugation yielded products with high concentrations of interleukin 8, interleukin 1β. The open system resulted in a product with high levels of interleukin 6. Other cytokines and chemokines measured were similar between systems. The product obtained with the open method showed higher levels of TGF-β1 in relation to other systems and low FGF-2 levels.

Conclusion:: The formed elements, growth factors and cytokines in samples of platelet-rich plasma varied according to the centrifugation technique used.

Objetivo:: Avaliar fatores de crescimento e citocinas em amostras de plasma rico em plaquetas obtidas por três diferentes métodos de centrifugação.

Métodos:: Foi coletado sangue periférico de seis indivíduos, sem doença hematológica, com idades entre 18 e 68 anos, para obtenção de plasma rico em plaquetas, utilizando o método aberto e sistemas comerciais das empresas Medtronic e Biomet. Os produtos obtidos com os diferentes tipos de centrifugação foram submetidos às análises laboratoriais, incluindo citocinas próinflamatórias e quimiocinas, por meio de ensaios de citometria de fluxo, concentração do fator de crescimento fibroblástico-2 (FGF-2) e fator de crescimento transformador-beta1 (TGF-β1).

Resultados:: As diferentes centrifugações geraram perfis sistematicamente diferentes referentes ao número de plaquetas e de leucócitos. O sistema da Medtronic originou produto com a maior concentração de plaquetas, e o método aberto com a menor concentração de plaquetas. Os resultados da análise de citocinas demonstraram que os diferentes tipos de centrifugação originaram produtos com elevadas concentrações de interleucina 8 e interleucina 1β. O sistema aberto resultou em produto com elevados níveis de interleucina 6. As demais citocinas e quimiocinas mensuradas foram similares entre os sistemas. O produto obtido com o método aberto apresentou níveis superiores de TGF-β1 em relação aos demais sistemas e reduzidos níveis de FGF-2.

Conclusão:: Os elementos figurados, fatores de crescimento e citocinas, em amostras de plasma rico em plaquetas, variaram conforme a técnica de centrifugação utilizada.
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http://dx.doi.org/10.1590/S1679-45082016AO3548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5234752PMC
July 2017

Autoimmune Hepatitis in Brazilian Children: IgE and Genetic Polymorphisms in Associated Genes.

J Immunol Res 2015 26;2015:679813. Epub 2015 Nov 26.

Departamento de Hepatologia, Instituto da Criança, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, 05403-000 São Paulo, SP, Brazil.

Pediatric autoimmune hepatitis (AIH) patients present hypergammaglobulinemia, periportal CD8(+) cytotoxic T cell infiltration, and cirrhosis. Autoantibody profile defines AIH types 1 and 2 in addition to strong association with HLA-DRB1. We previously detected increased IgE serum levels and sought to compare clinical and histological features according to IgE levels in AIH (n = 74, ages 1-14 years) patients. Additionally, we typed 117 patients and 227 controls for functional polymorphisms of IL4, IL13, IL5, and IL4RA genes involved in IgE switching and eosinophil maturation that might contribute to overall genetic susceptibility to AIH. Serum IgE levels were high in 55% of AIH-1, but only in 12% of AIH-2 (P = 0.003) patients. Liver IgE was present in 91.3% of AIH-1 patients. The A alleles at both IL13 rs20541 and IL4RA rs1805011 were associated with AIH-1 (P = 0.024, OR = 1.55 and P < 0.0001, OR = 2.15, resp.). Furthermore, individuals presenting homozygosis for the A allele at IL4RA rs1805011 and HLA-DRB1(∗)03 and/or (∗)13 allele had sixfold greater risk to develop the disease (OR = 14.00, P < 0.001). The novel association suggests an additional role for IgE-linked immune response genes in the pathogenesis of AIH.
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http://dx.doi.org/10.1155/2015/679813DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674601PMC
September 2016

Modulation of Hyaluronan Synthesis by the Interaction between Mesenchymal Stem Cells and Osteoarthritic Chondrocytes.

Stem Cells Int 2015 26;2015:640218. Epub 2015 Jul 26.

Hospital Israelita Albert Einstein, No. 627/701, 05652-900 São Paulo, SP, Brazil.

