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Claudia Durán-Aniotz,
Gabriela Segal,
Lorena Salazar,
Cristián Pereda,
Cristián Falcón,
Fabián Tempio,
Raquel Aguilera,
Rodrigo González,
Claudio Pérez,
Andrés Tittarelli, Diego Catalán,
Bruno Nervi,
Milton Larrondo,
Flavio Salazar-Onfray,
Mercedes N López
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ABSTRACT: INTRODUCTION: Immunization with autologous dendritic cells (DCs) loaded with a heat shock-conditioned allogeneic melanoma cell lysate caused lysate-specific delayed type hypersensitivity (DTH) reactions in a number of patients. These responses correlated with a threefold prolonged long-term survival of DTH(+) with respect to DTH(-) unresponsive patients. Herein, we investigated whether the immunological reactions associated with prolonged survival were related to dissimilar cellular and cytokine responses in blood. MATERIALS AND METHODS: Healthy donors and melanoma patient's lymphocytes obtained from blood before and after vaccinations and from DTH biopsies were analyzed for T cell population distribution and cytokine release. RESULTS/DISCUSSION: Peripheral blood lymphocytes from melanoma patients have an increased proportion of Th3 (CD4(+) TGF-β(+)) regulatory T lymphocytes compared with healthy donors. Notably, DTH(+) patients showed a threefold reduction of Th3 cells compared with DTH(-) patients after DCs vaccine treatment. Furthermore, DCs vaccination resulted in a threefold augment of the proportion of IFN-γ releasing Th1 cells and in a twofold increase of the IL-17-producing Th17 population in DTH(+) with respect to DTH(-) patients. Increased Th1 and Th17 cell populations in both blood and DTH-derived tissues suggest that these profiles may be related to a more effective anti-melanoma response. CONCLUSIONS: Our results indicate that increased proinflammatory cytokine profiles are related to detectable immunological responses in vivo (DTH) and to prolonged patient survival. Our study contributes to the understanding of immunological responses produced by DCs vaccines and to the identification of follow-up markers for patient outcome that may allow a closer individual monitoring of patients.
Cancer Immunology and Immunotherapy 12/2012; · 3.70 Impact Factor
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David Gárate,
Nicole Rojas-Colonelli,
Corina Peña,
Lorena Salazar,
Paula Abello,
Bárbara Pesce,
Octavio Aravena,
Paulina García-González,
Carolina H Ribeiro,
María C Molina, Diego Catalán,
Juan C Aguillón
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ABSTRACT: OBJECTIVE: Dendritic cells (DCs) modulated with lipopolysaccharide (LPS) are able to reduce inflammation when therapeutically administered into mice with collagen-induced arthritis (CIA). The aim of this study was to uncover the mechanisms that define the tolerogenic effect of short-term LPS-modulated DCs on CIA. METHODS: Bone marrow-derived DCs were stimulated for 4 hours with LPS (4hLPS/DCs) and characterized for their maturation markers expression and cytokine secretion profile. Either 4hLPS/DCs inhibited for the p38 and the transforming growth factor-β (TGF-β) receptor pathways with SB203580 and SB431542, respectively, or 4hLPS/DCs left unmodified, were inoculated into mice at day 35 after CIA induction, and disease severity was clinically evaluated. CD4+ T cell populations were determined in spleen and lymph nodes of inoculated or untreated CIA mice. Splenic CD4+ T cells were transferred from 4hLPS/DCs-treated or untreated CIA mice into other CIA mice at day 35. RESULTS: 4hLPS/DCs treatment increased interleukin (IL)-10 and TGF-β-secreting CD4+ T cells, while decreased Th17 cell population. Adoptive transfer of CD4+ T cells from treated CIA mice reproduced the inhibition of active CIA accomplished with 4hLPS/DCs. The therapeutic effect of 4hLPS/DCs and their influence on T cell populations were abolished when the p38 and the TGF-β receptor pathways were inhibited. CONCLUSION: We demonstrate that short-term LPS-modulated DCs are able to confer a sustained cure of mice with established arthritis by reeducating CD4+ T cell populations. This effect is dependent on the p38 and the TGF-β receptor signaling pathways, suggesting the participation of IL-10 and TGF-β on tolerance recovering. © 2012 American College of Rheumatology.
