Production of the main celiac disease autoantigen by transient expression in Nicotiana benthamiana

Celiac Disease (CD) is a gluten sensitive enteropathy that remains widely undiagnosed and implementation of massive screening tests is needed to reduce the long term complications associated to untreated CD. The main CD autoantigen, human tissue transglutaminase (TG2), is a challenge for the different expression systems available since its cross-linking activity affects cellular processes. Plant-based transient expression systems can be an alternative for the production of this protein. In this work, a transient expression system for the production of human TG2 in Nicotiana benthamiana leaves was optimized and reactivity of plant-produced TG2 in CD screening test was evaluated. First, a subcellular targeting strategy was tested. Cytosolic, secretory, endoplasmic reticulum (C-terminal SEKDEL fusion) and vacuolar (C-terminal KISIA fusion) TG2 versions were transiently expressed in leaves and recombinant protein yields were measured. ER-TG2 and vac-TG2 levels were 9- to 16-fold higher than their cytosolic and secretory counterparts. As second strategy, TG2 variants were co-expressed with a hydrophobic elastin-like polymer (ELP) construct encoding for 36 repeats of the pentapeptide VPGXG in which the guest residue X were V and F in ratio 8:1. Protein bodies (PB) were induced by the ELP, with a consequent two-fold-increase in accumulation of both ER-TG2 and vac-TG2. Subsequently, ER-TG2 and vac-TG2 were produced and purified using immobilized metal ion affinity chromatography. Plant purified ER-TG2 and vac-TG2 were recognized by three anti-TG2 monoclonal antibodies that bind different epitopes proving that plant-produced antigen has immunochemical characteristics similar to those of human TG2. Lastly, an ELISA was performed with sera of CD patients and healthy controls. Both vac-TG2 and ER-TG2 were positively recognized by IgA of CD patients while they were not recognized by serum from non-celiac controls. These results confirmed the usefulness of plant-produced TG2 to develop screening assays. In conclusion, the combination of subcellular sorting strategy with co-expression with a PB inducing construct was sufficient to increase TG2 protein yields. This type of approach could be extended to other problematic proteins, highlighting the advantages of plant based production platforms.Fil: Marin Viegas, Vanesa Soledad. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; ArgentinaFil: Acevedo, Gonzalo Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Bayardo, Mariela Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Chirdo, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Petruccelli, Silvana. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Criotecnología de Alimentos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Criotecnología de Alimentos; Argentin

Transglutaminase 2 expression is enhanced synergistically by interferon-γ and tumour necrosis factor-α in human small intestine

Transglutaminase 2 (TG2) is expressed ubiquitously, has multiple physiological functions and has also been associated with inflammatory diseases, neurodegenerative disorders, autoimmunity and cancer. In particular, TG2 is expressed in small intestine mucosa where it is up-regulated in active coeliac disease (CD). The aim of this work was to investigate the induction of TG2 expression by proinflammatory cytokines [interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and IL-15] and the signalling pathways involved, in human epithelial and monocytic cells and in intestinal tissue from controls and untreated CD patients. Here we report that IFN-γ was the most potent inducer of TG2 expression in the small intestinal mucosa and in four [Caco-2, HT-29, Calu-6 and human acute monocytic leukaemia cell line (THP-1)] of five cell lines tested. The combination of TNF-α and IFN-γ produced a strong synergistic effect. The use of selective inhibitors of signalling pathways revealed that induction of TG2 by IFN-γ was mediated by phosphoinositide 3-kinase (PI3K), while c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were required for TNF-α activation. Quantitative polymerase chain reaction (PCR), flow cytometry and Western blot analysis showed that TG2 expression was blocked completely when stimulation by either TNF-α or IFN-γ was performed in the presence of nuclear factor (NF)-κB inhibitors (sulphasalazine and BAY-117082). TG2 was up-regulated substantially by TNF-α and IFN-γ in intestinal mucosa in untreated CD compared with controls. This study shows that IFN-γ, a dominant cytokine in intestinal mucosa in active CD, is the most potent inducer of TG2, and synergism with TNF-α may contribute to exacerbate the pathogenic mechanism of CD. Selective inhibition of signalling pathways may be of therapeutic benefit. © 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.Fil: Bayardo, Mariela Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Punzi, F.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Bondar, Constanza María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Chopita, Nestor. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Chirdo, Fernando Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; Argentin

Transglutaminase 2 expression is enhanced synergistically by interferon-γ and tumour necrosis factor-α in human small intestine