Bone marrow mesenchymal stem cells (BM-MSCs) are considered a good source for cellular therapy in cartilage repair. But, their potential to repair the extracellular matrix, in an osteoarthritic environment, is still controversial. In osteoarthritis (OA), anti-inflammatory action and extracellular matrix production are important steps for cartilage healing. This study examined the interaction of BM-MSC and OA-chondrocyte on the production of hyaluronan and inflammatory cytokines in a Transwell system. We compared cocultured BM-MSCs and OA-chondrocytes with the individually cultured controls (monocultures). There was a decrease in BM-MSCs cell count in coculture with OA-chondrocytes when compared to BM-MSCs alone. In monoculture, BM-MSCs produced higher amounts of hyaluronan than OA-chondrocytes and coculture of BM-MSCs with OA-chondrocytes increased hyaluronan production per cell. Hyaluronan synthase-1 mRNA expression was upregulated in BM-MSCs after coculture with OA-chondrocytes, whereas hyaluronidase-1 was downregulated. After coculture, lower IL-6 levels were detected in BM-MSCs compared with OA-chondrocytes. These results indicate that, in response to coculture with OA-chondrocytes, BM-MSCs change their behavior by increasing production of hyaluronan and decreasing inflammatory cytokines. Our results indicate that BM-MSCs per se could be a potential tool for OA regenerative therapy, exerting short-term effects on the local microenvironment even when cell:cell contact is not occurring.
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http://dx.doi.org/10.1155/2015/640218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4529975PMC
August 2015

Comparison between isolation protocols highlights intrinsic variability of human umbilical cord mesenchymal cells.

Cell Tissue Bank 2016 Mar 9;17(1):123-36. Epub 2015 Jul 9.

Hospital Israelita Albert Einstein, IIEP, São Paulo, Brazil.

Mesenchymal stem cells (MSCs) though multipotent exhibit limited lifespan in vitro, with progressive reduction in capacity for self-renewal leading to irreversible arrest of cell division, which limits their use for therapeutic purposes. Human umbilical cord wall MSCs are easy to process and proliferate rapidly in culture, but variability of individual samples and impact upon in vitro expansion and aging processes is unknown. We compared isolation protocols to determine which one yields the highest number of viable cells with the best proliferation capacity. Three different protocols were tested: two were enzymatic procedures and one explant method. Isolated cells were evaluated in terms of proliferation, differentiation capacity, and phenotype. All samples were processed using one or more protocols. After passage 2 adherent cells displayed standard phenotypic and differentiation characteristics of MSCs, but our results show that isolating cells directly from Wharton's jelly is more advantageous. Cells obtained from explants presented similar characteristics to those from enzymatic protocols, but always reached proliferation arrest earlier, irrespective of initial population doubling times. From the same sample, cells obtained with enzymatic protocol ii reached later passages while exhibiting shorter doubling times in culture than cells from other protocols, that is, took longer to reach senescence. More important, each individual MSC sample exhibited different population doubling rates and reached senescence at different passages, irrespective of protocol. Thus, even when in strict conformity with procedures and quality control, each cord sample shows a unique behavior, a finding that should be taken into account when planning for therapeutic approaches.
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http://dx.doi.org/10.1007/s10561-015-9525-6DOI Listing
March 2016

MHC structure and function – antigen presentation. Part 1.

Einstein (Sao Paulo) 2015 Jan-Mar;13(1):153-6. Epub 2015 Mar 24.

Hospital Israelita Albert Einstein, São Paulo, SP, Brazil.

The setting for the occurrence of an immune response is that of the need to cope with a vast array of different antigens from both pathogenic and non-pathogenic sources. When the first barriers against infection and innate defense fail, adaptive immune response enters the stage for recognition of the antigens by means of extremely variable molecules, namely immunoglobulins and T-cell receptors. The latter recognize the antigen exposed on cell surfaces, in the form of peptides presented by the HLA molecule. The first part of this review details the central role played by these molecules, establishing the close connection existing between their structure and their antigen presenting function.
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http://dx.doi.org/10.1590/S1679-45082015RB3122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4977602PMC
September 2015

MHC structure and function - antigen presentation. Part 2.

Einstein (Sao Paulo) 2015 Jan-Mar;13(1):157-62. Epub 2015 Mar 24.

Hospital Israelita Albert Einstein, São Paulo, SP, Brazil.

The second part of this review deals with the molecules and processes involved in the processing and presentation of the antigenic fragments to the T-cell receptor. Though the nature of the antigens presented varies, the most significant class of antigens is proteins, processed within the cell to be then recognized in the form of peptides, a mechanism that confers an extraordinary degree of precision to this mode of immune response. The efficiency and accuracy of this system is also the result of the myriad of mechanisms involved in the processing of proteins and production of peptides, in addition to the capture and recycling of alternative sources aiming to generate further diversity in the presentation to T-cells.
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http://dx.doi.org/10.1590/S1679-45082015RB3123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4977603PMC
September 2015

Immunotherapeutic strategies in autoimmune uveitis.

Autoimmun Rev 2014 Sep 12;13(9):909-16. Epub 2014 May 12.

