RESUMO
OBJECTIVE: A GGGGCC repeat expansion in the C9orf72 gene is the most common cause of genetic frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). As potential therapies targeting the repeat expansion are now entering clinical trials, sensitive biomarker assays of target engagement are urgently required. Our objective was to develop such an assay. METHODS: We used the single molecule array (Simoa) platform to develop an immunoassay for measuring poly(GP) dipeptide repeat proteins (DPRs) generated by the C9orf72 repeat expansion in cerebrospinal fluid (CSF) of people with C9orf72-associated FTD/ALS. RESULTS AND CONCLUSIONS: We show the assay to be highly sensitive and robust, passing extensive qualification criteria including low intraplate and interplate variability, a high precision and accuracy in measuring both calibrators and samples, dilutional parallelism, tolerance to sample and standard freeze-thaw and no haemoglobin interference. We used this assay to measure poly(GP) in CSF samples collected through the Genetic FTD Initiative (N=40 C9orf72 and 15 controls). We found it had 100% specificity and 100% sensitivity and a large window for detecting target engagement, as the C9orf72 CSF sample with the lowest poly(GP) signal had eightfold higher signal than controls and on average values from C9orf72 samples were 38-fold higher than controls, which all fell below the lower limit of quantification of the assay. These data indicate that a Simoa-based poly(GP) DPR assay is suitable for use in clinical trials to determine target engagement of therapeutics aimed at reducing C9orf72 repeat-containing transcripts.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Esclerose Lateral Amiotrófica/líquido cefalorraquidiano , Esclerose Lateral Amiotrófica/diagnóstico , Esclerose Lateral Amiotrófica/genética , Biomarcadores/líquido cefalorraquidiano , Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Demência Frontotemporal/diagnóstico , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , HumanosRESUMO
A GGGGCC hexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense repeat-containing transcripts undergo repeat-associated non-AUG-initiated translation to produce five dipeptide proteins (DPRs). The polyGR and polyPR DPRs are extremely toxic when expressed in Drosophila neurons. To determine the mechanism that mediates this toxicity, we purified DPRs from the Drosophila brain and used mass spectrometry to identify the in vivo neuronal DPR interactome. PolyGR and polyPR interact with ribosomal proteins, and inhibit translation in both human iPSC-derived motor neurons, and adult Drosophila neurons. We next performed a screen of 81 translation-associated proteins in GGGGCC repeat-expressing Drosophila to determine whether this translational repression can be overcome and if this impacts neurodegeneration. Expression of the translation initiation factor eIF1A uniquely rescued DPR-induced toxicity in vivo, indicating that restoring translation is a potential therapeutic strategy. These data directly implicate translational repression in C9orf72 repeat-induced neurodegeneration and identify eIF1A as a novel modifier of C9orf72 repeat toxicity.
Assuntos
Proteína C9orf72/metabolismo , Fator de Iniciação 1 em Eucariotos/metabolismo , Neurônios/metabolismo , Biossíntese de Proteínas/fisiologia , Esclerose Lateral Amiotrófica/genética , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Proteína C9orf72/genética , Expansão das Repetições de DNA , Dipeptídeos/metabolismo , Drosophila , Demência Frontotemporal/genética , HumanosRESUMO
Small ruminant lentivirus (SRLV) infection causes losses in the small ruminant industry due to reduced animal production and increased replacement rates. Infection of wild ruminants in close contact with infected domestic animals has been proposed to play a role in SRLV epidemiology, but studies are limited and mostly involve hybrids between wild and domestic animals. In this study, SRLV seropositive red deer, roe deer and mouflon were detected through modified ELISA tests, but virus was not successfully amplified using a set of different PCRs. Apparent restriction of SRLV infection in cervids was not related to the presence of neutralizing antibodies. In vitro cultured skin fibroblastic cells from red deer and fallow deer were permissive to the SRLV entry and integration, but produced low quantities of virus. SRLV got rapidly adapted in vitro to blood-derived macrophages and skin fibroblastic cells from red deer but not from fallow deer. Thus, although direct detection of virus was not successfully achieved in vivo, these findings show the potential susceptibility of wild ruminants to SRLV infection in the case of red deer and, on the other hand, an in vivo SRLV restriction in fallow deer. Altogether these results may highlight the importance of surveilling and controlling SRLV infection in domestic as well as in wild ruminants sharing pasture areas, and may provide new natural tools to control SRLV spread in sheep and goats.
