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BACKGROUND: The molecular basis of the D variant phenotype in the Chinese differs greatly from that of the Caucasian. Adapting a specific D typing strategy to the spectrum of prevalent RHD variant alleles is necessary. STUDY DESIGN AND METHODS: Blood samples with ambiguous D phenotypes were collected in the Southern Chinese population. A special three-step typing strategy was applied. First, the common DVI type 3 was identified from epitope profiles of D antigen. Then, another common weak D type 15 (RHD*845A) was identified by epitope profiles of D antigen and Sanger sequencing of RHD exon 6. Finally, the remaining D variants were genotyped mainly by Sanger sequencing. For the novel RHD alleles in the coding region and exon-intron junction, in vitro transfection and minigene splicing assays were performed, respectively. The anti-D investigation was performed. RESULTS: DVI type 3 (65/253, 25.7%) and weak D type 15 (62/253, 24.5%) were common Chinese D variants, and RHD*960A, DFR, RHD*weak D type 25, 72, and 136 were frequent variant RHD alleles. Besides, twenty-two sporadic and seven novel RHD alleles (RHD*188A; RHD*688C; RHD*782 T; RHD*1181C; RHD*165 T, 993A; RHD*148 + 3G > T and RHD*1227 + 5G > C) were identified. The deleterious effect of the novel RHD alleles on D antigen or mRNA expression was confirmed. Anti-D was detected in two DVI type 3 pregnant women. DISCUSSION: The three-step typing strategy provides an effective approach for Chinese D variant typing. It can be anticipated that commercially available RHD genotyping kits have limitations for testing Chinese D variants, as some of the frequent variants are not interrogated.
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População do Leste Asiático , Sistema do Grupo Sanguíneo Rh-Hr , Gravidez , Feminino , Humanos , Alelos , Genótipo , Fenótipo , Epitopos , Sistema do Grupo Sanguíneo Rh-Hr/genéticaRESUMO
Hemochromatosis type 4, or ferroportin disease, is considered as the second leading cause of primary iron overload after HFE-related hemochromatosis. The disease, which is predominantly associated with missense variations in the SLC40A1 gene, is characterized by wide clinical heterogeneity. We tested the possibility that some of the reported missense mutations, despite their positions within exons, cause splicing defects. Fifty-eight genetic variants were selected from the literature based on two criteria: a precise description of the nucleotide change and individual evidence of iron overload. The selected variants were investigated by different in silico prediction tools and prioritized for midigene splicing assays. Of the 15 variations tested in vitro, only two were associated with splicing changes. We confirm that the c.1402G>A transition (p.Gly468Ser) disrupts the exon 7 donor site, leading to the use of an exonic cryptic splicing site and the generation of a truncated reading frame. We observed, for the first time, that the p.Gly468Ser substitution has no effect on the ferroportin iron export function. We demonstrate alternative splicing of exon 5 in different cell lines and show that the c.430A>G (p.Asn144Asp) variant promotes exon 5 inclusion. This could be part of a gain-of-function mechanism. We conclude that splicing mutations rarely contribute to hemochromatosis type 4 phenotypes. An in-depth investigation of exon 5 auxiliary splicing sequences may help to elucidate the mechanism by which splicing regulatory proteins regulate the production of the full length SLC40A1 transcript and to clarify its physiological importance.
