Novel erythropoietin-based therapeutic candidates with extra N-glycan sites that block hematopoiesis but preserve neuroplasticity

Neurological disorders affect millions of people causing behavior-cognitive disabilities. Nowadays they have no effective treatment. Human erythropoietin (hEPO) has been clinically used because of its neurotrophic and cytoprotective properties. However, the erythropoietic activity (EA) should be considered as a side effect. Some analogs like non-sialylated EPO, carbamylated EPO, or EPO peptides have been developed showing different weaknesses: erythropoiesis preservation, low stability, potential immunogenicity, or fast clearance. Herein, we used a novel strategy that blocks the EA but preserves hEPO neurobiological actions. N-glycoengineering was accomplished to add a new glycosylation site within the hEPO sequence responsible for its EA. hEPO-derivatives were produced by CHO.K1 cells, affinity-purified and functionally analyzed studying their in vitro and in vivo EA, their in vitro neuronal plasticity in hippocampal neurons and their neuroprotective action by rescuing hippocampal neurons from apoptosis. Muteins Mut 45_47 (K45 > N45 + N47 > T47), Mut 104 (S104 > N104), and Mut 151_153 (G151 > N151 + K153 > T153) lost their EA but preserved their neuroprotection activity and enhanced neuroplasticity more efficiently than hEPO. Interestingly, Mut 45_47 resulted in a promising candidate to explore as neurotherapeutic considering not only its biopotency but also its pharmacokinetic potential due to the hyperglycosylation.Fil: Bürgi Fissolo, María de Los Milagros. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Aparicio, Gabriela Inés. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Dorella, Aquiles. Universidad Nacional del Litoral; ArgentinaFil: Kratje, Ricardo Bertoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Scorticati, Camila. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Oggero, Marcos. Universidad Nacional del Litoral; Argentin

Crosslinked casein micelles bound paclitaxel as enzyme activated intracellular drug delivery systems for cancer therapy

Nanomedicine for cancer therapy is a successful tool to diminish the side effect of chemotherapeutics such as paclitaxel (PTX). In this regard, Abraxane®, a human serum albumin (HSA)-based nanomedicine system has shown lesser side effects than Taxol®. However, the large-scale production of HSA protein is limited and expensive, which is traduced in a high cost of the treatments in clinical applications. Thus, the use of easily-available alternative nanocarriers could increment the accessibility of patients to nanomedicine for cancer treatments. Casein is a low-cost protein able to self-assemble into micelles which could efficiently encapsulate PTX into their structure. In this work, the synthesis of chemically crosslinked casein micelles (CCM), used to prepare PTX-based nanoformulations, is presented. CCM@PTX nanoformulations showed promising results in vitro to be applied as nanomedicine for cancer therapy. Thus, the obtained nanoformulations are great candidates to be parenterally administered, accumulate in tumor by passive targeting without leakage of PTX in plasma, and release the drug within the tumor microenvironment, in response to overexpressed proteases such as trypsin.Fil: Cuggino, Julio César. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Picchio, Matías Luis. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; ArgentinaFil: Gugliotta, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bürgi Fissolo, María de Los Milagros. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ronco, Ludmila Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Calderón, Marcelo. Polymat; EspañaFil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; ArgentinaFil: Alvarez Igarzabal, Cecilia Ines. Universidad Nacional de Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada; ArgentinaFil: Minari, Roque Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; ArgentinaFil: Gugliotta, Luis Marcelino. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química. Universidad Nacional del Litoral. Instituto de Desarrollo Tecnológico para la Industria Química; Argentin

Natural and Synthetic compounds that module the biological activity of human type I interferons: analysis using a new biological tool

