976 resultados para cell-free antigens


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RESUMO: As células dendríticas (DCs) têm a capacidade única de induzir respostas imunitárias contra as células tumorais, fagocitando antigénios tumorais e apresentando-os às células T, provocando respostas imunitárias específicas que conduzem à eliminação de células de tumorais. Por induzirem memória imunológica de longa duração, as DCs são uma estratégia atrativa para o tratamento e/ou prevenção do cancro. No entanto, os resultados terapêuticos obtidos em ensaios clínicos com DCs são escassos e pouco eficientes. O nosso grupo demonstrou que ácidos siálicos que contêm glicanos desempenham um papel funcional importante em DCs geradas ex vivo. Com o objetivo de estabelecer um modelo in vitro para avaliar a resposta anti-tumoral específica realizou-se um tratamento enzimático a DCs derivadas de monócitos (moDCs) com sialidase, enzima que cliva ácidos siálicos na superfície celular. O perfil de maturação de moDCs foi caracterizado por citometria de fluxo e expressão de citocinas. Os resultados mostram que a sialidase pode regular positivamente a expressão de moléculas co-estimuladoras na superfície de moDCs estimuladas com agonistas de Toll like receptors (TLRs). Para percebermos se o tratamento com sialidase afeta a sinalização dos TLRs foram usadas células HEK transfectadas de forma estável com TLRs 2, 4 and 7/8. Os dados mostraram que a desialilação não afeta a sinalização através estes recetores. Para investigar o impacto funcional da sialidase na capacidade de moDCs em apresentar um antigénio e ativar células T, moDCs foram tratadas, ou não, com sialidase e cultivadas com clones de células T CD8+ específicas para os péptidos derivados do antigénio tumoral gp100. Os resultados mostram que DCs HLA*02:01+ desialiladas exibem maior cross-presentation do péptido gp100280-288 às células T CD8+ específicas. Além disso o tratamento com sialidase também aumenta a capacidade de DCs de induzir a proliferação de células T CD4+. Em conjunto, os resultados indicam que moDCs com menos ácidos siálicos na superfície, têm melhor potencial imuno-estimulador, com maior capacidade de induzir respostas imunes anti-tumorais.--------------------- ABSTRACT: Dendritic cells (DCs) have a unique capacity to induce immune responses against tumor cells. They can phagocyte tumor antigens, maturate and present them to T cells, triggering antigen-specific immune responses that may lead to the elimination of tumor cells. Since they induce long-lasting immunological memory, DCs become an attractive strategy as cellular targets for vaccines in the treatment and/or prevention of cancer. However, the therapeutic results obtained in clinical trials with DCs are scarce and only few patients effectively respond to the DC vaccines. Our group has shown that sialic acid containing glycans play an important functional role in ex vivo generated DC. Here we aimed to establish an in vitro model to assess specific antitumor responses. To achieve this, an enzymatic treatment of monocyte-derived DCs (moDCs) was performed using sialidase to cleave surface sialic acids. The maturation profile of the moDCs was characterized by flow cytometry and cytokine expression. The results show that sialidase treatment can upregulate co-stimulatory molecules on surface of moDCs stimulated with Toll like receptor (TLR) agonists. To understand whether sialidase treatment affected the TLR signaling, we have used HEK cells stably transfected with TLRs 2, 4 and 7/8. The data showed that desialylation of moDCs does not affect the signaling via these receptors. To investigate the functional impact of sialidase treatment in the capacity of moDCs to present antigen and to activate antigen specific T cells, sialidase treated and untreated moDCs were co-cultured with CD8+ T cell clones specific for peptides derived from the gp100 tumor antigen. Our results show that desialylated HLA02:01+ DCs are superior in cross-presentation of the peptide to gp100280–288 specific CD8+ T cells. In addition, sialidase treatment also increased the DC capacity to induce CD4+ T cells proliferation. Together, these data indicate that moDCs with altered cell surface sialic acids, through a sialidase treatment, have a better immunostimulatory potential which could improve anti-tumor immune responses.