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Processamento Alternativo , Proteínas de Transporte de Cátions/deficiência , Hemocromatose/genética , Mutação de Sentido Incorreto , Proteínas de Transporte de Cátions/genética , Éxons , Genômica , Células Hep G2 , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Although D variant phenotype is known to be due to genetic defects, including rare missense single nucleotide variants (SNVs), within the RHD gene, few studies have addressed the molecular and cellular mechanisms driving this altered expression. We and others showed previously that splicing is commonly disrupted by SNVs in constitutive splice sites and their vicinity. We thus sought to investigate whether rare missense SNVs located in "deep" exonic regions could also impair this mechanism. STUDY DESIGN AND METHODS: Forty-six missense SNVs reported within exons 6 and 7 were first selected from the Human RhesusBase. Their respective effect on splicing was assessed by using an in vitro assay. An RhD-negative cell model was further generated by using the CRISPR-Cas9 approach. RhD-mutated proteins were overexpressed in the newly created model, and cell membrane expression of the D antigen was measured by flow cytometry. RESULTS: Minigene splicing assay showed that 14 of 46 (30.4%) missense SNVs alter splicing. Very interestingly, further investigation of two missense SNVs, which both affect codon 338 and confer a weak D phenotype, showed various mechanisms: c.1012C>G (p.Leu338Val) disrupts splicing only, while c.1013T>C (p.Leu338Pro) alters only the protein structure, in agreement with in silico prediction tools and 3D protein structure visualization. CONCLUSION: Our functional data set suggests that missense SNVs damage quantitatively D antigen expression by, at least, two different mechanisms (splicing alteration and protein destabilization) that may act independently. These data thereby contribute to extend the current knowledge of the molecular mechanisms governing weakened D expression.
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Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único , Sistema do Grupo Sanguíneo Rh-Hr/genética , Expressão Gênica , Humanos , Células K562 , Modelos Moleculares , Splicing de RNA , Sistema do Grupo Sanguíneo Rh-Hr/químicaRESUMO
BACKGROUND: In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. MATERIALS AND METHODS: After long-range PCR amplification spanning the whole ACKR1 gene locus (â¼2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. RESULTS: High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. DISCUSSION: Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a "longitudinal" overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.
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The Brown Ring Disease is an infection caused by the bacterium Vibrio tapetis on the Manila clam Ruditapes philippinarum. The process of infection, in the extrapallial fluids (EPFs) of clams, involves alteration of immune functions, in particular on hemocytes which are the cells responsible of phagocytosis. Disorganization of the actin-cytoskeleton in infected clams is a part of what leads to this alteration. This study is the first transcriptomic approach based on collection of extrapallial fluids on living animals experimentally infected by V. tapetis. We performed differential gene expression analysis of EPFs in two experimental treatments (healthy-against infected-clams by V. tapetis), and showed the deregulation of 135 genes. In infected clams, a downregulation of transcripts implied in immune functions (lysosomal activity and complement- and lectin-dependent PRR pathways) was observed during infection. We also showed a deregulation of transcripts encoding proteins involved in the actin cytoskeleton organization such as an overexpression of ß12-Thymosin (which is an actin sequestration protein) or a downregulation of proteins that closely interact with capping proteins such as Coactosin, that counteract action of capping proteins, or Profilin. We validated these transcriptomic results by cellular physiological analyses that showed a decrease of the lysosome amounts and the disorganization of actin cytoskeleton in infected hemocytes.
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Bivalves/imunologia , Citoesqueleto/microbiologia , Imunidade Inata/genética , Transcriptoma/imunologia , Vibrio/fisiologia , Animais , Bivalves/genética , Perfilação da Expressão GênicaRESUMO
Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three-dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)-supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%-increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two-fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and "stemness." These features will be further tested within three-dimensional scaffolds.
Assuntos
Substitutos Sanguíneos/farmacologia , Técnicas de Cultura de Células/métodos , Células Cultivadas/metabolismo , Hemoglobinas/farmacologia , Células-Tronco Mesenquimais/metabolismo , Oxigênio/metabolismo , Organismos Aquáticos , Técnicas de Cultura Celular por Lotes/métodos , Plaquetas/química , Células da Medula Óssea , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Meios de Cultura/química , Citometria de Fluxo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Engenharia Tecidual/métodosRESUMO
Manila clams, Venerupis philippinarum (Adams and Reeve, 1850), were experimentally infected with two different bacterial strains and challenged with two different temperatures. Bacterial strains used in this study were Vibrio tapetis strain CECT4600(T), the causative agent of Brown Ring Disease (BRD) and V. tapetis strain LP2, supposed less virulent to V. philippinarum. V. tapetis is considered to proliferate at low temperatures, i.e. under 21 °C. In a global warming context we could hypothesize a decrease of mass mortalities caused by V. tapetis but these thermal changes could also directly impact the immune system of the host V. philippinarum. Thus, the aim of this study was to investigate the effects of the extrapallial injection with V. tapetis combined with temperature challenge on two enzymes activities in V. philippinarum. More precisely, after infection, phenoloxidase (PO) and superoxide dismutase (SOD), two major enzymes involved in immune response, were studied for 30 days in two compartments: the mantle and the hemolymph. Conchyolin Deposit Stages (CDS) and Shell Repair Stages (SRS) were also determined 30 days post-injection as a proxy of the virulence of the tested strains. In this study, we highlighted that host-pathogen interaction in a varying environment affects the enzymatic response of the host. The coupled effect of V. tapetis injection and temperature challenge was detected 30 days post injection and resulted in virulence differences. These findings were supported by CDS and SRS determination in clams and lead to the conclusion that clam's immunity could be enhanced at 22 °C while V. tapetis virulence is lowered at this temperature. Another result of our study was the increase of PO and SOD basal activities as clams are exposed to warmer temperature.
