Increased brain cytokine level associated impairment of vigilance and memory in aged rats can be alleviated by alpha7 nicotinic acetylcholine receptor agonist treatment.

Age-related neurocognitive disorders are common problems in developed societies. Aging not only affects memory processes, but may also disturb attention, vigilance, and other executive functions. In the present study, we aimed to investigate age-related cognitive deficits in rats and associated molecular alterations in the brain. We also aimed to test the effects of the alpha7 nicotinic acetylcholine receptor (nAChR) agonist PHA-543613 on memory as well as on the sustained attention and vigilance of aged rats. Short- and long-term spatial memories of the rats were tested using the Morris water maze (MWM) task. To measure attention and vigilance, we designed a rat version of the psychomotor vigilance task (PVT) that is frequently used in human clinical examinations. At the end of the behavioral experiments, mRNA and protein expression of alpha7 nAChRs, cytokines, and brain-derived neurotrophic factor (BDNF) were quantitatively measured in the hippocampus, frontal cortex, striatum, and cerebellum. Aged rats showed marked cognitive deficits in both the MWM and the PVT. The deficit was accompanied by increased IL-1beta and TNFalpha mRNA expression and decreased BDNF protein expression in the hippocampus. PHA-543613 significantly improved the reaction time of aged rats in the PVT, especially for unexpectedly appearing stimuli, while only slightly (non-significantly) alleviating spatial memory deficits in the MWM. These results indicate that targeting alpha7 nAChRs may be an effective strategy for the amelioration of attention and vigilance deficits in age-related neurocognitive disorders.

Distinct Uptake Routes Participate in Silver Nanoparticle Engulfment by Earthworm and Human Immune Cells.

The consequences of engineered silver nanoparticle (AgNP) exposure and cellular interaction with the immune system are poorly understood. The immunocytes of the Eisenia andrei earthworm are frequently applied in ecotoxicological studies and possess functional similarity to vertebrate macrophages. Hence, we characterized and compared the endocytosis mechanisms for the uptake of 75 nm AgNPs by earthworm coelomocytes, human THP-1 monocytes, and differentiated THP-1 (macrophage-like) cells. Our results indicate that microtubule-dependent, scavenger-receptor, and PI3K signaling-mediated macropinocytosis are utilized during AgNP engulfment by human THP-1 and differentiated THP-1 cells. However, earthworm coelomocytes employ actin-dependent phagocytosis during AgNPs uptake. In both human and earthworm immunocytes, AgNPs were located in the cytoplasm, within the endo-/lysosomes. We detected that the internalization of AgNPs is TLR/MyD88-dependent, also involving the bactericidal/permeability-increasing protein (BPI) in the case of human immunocytes. The exposure led to decreased mitochondrial respiration in human immunocytes; however, in coelomocytes, it enhanced respiratory parameters. Our findings provide more data about NP trafficking as nano-carriers in the nanomedicine field, as well as contribute to an understanding of the ecotoxicological consequences of nanoparticle exposure.

Bacterial Engulfment Mechanism Is Strongly Conserved in Evolution Between Earthworm and Human Immune Cells.

Invertebrates, including earthworms, are applied to study the evolutionarily conserved cellular immune processes. Earthworm immunocytes (so-called coelomocytes) are functionally similar to vertebrate myeloid cells and form the first line of defense against invading pathogens. Hereby, we compared the engulfment mechanisms of THP-1 human monocytic cells, differentiated THP-1 (macrophage-like) cells, and Eisenia andrei coelomocytes towards Escherichia coli and Staphylococcus aureus bacteria applying various endocytosis inhibitors [amantadine, 5-(N-ethyl-N-isopropyl) amiloride, colchicine, cytochalasin B, cytochalasin D, methyl-ß-cyclodextrin, and nystatin]. Subsequently, we investigated the messenger RNA (mRNA) expressions of immune receptor-related molecules (TLR, MyD88, BPI) and the colocalization of lysosomes with engulfed bacteria following uptake inhibition in every cell type. Actin depolymerization by cytochalasin B and D has strongly inhibited the endocytosis of both bacterial strains in the studied cell types, suggesting the conserved role of actin-dependent phagocytosis. Decreased numbers of colocalized lysosomes/bacteria supported these findings. In THP-1 cells TLR expression was increased upon cytochalasin D pretreatment, while this inhibitor caused a dropped LBP/BPI expression in differentiated THP-1 cells and coelomocytes. The obtained data reveal further insights into the evolution of phagocytes in eukaryotes. Earthworm and human phagocytes possess analogous mechanisms for bacterial internalization.

