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Evaluation of the host cell response following Leishmania infantum infection
Full text linkThe Leishmaniases are a group of parasitic diseases caused by protozoa belonging to the Leishmania
genus and transmitted by the bite of infected female phlebotomine sandflies, affecting both humans
and other vertebrates, especially dogs. Mammalian leishmaniasis shows a worldwide distribution.
Leishmania is endemic in at least 98 countries, with the highest number of cases in developing
nations.
There are three forms of the disease: cutaneous leishmaniasis (CL) is the most common form of
leishmaniasis, mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL). The main species
in Italy is Leishmania infantum responsible for both cutaneous and visceral form of the disease.
The aim of this project was to investigate molecular response in macrophages infected with L.
infantum, to better understand and characterize the interaction between Leishmania infantum and
the host cells.
The reference strain of L. infantum MHOM/TN/80/IPT1 was provided by the OIE Reference
Laboratory National Reference Center for Leishmaniasis in Palermo. The in vitro infection model has
been developed from a human monocytic cell lines U937 and THP-1 after differentiation with
phorbol myristic acid and from murine primary macrophages.
Cells were infected with a parasite-to-cell ratio of 10:1 showing about 30% and 60% of infected cells
after 8 and 24 h, respectively, evaluated by cell counting and a quantitative PCR assay targeting the
Leishmania kinetoplastid DNA.
Using this infection protocol it was possible to analyze the molecular response in infected
macrophages, revealing that Leishmania induced Akt phosphorylation and inhibited the caspase-3
cleavage, promoting host cell survival. Moreover, gene expression analysis of ER stress-related
genes showed a mild but significant induction of the UPR markers. On the contrary, results showed
that Leishmania inhibited the induction of UPR markers following treatment with a strong inducer
of ER stress (tunicamycin and DTT).
Additionally, to explore the gene expression modulation in host cells following L. infantum infection,
experiments of RNA sequencing targeting total RNA of non-infected and infected U937 and
Leishmania RNA (Splice Leader protocol) were performed at the Center for Infectious Disease
Research in Seattle.
Another mechanism triggered in response to ER stress involves the induction of microRNA by UPR.
Among different miRNA found to be related to the UPR, we investigate the induction of mir-346
following the increase of the spliced XBP1, revealing a significant upregulation of mir-346 in both
U937 and THP-1 derived macrophages infected with L. infantum reference strain and two canine
clinical isolates.
In conclusion, we suggested that L. infantum could promote host cells survival by inducing a mild ER
stress response (subtle but significant increase in ER stress expression markers, delay/attenuation
of the effects of ER stress inducers).
Moreover, a significant up-regulation of the miR-346 has been detected, in both U937 and THP1-
derived macrophages infected with L. infantum.
However, to develop more effective/new therapeutic approaches targeting these molecular
pathways, we need to deeply understand the intricated interactions between host molecular
pathways and Leishmania parasites
Endoplasmic reticulum stress and unfolded protein response in infection by intracellular parasites
Full text linkPerturbations of the physiological status of the endoplasmic reticulum (ER) trigger a
specific response known as the ER stress response or unfolded protein response (UPR).
