Preclinical Studies of NEAST (Neutralizing Equine Anti-Shiga Toxin): A Potential Treatment for Prevention of Stec-Hus

STEC-HUS is a clinical syndrome characterized by the triad of thrombotic microangiopathy, thrombocytopenia and acute kidney injury. Despite the magnitude of the social and economic problems caused by STEC infections, there are currently no specific therapeutic options on the market. HUS is a toxemic disorder and the therapeutic effect of the early intervention with anti-toxin neutralizing antibodies has been supported in several animal models. We have designed novel protein nanoparticles with very strong immunogenic capacity by inserting Stx1B and Stx2B subunits of Shiga toxin to Brucella abortus Lumazine Synthase (BLS). The chimeric BLS-StxBs particles elicited strong neutralizing antibodies against Shiga toxins in horses. Based on these responses, we produced NEAST (Neutralizing Equine Anti-Shiga Toxin), composed of purified F(ab’)2 fragments. In this report we present the preclinical analysis of this new treatment. As a general conclusion, NEAST shows a very strong neutralizing capacity against 8 Shiga toxin variants in preclinical models of STEC-HUS. NEAST is also able to neutralize pathologic effects after previous exposure to Shiga Toxin, defining a potential therapeutic window. Besides, NEAST shows an excellent pharmacokinetics and safety profile in animal models. Overall, these results indicate that NEAST is a very good drug candidate for preventing the onset of HUS in STEC-infected patients and validate the conduction of a first in human clinical study at the Hospital Italiano de Buenos Aires in order to evaluate its safety and pharmacokinetic profiles in healthy adult volunteers.Fil: Hiriart, Yanina. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Pardo, Romina Paola. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bukata, Lucas. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lauché, Constanza Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Muñoz, Luciana. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Berengeno, Andrea Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Colonna, Mariana. Inmunova S.A; ArgentinaFil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Goldbaum, Fernando Alberto. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Sanguineti, Santiago. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina. Inmunova S.A; ArgentinaFil: Zylberman, Vanesa. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentin

Generation of a colony of homozygous K18-hACE2 transgenic mice for the evaluation of vaccine and therapeutic candidates against SARS-CoV-2

EL SARS-CoV-2 es el agente etiológico responsable de la enfermedad COVID-19 que inició una pandemia desde finales de 2019. Existen distintos modelos animales para esta enfermedad siendo el modelo de ratón transgénico K18-hACE2, originalmente desarrollado para el estudio del SARS CoV-1, de gran relevancia en el contexto actual. Considerando que los ratones se comercializan como hemicigotas y dada la ausencia de ratones homocigotas para el transgén K18-hACE2, el objetivo de este trabajo fue generar una colonia de ratones homocigotas K18-hACE2-Tg/Tg bajo condiciones ambientales controladas. Para la F1 se utilizaron 10 ratones hemicigotas (7 hembras y 3 machos), de 6-8 semanas de edad, provenientes de The Jackson Laboratory (USA). De la F1 se identificaron mediante genotipificación por PCR 78 animales: 62 hemicigotas (Tg/0) y 16 salvajes (0/0). Aquellos animales identificados como transgénicos se cruzaron con ratones C57BL/6J para seleccionar los parentales homocigotas que generaron 100% de hemicigotas en la F2. De 224 crías analizadas, se obtuvieron 7 parentales homocigotas K18-hACE2-Tg/Tg que fueron utilizados para la colonia fundación de ratones homocigotas K18-hACE2-Tg/Tg. El modelo de ratón homocigota K18 hACE2-Tg/Tg desarrollado en este trabajo podrá ser empleado para el estudio de la patogénesis de la enfermedad y la evaluación de posibles terapéuticos contra el SARS-CoV-2.SARS-CoV-2 is the causative etiological agent of the COVID-19 disease that started a pandemic in late 2019. Among different animal models for this disease, the K8-hACE2 transgenic mouse model, originally developed for the study of SARS-CoV-1, proved to be of great relevance in the current context. Considering that those mice are marketed as hemizygous and given that homozygous mice for the K18-hACE2 transgene are not commercially available, this work aimed to describe the creation of a colony of homozygous K18-hACE2-Tg/Tg mice under controlled environmental conditions. For the F1 progeny 10 hemizygous mice (7 females and 3 males), between 6-8 weeks of age, from The Jackson Laboratory (USA) were used. From the F1, 78 animals were identified by PCR genotyping: 62 hemizygous (Tg/0) and 16 wild types (0/0). Those animals identified as transgenic were crossed with C57BL/6J mice to select the homozygous parents that generated 100% hemizygous in F2. From a total of 224 analyzed offspring, 7 homozygous K18-hACE2-Tg/Tg parents were obtained, which were used for the foundation colony of homozygous K18-hACE2-Tg/Tg mice. The homozygous K18-hACE2-Tg/Tg mouse colony developed in this work will be able to use for the study of pathogenesis and the evaluation of possible therapeutics against SARS-CoV-2.Fil: Berengeno, Andrea Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Matiller, Valentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Díaz, Pablo Uriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Rebelindo, Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Rodríguez, Fernanda Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Anweg, Ayelen. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Silvestrini, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Cattaneo, M. L.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Peralta, María Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Durante, Leandro Ivan. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Notaro, Ulises Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Cainelli, Sofía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Taborda, Paula Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Stalder, Veronica Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Etchevers, Lucas. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Rey, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Salvetti, Natalia Raquel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentin

Modulation of Mrp1 (ABCc1) and Pgp (ABCb1) by Bilirubin at the Blood-CSF and Blood-Brain Barriers in the Gunn Rat

Accumulation of unconjugated bilirubin (UCB) in the brain causes bilirubin encephalopathy. Pgp (ABCb1) and Mrp1 (ABCc1), highly expressed in the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSFB) respectively, may modulate the accumulation of UCB in brain. We examined the effect of prolonged exposure to elevated concentrations of UCB on expression of the two transporters in homozygous, jaundiced (jj) Gunn rats compared to heterozygous, not jaundiced (Jj) littermates at different developmental stages (2, 9, 17 and 60 days after birth). BBB Pgp protein expression was low in both jj and Jj pups at 9 days (about 16–27% of adult values), despite the up-regulation in jj animals (2 and 1.3 fold higher than age matched Jj animals at P9 and P17–P60, respectively); Mrp1 protein expression was barely detectable. Conversely, at the BCSFB Mrp1 protein expression was rather high (60–70% of the adult values) in both jj and Jj at P2, but was markedly (50%) down-regulated in jj pups starting at P9, particularly in the 4th ventricle choroid plexuses: Pgp was almost undetectable. The Mrp1 protein down regulation was accompanied by a modest up-regulation of mRNA, suggesting a translational rather than a transcriptional inhibition. In vitro exposure of choroid plexus epithelial cells obtained from normal rats to UCB, also resulted in a down-regulation of Mrp1 protein. These data suggest that down-regulation of Mrp1 protein at the BSCFB, resulting from a direct effect of UCB on epithelial cells, may impact the Mrp1-mediated neuroprotective functions of the blood-cerebrospinal fluid barrier and actually potentiate UCB neurotoxicity

