Ruthenium(II)-Arene Curcuminoid Complexes as Photosensitizer Agents for Antineoplastic and Antimicrobial Photodynamic Therapy: In Vitro and In Vivo Insights.

Photodynamic therapy (PDT) is an anticancer/antibacterial strategy in which photosensitizers (PSs), light, and molecular oxygen generate reactive oxygen species and induce cell death. PDT presents greater selectivity towards tumor cells than conventional chemotherapy; however, PSs have limitations that have prompted the search for new molecules featuring more favorable chemical-physical characteristics. Curcumin and its derivatives have been used in PDT. However, low water solubility, rapid metabolism, interference with other drugs, and low stability limit curcumin use. Chemical modifications have been proposed to improve curcumin activity, and metal-based PSs, especially ruthenium(II) complexes, have attracted considerable attention. This study aimed to characterize six Ru(II)-arene curcuminoids for anticancer and/or antibacterial PDT. The hydrophilicity, photodegradation rates, and singlet oxygen generation of the compounds were evaluated. The photodynamic effects on human colorectal cancer cell lines were also assessed, along with the ability of the compounds to induce ROS production, apoptotic, necrotic, and/or autophagic cell death. Overall, our encouraging results indicate that the Ru(II)-arene curcuminoid derivatives are worthy of further investigation and could represent an interesting option for cancer PDT. Additionally, the lack of significant in vivo toxicity on the larvae of Galleria mellonella is an important finding. Finally, the photoantimicrobial activity of HCurc I against Gram-positive bacteria is indeed promising.

Susceptibility of Drosophila suzukii larvae to the combined administration of the entomopathogens Bacillus thuringiensis and Steinernema carpocapsae.

Non-native pests are often responsible for serious crop damage. Since Drosophila suzukii has invaded North America and Europe, the global production of soft, thin-skinned fruits has suffered severe losses. The control of this dipteran by pesticides, although commonly used, is not recommended because of the negative impact on the environment and human health. A possible alternative is the use of bio-insecticides, including Bacillus thuringiensis and entomopathogenic nematodes, such as Steinernema carpocapsae. These biological control agents have a fair effectiveness when used individually on D. suzukii, but both have limits related to different environmental, methodological, and physiological factors. In this work, we tested various concentrations of B. thuringiensis and S. carpocapsae to evaluate their efficacy on D. suzukii larvae, when administered individually or in combination by using agar traps. In the combined trials, we added the nematodes after 16 h or concurrently to the bacteria, and assessed larvae lethality from 16 to 48 h. The assays demonstrated a higher efficacy of the combined administration, both time-shifted and concurrent; the obtained data also showed a relevant decrease of the time needed to kill the larvae. Particularly, the maximum mortality rate, corresponding to 79% already at 16 h, was observed with the highest concentrations (0.564 µg/mL of B. thuringiensis and 8 × 102 IJs of S. carpocapsae) in the concurrent trials. This study, conducted by laboratory tests under controlled conditions, is a good starting point to develop a further application step through field studies for the control of D. suzukii.

In Vivo Effects of A Pro-PO System Inhibitor on the Phagocytosis of Xenorhabdus Nematophila in Galleria Mellonella Larvae.

Xenorhabdus nematophila is a Gram-negative bacterium symbiont of the entomopathogen nematode Steinernema carpocapsae whose immunosuppressive properties over host's immune response have been thoroughly investigated. In particular, live X. nematophila actively impairs phagocytosis in host's hemocytes through the secretion of inhibitors of eicosanoids synthesis. In this article we have investigated the cell surface structural features of X. nematophila responsible for the elusion from phagocytosis. To this end we have studied the uptake of heat-killed (hk), fluorescein isothiocyanate (FITC)-labeled X. nematophila by phagocytes from both a host insect and a mammalian species. In vitro dead X. nematophila passively resists engulfment by insect hemocytes without impairing the phagocytosis machinery whereas, unexpectedly, in vivo a significant phagocytosis of dead X. nematophila was observed. X. nematophila in vivo phagocytosis was increased by the co-injection of the specific inhibitor of pro-phenoloxidase (PO) system phenylthiourea (PTU), even if these effects were not observed in in vitro tests. Furthermore, biochemical modifications of X. nematophila cell wall implement in vivo phagocytosis, suggesting that this bacterium avoid phagocytosis because the ligand of phagocytic receptors is somehow buried or disguised in the cell wall. Finally, dead X. nematophila escapes engulfment even by human phagocytes suggesting that X. nematophila could be a useful model to investigate escape from phagocytosis by mammalian macrophages.