Arthritis & Rheumatism 09/2012; · 7.87 Impact Factor
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ABSTRACT: Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.
mAbs 07/2012; 4(4):542-50.
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Diego Catalán,
Octavio Aravena,
Roberto Zúñiga,
Natalia Silva,
Alejandro Escobar,
Francisca Sabugo,
Pamela Wurmann,
Lilian Soto,
Rodrigo González,
Jorge Alfaro,
Milton Larrondo,
Miguel Cuchacovich,
Juan Carlos Aguillón
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ABSTRACT: Citrullinated vimentin (cVIM) is one of the antigens specifically targeted by anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) patients. The association between ACPA and certain HLA-DRB1 alleles, those coding for the shared epitope (SE), suggests that this response could be T-cell mediated. HLA-DR9 alleles, which do not code for the SE, have recently been associated with ACPA (+) RA. The objective of this work was to study CD4+ T cell responses to cVIM in RA patients and healthy controls carrying HLA-DR9 alleles. Fourteen RA patients and ten healthy controls previously genotyped for HLA-DRB1 were studied for the presence of serum anti-cVIM antibodies by Western blot and ELISA. Peripheral blood mononuclear cells were stimulated with native vimentin and cVIM, and CD4+ T cells proliferation was assessed by flow cytometry. Citrulline-specific CD4+ T cells proliferation was found not only in RA patients but also in healthy controls. Although most patients carrying HLA-DR9 alleles present anti-cVIM antibodies, HLA-DR9 alleles were associated with weaker cVIM-driven CD4+ T-cell responses among RA patients. These results suggest that HLA-DR9 alleles could exert a protective effect on the recognition of cVIM epitopes by CD4+ T cells. In this context, other citrullinated proteins may break T and B cell tolerance, with cVIM only acting as a cross-reactive target for ACPA.
Rheumatology International 07/2011; 32(6):1819-25. · 1.88 Impact Factor
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Octavio Aravena,
Bárbara Pesce,
Lilian Soto,
Natalia Orrego,
Francisca Sabugo,
Pamela Wurmann,
María Carmen Molina,
Jorge Alfaro,
Miguel Cuchacovich,
Juan Carlos Aguillón, Diego Catalán
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ABSTRACT: The aim of this work was to study the effect of anti-TNF treatment on CD4+ Th1, Th17 and regulatory T cells (Tregs), together with CD8+ T cells and NK cells from rheumatoid arthritis (RA) patients. For this purpose, 18 RA patients received adalimumab during 16weeks and their peripheral blood lymphocytes were assessed by flow cytometry at the beginning and at the end of the study. We found that the proportion of Th17 cells was directly correlated with Th1 cells, but inversely correlated with IFN-γ-producing NK cells. A decrease was observed in Th1, Th17 cells and IFN-γ-producing CD8+ T cells by anti-TNF therapy. Conversely, the proportion of Tregs increased, as did the percentage of IFN-γ-producing NK cells. We postulate that a rise in IFN-γ production due to recovery of NK cells' function, together with expanded Tregs, contribute to decrease the Th17 response in anti-TNF-treated RA patients.
Immunobiology 07/2011; 216(12):1256-63. · 3.20 Impact Factor
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ABSTRACT: Several molecules help preserve peripheral B cell tolerance, but when altered, they may predispose to autoimmunity. This work studied the expression of the costimulatory molecule CD86 and the inhibitory receptor for IgG immune complexes FcgammaRIIb (CD32b), on B cells from rheumatoid arthritis (RA) patients, and the influence of anti-tumor necrosis factor (TNF) therapy.
Peripheral B cells from 18 RA patients and 13 healthy donors were characterized using flow cytometry. Eleven patients who underwent a six-month adalimumab therapy were further assessed for phenotypic changes on their B cells.
RA patients exhibited a high percentage of naïve and memory B cells expressing CD86. In contrast, expression of FcgammaRIIb was significantly reduced on RA memory B cells and plasmablasts as compared to healthy donors, probably due to downregulation of this receptor when differentiating from naïve to memory cells. These alterations on FcgammaRIIb were associated with high levels of anti-citrullinated vimentin autoantibodies. In addition, treatment with adalimumab normalized the expression of CD86 on memory B cells and reduced the expression of FcgammaRIIb, mainly on naïve B cells.
