The maintenance of differentiated hepatocyte phenotype in vitro depends on several factors—in particular, on extracellular matrix interactions, for example, with three-dimensional (3D) matrices. Alginate hydrogel provides the cells with a good extracellular matrix due to the formation of a massive capsule with semi-permeable properties that allows for diffusion of the medium components into the cells as well as efficient waste product elimination. Simultaneously, alginate protects the cells from shear stress caused by the hydrodynamics when cultured in stirred systems such as bioreactors. We have previously developed a hepatocyte aggregate 3D culture system in a bioreactor where improved hepatocyte functionality could be maintained over longer periods (21 days). In this work, ultra-high-viscosity alginate was used for hepatocyte aggregates entrapment. Hepatocyte biotransformation (phase I and II enzymes), CYP450 inducibility, and secretory capacity (albumin and urea production) were monitored. The analyses were performed in both spinner vessels and bioreactors to test the effect of the pO ₂ control, unavailable in the spinners. Performance of alginate-encapsulated hepatocyte aggregates in culture was compared with nonencapsulated aggregate cultures in both bioreactor (controlled environment) and spinner vessels. For both culture systems, hepatocytes' metabolic and biotransformation capacities were maintained for up to 1 month, and encapsulated cells in bioreactors showed the best performance. In particular, albumin production rate increased 2- and 1.5-fold in encapsulated aggregates compared with nonencapsulated aggregates in bioreactor and spinner vessels, respectively. Urea production rate increased twofold in encapsulated cultures compared with nonencapsulated cells, in both bioreactor and spinner vessels. Similarly, in both the bioreactor and the spinner system, cell encapsulation resulted in a 1.5- and 2.8-fold improvement of hepatocyte 7-ethoxycoumarin and uridine diphosphate glucuronosyltransferases (UGT) activities, respectively. For all parameters, but for UGT activity, the bioreactor system resulted better than the spinner vessels; for UGT activity no difference was observed between the two. Furthermore, both encapsulated and nonencapsulated 3D culture systems were inducible by 3-methylcholanthrene and dexamethasone. The encapsulated systems consistently showed improved performance over the nonencapsulated cells, indicating that the protection conferred by the alginate matrix plays a relevant role in maintaining the hepatocyte functionalities in vitro .

Ovarian cancer is the second most common gynaecologic malignancy and the main cause of death from gynaecologic cancer, due to late diagnosis and chemoresistance. Studies have reported the role of cysteine in cancer, by contributing for hydrogen sulphide (H₂S) generation and as a precursor of glutathione (GSH). However, the role of cysteine in the adaptation to hypoxia and therapy response remains unclear. We used several ovarian cancer cell lines, ES2, OVCAR3, OVCAR8, A2780 and A2780cisR, to clarify cysteine relevance in ovarian cancer cells survival upon hypoxia and carboplatin. Results show that ES2 and OVCAR8 cells presented a stronger dependence on cysteine availability upon hypoxia and carboplatin exposure than OVCAR3 cells. Interestingly, the A2780 cisR, but not A2780 parental cells, benefits from cysteine upon carboplatin exposure, showing that cysteine is crucial for chemoresistance. Moreover, GSH degradation and subsequent cysteine recycling pathway is associated with ovarian cancer as seen in peripheral blood serum from patients. Higher levels of total free cysteine (Cys) and homocysteine (HCys) were found in ovarian cancer patients in comparison with benign tumours and lower levels of GSH were found in ovarian neoplasms patients in comparison with healthy individuals. Importantly, the total and S-Homocysteinylated levels distinguished blood donors from patients with neoplasms as well as patients with benign from patients with malignant tumours. The levels of S-cysteinylated proteins distinguish blood donors from patients with neoplasms and the free levels of Cys in serum distinguish blood from patients with benign tumours from patients with malignant tumours. Herein we disclosed that cysteine contributes for a worse disease prognosis, allowing faster adaptation to hypoxia and protecting cells from carboplatin. The measurement of serum cysteine levels can be an effective tool for early diagnosis, for outcome prediction and follow up of disease progression.

Primary cultures of human hepatocytes are a reference cellular model, because they maintain key features of liver cells in vivo , such as expression of drug-metabolizing enzymes, response to enzyme inducers, and generation of hepatic metabolites. However, there is a restricted availability of primary hepatocytes, and they show phenotypic instability in culture. Thus, different alternatives have been developed to overcome the culture limitations and to mimic in vivo tissue material. Herein, culture conditions, such as medium composition, impeller type, and cell inoculum concentration, were optimized in stirred culture vessels and applied to a three-dimensional (3D) bioreactor system. Cultures of rat hepatocytes as 3D structures on bioreactor, better resembling in vivo cellular organization, were compared to traditional monolayer cultures. Liver-specific functions, such as albumin and urea secretion, phase I and phase II enzyme activities, and the capacity to metabolize diphenhydramine and troglitazone, were measured over time. Hepatocyte functions were preserved for longer time in the 3D bioreactor than in the monolayer system. Moreover, rat hepatocytes grown in 3D system maintained the ability to metabolize such compounds, as well as in vivo . Our results indicate that hepatocytes cultured as 3D structures are a qualified model system to study hepatocyte drug metabolism over a long period of time. Moreover, these cultures can be used as feeding systems to obtain cells for other tests in a short time.