Hospital Israelita Albert Einstein, Av. Albert Einstein 627-701, 2-SS Bloco A, 05651-901 São Paulo, Brazil; Instituto de Investigação em Imunologia, Instituto Nacional de Ciência e Tecnologia (iii-INCT), Brazil. Electronic address:

Autoimmune uveitis is an organ-specific disorder characterized by irreversible lesions to the eye that predominantly affect people in their most productive years and is among the leading causes of visual deficit and blindness. Currently available therapies are effective in the treatment of a wide spectrum of uveitis, but are often associated with severe side effects. Here, we review ongoing research with promising immunomodulatory therapeutic strategies, describing their specific features, interactions and the responses triggered by the targeted immune molecules that aim to minimize clinical complications and the likelihood of disease relapse. We first review the main features of the disease, diagnostic tools, and traditional forms of therapy, as well as the animal models predominantly used to understand the pathogenesis and test the novel intervention approaches aiming to control the acute immune and inflammatory responses and to dampen chronic responses. Both exploratory research and clinical trials have targeted either the blockade of effector pathways or of their companion co-stimulatory molecules. Examples of targets are T cell receptors (CD3), their co-stimulatory receptors (CD28, CTLA-4) and corresponding ligands (B7-1 and B7-2, also known as CD80 and CD86), and cytokines like IL-2 and their receptors. Here, we summarize the available evidence on effectiveness of these treatments in human and experimental uveitis and highlight a novel CD28 antagonist monovalent Fab' antibody, FR104, which has shown preclinical efficacy suppressing effector T cells while enhancing regulatory T cell function and immune tolerance in a humanized graft-versus-host disease (GVHD) mice model and is currently being tested in a mouse autoimmune uveitis model with encouraging results.
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http://dx.doi.org/10.1016/j.autrev.2014.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4181827PMC
September 2014

Application of the enterprise management tools Lean Six Sigma and PMBOK in developing a program of research management.

Einstein (Sao Paulo) 2012 Oct-Dec;10(4):480-90

Hospital Israelita Albert Einstein, São Paulo, SP, Brazil.

Objective: Introduce a program for the management of scientific research in a General Hospital employing the business management tools Lean Six Sigma and PMBOK for project management in this area.

Methods: The Lean Six Sigma methodology was used to improve the management of the institution's scientific research through a specific tool (DMAIC) for identification, implementation and posterior analysis based on PMBOK practices of the solutions found.

Results: We present our solutions for the management of institutional research projects at the Sociedade Beneficente Israelita Brasileira Albert Einstein. The solutions were classified into four headings: people, processes, systems and organizational culture. A preliminary analysis of these solutions showed them to be completely or partially compliant to the processes described in the PMBOK Guide.

Conclusion: In this post facto study, we verified that the solutions drawn from a project using Lean Six Sigma methodology and based on PMBOK enabled the improvement of our processes dealing with the management of scientific research carried out in the institution and constitutes a model to contribute to the search of innovative science management solutions by other institutions dealing with scientific research in Brazil.
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http://dx.doi.org/10.1590/s1679-45082012000400015DOI Listing
December 2013

Isolation and characterization of mesenchymal stem cells obtained from reusable and disposable bone marrow collection filters.

Einstein (Sao Paulo) 2012 Jul-Sep;10(3):296-301

Hospital Israelita Albert Einstein, São Paulo, SP, Brasil.

Objective: To compare human mesenchymal stem cells obtained from reusable and disposable filters and to characterize them according to the criteria of the International Society of Cellular Therapy.

Methods: Human mesenchymal stem cells were isolated from bone marrow collection reusable sets and compared with those obtained from disposable sets by washing the filters with cell culture media. The isolated cells were characterized according to the criteria of the International Society of Cellular Therapy using flow cytometry, differentiation in vitro, and cytochemistry techniques.

Results: Samples were obtained from disposable (n=3) and from reusable collection sets (n=3). All samples obtained from bone marrow disposable sets successfully produced mesenchymal stem cells. All bone marrow derived mesenchymal stem cells were characterized and fulfilled the criteria established by International Society of Cellular Therapy.

Conclusion: This study showed that mesenchymal stem cells can also be obtained from reusable collection sets (which are still used in several centers around the world) to be employed in research as an alternative and ethical source.
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http://dx.doi.org/10.1590/s1679-45082012000300007DOI Listing
December 2013

Frequency of single nucleotide polymorphisms of some immune response genes in a population sample from São Paulo, Brazil.

Einstein (Sao Paulo) 2011 Sep;9(3):359-66

Instituto do Coração, Faculdade de Medicina, Universidade de São Paulo - USP, São Paulo, SP, BR.