Our findings show that peripheral B cells from RA patients have an altered expression of key molecules, such as CD86 and FcgammaRIIb. Because this latter receptor is required for feedback inhibition, a deficient expression might contribute to humoral autoimmune responses. Furthermore, these molecules are likely to be influenced by inflammatory factors, since they were modulated by TNF inhibition.
Arthritis research & therapy 01/2010; 12(2):R68. · 4.27 Impact Factor
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L Salazar,
O Aravena,
P Abello,
A Escobar,
J Contreras-Levicoy,
N Rojas-Colonelli, D Catalán,
A Aguirre,
R Zúñiga,
B Pesce,
C González,
R Cepeda,
M Cuchacovich,
M C Molina,
F Salazar-Onfray,
M Delgado,
R E Toes,
J C Aguillón
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ABSTRACT: The use of regulatory or immature dendritic cells (DCs) as tools for modulating experimental rheumatoid arthritis is very recent. Tumour necrosis factor (TNF)-stimulated DCs have been shown to restore tolerance in experimental autoimmune encephalomyelitis and collagen-induced arthritis (CIA).
We investigated the capacity of short-term lipopolysaccharide (LPS)-stimulated DCs pulsed with type II collagen (CII) to induce tolerance against established CIA.
Bone marrow-derived DCs were generated in the presence of granulocyte monocyte colony-stimulating factor (GM-CSF). After CIA induction, mice were injected at day 35 with a single dose of 4- or 24-h LPS-stimulated DCs that had been loaded with CII (4hLPS/CII/DCs or 24hLPS/CII/DCs). Arthritis progression was monitored by clinical and histological evaluations.
Flow cytometry of 4hLPS/CII/DCs showed intermediate CD40 and CD86 expression, lower than that of 24hLPS/CII/DCs (fully mature) and higher than that of CII/DCs (immature). A functional assay showed that 4hLPS/CII/DCs display increased endocytosis ability with respect to 24hLPS/CII/DCs, indicating a semimature state. The single inoculation of 4hLPS/CII/DCs in mice with established CIA reduced disease severity significantly over time. Histological evaluation of mice treated with 4hLPS/CII/DCs revealed diminished inflammatory synovitis, cartilage damage and fibrosis. Co-cultures of DCs with splenocytes from CIA mice showed that collagen-specific interferon (IFN)gamma production was dramatically inhibited by 4hLPS/CII/DCs. 4hLPS/CII/DCs were high IL10 producers, which could explain the inhibition of arthritis progression in mice receiving this treatment because neither antibodies nor regulatory CD4+CD25+Foxp3+ T lymphocytes were demonstrated to be involved.
Short-term LPS-modulated DCs inoculation interferes with CIA progression when loaded with CII.
Annals of the rheumatic diseases 12/2007; 67(9):1235-41. · 8.11 Impact Factor
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ABSTRACT: Using the murine model of type II collagen-induced arthritis (CIA), we studied its evolution over time by histopathological, immunohistochemical and clinical evaluations. The first clinical symptoms appeared 28 days post-inoculation (dpi), with bovine type II collagen, with an average arthritic index of 1.00 +/- 0.48 corresponding to erythema of the articulation. The disease progressed, and by 70 dpi showed an average arthritic index of 3.83 +/- 0.27 corresponding to edema and maximum deformation, with ankylosis. Computed morphometry demonstrated that, in comparison to controls, the induction of CIA, produces a significant and increasing accumulation of inflammatory cells, fibrosis (p < 0.0001) and cartilage destruction (p = 0.0029). Likewise, the area of von Willebrand factor (vWF) immunostaining, as an indicator of endothelial proliferation, increased significantly from 28 dpi (p < 0.0001), in CIA mice compared to controls. However, the effective synovial vascularization, calculated as the synovial vascular bed area index, significantly increased by 42 dpi (p = 0.0014). This indicates that the activation and proliferation of endothelium becomes significant before an effective vascularization area is formed. The apoptosis index was also an earlier indicator of cartilage damage, becoming significant from 28 dpi in comparison to controls (p < 0.0001). Finally, it was observed that the increase in the arthritic index showed a strong correlation with the increase in both angiogenesis (r = 0.95; p = 0.0021) and apoptosis (r = 0.90; p = 0.0015). In conclusion, a robust correlation between synovial membrane inflammation, angiogenesis and chondrocyte apoptosis, with respect to the increase in the clinical severity of CIA, has been demonstrated by a quantitative computer-assisted immunomorphometric analysis.