Los interferones son importantes glicoproteínas del sistema inmunológico, ampliamente utilizados como biofármacos para el tratamiento de diversas patologías. En este trabajo de tesis se desarrolló un ensayo de gen reportero basado en líneas celulares reporteras Mx2/EGFP para determinar la actividad de los hIFNs I. En consecuencia, el porcentaje de células verdes positivas se correlaciona directamente con la cantidad de IFN presente en la muestra. El IFN se presenta como una molécula central cuyo accionar debe ser regulado. Así, resulta indispensable incrementar su eficacia terapéutica en aquellas patologías que requieran de su administración exógena y, contrariamente, bloquear sus efectos nocivos en enfermedades donde su producción constituye parte de la etiología de las mismas. Consecuentemente, se ha propuesto la identificación y caracterización de nuevas moléculas, que actúen en forma sinérgica o antagónica con la actividad de los IFNs-I. Con ese fin, se emplearon las líneas celulares reporteras para monitorear 552 compuestos provenientes de bibliotecas de compuestos naturales y sintéticos, en presencia de rhIFN-α2a o rhIFN β1a mediante el ensayo de gen reportero desarrollado. Utilizando este sistema pudieron identificarse 20 compuestos sintéticos inhibidores de la actividad de los hIFNs-I y 5 compuestos naturales, 4 de ellos inhibidores y 1 potenciador de los hIFNs I. Todos ellos fueron estudiados con el fin de validar el nuevo ensayo. Finalmente, en este trabajo de tesis se logró desarrollar 4 líneas celulares reporteras capaces de cuantificar la actividad de los hIFNs-I, que fueron utilizadas como herramientas biológicas para monitorear bibliotecas de compuestos sintéticos y naturales como posibles moduladores de la actividad de los hIFN-I.Interferons are important glycoprotein from the immune system, widely used as biopharmaceuticals for some pathology treatments. In this work, a new reporter gene assay was developed using Mx2/EGFP reporter cell lines to determine the hIFNs-I potency. Therefore, the expressed EGFP is quantified by flow cytometry. The positive green cells percentage is directly correlated with the IFN quantity in the sample. Thus, IFN is presented as a central molecule whose activity must be regulated. In this way, it is essential to increase its therapeutic efficacy in those pathologies where exogenous IFN administration is necessary and, on the other hand, to block its harmful effect on those illnesses where the endogenous IFN production constitutes a part of etiology. Consequently, it has been proposed the identification and characterization of new molecules that act synergistically or antagonistically as regards IFNs-I activity. In the second stage of this thesis, reporter cell lines were employed to screen 552 compounds from natural and synthetic libraries. They were analyzed in presence of rhIFN-alpha2a and rhIFN-beta1a using the reporter gene assay previously developed. This system allowed the identification of 20 synthetic compounds that inhibit the hIFNs-I activity and 5 natural compounds, 4 of them are inhibitors and 1 of them is an enhancer of hIFNs-I activity. All of them were analyzed with the purpose of validating our new reporter gene assay. Finally, in this work 4 reporter cell lines, capable of quantifying the IFNs-I activity were achieved. They were used as biological tools to screen natural and synthetic libraries compounds, possible modulators of hIFN-I activity.Consejo Nacional de Investigaciones Científicas y Técnica

Novel reactive PEG for amino group conjugation

Activated mPEG carbonates are important reagents that have been widely used for the PEGylation of several peptides and proteins by means of stable urethane linkages. In fact, mPEG-N-hydroxysuccinimidyl carbonate and mPEG-p-nitrophenyl carbonate are among the most used reagents in PEGylation technology. However, the synthesis and storage of these reagents are not always easy to resolve. With the aim of surpassing some of the drawbacks associated with the use of activated mPEG-carbonates we have prepared and evaluated a new mPEG-carbonylimidazolium iodide, which can be used for the conjugation of the NH2 group by means of urethane linkages, as an interesting alternative to the known reagents. It is noteworthy that the novel reagent is prepared by a simple two-step procedure under mild experimental conditions. Moreover, we performed a detailed study of the conjugation reaction of interferon-α2b with the carbonylimidazolium derivative, and evaluated the conjugate in in vitro and in vivo studies.Fil: Gonzalez, Marianela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); ArgentinaFil: Ceaglio, Natalia Analia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bürgi Fissolo, María de Los Milagros. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Etcheverrigaray, Marina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Kratje, Ricardo Bertoldo. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Vaillard, Santiago Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Desarrollo Tecnológico para la Industria Química (i); Argentin

Novel erythropoietin‐based therapeutic candidates with extra N

Neurological disorders affect millions of people causing behavior-cognitive disabilities. Nowadays they have no effective treatment. Human erythropoietin (hEPO) has been clinically used because of its neurotrophic and cytoprotective properties. However, the erythropoietic activity (EA) should be considered as a side effect. Some analogs like non-sialylated EPO, carbamylated EPO, or EPO peptides have been developed showing different weaknesses: erythropoiesis preservation, low stability, potential immunogenicity, or fast clearance. Herein, we used a novel strategy that blocks the EA but preserves hEPO neurobiological actions. N-glycoengineering was accomplished to add a new glycosylation site within the hEPO sequence responsible for its EA. hEPO-derivatives were produced by CHO.K1 cells, affinity-purified and functionally analyzed studying their in vitro and in vivo EA, their in vitro neuronal plasticity in hippocampal neurons and their neuroprotective action by rescuing hippocampal neurons from apoptosis. Muteins Mut 45_47 (K45 > N45 + N47 > T47), Mut 104 (S104 > N104), and Mut 151_153 (G151 > N151 + K153 > T153) lost their EA but preserved their neuroprotection activity and enhanced neuroplasticity more efficiently than hEPO. Interestingly, Mut 45_47 resulted in a promising candidate to explore as neurotherapeutic considering not only its biopotency but also its pharmacokinetic potential due to the hyperglycosylation.Fil: Bürgi Fissolo, María de Los Milagros. Universidad Nacional del Litoral; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Aparicio, Gabriela Inés. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Dorella, Aquiles. Universidad Nacional del Litoral; ArgentinaFil: Kratje, Ricardo Bertoldo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral; ArgentinaFil: Scorticati, Camila. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Oggero, Marcos. Universidad Nacional del Litoral; Argentin