Assuntos
Bivalves , Imunidade Inata , Monofenol Mono-Oxigenase/imunologia , Superóxido Dismutase/imunologia , Vibrio/fisiologia , Animais , Bivalves/enzimologia , Bivalves/imunologia , Bivalves/microbiologia , Temperatura , Vibrio/genética , Vibrio/imunologiaRESUMO
Manila clams, Venerupis philippinarum (Adams and Reeve, 1850), were experimentally challenged with two Vibrio tapetis strains: CECT4600T, the causative agent of Brown Ring Disease (BRD); and LP2 supposedly non-pathogenic in V. philippinarum. Changes in phenoloxidase (PO) and superoxide dismutase (SOD), two major enzymes involved in immunity, were studied in two tissues, the mantle and hemolymph for 30 days after infection in the extrapallial cavity. Bacterial infection in V. philippinarum resulted in modulation of PO and SOD activities that was both tissue- and time-dependent. A response at early times was detected in the mantle and was associated with significant increases in PO and SOD activities in LP2- and CECT4600T-challenged clams 36 h post injection. This first response in the mantle could be explained by the proximity to the injection region (extrapallial cavity). In the hemolymph the response occurred at later times and was associated with an increase in PO activity and a decrease in SOD activity. As hemolymph is a circulating fluid, this response delay could be due to an "integration time" needed by the organism to counteract the infection. Injections also impacted PO and SOD activities in both tissues and confirmed a difference in pathogenicity between the two V. tapetis strains.
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Bivalves/enzimologia , Bivalves/imunologia , Bivalves/microbiologia , Monofenol Mono-Oxigenase/imunologia , Superóxido Dismutase/imunologia , Vibrio/imunologia , Animais , Proteínas de Artrópodes , Hemolinfa/imunologia , Oligopeptídeos , Fatores de Tempo , Vibrio/patogenicidadeRESUMO
BACKGROUND: Alzheimer's disease and related disorders are characterized by cognitive impairment associated with behavioral and psychological symptoms of dementia. These symptoms have significant consequences for both the patient and his family environment. While risk factors for behavioral disorders have been identified in several studies, few studies have focused on the evolution of these disorders. Moreover, it is important to identify factors linked to the long-term evolution of behavioral disorders, as well as patients' and caregivers' quality of life. Our purpose is to present the methodology of the EVITAL study, which primary objective is to determine the factors associated with the evolution of behavioral disorders among patients with Alzheimer's disease and related disorders during the year following their hospitalization in cognitive and behavioral units. Secondary objectives were 1) to assess the factors related to the evolution of behavioral disorders during hospitalization in cognitive and behavioral units; 2) to identify the factors linked to patients' and caregivers' quality of life, as well as caregivers' burden; 3) to assess the factors associated with rehospitalization of the patients for behavioral disorders in the year following their hospitalization in cognitive and behavioral units. METHOD/DESIGN: A multicenter, prospective cohort of patients with Alzheimer's disease and related disorders as well as behavioral disorders who are hospitalized in cognitive and behavioral units. The patients will be included in the study for a period of 24 months and followed-up for 12 months. Socio-demographic and environmental data, behavioral disorders, medications, patients and caregivers quality of life as well as caregivers burden will be assessed throughout hospitalization in cognitive and behavioral units. Follow-up will be performed at months 3, 6 and 12 after hospitalization. Socio-demographic and environmental data, behavioral disorders, medications, patients and caregivers quality of life, unplanned rehospitalization as well as caregivers burden will also be assessed at each follow-up interview. DISCUSSION: The present study should help better identify the factors associated with reduction or stabilization of the behavioral and psychological symptoms of dementia in patients with Alzheimer's disease. It could therefore help clinicians to better manage these symptoms. TRIAL REGISTRATION: Clinical Trials NCT01901263. Registered July 9, 2013.