European cytokine network 09/2007; 18(3):127-35. · 1.73 Impact Factor
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Juan C Aguillón,
Andrea Cruzat,
Juan Contreras-Levicoy,
Andrés Dotte,
Bárbara Pesce,
Octavio Aravena,
Lorena Salazar, Diego Catalán,
Paula Abello,
Adam Aguirre,
Carolina Llanos,
Miguel Cuchacovich
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ABSTRACT: The use of biological agents such as etanercept, infliximab, adalimumab and anakinra has been recently approved for the treatment of rheumatoid arthritis. All are effective controlling signs and symptoms and inhibiting disease progression. To overcome the problems generated by their high costs and possible participation in reactivating latent infections, other therapeutic tools are being developed. Gene therapy using expression vectors carrying genes coding for specific proteins, may interfere in key points involved in the pathogenesis of the disease. Intra-articular administration of cDNA coding for soluble TNF receptors, IL-1, or IL-1Ra decreases signs of the disease in animal models. Vectors, expressing inhibitors of signal transduction pathways involving to NF-kB and JAK-STAT-3, are effective in modulating joint inflammation in mice. The use of antigen-pulsed antigen presenting cells or dendritic cells (DC) bound to apoptosis-inducing molecules, specifically eliminates autoreactive T cells. Other novel approach attempts the development of T regulatory-inducing tolerogenic DC-based vaccines that inhibit autoreactive T cells, through the secretion of suppressing cytokines or by other mechanisms to be elucidated. Oral tolerance induction to auto-antigens is also a successful experimental strategy under study. Current research aims to control peripheral tolerance in rheumatoid arthritis patients.
Revista medica de Chile 09/2005; 133(8):969-76. · 0.33 Impact Factor
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Irene Schiattino,
Rodrigo Villegas,
Andrea Cruzat,
Jimena Cuenca,
Lorena Salazar,
Octavio Aravena,
Bárbara Pesce, Diego Catalán,
Carolina Llanos,
Miguel Cuchacovich,
Juan C Aguillón
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ABSTRACT: Longitudinal studies aimed at evaluating patients clinical response to specific therapeutic treatments are frequently summarized in incomplete datasets due to missing data. Multivariate statistical procedures use only complete cases, deleting any case with missing data. MI and MIANALYZE procedures of the SAS software perform multiple imputations based on the Markov Chain Monte Carlo method to replace each missing value with a plausible value and to evaluate the efficiency of such missing data treatment. The objective of this work was to compare the evaluation of differences in the increase of serum TNF concentrations depending on the -308 TNF promoter genotype of rheumatoid arthritis (RA) patients receiving anti-TNF therapy with and without multiple imputations of missing data based on mixed models for repeated measures. Our results indicate that the relative efficiency of our multiple imputation model is greater than 98% and that the related inference was significant (p-value < 0.001). We established that under both approaches serum TNF levels in RA patients bearing the G/A -308 TNF promoter genotype displayed a significantly (p-value < 0.0001) increased ability to produce TNF over time than the G/G patient group, as they received successively doses of anti-TNF therapy.
Biological research 02/2005; 38(1):7-12. · 1.03 Impact Factor
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Juan C Aguillón,
Juan Contreras,
Andrés Dotte,
Andrea Cruzat, Diego Catalán,
Lorena Salazar,
María Carmen Molina,
Julia Guerrero,
Mercedes López,
Lilian Soto,
Flavio Salazar-Onfray,
Miguel Cuchacovich
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ABSTRACT: The fusion of a murine B cell and a myeloma cell generates a hybridoma that produces monoclonal antibody (mAb). These murine mAb induce the HAMA (human anti-mouse antibodies) response. Murine mAb have been modified by genetic engineering, producing molecules with a higher proportion of human protein. At present, chimeric, humanized and fully human mAb are available. mAb block interactions between target molecules and their ligands or trigger the lyses of mAb-coated tumor cells. Numerous mAb have been developed using the recombinant DNA technology and several are available in the market. Trastuzumab, against HER2/neu, is useful in breast cancer; rituximab, against CD20 in B lymphocytes is useful in lymphoma; alemtuzumah, against CD52 is used in lymphoma and leukemia; daclizumab and basiliximab block the IL-2 receptor interaction and reduce acute rejection in kidney transplantation; abciximab, an antagonist of GPIIb/IIIa platelet receptor, is used in patients undergoing acute coronary syndromes. In autoimmunity diseases, blocking tumor necrosis factor by infliximab and adalimumab has demonstrated excellent results. Thus, infliximab is useful in the treatment of rheumatoid arthritis (RA), Crohn's disease and ulcerative colitis while adalimumab is the first fully human mAb available for RA. Infliximab and adalimumab reduce signs and symptoms in RA and they also interfere with progression of joint damage. Finally, the direct benefits of antagonist treatment can occur at the expense of a major adverse effect in some other biological function.