Assuntos
Cervos , Fibroblastos/virologia , Infecções por Lentivirus/veterinária , Lentivirus/fisiologia , Doenças dos Ovinos/virologia , Carneiro Doméstico , Replicação Viral/fisiologia , Animais , Animais Selvagens , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Infecções por Lentivirus/sangue , Infecções por Lentivirus/transmissão , Ovinos , Doenças dos Ovinos/sangue , Doenças dos Ovinos/transmissão , Internalização do VírusRESUMO
Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-ß1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-ß1 followed by COL6A1. Knockdown of TGF-ß1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-ß1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Camundongos , Demência Frontotemporal/patologia , Esclerose Lateral Amiotrófica/metabolismo , Fator de Crescimento Transformador beta1 , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Drosophila , Matriz Extracelular/metabolismo , Dipeptídeos/metabolismo , Expansão das Repetições de DNA/genéticaRESUMO
Small ruminant lentiviruses (SRLV) infect the monocyte/macrophage lineage inducing a long-lasting infection affecting body condition, production and welfare of sheep and goats all over the world. Macrophages play a pivotal role on the host's innate and adaptative immune responses against parasites by becoming differentially activated. Macrophage heterogeneity can tentatively be classified into classically differentiated macrophages (M1) through stimulation with IFN-γ displaying an inflammatory profile, or can be alternatively differentiated by stimulation with IL-4/IL-13 into M2 macrophages with homeostatic functions. Since infection by SRLV can modulate macrophage functions we explored here whether ovine and caprine macrophages can be segregated into M1 and M2 populations and whether this differential polarization represents differential susceptibility to SRLV infection. We found that like in human and mouse systems, ovine and caprine macrophages can be differentiated with particular stimuli into M1/M2 subpopulations displaying specific markers. In addition, small ruminant macrophages are plastic since M1 differentiated macrophages can express M2 markers when the stimulus changes from IFN-γ to IL-4. SRLV replication was restricted in M1 macrophages and increased in M2 differentiated macrophages respectively according to viral production. Identification of the infection pathways in macrophage populations may provide new targets for eliciting appropriate immune responses against SRLV infection.
Assuntos
Citocinas/genética , Regulação da Expressão Gênica , Doenças das Cabras/imunologia , Infecções por Lentivirus/veterinária , Lentivirus/fisiologia , Macrófagos/imunologia , Doenças dos Ovinos/imunologia , Animais , Células CHO , Cricetulus , Citocinas/metabolismo , Marcadores Genéticos , Doenças das Cabras/virologia , Cabras , Células HEK293 , Humanos , Infecções por Lentivirus/imunologia , Infecções por Lentivirus/virologia , Lipopolissacarídeos , Macrófagos/citologia , Macrófagos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Ovinos , Doenças dos Ovinos/virologiaRESUMO
An intronic GGGGCC repeat expansion in C9orf72 is a common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeats are transcribed in both sense and antisense directions to generate distinct dipeptide repeat proteins, of which poly(GA), poly(GR), and poly(PR) have been implicated in contributing to neurodegeneration. Poly(PR) binding to RNA may contribute to toxicity, but analysis of poly(PR)-RNA binding on a transcriptome-wide scale has not yet been carried out. We therefore performed crosslinking and immunoprecipitation (CLIP) analysis in human cells to identify the RNA binding sites of poly(PR). We found that poly(PR) binds to nearly 600 RNAs, with the sequence GAAGA enriched at the binding sites. In vitro experiments showed that poly(GAAGA) RNA binds poly(PR) with higher affinity than control RNA and induces the phase separation of poly(PR) into condensates. These data indicate that poly(PR) preferentially binds to poly(GAAGA)-containing RNAs, which may have physiological consequences.
