The impact of epigenetic mechanisms on molecular and cellular processes of regeneration remains a less-investigated field, especially concerning earthworm segment restoration. To evaluate distinct methylated cytosines under different conditions, earthworm segments were collected: decitabine-treated and controls; anterior and posterior amputation; intact and regenerated (2- and 4-week). 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) distribution in tissues was assessed by immunohistochemistry (IHC), and in genomic DNA by dot blot. DNA-methyltransferase (DNMT) and ten-eleven translocation (TET) dioxygenase activity was determined by immuno-based colorimetry. DNMT1 and TET gene expressions were verified with real-time PCR. Decitabine treatment reduced 5mC levels in most tissues, persisting in the anterior coelomic cavity (intact and blastemas). 5hmC remained elevated in most tissues, remarkably in 2-week blastemas. In the same period, DNMT and TET presented the highest activity in anterior treated samples compared to other treated periods. Concurrently, DNMT1 gene expression increased prominently in anterior segments, while TET showed the highest expression in both anterior and posterior 2-week treated groups. Thus far, our observations indicate that, despite decitabine’s effect on the DNA methylation machinery of the earthworm model, the region of amputation and the regeneration period also interfere with activity and expression of epigenetic enzymes, affecting 5mC and 5hmC distribution.

BackgroundNeuroinflammation and systemic immune dysregulation are increasingly recognized as key contributors to secondary brain injury after aneurysmal subarachnoid hemorrhage (aSAH). However, the temporal relationship between circulating cytokine responses and innate-like lymphocyte populations, particularly mucosal-associated invariant T (MAIT) cells, remains poorly characterized.MethodsIn this prospective observational study, peripheral blood cytokine profiles were longitudinally analyzed in 48 patients with aSAH on days 1, 5, and 9 after ictus and compared with healthy controls. Serum concentrations of IL-6, IL-7, IL-12p40, IL-12p70, IL-15, IL-17A, IL-18, IL-22, IP-10, and CXCL-9 were measured using multiplex immunoassay. Flow cytometric immunophenotyping was performed in a subgroup of 16 patients to evaluate MAIT cells, NK cells, γδ T cells, iNKT cells, and NKT-like cells. Associations between immune parameters and 3-month functional outcome were also assessed.ResultsPatients with aSAH demonstrated persistently elevated IL-6, IL-18, and IL-15 levels across all examined time points, while IL-7, IL-12p40, IP-10, and CXCL-9 showed delayed increases during the subacute phase. IL-6 and IL-15 concentrations were significantly higher in patients with unfavorable outcome, whereas IL-22 levels were increased in patients with favorable recovery. However, these associations were not independent of disease severity after adjustment for WFNS score and age. Flow cytometry revealed a persistent reduction in circulating MAIT-cell frequencies following aSAH, while other innate-like lymphocyte populations remained largely unchanged. DN MAIT-cell frequencies demonstrated significant negative correlations with circulating IL-18 levels across all examined time points.ConclusionaSAH is associated with sustained systemic inflammatory activation and selective alterations of MAIT-cell populations. Although several cytokines were associated with functional outcome in univariate analyses, these associations were attenuated after adjustment for WFNS score and age, suggesting that they primarily reflect disease severity rather than independent prognostic effects. The observed relationship between MAIT-cell dysregulation and IL-18–associated inflammation provides further evidence of altered innate-like immune responses following aSAH.

