RESUMEN
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.
Asunto(s)
Inmunidad Innata , Oligoquetos/fisiología , Regeneración , Heridas y Lesiones , Animales , Apoptosis , Proliferación Celular , Regulación de la Expresión Génica , Oligoquetos/genética , Oligoquetos/inmunología , Toxinas BiológicasRESUMEN
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.
Asunto(s)
Células T Invariantes Asociadas a Mucosa/metabolismo , Células T Asesinas Naturales/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/sangre , Adulto , Femenino , Citometría de Flujo/métodos , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Células T Invariantes Asociadas a Mucosa/inmunología , Células T Asesinas Naturales/inmunología , Enfermedad Pulmonar Obstructiva Crónica/diagnóstico , Enfermedad Pulmonar Obstructiva Crónica/inmunología , Esputo/inmunología , Esputo/metabolismoRESUMEN
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.
Asunto(s)
Linfocitos B/inmunología , Memoria Inmunológica , Activación de Linfocitos , Esclerodermia Difusa/inmunología , Esclerodermia Sistémica/inmunología , Adulto , Anciano , Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Linfocitos B/metabolismo , Biomarcadores/metabolismo , Estudios de Casos y Controles , Separación Celular/métodos , Femenino , Citometría de Flujo , Humanos , Inmunofenotipificación/métodos , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Fenotipo , Esclerodermia Difusa/sangre , Esclerodermia Difusa/diagnóstico , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/diagnóstico , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Receptor fas/inmunología , Receptor fas/metabolismoRESUMEN
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.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo , Receptor Nicotínico de Acetilcolina alfa 7 , Humanos , Ratas , Animales , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Citocinas/metabolismo , Encéfalo/metabolismo , ARN MensajeroRESUMEN
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.
Asunto(s)
Comunicación Celular , Nanopartículas/química , Oligoquetos/citología , Proteínas/química , Animales , Bovinos , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Humanos , Peso Molecular , Oligoquetos/metabolismo , Proteínas/metabolismo , Plata/química , Especificidad de la Especie , Toxinas Biológicas/químicaRESUMEN
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.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Fenantrenos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Acetilcisteína/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Carcinoma Hepatocelular/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Necrosis , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Interferencia de ARN , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Recently described forkhead box protein 3 (FoxP3) transcription factor is a key molecule in CD4+ CD25hi+ T-cell characterization. Invariant NK T (iNKT) cells are also characterized as regulatory cells modulating the immune response by rapidly producing T(h)1 and T(h)2 cytokines. We aimed to analyze cellular markers important in regulatory features of human iNKT cells and to study their role in functional assays. iNKT cells were single cell sorted from peripheral mononuclear cells of healthy individuals after immunostaining of invariant TCR α-chain. We found FoxP3 expression in human iNKT clones. Randomly selected iNKT cell clones (CD4+, double negative, CD8+) expressed FoxP3 mRNA and protein at different levels upon stimulation as supported by various approaches. FoxP3 mRNA and protein expression was detected in unstimulated iNKT cells as well. Furthermore, different stimulations changed the FoxP3 expression in iNKT cells over time and the most dramatic changes were observed upon anti-CD3 stimulation. Both the supernatant of iNKT cells and iNKT cells themselves exerted similar stimulation effects on PBMC proliferation in functional assays and these stimulations showed a negative correlation with FoxP3 expression. Our data indicate that the FoxP3 expression in iNKT cells may be a key transcriptional factor in controlling the regulatory function of the iNKT cells.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Células T Asesinas Naturales/inmunología , Proliferación Celular , Células Cultivadas , Citocinas/biosíntesis , Epítopos/inmunología , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/inmunología , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Activación de Linfocitos/inmunología , Células T Asesinas Naturales/metabolismo , ARN Mensajero/genética , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
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.
Asunto(s)
Evolución Biológica , Inmunidad/efectos de los fármacos , Nanopartículas del Metal/toxicidad , Oligoquetos/efectos de los fármacos , Oligoquetos/inmunología , Plata/toxicidad , Estrés Fisiológico/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Línea Celular , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunidad/genética , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Nanopartículas del Metal/ultraestructura , Especies Reactivas de Oxígeno/metabolismo , Estrés Fisiológico/genética , Factores de TiempoRESUMEN
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.
Asunto(s)
Vasos Sanguíneos/citología , Calcio/metabolismo , Gránulos Citoplasmáticos/metabolismo , Oligoquetos/metabolismo , Fósforo/metabolismo , Estrés Fisiológico , Fosfatasa Ácida/metabolismo , Animales , Frío , Gránulos Citoplasmáticos/enzimología , Gránulos Citoplasmáticos/ultraestructura , Sistema Digestivo/irrigación sanguínea , Oligoquetos/citología , Oligoquetos/fisiología , Forma de los OrgánulosRESUMEN
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.
RESUMEN
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.