Assuntos
Perfilação da Expressão Gênica , Transcriptoma , Humanos , Transcriptoma/genética , Proteína C9orf72/genética , Poli A , Dipeptídeos , RNA/genéticaRESUMO
Thirty-one sheep naturally infected with small ruminant lentiviruses (SRLV) of known genotype (A or B), and clinically affected with neurological disease, pneumonia or arthritis were used to analyse mannose receptor (MR) expression (transcript levels) and proviral load in virus target tissues (lung, mammary gland, CNS and carpal joints). Control sheep were SRLV-seropositive asymptomatic (n = 3), seronegative (n = 3) or with chronic listeriosis, pseudotuberculosis or parasitic cysts (n = 1 in each case). MR expression and proviral load increased with the severity of lesions in most analyzed organs of the SRLV infected sheep and was detected in the affected tissue involved in the corresponding clinical disease (CNS, lung and carpal joint in neurological disease, pneumonia and arthritis animal groups, respectively). The increased MR expression appeared to be SRLV specific and may have a role in lentiviral pathogenesis.
Assuntos
Regulação da Expressão Gênica , Lectinas Tipo C/genética , Infecções por Lentivirus/veterinária , Lentivirus Ovinos-Caprinos/isolamento & purificação , Lectinas de Ligação a Manose/genética , Provírus/isolamento & purificação , Receptores de Superfície Celular/genética , Doenças dos Ovinos/genética , Carga Viral/veterinária , Animais , Artrite/genética , Artrite/veterinária , Artrite/virologia , Encefalite/genética , Encefalite/veterinária , Encefalite/virologia , Feminino , Lectinas Tipo C/metabolismo , Infecções por Lentivirus/genética , Infecções por Lentivirus/virologia , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Especificidade de Órgãos , Pneumonia/genética , Pneumonia/veterinária , Pneumonia/virologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Receptores de Superfície Celular/metabolismo , Ovinos , Doenças dos Ovinos/virologia , EspanhaRESUMO
BACKGROUND: A central nervous system (CNS) disease outbreak caused by small ruminant lentiviruses (SRLV) has triggered interest in Spain due to the rapid onset of clinical signs and relevant production losses. In a previous study on this outbreak, the role of LTR in tropism was unclear and env encoded sequences, likely involved in tropism, were not investigated. This study aimed to analyze heterogeneity of SRLV Env regions--TM amino terminal and SU V4, C4 and V5 segments--in order to assess virus compartmentalization in CNS. RESULTS: Eight Visna (neurologically) affected sheep of the outbreak were used. Of the 350 clones obtained after PCR amplification, 142 corresponded to CNS samples (spinal cord and choroid plexus) and the remaining to mammary gland, blood cells, bronchoalveolar lavage cells and/or lung. The diversity of the env sequences from CNS was 11.1-16.1% between animals and 0.35-11.6% within each animal, except in one animal presenting two sequence types (30% diversity) in the CNS (one grouping with those of the outbreak), indicative of CNS virus sequence heterogeneity. Outbreak sequences were of genotype A, clustering per animal and compartmentalizing in the animal tissues. No CNS specific signature patterns were found. CONCLUSIONS: Bayesian approach inferences suggested that proviruses from broncoalveolar lavage cells and peripheral blood mononuclear cells represented the common ancestors (infecting viruses) in the animal and that neuroinvasion in the outbreak involved microevolution after initial infection with an A-type strain. This study demonstrates virus compartmentalization in the CNS and other body tissues in sheep presenting the neurological form of SRLV infection.