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Doença de Alzheimer/diagnóstico , Doença de Alzheimer/psicologia , Readmissão do Paciente/estatística & dados numéricos , Qualidade de Vida/psicologia , Doença de Alzheimer/tratamento farmacológico , Cuidadores/psicologia , Estudos de Coortes , Efeitos Psicossociais da Doença , Progressão da Doença , Feminino , Seguimentos , França , Hospitalização , Humanos , Entrevistas como Assunto/métodos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Psicotrópicos/uso terapêutico , Projetos de Pesquisa , Fatores de RiscoRESUMO
The aim of this study was to assess 1) the satisfaction of caregivers of patients with Alzheimer's disease or related diseases regarding a new collective support intervention called "Forum Passerelle"(Gateway forum), and 2) to assess the impact of "Forum Passerelle" on the implementation of services and support for patients or caregivers. This is an observational study carried out on the basis of 3 "Forum Passerelle" (between October 2018 and February 2020). Following each "Forum Passerelle", the caregivers' satisfaction was assessed and the number of services and support was counted. In total, 78 caregivers took part in these first "Forum passerelle". The satisfaction survey showed that 98% of caregivers were satisfied with the topics covered by "Forum Passerelle". The forum fully met the expectations of 61% of caregivers and partially for 39%. Among the caregivers contacted 3 or 6 months after the forum, 68% had set up a new service/support (85 new services/support for the sick relatives and 32 for caregivers) and 59% declared that these changes had taken place thanks to the "Forum Passerelle". To conclude, the first 3 "Forums Passerelle" were received very favorably by caregivers and contributed to the implementation of new services to optimize home support for the sick relative.
Assuntos
Doença de Alzheimer/terapia , Cuidadores , Cuidadores/educação , Cuidadores/psicologia , Família , Humanos , Inquéritos e QuestionáriosRESUMO
Tissue engineering strategies have been developed to optimize osseointegration in dental implant surgery. One of the major problems is the non-homogeneous spatial cell distribution in the scaffold, as well as subsequent matrix production. Insufficient nutrient and oxygen supplies inside the scaffold are factors in this phenomenon. To mediate this gradient formation, we have implemented a perfusion culture method to seed human bone marrow mesenchymal stem cells (MSCs) into three-dimensional (3-D)-allogenic bone scaffolds in combination with a marine haemoglobin, HEMOXCell®, for oxygen delivery. Cell culture was performed under static and perfusion conditions, with standard and osteogenic media, with and without HEMOXCell®. The cell seeding efficiency, as well as MSC/scaffold cytocompatibly were assessed using viability and proliferation assays. Scaffolds' cellularization and extracellular matrix (ECM) formation were analyzed using scanning electron microscopy and histological staining. Cell differentiation was investigated with osteogenic biomarkers gene expression analysis. The perfusion culture was observed to significantly promote MSC proliferation and differentiation throughout the scaffolds, especially when using the induction medium w/HEMOXCell®. Our data suggest that perfusion culture of MSC into allogenic bone substitute with HEMOXCell® as a natural oxygen carrier is promising for tissue engineering applications to oxygenate hypoxic areas and to promote cellular proliferation.