Injury-Induced Innate Immune Response During Segment Regeneration of the Earthworm, Eisenia andrei.

Regeneration of body parts and their interaction with the immune response is a poorly understood aspect of earthworm biology. Consequently, we aimed to study the mechanisms of innate immunity during regeneration in Eisenia andrei earthworms. In the course of anterior and posterior regeneration, we documented the kinetical aspects of segment restoration by histochemistry. Cell proliferation peaked at two weeks and remitted by four weeks in regenerating earthworms. Apoptotic cells were present throughout the cell renewal period. Distinct immune cell (e.g., coelomocyte) subsets were accumulated in the newly-formed blastema in the close proximity of the apoptotic area. Regenerating earthworms have decreased pattern recognition receptors (PRRs) (e.g., TLR, except for scavenger receptor) and antimicrobial peptides (AMPs) (e.g., lysenin) mRNA patterns compared to intact earthworms. In contrast, at the protein level, mirroring regulation of lysenins became evident. Experimental coelomocyte depletion caused significantly impaired cell divisions and blastema formation during anterior and posterior regeneration. These obtained novel data allow us to gain insight into the intricate interactions of regeneration and invertebrate innate immunity.

Nanomaterials and Annelid Immunity: A Comparative Survey to Reveal the Common Stress and Defense Responses of Two Sentinel Species to Nanomaterials in the Environment.

Earthworms and leeches are sentinel animals that represent the annelid phylum within terrestrial and freshwater ecosystems, respectively. One early stress signal in these organisms is related to innate immunity, but how nanomaterials affect it is poorly characterized. In this survey, we compare the latest literature on earthworm and leeches with examples of their molecular/cellular responses to inorganic (silver nanoparticles) and organic (carbon nanotubes) nanomaterials. A special focus is placed on the role of annelid immunocytes in the evolutionarily conserved antioxidant and immune mechanisms and protein corona formation and probable endocytosis pathways involved in nanomaterial uptake. Our summary helps to realize why these environmental sentinels are beneficial to study the potential detrimental effects of nanomaterials.

Genetically highly divergent RNA virus with astrovirus-like (5'-end) and hepevirus-like (3'-end) genome organization in carnivorous birds, European roller (Coracias garrulus).

Astroviruses (family Astroviridae) and hepeviruses (family Hepeviridae) are small, non-enveloped viruses with genetically diverse +ssRNA genome thought to be enteric pathogens infecting vertebrates including humans. Recently, many novel astro- and hepatitis E virus-like +ssRNA viruses have been described from lower vertebrate species. The non-structural proteins of astro- and hepeviruses are highly diverse, but the structural/capsid proteins represent a common phylogenetic position shed the light of their common origin by inter-viral recombination. In this study, a novel astrovirus/hepevirus-like virus with +ssRNA genome (Er/SZAL5/HUN/2011, MK450332) was serendipitously identified and characterized from 3 (8.5%) out of 35 European roller (Coracias garrulus) faecal samples by RT-PCR in Hungary. The complete genome of Er/SZAL5/HUN/2011 (MK450332) is 8402 nt-long and potentially composed three non-overlapping open reading frames (ORFs): ORF1a (4449 nt/1482aa), ORF1b (1206 nt/401aa) and ORF2 (1491 nt/496aa). The ORF1ab has an astrovirus-like genome organization containing the non-structural conserved elements (TM, CC, NLS, VPg) and enzyme residues (trypsine-like protease, RNA-dependent RNA-polymerase) with low amino acid sequence identity, 15% (ORF1a) and 44% (ORF1b), to astroviruses. Supposedly the ORF2 is a capsid protein but neither the astrovirus-like subgenomic RNA promoter (sgRNA) nor the astrovirus-like capsid characteristics have been identifiable. However, the predicted capsid protein (ORF2) showed 26% identity to the corresponding protein of hepevirus-like novel Rana hepevirus (MH330682). This novel +ssRNA virus strain Er/SZAL5/HUN/2011 with astrovirus-like genome organization in the non-structural genome regions (ORF1a and ORF1b) and Rana hepevirus-related capsid (ORF2) protein represent a potentially recombinant virus species and supports the common origin hypothesis, although, the taxonomic position of the studied virus is still under discussion.