Earthworm coelomocytes (e.g. amoebocytes and eleocytes) participate in diverse physiological processes from nutrition storage to immune defense. While the morphology and functional attributes of adult coelomocytes have been extensively studied, their origin and ontogeny are largely unexplored during earthworm development. To uncover this knowledge gap, we examined the emergence, distribution, and molecular characteristics of amoebocytes vs. eleocytes during embryogenesis (E1-E4 stages) of Eisenia andrei earthworms. Applying light-microscopy and flow cytometry, we observed a sequential manifestation of coelomocyte subsets: amoebocytes predominated in E3 stage, whereas eleocytes became more abundant in E4; although amoebocytes remain prevalent overall. In parallel, the expression dynamics of key earthworm immune-related genes evidenced characteristic differences. The transcripts of evolutionarily conserved TLR and MyD88 displayed constitutive expression throughout all embryonic stages (E1-E4). Conversely, LBP/BPI gene expression decreased, while CCF mRNA level increased during Eisenia embryogenesis. Moreover, antimicrobial effectors (lysenin and lysozyme) were progressively increased from E1 to E4 stages. Our novel experimental data shed more light at molecular and cellular levels into the development and maturation of immune cell populations during earthworm embryogenesis.

Sindbis virus (SINV) is a mosquito-borne alphavirus capable of causing neurological and immunological symptoms in humans, yet its effects on neural/immune systems remain insufficiently characterized. This study aimed to examine SINV replication, UV-C light inactivation, apoptosis induction, and immune gene modulation in human SH-SY5Y neuroblastoma cells. Following viral adaptation and infectious dose determination, SINV replication and inactivation were assessed using RT-qPCR and dsRNA immunofluorescence. Apoptotic markers (caspase-3, Bax, Bcl-2) were analyzed by immunofluorescence and immune genes expression kinetics (TLR3/7, RIGI, MDA5, IL-1β, IL-6, TNFα, IL-10, IFNβ and β-catenin) were measured at defined time points post-infection by RT-qPCR. SH-SY5Y cells supported productive SINV infection, with viral RNA detectable as early as 3 hpi and marked cytopathic effects by 24 hpi. A custom-built UV-C chamber achieved complete viral inactivation following 3 × 30 s exposures. We observed SINV time-course replication and UV-C inactivation with conspicuous morphological alterations in SH-SY5Y cells. Furthermore, SINV triggered caspase-dependent apoptosis and robust transcriptional upregulation of innate immune genes, peaking between 12–16 hpi and declining by 30 hpi. These findings elucidate the temporal dynamics of SINV replication, cell death mechanisms, and immune activation in a neuronal context, contributing to a better understanding of SINV neuropathogenesis.

BACKGROUND:Combination treatments based on pharmacological interactions at α7 nicotinic acetylcholine receptors (nAChRs) are promising therapeutic approaches for neurocognitive disorders. METHODS:Here, we tested the cognitive efficacy of combinations of memantine with an α7 nAChR-selective agonist (PHA-543613) in naturally aged rats. Age-related changes in the expression of some key genes and proteins were also measured using quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). RESULTS:Aged rats showed marked cognitive decline in the novel object recognition test, and they also exhibited cholinergic changes such as mRNA upregulation of α7 nAChRs. Upregulation of interleukin-1β, macrophage inflammatory protein 1α, CX3CL1, intercellular adhesion molecule 1, and ciliary neurotrophic factor mRNA was also detected in aged rats. Combination treatment of memantine and PHA-543613 successfully alleviated the age-related decline of recognition memory of rats by exceeding the effects of the corresponding monotreatments. CONCLUSIONS:Results indicate a positive interaction between memantine and PHA-543613, which also reflects a putative role of α7 nAChRs in the cognitive enhancer effects of memantine. These findings may facilitate the development of combination therapies for age-related neurocognitive disorders.