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Tendon's regeneration is limited, demanding for cell-based strategies to fully restore their functionality upon injury. The concept of magnetic force-based TE(1), generally using magnetic nanoparticles may enable, for example, stem cell stimulation and/or remote control over TE constructs. Thus, we originally propose the development of magnetic cell sheets (magCSs) with tenogenic capability, aimed at promoting tendon's regeneration. A Tenomodulin (TNMD+) subpopulation was sorted from human adipose stem cells (hASCs), using TNMD-coated immunomagnetic beads(2) and used as cell source for the development of magCSs. Briefly, cells were labeled with iron oxide composite particles (Micromod) and cultured for 7 days in α-MEM medium with or without magnetic stimulation provided by a magnetic device (nanoTherics). CSs were retrieved from the plates using magnet attraction as contiguous sheets of cells within its own deposited ECM.

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Tese de Doutoramento Biologia Molecular e Ambiental - Especialidade em Biologia Celular e Saúde

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In tissue engineering of cartilage, polymeric scaffolds are implanted in the damaged tissue and subjected to repeated compression loading cycles. The possibility of failure due to mechanical fatigue has not been properly addressed in these scaffolds. Nevertheless, the macroporous scaffold is susceptible to failure after repeated loading-unloading cycles. This is related to inherent discontinuities in the material due to the micropore structure of the macro-pore walls that act as stress concentration points. In this work, chondrogenic precursor cells have been seeded in Poly-ε-caprolactone (PCL) scaffolds with fibrin and some were submitted to free swelling culture and others to cyclic loading in a bioreactor. After cell culture, all the samples were analyzed for fatigue behavior under repeated loading-unloading cycles. Moreover, some components of the extracellular matrix (ECM) were identified. No differences were observed between samples undergoing free swelling or bioreactor loading conditions, neither respect to matrix components nor to mechanical performance to fatigue. The ECM did not achieve the desired preponderance of collagen type II over collagen type I which is considered the main characteristic of hyaline cartilage ECM. However, prediction in PCL with ECM constructs was possible up to 600 cycles, an enhanced performance when compared to previous works. PCL after cell culture presents an improved fatigue resistance, despite the fact that the measured elastic modulus at the first cycle was similar to PCL with poly(vinyl alcohol) samples. This finding suggests that fatigue analysis in tissue engineering constructs can provide additional information missed with traditional mechanical measurements.

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Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Biomateriais, Reabilitação e Biomecânica)

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Renal cell tumors (RCTs) are the most lethal of the common urological cancers. The widespread use of imaging entailed an increased detection of small renal masses, emphasizing the need for accurate distinction between benign and malignant RCTs, which is critical for adequate therapeutic management. Histone methylation has been implicated in renal tumorigenesis, but its potential clinical value as RCT biomarker remains mostly unexplored. Hence, the main goal of this study was to identify differentially expressed histone methyltransferases (HMTs) and histone demethylases (HDMs) that might prove useful for RCT diagnosis and prognostication, emphasizing the discrimination between oncocytoma (a benign tumor) and renal cell carcinoma (RCC), especially the chromophobe subtype (chRCC). We found that the expression levels of three genes-SMYD2, SETD3, and NO66-was significantly altered in a set of RCTs, which was further validated in a large independent cohort. Higher expression levels were found in RCTs compared to normal renal tissues (RNTs) and in chRCCs comparatively to oncocytomas. SMYD2 and SETD3 mRNA levels correlated with protein expression assessed by immunohistochemistry. SMYD2 transcript levels discriminated RCTs from RNT, with 82.1% sensitivity and 100% specificity (AUC=0.959), and distinguished chRCCs from oncocytomas, with 71.0% sensitivity and 73.3% specificity (AUC: 0.784). Low expression levels of SMYD2, SETD3, and NO66 were significantly associated with shorter disease-specific and disease-free survival, especially in patients with non-organ confined tumors. We conclude that expression of selected HMTs and HDMs might constitute novel biomarkers to assist in RCT diagnosis and assessment of tumor aggressiveness.

