Establishment of a Greek food database for palaeodiet reconstruction:Case study of human and fauna remains from Neolithic to Late Bronze Age from Greece

We review the stable isotopic data of recovered Greek bones from the Early Neolithic to the Late Bronze period in order to examine dietary changes over time. As an isotopic baseline we use the published fauna data of the periods. The analysis revealed a diet that included a significant proportion of foods based on C3 plants, and the bulk of the animal protein must have been provided by terrestrial mammals with a small but detectable proportion of marine protein for coastal and island populations. A more significant contribution of marine protein is observed for Bronze Age populations while the enrichment in both C and N isotopes is connected, for some areas, to the introduction of millet during the Bronze Age, and to freshwater consumption. An extensive database of Greek food sources is presented and compared to the fauna from the prehistoric periods (Early Neolithic to Late Bronze Age) of the literature. We propose that this database can be used in palaeodiet reconstruction studies

Quantification of Nitric Oxide and Reactive Oxygen Species in Leishmania-infected J774A.1 Macrophages as a Response to the in vitro treatment with a Natural Product Compound

Leishmaniasis is a parasitic disease caused by the obligatory intracellular protozoa Leishmania spp. Current therapeutic options are limited and thus, drug discovery against leishmaniasis is very important. Nevertheless, there is a great difficulty to develop therapeutic strategies against the disease because the parasite deploys various mechanisms to evade the immune system and multiply inside the host. Among the main factors of the immunity that are recruited to confront the Leishmania infection are the macrophages (M Phi s) that produce effector molecules such as Nitric Oxide (NO) and Reactive Oxygen Species (ROS). Therefore, efficient drug agents should combine the antileishmanial effect of these gaseous transmitters along with the enhancement of the host's adaptive immunity. In the quest of therapeutic alternatives, natural products have been extensively studied and are considered as candidate antileishmanial agents since they exhibit specific properties in modulating the host's immune response towards an effective anti-leishmanial cell-mediated immunity capable to eliminate parasitic dissemination. In the current protocol, Leishmania-infected MFs (J774A.1 cell line) that have been treated with various increasing concentrations of a natural compound, are tested for the production of the aforementioned molecules. In order to detect NO production, we employ the Griess colorimetric nitrite assay and quantification relies on the construction of an accurate standard curve using appropriate standards of known concentration. ROS detection and quantification is achieved by flow cytometry and relies on the use of carboxy-H(2)DCFDA, an indicator that converts to a fluorescent form when interacts with ROS molecules

Leishmania eukaryotic initiation factor (LeIF) inhibits parasite growth in murine macrophages.

The leishmaniases constitute neglected global public health problems that require adequate control measures, prophylactic clinical vaccines and effective and non-toxic drug treatments. In this study, we explored the potential of Leishmania infantum eukaryotic initiation factor (LieIF), an exosomal protein, as a novel anti-infective therapeutic molecule. More specifically, we assessed the efficacy of recombinant LieIF, in combination with recombinant IFN-γ, in eliminating intracellular L. donovani parasites in an in vitro macrophage model. J774A.1 macrophages were initially treated with LieIF/IFN-γ prior to in vitro infection with L. donovani stationary phase promastigotes (pre-infection treatment), and resistance to infection was observed 72 h after infection. J774A.1 macrophages were also treated with LieIF/IFN-γ after L. donovani infection (post-infection treatment), and resistance to infection was also observed at both time points tested (19 h and 72 h) after infection. To elucidate the LieIF/IFN-γ-induced mechanism(s) that mediate the reduction of intracellular parasite growth, we examined the generation of potent microbicidal molecules, such as nitric oxide (NO) and reactive oxygen species (ROS), within infected macrophages. Furthermore, macrophages pre-treated with LieIF/IFN-γ showed a clear up-regulation in macrophage inflammatory protein 1α (MIP-1α) as well as tumor necrosis factor alpha (TNF-α) expression. However, significant different protein levels were not detected. In addition, macrophages pre-treated with LieIF/IFN-γ combined with anti-TNF-α monoclonal antibody produced significantly lower amounts of ROS. These data suggest that during the pre-treatment state, LieIF induces intramacrophage parasite growth inhibition through the production of TNF-α, which induces microbicidal activity by stimulating NO and ROS production. The mechanisms of NO and ROS production when macrophages are treated with LieIF after infection are probably different. Overall, these results indicate that LieIF is a good candidate for use as an anti-leishmanial molecule

New Insights on the Adjuvant Properties of the Leishmania infantum Eukaryotic Initiation Factor

Vaccination is the most effective tool against infectious diseases. Subunit vaccines are safer compared to live-attenuated vaccines but are less immunogenic and need to be delivered with an adjuvant. Adjuvants are essential for enhancing vaccine potency by improving humoral and cell-mediated immune responses. Only a limited number of adjuvants are licensed for human vaccines, and their mode of action is still not clear. Leishmania eukaryotic initiation factor (LeIF) has been described having a dual role, as a natural adjuvant and as an antigen that possesses advantageous immunomodulatory properties. In this study, we assessed the adjuvant properties of recombinant Leishmania infantum eukaryotic initiation factor (LieIF) through in vitro and in vivo assays. LieIF was intraperitoneally administered in combination with the protein antigen ovalbumin (OVA), and the widely used alum was used as a reference adjuvant. Our in vitro studies using J774A.1 macrophages showed that LieIF induced stimulatory effects as demonstrated by the enhanced surface expression of CD80 and CD86 co-stimulatory molecules and the induced production of the immune mediators NO and MIP-1α. Additionally, LieIF co-administration with OVA in an in vivo murine model induced a proinflammatory environment as demonstrated by the elevated expression of TNF-α, IL-1β, and NF-κB2 genes in peritoneal exudate cells (PEC). Furthermore, PEC derived from OVA-LieIF-immunized mice exhibited elevated expression of CD80 molecule and production of NO and MIP-1α in culture supernatants. Moreover, LieIF administration in the peritoneum of mice resulted in the recruitment of neutrophils and monocytes at 24 h post-injection. Also, we showed that this immunopotentiating effect of LieIF did not depend on the induction of uric acid danger signal. These findings suggest the potential use of LieIF as adjuvant in new vaccine formulations against different infectious diseases