Identification of novel lumbricin homologues in Eisenia andrei earthworms.

Lumbricin and its orthologue antimicrobial peptides were typically isolated from annelids. In this report, mRNA for lumbricin and -serendipitously- a novel lumbricin-related mRNA sequence were identified in Eisenia andrei earthworms. The determined mRNA sequences of E. andrei lumbricin and lumbricin-related peptide consist of 477 and 575 nucleotides. The precursors of proline-rich E. andrei lumbricin and the related peptide contain 63 and 59 amino acids, respectively. Phylogenetic analysis indicated close relationship with other annelid lumbricins. Highest expression of both mRNAs appeared in the proximal part of the intestine (pharynx, gizzard), while other tested organs had moderate (body wall, midgut, ovary, metanephridium, seminal vesicles, ventral nerve cord) or low (coelomocytes) levels. During ontogenesis their expression revealed continuous increase in embryos. Following 48 h of in vivo Gram-positive bacteria challenge both mRNAs were significantly elevated in coelomocytes, while Gram-negative bacteria or zymosan stimulation had no detectable effects.

Multiple divergent picobirnaviruses with functional prokaryotic Shine-Dalgarno ribosome binding sites present in cloacal sample of a diarrheic chicken.

Picobirnaviruses (PBVs) of family Picobirnaviridae have bisegmented (S1 and S2 segments), double-stranded RNA genomes. In this study a total of N = 12 complete chicken PBVs (ChPBV) segments (N = 5 of S1 and N = 7 of S2, Acc. Nos.: MH425579-90) were determined using viral metagenomic and RT-PCR techniques from a single cloacal sample of a diarrheic chicken. The identified ChPBV segments are unrelated to each other and distant from all of the currently known PBVs. In silico sequence analyses revealed the presence of conserved prokaryotic Shine-Dalgarno-like (SD-like) sequences upstream of the three presumed open reading frames (ORFs) of the S1 and a single presumed ORF of the S2 segments. According to the results of expression analyses in E. coli using 6xHis-tagged recombinant ChPBV segment 1 construct and Western blot these SD-like sequences are functional in vivo suggesting that S1 of study PBVs can contain three ORFs and supporting the bacteriophage-nature of PBVs.

Acute toxicity of selenate and selenite and their impacts on oxidative status, efflux pump activity, cellular and genetic parameters in earthworm Eisenia andrei.