Assuntos
Vírus Visna-Maedi/genética , Visna/virologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sistema Nervoso Central/virologia , Surtos de Doenças/veterinária , Genótipo , Glândulas Mamárias Animais/virologia , Dados de Sequência Molecular , Filogenia , Ovinos , Espanha/epidemiologia , Visna/epidemiologiaRESUMO
This study aims to characterize the mannose receptor (MR) gene in sheep and its role in ovine visna/maedi virus (VMV) infection. The deduced amino acid sequence of ovine MR was compatible with a transmembrane protein having a cysteine-rich ricin-type amino-terminal region, a fibronectin type II repeat, eight tandem C-type lectin carbohydrate-recognition domains (CRD), a transmembrane region, and a cytoplasmic carboxy-terminal tail. The ovine and bovine MR sequences were closer to each other compared to human or swine MR. Concanavalin A (ConA) inhibited VMV productive infection, which was restored by mannan totally in ovine skin fibroblasts (OSF) and partially in blood monocyte-derived macrophages (BMDM), suggesting the involvement of mannosylated residues of the VMV ENV protein in the process. ConA impaired also syncytium formation in OSF transfected with an ENV-encoding pN3-plasmid. MR transcripts were found in two common SRLV targets, BMDM and synovial membrane (GSM) cells, but not in OSF. Viral infection of BMDM and especially GSM cells was inhibited by mannan, strongly suggesting that in these cells the MR is an important route of infection involving VMV Env mannosylated residues. Thus, at least three patterns of viral entry into SRLV-target cells can be proposed, involving mainly MR in GSM cells (target in SRLV-induced arthritis), MR in addition to an alternative route in BMDM (target in SRLV infections), and an alternative route excluding MR in OSF (target in cell culture). Different routes of SRLV infection may thus coexist related to the involvement of MR differential expression.
Assuntos
Concanavalina A/farmacologia , Células Gigantes/virologia , Lectinas Tipo C/genética , Lectinas de Ligação a Manose/genética , Pneumonia Intersticial Progressiva dos Ovinos/imunologia , Receptores de Superfície Celular/genética , Vírus Visna-Maedi/fisiologia , Animais , Western Blotting/veterinária , Imuno-Histoquímica/veterinária , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/metabolismo , Dados de Sequência Molecular , Pneumonia Intersticial Progressiva dos Ovinos/metabolismo , Pneumonia Intersticial Progressiva dos Ovinos/virologia , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Receptores Virais/química , Receptores Virais/genética , Receptores Virais/metabolismo , Análise de Sequência de Proteína/veterinária , OvinosRESUMO
Small ruminant lentiviruses (SRLV) cause an incurable multiorganic disease widely spread in sheep and goats that disturbs animal welfare and production. In the absence of a vaccine, control measures have been traditionally based on early diagnosis and breeding with virus-inactivated colostrum with segregation of seropositive animals. However, antigenic heterogeneity, poor antibody production due to low viral load, and single strain design of most available ELISA, pose a threat to SRLV diagnosis. Genome-wide association studies have described TMEM154 E35K polymorphism as a good genetic marker for selection of resistant animals in some American and European breeds. In this study, a multitargeted serological and virological screening of more than 500 animals from four different breeds (latxa, raza Navarra, assaf, and churra) attending to SRLV infection status was performed. Then, animals were genotyped to characterize TMEM154 E35K polymorphism. ELISA procedures, individually considered, only identified a proportion of the seropositive animals, and PCR detected a fraction of seronegative animals, globally offering different animal classifications according to SRLV infection status. TMEM154 allele frequency differed substantially among breeds and a positive association between seroprevalence and TMEM154 genotype was found only in one breed. Selection based on TMEM154 may be suitable for specific ovine breeds or SRLV strains, however generalization to the whole SRLV genetic spectrum, ovine breeds, or epidemiological situation may need further validation.
RESUMO
G4C2 repeat expansions within the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The repeats undergo repeat-associated non-ATG translation to generate toxic dipeptide repeat proteins. Here, we show that insulin/IGF signalling is reduced in fly models of C9orf72 repeat expansion using RNA sequencing of adult brain. We further demonstrate that activation of insulin/IGF signalling can mitigate multiple neurodegenerative phenotypes in flies expressing either expanded G4C2 repeats or the toxic dipeptide repeat protein poly-GR. Levels of poly-GR are reduced when components of the insulin/IGF signalling pathway are genetically activated in the diseased flies, suggesting a mechanism of rescue. Modulating insulin signalling in mammalian cells also lowers poly-GR levels. Remarkably, systemic injection of insulin improves the survival of flies expressing G4C2 repeats. Overall, our data suggest that modulation of insulin/IGF signalling could be an effective therapeutic approach against C9orf72 ALS/FTD.
