RESUMEN
Stromal cells promote extensive fibrosis in pancreatic ductal adenocarcinoma (PDAC), which is associated with poor prognosis and therapeutic resistance. We report here for the first time that loss of the RNA-binding protein human antigen R (HuR, ELAVL1) in PDAC cells leads to reprogramming of the tumor microenvironment. In multiple in vivo models, CRISPR deletion of ELAVL1 in PDAC cells resulted in a decrease of collagen deposition, accompanied by a decrease of stromal markers (i.e. podoplanin, α-smooth muscle actin, desmin). RNA-sequencing data showed that HuR plays a role in cell-cell communication. Accordingly, cytokine arrays identified that HuR regulates the secretion of signaling molecules involved in stromal activation and extracellular matrix organization [i.e. platelet-derived growth factor AA (PDGFAA) and pentraxin 3]. Ribonucleoprotein immunoprecipitation analysis and transcription inhibition studies validated PDGFA mRNA as a novel HuR target. These data suggest that tumor-intrinsic HuR supports extrinsic activation of the stroma to produce collagen and desmoplasia through regulating signaling molecules (e.g. PDGFAA). HuR-deficient PDAC in vivo tumors with an altered tumor microenvironment are more sensitive to the standard of care gemcitabine, as compared to HuR-proficient tumors. Taken together, we identified a novel role of tumor-intrinsic HuR in its ability to modify the surrounding tumor microenvironment and regulate PDGFAA.
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INTRODUCTION: Standard-of-care systemic chemotherapies for pancreatic ductal adenocarcinoma (PDAC) currently have limited clinical benefits, in addition to causing adverse side effects in many patients. One factor known to contribute to the poor chemotherapy response is the poor drug diffusion into PDAC tumors. Novel treatment methods are therefore drastically needed to improve targeted delivery of treatments. Here, we evaluated the efficacy of the 3DNA® Nanocarrier (3DNA) platform to direct delivery of therapeutics to PDAC tumors in vivo. MATERIALS AND METHODS: A panel of PDAC cell lines and a patient tissue microarray were screened for established tumor-specific proteins to identify targeting moieties for active targeting of the 3DNA. NRG mice with or without orthotopic MIA PaCa-2-luciferase PDAC tumors were treated intraperitoneally with 100 µl of fluorescently labeled 3DNA. RESULTS: Folic acid and transferrin receptors were significantly elevated in PDAC compared to normal pancreas. Accordingly, both folic acid- and transferrin-conjugated 3DNA treatments significantly increased delivery of 3DNA specifically to tumors in comparison to unconjugated 3DNA treatment. In the absence of tumors, there was an increased clearance of both folic acid-conjugated 3DNA and unconjugated 3DNA, compared to the clearance rate in tumor-bearing mice. Lastly, delivery of siLuciferase by folic acid-conjugated 3DNA in an orthotopic model of luciferase-expressing PDAC showed significant and prolonged suppression of luciferase protein expression and activity. CONCLUSION: Our study progresses the 3DNA technology as a reliable and effective treatment delivery platform for targeted therapeutic approaches in PDAC.