Metaprofiling of the bacterial community in sorghum silages inoculated with lactic acid bacteria

To characterize the fermentation process and bacterial diversity of sorghum silage inoculated with Lactiplantibacillus plantarum LpAv, Pediococcus pentosaceus PpM and Lacticaseibacillus paracasei LcAv.EEA RafaelaFil: Puntillo, Melisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Lactología Industrial (INLAIN); ArgentinaFil: Puntillo, Melisa. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactologia Industrial (INLAIN); ArgentinaFil: Peralta, Guillermo H. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Lactologia Industrial (INLAIN); ArgentinaFil: Peralta, Guillermo H. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactologia Industrial (INLAIN); ArgentinaFil: Bürgi, María D. Milagros. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas, Centro Biotecnológico del Litoral; ArgentinaFil: Bürgi, María D. Milagros. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Biotecnológico del Litoral, Santa Fe; ArgentinaFil: Huber, Paula. Universidad Nacional del Litoral. Instituto Nacional de Limnología (INALI). Laboratorio de Plancton; ArgentinaFil: Huber, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto Nacional de Limnología (INALI). Laboratorio de Plancton; ArgentinaFil: Huber, Paula. Universidade Federal de São Carlos. Departamento de Hydrobiologia. São Carlos; BrasilFil: Gaggiotti, Mónica C. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Gaggiotti, Mónica C. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina.Fil: Gaggiotti, Mónica C. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación de la Cadena Láctea (IDICAL); Argentina.Fil: Binetti, Ana G. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Lactologia Industrial (INLAIN); ArgentinaFil: Binetti, Ana G. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactologia Industrial; ArgentinaFil: Vinderola, Celso Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Santa Fe. Instituto de Lactologia Industrial (INLAIN); ArgentinaFil: Vinderola, Celso Gabriel. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Lactologia Industrial (INLAIN); Argentin

Artichoke extracts with potential application in chemoprevention and inflammatory processes

The aim of this work is to study three cultivars of artichoke (Cynara cardunculus var. scolymus): Gauchito, Guri and Oro Verde in terms of their in vitro chemoprevention and anti-inflammatory properties. These cultivars show good productive performance. The phenolic composition of their fresh leaves and edible bracts was analyzed by high performance liquid chromatography and high resolution mass spectrometry (HPLC-HRMS), showing mainly caffeoylquinic acids and flavonoids. Caffeoylquinic acids were quantified and the highest content was found in Gauchito cultivar. In this cultivar, the content of dicaffeoylquinic acids in fresh bracts was six times higher than that in fresh leaves (10064.5 ± 378.3 mg/kg versus 1451.0 ± 209.3 mg/kg respectively). Luteolin flavonoids were detected in leaves. The extracts from fresh bracts and leaves were assessed in their in vitro bioactivity against human neuroblastoma cells (SH-SY5Y). Inhibition of SH-SY5Y cells proliferation by Gauchito and Guri leaf extracts (8 µg/mL) was higher than 50 %. The leaf extracts of the same cultivars showed an inhibitory effect on human interferon IFN-I, decreasing its activity 50% at 40 µg/mL. Interestingly, the bract extracts did not show in vitro bioactivity at these concentrations, nor did the pure compounds chlorogenic acid, cynarin, apigenin and luteolin (at 2 µg/mL). These results suggest that Gauchito and Guri leaf extracts have potential for human neuroblastoma chemoprevention and treatment of inflammatory processes

Screening and characterization of molecules that modulate the biological activity of IFNs-I.

Type I Interferons (IFNs-I) are species-specific glycoproteins which play an important role as primary defence against viral infections and that can also modulate the adaptive immune system. In some autoimmune diseases, interferons (IFNs) are over-produced. IFNs are widely used as biopharmaceuticals for a variety of cancer indications, chronic viral diseases, and for their immuno-modulatory action in patients with multiple sclerosis; therefore, increasing their therapeutic efficiency and decreasing their side effects is of high clinical value. In this sense, it is interesting to find molecules that can modulate the activity of IFNs. In order to achieve that, it was necessary to establish a simple, fast and robust assay to analyze numerous compounds simultaneously. We developed four reporter gene assays (RGAs) to identify IFN activity modulator compounds by using WISH-Mx2/EGFP, HeLa-Mx2/EGFP, A549-Mx2/EGFP, and HEp2-Mx2/EGFP reporter cell lines (RCLs). All of them present a Z' factor higher than 0.7. By using these RGAs, natural and synthetic compounds were analyzed simultaneously. A total of 442 compounds were studied by the Low Throughput Screening (LTS) assay using the four RCLs to discriminate between their inhibitory or enhancing effects on IFN activity. Some of them were characterized and 15 leads were identified. Finally, one promising candidate with enhancing effect on IFN-α/-β activity and five compounds with inhibitory effect were described