Assuntos
Esclerose Lateral Amiotrófica/genética , Proteína C9orf72/toxicidade , Expansão das Repetições de DNA , Drosophila melanogaster/fisiologia , Demência Frontotemporal/genética , Insulina/fisiologia , Transdução de Sinais , Animais , Proteína C9orf72/genética , FemininoRESUMO
A C9orf72 repeat expansion is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis. One of the suggested pathomechanisms is toxicity from dipeptide repeat proteins (DPRs), which are generated via unconventional translation of sense and antisense repeat transcripts with poly-GA, poly-GP and poly-GR being the most abundant dipeptide proteins. Animal and cellular studies highlight a neurotoxic role of poly-GR and poly-PR and to a lesser degree of poly-GA. Human post-mortem studies in contrast have been much less clear on a potential role of DPR toxicity but have largely focused on immunohistochemical methods to detect aggregated DPR inclusions. This study uses protein fractionation and sensitive immunoassays to quantify not only insoluble but also soluble poly-GA, poly-GP and poly-GR concentrations in brain homogenates of FTD patients with C9orf72 mutation across four brain regions. We show that soluble DPRs are less abundant in clinically affected areas (i.e. frontal and temporal cortices). In contrast, the cerebellum not only shows the largest DPR load but also the highest relative DPR solubility. Finally, poly-GR levels and poly-GP solubility correlate with clinical severity. These findings provide the first cross-comparison of soluble and insoluble forms of all sense DPRs and shed light on the distribution and role of soluble DPRs in the etiopathogenesis of human C9orf72-FTD.
Assuntos
Encéfalo/metabolismo , Dipeptídeos/metabolismo , Demência Frontotemporal/metabolismo , Polímeros/metabolismo , Proteínas/metabolismo , Idoso , Proteína C9orf72/genética , Expansão das Repetições de DNA/genética , Feminino , Demência Frontotemporal/genética , Humanos , Masculino , Pessoa de Meia-Idade , Sequências Repetitivas de Aminoácidos/genética , SolubilidadeRESUMO
Small ruminant lentiviruses (SRLVs) are endemic in most areas of Europe, causing a chronic infection and a multisystemic disease affecting the udder, carpal joints, lungs, and central nervous system. Due to the lack of treatments and protective vaccination strategies, infection control is focused on the identification of infected animals through serological or molecular techniques. However, antigenic and genetic heterogeneity of SRLVs represent a clear drawback for diagnosis. Infected animals may present lower animal production parameters such as birth weight or milk production and quality, depending on productive systems considered and, likely, to the diagnostic method applied. In this study, four sheep flocks dedicated to dairy or meat production were evaluated using three different ELISA and two PCR strategies to classify animal population according to SRLV infection status. Productive parameters were recorded along one whole lactation or reproductive period and compared between positive and negative animals. SRLV was present in 19% of the total population, being unequally distributed in the different flocks. Less than half of the infected animals were detected by a single diagnostic method, highlighting the importance of combining different diagnostic techniques. Statistical analysis employing animal classification using all the diagnostic methods associated lambing size, lamb weight at birth, and daily weight gain with SRLV infection status in meat flocks. Milk production, somatic cell count, fat, and protein content in the milk were associated with SRLV infection in dairy flocks, to a greater extent in the flock showing higher seroprevalence. A multi-platform SRLV diagnostic strategy was useful for ensuring correct animal classification, thus validating downstream studies investigating production traits.
RESUMO
Nucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Unconventional translation (RAN translation) of C9orf72 repeats generates dipeptide repeat proteins that can cause neurodegeneration. We performed a genetic screen for regulators of RAN translation and identified small ribosomal protein subunit 25 (RPS25), presenting a potential therapeutic target for C9orf72-related amyotrophic lateral sclerosis and frontotemporal dementia and other neurodegenerative diseases caused by nucleotide repeat expansions.
