First report of Anaplasma phagocytophilum and its co-infections with Borrelia burgdorferi sensu lato in Ixodes ricinus ticks (Acari: Ixodidae) from Republic of Moldova

We examined 198 questing Ixodes ricinus ticks collected in Chisinau City, Republic of Moldova by PCR assays for Anaplasma phagocytophilum, Borrelia burgdorferi sensu lato and co-infection of both pathogens, which were detected in 9%, 25.2% and 2.5% of tested ticks, respectively. B. burgdorferi s.l. genotyping revealed the presence of five genospecies with dominance of B. garinii. Our preliminary study provides evidence about occurrence of both pathogens in this populated area, which represent a potential health risk for inhabitants.     

Unveiling Ga(III) phthalocyanine—a different photosensitizer in neuroblastoma cellular model

Phthalocyanines (Pc) and their metallated derivatives are strongly considered for photodynamic therapy (PDT) possessing unique properties as possible new photosensitizers (PS). We have used toxicological assessments, real‐time monitoring of cellular impedance, and imagistic measurements for assessing the in vitro dark toxicity and PDT efficacy of Ga(III)‐Pc in SHSy5Y neuroblastoma cells. We have established the non‐toxic concentration range of Ga(III)‐Pc, a compound which shows a high intracellular accumulation, with perinuclear distribution in confocal microscopy. By choosing Ga(III)Pc non‐toxic dose, we performed in vitro experimental PDT hampering cellular proliferation. Our proposed Ga(III)‐Pc could complete a future PS panel for neuroblastoma alternate therapy.     

Age‐dependent effect between MARCO and TLR4 on PMMA particle phagocytosis by macrophages

Progressive generation of total joint implant‐derived wear particles is one of the major risk factors in development of peri‐prosthetic osteolysis especially in the aging society. It is commonly accepted that macrophages predominantly drive the inflammatory response to wear debris particles. Among various surface receptors that activate the macrophages to phagocytize particles, it is believed that the Toll‐like receptor 4 (TLR4) and the scavenger macrophage receptor with collagenous structure (MARCO) play key roles in recognition of wear debris particles. However, a strong body of evidence indicates an age‐dependent diminished function of human TLRs. Thus, we hypothesized that the MARCO receptor may be more engaged than TLRs in the phagocytosis of wear debris particles which in turn up‐regulate production of pro‐inflammatory cytokines from aged macrophages. We demonstrated that peritoneal macrophages isolated from aged mice show elevated expression of MARCO receptor compared to that from young mice. In contrast the expression of TLR4 was significantly decreased on the surface of aged macrophages. Furthermore, using anti‐MARCO and anti‐TLR4 neutralizing mAbs, we demonstrated the age‐dependent pathogenic role of MARCO, but not TLR4, receptor in promoting poly‐methyl methacrylate (PMMA) bone cement particles phagocytosis by macrophages leading to the release of pro‐inflammatory cytokines migration inhibitory factor and tumour necrosis factor in vitro. These data also suggest that the approach to neutralize MARCO may lead to the development of therapeutic regimen for the prevention of particle‐induced osteolysis in aged patients.     

Genotoxicity Induced by Foetal and Infant Exposure to Magnetic Fields and Modulation of Ionising Radiation Effects

Background

Few studies have investigated the toxicity and genotoxicity of extremely low frequency magnetic fields (ELF-MF) during prenatal and neonatal development. These phases of life are characterized by cell proliferation and differentiation, which might make them sensitive to environmental stressors. Although in vitro evidences suggest that ELF-MF may modify the effects of ionizing radiation, no research has been conducted so far in vivo on the genotoxic effects of ELF-MF combined with X-rays.

Aim and methods

Aim of this study was to investigate in somatic and germ cells the effects of chronic ELF-MF exposure from mid gestation until weaning, and any possible modulation produced by ELF-MF exposure on ionizing radiation-induced damage. Mice were exposed to 50 Hz, 65 μT magnetic field, 24 hours/day, for a total of 30 days, starting from 12 days post-conception. Another group was irradiated with 1 Gy X-rays immediately before ELF-MF exposure, other groups were only X-irradiated or sham-exposed. Micronucleus test on blood erythrocytes was performed at multiple times from 1 to 140 days after birth. Additionally, 42 days after birth, genotoxic and cytotoxic effects on male germ cells were assessed by comet assay and flow cytometric analysis.