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Materiais Biocompatíveis/farmacologia , Substitutos Ósseos/farmacologia , Adesão Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Substitutos Ósseos/química , Diferenciação Celular/efeitos dos fármacos , Hemoglobinas/química , Hemoglobinas/metabolismo , Humanos , Oxigênio/metabolismo , PerfusãoRESUMO
We investigated the effect of brown ring disease (BRD) development and algal diet on energy reserves and activity of enzymes related to energy metabolism, antioxidant system and immunity in Manila clam, Ruditapes philippinarum. We found that algal diet did not impact the metabolic response of clams exposed to Vibrio tapetis. At two days post-injection (dpi), activities of superoxide dismutase and glutathione peroxidase (GPx) decreased whereas activities of nitric oxide synthase (iNOS) and catalase increased in infected clams, although no clinical signs were visible (BRD-). At 7 dpi, activities of several antioxidant and immune-related enzymes were markedly increased in BRD-likely indicating an efficient reactive oxygen species (ROS) scavenging compared to animals which developed clinical signs of BRD (BRD+). Therefore, resistance to BRD clinical signs appearance was associated with higher detoxification of ROS and enhancement of immune response. This study provides new biochemical indicators of disease resistance and a more comprehensive view of the global antioxidant response of clam to BRD development.
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Bivalves/metabolismo , Vibrio/fisiologia , Animais , Benzamidas/metabolismo , Bivalves/imunologia , Bivalves/microbiologia , Catalase/metabolismo , Dieta , Glutationa Peroxidase/metabolismo , Interações Hospedeiro-Patógeno , Imunidade Inata , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Compostos Organosselênicos/metabolismo , Superóxido Dismutase/metabolismoRESUMO
The Gram-negative bacterium Vibrio tapetis is known as the causative agent of Brown Ring Disease (BRD) in the Manila clam Venerupis (=Ruditapes) philippinarum. This bivalve is the second most important species produced in aquaculture and has a high commercial value. In spite of the development of several molecular methods, no survey has been yet achieved to rapidly quantify the bacterium in the clam. In this study, we developed a Taqman real-time PCR assay targeting virB4 gene for accurate and quantitative identification of V. tapetis strains pathogenic to clams. Sensitivity and reproducibility of the method were assessed using either filtered sea water or extrapallial fluids of clam injected with the CECT4600(T) V. tapetis strain. Quantification curves of V. tapetis strain seeded in filtered seawater (FSW) or extrapallial fluids (EF) samples were equivalent showing reliable qPCR efficacies. With this protocol, we were able to specifically detect V. tapetis strains down to 1.125 10(1) bacteria per mL of EF or FSW, taking into account the dilution factor used for appropriate template DNA preparation. This qPCR assay allowed us to monitor V. tapetis load both experimentally or naturally infected Manila clams. This technique will be particularly useful for monitoring the kinetics of massive infections by V. tapetis and for designing appropriate control measures for aquaculture purposes.
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Brest harbor (Bay of Brest, Brittany, France) has a severe past of anthropogenic chemical contamination, but inputs tended to decrease, indicating a reassessment of its ecotoxicological status should be carried out. Here, native and caged mussels (Mytilus spp.) were used in combination to evaluate biological effects of chronic chemical contamination in Brest harbor. Polycyclic aromatic hydrocarbon (PAH) contamination was measured in mussel tissues as a proxy of harbor and urban pollution. Biochemical biomarkers of xenobiotic biotransformation, antioxidant defenses, generation of reducing equivalents, energy metabolism and oxidative damage were studied in both gills and digestive glands of native and caged mussels. In particular, activities of glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), NADP-dependent isocitrate dehydrogenase (IDP), pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) were measured and lipid peroxidation was assessed by malondialdehyde (MDA) quantification. In addition, a condition index was calculated to assess the overall health of the mussels. Moderate PAH contamination was detected in digestive glands of both native and caged individuals from the exposed site. Modulations of biomarkers were detected in digestive glands of native harbor mussels indicating the presence of a chemical pressure. In particular, results suggested increased biotransformation (GST), antioxidant defenses (CAT), NADPH generation (IDP) and gluconeogenesis (PEPCK), which could represent a coordinated response against chemically-induced cellular stress. Lipid peroxidation assessment and condition index indicated an absence of acute stress in the same mussels suggesting metabolic changes could, at least partially, offset the negative effects of contamination. In caged mussels, only GR was found modulated compared to non-exposed mussels but significant differences in oxidative stress and energy-related biomarkers were observed compared to native harbor mussels. Overall, these results suggested mussels chronically exposed to contamination have set up metabolic adaptation, which may contribute to their survival in the moderately contaminated harbor of Brest. Whether these adaptive traits result from phenotypic plasticity or genetic adaptation needs to be further investigated.