Assuntos
Proteína C9orf72/genética , Doenças Neurodegenerativas/genética , Proteínas Ribossômicas/genética , Animais , Expansão das Repetições de DNA/genética , Humanos , Biossíntese de ProteínasRESUMO
In small ruminant lentivirus infections, cellular immune responses are diminished in clinically affected animals. The underlying mechanisms for this are unknown. In this study, we tested the hypothesis that alterations in expression of the co-stimulatory molecules B7-1 and B7-2 are involved in infections with Visna/Maedi virus (VMV), the prototype lentivirus of sheep. We studied B7 expression levels ex vivo in peripheral blood mononuclear cells (PBMCs), determining B7 RNA levels by real time reverse transcriptase polymerase chain reaction in asymptomatic as well as clinically affected VMV-seropositive sheep. The levels of both B7 molecules were increased in VMV-seropositive asymptomatic sheep. However, in VMV clinically affected sheep, the level of CD80 (but not CD86) was low compared with the level in uninfected sheep (p < 0.05). CD80 and CD86 RNA levels were associated with the ability of PBMCs to respond to VMV gag antigens (p14, p17, and p25) by proliferation, with most seropositive asymptomatic sheep showing positive proliferative responses but clinically affected sheep showing no response. The response to p25 in clinically affected animals was increased by the addition of interleukin-2 to the cultures. Decreased recall responses to unrelated antigens (assessed by production of interferon-gamma) were also found in clinically affected sheep. Thus, among seropositive sheep, decreased B7-1 (CD80) RNA levels and diminished antigen-specific cellular immune responses in PBMCs point to a VMV disease status, whereas increased CD80 and CD86 levels and augmented cellular responses are linked to asymptomatic infection.
Assuntos
Antígeno B7-1/imunologia , Antígeno B7-2/imunologia , Infecções por Lentivirus/veterinária , Doenças dos Ovinos/imunologia , Vírus Visna-Maedi/imunologia , Animais , Antígeno B7-1/genética , Antígeno B7-2/genética , Produtos do Gene gag/genética , Produtos do Gene gag/imunologia , Interferon gama/imunologia , Interleucina-2/imunologia , Infecções por Lentivirus/imunologia , Infecções por Lentivirus/virologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Proteínas Recombinantes/imunologia , Ovinos , Doenças dos Ovinos/virologia , Reino Unido , Regulação para Cima , Carga ViralRESUMO
ß-glucans exhibited in cell walls of several pathogens as bacteria or fungi are sensed by pathogen recognition receptors such as scavenger receptors present in antigen presenting cells, i.e., macrophages. ß-glucans obtained from Shiitake mushrooms were chemically characterized. A ß-glucan supplemented diet was assayed for 30 days in rabbits aiming to characterize the immune response elicited in blood-derived macrophages. M1 and M2 profiles of macrophage differentiation were confirmed in rabbits by in vitro stimulation with IFN-γ and IL-4 and marker quantification of each differentiation pathway. Blood derived macrophages from rabbits administered in vivo with the ß-glucan supplemented diet showed higher IL-4, IFN-γ and RAGE together with lower IL-10 relative expression, indicative of an ongoing immune response. Differences in IL-1ß, IL-13 and IL-4 expression were also found in rabbit sera by ELISA suggesting further stimulation of the adaptive response. Recent challenges in the rabbit industry include the search of diet supplements able to elicit an immune stimulation with particular interest in facing pathogens such as viruses or bacteria. ß-glucans from fungi may contribute to maintain an immune steady state favouring protection and thus reducing antibiotic treatment.
RESUMO
Intrinsic factors of the innate immune system include the apolipoprotein B editing enzyme catalytic polypeptide-like 3 (APOBEC3) protein family. APOBEC3 inhibits replication of different virus families by cytosine deamination of viral DNA and a not fully characterized cytosine deamination-independent mechanism. Sheep are susceptible to small ruminant lentivirus (SRLVs) infection and contain three APOBEC3 genes encoding four proteins (A3Z1, Z2, Z3 and Z2-Z3) with yet not deeply described antiviral properties. Using sheep blood monocytes and in vitro-derived macrophages, we found that A3Z1 expression is associated with lower viral replication in this cellular type. A3Z1 transcripts may also contain spliced variants (A3Z1Tr) lacking the cytidine deaminase motif. A3Z1 exogenous expression in fully permissive fibroblast-like cells restricted SRLVs infection while A3Z1Tr allowed infection. A3Z1Tr was induced after SRLVs infection or stimulation of blood-derived macrophages with interferon gamma (IFN-γ). Interaction between truncated isoform and native A3Z1 protein was detected as well as incorporation of both proteins into virions. A3Z1 and A3Z1Tr interacted with SRLVs Vif, but this interaction was not associated with degradative properties. Similar A3Z1 truncated isoforms were also present in human and monkey cells suggesting a conserved alternative splicing regulation in primates. A3Z1-mediated retroviral restriction could be constrained by different means, including gene expression and specific alternative splicing regulation, leading to truncated protein isoforms lacking a cytidine-deaminase motif.