Objective: To present the frequency of single nucleotide polymorphisms of a few immune response genes in a population sample from São Paulo City (SP), Brazil.

Methods: Data on allele frequencies of known polymorphisms of innate and acquired immunity genes were presented, the majority with proven impact on gene function. Data were gathered from a sample of healthy individuals, non-HLA identical siblings of bone marrow transplant recipients from the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, obtained between 1998 and 2005. The number of samples varied for each single nucleotide polymorphism analyzed by polymerase chain reaction followed by restriction enzyme cleavage.

Results: Allele and genotype distribution of 41 different gene polymorphisms, mostly cytokines, but also including other immune response genes, were presented.

Conclusion: We believe that the data presented here can be of great value for case-control studies, to define which polymorphisms are present in biologically relevant frequencies and to assess targets for therapeutic intervention in polygenic diseases with a component of immune and inflammatory responses.
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http://dx.doi.org/10.1590/S1679-45082011AO1866DOI Listing
September 2011

TGFB1 and IL8 gene polymorphisms and susceptibility to visceral leishmaniasis.

Infect Genet Evol 2011 Jul 2;11(5):912-6. Epub 2011 Mar 2.

Laboratory of Immunology, Heart Institute, InCor, HC-FMUSP, SP, Brazil.

Visceral leishmaniasis (VL) or Kala-azar is a serious protozoan infectious disease caused by an obligate intracellular parasite. Cytokines have a major role in determining progression and severity of clinical manifestations in VL. We investigated polymorphisms in the TGFB1and IL8 genes, which are cytokines known to have a role in onset and severity of the disease. Polymorphisms at TGFB1 -509 C/T and +869 T/C, and IL8 -251 A/T were analyzed by a PCR-RFLP technique, in 198 patients with VL, 98 individuals with asymptomatic infection positive for a delayed-type hypersensitivity test (DTH+) and in 101 individuals with no evidence of infection (DTH-). The presence of the T allele in position -509 of the TGFB1 gene conferred a two-fold risk to develop infection both when including those with clinical symptoms (DTH+ and VL, grouped) or when considering DTH+ only, respectively p = 0.007, OR = 1.9 [1.19-3.02] and p = 0.012, OR = 2.01 [1.17-3.79], when compared with DTH- individuals. In addition, occurrence of hemorrhage was associated with TGFB1 -509 T allele. We suggest that the -509 T allele of the TGFB1 gene, a cytokine with a biologically relevant role in the natural history of the disease, may contribute to overall susceptibility to infection by Leishmania and to severity of the clinical disease.
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http://dx.doi.org/10.1016/j.meegid.2011.02.014DOI Listing
July 2011

Contrasting roles of donor and recipient TGFB1 and IFNG gene polymorphic variants in chronic kidney transplant rejection.

Einstein (Sao Paulo) 2011 Mar;9(1):46-51

Instituto de Investigação em Imunologia, Institutos Nacionais de Ciência e Tecnologia, BR.

Objective: To assess the long-term impact (minimum of 3 years follow-up) of polymorphisms in cytokine genes in donor:recipient pairs on the results of the transplant.

Methods: We compared genetic cytokine polymorphisms and the primary factors of risk for the development of chronic rejection in paired groups of renal transplant patients with and without chronic allograft nephropathy [CAN].

Results: Multivariate analysis indicated that the presence of the high-production TT genotype (codon 10) of the transforming growth factor beta-1 (TGFB1) was protective in receptors (p=0.017), contrasting with the increased risk when present in donor samples (p=0.049). On the other hand, in the case of the gamma interferon studied, the greater frequency of the high production allele was protective in the analysis of the donor group (p=0.013), increasing the risk of chronic nephropathy of the allograft when present in the recipients (p=0.036).

Conclusion: Our results highlight the importance of TGFB1 genotyping in donors, and indicate that polymorphisms in the gene of this cytokine in donor cells might contribute to the development of chronic allograft nephropathy.
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http://dx.doi.org/10.1590/S1679-45082011AO1852DOI Listing
March 2011

Autoimmune hepatitis, HLA and extended haplotypes.

Autoimmun Rev 2011 Feb 7;10(4):189-93. Epub 2010 Oct 7.

Laboratório de Pediatria Clinica (LIM-36), Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, Av. Dr. Eneas de Carvalho Aguiar 44, São Paulo, Brazil.