Susceptibility to entomopathogens and modulation of basal immunity in two insect models at different temperatures.

In this work, we analysed the efficacy of different commercial bio-insecticides (Steinernema feltiae, Steinernema carpocapsae, Heterorhabditis bacteriophora and Bacillus thuringiensis) by valuating the mortality induced on two insect models, Galleria mellonella (Lepidoptera) and Sarcophaga africa (Diptera) after exposure to different temperatures (10, 20 and 30 °C). Moreover, we investigated the effects of temperature on the basal humoral immunity of the two target insects; particularly, phenoloxidase (PO) and lysozyme activity. Our results show that G. mellonella is susceptible to all bio-insecticides at all the examined temperatures, except when infected at 10 °C with S. carpocapsae and at 30 °C with S. feltiae and B. thuringiensis. S. africa is more susceptible at 30 °C to all bioinsecticides; whereas, when infected at 10 and 20 °C, H. bacteriophora is the most efficient. Temperature modulates PO activity of both G. mellonella and S. africa, otherwise variations in lysozyme activity is observed only in G. mellonella. Except for a possible correlation between the increased lysozyme activity and the delayed Bt efficacy recorded on G. mellonella at 30 °C, a different resistance to bio-insecticides at different temperatures does not seem to be associated to variations of the host basal immunity, probably due to immunoevasive and immunodepressive strategies of these entomopathogens.

Effects of an entomopathogen nematode on the immune response of the insect pest red palm weevil: Focus on the host antimicrobial response.

Relationships between parasites and hosts can be drastic, depending on the balance between parasite strategies and the efficiency of the host immune response. In the case of entomopathogenic nematodes and their insect hosts, we must also consider the role of bacterial symbionts, as the interaction among them is tripartite and each component plays a critical role in death or survival. We analyzed the effects induced by the nematode-bacteria complex Steinernema carpocapsae, against red palm weevil (RPW) larvae, Rhynchophorus ferrugineus. We examined the antimicrobial response of the insect when in the presence of nematocomplexes or of its symbionts, Xenorhabdus nematophila. In detail, we investigated the potential interference of live and dead S. carpocapsae, their isolated cuticles, live or dead bacterial symbionts and their lipopolysaccharides, on the synthesis and activity of host antimicrobial peptides. Our data indicate that both live nematodes and live bacterial symbionts are able to depress the host antimicrobial response. When nematodes or symbionts were killed, they lacked inhibitory properties, as detected by the presence of antimicrobial peptides (AMPs) in the host hemolymph and by assays of antimicrobial activity. Moreover, we isolated S. carpocapsae cuticles; when cuticles were injected into hosts they revealed evasive properties because they were not immunogenic and were not recognized by the host immune system. We observed that weevil AMPs did not damage X. nematophila, and the lipopolysaccharides purified from symbionts seemed to be non-immunogenic. We believe that our data provide more information on the biology of entomopathogenic nematodes, in particular concerning their role and the activity mediated by symbionts in the relationship with insect hosts.

Inducible factors with antimicrobial activity after immune challenge in the haemolymph of Red Palm Weevil (Insecta).

Insects are capable of innate immune responses elicited after microbial infection. In this process, the receptor-mediated recognition of foreign bodies and the subsequent activation of immunocompetent cells lead to the synthesis ex novo of a peptide pool with antimicrobial activity. We investigated the inducible immune response of a coleopteran, Rhynchophorus ferrugineus, challenged with both Gram-negative and Gram-positive bacteria. After immunization, we evaluated the presence of antimicrobial peptides using either biochemical analyses or microbiological techniques. The antimicrobial properties of the newly synthesized protein pool, detectable in haemolymph fractions of low molecular mass, showed strong antibacterial activity against various bacterial strains (Escherichia coli, Pseudomonas sp. OX1, Bacillus subtilis and Micrococcus luteus). In addition to the preliminary study of the mechanism of action of the pool of antimicrobial peptides, we also investigated its effects on bacterial cell walls by means of fluorescence microscopy and scanning electron microscopy. The data suggest that the main effects seem to be directed at destabilizing and damaging the bacterial wall. This study provides data that help us to understand some aspects of the inducible innate immunity in a system model that lacks anticipatory responses. However, the weevil has finely tuned its defensive strategies to counteract effectively microbial infection.