Selenium (Se) is an essential element for humans, animals, and certain lower plants, but can be toxic at high concentration. Even though Se is potentially toxic, little information is available about the effects of Se on soil animals. The aim of this study was to assess the impact of different concentrations of two Se forms, selenate and selenite, on earthworm Eisenia andrei. In order to obtain comprehensive overview on the Se effects, different parameters were measured. Namely, acute toxicity, apoptosis, efflux pump activity, different enzymatic and non-enzymatic biomarkers (acetylcholinesterase, carboxylesterase, glutathione S-transferase, catalase, glutathione reductase and superoxide dismutase activities, lipid peroxidation level and GSH/GSSG ratio) and expression of genes involved in oxidative and immune response have been investigated. Additionally, measurement of metallothioneins concentration and concentration of Se in exposed earthworms has been also performed. The assessment of acute toxicity showed a greater sensitivity of E. andrei to selenite exposure, whereas Se concentration measurements in earthworms showed higher accumulation of selenate form. Both Se forms caused inhibition of the efflux pump activity. Decrease in superoxide dismutase activity and increase in lipid peroxidation and glutathione reductase activity indicate that Se has a significant impact on the oxidative status of earthworms. Selenate exposure caused an apoptotic-like cell death in the coelomocytes of exposed earthworms, whereas decreased mRNA levels of stress-related genes and antimicrobial factors were observed upon the exposure to selenite. The obtained data give insight into the effects of two most common forms of Se in soil on the earthworm E. andrei.

Deficiency of innate-like T lymphocytes in chronic obstructive pulmonary disease.

BACKGROUND: Based on the phenotypic and functional characteristics unconventional T-lymphocytes such as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells link the innate and adaptive immune responses. Up to now data are scarce about their involvement in pulmonary disorders including chronic obstructive pulmonary disease (COPD). This study explores simultaneously the frequencies of iNKT and MAIT cells in the peripheral blood and sputum of stable and exacerbating COPD patients. METHODS: By means of multicolor flow cytometry frequencies of total iNKT and MAIT cells and their subsets were enumerated in peripheral blood and sputum samples of healthy controls, and COPD patients. In addition, gene expression of TCR for iNKT, MAIT cells, and CD1d, MR1 were assessed by qPCR in the study cohorts. RESULTS: Percentages of total iNKT and MAIT cells were dramatically dropped in blood, and reduced numbers of iNKT cells were observed in the sputum of COPD patients. Furthermore decreased DN and increased CD4+ iNKT subsets, while increased DN and decreased CD8+ MAIT subpopulations were measured in the blood of COPD patients. Reduced invariant TCR mRNA levels in COPD patients had confirmed these previous findings. The mRNA expression of CD1d and MR1 were increased in stable and exacerbating COPD patients; however both molecules were decreased upon antibiotic and systemic steroid treatments. CONCLUSIONS: Our results support the notion that both invariant T-cell populations are affected in COPD. Further detailed analysis of invariant T cells could shed more light into the complex interactions of these lymphocyte groups in COPD pathogenesis.

Phenotypic and functional characterization of earthworm coelomocyte subsets: Linking light scatter-based cell typing and imaging of the sorted populations.

Flow cytometry is a common approach to study invertebrate immune cells including earthworm coelomocytes. However, the link between light-scatter- and microscopy-based phenotyping remains obscured. Here we show, by means of light scatter-based cell sorting, both subpopulations (amoebocytes and eleocytes) can be physically isolated with good sort efficiency and purity confirmed by downstream morphological and cytochemical applications. Immunocytochemical analysis using anti-EFCC monoclonal antibodies combined with phalloidin staining has revealed antigenically distinct, sorted subsets. Screening of lectin binding capacity indicated wheat germ agglutinin (WGA) as the strongest reactor to amoebocytes. This is further evidenced by WGA inhibition assays that suggest high abundance of N-acetyl-d-glucosamine in amoebocytes. Post-sort phagocytosis assays confirmed the functional differences between amoebocytes and eleocytes, with the former being in favor of bacterial engulfment. This study has proved successful in linking flow cytometry and microscopy analysis and provides further experimental evidence of phenotypic and functional heterogeneity in earthworm coelomocyte subsets.

Reduced non-switched memory B cell subsets cause imbalance in B cell repertoire in systemic sclerosis.

