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

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

Jejunum-derived NF-κB reporter organoids as 3D models for the study of TNF-alpha-induced inflammation

Inflammation is an important process for epithelial barrier protection but when uncontrolled, it can also lead to tissue damage. The nuclear factor-kappa light chain enhancer of activated B cells (NF-κB) signaling pathway is particularly relevant in the intestine, as it seems to play a dual role. Whereas NF-κB protects intestinal epithelium against various noxious stimuli, the same pathway mediates intestinal inflammatory diseases by inducing pro-inflammatory gene expression. The availability of appropriate in vitro models of the intestinal epithelium is crucial for further understanding the contribution of NF-κB in physiological and pathological processes and advancing in the development of drugs and therapies against gut diseases. Here we established, characterized, and validated three-dimensional cultures of intestinal organoids obtained from biopsies of NF-κB-RE-Luc mice. The NF-κB-RE-Luc intestinal organoids derived from different intestine regions recreated the cellular composition of the tissue and showed a reporter responsiveness similar to the in vivo murine model. When stimulated with TNF-α, jejunum-derived NF-κB-RE-Luc-reporter organoids, provided a useful model to evaluate the anti-inflammatory effects of natural and synthetic compounds. These reporter organoids are valuable tools to explore the epithelial TNF-α-induced NF-κB contribution in the small intestine, being a reliable alternative method while helping to reduce the use of laboratory animals for experimentation.Agencia Nacional de Investigación e InnovaciónFOCEM (MERCOSUR Structural Convergence Fund

Potencial de los organoides intestinales murinos para el estudio de la enfermedad de Chagas

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

What have we learned from a case of convalescent plasma treatment in a two-time kidney transplant recipient COVID-19 patient? A case report from the perspective of viral load evolution and immune response

Coronavirus disease 2019 (COVID-19), an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, can have a wide range of clinical manifestations, ranging from asymptomatic disease to potentially life-threatening complications. Convalescent plasma therapy has been proposed as an effective alternative for the treatment of severe cases. The aim of this study was to follow a two-time renal transplant patient with severe COVID-19 treated with convalescent plasma over time from an immunologic and virologic perspective. A 42-year-old female patient, who was a two-time kidney transplant recipient, was hospitalized with COVID-19. Due to worsening respiratory symptoms, she was admitted to the intensive care unit, where she received two doses of convalescent plasma. We analyzed the dynamics of viral load in nasopharyngeal swab, saliva, and tracheal aspirate samples, before and after convalescent plasma transfusion. The levels of pro-inflammatory cytokines and antibody titers were also measured in serum samples. A significant decrease in viral load was observed after treatment in the saliva and nasopharyngeal swab samples, and a slight decrease was observed in tracheal aspirate samples. In addition, we found evidence of an increase in antibody titers after transfusion, accompanied by a decrease in the levels of several cytokines responsible for cytokine storm

Organoides intestinales murinos: diferentes aproximaciones metodológicas

Los organoides intestinales constituyen modelos tridimensionales de complejidad intermedia entre los cultivos de líneas celulares y los modelos in vivo, permitiendo aproximaciones de estudio fisiológicamente más relevantes y mejorando el poder predictivo de los ensayos in vitro. Sin embargo, existe una diferencia evidente con el intestino y es que en los organoides el lumen queda contenido en el interior de la estructura celular, limitando el acceso a la cara apical del epitelio. Para superar esta limitación se han desarrollado metodologías que permiten el acceso al lumen de los organoides, incrementando la versatilidad de este modelo. En este video presentamos 3 metodologías para acceder a la cara apical de los organoides: la ruptura parcial de los organoides, la microinyecciòn y el cultivo de organoides en monocapa.UNU-Biola

Organoides intestinales murinos: obtención, manipulación y conservación

Los organoides intestinales son estructuras multicelulares tridimensionales que poseen un poder replicativo ilimitado natural. Están constituidos por células madre que se diferencian a distintos tipos celulares y recrean en buena medida las características del tejido in vivo. En este video describimos paso a paso el procedimiento para la obtención de un cultivo de organoides intestinales murinos a partir del aislamiento de criptas, su expansión, criopreservación y descongelado.UNU-Biola

The Anticancer Peptide CIGB-552 Exerts Anti-Inflammatory and Anti-Angiogenic Effects through COMMD1

CIGB-552 is a synthetic anti-tumor peptide capable of reducing tumor size and increasing the lifespan of tumor-bearing mice. Part of its anti-cancer effects consists of inducing apoptosis, modulating NF-kB signaling pathway, and the angiogenesis process. Although one of its major mediators, the COMMD1 protein, has been identified, the mechanism by which CIGB-552 exerts such effects remains elusive. In the present study, we show the role of COMMD1 in CIGB-552 mechanism of action by generating the COMMD1 knock-out from the human lung cancer cell line NCI-H460. A microarray was performed to analyze both wild-type and KO cell lines with regard to CIGB-552 treatment. Additionally, different signaling pathways were studied in both cell lines to validate the results. Furthermore, the interaction between CIGB-552 and COMMD1 was analyzed by confocal microscopy. By signaling pathway analysis we found that genes involved in cell proliferation and apoptosis, oncogenic transformation, angiogenesis and inflammatory response are potentially regulated by the treatment with CIGB-552. We then demonstrated that CIGB-552 is capable of modulating NF-kB in both 2D and 3D cell culture models. Finally, we show that the ability of CIGB-552 to negatively modulate NF-kB and HIF-1 pathways is impaired in the COMMD1 knock-out NCI-H460 cell line, confirming that COMMD1 is essential for the peptide mechanism of action

Organoides intestinales: una herramienta versátil para el estudio in vitro de patologías del epitelio intestinal

Los organoides intestinales son estructuras multicelulares tridimensionales que derivan de células madre y tienen la capacidad de auto-organizarse. Recrean varios aspectos de la morfología, composición celular y fisiología del intestino, constituyendo modelos del epitelio intestinal de mayor relevancia que las líneas celulares tradicionales. El objetivo de este trabajo consistió en implementar el cultivo de organoides intestinales murinos, bovinos y ovinos, a partir de células madre adultas. Para posteriormente emplearlos como herramientas de reducción del uso de animales de experimentación y para el estudio de patologías asociadas al epitelio intestinal.Agencia Nacional de Investigación e Innovació