Showing papers by "María Isabel Colombo published in 2018"

Autophagic Induction Greatly Enhances Leishmania major Intracellular Survival Compared to Leishmania amazonensis in CBA/j-Infected Macrophages.

Abstract: CBA mouse macrophages control Leishmania major infection yet are permissive to Leishmania amazonensis. Few studies have been conducted to assess the role played by autophagy in Leishmania infection. Therefore, we assessed whether the autophagic response of infected macrophages may account for the differential behavior of these two parasite strains. After 24 h of infection, the LC3-II/Act ratio increased in both L. amazonensis- and L. major-infected macrophages compared to uninfected controls, but less than in chloroquine-treated cells. This suggests that L. amazonensis and L. major activate autophagy in infected macrophages, without altering the autophagic flux. Furthermore, L. major-infected cells exhibited higher percentages of DQ-BSA-labeled parasitophorous vacuoles (50%) than those infected by L. amazonensis (25%). However, L. major- and L. amazonensis-induced parasitophorous vacuoles accumulated LysoTracker similarly, indicating that the acidity in both compartment was equivalent. At as early as 30 min, endogenous LC3 was recruited to both L. amazonensis- and L. major-induced parasitophorous vacuoles, while after 24 h a greater percentage of LC3 positive vacuoles was observed in L. amazonensis-infected cells (42.36%) compared to those infected by L. major (18.10%). Noteworthy, principal component analysis (PCA) and an hierarchical cluster analysis completely discriminated L. major-infected macrophages from L. amazonensis-infected cells accordingly to infection intensity and autophagic features of parasite-induced vacuoles. Then, we evaluated whether the modulation of autophagy exerted an influence on parasite infection in macrophages. No significant changes were observed in both infection rate or parasite load in macrophages treated with the autophagic inhibitors wortmannin, chloroquine or VPS34-IN1, as well as with the autophagic inducers rapamycin or physiological starvation, in comparison to untreated control cells. Interestingly, both autophagic inducers enhanced intracellular L. amazonensis and L. major viability, while the pharmacological inhibition of autophagy exerted no effects on intracellular parasite viability. We also demonstrated that autophagy induction reduced NO production by L. amazonensis- and L. major-infected macrophages but not alters arginase activity. These findings provide evidence that although L. amazonensis-induced parasitophorous vacuoles recruit LC3 more markedly, L. amazonensis and L. major similarly activate the autophagic pathway in CBA macrophages. Interestingly, the exogenous induction of autophagy favors L. major intracellular viability to a greater extent than L. amazonensis related to a reduction in the levels of NO.

Infectious Bursal Disease Virus Hijacks Endosomal Membranes as the Scaffolding Structure for Viral Replication.