In mammalian cells, the UPR is mediated by three ER transmembrane proteins (IRE1,
PERK and ATF6) which activate three signaling cascades to restore ER homeostasis. In
recent years, a cross-talk between UPR, inflammatory and microbial sensing pathways
has been elucidated. Pathogen infection can lead to UPR activation; moreover, several
pathogens subvert the UPR to promote their survival and replication. While the UPR
in viral and bacterial infection has been characterized, little is known about the role
of UPR in intracellular parasite infection. Here, we review recent findings on UPR
induction/modulation by intracellular parasites in host cells
Real-time PCR applications for diagnosis of leishmaniasis
Full text linkLeishmaniasis is a vector-borne disease caused by many Leishmania species, which can infect both humans and other mammals. Leishmaniasis is a complex disease, with heterogeneous clinical manifestations ranging from asymptomatic infections to lesions at cutaneous sites (cutaneous leishmaniasis), mucosal sites (mucocutaneous leishmaniasis) or in visceral organs (visceral leishmaniasis), depending on the species and host characteristics. Often, symptoms are inconclusive and leishmaniasis can be confused with other co-endemic diseases. Moreover, co-infections (mainly with HIV in humans) can produce atypical clinical presentations. A correct diagnosis is crucial to apply the appropriate treatment and the use of molecular techniques in diagnosis of leishmaniasis has become increasingly relevant due to their remarkable sensitivity, specificity and possible application to a variety of clinical samples. Among them, real-time PCR (qPCR)-based approaches have become increasingly popular in the last years not only for detection and quantification of Leishmania species but also for species identification. However, despite qPCR-based methods having proven to be very effective in the diagnosis of leishmaniasis, a standardized method does not exist. This review summarizes the qPCR-based methods in the diagnosis of leishmaniasis focusing on the recent developments and applications in this field
Metformin prevents cell tumorigenesis through autophagy-related cell death
Full text linkAutophagy is a cellular mechanism by which cells degrade intracellular components in lysosomes, maintaining cellular homeostasis. It has been hypothesized that autophagy could have a role in cancer prevention through the elimination of damaged proteins and organelles; this could explain epidemiological evidence showing the chemopreventive properties of the autophagy-inducer metformin. In this study, we analyzed the autophagy-related effect of metformin in both cancer initiation and progression in non-tumorigenic cells. We also analyzed the induction of tumorigenesis in autophagy-deficient cells, and its correlation with the ER stress. Our results showed that metformin induced massive cell death in preneoplastic JB6 Cl 41-5a cells treated with tumor promoter (phorbol) and in NIH/3T3 treated with H2O2. Inhibiting autophagy with wortmannin or ATG7 silencing, the effect of metformin decreased, indicating an autophagy-related cytotoxic activity under stress conditions. We also found an induction of tumorigenesis in ATG7-silenced NIH/3T3 cell clone (3T3-619C3 cells), but not in wild-type and in scrambled transfected cells, and an upregulation of unfolded protein response (UPR) markers in 3T3-619C3 cells treated with H2O2. These findings suggest that autophagic cell death could be considered as a new mechanism by which eliminate damaged cells, representing an attractive strategy to eliminate potential tumorigenic cells
Extracellular embryo genomic DNA and its potential for genotyping applications
Full text linkBackground: Preimplantation genetic diagnosis (PGD) currently relies on biopsy of one or few embryo cells. Our aim was to evaluate the embryo extracellular matrices (spent medium and blastocoele fluid) as source of DNA for embryo genotyping. Results/methodology: We first evaluated the amplifiability and the amount of genomic DNA in spent embryo culture media from day 3 (n = 32) and day 5/6 (n = 54). Secondly, we evaluated the possibility to genotype the MTHFR polymorphism C677T from media at day 5/6 (n = 8) and blastocoele fluids (n = 9) by direct sequencing. The C677T polymorphism detection rate was 62.5 and 44.4% in medium and fluid, respectively. Conclusion: A noninvasive approach for embryo genotyping was possible, but still with limitations due to low detection rate and possible allele dropout
The host micro-RNA cfa-miR-346 is induced in canine leishmaniasis
Full text linkBackground
Leishmaniases are a group of anthropo-zoonotic parasitic diseases caused by a protozoan of the Leishmania genus, affecting both humans and other vertebrates, including dogs. L. infantum is responsible for the visceral and occasionally cutaneous form of the disease in humans and canine leishmaniasis. Previously, we have shown that L. infantum induces a mild but significant increase in endoplasmic reticulum (ER) stress expression markers to promote parasites survival in human and murine infected macrophages. Moreover, we demonstrated that the miRNA hsa-miR-346, induced by the UPR-activated transcription factor sXBP1, was significantly upregulated in human macrophages infected with different L. infantum strains. However, the ER stress response in infected dogs, which represent an important reservoir for Leishmania parasite, was described once recently, whereas the miR-346 expression was not reported before. Therefore, this study aimed to investigate these pathways in the canine macrophage-like cell line DH82 infected by Leishmania spp. and to evaluate the presence of cfa-miR-346 in plasma of non-infected and infected dogs.
The DH82 cells were infected with L. infantum and L. braziliensis parasites and the expression of cfa-mir-346 and several ER stress markers was evaluated by quantitative PCR (qPCR) at different time points. Furthermore, the cfa-miR-346 was monitored in plasma collected from non-infected dogs (n = 11) and dogs naturally infected by L. infantum (n = 18).