Autoimmune hepatitis (AIH) is a chronic inflammatory liver disease. Characteristic liver-infiltrating immune cells in portal and periportal areas, hypergammaglobulinemia and typical autoantibodies indicate an ongoing autoimmune reaction against liver self antigens, which lead to irreversible cellular damage and ultimately to severe hepatic failure. A significant part of adult, but not pediatric AIH patients, exhibit concurrent autoimmune diseases, further strengthening the immunological etiology of the disease. Genetic susceptibility to autoimmune hepatitis is strongly associated with HLA-DRB1 alleles. In Caucasian European and North American patients, AIH-1 is associated with the presence of DRB1*0301, DRB3*0101 and DRB1*0401 alleles, while AIH-2 is associated with DRB1*0301 or DRB1*07. In Brazil, the primary susceptibility allele for AIH-1 is DRB1*1301, but a secondary association with DRB1*0301 has also been identified. We looked for additional susceptibility factors in the extended MHC region. We genotyped 107 AIH-1 children and up to 326 healthy subjects for TNFA G-308A, TNFA G-238A, LTA A+252G, LTA A+80C, NFKBIL1 T-63A, BAT1 C-348T, BAT1 G-22C, MICA, and HLA-B polymorphisms. The TNFA-308 A allele was significantly increased in AIH-1 when compared with healthy controls, confirming data from other studies. Linkage disequilibrium analysis was carried out. The ancestral haplotype comprising TNFA-308A, TNFA-238G, LTA+252G, LTA+80C, NFKBIL1-63A, BAT1-348C, BAT1-22C, HLA-B*08, MICA*08 was more common in DRB1*03 positive patients than in controls (40% vs. 14%), showing a seven-fold increased risk for the disease [OR=7.8 (95%CI 2.04-29.9.2, p=0.0021). In contrast, the remaining patients carrying DRB1*03 exhibited varied haplotypes. Finally, a variety of class III haplotypes was also present in HLA-DRB1*13 patients, without a predominant pattern. The most common of the 98 haplotypes present in patients were completely absent in controls. The extended haplotype analysis in this sample of AIH-1 patients highlights not only the genetic diversity present in the Brazilian population, but is also in accordance with the previously documented microdiversity within the MHC region. The present knowledge of AIH suggests that the same or a very similar disease can be induced by yet unknown, but different, triggers followed by presentation on different HLA-DR molecules of the epitopes derived from the corresponding autoantigens, characterizing a much more complex disease than previously thought.
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http://dx.doi.org/10.1016/j.autrev.2010.09.024DOI Listing
February 2011

Immunological and non-immunological effects of cytokines and chemokines in the pathogenesis of chronic Chagas disease cardiomyopathy.

Mem Inst Oswaldo Cruz 2009 Jul;104 Suppl 1:252-8

Laboratório de Imunologia, Instituto do Coração, Hospital das Clínicas, São Paulo, SP, Brasil.

The pathogenesis of Chagas disease cardiomyopathy (CCC) is not well understood. Since studies show that myocarditis is more frequent during the advanced stages of the disease, and the prognosis of CCC is worse than that of other dilated cardiomyopathies of non-inflammatory aetiology, which suggest that the inflammatory infiltrate plays a major role in myocardial damage. In the last decade, increasing evidence has shown that inflammatory cytokines and chemokines play a role in the generation of the inflammatory infiltrate and tissue damage. CCC patients have an increased peripheral production of the inflammatory Th1 cytokines IFN-gamma and TNF-alpha when compared to patients with the asymptomatic/indeterminate form. Moreover, Th1-T cells are the main producers of IFN-gamma and TNF-alpha and are frequently found in CCC myocardial inflammatory infiltrate. Over the past several years, our group has collected evidence that shows several cytokines and chemokines produced in the CCC myocardium may also have a non-immunological pathogenic effect via modulation of gene and protein expression in cardiomyocytes and other myocardial cell types. Furthermore, genetic polymorphisms of cytokine, chemokine and innate immune response genes have been associated with disease progression. We will review the molecular and immunological mechanisms of myocardial damage in human CCC in light of recent findings.
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http://dx.doi.org/10.1590/s0074-02762009000900032DOI Listing
July 2009

Analysis of HFE and non-HFE gene mutations in Brazilian patients with hemochromatosis.

Clinics (Sao Paulo) 2009 ;64(9):837-41

Portuguese Hospital, Salvador/BA, Brasil.

Background: Approximately one-half of Brazilian patients with hereditary hemochromatosis (HH) are neither homozygous for the C282Y mutation nor compound heterozygous for the H63D and C282Y mutations that are associated with HH in Caucasians. Other mutations have been described in the HFE gene as well as in genes involved in iron metabolism, such as transferrin receptor 2 (TfR2) and ferroportin 1 (SCL40A1).

Aims: To evaluate the role of HFE, TfR2 and SCL40A1 mutations in Brazilian subjects with HH.