RESUMEN
Yes-associated protein 1 (YAP1) is indispensable for the development of mutant KRAS-driven pancreatic ductal adenocarcinoma (PDAC). High YAP1 mRNA is a prognostic marker for worse overall survival in patient samples; however, the regulatory mechanisms that mediate its overexpression are not well understood. YAP1 genetic alterations are rare in PDAC, suggesting that its dysregulation is likely not due to genetic events. HuR is an RNA-binding protein whose inhibition impacts many cancer-associated pathways, including the "conserved YAP1 signature" as demonstrated by gene set enrichment analysis. Screening publicly available and internal ribonucleoprotein immunoprecipitation (RNP-IP) RNA sequencing (RNA-Seq) data sets, we discovered that YAP1 is a high-confidence target, which was validated in vitro with independent RNP-IPs and 3' untranslated region (UTR) binding assays. In accordance with our RNA sequencing analysis, transient inhibition (e.g., small interfering RNA [siRNA] and small-molecular inhibition) and CRISPR knockout of HuR significantly reduced expression of YAP1 and its transcriptional targets. We used these data to develop a HuR activity signature (HAS), in which high expression predicts significantly worse overall and disease-free survival in patient samples. Importantly, the signature strongly correlates with YAP1 mRNA expression. These findings highlight a novel mechanism of YAP1 regulation, which may explain how tumor cells maintain YAP1 mRNA expression at dynamic times during pancreatic tumorigenesis.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Regiones no Traducidas 3'/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Proteína 1 Similar a ELAV/genética , Proteína 1 Similar a ELAV/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , ARN Mensajero/genética , ARN Interferente Pequeño , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Señalizadoras YAP , Neoplasias PancreáticasRESUMEN
Analyses of somatic hypermutation (SHM) patterns in B cell Ig sequences have important basic science and clinical applications, but they are often confounded by the intrinsic biases of SHM targeting on specific DNA motifs (i.e., hot and cold spots). Modeling these biases has been hindered by the difficulty in identifying mutated Ig sequences in vivo in the absence of selection pressures, which skew the observed mutation patterns. To generate a large number of unselected mutations, we immunized B1-8 H chain transgenic mice with nitrophenyl to stimulate nitrophenyl-specific λ+ germinal center B cells and sequenced the unexpressed κ L chains using next-generation methods. Most of these κ sequences had out-of-frame junctions and were presumably uninfluenced by selection. Despite being nonfunctionally rearranged, they were targeted by SHM and displayed a higher mutation frequency than functional sequences. We used 39,173 mutations to construct a quantitative SHM targeting model. The model showed targeting biases that were consistent with classic hot and cold spots, yet revealed additional highly mutable motifs. We observed comparable targeting for functional and nonfunctional sequences, suggesting similar biological processes operate at both loci. However, we observed species- and chain-specific targeting patterns, demonstrating the need for multiple SHM targeting models. Interestingly, the targeting of C/G bases and the frequency of transition mutations at C/G bases was higher in mice compared with humans, suggesting lower levels of DNA repair activity in mice. Our models of SHM targeting provide insights into the SHM process and support future analyses of mutation patterns.
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Linfocitos B/inmunología , Centro Germinal/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Modelos Genéticos , Hipermutación Somática de Inmunoglobulina/genética , Animales , Células Cultivadas , Selección Clonal Mediada por Antígenos , Reparación del ADN , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Mutación/genética , Tasa de MutaciónRESUMEN
Autoantibodies specific for the enzyme transglutaminase 2 (TG2) are a hallmark of the gluten-sensitive enteropathy celiac disease. Production of the Abs is strictly dependent on exposure to dietary gluten proteins, thus raising the question how a foreign Ag (gluten) can induce an autoimmune response. It has been suggested that TG2-reactive B cells are activated by gluten-reactive T cells following receptor-mediated uptake of TG2-gluten complexes. In this study, we propose a revised model that is based on the ability of the BCR to serve as a substrate to TG2 and become cross-linked to gluten-derived peptides. We show that TG2-specific IgD molecules are preferred in the reaction and that binding of TG2 via a common epitope targeted by cells using the IgH variable gene segment (IGHV)5-51 results in more efficient cross-linking. Based on these findings we hypothesize that IgD-expressing B cells using IGHV5-51 are preferentially activated, and we suggest that this property can explain the previously reported low number of somatic mutations as well as the overrepresentation of IGHV5-51 among TG2-specific plasma cells in the celiac lesion. The model also couples gluten peptide uptake by TG2-reactive B cells directly to peptide deamidation, which is necessary for the activation of gluten-reactive T cells. It thereby provides a link between gluten deamidation, T cell activation, and the production of TG2-specific Abs. These are all key events in the development of celiac disease, and by connecting them the model may explain why the same enzyme that catalyzes gluten deamidation is also an autoantigen, something that is hardly coincidental.
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Autoanticuerpos/inmunología , Enfermedad Celíaca/inmunología , Proteínas de Unión al GTP/inmunología , Glútenes/inmunología , Inmunoglobulina D/inmunología , Transglutaminasas/inmunología , Animales , Autoanticuerpos/biosíntesis , Autoantígenos/inmunología , Linfocitos B/inmunología , Línea Celular Tumoral , Epítopos de Linfocito T/inmunología , Glútenes/metabolismo , Humanos , Inmunoglobulina A/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Activación de Linfocitos/inmunología , Ratones , Proteína Glutamina Gamma Glutamiltransferasa 2 , Receptores de Antígenos de Linfocitos B/inmunología , Linfocitos T/inmunologíaRESUMEN
The B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%-2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies.