Impact of unconjugated bilirubin on brain parenchyma of the Gunn rat

2009/2010In infants and in the Crigler-Najjar syndrome type I patients, severe hyperbilirubinemia due to high levels of unconjugated bilirubin (UCB) may cause Kernicterus, leading to an irreversible and selective brain damage. The Gunn rat is the animal model for the study of these pathologies. It has been suggested that different enzymes of the phase I (cytochrome P-450-dependent mixed function oxygenases 1A1, 1A2, 2A3), phase II (glutathione-S-transferases α2, α3, µ3, µ4, π) and phase III transporters (particularly, Mrp1) seems to be involved in UCB detoxification pathways. However, to date, their in vivo brain expression has been evidenced only at the blood-brain interfaces, while remains largely unexplored in brain parenchyma. Particularly for Mrp1, in vitro evidence reported a role of this transporter in protection of neural primary cultures from dissected cortex, by extruding bilirubin out of the cell. The aim of this study is establish the developmental profile of these genes in brain parenchyma, and assess their alteration in hyperbilirubinemic jj animals. Due to the high regional selectivity of UCB-induced neurotoxicity, cerebellum (Cll), striatum (St), hippocampus (Hip) and cerebral cortex (Cx) were chosen for this study. Our results regard the Mrp1 protein in cerebral cortex of normobilirubinemic (JJ) rats showed that its expression varied during the post-natal age, reaching the highest levels at 9 days after birth. No changes were found between JJ and Jj (having a temporary hyperbilirubinemia in the first week of life) rat for all ages analyzed. Similarly, no differences were detected among JJ/Jj and jj (hyperbilirubinemic) rats at P2, P17 and P60, while a significant increase (p < 0.005) was evidenced in P9 jj rats as compared to age-matched JJ animals. Our Mrp1 mRNA analysis in four regions of P9 animals by Real Time-qPCR revealed the absence of differences among Cx, Cll, St and Hip of P9 normobilirubinemic JJ rats. Moreover, no variations between jj and JJ control animals were detected. Regarding the Mrp1 protein expression in the same four regions by Western blot analysis, our results showed that the levels of this transporter in normobilirubinemic JJ rats were lower in Cx, similar in Cll, St and Hip (p < 0.05 vs Cx). Comparing genotypes, a reduction on Mrp1 in jj animals (compared to Mrp1 amount in the same region of JJ pups) was detected in Cll, St, but reached the statistical significance only in Hip (p < 0.05 vs Hip JJ). The analysis of CYPs gene expression in P9 Gunn rats indicate that CYP1A1, 1A2 and 2A3 mRNA were differently expressed among Cx, Cll, St and Hip of JJ rat. Similarly a region-specific modulation of CYPs expression in jj Gunn rats (compared to JJ) was pointed-out. Surprisingly, UCB seems to generate a plateau effect on CYPs mRNA levels among brain regions of jj rats. In P60 JJ Gunn rats the CYPs expression is higher than in P9 animals, with the following pattern among regions: Cx CllSt Hip. A down-regulation (p < 0.05) in St of P60 jj compared to normal animals was observed. Analyzing the GSTs expression in P9 animals, higher variability in the GSTs expression among the four brain areas was evidenced. In hyperbilirubinemic (jj) rats (compared to JJ), statistically relevant down-regulations were detected for GSTα2 (in St;p < 0.05), GSTα3 (in Hip;p < 0.05), µ3 (in Cx;p < 0.01), µ4 (in Cx; p < 0.05) and π (in Cll: p < 0.05); while GSTµ4 was up-regulated in St (p < 0.05). From P9 to P60, in JJ animals: GSTα3 expression increased (13-75-fold depending on the region); while GSTα2 (5-fold), µ3 (p < 0.05), µ4 (2-fold) and π (2-fold) mRNA amounts decreased. In P60 jj Gunn rats, compared to controls (JJ):a relevant up-regulation of GSTα3 was observed in Cll (p < 0.005) and Hip (p < 0.05), while GSTµ3 in jj was down-regulated (p < 0.05). The Mrp1 results obtained in the present in vivo study seems not to be in agreement with the in vitro data reported, to date. Thus, the Mrp1 expression is low in brain parenchyma and bilirubin affect (up-regulation) only marginally the protein amounts in cortex of P9 animals, while in other regions Mrp1 is not modulated, indicating a marginal role in vivo in bilirubin clearance. Similarly, while in liver GSTα2 and α3 act together as ligandin, this seems not happens in brain where the two subunit are expressed at very low levels (P9: α2 77000- α3 1500 fold difference; P60: α2 112000-α3 2200 fold difference with respect to age matched livers). For all genes under analysis, a very complex and variable pattern of expression among brain areas was evidenced. Consequently, no general rules concerning bilirubin-induced modulation could be drawn, as both up and down-regulation were observed. Additionally, in Cx of P9 jj animals, a translational control of Mrp1 might be hypothesized due to a significant increase in Mrp1 protein, without changes in mRNA level. Therefore, the genomic screening made in this work provides the first general overview on the mRNA developmental profiles of several CYPs and GSTs genes in brain parenchyma (specifically Cx, Cll, St and Hip) of normal rats, and of animals suffering from hyperbilirubinemia, underlying the necessity to find functional evidence to finally understand the role of these enzymes associated with the kernicterus and Crigler-Najjar type I syndrome pathologies.XXIII Ciclo197

In vitro and in vivo antitumor activity of Yerba Mate extract in colon cancer models