Results

ELF-MF exposure had no teratogenic effect and did not affect survival, growth and development. The micronucleus test indicated that ELF-MF induced a slight genotoxic damage only after the maximum exposure time and that this effect faded away in the months following the end of exposure. ELF-MF had no effects on ionizing radiation (IR)-induced genotoxicity in erythrocytes. Differently, ELF–MF appeared to modulate the response of male germ cells to X-rays with an impact on proliferation/differentiation processes. These results point to the importance of tissue specificity and development on the impact of ELF-MF on the early stages of life and indicate the need of further research on the molecular mechanisms underlying ELF-MF biological effects.     

Sterol and pH interdependence in the binding, oligomerization, and pore formation of Listeriolysin O

Listeriolysin O (LLO) is the most important virulence factor of the intracellular pathogen Listeria monocytogenes. Its main task is to enable escape of bacteria from the phagosomal vacuole into the cytoplasm. LLO belongs to the cholesterol-dependent cytolysin (CDC) family but differs from other members, as it exhibits optimal activity at low pH. Its pore forming ability at higher pH values has been largely disregarded in Listeria pathogenesis. Here we show that high cholesterol concentrations in the membrane restore the low activity of LLO at high pH values. LLO binds to lipid membranes, at physiological or even slightly basic pH values, in a cholesterol-dependent fashion. Binding, insertion into lipid monolayers, and permeabilization of calcein-loaded liposomes are maximal above approximately 35 mol % cholesterol, a concentration range typically found in lipid rafts. The narrow transition region of cholesterol concentration separating low and high activity indicates that cholesterol not only allows the binding of LLO to membranes but also affects other steps in pore formation. We were able to detect some of these by surface plasmon resonance-based assays. In particular, we show that LLO recognition of cholesterol is determined by the most exposed 3beta-hydroxy group of cholesterol. In addition, LLO binds and permeabilizes J774 cells and human erythrocytes in a cholesterol-dependent fashion at physiological or slightly basic pH values. The results clearly show that LLO activity at physiological pH cannot be neglected and that its action at sites distal to cell entry may have important physiological consequences for Listeria pathogenesis.     

Type I IFNs stimulate nitric oxide production and resistance to Trypanosoma cruzi infection

The participation of type I IFNs (IFN-I) in NO production and resistance to Trypanosoma cruzi infection was investigated. Adherent cells obtained from the peritoneal cavity of mice infected by the i.p. route produced NO and IFN-I. Synthesis of NO by these cells was partially inhibited by treatment with anti-IFN-alphabeta or anti-TNF-alpha Abs. Compared with susceptible BALB/c mice, peritoneal cells from parasite-infected resistant C57BL/6 mice produced more NO (2-fold), IFN-I (10-fold), and TNF-alpha (3.5-fold). Later in the infection, IFN-I levels measured in spleen cell (SC) cultures from 8-day infected mice were greater in C57BL/6 than in infected BALB/c mice, and treatment of the cultures with anti-IFN-alphabeta Ab reduced NO production. IFN-gamma or IL-10 production by SCs was not different between the two mouse strains; IL-4 was not detectable. Treatment of C57BL/6 mice with IFN-I reduced parasitemia levels in the acute phase of infection. Mice deprived of the IFN-alphabetaR gene developed 3-fold higher parasitemia levels in the acute phase in comparison with control 129Sv mice. Production of NO by peritoneal macrophages and SCs was reduced in mice that lacked signaling by IFN-alphabeta, whereas parasitism of macrophages was heavier than in control wild-type mice. We conclude that IFN-I costimulate NO synthesis early in T. cruzi infection, which contributes to a better control of the parasitemia in resistant mice.     