Asunto(s)
Infecciones por Escherichia coli/inmunología , Escherichia coli/fisiología , Macrófagos/fisiología , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/fisiología , Amantadina/farmacología , Animales , Evolución Biológica , Diferenciación Celular , Endocitosis , Humanos , Inmunidad Celular , Inmunidad Innata , Oligoquetos , Células THP-1RESUMEN
Pituitary adenylate cyclase activating polypeptide (PACAP)-like molecules have been shown to be present in cocoon albumin and in Eisenia fetida embryos at an early developmental stage (E1) by immunocytochemistry and radioimmunoassay. Here, we focus on detecting the stage at which PAC1 receptor (PAC1R)-like immunoreactivity first appears in germinal layers and structures, e.g., various parts of the central nervous system (CNS), in developing earthworm embryos. PAC1R-like immunoreactivity was revealed by Western blot and Far Western blot as early as the E2 developmental stage, occurring in the ectoderm and later in specific neurons of the developing CNS. Labeled CNS neurons were first seen in the supraesophageal ganglion (brain) and subsequently in the subesophageal and ventral nerve cord ganglia. Ultrastructurally, PAC1Rs were located mainly on plasma membranes and intracellular membranes, especially on cisternae of the endoplasmic reticulum. Therefore, PACAP-like compounds probably influence the differentiation of germinal layers (at least the ectoderm) and of some neurons and might act as signaling molecules during earthworm embryonic development.
Asunto(s)
Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Oligoquetos/embriología , Oligoquetos/metabolismo , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Ganglios/citología , Ganglios/embriología , Ganglios/metabolismo , Ganglios/ultraestructura , Ratones , Ratones Endogámicos BALB C , Especificidad de Órganos , Hipófisis/metabolismo , Extractos de Tejidos/metabolismoRESUMEN
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.
RESUMEN
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 8402â¯nt-long and potentially composed three non-overlapping open reading frames (ORFs): ORF1a (4449â¯nt/1482aa), ORF1b (1206â¯nt/401aa) and ORF2 (1491â¯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.
Asunto(s)
Aves/virología , Animales , Astroviridae/genética , Genoma Viral , Hepevirus/genética , Hungría , Filogenia , Virus ARN/clasificación , Virus ARN/genética , Virus ARN/aislamiento & purificaciónRESUMEN
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 48â¯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.
Asunto(s)
Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Grampositivas/inmunología , Proteínas del Helminto/genética , Intestinos/fisiología , Oligoquetos/inmunología , Péptidos/genética , Animales , Antiinfecciosos/metabolismo , Clonación Molecular , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas del Helminto/metabolismo , Inmunidad Innata , Oligoquetos/genética , Oligoquetos/microbiología , Péptidos/metabolismo , Filogenia , TranscriptomaRESUMEN
Invariant NKT (iNKT) cells are considered to be important in some autoimmune diseases including Type 1 diabetes mellitus (T1DM). So far, the published data are contradictory in regard to the role of iNKT cells in T1DM. We aimed to study iNKT cell frequency and the function of different iNKT cell subgroups in T1DM. We compared the results of four subject groups: healthy (H), long-term T2DM (ltT2DM; more than 1 year), newly diagnosed T1DM (ndT1DM; less than 3 months), and ltT1DM (more than 1 year) individuals. We measured the iNKT cell frequencies by costaining for the invariant TCR alpha-chain with 6B11-FITC and Valpha24-PE. After sorting the Valpha24+6B11+ cells, the generated iNKT clones were characterized. We tested CD4, CD8, and CD161 expression and IL-4 and IFN-gamma production on TCR stimulation. The CD4+ population among the iNKT cells was decreased significantly in ltT1DM versus ndT1DM, ltT2DM, or H individuals. The T1DM iNKT cell cytokine profile markedly shifted to the Th1 direction. There was no difference in the frequency of iNKT cells in PBMC among the different patient groups. The decrease in the CD4+ population among the iNKT cells and their Th1 shift indicates dysfunction of these potentially important regulatory cells in T1DM.
Asunto(s)
Antígenos CD4/biosíntesis , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Células Asesinas Naturales/inmunología , Células TH1/inmunología , Adulto , Antígenos de Superficie/biosíntesis , Antígenos CD8/biosíntesis , Recuento de Células , Células Cultivadas , Células Clonales , Femenino , Humanos , Interferón gamma/biosíntesis , Interleucina-4/biosíntesis , Células Asesinas Naturales/clasificación , Lectinas Tipo C/biosíntesis , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Subfamilia B de Receptores Similares a Lectina de Células NK , Receptores de Antígenos de Linfocitos T/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Picobirnaviruses (PBVs) of family Picobirnaviridae have bisegmented (S1 and S2 segments), double-stranded RNA genomes. In this study a total of Nâ¯=â¯12 complete chicken PBVs (ChPBV) segments (Nâ¯=â¯5 of S1 and Nâ¯=â¯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.
Asunto(s)
Pollos/virología , Cloaca/virología , Diarrea/veterinaria , Picobirnavirus/aislamiento & purificación , Enfermedades de las Aves de Corral/virología , Ribosomas , Animales , Clonación Molecular , Diarrea/virología , Filogenia , Picobirnavirus/metabolismo , Unión Proteica , ARN Viral/genéticaRESUMEN
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.
Asunto(s)
Oligoquetos/efectos de los fármacos , Ácido Selénico/toxicidad , Ácido Selenioso/toxicidad , Contaminantes del Suelo/toxicidad , Animales , Peroxidación de Lípido/efectos de los fármacos , Oligoquetos/enzimología , Oligoquetos/metabolismo , Estrés Oxidativo/efectos de los fármacos , Suelo/químicaRESUMEN
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.
Asunto(s)
Nanopartículas del Metal/toxicidad , Oligoquetos/efectos de los fármacos , Oligoquetos/genética , Corona de Proteínas/metabolismo , Proteínas/genética , Proteínas/metabolismo , Plata/toxicidad , Animales , Perfilación de la Expresión Génica , Nanopartículas del Metal/química , Oligoquetos/metabolismo , Plata/química , Receptores Toll-Like/metabolismo , Toxinas Biológicas/metabolismo , Transcriptoma/efectos de los fármacosRESUMEN
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.