OBJECTIVES: Analysis of peripheral blood B lymphocytes in patients with systemic sclerosis (SSc) has provided evidence for specific alterations in naive and memory B cell balance. However, memory B cell subsets in SSc have not been thoroughly investigated. This study sought to identify phenotypic abnormalities and activation markers in peripheral blood memory B cells in SSc subtypes. METHODS: Blood samples were obtained from 28 SSc patients with early form of disease (9 limited (lcSSc), 19 diffuse cutaneous SSc (dcSSc)) and 15 healthy controls. After magnetic bead separation of CD19+ B cells, multiparametric flow cytometry was performed and CD19+CD27- IgD+ naive, CD19+CD27+ memory, CD19+CD27+IgD+ non-switched memory CD19+CD27+IgD- switched memory, CD19+CD27-IgD- double negative (DN) memory, CD80+ or CD95+ activated cells were identified. RESULTS: The proportion of naive B cells was higher (p=0.046) in SSc than in controls, with a decreased percentage of memory (p=0.018), especially non-switched memory B cells (p=0.015). The dcSSc patients had a significantly higher frequency of switched memory and DN memory B cells compared to lcSSc patients (p=0.025 and p=0.031). Percentage of CD95+CD27+ memory and CD95+ DN memory B cells was also significantly elevated in dcSSc compared to lcSSc patients (p=0.038 and p=0.045). CONCLUSIONS: We conclude that the decreased proportion of memory B cells in SSc is due to reduction of non- switched memory B cells, resulting in an imbalance between the tolerogenic and activated memory B cell types. Elevated switched and activated CD95+ DN memory B cells may serve as a biomarker for dcSSc and can have a pathogenic potential by cytokine and autoantibody production.

Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona.

Previously we have identified lysenin as a key protein constituent of the secretome from Eisenia fetida coelomocytes and revealed its critical importance in priming interactions between the cells and the protein corona around nanosilver. As alterations of the protein environment can directly affect the corona composition, the extent to which nanoparticles influence the cells' protein secretion profile is of remarkable interest that has rarely acquired attention. Here, we have probed transcriptional responses of E. fetida coelomocytes to the representative nanosilver NM-300K (15 nm) in a time-dependent manner (2, 4, 8 and 24 h at a low-cytotoxic concentration), and examined the implication of the temporal changes in transcriptional profiles of secretory proteins with a particular reference to that of lysenin. NM-300K was accumulated in/at the cells and lysenin was, after transient induction, gradually suppressed over time indicating a negative feedback cycle. This may limit further enrichment of lysenin in the corona and thereby decrease the lysenin-assisted uptake of the nanoparticles. Other differentially expressed genes were those involved in metal stress (likewise in AgNO3-stressed cells) and in Toll-like receptor (TLR) signaling. This offers an intriguing perspective of the nanosilver pathophysiology in earthworms, in which the conserved pattern recognition receptor TLRs may play an effector role.

Induction of apoptosis-like cell death by coelomocyte extracts from Eisenia andrei earthworms.

Earthworm's innate immunity is maintained by cellular and humoral components. Our objective was to characterize the cytotoxicity leading to target cell death caused by earthworm coelomocytes. Coelomocyte lysates induced strong cytotoxicity in tumor cell lines. Transmission electron microscopy revealed cell membrane and intracellular damage in cells treated with coelomocyte lysates. Using TUNEL-assay, within 5 min of incubation we detected DNA fragmentation. Moreover, we found phosphatidylserine translocation in target cell-membranes. Furthermore, we detected dose-dependent Ca(2+) influx and decrease of mitochondrial membrane potential in coelomocyte lysate-treated cells. Interestingly, caspase 3/8 activation was undetectable in exposed tumor cells. One such cytotoxic molecule, lysenin identified in earthworms binds to sphingomyelin and causes target cell lysis in vertebrates. Pretreatment with our anti-lysenin monoclonal antibody rescued the majority but not all target cells from coelomocyte induced death. These data suggest that, not only lysenin but also other factors participate in the caspase-independent apoptosis induced by coelomocytes.

Longitudinal in vivo MR imaging of live earthworms.