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Linfocitos B/inmunología , Selección Clonal Mediada por Antígenos/inmunología , Centro Germinal/inmunología , Salmonella typhimurium/inmunología , Hipermutación Somática de Inmunoglobulina/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Selección Clonal Mediada por Antígenos/genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/inmunología , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/microbiología , Hipermutación Somática de Inmunoglobulina/genética , Bazo/citología , Bazo/inmunologíaRESUMEN
Anti-idiotype antibodies have potential therapeutic applications in many fields, including autoimmune diseases. Herein we report the isolation and characterization of AIM2, an anti-idiotype antibody elicited in a mouse model upon expression of the celiac disease-specific autoantibody MB2.8 (directed against the main disease autoantigen type 2 transglutaminase, TG2). To characterize the interaction between the two antibodies, a 3D model of the MB2.8-AIM2 complex has been obtained by molecular docking. Analysis and selection of the different obtained docking solutions was based on the conservation within them of the inter-residue contacts. The selected model is very well representative of the different solutions found and its stability is confirmed by molecular dynamics simulations. Furthermore, the binding mode it adopts is very similar to that observed in most of the experimental structures available for idiotype-anti-idiotype antibody complexes. In the obtained model, AIM2 is directed against the MB2.8 CDR region, especially on its variable light chain. This makes the concurrent formation of the MB2.8-AIM2 complex and of the MB2.8-TG2 complex incompatible, thus explaining the experimentally observed inhibitory effect on the MB2.8 binding to TG2.
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Anticuerpos Antiidiotipos/química , Enfermedad Celíaca/inmunología , Simulación del Acoplamiento Molecular , Secuencia de Aminoácidos , Animales , Anticuerpos Antiidiotipos/metabolismo , Autoanticuerpos/química , Autoanticuerpos/metabolismo , Sitios de Unión de Anticuerpos , Proteínas de Unión al GTP/inmunología , Humanos , Ratones , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/inmunologíaRESUMEN
The gluten-sensitive enteropathy celiac disease is tightly associated with the production of autoantibodies specific for the enzyme transglutaminase 2 (TG2). The mechanisms underlying the activation of autoreactive B cells, however, are not well defined. To gain more insight into this autoimmune response we have characterized the binding of TG2 by a panel of human mAbs generated by expression cloning of Ig genes from single plasma cells of the celiac disease lesion. The Abs were highly specific to TG2 and bound preferentially to the open, Ca(2+)-activated enzyme conformation. Epitope mapping revealed that they recognize few distinct conformational epitopes that cluster in the N-terminal half of the enzyme. Two of the epitopes were overlapping with the fibronectin binding site in TG2, and none of the epitopes was accessible when TG2 was in a cell surface-bound form. Based on our findings, we propose that the autoantibodies are generated against the soluble, catalytically active enzyme, whereas Abs reactive with cell surface-associated TG2 are absent from the response due to negative selection of B cells recognizing membrane-bound self-Ag. The findings give insight into the mechanisms controlling the formation of anti-TG2 autoantibodies in celiac disease.
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Autoanticuerpos/inmunología , Enfermedad Celíaca/inmunología , Epítopos de Linfocito B/inmunología , Transglutaminasas/inmunología , Animales , Especificidad de Anticuerpos , Autoantígenos/química , Autoantígenos/inmunología , Autoinmunidad/inmunología , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Epítopos de Linfocito B/química , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteínas de Unión al GTP , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/químicaRESUMEN
The aim of this study was to dissect the autoantibody response in celiac disease (CD) that remains largely unknown, with the goal of identifying the disease-specific autoantigenic protein pattern or the so called epitome. Sera from CD patients were used to select immunoreactive antigens from a cDNA phage-display library. Candidate genes were identified, the corresponding proteins produced and their immunoreactivity validated with sera from CD patients and controls. Thirteen CD-specific antigens were identified and further validated by protein microarray. The specificity for 6 of these antigens was confirmed by ELISA. Furthermore we showed that this antibody response was not abolished on a gluten free diet and was not shared with other autoimmune diseases. These antigens appear to be CD specific and independent of gluten induction. The utility of this panel extends beyond its diagnostic value and it may drive the attention to new targets for unbiased screens in autoimmunity research.