Evolutionarily, Wnt/(3-catenin signaling is well-conserved and supports several key cell-biological processes (e.g. adhesion and proliferation). Its crucial component, (3-catenin, has been described in several organisms, however, its identification and characterization are notably lacking in annelid earthworms. Here, we report a novel (3-catenin homologue from the earthworm Eisenia andrei, termed Ea-(3-catenin. The fulllength 3253 nt Ea-13-catenin mRNA includes an open reading frame of 2499 nt encoding a putative protein with 833 amino acid residues that comprise 11 classical armadillo-repeat regions. Phylogenetic analysis indicates that Ea-(3-catenin shows strong homology with Lophotrochozoan (3-catenins. Ubiquitous, but variable expressions of Ea-(3-catenin were observed in distinct earthworm tissues. During embryogenesis, Ea-13-catenin mRNA gradually increased from the E1 to E4 developmental stages. Regeneration experiments revealed an inverse correlation between Ea-13-catenin mRNA levels and the rate of EdU+/PY489-(3-catenin+ proliferating cells during the second week of the posterior blastema formation. In vitro exposures to poly(I:C) and zymosan significantly increased Ea13-catenin mRNA levels, while small molecule Wnt-pathway modulators such as LiCl or iCRT14 increased or decreased Ea-13-catenin mRNA expression, and nuclear translocation of PY489-(3-catenin, respectively. These novel results pave the way for follow-up studies aimed at characterizing additional members of the Wnt/ (3-catenin pathway that may be involved in embryonic and/or postembryonic development, as well as innate immunity in earthworms.

Distinct gene expression patterns are important to various biological functions, spanning developmental processes, wound healing, and the restoration of body parts. Moreover, immunity intertwines with these processes, as researchers propose links between immune system evolution and the variable regenerative capacities seen across different organisms. Concomitantly, elements that influence gene expression can also affect regeneration, since DNA methylation is a key epigenetic mechanism that emerges as a critical regulator of cellular fate and behavior. While various studies propose methodologies for detecting and quantifying DNA methylation under diverse experimental settings, its interaction with regeneration remains relatively unexplored, particularly in annelids. This review aims to address this gap through exploring the connections between immunity, regeneration, and epigenetics by compiling information from studies conducted in different organisms and focusing on annelids as regenerative models. Additionally, it also provides an overview of protocols applying monoclonal antibodies to target specific DNA methylation forms.

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.

Combination treatments based on pharmacological interactions at α7 nAChRs are promising therapeutic approaches for neurocognitive disorders (NCDs). Memantine, an already approved medication in some NCDs, may not only act as an antagonist of the glutamatergic NMDA receptors (NMDAR) but also serve as an antagonist of the cholinergic α7 nAChRs. Here we set out to utilize a combination treatment regime with an α7 nAChR-selective agonist (PHA-543613) and a novel proprietary α7 nAChR ligand with marked positive allosteric modulator (PAM) activity (CompoundX). The cognitive efficacy of combination treatments was tested in a naturally aged rat model. Naturally aged rats showed marked cognitive decline in the novel object recognition (NOR) test, and they displayed pathological changes at the molecular level in terms of various inflammatory markers. In addition, aged rats also exhibited cholinergic changes such as mRNA upregulation of α7 nAChRs. Memantine-PHA-543613 and memantine-CompoundX combination treatments successfully alleviated the age-related decline of recognition memory of rats by exceeding the null-effects of the corresponding subtherapeutic levels of monotreatments. Results indicate a positive interaction between memantine and α7-nAChR agonists and PAMs, which reflects a prominent role of α7 nAChRs in the cognitive enhancement of combination treatments especially in age-related cognitive decline. Moreover, the putative direct action of memantine on α7 nAChRs may also contribute to the observed synergistic interaction between memantine and other selective α7 nAChR compounds.

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.

A T1DM-et az inzulint termelő hasnyálmirigy β-sejtjeinek T-sejt-függő pusztulása jellemzi. A feltételezett célantigének száma továbbra is növekszik, de nincs egyetértés az elsődleges antigén természetével kapcsolatban. Legnagyobb valószínűséggel ez az antigén maga az inzulin vagy a proinzulin. A teljes hosszúságú inzulin nagy dózisban nem alkalmazható intervenciós vizsgálatokban, annak metabolikus hatása miatt, ezért a pontos epitóp azonosítása elengedhetetlen. Korábban a hasnyálmirigyet körülvevő nyirokcsomókból izoláltak és oligoklonálisan tenyésztettek T-sejteket, amelyek felismerték az inzulin A-láncának 1–15. aminosavát. A diabetesspecifikus autoantigéneket azonosító publikációk száma nagyon korlátozott. Ennek ellenére több klinikai vizsgálat során számos különféle antigént alkalmaztak a β-sejtek pusztulásának lassítása vagy blokkolása céljából. Feltételeztük, hogy újonnan diagnosztizált T1DM-es betegek perifériás véréből ezen auto­reaktív T-sejtek azonosíthatóak. Célul tűztük ki továbbá, hogy meghatározzuk ezen sejtek antigénspecificitását.