Modulation of immune responses of Rhynchophorus ferrugineus (Insecta: Coleoptera) induced by the entomopathogenic nematode Steinernema carpocapsae (Nematoda: Rhabditida).

Aim of this study was to investigate relationships between the red palm weevil (RPW) Rhynchophorus ferrugineus (Olivier) and the entomopathogenic nematode Steinernema carpocapsae (EPN); particularly, the work was focused on the immune response of the insect host in naive larvae and after infection with the EPN. Two main immunological processes have been addressed: the activity and modulation of host prophenoloxidase-phenoloxidase (proPO) system, involved in melanization of not-self and hemocytes recognition processes responsible for not-self encapsulation. Moreover, immune depressive and immune evasive strategies of the parasite have been investigated. Our results suggest that RPW possess an efficient immune system, however in the early phase of infection, S. carpocapsae induces a strong inhibition of the host proPO system. In addition, host cell-mediated mechanisms of encapsulation, are completely avoided by the parasite, the elusive strategies of S. carpocapsae seem to be related to the structure of its body-surface, since induced alterations of the parasite cuticle resulted in the loss of its mimetic properties. S. carpocapsae before the release of its symbiotic bacteria, depress and elude RPW immune defenses, with the aim to arrange a favorable environment for its bacteria responsible of the septicemic death of the insect target.

A pathogenic parasite interferes with phagocytosis of insect immunocompetent cells.

Phagocytosis activity of hemocytes of the host Galleria mellonella (Lepidoptera) was modulated by the infection of the entomopathogenic nematode Steinernema feltiae (Rahbditida) and was found to be correlated with the opsonization of bacteria by hemolymph factors. The presence of nematodes resulted in a significative decrease in phagocytosis of bacteria by host hemocytes, both in in vivo and in in vitro assays. Host interacting proteins (HIPs), which appear to function as opsonic factors and are essential to perform immune responses, were removed by S. feltiae from host hemolymph, by means of its epicuticle binding properties. Host humoral factors sequestered by the parasite have been identified by monodimensional and 2D electrophoretic analysis. The data suggest that S. feltiae, living in association with symbiontic bacteria (Xenorhabdus nematophilus), develop an immune suppressive strategy to support its bacteria, which diminished the effectiveness of immunological surveillance by the host.

Cuticular surface lipids are responsible for disguise properties of an entomoparasite against host cellular responses.

Entomopathogenic nematodes are widely used as alternatives to chemicals for the biological control of insects. These endoparasites are symbiotically associated with bacteria that are lethal for the host; however, parasites need to overcome the host immune defences to complete a successful life cycle. The processes parasites employ to escape or depress host immunity are targeted at deceiving non-self recognition as well as inactivating defence reactions. The purpose of this paper is to investigate the interactions between the entomopathogenic nematode Steinernema feltiae and the lepidopteran Galleria mellonella, focusing on the role of the parasite's body-surface compounds in the immunoevasion of host cell-mediated responses. To evaluate host self/non-self discrimination and encapsulation efficiency, we carried out a series of interaction assays between cultured host hemocytes and parasites or isolated cuticles. The data obtained suggest that the parasite cuticular lipids (PCLs) are able to bind a variety of host hemolymph molecules; PCLs attract host proteins from the hemolymph creating a coat around the parasite, thus, enabling Steinernema to disguise itself against hemocytes recognition. The role of parasite lipids in the disguise process was also investigated by simulating the nematode body surface with agarose microbeads covered with purified cuticular components; when the beads were coated with cuticular lipids, host hemocytes were not able to recognize and encapsulate. Results suggest that by means of attracting host hemolymph components onto its cuticular surface, S. feltiae prevents hemocytes attachment to its cuticle and inhibits melanization by depleting hemolymph components.

The role of Steinernema feltiae body-surface lipids in host-parasite immunological interactions.