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Autoanticuerpos/inmunología , Autoantígenos/inmunología , Enfermedad Celíaca/inmunología , Adolescente , Adulto , Autoanticuerpos/sangre , Autoantígenos/genética , Enfermedad Celíaca/sangre , Enfermedad Celíaca/diagnóstico , Enfermedad Celíaca/genética , Técnicas de Visualización de Superficie Celular , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis por Matrices de Proteínas , Curva ROC , Adulto JovenRESUMEN
The function of intestinal immunity is to provide protection toward pathogens while preserving the composition of the microflora and tolerance to orally fed nutrients. This is achieved via a number of tightly regulated mechanisms including production of IgA antibodies by intestinal plasma cells. Celiac disease is a common gut disorder caused by a dysfunctional immune regulation as signified, among other features, by a massive intestinal IgA autoantibody response. Here we review the current knowledge of this B-cell response and how it is induced, and we discuss key questions to be addressed in future research.
RESUMEN
Celiac disease is an immune-mediated disorder in which mucosal autoantibodies to the enzyme transglutaminase 2 (TG2) are generated in response to the exogenous antigen gluten in individuals who express human leukocyte antigen HLA-DQ2 or HLA-DQ8 (ref. 3). We assessed in a comprehensive and nonbiased manner the IgA anti-TG2 response by expression cloning of the antibody repertoire of ex vivo-isolated intestinal antibody-secreting cells (ASCs). We found that TG2-specific plasma cells are markedly expanded within the duodenal mucosa in individuals with active celiac disease. TG2-specific antibodies were of high affinity yet showed little adaptation by somatic mutations. Unlike infection-induced peripheral blood plasmablasts, the TG2-specific ASCs had not recently proliferated and were not short-lived ex vivo. Altogether, these observations demonstrate that there is a germline repertoire with high affinity for TG2 that may favor massive generation of autoreactive B cells. TG2-specific antibodies did not block enzymatic activity and served as substrates for TG2-mediated crosslinking when expressed as IgD or IgM but not as IgA1 or IgG1. This could result in preferential recruitment of plasma cells from naive IgD- and IgM-expressing B cells, thus possibly explaining why the antibody response to TG2 bears signs of a primary immune response despite the disease chronicity.
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Células Productoras de Anticuerpos/inmunología , Autoanticuerpos/inmunología , Enfermedad Celíaca/inmunología , Proteínas de Unión al GTP/inmunología , Proteínas de Unión al GTP/metabolismo , Inmunoglobulina A/inmunología , Transglutaminasas/inmunología , Transglutaminasas/metabolismo , Autoanticuerpos/sangre , Linfocitos B/inmunología , Enfermedad Celíaca/sangre , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Unión al GTP/sangre , Glútenes/inmunología , Antígenos HLA-DQ/inmunología , Humanos , Inmunoglobulina A/sangre , Inmunoglobulina D/sangre , Inmunoglobulina D/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Mucosa Intestinal/enzimología , Mucosa Intestinal/inmunología , Mutación , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Análisis de la Célula Individual , Hipermutación Somática de Inmunoglobulina , Transglutaminasas/sangreRESUMEN
To understand the biology of Ab-secreting cells in the human small intestine, we examined Ab production of intestinal biopsies kept in culture. We found sustained IgA and IgM secretion as well as viable IgA- or IgM-secreting cells after >4 wk of culture. The Ab-secreting cells were nonproliferating and expressing CD27 and CD138, thus having a typical plasma cell phenotype. Culturing of biopsies without tissue disruption gave the highest Ab production and plasma cell survival suggesting that the environment regulates plasma cell longevity. Cytokine profiling of the biopsy cultures demonstrated a sustained presence of IL-6 and APRIL. Blocking of the activity of endogenous APRIL and IL-6 with BCMA-Fc and anti-human IL-6 Ab demonstrated that both these factors were essential for plasma cell survival and Ab secretion in the biopsy cultures. This study demonstrates that the human small intestine harbors a population of nonproliferating plasma cells that are instructed by the microenvironment for prolonged survival and Ab secretion.