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.

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.

Two closely-related earthworm species (Eisenia spp.) have long been used as model organisms in ecotoxicology. The same nanoparticles (NPs) may affect the two species differently, not only because of the inherent differences in susceptibility but also due to how the immune system could recognize NPs. In a comparative approach using E. andrei and E. fetida, we study various immune-related parameters of earthworm coelomocytes following in vitro exposure to 10 nm NPs (silver, Ag; and gold, Au) or dissolved Ag (AgNO3). In general, E. fetida coelomocytes were more susceptible to AgNPs and AgNO3 while AuNPs did not show cytotoxicity. At the sub-cellular level, AgNPs similarly affected cellular redox reactions in both species; however, E. fetida showed greater responses for apoptosis-related endpoints. At the molecular level, AgNPs (at 24 h LC20) induced a significantly high level of superoxide dismutase in E. andrei coelomocytes while E. fetida was additionally characterized by consistent induction of metallothionein and differential capacity for redox/metal regulation. Although AuNPs were not cytotoxic, both NP types (Ag and Au) seemed to alter the expression pattern of immune-related genes (toll-like receptor and lysenin) in both species, but more clearly in E. fetida. We further observed that lysenin proteins, while secreted differentially between the two species, bind only to AgNPs resulting in negative secretion feedback. Our findings support the general preference for E. fetida in ecotoxicology, and reveal the potential roles of protective and immune mechanisms optimized for each species in its own ecological niche.

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.

Abstract Innate immune processes arose from specific host–pathogen coevolutive interactions and the ability to recognise self and nonself structures is extremely conserved from invertebrates to vertebrates. In this context, phagocytes represent the most ancient defensive line against foreign materials, already present in earliest protostomes. Moreover, although more complex functions evolved in vertebrates, both cellular and humoral innate immune mechanisms are extremely conserved, leading to identify some forms of innate immune memory also in invertebrates. Key Concepts Phagocytosis represents the first mechanism of defence in metazoans. Immune system has evolved to discriminate self and nonself structures. Immune mechanisms present a high level of conservation throughout phyla . Vertebrate innate immune memory derives from ancient forms and mechanisms already present in invertebrates. Alternative animal models are crucial for investigating the molecular basis of the immune processes.

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.

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.

During phylogenesis different types of immunocytes such as amoebocytes and eleocytes have developed in earthworms to defend the host against microbial pathogens. Previously we applied a cell sorting-based approach to untangle the morphological and functional properties of these aforementioned coelomocyte subsets. In order to compare their constitutive gene expression patterns, cell-sorting was performed and followed by semiquantitative RT-PCR in the distinct, separated coelomocyte subpopulations of unmanipulated Eisenia andrei earthworms. We targeted a variety of genes with diverse functions ranging from pattern recognition through intracellular signaling to oxidative stress. Several immune-related genes (CCF, TLR, lumbricin, LuRP, MyD88) were only manifested in the amoebocytes. In contrast, other immune response genes (lysozyme, lysenin), lysosomal hydrolases (cathepsin L and cathepsin C) and cystatin B were expressed in both subpopulations. In addition, cell signaling molecules (MyD88, PKC1) and oxidative stress-related genes (Cu/ZnSOD, MnSOD) were mainly observed in amoebocytes, while other stress-related genes (Cd-metallothionein, catalase) were apparent in both subsets. We conclude that these characteristic differences of the molecular signatures manifest in the functional heterogeneity of distinct coelomocyte subtypes.