Patients And Methods: Nineteen male subjects (median age 42 [range: 20-72] years) with HH were evaluated using the Haemochromatosis StripAssay A. This assay is capable of detecting twelve HFE mutations, which are V53M, V59M, H63D, H63H, S65C, Q127H, P160delC, E168Q, E168X, W169X, C282Y and Q283, four TfR2 mutations, which are E60X, M172K, Y250X, AVAQ594-597del, and two SCL40A1 mutations, which are N144H and V162del.

Results: In our cohort, nine (47%) patients were homozygous for the C282Y mutation, two (11%) were heterozygous for the H63D mutation, and one each (5%) was either heterozygous for C282Y or compound heterozygous for C282Y and H63D. No other mutations in the HFE, TfR2 or SCL40A1 genes were observed in the studied patients.

Conclusions: One-third of Brazilian subjects with the classical phenotype of HH do not carry HFE or other mutations that are currently associated with the disease in Caucasians. This observation suggests a role for other yet unknown mutations in the aforementioned genes or in other genes involved in iron homeostasis in the pathogenesis of HH in Brazil.
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http://dx.doi.org/10.1590/S1807-59322009000900003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2745139PMC
December 2010

Molecular markers of susceptibility to ocular toxoplasmosis, host and guest behaving badly.

Clin Ophthalmol 2008 Dec;2(4):837-48

Oswaldo Cruz Institution (IOC), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, RJ, Brazil.

Infection with Toxoplasma gondii results in retinochoroiditis in 6% to 20% of immunocompetent individuals. The outcome of infection is the result of a set of interactions involving host genetic background, environmental, and social factors, and the genetic background of the parasite, all of which can be further modified by additional infections or even reinfection. Genes that encode several components of the immune system exhibit polymorphisms in their regulatory and coding regions that affect level and type of expression in response to stimuli, directing the immune response into different pathways. These variant alleles have been associated with susceptibility to immune-mediated diseases and with severity of pathology. We have investigated polymorphisms in several of these genes, identified as candidates for progression to retinochoroiditis caused by toxoplasmosis, namely chemokine (C-C motif) receptor 5 (CCR5), toll-like receptor-2 (TLR2), and TLR4. Furthermore, because interleukin-12 (IL-12) has been shown to be fundamental both in mice and in man to control a protective response against T. gondii, molecules that have a key function in IL-12 production will be emphasized in this review, in addition to discussing the importance of the genetic background of the parasite in the establishment of ocular disease.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699811PMC
http://dx.doi.org/10.2147/opth.s1629DOI Listing
December 2008

Leptin and the immune response: an active player or an innocent bystander?

Ann N Y Acad Sci 2009 Feb;1153:184-92

Cell and Molecular Therapy Center, University of São Paulo, São Paulo, Brazil.

Leptin is involved in the control of energy storage by the body. Low serum leptin levels, as seen in starvation, are associated with impaired inflammatory T cell responses that can be reversed by exogenous leptin. Common variable immunodeficiency (CVID) is characterized by hypogammaglobulinemia and recurrent infections. Several defects in T cell function have also been described, and allergy, autoimmune disease, and lymphomas or other malignancies can be present. Previous studies in Brazilian CVID patients have shown that, in contrast with mononuclear cells from healthy controls, CVID cells cultured with phytohemagglutinin and added leptin increased the proliferative response and decreased activation-induced apoptosis. Interleukin (IL)-2 and especially IL-4 production also increased significantly, although the effects of exposure to leptin were not observed uniformly in CVID patients. The majority, however, responded in some degree, and some exhibited completely restored values of the four parameters.These remarkable results indicate leptin could be used to improve immune function in these patients. On the other hand, we found no specific correlation between serum leptin levels and the number of infectious events over a 24-month period, presence of autoimmunity, allergies, or cancer in these patients. The results suggest that the absolute value of serum leptin does not determine the clinical behavior of patients or responses to leptin in vitro. Of note is the divergence between serum leptin, response to leptin in vitro, and the presence of autoimmunity, indicating the need to identify the cellular and molecular players involved in the regulation of the immune response by leptin in CVID.
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http://dx.doi.org/10.1111/j.1749-6632.2008.03971.xDOI Listing
February 2009

Revised diagnostic criteria for vogt-koyanagi-harada disease: considerations on the different disease categories.

Am J Ophthalmol 2009 Feb 7;147(2):339-345.e5. Epub 2008 Nov 7.

Department of Ophthalmology, Uveitis Service, Hospital das Clínicas, University of São Paulo School of Medicine, São Paulo, Brazil.