Revista medica de Chile 01/2004; 131(12):1445-53. · 0.33 Impact Factor
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Claudio Pérez,
Fermín E González,
Violeta Pavez,
Aida V Araya,
Adam Aguirre,
Andrea Cruzat,
Juan Contreras-Levicoy,
Andrés Dotte,
Octavio Aravena,
Lorena Salazar, Diego Catalán,
Jimena Cuenca,
Arturo Ferreira,
Irene Schiattino,
Juan C Aguillón
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ABSTRACT: Several single-nucleotide polymorphisms (SNPs) have been identified in the TNF-alpha gene promoter. The transition G-->A at position -308 generates the TNF-alpha1 (G/G) and TNF-alpha2 (G/A or A/A) alleles, where the polymorphic TNF-alpha2 allele is associated with a high, in vitro TNF-alpha expression and an increased susceptibility to diverse illnesses. Here we study the association of the -308 TNF-alpha SNP with the susceptibility for developing aggressive periodontitis (AP), AP combined with type 1 diabetes mellitus (DM) and DM. We also explore the TNF-alpha capability expression and the presence of the -308 polymorphism. For this purpose we recruited 27 individuals with AP (AP+ group), 27 individuals with AP combined with DM (AP+/DM+ group), and 27 individuals with DM without signs of periodontitis upon clinical examination (DM+ group). The control group was comprised of 30 subjects. Genotyping for TNF-alpha promoter was performed by PCR-RFLP analysis. For TNF-alpha expression we used a blood culture system.
European cytokine network 15(4):364-70. · 1.73 Impact Factor
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ABSTRACT: Publicación ISI Email : jaguillo@med.uchile.cl Dendritic cells (DCs) are professional, antigen-presenting cells, which induce and regulate T cell reactivity. DCs are crucial in innate and adaptive immune responses, and are also involved in central and peripheral tolerance induction. Tolerance can be mediated by immature and semi-mature DCs expressing low levels of co-stimulator and major histocompatibility complex (MHC) molecules. The aim of this study was to investigate the ability of short-term lipopolysaccharide (LPS) stimulation to modulate the stage of differentiation of bone marrow-derived DCs. For this purpose, DCs obtained from DBA1/lacJ mice were stimulated for four (4hLPS/DCs) or 24 (24hLPS/DCs) hours with LPS, using DCs without stimulation (0hLPS/DCs) as a control. Flow cytometry analysis of 4hLPS/DCs showed intermediate CD40 and MHC class II expression, lower than that of 24hLPS/DCs (fully mature), and greater than that of 0hLPS/DCs (immature). A functional assay showed that 4hLPS/DCs displayed increased endocytotic ability compared to 24hLPS/DCs, indicating a semi-mature state. 4hLPS/DCs were greater producers of IL-10 protein and TGF beta 1 mRNA than 24hLPS/DCs and immature DCs, displaying a cytokine production pattern that is characteristic of tolerogenic DCs. An assay for antigen-presenting capacity demonstrated that 4hLPS/DCs induced secretion of IL-2 from an OTH4 T cell hybridoma, indicating a functional presenting activity. Finally, the tolerogenic phenotype of 4hLPS/DCs was demonstrated by their ability to interfere with the progression of bovine type 11 collagen (bII)-induced arthritis (CIA) when they were loaded with bCII antigen and injected into mice with established CIA. We conclude that the stimulation of murine bone marrow-derived DCs with LPS for four hours generates semi-mature DCs with tolerogenic capability.