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Adaptação Fisiológica/efeitos dos fármacos , Monitoramento Ambiental/métodos , Mytilus/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Poluentes Químicos da Água/toxicidade , Poluição Química da Água/efeitos adversos , Animais , Biomarcadores/metabolismo , Biotransformação , Metabolismo Energético/efeitos dos fármacos , França , Peroxidação de Lipídeos/efeitos dos fármacos , Mytilus/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , Poluentes Químicos da Água/metabolismoRESUMO
After Mycobacterium avium subsp. paratuberculosis (Map) infection the cell-mediated immune (CMI) response indicative of early Th1 activation may be detected using interferon-gamma release assay (IGRA). Currently, the purified protein derivatives (PPDs), i.e., the total extract of mycobacteria antigens are used to recall CMI responses against Map. This study aimed to assess the ability of the chemically synthesized Map specific cell wall lipopentapeptide L5P to induce CMI response in cows infected by Map compared to PPD. L5P and PPD elicited an IFN-γ response in 12 and 35 animals from two Map infected herds respectively, but IFN-γ was not detected in the 13 cows recruited from a non-infected herd. Levels of IFN-γ detected were higher with PPD than with L5P. There was no correlation between the IFN-γ response and the humoral response to Map or faecal culture.
Assuntos
Antígenos de Bactérias/imunologia , Doenças dos Bovinos/imunologia , Interferon gama/metabolismo , Mycobacterium avium subsp. paratuberculosis/metabolismo , Paratuberculose/imunologia , Animais , Bovinos , Doenças dos Bovinos/microbiologia , Feminino , Imunidade Celular/imunologia , Mycobacterium avium subsp. paratuberculosis/imunologia , Paratuberculose/microbiologia , TuberculinaRESUMO
Manila clam stock from Arcachon Bay, France, is declining, as is commercial harvest. To understand the role of environmental biotic interactions in this decrease, effects of a toxic dinoflagellate, Alexandrium ostenfeldii, which blooms regularly in Arcachon bay, and the interaction with perkinsosis on clam physiology were investigated. Manila clams from Arcachon Bay, with variable natural levels of perkinsosis, were exposed for seven days to a mix of the nutritious microalga T-Iso and the toxic dinoflagellate A. ostenfeldii, a producer of spirolides, followed by seven days of depuration fed only T-Iso. Following sacrifice and quantification of protozoan parasite Perkinsus olseni burden, clams were divided into two groups according to intensity of the infection ("Light-Moderate" and "Moderate-Heavy"). Hemocyte and plasma responses, digestive enzyme activities, antioxidant enzyme activities in gills, and histopathological responses were analyzed. Reactive oxygen species (ROS) production in hemocytes and catalase (CAT) activity in gills increased with P. olseni intensity of infection in control clams fed T-Iso, but did not vary among A. ostenfeldii-exposed clams. Exposure to A. ostenfeldii caused tissue alterations associated with an inflammatory response and modifications in hemocyte morphology. In the gills, superoxide dismutase (SOD) activity decreased, and an increase in brown cell occurrence was seen, suggesting oxidative stress. Observations of hemocytes and brown cells in tissues during exposure and depuration suggest involvement of both cell types in detoxication processes. Results suggest that exposure to A. ostenfeldii disrupted the pro-/anti-oxidant response of clams to heavy P. olseni intensity. In addition, depressed mitochondrial membrane potential (MMP) in hemocytes of clams exposed to A. ostenfeldii suggests that mitochondrial functions are regulated to maintain homeostasis of digestive enzyme activity and condition index.