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Antecedentes: En nuestro laboratorio hemos demostrado que antígenos (Ags) de Fasciola hepatica inducen en células dendríticas murinas (CD), diferentes propiedades tolerogénicas como la incapacidad por si mismos de inducir la maduración de las células, la resistencia a la maduración por ligandos de TLR, el incremento en la producción de IDO y también la capacidad de esta estas células de dirigir la respuesta inmune hacia un perfil Th2 y T reg. Por otra parte ha sido bien documentado que CD con características tolerogénicas, ya sea inmaduras o semimaduras, son útiles para reducir respuestas inflamatorias excesivas tales como las que ocurren en enfermedades autoinmunes. Además hemos demostrado que CD tratadas con Ags del parásito en conjunto con un ligando Toll (CpG-ODN) producen altos niveles de citoquinas anti-inflamatorias (IL-10 y TGF-) bajos de citoquinas proinflamatorias (TNF, IL-6, IL-12). Hipótesis: El fenotipo semimaduro alcanzado en las CDpodría ser utilizado para reducir la inflamación en un modelo de enfermedad autoinmune en donde existe una exacerbada respuesta Th1 y Th17, ya que la producción elevada de IL-10 y TGF- podría inhibir o controlar estas respuestas de manera directa o a través de la inducción de células T regulatorias. Objetivos: En este proyecto nosotros proponemos la inmunización de animales susceptibles (ratones DBA1/j), al desarrollo de artritis inducida por colágeno (AIC) con CD tratadas con Ags de F. hepatica en conjunto con CpG-ODN para reducir los síntomas clínicos de la enfermedad. Materiales a utilizar: En nuestro laboratorio hemos desarrollado un modelo de artritis inducida por colágeno (AIC) mediante dos inmunizaciones de ratones DBA1/j con colágeno tipo II bovino y adyuvante de Freund. El modelo permitió establecer un índice clínico mediante la hinchazón en las patas de los animales. Doce días posteriores a la primera inmunización los animales serán inyectados con CD tratadas con: 1. PBS, 2.Extracto total de F.hepatica (TE) + CII, 3. CpG + CII, 4. TE+CpG+CII Se realizará la observación macroscópica diaria, a partir de los 7 días de la 2a inmunización Luego del sacrificio las articulaciones de las patas se prepararán para realizar un análisis histológico. Se detectará en suero los niveles de anticuerpos IgG1 (perfil Th2) y de IgG2a (perfil Th1) mediante la técnica de ELISA. Se detectará también el perfil de citoquinas en los nódulos drenantes por la técnica de ELISA y adicionalmente la poblaciónes celulares de células T regulatorias (Treg) CD4+CD25+Foxp3 o células Tr1. Resultados esperados: Pensamos que el tratamiento de los animales que desarrollan AIC con CD semimaduras (por el tratamiento con TE y CpG), serán capaces de migrar a los órganos linfaticos y secretar TGF-be(inductora de células T reg), IL-10 (inductoras de células Tr1), IDO inhibitoria de la respuesta de Li T y promotor de células T reg, también podría generarse una respuesta Th2 (por la presencia de antígenos del parásito), y estas respuestas aisladas o en forma sinérgica podrían inhibir las respuestas de tipo Th17 y Th1 asociadas a la patología en esta enfermedad. Importancia del proyecto: En el desarrollo de la artritis existe un aumento de la inmunidad mediada por células, asi como de la respuesta inmune humoral hacia componentes de la matriz del cartílago. El tratamiento convencional de la artritis recae en general en el uso de inmunosupresores no-específicos, los cuales poseen una variedad de efectos adversos y la inhibición de la respuesta inflamatoria no es específica. En este proyecto proponemos el uso de CD tratadas con antígenos del helminto F. hepatica y CpG ligando Tol que capacita a estas células para generar una respuesta adaptativa de tipo regulatoria, útil en la inhibición de las respuestas inflamatorias como la que ocurre durante la progresión de artritis reumatoidea en un modelo experimental en ratones. We have shown that F. hepatica Ags-treated dendritic cells (DC) together with a TLRl ligand (CpG-ODN) produce high levels of anti-inflammatory cytokines (IL-10 and TGF-Beta) and low of proinflammatory cytokines (TNF, IL-6, IL -12). Hypothesis: The semimature phenotype achieved by DC, could be used to reduce inflammation in a model of autoimmune disease. The high production of IL-10 and TGF-Beta by these cells could directly or through the induction of T reg cells inhibit the inflammatory response. Objective: In this project we propose the immunization of DBA1 / j mice, susceptible to the development of collagen-induced arthritis (CIA) with F. hepatica-treated DC in conjunction with CpG-ODN to reduce clinical signs of disease. Materials: In our laboratory, we developed the CIA model by two immunizations of DBA1 / j mice with bovine type II collagen and Freund's adjuvant. The model allowed to stablish a clinical index by swelling in the legs of animals. Twelve days after the first immunization the animals are injected with DC treated with: 1. PBS 2. F.hepatica Extract (TE) + CII, 3. CpG + CII, 4. TE + CpG + CII Macroscopic observation will take place daily from 7 days of the 2nd immunization. After sacrifice the joints of the legs will be prepared for histological analysis. Serum levels of IgG1 antibodies (Th2 profile) and IgG2a (Th1 profile) will be detected by ELISA. It will also detected the cytokine profile in draining lymph nodes by ELISA and additionally the cell populations of regulatory T cells (Treg) CD4 + CD25 + Foxp3 or Tr1 cells. Expected results: We believe that the treatment of animals that had developed CIA with DC will be able to migrate to lymphatic organs and secrete TGF-B (T reg cell-inducing), IL-10 (inducing Tr1 cells), IDO (inhibitory of T cells and inducing of T reg cells) could alone or in synergy inhibit Th17-type responses and Th1 associated with the pathology in this disease.