Earthworm (Oligochaeta, Lumbricidae) species are used widely in eco-toxicological tests especially with contaminated soils. These long-term tests are reliable, but a high sample size is needed. Magnetic resonance imaging (MRI) can produce fast, robust, sensitive, and longitudinal morphological results using a small sample size. Performing longitudinal in vivo examinations of earthworms using MRI requires the need for anesthetics to completely avoid earthworm's moving. Our goal was to develop a simple and non-invasive method to anesthetize earthworms for in vivo longitudinal imaging studies. We investigated a number of different anesthesia methods and found that propan-2-ol and its vapor was optimal. We used a commercial sequential nanoScan® PET/MRI system (Mediso Ltd, Hungary, Budapest) to explore feasibility of MR imaging in immobilized earthworms. It was possible to visualize via micro MRI the brain, gastrointestinal tract, seminal vesicles, calciferous gland (Morren gland), and main blood vessels of the circulatory system. Our findings show the possibilities to examine changes in morphology using MRI of certain organs using a reversible, long-term immobilization method.

Species differences take shape at nanoparticles: protein corona made of the native repertoire assists cellular interaction.

Cells recognize the biomolecular corona around a nanoparticle, but the biological identity of the complex may be considerably different among various species. This study explores the importance of protein corona composition for nanoparticle recognition by coelomocytes of the earthworm Eisenia fetida using E. fetida coelomic proteins (EfCP) as a native repertoire and fetal bovine serum (FBS) as a non-native reference. We have profiled proteins forming the long-lived corona around silver nanoparticles (75 nm OECD reference materials) and compared the responses of coelomocytes to protein coronas preformed of EfCP or FBS. We find that over time silver nanoparticles can competitively acquire a biological identity native to the cells in situ even in non-native media, and significantly greater cellular accumulation of the nanoparticles was observed with corona complexes preformed of EfCP (p < 0.05). An EfCP-nanoparticle mimicry made with a recombinant protein, lysenin, revealed its critical contribution in the observed cell-nanoparticle response. This confirms the determinant role of the recognizable biological identity during invertebrate in vitro testing of nanoparticles. Our finding shows a case of species-specific formation of biomolecular coronas, and this suggests that the use of representative species may need careful consideration in assessing the risks associated with nanoparticles.

Revising lysenin expression of earthworm coelomocytes.

Lysenin is a species-specific bioactive molecule of Eisenia andrei earthworms. This protein is a potent antimicrobial factor; however its cellular expression and induction against pathogens are still not fully understood. We developed a novel monoclonal antibody against lysenin and applied this molecular tool to characterize its production and antimicrobial function. We demonstrated by flow cytometry and immunocytochemistry that one subgroup of earthworm immune cells (so called coelomocytes), the chloragocytes expressed the highest amount of lysenin. Then, we compared lysenin expression with earlier established coelomocyte (EFCC) markers. In addition, we determined by immunohistology of earthworm tissues that lysenin production is only restricted to free-floating chloragocytes. Moreover, we observed that upon in vitro Staphylococcus aureus but not Escherichia coli challenged coelomocytes over-expressed and then secreted lysenin. These results indicate that among subpopulations of coelomocytes, lysenin is mainly produced by chloragocytes and its expression can be modulated by Gram-positive bacterial exposure.

Cold-stress induced formation of calcium and phosphorous rich chloragocyte granules (chloragosomes) in the earthworm Eisenia fetida.