Yerba Mate (Ilex paraguariensis St. Hill. Aquifoliaceae) is a native South American tree and has a large amount of bioactive compounds. Colorectal cancer (CRC) is one of the so-called westernized diseases and is the third most common cancer in both men and women. Efficient strategies for the treatment of CRC are extensively being explored including dietary intervention. The objective of our research was to evaluate the effects of Yerba Mate extract on cell proliferation, invasive capacity of tumor cells, and angiogenesis. For this, in vitro and in vivo experimentation was carried out using CRC models. The extract was generated by aqueous extraction and prepared according to traditional American procedure of preparing mate infusion. In vitro results showed that the Yerba Mate extract inhibits CT26 and COLO 205 cell proliferation with IC50 values of 0.25 and 0.46 mg/mL, respectively. We demonstrated by TUNEL assay that one of the mechanisms by which Yerba Mate extract decreases cell proliferation is by induction of apoptosis. In a murine syngeneic tumor model, oral administration of Yerba Mate extract in a dose of 1.6 g/kg/day significantly inhibited angiogenesis and tumor growth without affecting biological parameters or body weight. Our findings suggest that Yerba Mate may be a promising agent for the treatment of colon cancer and could be used as an herbal medicine or functional food ingredient. Practical Application: Considering the chemical composition and presence of phenolic compounds with their free-radical scavenging activities and bioactivities against colon cancer cells, Yerba Mate can be a promising candidate as healthy food sources in human nutrition, and also be considered a natural source of potential antitumor agents. Taking into account the economic importance of Yerba Mate in Argentina, this vegetable would have a greater commercial value as a functional food.Fil: Garcia Lazaro, Rocio Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Lamdan Ordas, Humberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Caligiuri, Lorena Gisel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Lorenzo Pérez, Norailys. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Berengeno, Andrea Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Alonso, Daniel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Farina, Hernán Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentin

In vitro

Yerba Mate (Ilex paraguariensis St. Hill. Aquifoliaceae) is a native South American tree and has a large amount of bioactive compounds. Colorectal cancer (CRC) is one of the so-called westernized diseases and is the third most common cancer in both men and women. Efficient strategies for the treatment of CRC are extensively being explored including dietary intervention. The objective of our research was to evaluate the effects of Yerba Mate extract on cell proliferation, invasive capacity of tumor cells, and angiogenesis. For this, in vitro and in vivo experimentation was carried out using CRC models. The extract was generated by aqueous extraction and prepared according to traditional American procedure of preparing mate infusion. In vitro results showed that the Yerba Mate extract inhibits CT26 and COLO 205 cell proliferation with IC50 values of 0.25 and 0.46 mg/mL, respectively. We demonstrated by TUNEL assay that one of the mechanisms by which Yerba Mate extract decreases cell proliferation is by induction of apoptosis. In a murine syngeneic tumor model, oral administration of Yerba Mate extract in a dose of 1.6 g/kg/day significantly inhibited angiogenesis and tumor growth without affecting biological parameters or body weight. Our findings suggest that Yerba Mate may be a promising agent for the treatment of colon cancer and could be used as an herbal medicine or functional food ingredient. Practical Application: Considering the chemical composition and presence of phenolic compounds with their free-radical scavenging activities and bioactivities against colon cancer cells, Yerba Mate can be a promising candidate as healthy food sources in human nutrition, and also be considered a natural source of potential antitumor agents. Taking into account the economic importance of Yerba Mate in Argentina, this vegetable would have a greater commercial value as a functional food.Fil: Garcia Lazaro, Rocio Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Lamdan Ordas, Humberto. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Caligiuri, Lorena Gisel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Lorenzo Pérez, Norailys. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Berengeno, Andrea Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Ortega, Hugo Hector. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; ArgentinaFil: Alonso, Daniel Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; ArgentinaFil: Farina, Hernán Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Oncología Molecular; Argentin

Blood brain barrier localization in rat brain, and microvessels isolated from rats.

<p>Cartoon representation of the lateral, 3^rd, 4^th ventricle choroid plexuses (LV CP, 3^rd V CP and 4^thV CP, respectively) and microvessels (MVs) localization in rat brain (A). Representative microvessels freshly isolated from rat brains. Freshly isolate MVs in 0.1% albumin in Krebbs-Ringer buffer were placed on a microscope slide and directly observed by phase contrast microscopy: In B scale bar 100 µm; in C, D, E, F and G scale bar 25 µm. P: post-natal age in days. jj: hyperbilirubinemic rats; Jj: controls.</p

Western blot analysis of Mrp1 protein in the choroidal epithelium following bilirubin treatment.

<p>Duplicate monolayers of choroidal epithelial cells were included for each experimental condition. Following daily basolateral treatment with 40 or 140 nM unbound bilirubin for 6 consecutive days, filters were treated in exactly the same conditions. The homogeneity in protein loading is reflected by the actin band revealed on the lower part of the membrane.</p

Comparison of Mrp1 mRNA and protein relative expression in choroid plexuses.

<p>White bars: heterozygous (Jj) P9 rats, black bars: hyperbilirubinemic homozygous (jj) P9 animals. The values are expressed as mean ± SD. Statistical significance is reported. NS: not significant difference.</p

Primers used for transcript analysis, amplicon length and amplification efficiency.

<p>Primers used for transcript analysis, amplicon length and amplification efficiency.</p