In Vitro Effects of Calcium Fructoborate upon Production of Inflammatory Mediators by LPS-stimulated RAW 264.7 Macrophages

The present study is supported by our previous findings suggesting that calcium fructoborate (CF) has anti-inflammatory and antioxidant properties. Thus, we investigated the effects of CF on a model for studying inflammatory disorders in vitro represented by lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. This investigation was performed by analyzing the levels of some mediators released during the inflammatory process: cytokines such as tumor necrosis factor-α (TNF-α), interleukins IL-1β and IL-6 as well as cyclooxygenase-2 (COX-2), the main enzyme responsible for endotoxin/LPS-induced prostaglandin synthesis by macrophages. We also measured production of nitric oxide (NO) that plays an important role in the cytotoxicity activity of macrophages towards microbial pathogens. After CF treatment of LPS-stimulated macrophages we found an up-regulation of TNF-α protein level in culture medium, no significant changes in the level of COX-2 protein expression and a decrease in NO production as well as in IL-1β and IL-6 release. Collectively, this series of experiments indicate that CF affect macrophage production of inflammatory mediators. However, further research is required in order to establish whether CF treatment can be beneficial in suppression of pro-inflammatory cytokine production and against progression of endotoxin-related diseases.     

The first study on bacterial flora of pest beetles Sciaphobus squalidus,Tatianaerhynchites aequatus and Byctiscus betulae in the Republic of Moldova

The present study was conducted to identify the microbial flora of pest weevils Sciaphobus squalidus (Gyll.), Tatianaerhynchites aequatus (L.) and Byctiscus betulae L., which may provide novel approaches for insect biocontrol. According to morphological, physiological, biochemical and molecular properties thirteen bacteria were revealed and characterized. Ten of these bacteria were identified at the species level and the rest at the genus level. Isolates were identified as Staphyloccocus haemolyticus (Ta5), Bacillus cereus (Ss1), Pseudomonas moraviensis (Ss4), Pseudomonas fluorescens (Ss2), Pantoea agglomerans (Ss3, Bb3), Klebsiella pneumoniae (Ta1, Ta3, and Ta4), Erwinia billingiae (Ta2) and Erwinia spp. (Bb1, Bb2, Bb4). This is the first record of bacterial isolates (Ss4, Ta2 and Ta5) from any insect. The pathogenicity of bacteria was tested against adults of S. squalidus, T. aequatus and B. betulae and three of them showed insecticidal activity. The highest activity, 93% had B. cereus on S. squalidus and 90% on B. betulae within five days. The maximum activities of other two isolates Ss2 and Ss4 were determined as 73% and 46% against S. squalidus. Our data can offer useful information for future investigations on bacterial agent development and implementation as insecticides in agricultural system.     

Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs,GDI1 and Pak1

Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes.     

Storage of Blood in the Mammalian Spleen: an Evolutionary Perspective

Splenic blood storage is usually considered a characteristic restricted to Carnivora, Perissodactyla, and Ruminantia. In these mammals, the red pulp comprises the major part of the organ and – within it – the cords show a vast extension, allowing the storage of a great quantity of erythrocytes. Moreover, well-muscularized capsule and trabeculae permit a strong contractility. In this way, a significant quantity of erythrocytes can be poured into the circulation in response to an increased demand of oxygen. These spleens are usually classified as the “storing type,” in contrast to the “defensive type,” characterized by negligible quantity of blood stored. Past evolutionary interpretations have seen the former type as derived from the latter one. This explanation was based on some ontogenetic observations, thus deriving from the approach of Haeckel’s biogenetic law, and caused some contradictions that remained unsolved. An in-depth examination of the literature reveals that almost all mammalian species can store erythrocytes in their spleen. What changes is the quantity of this storage and the efficiency of its release. This means that the dichotomy between “storage” and “defensive” types is just an approximation – and not very useful. Splenic storage, indeed, is not a recently acquired character by some mammals, rather a primitive one, that underwent different quantitative and qualitative changes during the evolution of the different mammalian lineages. This review offers a new hypothesis on the evolution of this function (viewing the presence of splenic erythropoiesis as a biological constraint) and proposes it as a case of exaptation.