The cytochemical and functional characteristics of chloragocytes of both 'control' and cold-stressed Eisenia fetida were examined. Flow cytometry revealed the heterogeneity of chloragocytes: the first group was characterized by low, the second one by high acid phosphatase (AcP) content. In 'control' animals the former, in cold-stressed ones the latter type were the dominant form. The elevated AcP-activity correlated with the accumulation of autophagic vacuoles (AVs) in chloragocytes. Both AVs and all small chloragosomes showed high AcP activity, while most of the large chloragosomes did not display any. Most 'control' granules (0.75-1.25 µm) contained high amounts of Ca and P, with less and variable quantities of S, Cl, K, Fe and Zn. Small chloragosomes with low Ca and P concentrations were seldom found. In cold-stressed animals the number of small granules (0.25-0.75 µm) increased up to 40% of total population. Their Ca and P contents were significantly lower; S and Fe concentrations were higher than those of large chloragosomes (1.0-1.5 µm). Our results prove that the formation and elemental composition of chloragosomes can be influenced by environmental stressors and suggest that the mature chloragosomes are tertiary lysosomes and their formation is coupled to autophagocytosis.

Protective effect of the poly(ADP-ribose) polymerase inhibitor PJ34 on mitochondrial depolarization-mediated cell death in hepatocellular carcinoma cells involves attenuation of c-Jun N-terminal kinase-2 and protein kinase B/Akt activation.

BACKGROUND: 2,4-Dimethoxyphenyl-E-4-arylidene-3-isochromanone (IK11) was previously described to induce apoptotic death of A431 tumor cells. In this report, we investigated the molecular action of IK11 in the HepG2 human hepatocellular carcinoma cell line to increase our knowledge of the role of poly (ADP-ribose)-polymerase (PARP), protein kinase B/Akt and mitogen activated protein kinase (MAPK) activation in the survival and death of tumor cells and to highlight the possible role of PARP-inhibitors in co-treatments with different cytotoxic agents in cancer therapy. RESULTS: We found that sublethal concentrations of IK11 prevented proliferation, migration and entry of the cells into their G2 phase. At higher concentrations, IK11 induced reactive oxygen species (ROS) production, mitochondrial membrane depolarization, activation of c-Jun N-terminal kinase 2 (JNK2), and substantial loss of HepG2 cells. ROS production appeared marginal in mediating the cytotoxicity of IK11 since N-acetyl cysteine was unable to prevent it. However, the PARP inhibitor PJ34, although not a ROS scavenger, strongly inhibited both IK11-induced ROS production and cell death. JNK2 activation seemed to be a major mediator of the effect of IK11 since inhibition of JNK resulted in a substantial cytoprotection while inhibitors of the other kinases failed to do so. Inhibition of Akt slightly diminished the effect of IK11, while the JNK and Akt inhibitor and ROS scavenger trans-resveratrol completely protected against it. CONCLUSIONS: These results indicate significant involvement of PARP, a marginal role of ROS and a pro-apoptotic role of Akt in this system, and raise attention to a novel mechanism that should be considered when cancer therapy is augmented with PARP-inhibition, namely the cytoprotection by inhibition of JNK2.

Earthworms and humans in vitro: characterizing evolutionarily conserved stress and immune responses to silver nanoparticles.

Little is known about the potential threats of silver nanoparticles (AgNPs) to ecosystem health, with no detailed report existing on the stress and immune responses of soil invertebrates. Here we use earthworm primary cells, cross-referencing to human cell cultures with a particular emphasis on the conserved biological processes, and provide the first in vitro analysis of molecular and cellular toxicity mechanisms in the earthworm Eisenia fetida exposed to AgNPs (83 ± 22 nm). While we observed a clear difference in cytotoxicity of dissolved silver salt on earthworm coelomocytes and human cells (THP-1 cells, differentiated THP-1 cells and peripheral blood mononuclear cells), the coelomocytes and differentiated (macrophage-like) THP-1 cells showed a similar response to AgNPs. Intracellular accumulation of AgNPs in the coelomocytes, predominantly in a phagocytic population, was evident by several methods including transmission electron microscopy. Molecular signatures of oxidative stress and selected biomarker genes probed in a time-resolved manner suggest early regulation of oxidative stress genes and subsequent alteration of immune signaling processes following the onset of AgNP exposure in the coelomocytes and THP-1 cells. Our findings provide mechanistic clues on cellular innate immunity toward AgNPs that is likely to be evolutionarily conserved across the animal kingdom.