Organoides: posibles aplicaciones en la Química Medicinal

Los avances en la biología de células madre y en el desarrollo de cultivos en tres dimensiones han permitido la generación de nuevos modelos celulares denominados organoides. Los organoides son estructuras multicelulares y autoorganizadas que recapitulan in vitro elementos clave de la arquitectura, composición, funcionalidad y perfil genético del órgano que representan. Estas características les confieren una ventaja en su capacidad predictiva frente a los cultivos celulares tradicionales, convirtiéndolos en herramientas prometedoras en diversas áreas de estudio. Desde el trabajo pionero en organoides intestinales murinos hasta la actualidad, se han establecido organoides de diversos tipos (intestino, páncreas, riñón, hígado, próstata, retina, cerebro) y de varias especies, incluyendo la humana. Los organoides han sido aplicados con éxito en el cribado de drogas en estudios piloto. Por otra parte, la posibilidad de modelar enfermedades humanas a través de la generación de organoides derivados de pacientes, abre la posibilidad de identificar nuevas dianas moleculares para el desarrollo de fármacos. Durante esta presentación se discutirán aspectos clave del cultivo de organoides y se presentarán ejemplos de diferentes organoides aplicados al modelado de enfermedades y el desarrollo de nuevas drogas. Si bien existen limitaciones técnicas como la estandarización, la reproducibilidad, la madurez celular alcanzado o la ausencia de vascularización, los hitos logrados hasta ahora demuestran que la tecnología de organoides tiene el potencial para mejorar la etapa pre-clínica del desarrollo de drogas y contribuir al Principio de las 3R, reduciendo el número de animales de experimentación.Agencia Nacional de Investigación e Innovació

Modeling the human placental barrier to understand Toxoplasma gondii´s vertical transmission

Toxoplasma gondii is a ubiquitous apicomplexan parasite that can infect virtually any warm-blooded animal. Acquired infection during pregnancy and the placental breach, is at the core of the most devastating consequences of toxoplasmosis. T. gondii can severely impact the pregnancy’s outcome causing miscarriages, stillbirths, premature births, babies with hydrocephalus, microcephaly or intellectual disability, and other later onset neurological, ophthalmological or auditory diseases. To tackle T. gondii’s vertical transmission, it is important to understand the mechanisms underlying hostparasite interactions at the maternal-fetal interface. Nonetheless, the complexity of the human placenta and the ethical concerns associated with its study, have narrowed the modeling of parasite vertical transmission to animal models, encompassing several unavoidable experimental limitations. Some of these difficulties have been overcome by the development of different human cell lines and a variety of primary cultures obtained from human placentas. These cellular models, though extremely valuable, have limited ability to recreate what happens in vivo. During the last decades, the development of new biomaterials and the increase in stem cell knowledge have led to the generation of more physiologically relevant in vitro models. These cell cultures incorporate new dimensions and cellular diversity, emerging as promising tools for unraveling the poorly understood T. gondii´s infection mechanisms during pregnancy. Herein, we review the state of the art of 2D and 3D cultures to approach the biology of T. gondii pertaining to vertical transmission, highlighting the challenges and experimental opportunities of these up-and-coming experimental platforms.Institut Pasteur International NetworkFOCE

Felinized murine intestinal organoids for gaining insight into sexual reproduction of Toxoplasma gondii