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Circulating antigens were detected in sera of mice experimentally infected with a high close of Trypanosoma cruzi by reaction with sera from chronically infected mice. The immunodiffusion reaction between homologous acute and chronic sera produced four precipitation lines. By reaction with chronic mouse serum, circulating antingens were detected in sera from heavily infected hamsters, dogs, rabbits and in sera from chagasic patients. A reaction was also found in urine from acutely infected mice and dogs. Trypanosoma cruzi exoantigen was detected in trypanosome culture medium and in the supernatant of infected cell cultures. Attempts to isolate the antigens are described.

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HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.

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The mechanism of CD8 cooperation with the TCR in antigen recognition was studied on live T cells. Fluorescence correlation measurements yielded evidence of the presence of two TCR and CD8 subpopulations with different lateral diffusion rate constants. Independently, evidence for two subpopulations was derived from the experimentally observed two distinct association phases of cognate peptide bound to class I MHC (pMHC) tetramers and the T cells. The fast phase rate constant ((1.7 +/- 0.2) x 10(5) M(-1) s(-1)) was independent of examined cell type or MHC-bound peptides' structure. Its value was much faster than that of the association of soluble pMHC and TCR ((7.0 +/- 0.3) x 10(3) M(-1) s(-1)), and close to that of the association of soluble pMHC with CD8 ((1-2) x 10(5) M(-1) s(-1)). The fast binding phase disappeared when CD8-pMHC interaction was blocked by a CD8-specific mAb. The latter rate constant was slowed down approximately 10-fold after cells treatment with methyl-beta-cyclodextrin. These results suggest that the most efficient pMHC-cell association route corresponds to a fast tetramer binding to a colocalized CD8-TCR subpopulation, which apparently resides within membrane rafts: the reaction starts by pMHC association with the CD8. This markedly faster step significantly increases the probability of pMHC-TCR encounters and thereby promotes pMHC association with CD8-proximal TCR. The slow binding phase is assigned to pMHC association with a noncolocalized CD8-TCR subpopulation. Taken together with results of cytotoxicity assays, our data suggest that the colocalized, raft-associated CD8-TCR subpopulation is the one capable of inducing T-cell activation.

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We have developed an in vitro model of granuloma formation for the purpose of studying the immunological components of delayed type hypersensitivity granuloma formation in patients infected with Schistosoma mansoni. Our data show that 1) granulomatous hypersensitivity can be studied by examining the cellular reactivity manifested as multiple cell layers surrounding the antigen conjugated beads; 2) this reactivity is a CD4 cell dependent, macrophage dependent, B cell independent response and 3) the in vitro granuloma response is antigenically specific for parasite egg antigens. Studies designed to investigate the immune regulation of granulomatous hypersensitivity using purified populations of either CD4 or CD8 T cells have demonstrated the complexity of cellular interactions in the suppression of granulomatous hypersensitivity. The anti-S. mansoni egg immune responses of individual patients with chronic intestinal schistosomiasis can be classified either as soluble egg antigen (SEA) hypersensitive with maximal granulomatous hypersensitivity or SEA suppressive with activation of the T cell suppressor pathway with effective SEA granuloma modulation. Our data suggest that T cell network interactions are active in the generation of effective granuloma modulation in chronic intestinal schistosomiasis patients.