Interactions between entomopathogenic nematodes (Steinernema feltiae) and insect host (Galleria mellonella) immune system were investigated. We focused on the immunosuppressive properties of the parasite cuticle and on its interaction with hemolymph humoral components. Effects of parasite cuticle against host proPO system enzymatic cascade were evaluated a short time after infection. The presence of parasite cuticles decreased both normal and LPS-elicited proPO system activity, suggesting that S. feltiae body surface plays a key role in the early parasitation phase, probably interfering with host proPO activation pathways. The data obtained showed that cuticle lipidic compounds are able to interact with host humoral components, removing them from the hemolymph. The depletion of these molecules, arbitrarily named host-interacting proteins (HIPs), seems to be responsible of the drastic decrease in proPO system activity. Moreover, hemolymph HIPs showed LPS-binding properties and parasite cuticle cross-reacted with anti-LPS antibodies. Finally, we also assessed the involvement of parasite body surface on immunoevasion strategies of S. feltiae against host cell-mediated encapsulation processes. We conclude that S. feltiae body surface is responsible for short-term immunosuppression and immunoevasion processes; since it is able to sequester host hemolymph compounds involved in proPO system activation and this process could be responsible for a molecular disguise strategy against cellular encapsulation.

Differentiation of slow and fast fibers in tentacles of Sepia officinalis (Mollusca).

The tentacles of Sepia officinalis are cylindrical muscular structures that can be quickly everted and elongated to capture prey. The combination of both velocity and extensive elongation of the tentacles is due to the presence of both cross-striated and helical muscles. The complex organization and differentiation of different fibers has been studied in cuttlefish extracted from egg gel coats at different developmental stages, and in completely developed animals. Tentacle muscles start to differentiate centrifugally from the area close to the axial nervous system, where two types of myocytes can be recognized. These populations of myocytes, which may be distinguished morphologically and which express different myosin isoforms, give rise to fast and slow muscles. The presence in molluscs of slow and fast muscles arising from different populations of myocytes, as in vertebrate muscle development, could be considered as an example of evolutionary conservation.

Muscle differentiation in tentacles of Sepia officinalis (Mollusca) is regulated by muscle regulatory factors (MRF) related proteins.

The tentacles of Sepia officinalis are muscular structures that can be quickly everted and 'super-elongated' to capture prey. The speed and super-elongation are achieved by the presence of both cross-striated and helical muscles. In the present study, the complex organization and differentiation of various fibers of the cuttlefish were examined from an early stage of development (stage 26), when the embryo is still inside the egg gel-coating, until the juvenile stage (two weeks after hatching). The muscles start to differentiate centrifugally from the area around the axial nervous system where two types of myoblasts can be recognized. Smooth fibers (referred to here as 'smooth-like' fibers because of their similarity to vertebrate smooth fibers) appear first, then bundles and layers of circomyarian helical and cross-striated fibers differentiate. In Sepia, two muscle-specific transcription factors (MRF), Myf5-like and MyoD-like, have been identified and they are differently expressed during development. Myf5 was detected at first in myoblasts, which give rise to helical smooth-like fibers, while MyoD was expressed later in the other population of myocytes from which circomyarian helical and cross-striated fibers derive. The effective role of these two MRF in tentacle muscle differentiation was confirmed by RNA interference experiments. Injection of double stranded (ds)RNA Myf5 inhibited differentiation of smooth-like fibers, whereas injection of dsRNA MyoD resulted in inhibition of cross-striated and circomyarian helical fibers.

Immune suppression of Galleria mellonella (Insecta, Lepidoptera) humoral defenses induced by Steinernema feltiae (Nematoda, Rhabditida): involvement of the parasite cuticle.

Immune depression of Galleria mellonella larvae was evaluated a short time after infection with the entomopathogenic nematode Steinernema feltiae. In the host the activity of the enzymatic cascade known as the proPO system was significantly reduced by the presence of either live or dead parasites. The presence of parasites decreased the LPS-elicited proPO system activity. In addition, this process seems to be related to a decrease in the activity of hemolymph proteases, more than to phenoloxidase damage. proPO inhibition was also achieved by injected isolated cuticle fragments, suggesting that the parasite body surface plays an important role in the early parasitation phase.