Purpose: To evalulate the applicability of the Revised Diagnostic Criteria for Vogt-Koyanagi-Harada (VKH) disease to Brazilian patients and to verify the association between different disease categories, clinical parameters, and the presence of HLA-DRB1*0405.

Design: A retrospective observational case series.

Methods: Medical charts of 67 patients (10 to 64 years in age; 12 men and 55 women), from the Uveitis Service, Hospital das Clínicas, University of São Paulo School of Medicine (HCFMUSP), São Paulo, Brazil were reviewed. Patients, previously diagnosed with VKH disease using criteria proposed by the American Uveitis Society, underwent retrospective classification based on the Revised Diagnostic Criteria. The degree of concordance was assessed. At presentation, 46 patients (69%) were in the early phase. In this group, the mean time from disease onset to treatment was 15 days (range, one to 30 days). Forty-eight patients (72%) were typed for HLA-DRB1*0405 by polymerase chain reaction-sequence specific primer and polymerase chain reaction-sequence-specific oligonucleotides primer. Disease categories, phase at initial presentation, and ocular complications were analyzed.

Results: There was a 100% of concordance between the two criteria. Disease was classified as complete in 10 patients (15%), incomplete in 37 patients (55%), and probable in 20 patients (30%). In each group, respectively, 90%, 76%, and 45% were in the early phase at presentation (P = .017). There was no association between disease categories, the presence of HLA-DRB1*0405, and clinical parameters.

Conclusion: The Revised Diagnostic Criteria proved useful for diagnosis of VKH disease in Brazilian patients. The present retrospective study did not find any association between disease category and severity parameters. To better understand the relevance of disease categories, a minimum follow-up period to categorize patients should be included in future prospective studies.
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http://dx.doi.org/10.1016/j.ajo.2008.08.034DOI Listing
February 2009

TNF-alpha polymorphisms are associated with obsessive-compulsive disorder.

Neurosci Lett 2008 Sep 12;442(2):86-90. Epub 2008 Jul 12.

Department of Psychiatry, Institute of Psychiatry, Clinical Hospital, University of São Paulo (USP), Brazil.

Introduction: Several lines of evidence support an immunologic involvement in obsessive-compulsive disorder (OCD): the increased prevalence of OCD in patients with rheumatic fever (RF), and the aggregation of obsessive-compulsive spectrum disorders among relatives of RF probands. Tumor necrosis factor alpha is a proinflammatory cytokine involved in RF and other autoimmune diseases. Polymorphisms in the promoter region of the TNFA gene have been associated with RF. Given the association between OCD and RF, the goal of the present study was to investigate a possible association between polymorphisms within the promoter region of TNFA and OCD.

Materials And Methods: Two polymorphisms were investigated: -308 G/A and -238 G/A. The allelic and genotypic frequencies of these polymorphisms were examined in 111 patients who fulfilled DSM-IV criteria for OCD and compared with the frequencies in 250 controls.

Results: Significant associations were observed between both polymorphisms and OCD. For -238 G/A, an association between the A allele and OCD was observed (chi(2)=12.05, p=0.0005). A significant association was also observed between the A allele of the -308 G/A polymorphism and OCD (chi(2)=7.09, p=0.007). Finally, a haplotype consisting of genotypes of these two markers was also examined. Significant association was observed for the A-A haplotype (p=0.0099 after correcting for multiple testing).

Discussion: There is association between the -308 G/A and -238 G/A TNFA polymorphisms and OCD in our Brazilian sample. However, these results need to be replicated in larger samples collected from different populations.
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http://dx.doi.org/10.1016/j.neulet.2008.07.022DOI Listing
September 2008

T-cell recognition of Paracoccidioides brasiliensis gp43-derived peptides in patients with paracoccidioidomycosis and healthy individuals.

Clin Vaccine Immunol 2007 Apr 28;14(4):474-6. Epub 2007 Feb 28.

Laboratory of Immunolgoy, Heart Institute (InCor), Millenium Institutes, São Paulo, Brazil.

Vaccines with synthetic peptides induce the immune response to epitopes that bind to several HLA alleles. By using a TEPITOPE algorithm, we selected and analyzed the T-cell responses of peripheral blood mononuclear cells from 29 paracoccidioidomycosis (PCM) patients to peptides of the immunodominant gp43 antigen of Paracoccidioides brasiliensis, the causative agent of PCM.
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http://dx.doi.org/10.1128/CVI.00458-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1865602PMC
April 2007

Lack of association of tumor necrosis factor-alpha polymorphisms with Chagas disease in Brazilian patients.

Immunol Lett 2007 Jan 27;108(1):109-11. Epub 2006 Nov 27.

Heart Institute (InCor), School of Medicine, University of São Paulo, São Paulo, Brazil.