Results
The results in DH82 cells showed that cfa-mir-346 was induced at both 24 h and 48 h post-infection with all Leishmania strains but not with tunicamycin, accounting for a mechanism of induction independent from sXBP1, unlike what was previously observed in human cell lines. Moreover, the cfa-miR-346 expression analysis on plasma revealed a significant increase in infected dogs compared to non-infected dogs.
Conclusions
Here for the first time, we report the upregulation of cfa-miR-346 induced by Leishmania infection in canine macrophage-like cells and plasma samples of naturally infected dogs. According to our results, the cfa-miR-346 appears to be linked to infection, and understanding its role and identifying its target genes could contribute to elucidate the mechanisms underlying the host–pathogen interaction in leishmaniasis
Exosome secretion by Leishmania infantum modulate the chemotactic behavior and cytokinic expression creating an environment permissive for early infection
No full textIn recent years, several studies demonstrated the role of exosomes in intercellular communications, several Leishmania species belonging to subgenera Leishmania and Viannia have been demonstrated to release exosomes, and their role in parasite-macrophage interactions and in leishmaniasis development has been investigated. However, the release of exosomes by Leishmania infantum has not been studied so far. The aim of this study was to isolate and characterize L. infantum exosomes, and to investigate the biological activity of these exosomes in macrophage cultures. To this end, exosomes were collected from both amastigote and promastigote L. infantum conditioned medium by ultracentrifugation. Exosomes were then characterized by monitoring the presence of HSP70, HSP83/90 and acetylcholinesterase activity. Moreover, extracellular vesicles-tracking analysis revealed that promastigote and amastigote exosomes had mean diameter of 122 ± 56 nm and 115 ± 65 nm, respectively. Human monocytic cell line U937-derived macrophages treated with promastigote and amastigote exosomes showed an increase in motility and an overproduction of interleukin IL-10 and IL-18 reduction, involved in immune response. Since L. infantum exosomes demonstrated the capacity to modulate the chemotactic behaviour of the cells studied and cytokines production, they could contribute in the disease establishment and may be considered an appropriate candidate for a vaccine therapy in prophylaxis and treatment
The use of conjunctival swabs in the diagnosis of human visceral leishmaniasis
No full textHuman visceral leishmaniasis (VL) is a severe disease whose diagnosis comprises immunological tests, microscopic biopsy examination, and biomolecular assays. In veterinary medicine, conjunctival swabs are widely used for detection of parasite DNA. Here, we describe the case of human VL in which conjunctival swabs were successfully used for Leishmania detection
Inhibition of Testosterone Aromatization by the Indole-3-carbinol Derivative CTet in CYP19A1-overexpressing MCF-7 Breast Cancer Cells
No full textNatural products such as aromatase inhibitors have been the object of growing attention in recent years because of their potential to inhibit aromatase with fewer side effects and the possible translation of their current use as chemotherapeutic agents to future clinical applications in breast cancer chemoprevention. We have previously investigated CTet, a novel anticancer agent obtained from the broccoli-derived compound indole-3- carbinol (I3C), that shows great anticancer potential in both in vitro and in vivo studies. Here we evaluated the potential of CTet as a chemopreventive agent in aromatase expressing MCF-7/AROM-1 breast cancer cells. The testosterone (TE) aromatization in estradiol (E2) was indirectly evaluated in terms of inhibition of TE-induced cell proliferation, ERα phosphorylation/activation and Bcl-2 and IGF-1R ERE-regulated protein accumulation. Our results showed that CTet inhibited TE-driven ERα phosphorylation of both cytosolic and nuclear ERα pools, suggesting an inhibitory effect of TE aromatization in E2. CTet did not inhibit E2-driven nuclear ERα phosphorylation, but partially inhibited E2-driven cytosolic ERα phosphorylation. Moreover, CTet inhibited Bcl-2 and IGF-1R accumulation induced by TE but not that which was induced by E2. A cell-free enzymatic assay showed that CTet did not inhibit aromatase activity directly; however, since CTet treatment induced endoplasmic reticulum stress, the TE aromatization could be affected because the aromatase enzyme is located within the endoplasmic reticulum. Finally, CTet and letrozole synergistically inhibited TE-induced cell proliferation. These results showed the potential of the I3C derivative CTet as a chemopreventive agent that interferes with aromatase activity
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