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Inmunidad Mucosa/inmunología , Inmunoglobulina A/biosíntesis , Inmunoglobulina M/biosíntesis , Células Plasmáticas/metabolismo , Biopsia , Separación Celular , Supervivencia Celular , Duodeno/citología , Duodeno/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Inmunoglobulina A/inmunología , Inmunoglobulina M/inmunología , Interleucina-6/inmunología , Interleucina-6/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Células Plasmáticas/inmunología , Técnicas de Cultivo de Tejidos , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismoRESUMEN
The gut mucosal surface is efficiently protected by Abs, and this site represents one of the richest compartments of Ab-secreting cells in the body. A simple and effective method to generate Ag-specific human monoclonal Abs (hmAbs) from such cells is lacking. In this paper, we describe a method to generate hmAbs from single Ag-specific IgA- or IgM-secreting cells of the intestinal mucosa. We found that CD138-positive plasma cells from the duodenum expressed surface IgA or IgM. Using eGFP-labeled virus-like particles, we harnessed the surface Ig expression to detect rotavirus-specific plasma cells at low frequency (0.03-0.35%) in 9 of 10 adult subjects. Single cells were isolated by FACS, and as they were viable, further testing of secreted Abs by ELISPOT and ELISA indicated a highly specific selection procedure. Ab genes from single cells of three donors were cloned, sequenced, and expressed as recombinant hmAbs. Of 26 cloned H chain Ab genes, 22 were IgA and 4 were IgM. The genes were highly mutated, and there was an overrepresentation of the VH4 family. Of 10 expressed hmAbs, 8 were rotavirus-reactive (6 with K(d) < 1 × 10(-10)). Importantly, our method allows generation of hmAbs from cells implicated in the protection of mucosal surfaces, and it can potentially be used in passive vaccination efforts and for discovery of epitopes directly relevant to human immunity.
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Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Técnicas Inmunológicas , Mucosa Intestinal/inmunología , Células Plasmáticas/inmunología , Rotavirus/inmunología , Adulto , Anciano , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Antígenos Virales/inmunología , Western Blotting , Proteínas de la Cápside/inmunología , Separación Celular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Humanos , Inmunoglobulina A/biosíntesis , Inmunoglobulina A/inmunología , Inmunoglobulina M/biosíntesis , Inmunoglobulina M/inmunología , Mucosa Intestinal/citología , Intestino Delgado/citología , Intestino Delgado/inmunología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Adulto JovenRESUMEN
We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120,000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.
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Mapeo de Interacción de Proteínas/métodos , Análisis de Secuencia de ADN/métodos , ADN Complementario/química , Proteínas de Unión al GTP/metabolismo , Humanos , Sistemas de Lectura Abierta , Biblioteca de Péptidos , Proteína Glutamina Gamma Glutamiltransferasa 2 , Dominios y Motivos de Interacción de Proteínas , Transglutaminasas/metabolismoRESUMEN
The advent of the serological identification of antigens by procedures such as cDNA cloning and recombinant protein expression has allowed the direct molecular definition of immunogenic proteins. The phage-display technology provides several advantages over conventional immunoscreening procedures based on plasmid or lambda-phage cDNA libraries. So far, attempts to display open reading frames, such as those encoded by cDNA fragments, on filamentous phages have not been very successful. We managed to develop a strategy based on "folding reporters" which allows filtering out open reading frames from DNA and displaying them on filamentous phages in such a way that they are amenable to subsequent selection or screening. Once the cDNA library of interest is created, phage-display technology is used for selection of novel putative antigens; these are then validated by printing isolated protein on microarray and screening with patients' sera.
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Autoanticuerpos/análisis , Biblioteca de Genes , Biblioteca de Péptidos , Autoanticuerpos/genética , Autoanticuerpos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Modelos Biológicos , Sistemas de Lectura Abierta , Análisis por Matrices de Proteínas/métodos , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunologíaRESUMEN
Protein fragment complementation assay (PCA) is based on the interaction between two protein partners (e.g. target antigen and antibody), which are genetically fused to the two halves of a dissected marker protein. Binding of the two partners reassembles the marker protein and hence reconstitutes its activity. In this work we have developed the first application of beta-lactamase-based PCA for the isolation of single chain Fv fragments (scFvs) binding to the human receptor RON from a naïve library. Specific scFvs with the ability to immunoprecipitate could be isolated after a single round of PCA selection from an scFv repertoire previously pre-selected by phage display. Furthermore, the PCA was used to successfully map the epitopes recognized by the selected scFvs by screening them against a small library of random RON fragments.