The apicomplexan parasite Toxoplasma gondii is the causative agent of Toxoplasmosis, a zoonotic disease affecting one-third of the human population which can cause severe fetal damage by vertical transmission in pregnancy. Toxoplasmosis has great impact in reproductive outcomes of productive species and thus economic losses worldwide. The life cycle of T. gondii encompasses sexual and asexual phases. The asexual cycle can occur in any warm-blooded animal while the sexual stage is restricted to felids. In the feline enterocytes, bradyzoites turn into merozoites, initiating sexual replication that will end in the formation of a zygote. Zygotes are key to the generation of diversity, as they allow the occurrence of genetic admixing and the generation of oocyst that will be disseminated in the environment with the feline feces. Until recently, limitations in the availability of appropriate experimental models had hindered the study of sexual stages despite its clear biological relevance. In the last year, the molecular basis involved in the species specificity of the sexual parasitic forms was identified: an excess of linoleic acid given by the lack of delta-6-desaturase activity in the felid´s intestine. Thus, mimicking these conditions in a murine intestine allowed T. gondii to sexually develop in a mouse model, providing the opportunity to answer biological questions relevant to T. gondii reproduction without the need of using feline animal models. This work aimed to set up culturing systems based on “felinized” murine intestinal organoids in order to trigger T. gondii’s differentiation into sexual stages in vitro. For this purpose, murine intestinal organoids generated from isolated intestinal stem cells and maintained in a 3D system inside a matrix, or trypsinized and seeded as a monolayer, were incubated in the presence of 20 µM delta-6-desaturase inhibitor and 200 µM linoleic acid. The cytotoxicity of felinizing compounds in 2D and 3D cultures was assessed showing no cytotoxicity for 5 days of culture. Optimization of the infection assays was performed by incubating intestinal organoids with bradyzoites, at two multiplicities of infection (1:1 and 1:10). The presence of the parasite was evaluated after 5 days of culture by immunofluorescence. Kinetic studies of the sexual differentiation of T. gondii were carried out and the evaluation of parasite switching efficiency to sexual stages is ongoing, both by immunofluorescence and qRT-PCR. We put forward in vitro felinized intestinal organoids as a valuable tool for answering biological questions relevant to persistence and dissemination of T. gondii.Agencia Nacional de Investigación e Innovació

Exploring Toxoplasma gondii´s Biology within the Intestinal Epithelium: intestinal-derived models to unravel sexual differentiation

A variety of intestinal-derived culture systems have been developed to mimic in vivo cell behavior and organization, incorporating different tissue and microenvironmental elements. Great insight into the biology of the causative agent of toxoplasmosis, Toxoplasma gondii, has been attained by using diverse in vitro cellular models. Nonetheless, there are still processes key to its transmission and persistence which remain to be elucidated, such as the mechanisms underlying its systemic dissemination and sexual differentiation both of which occur at the intestinal level. Because this event occurs in a complex and specific cellular environment (the intestine upon ingestion of infective forms, and the feline intestine, respectively), traditional reductionist in vitro cellular models fail to recreate conditions resembling in vivo physiology. The development of new biomaterials and the advances in cell culture knowledge have opened the door to a next generation of more physiologically relevant cellular models. Among them, organoids have become a valuable tool for unmasking the underlying mechanism involved in T. gondii sexual differentiation. Murine-derived intestinal organoids mimicking the biochemistry of the feline intestine have allowed the generation of pre-sexual and sexual stages of T. gondii for the first time in vitro, opening a window of opportunity to tackling these stages by “felinizing” a wide variety of animal cell cultures. Here, we reviewed intestinal in vitro and ex vivo models and discussed their strengths and limitations in the context of a quest for faithful models to in vitro emulate the biology of the enteric stages of T. gondii.Pasteur NetworkFondo para la Convergencia Estructural del MERCOSURAgencia Nacional de Investigación e InnovaciónPrograma de Desarrollo de las Ciencias Básicas (PEDEClBA

Effects of heme oxygenase isozymes on Leydig cells steroidogenesis

In the present study, we demonstrate the expression of heme oxygenase (HO) isozymes, HO-1 and HO-2 (listed as HMOX1 and HMOX2 in the MGI Database), in MA-10 Leydig tumor cells and its effect on steroidogenesis. The well-known HO inducer, hemin, increased both HO-1 and HO-2 protein levels and HO-specific activity. Induction of HO by hemin inhibited basal, hCG-, and dibutyryl cAMP (db-cAMP)-induced steroidogenesis in a reversible way. When we studied the effect of HO isozymes along the steroid synthesis, we found that steroidogenic acute regulatory protein levels were decreased, and the conversion of cholesterol to pregnenolone was inhibited by hemin treatment, with no changes in the content of cholesterol side-chain cleavage enzyme (P450scc). hCG and db-cAMP also stimulated the expression of HO-1 and HO-2, and HO enzymatic activity in MA-10 cells. Basal and hCG-stimulated testosterone synthesis was also inhibited by hemin in rat normal Leydig cells. Taken together, these results suggest that: i) at least one of HO products (presumably carbon monoxide) inhibits cholesterol transport to the inner mitochondrial membrane and Leydig cell steroidogenesis by binding to the heme group of the cytochrome P450 enzymes, in a similar way as we described for nitric oxide, and ii) hCG stimulation results in the induction of an antioxidant enzymatic system (HO) acting as a cytoprotective mechanism in Leydig cells, as already demonstrated in the adrenal gland.Fil: Piotrkowski, Barbara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Monzón, Casandra Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pagotto, Romina María del Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Reche, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Besio Moreno, Marcos Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Cymeryng, Cora Betriz. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pignataro, Omar Pedro. Universidad de Buenos Aires. Facultad de Medicina. Departamento de Bioquímica Humana; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentin