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We have designed a vaccine model based on induction of cell-mediated immunity and shown that it protects mice against Schistosoma mansoni infection. Mice are immunized by intradermal injection with schistosome antigens plus BCG. Resistance is dependent on the route of antigen presentation and the adjuvant chosen. The pattern of resistance correlates with sensitization of T lymphocytes for production of gamma interferon, a macrophage activating lymphokine that stimulates the cellular effector mechanism of protection. Purified schistosome paramyosin, a muscle cell component present in soluble parasite antigenic preparations, is immunogenic for T lymphocytes and induces resistance when given intradermally with BCG. It is likely that this protein, and possibly other soluble molecules that are released by the parasites of a challenge infection, induce a cellular inflammatory response resulting in larval trapping and/or killing by activated macrophages. These results verify the feasibility of a vaccine against schistosomiasis based on induction of cell-mediated immune resistance mechanisms.

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The activation of B cell dependent T cells during Leishmania infection cannot be considered a trivial event, because their removal profoundly alters the course and outcome of infection within genetically susceptible and resistant mouse strains. The demonstration that idiotype recognizing T cells also appear within human populations sensitized to leishmanial antigens as a result of asymptomatic or subclinical infections supports a role for these cells in immunity. These cells are not demonstrable in patients with active visceral disease, so that their role in promoting specific unresponsiveness has not been extended to humans. Whether B cell dependent, idiotype specific T cells represent a functionally distinct T lymphocyte subset with unique regulatory activities remains to be determined.

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To directly assess the binding of exogenous peptides to cell surface-associated MHC class I molecules at the single cell level, we examined the possibility of combining the use of biotinylated peptide derivatives with an immunofluorescence detection system based on flow cytometry. Various biotinylated derivatives of the adenovirus 5 early region 1A peptide 234-243, an antigenic peptide recognized by CTL in the context of H-2Db, were first screened in functional assays for their ability to bind efficiently to Db molecules on living cells. Suitable peptide derivatives were then tested for their ability to generate positive fluorescence signals upon addition of phycoerythrin-labeled streptavidin to peptide derivative-bearing cells. Strong fluorescent staining of Db-expressing cells was achieved after incubation with a peptide derivative containing a biotin group at the C-terminus. Competition experiments using the unmodified parental peptide as well as unrelated peptides known to bind to Kd, Kb, or Db, respectively, established that binding of the biotinylated peptide to living cells was Db-specific. By using Con A blasts derived from different H-2 congenic mouse strains, it could be shown that the biotinylated peptide bound only to Db among > 20 class I alleles tested. Moreover, binding of the biotinylated peptide to cells expressing the Dbm13 and Dbm14 mutant molecules was drastically reduced compared to Db. Binding of the biotinylated peptide to freshly isolated Db+ cells was readily detectable, allowing direct assessment of the relative amount of peptide bound to distinct lymphocyte subpopulations by three-color flow cytometry. While minor differences between peripheral T and B cells could be documented, thymocytes were found to differ widely in their peptide binding activity. In all cases, these differences correlated positively with the differential expression of Db at the cell surface. Finally, kinetic studies at different temperatures strongly suggested that the biotinylated peptide first associated with Db molecules available constitutively at the cell surface and then with newly arrived Db molecules.

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The concept of cellular schwannoma as an unusual benign tumor is well established for peripheral nerves but has never been tested in neurosurgical series. In order to test the validity of this concept in cranial nerves and spinal roots we performed an analysis of the clinical and morphological characteristics of 12 cellular and 166 classical benign schwannomas. Immunohistochemical detection of antigen expression in Schwann cells including proliferating cell nuclear antigen (PCNA) was also performed. This study shows that cellular schwannomas in neurosurgical series manifest at a lower age than the classical benign variant and occur mainly in the spinal roots. Mitotic activity and sinusoidal vessels appear more frequently in cellular schwannomas and constitute with high cellularity, the most valuable criteria separating both entities. The postoperative course in both types of tumors was free of metastases or sarcomatous changes. Immunoexpression of S-100 protein, vimentin, epithelial membrane antigen and glial fibrillary acidic protein is not statistically different between the two variants. In contrast, PCNA is more highly expressed in cellular schwannomas. These These results confirm the concept that cellular schwannomas are a clinico-pathological variant of benign schwannomas and provide significant support for the introduction of this entity in neurosurgical oncology.