Transglutaminase 2 (TG2) is expressed ubiquitously, has multiple physiological functions and has also been associated with inflammatory diseases, neurodegenerative disorders, autoimmunity and cancer. In particular, TG2 is expressed in small intestine mucosa where it is up-regulated in active coeliac disease (CD). The aim of this work was to investigate the induction of TG2 expression by proinflammatory cytokines [interleukin (IL)-1, IL-6, tumour necrosis factor (TNF)-α, interferon (IFN)-γ and IL-15] and the signalling pathways involved, in human epithelial and monocytic cells and in intestinal tissue from controls and untreated CD patients. Here we report that IFN-γ was the most potent inducer of TG2 expression in the small intestinal mucosa and in four [Caco-2, HT-29, Calu-6 and human acute monocytic leukaemia cell line (THP-1)] of five cell lines tested. The combination of TNF-α and IFN-γ produced a strong synergistic effect. The use of selective inhibitors of signalling pathways revealed that induction of TG2 by IFN-γ was mediated by phosphoinositide 3-kinase (PI3K), while c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were required for TNF-α activation. Quantitative polymerase chain reaction (PCR), flow cytometry and Western blot analysis showed that TG2 expression was blocked completely when stimulation by either TNF-α or IFN-γ was performed in the presence of nuclear factor (NF)-κB inhibitors (sulphasalazine and BAY-117082). TG2 was up-regulated substantially by TNF-α and IFN-γ in intestinal mucosa in untreated CD compared with controls. This study shows that IFN-γ, a dominant cytokine in intestinal mucosa in active CD, is the most potent inducer of TG2, and synergism with TNF-α may contribute to exacerbate the pathogenic mechanism of CD. Selective inhibition of signalling pathways may be of therapeutic benefit.Laboratorio de Investigaciones del Sistema Inmun

Nitrogen fertilization increases ammonium accumulation during senescence of barley leaves

Leaf senescence is a developmental process characterized by two events: (1) dismantling of the photosynthetic apparatus and, (2) nitrogen remobilization to other parts of the plant. Ammonium accumulation during senescence of barley leaves was produced by chloroplast proteins degradation. Although most of the ammonium is remobilized as amino acids from senescing leaves, a minor part is lost as NH3emitted across stomata. The amount of ammonia emitted depends on the amount of NH3accumulated in the substomatal cavity, which is continuously re-supplied with NH4 1+from the cytoplasm of neighboring cells. Ammonia accumulation in tissues could increase the possibility of loss of N as NH3emitted. In this report we analyzed the effect of N fertilization on nitrogen metabolism during senescence of barley leaves during the vegetative and reproductive stages of development. During senescence of barley leaves protein degradation was accompanied by transient ammonium accumulation at both stages of development. The peak of ammonium occurred immediately after major protein degradation in all samples analyzed, thereafter levels of ammonium clearly decreased. N accumulated as ammonium during senescence of barley leaves represented a high percentage of protein-N, i.e., approximately 16 % in primary leaves and 23 % in flag leaves. A significant increase of ammonium peak concentration was observed when doses of N fertilizer increased, mainly at the reproductive stage, where the percentage of N accumulated as ammonium reached near 35 % of protein-N at that stage. Vascular cytosolic glutamine synthetase (Hv GS1_1) transcript levels were upregulated during senescence of the flag leaf, but they were downregulated by increases in N availability. These results suggest that the decreases of ammonia levels after its peak may be more closely related to NH3emission than to N re-assimilation by GS.Fil: Rolny, Nadia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Bayardo, Mariela Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Guiamet, Juan José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Costa, M.lorenza. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales; Argentin

Evaluación de la vida postcosecha de una nueva fruta nativa Campomanesia guazumifolia “sietecapotes” y la aplicación de un tratamiento térmico para disminuir las pérdidas postcosecha y la ocurrencia de daño por frío