Murine colon organoids as a novel model to study Trypanosoma cruzi infection and interactions with the intestinal epithelium

Chagas disease (CD) is a life-threatening illness caused by the parasite Trypanosoma cruzi (T. cruzi). With around seven million people infected worldwide and over 50,000 deaths per year, CD is a major public health issue in Latin America. The main route of transmission to humans is through a triatomine bug (vector-borne), but congenital and oral transmission have also been reported. The acute phase of CD presents mild symptoms but may develop into a long-lasting chronic illness, characterized by severely impaired cardiac, digestive, and neurological functions. The intestinal tissue appears to have a key role during oral transmission and chronic infection of CD. In this immune-privileged reservoir, dormant/quiescent parasites have been suggested to contribute to disease persistence, infection relapse, and treatment failure. However, the interaction between the intestinal epithelium and T. cruzi has not been examined in depth, in part, due to the lack of in vitro models that approximate to the biological and structural complexity of this tissue. Therefore, to understand the role played by the intestinal tissue during transmission and chronic infection, physiological models resembling the organ complexity are needed. Here we addressed this issue by establishing and characterizing adult stem cell-derived colonoid infection models that are clinically relevant for CD. 3D and 2D systems of murine intestinal organoids infected with T. cruzi Dm28c (a highly virulent strain associated with oral outbreaks) were analyzed at different time points by confocal microscopy. T. cruzi was able to invade and replicate in intestinal epithelial primary cells grown as intact organoids (3D) and monolayers (2D). The permissiveness to pathogen infection differed markedly between organoids and cell lines (primate and intestinal human cell lines). So far, this represents the first evidence of the potential that these cellular systems offer for the study of host-pathogen interactions and the discovery of effective anti-chagasic drugs.Agencia Nacional de Investigación e InnovaciónPasteur NetworkFOCEM (MERCOSUR Structural Convergence Fund

Modeling host-parasite interaction in chagas disease with murine intestinal organoids

Chagas disease (CD) is a potentially life-threatening illness caused by the parasite Trypanosoma cruzi (T. cruzi). With around seven million people infected worldwide and over 10,000 deaths per year, CD is a major public health issue in Latin America. The main route of transmission to humans is through a triatomine bug (vector-borne) and, to a minor extent, by blood transfusion, organ transplantation, laboratory accidents, congenitally and orally (food-borne). The acute phase of CD presents mild symptoms. If left untreated, it develops into a long-lasting chronic illness, characterized by severely impaired cardiac, digestive, and neurological functions. The intestinal tissue appears to have a key role during oral transmission and chronic infection of CD. In these immune-privileged reservoirs, dormant/quiescent parasites have been suggested to contribute to disease persistence, infection relapse, and treatment failure. However, the interaction between the intestinal epithelium and T. cruzi has not been examined in depth, in part, due to the lack of in vitro models resembling the biological and structural complexity of this organ. Therefore, to understand the pathophysiological role played by the intestinal tissue during transmission and chronic infection, we evaluated the progression of T. cruzi infection of murine colon organoids. In order to model CD, 3D and 2D systems of murine intestinal organoids were infected with T. cruzi Dm28c, a strain that has been associated with high virulence and oral outbreaks. At different time points, the presence and load of parasites in the organoids, as well as the host cell morphology were evaluated by confocal microscopy, and compared to those obtained with a classical infection model (Vero cells). We show that the parasite invades and replicates in intestinal epithelial primary cells grown as intact organoids (3D) and monolayers (2D). The permissiveness to pathogen infection differed markedly between the primary and the tumoral (Vero) cells. So far, this represents the first evidence of the potential of these nearly physiological cellular systems to study host-pathogen interaction for CD and/or for the future evaluation of anti-chagasic drugs.Agencia Nacional de Investigación e Innovación (ANII)FOCEM (MERCOSUR Structural Convergence Fund

Histamine inhibits adrenocortical cell proliferation but does not affect steroidogenesis.

