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

Adipose Tissue Dysfunction in Obesity: Role of Mineralocorticoid Receptor

The mineralocorticoid receptor (MR) acts as an essential regulator of blood pressure, volume status, and electrolyte balance. However, in recent decades, a growing body of evidence has suggested that MR may also have a role in mediating pro-inflammatory, pro-oxidative, and pro-fibrotic changes in several target organs, including the adipose tissue. The finding that MR is overexpressed in the adipose tissue of patients with obesity has led to the hypothesis that this receptor can contribute to adipokine dysregulation and low-grade chronic inflammation, alterations that are linked to the development of obesity-related metabolic and cardiovascular complications. Moreover, several studies in animal models have investigated the role of MR antagonists (MRAs) in preventing the metabolic alterations observed in obesity. In the present review we will focus on the potential mechanisms by which MR activation can contribute to adipose tissue dysfunction in obesity and on the possible beneficial effects of MRAs in this setting

Atrial Fibrillation and Aortic Ectasia as Complications of Primary Aldosteronism: Focus on Pathophysiological Aspects

Primary aldosteronism (PA) is the most common cause of secondary hypertension. A growing body of evidence has suggested that, beyond its well-known effects on blood pressure and electrolyte balance, aldosterone excess can exert pro-inflammatory, pro-oxidant and pro-fibrotic effects on the kidney, blood vessels and heart, leading to potentially harmful pathophysiological consequences. In clinical studies, PA has been associated with an increased risk of cardiovascular, cerebrovascular, renal and metabolic complication compared to essential hypertension, including atrial fibrillation (AF) and aortic ectasia. An increased prevalence of AF in patients with PA has been demonstrated in several clinical studies. Aldosterone excess seems to be involved in the pathogenesis of AF by inducing cardiac structural and electrical remodeling that in turn predisposes to arrhythmogenicity. The association between PA and aortic ectasia is less established, but several studies have demonstrated an effect of aldosterone on aortic stiffness, vascular smooth muscle cells and media composition that, in turn, might lead to an increased risk of aortic dilation and dissection. In this review, we focus on the current evidence regarding the potential role of aldosterone excess in the pathogenesis of AF and aortic ectasia

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

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

Primary Aldosteronism and Resistant Hypertension: A Pathophysiological Insight

Primary aldosteronism (PA) is a pathological condition characterized by an excessive aldosterone secretion; once thought to be rare, PA is now recognized as the most common cause of secondary hypertension. Its prevalence increases with the severity of hypertension, reaching up to 29.1% in patients with resistant hypertension (RH). Both PA and RH are “high-risk phenotypes”, associated with increased cardiovascular morbidity and mortality compared to non-PA and non-RH patients. Aldosterone excess, as occurs in PA, can contribute to the development of a RH phenotype through several mechanisms. First, inappropriate aldosterone levels with respect to the hydro-electrolytic status of the individual can cause salt retention and volume expansion by inducing sodium and water reabsorption in the kidney. Moreover, a growing body of evidence has highlighted the detrimental consequences of “non-classical” effects of aldosterone in several target tissues. Aldosterone-induced vascular remodeling, sympathetic overactivity, insulin resistance, and adipose tissue dysfunction can further contribute to the worsening of arterial hypertension and to the development of drug-resistance. In addition, the pro-oxidative, pro-fibrotic, and pro-inflammatory effects of aldosterone may aggravate end-organ damage, thereby perpetuating a vicious cycle that eventually leads to a more severe hypertensive phenotype. Finally, neither the pathophysiological mechanisms mediating aldosterone-driven blood pressure rise, nor those mediating aldosterone-driven end-organ damage, are specifically blocked by standard first-line anti-hypertensive drugs, which might further account for the drug-resistant phenotype that frequently characterizes PA patients

Clinical and Pathological Tools for Predicting Recurrence and/or Metastasis in Patients with Pheochromocytoma and Paraganglioma

Pheochromocytomas and paragangliomas are endocrine tumors belonging to the family of neural crest cell-derived neoplasms. They have an extremely variable clinical course, characterized by a non-negligible percentage of relapse and/or metastasis after radical surgery. To date, there are no reliable methods to predict the metastatic potential of these neoplasms, despite several clinical, molecular, and histopathological factors that have been extensively studied in the literature as predictors of the recurrence and/or metastasis in these neoplasms with different performances and results. In this review, we aimed to discuss and analyze the most important clinical and histopathological tools for predicting recurrence risk in patients affected by pheochromocytomas or paragangliomas. Thus, we compared the main available predictive models, exploring their applications in stratifying patients’ risks. In conclusion, we underlined the importance of simple and validated tools to better define disease aggressiveness and establish tailored patients’ treatments and follow-ups

Characterization of Oct4-GFP transgenic mice as a model to study the effect of environmental estrogens on the maturation of male germ cells by using flow cytometry

Oct4 is involved in regulation of pluripotency during normal development and is down-regulated during formation of postnatal reservoir of germ cells. We propose thatOct4/GFP transgenic mouse, which mimics the endogenous expression pattern of Oct4, could be used as a mammalian model to study the effects of environmental estrogens on the development of male germ cells. Oct4/GFP maturation profile was assessed during postnatal days -PND- 3, 5, 7, 10, 14 and 80, using flow cytometry. Then, we exposed pregnant mothers to 17a-ethinylestradiol (EE2) from day post coitum (dpc) 5 to PND7. Percentage of Oct4/GFP-expressing cells and levels of expression of Oct4/GPF were increased in PND7 after EE2 exposure. These observations were confirmed by analysis of GFP and endogenous Oct4 protein in the seminiferous tubules and by a reduction in epididymal sperm count in adult mice. We introduced Oct4/ GFP mouse together with flow cytometry as a tool to evaluate changes in male germ cells development.Agencia Nacional de Investigación e InnovaciónFONCyT (Fondo para la InvestigaciónCientífica y Tecnológica de la Agencia Nacional de PromociónCientífica y Tecnológica, ANPCyT, Argentina)FOCEM (MERCOSUR Structural Convergence Fund), COF 03/11CONICET (becas doctoral y postdoctoral

Il prelievo selettivo dalle vene surrenaliche nella diagnosi di sottotipo dell'iperaldosteronismo primario

SommarioL'iperaldosteronismo primario è la causa più frequente di ipertensione arteriosa secondaria e si associa ad aumentato rischio cardiovascolare. Il prelievo venoso selettivo surrenalico costituisce il gold standard nella diagnosi di sottotipo tra forme bilaterali e unilaterali, consentendo un adeguato approccio terapeutico. La sua diffusione è limitata in quanto si tratta di un esame di notevole difficoltà tecnica. Pertanto, risulta necessario eseguire tale procedura in centri di riferimento