Abstract: Birnaviruses are unconventional members of the group of double-stranded RNA (dsRNA) viruses that are characterized by the lack of a transcriptionally active inner core. Instead, the birnaviral particles organize their genome in ribonucleoprotein complexes (RNPs) composed by dsRNA segments, the dsRNA-binding VP3 protein, and the virally encoded RNA-dependent RNA polymerase (RdRp). This and other structural features suggest that birnaviruses may follow a completely different replication program from that followed by members of the Reoviridae family, supporting the hypothesis that birnaviruses are the evolutionary link between single-stranded positive RNA (+ssRNA) and dsRNA viruses. Here we demonstrate that infectious bursal disease virus (IBDV), a prototypical member of the Birnaviridae family, hijacks endosomal membranes of infected cells through the interaction of a viral protein, VP3, with the phospholipids on the cytosolic leaflet of these compartments for replication. Employing a mutagenesis approach, we demonstrated that VP3 domain PATCH 2 (P2) mediates the association of VP3 with the endosomal membranes. To determine the role of VP3 P2 in the context of the virus replication cycle, we used avian cells stably overexpressing VP3 P2 for IBDV infection. Importantly, the intra- and extracellular virus yields, as well as the intracellular levels of VP2 viral capsid protein, were significantly diminished in cells stably overexpressing VP3 P2. Together, our results indicate that the association of VP3 with endosomes has a relevant role in the IBDV replication cycle. This report provides direct experimental evidence for membranous compartments such as endosomes being required by a dsRNA virus for its replication. The results also support the previously proposed role of birnaviruses as an evolutionary link between +ssRNA and dsRNA viruses.IMPORTANCE Infectious bursal disease (IBD; also called Gumboro disease) is an acute, highly contagious immunosuppressive disease that affects young chickens and spreads worldwide. The etiological agent of IBD is infectious bursal disease virus (IBDV). This virus destroys the central immune organ (bursa of Fabricius), resulting in immunosuppression and reduced responses of chickens to vaccines, which increase their susceptibility to other pathogens. IBDV is a member of Birnaviridae family, which comprises unconventional members of dsRNA viruses, whose replication strategy has been scarcely studied. In this report we show that IBDV hijacks the endosomes of the infected cells for establishing viral replication complexes via the association of the ribonucleoprotein complex component VP3 with the phospholipids in the cytosolic leaflet of endosomal membranes. We show that this interaction is mediated by the VP3 PATCH 2 domain and demonstrate its relevant role in the context of viral infection.

Chronic Infections: A Possible Scenario for Autophagy and Senescence Cross-Talk

Abstract: Multiple tissues and systems in the organism undergo modifications during aging due to an accumulation of damaged proteins, lipids, and genetic material. To counteract this process, the cells are equipped with specific mechanisms, such as autophagy and senescence. Particularly, the immune system undergoes a process called immunosenescence, giving rise to a chronic inflammatory status of the organism, with a decreased ability to counteract antigens. The obvious result of this process is a reduced defence capacity. Currently, there is evidence that some pathogens are able to accelerate the immunosenescence process for their own benefit. Although to date numerous reports show the autophagy–senescence relationship, or the connection between pathogens with autophagy or senescence, the link between the three actors remains unexplored. In this review, we have summarized current knowledge about important issues related to aging, senescence, and autophagy.

Clinical features and outcomes of hepatocellular carcinoma in Caucasian cirrhotic patients on long-term analogue therapy for hepatitis B.

Abstract: Background Long-term oral nucleos(t)ide analogue (NUC) therapy in hepatitis B virus (HBV)-related compensated cirrhotics prevents clinical decompensation but not hepatocellular carcinoma (HCC) development. Aims To define the clinical features and outcomes of HCC in long-term NUC-treated HBV patients. Methods All HCCs developing between 2005 and 2016 in NUC-treated HBV patients under surveillance were studied, excluding those that occurred within the first 6 months of therapy. Clinical features of HCC, alpha faetoprotein (AFP) patterns and patients' outcome were studied. Results Seventy-six HCC patients were included. Median age was 67 (40-83) years, 84% males, 96% Caucasian, 95% HBeAg-negative, 96% with undetectable HBV DNA, 83% with normal ALT levels, and 92% with compensated cirrhosis. Median serum AFP levels were 4 (1-3615) ng/mL (>7 ng/mL in 36%). HCC was monofocal in 78%, had a median diameter of 20 (6-57) mm and was in its early stage in 92% which allowed potentially curative treatments in 78% (39% ablation, 28% surgical resection, 11% liver transplantation). Overall, a complete response was obtained in 61 (80%) patients: in 40 after a first-line treatment, in 3 after the second-line treatment, in 2 after the third-line treatment, while 16 underwent liver transplantation (8 as second line). During 45 (7-144) months after HCC diagnosis, 19 patients died, 84% from HCC progression. The median time to recurrence was 20.2 (3-53) months, and the cumulative 5-year liver-related survival was 74%. Conclusions HCCs developing in patients under long-term NUC treatment were single, small tumours, amenable to curative therapies able to confer excellent 5-year survival rates.