One third of Trypanosoma cruzi-infected individuals develop chronic Chagas disease cardiomyopathy (CCC) while the majority remains asymptomatic (ASY). About 30% of CCC patients develop heart failure due to end-stage inflammatory dilated cardiomyopathy. Increased production of tumor necrosis factor (TNF)-alpha has been described in all clinical forms of Chagas disease, and the highest levels are detected in CCC patients with severe ventricular dysfunction. Genetic susceptibility may play a role in the clinical outcome of Chagas disease. We investigated TNF as a candidate gene for susceptibility to development and/or progression of CCC. We analyzed the TNFa microsatellite and the -308 TNF promoter polymorphisms, in 166 CCC compared to 80 ASY geographically and age-matched patients in an association study. To analyze the association of TNF polymorphisms with progression of the cardiomyopathy, CCC patients were also grouped in three categories according to degree of left ventricular (LV) dysfunction into severe (n=57), mild to moderate (n=21) and absent (n=88). Our results show no significant differences either between CCC and ASY patients, or among CCC patients according to severity of cardiomyopathy with respect to TNFa or -308 TNF promoter polymorphisms. These results indicate that TNF polymorphisms are associated neither to CCC development nor to progression to more severe forms of cardiomyopathy in Brazilian Chagas disease patients.
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http://dx.doi.org/10.1016/j.imlet.2006.10.008DOI Listing
January 2007

BAT1, a putative anti-inflammatory gene, is associated with chronic Chagas cardiomyopathy.

J Infect Dis 2006 May 10;193(10):1394-9. Epub 2006 Apr 10.

Heart Institute (InCor), University of São Paulo, Brazil.

Background: It is not understood why only a subset of individuals infected with Trypanosoma cruzi develop chronic Chagas cardiomyopathy (CCC). Patients with CCC display high levels of circulating proinflammatory cytokines. Heart-infiltrating lymphocytes from patients with CCC also express proinflammatory cytokines (tumor necrosis factor- alpha and interferon- gamma ) that are detectable in biopsy samples and surgical heart-tissue samples. BAT1, a putative anti-inflammatory gene, presents functional polymorphisms in its promoter region that influence its transcriptional level.

Methods: We assessed, by polymerase chain reaction restriction fragment-length polymorphism analysis, BAT1 variants in the promoter region at positions -22C/G and -348C/T in 154 patients with CCC and in 76 T. cruzi-infected but asymptomatic (ASY) patients.

Results: Of the patients with CCC, 16% were homozygous for the -22C allele, compared with 4% of the ASY patients (P=.004; odds ratio [OR], 4.7 [95% confidence interval {CI}, 1.4-16]). A similar trend was observed for the -348C homozygotes (P=.01; OR, 1.9 [95% CI, 1.0-3.5]). Susceptibility to CCC was conferred by the C variants at nt -22 (P=.003; OR, 1.8 [95% CI, 1.2-2.8]) and at nt -348 (P=.02; OR, 1.7 [95% CI, 1.0-2.8]).

Conclusions: BAT1 variants previously associated with reduced expression of HLA-B-associated transcript 1 are predictive of the development of CCC. These variants may be less efficient in down-regulating inflammatory responses and may contribute to the elevated production of proinflammatory cytokines in patients with CCC.
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http://dx.doi.org/10.1086/503368DOI Listing
May 2006

TNF gene polymorphisms are associated with reduced survival in severe Chagas' disease cardiomyopathy patients.

Microbes Infect 2006 Mar 27;8(3):598-603. Epub 2005 Dec 27.

Heart Institute (InCor), School of Medicine, University of São Paulo, Av. Dr. Enéas de Carvalho Aguiar 44, bloco 2, 9 andar, CEP: 05403-001 São Paulo, Brazil.

Chronic Chagas' disease cardiomyopathy (CCC) is the most important clinical outcome of infection by the parasite Trypanosoma cruzi, affecting 18 million individuals in Latin America. One-third of CCC patients develop heart failure due to end-stage dilated cardiomyopathy, and their survival is reduced by 50% compared to patients with other cardiomyopathies. Genetic susceptibility may play a role in the differential survival of severe CCC patients. Given the role of TNF-alpha in the progression of heart failure, and the increased TNF-alpha plasma and heart tissue levels observed in these patients, we chose TNF as a candidate gene for increased mortality in severe CCC patients. We typed the TNFa microsatellite and the -308 TNF promoter polymorphism and then analyzed the survival curves of 42 patients with severe ventricular dysfunction (left ventricular ejection fraction
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http://dx.doi.org/10.1016/j.micinf.2005.08.009DOI Listing
March 2006