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Anticuerpos/inmunología , Fragmentos de Péptidos/inmunología , Análisis por Matrices de Proteínas/métodos , Proteínas Recombinantes de Fusión/inmunología , beta-Lactamasas/genética , Animales , Anticuerpos/genética , Anticuerpos/metabolismo , Afinidad de Anticuerpos/genética , Antígenos/genética , Antígenos/inmunología , Antígenos/metabolismo , Línea Celular , Mapeo Epitopo , Escherichia coli/genética , Humanos , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/inmunología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN , beta-Lactamasas/metabolismoRESUMEN
Celiac disease is an autoimmune illness characterized by intestinal mucosal injury and malabsorption precipitated by dietary exposure to gluten of some cereals. The immune response is based on both cellular and humoral components, although the former seem to be more important in the pathogenesis. The autoantibody response is directed at the enzyme tissue transglutaminase, tTG or TG2, which possibly play a role in the onset of the disease. In this study we sought to develop an animal model in which to analyze the immunological regulation and significance of anti-TG2 antibodies, by expressing specific human single-chain antibody fragments in mice using adeno-associated virus vectors. Upon vector injection in the skeletal muscles, high and persistent systemic levels of anti-TG2 antibodies were obtained. Mice injected with vectors encoding antibodies also recognizing rodent TG2, also developed a strong anti-idiotypic response. This finding raises the question of whether an anti-idiotypic response to anti-TG2 antibodies is a factor associated with celiac disease.
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Autoanticuerpos/biosíntesis , Proteínas de Unión al GTP/inmunología , Transglutaminasas/inmunología , Animales , Anticuerpos Antiidiotipos/biosíntesis , Autoanticuerpos/genética , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/patología , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos , Humanos , Ratones , Especificidad de Órganos , Proteína Glutamina Gamma Glutamiltransferasa 2RESUMEN
BACKGROUND: Phage display antibody libraries have been made from the lymphocytes of patients suffering from autoimmune diseases in which the antibodies are known to play a role in the pathogenesis or are important for the diagnosis of the disease. In the case of Celiac Disease, the immune response is directed against the autoantigen tissue transglutaminase. However, despite numerous studies, the role of these antibodies in the pathogenesis of this disease has not been elucidated. RESULTS: We were able to engineer specific anti-transglutaminase antibody fragments in the form called "miniantibody". These are produced by genetic fusion of anti-tTG scFv to Human, Mouse or Rat Fc domains, making them suitable for in vivo expression. The results obtained here indicate that the miniantibody molecule is efficiently secreted, and that the reactivity to the antigen is retained even after fusion to heterologous Fc domains. Further analysis demonstrate that the molecule is secreted as homodimeric, mimicking original antibody structure. Finally, the in vivo expression in mice leads to detectable serum levels with no apparent gross immune response by the host. CONCLUSION: In this work we demonstrated the usefulness of a method for the in vivo expression of miniantibodies specific to transglutaminase, corresponding to the autoimmune specificity of Celiac Disease. This can be proposed as a general method to study the pathogenic role of autoimmune antibodies in autoimmune diseases.
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Anticuerpos Monoclonales/inmunología , Autoinmunidad/inmunología , Enfermedad Celíaca/inmunología , Modelos Animales de Enfermedad , Inmunidad Innata/inmunología , Ingeniería de Proteínas/métodos , Transglutaminasas/inmunología , Animales , Enfermedades Autoinmunes/inmunología , Humanos , Ratones , Biblioteca de Péptidos , Transglutaminasas/genéticaRESUMEN
In the present paper, we describe a novel approach to map monoclonal antibody epitopes, using three new monoclonal antibodies that recognize h-TG2 (human transglutaminase 2) as an example. The target gene was fragmented and cloned upstream of an antibiotic-resistance gene, in the vector pPAO2, to select for in-frame polypeptides. After removal of the antibiotic-resistance gene by Cre/Lox recombination, an antigen fragment phage display library was created and selected against specific monoclonal antibodies. Using the h-TG2 fragment library, we were able to identify epitopes. This technique can also be broadly applied to the study of protein-protein interactions.