Los productos forestales no madereros resultan promisorios para el desarrollo de las economías regionales. Dentro de ellos, se destacan los frutales nativos como “sietecapotes”, Campomanesia guazumifolia (Cambess.) O. Berg. Además de su consumo fresco, se obtienen productos tales como dulces, vinagres, licores, pulpa congelada o jugos concentrados que permiten el agregado de valor. El consumo como fruta fresca de “sietecapotes” se ve limitado por su corta vida postcosecha, además, al tratarse de una especie subtropical, la conservación a bajas temperaturas puede estar limitada por el daño por frío. El objetivo del trabajo fue describir la vida postcosecha del fruto a temperatura ambiente y refrigerado. Posteriormente se analizó el efecto de la aplicación de un tratamiento térmico sobre la sintomatología de daño por frío. Los frutos fueron cosechados en la localidad de San Antonio, refrigerados (rango entre 8-10 °C) y transportados al laboratorio donde se almacenaron a temperatura ambiente y refrigerados durante 11 d. Se determinó cada 2 d el color superficial (CIELAB), la acidez titulable (AT) por volumetría ácido-base, los sólidos solubles totales (SST) por refractometría y la firmeza con un texturómetro portátil. Posteriormente se cosechó otra muestra de frutos para evaluar el efecto de un tratamiento térmico (TT) sobre el daño por frío de los frutos. En esta oportunidad los frutos se desinfectaron con NaClO 200 ppm. La mitad de los frutos se trataron por inmersión en agua caliente (55-60°C, 3 min) previo a la refrigeración y transporte. En el laboratorio los frutos se almacenaron refrigerados y cada 2 d se transfirieron muestras a temperatura ambiente durante 48 h para para evaluar el desarrollo de daño por frio. Se determinó el color superficial, color de pulpa, la pérdida de electrolitos (PE), la firmeza como el trabajo de compresión por texturometría y el porcentaje de frutos afectados por hongos. Durante el almacenamiento no se observan cambios en el color superficial de estos frutos que se asocien con su maduración postcosecha. Se verificó un aumento de 1,5% Brix y de 0,38 meq de ácido cítrico en 100 mL de jugo luego de 6 d independientemente de la condición de almacenamiento. Los frutos refrigerados acusaron un comportamiento anómalo de la firmeza, pardeamiento de los frutos y ataque fúngico lo cual sugiere la ocurrencia de daño por frio. En el segundo ensayo, se evaluó el efecto del tratamiento térmico para disminuir síntomas asociados a daño por frío. Los frutos tratados térmicamente resultaron 70% más firmes, con un 50% menos de ataque fúngico respecto a los controles. La PE en los frutos tratados resultó menor que en los controles. Los resultados sugieren que el TT sería una estrategia adecuada para prevenir el daño por frio en estos frutos, pero es necesario ajustar el protocolo.Facultad de Ciencias Agrarias y Forestale

Postharvest life evaluation of a new native fruit - Campomanesia guazumifolia “Sietecapotes” - and application of a heat treatment to decrease postharvest loss and chilling injury

“Sietecapotes” (Campomanesia guazumifolia (Cambess.) O. Berg.), es un fruto nativo recientemente incorporado al Código Alimentario Argentino. Se define como fruto tropical de vida postcosecha corta. En este trabajo se analizaron los cambios postcosecha de “Sietecapotes” almacenados a temperatura ambiente y refrigerados. Se determinó el color superficial, la acidez titulable, los sólidos solubles totales y la firmeza de los frutos. Independientemente de la condición de almacenamiento, no se observaron cambios de color superficial y de sólidos solubles asociados con la maduración mientras la acidez aumentó. Los frutos refrigerados acusaron un comportamiento anómalo de firmeza, pardeamiento de la pulpa y ataque fúngico, lo cual sugiere posible daño por frio. Por lo tanto, en un segundo ensayo se evaluó si un tratamiento térmico sería efectivo para disminuir estos síntomas. Se trabajó con frutos desinfectados y tratados por inmersión en agua caliente (55-60°C, 3 minutos) previo a la refrigeración. Los frutos se almacenaron refrigerados y cada 2 días se transfirieron muestras a temperatura ambiente para evaluar daño por frío. Los frutos tratados térmicamente resultaron más firmes, presentaron menos daño y menor pérdida de electrolitos respecto a los controles. El tratamiento térmico mejoró los posibles síntomas de daño por frío evaluados.“Sietecapotes” (Campomanesia guazumifolia (Cambess.) O. Berg.) is a native fruit recently incorporated into the Argentine Food Code. “Sietecapotes” is defined as a tropical fruit with a short post-harvest life. In this work, postharvest changes of “Sietecapotes” stored at room temperature and refrigerated were analyzed. Skin color, titratable acidity, total soluble solids and fruit firmness were determined. Regardless of storage condition, no changes in surface color and soluble solids associated with ripening were observed while acidity increased. Refrigerated fruit showed anomalous firmness behavior, flesh browning and fungal attack, suggesting possible chilling injury. Therefore, in a second trial, we evaluated whether heat treatment would be effective in reducing these symptoms. The fruits were disinfected and treated by immersion in hot water (55-60°C, 3 minutes) prior to refrigeration. Fruits were stored refrigerated and every 2 days samples were transferred to room temperature to evaluate chilling injury. Heat-treated fruit were firmer, showed less damage and less electrolyte loss than controls. The heat treatment improved the possible symptoms of chilling injury evaluated.Facultad de Ciencias Agrarias y Forestale