ROS generation by J774A.1 macrophages pre- and post- infection treated (A and B, respectively) with recombinant LieIF (10 µg/ml) and recombinant IFN-γ (1 ng/ml).

<p>ROS generation was determined at the late time point (72 h) after <i>L. donovani</i> infection. Cells were labelled with 5 µM H₂DCFDA fluorescent probe and fluorescence of cells reacting with the probe was estimated by FACS analysis. All data are presented as the mean ± S.D of at least three independent experiments. Asterisks indicate statistically significant production of ROS (p≤0.05) compared to the control group of infected J774A.1 cells stimulated with IFN-γ (J774 + MON2 + IFN-γ). NL: for medium only; MON2: for the MHOM/IN/1996/THAK35 <i>L. donovani</i> strain used.</p

Effect of pre-infection treatment of J774A.1 macrophages with LieIF/IFN-γ on intracellular <i>L. donovani</i> growth inhibition.

<p>(A) at early (4 h) and (B) late (72 h) time points after infection. J774A.1 cells were stimulated with recombinant LieIF (10 µg/ml) and recombinant ΙFN-γ (1 ng/ml). <i>L. donovani</i> growth inhibition at the late time point (72 h) is associated with reduction of the infection rate (C) and the parasite load (D). The parasite growth inhibition was determined by the colorimetric method alamarBlue and OD was measured at 570/630 nm. Infection rate and parasite load were determined by microscopic observation upon Giemsa's staining of slides to count the mean number of infected macrophages considering 200 macrophages and the mean number of intracellular amastigotes in 200 infected macrophages, respectively. Data are presented as the mean ± S.D of at least three independent experiments. Asterisks indicate statistically significant differences (p≤0.05).</p

Nitric oxide production (ng/ml) by J774A.1 macrophages upon treatment with LieIF/IFN-γ at late (A, B) and early (C, D) time points after <i>L. donovani</i> infection.

<p>J774A.1 cells were treated pre-infection (A, C) and post-infection (B, D) with recombinant LieIF (10 µg/ml) and recombinant IFN-γ (1 ng/ml). Nitrite accumulation in the medium was measured by addition of Griess reagent, absorbance reading of the reaction product at 570 nm. All data are presented as the mean ± S.D of at least three independent experiments. Asterisks indicate statistically significant production of NO (p≤0.05).</p

Total Phenolic Fraction (TPF) from Extra Virgin Olive Oil: Induction of apoptotic-like cell death in Leishmania spp. promastigotes and in vivo potential of therapeutic immunomodulation.

BackgroundLeishmaniasis is a serious multifactorial parasitic disease with limited treatment options. Current chemotherapy is mainly consisted of drugs with serious drawbacks such as toxicity, variable efficacy and resistance. Alternative bioactive phytocompounds may provide a promising source for discovering new anti-leishmanial drugs. Extra Virgin Olive Oil (EVOO), a key-product in the Mediterranean diet, is rich in phenols which are associated with anti-inflammatory, anti-cancer and anti-microbial effects. In this study, we investigate the anti-leishmanial effect of Total Phenolic Fraction (TPF) derived from EVOO in both in vitro and in vivo systems by investigating the contributing mechanism of action.Methodology/principal findingsWe tested the ability of TPF to cause apoptotic-like programmed cell death in L. infantum and L. major exponential-phase promastigotes by evaluating several apoptotic indices, such as reduction of proliferation rate, sub-G0/G1 phase cell cycle arrest, phosphatidylserine externalization, mitochondrial transmembrane potential disruption and increased ROS production, by using flow cytometry and microscopy techniques. Moreover, we assessed the therapeutic effect of TPF in L. major-infected BALB/c mice by determining skin lesions, parasite burden in popliteal lymph nodes, Leishmania-specific antibodies and biomarkers of tissue site cellular immune response, five weeks post-treatment termination. Our results show that TPF triggers cell-cycle arrest and apoptotic-like changes in Leishmania spp. promastigotes. Moreover, TPF treatment induces significant reduction of parasite burden in draining lymph nodes together with an antibody profile indicative of the polarization of Th1/Th2 immune balance towards the protective Th1-type response, characterized by the presence of IFN-γ-producing CD4+ T-cells and increased Tbx21/GATA-3 gene expression ratio in splenocytes.Conclusions/significanceTPF exhibits chemotherapeutic anti-leishmanial activity by inducing programmed cell death on cell-free promastigotes and immunomodulatory properties that induce in vivo T cell-mediated responses towards the protective Th1 response in experimental cutaneous leishmaniasis. These findings enable deeper understanding of TPF's dual mode of action that encourages further studies