Chagas disease (CD) is a vector-borne Neglected Zoonotic Disease (NZD) caused by a flagellate protozoan, Trypanosoma cruzi, that affects various mammalian species across America, including humans and domestic animals. However, due to an increase in population movements and new routes of transmission, T. cruzi infection is presently considered a worldwide health concern, no longer restricted to endemic countries. Dogs play a major role in the domestic cycle by acting very efficiently as reservoirs and allowing the perpetuation of parasite transmission in endemic areas. Despite the significant progress made in recent years, still there is no vaccine against human and animal disease, there are few drugs available for the treatment of human CD, and there is no standard protocol for the treatment of canine CD. In this review, we highlight human and canine Chagas Disease in its different dimensions and interconnections. Dogs, which are considered to be the most important peridomestic reservoir and sentinel for the transmission of T. cruzi infection in a community, develop CD that is clinically similar to human CD. Therefore, an integrative approach, based on the One Health concept, bringing together the advances in genomics, immunology, and epidemiology can lead to the effective development of vaccines, new treatments, and innovative control strategies to tackle CD.

The biochemical demands associated with tumor proliferation prompt neoplastic cells to augment the import of nutrients to sustain their survival and fuel cell growth, with a consequent metabolic remodeling. Fatty acids (FA) are crucial in this process, since they have a dual role as energetic coins and building blocks. Recently, our team has shown that FATP1 has a pivotal role in FA transfer between breast cancer cells (BCCs) and non-cancerous cells in the microenvironment. We aimed to investigate the role of FATP1 in BCCs and also to explore if FATP1 inhibition is a promising therapeutic strategy. In patients’ data, we showed a higher expression of FATP1/SLC27A1 in TNBC, which correlated with a significant decreased overall survival (OS). In vitro, we verified that FA and estradiol stimulated FATP1/SLC27A1 expression in BCCs. Additionally, experiments with estradiol and PHTPP (ER-β antagonist) showed that estrogen receptor-β (ER-β) regulates FATP1/SLC27A1 expression, the uptake of FA and cell viability, in four BCC lines. Furthermore, the inhibition of FATP1 with arylpiperazine 5k (DS22420314) interfered with the uptake of FA and cell viability. Our study, unraveled FATP1 as a putative therapeutic target in breast cancer (BC).

Canine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic disease of global concern. Antileishmanial drug therapies commonly used to treat sick dogs improve their clinical condition, although when discontinued relapses can occur. Thus, the current study aims to evaluate the effect of CanL treatments in peripheral blood, lymph node, and bone marrow cytokine profile associated with clinical recovery. Two groups of six dogs diagnosed with CanL were treated with miltefosine combined with allopurinol and meglumine antimoniate combined with allopurinol (MT+A and MG+A), respectively. At diagnosis and after treatment, during a 3-month follow-up, clinical signs, hematological and biochemical parameters, urinalysis results and antileishmanial antibody titers were registered. Furthermore, peripheral blood, popliteal lymph node, and bone marrow samples were collected to assess the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-β, and IFN-γ by qPCR. In parallel, were also evaluated samples obtained from five healthy dogs. Both treatment protocols promoted the remission of clinical signs as well as normalization of hematological and biochemical parameters and urinalysis values. Antileishmanial antibodies returned to non-significant titers in all dogs. Sick dogs showed a generalized upregulation of IFN-γ and downregulation of IL-2, IL-4, and TGF-β, while gene expression of IL-12, TNF-α, IL-5, and IL-10 varied between groups and according to evaluated tissue. A trend to the normalization of cytokine gene expression was induced by both miltefosine and meglumine antimoniate combined therapies. However, IFN-γ gene expression was still up-regulated in the three evaluated tissues. Furthermore, the effect of treatment in the gene expression of cytokines that were not significantly changed by infection, indicates that miltefosine and meglumine antimoniate combined therapy directly affects cytokine generation. Both combined therapies are effective in CanL treatment, leading to sustained pro-inflammatory immune environments that can compromise parasite survival and favor dogs' clinical cure. In the current study, anti-inflammatory and regulatory cytokines do not seem to play a prominent role in CanL or during clinical recovery.