Histamine (HA) is a neurotransmitter synthesized in most mammalian tissues exclusively by histidine decarboxylase enzyme. Among the plethora of actions mediated by HA, the modulatory effects on steroidogenesis and proliferation in Leydig cells (LCs) have been described recently. To determine whether the effects on LCs reported could be extrapolated to all steroidogenic systems, in this study, we assessed the effect of this amine on adrenal proliferation and steroidogenesis, using two adrenocortical cell lines as experimental models, murine Y1 cells and human NCI-H295R cells. Even when steroidogenesis was not modified by HA in adrenocortical cells, the biogenic amine inhibited the proliferation of H295R cells. This action was mediated by the activation of HRH1 subtype and an increase in the production of inositol phosphates as second messengers, causing cell-cycle arrest in the G2/M phase. These results indicate a new role for HA in the proliferation of human adrenocortical cells that could contribute to a better understanding of tumor pathology as well as to the development of new therapeutic agents.Fil: Pagotto, Romina María del Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Pereyra, Elba Nora. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Monzón, Casandra Margarita. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Mondillo, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); ArgentinaFil: Pignataro, Omar Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental (i); Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentin

Mammal intestinal organoids for studying zoonotic pathogens.

Intestinal organoids are self-organized three dimensional (3D) structures composed of a layer of polarized intestinal epithelial cells surrounding a hollow lumen. They recapitulate in vitro the intestinal multicelular composition, architecture and physiology. The aim of this work was to set up organoid models for studying zoonotic pathogens such as Salmonella and Toxoplasma gondii. T. gondii’s sexual cycle is restricted to felid’s intestines, which are characterized by an excess of linoleic acid given by the lack of delta-6-desaturase activity. “Felinized” murine intestinal organoids were generated for triggering T. gondii’s sexual differentiation in vitro. For this purpose, murine intestinal organoids from C57BL/6 mice were established from crypt isolated intestinal stem cells (2D or 3D) and incubated in the presence of 20 μM delta-6-desaturase inhibitor and 200 μM linoleic acid. Under these conditions no cytotoxicity of felinizing compounds was observed until 5 days of incubation. To optimize T. gondii’s infection, intestinal organoids were incubated with tachyzoites (at three distinct multiplicities of infection, MOIs) and evaluated by immunofluorescence assays (IFAs) at three time points post-infection. In order to set up a Salmonella infection model, intestinal organoids from farm animals (cow and sheep) were established and characterized by light microscopy and RT-PCR of specific markers. Forward steps will involve bovine intestinal organoids exposure to Salmonella enterica reporter strains at different MOIs, and bacteria invasion/proliferation evaluation at two time points after infection by extra and intracellular bacteria quantification and IFAs. Our results highlight the versatile uses of intestinal organoids as a powerful in vitro tool for modeling zoonotic diseases, contributing to the principle of reducing the use of experimental animal models.Agencia Nacional de Investigación e Innovación (ANII)FOCEM (MERCOSUR Structural Convergence Fund

Study of 6-propyl-2-thiouracil as a radioprotector for the thyroid gland

The objective of the paper was to study the application of 6-propyl-2-thiouracil (PTU) as a radioprotector for the thyroid gland. Rat thyroid epithelial cells (FRTL-5) and human colon cancer cells (ARO81-1) were exposed to γ-irradiation with or without 1 mM PTU. Radiation response was analysed by clonogenic survival assay. Cyclic AMP levels were measured by Radioimmunoassay (RIA). The results showed that PTU increased the Surviving Cell Fraction (SF) at 2 Gy significantly (p < 0.05) in both cell lines. PTU increased extracellular levels of cAMP in all the treatments in a dose- and time-dependent manner for FRTL-5 cells. In ARO81-1 cells, a peak was observed at 24 hours in extracellular levels incubated with 1 mM PTU (36.97 ± 6.74 fmol/μg prot vs. control: 17.53 ± 3.9 fmol/μg prot, p < 0.001). Forskolin and dibutyril cAMP mimicked the effect of PTU on SF. Thus PTU appears to be a radioprotector for thyroid cells and could exert its effect through cAMP.Fil: Perona, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); ArgentinaFil: Dagrosa, María Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica. Gerencia de Area de Aplicaciones de la Tecnología Nuclear. Gerencia de Radiobiología (Centro Atómico Constituyentes); ArgentinaFil: Pagotto, Romina. Fundación de Instituto de Biología y Medicina Experimental; ArgentinaFil: Casal, Mariana. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Pignataro, Omar Pedro. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Pisarev, Mario Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Oncología "Ángel H. Roffo"; ArgentinaFil: Juvenal, Guillermo Juan. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Constituyentes; Argentin