Assuntos
Citosina Desaminase/genética , Lentivirus/fisiologia , Replicação Viral , Processamento Alternativo/genética , Animais , Citosina Desaminase/química , Citosina Desaminase/metabolismo , Regulação da Expressão Gênica , Haplorrinos , Humanos , Interferon gama/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Motivos de Nucleotídeos/genética , Isoformas de Proteínas/genética , OvinosRESUMO
The major challenges in diagnosing small ruminant lentivirus (SRLV) infection include early detection and genotyping of strains of epidemiological interest. A longitudinal study was carried out in Rasa Aragonesa sheep experimentally infected with viral strains of genotypes A or B from Spanish neurological and arthritic SRLV outbreaks, respectively. Sera were tested with two commercial ELISAs, three based on specific peptides and a novel combined peptide ELISA. Three different PCR assays were used to further assess infection status. The kinetics of anti-viral antibody responses were variable, with early diagnosis dependent on the type of ELISA used. Peptide epitopes of SRLV genotypes A and B combined in the same ELISA well enhanced the overall detection rate, whereas single peptides were useful for genotyping the infecting strain (A vs. B). The results of the study suggest that a combined peptide ELISA can be used for serological diagnosis of SRLV infection, with single peptide ELISAs useful for subsequent serotyping.
Assuntos
Ensaio de Imunoadsorção Enzimática/veterinária , Infecções por Lentivirus/veterinária , Lentivirus/genética , Peptídeos/química , Doenças dos Ovinos/virologia , Animais , Anticorpos Antivirais/sangue , Genótipo , Lentivirus/classificação , Infecções por Lentivirus/diagnóstico , Infecções por Lentivirus/virologia , Masculino , Reação em Cadeia da Polimerase/veterinária , Testes Sorológicos , Ovinos , Doenças dos Ovinos/diagnósticoRESUMO
Production and excretion of small ruminant lentiviruses (SRLVs) varies with the stage of the host reproductive cycle, suggesting hormonal involvement in this variation. Stress may also affect viral expression. To determine if hormones affect SRLV transcriptional activity, the expression of green fluorescent protein (GFP) driven by the promoters in the U3-cap region of the long terminal repeats (LTRs) of different strains of SRLV was assessed in cell culture. High concentrations of steroids (progesterone, cortisol and dehydroepiandrosterone) inhibited expression of GFP driven by SRLV promoters. This effect decreased in a dose-dependent manner with decreasing concentrations of steroids. In some strains, physiological concentrations of cortisol or dehydroepiandrosterone (DHEA) induced the expression of GFP above the baseline. There was strain variation in sensitivity to hormones, but this differed for different hormones. The presence of deletions and a 43 base repeat in the U3 region upstream of the TATA box of the LTR made strain EV1 less sensitive to DHEA. However, no clear tendencies or patterns were observed when comparing strains of different genotypes and/or subtypes, or those triggering different forms of disease.
Assuntos
Desidroepiandrosterona/metabolismo , Regulação Viral da Expressão Gênica , Hidrocortisona/metabolismo , Progesterona/metabolismo , Regiões Promotoras Genéticas , Sequências Repetidas Terminais , Vírus Visna-Maedi/genética , Animais , Sequência de Bases , Proteínas de Fluorescência Verde/genética , Dados de Sequência Molecular , Plasmídeos/genética , Pneumonia Intersticial Progressiva dos Ovinos/virologia , Ovinos , Doenças dos Ovinos/virologia , Visna/virologia , Vírus Visna-Maedi/metabolismoRESUMO
The presence of proviral DNA, mRNA transcripts and/or viral proteins in small ruminant lentiviral infections may be intermittent. The aim of this study was to identify methods of avoiding small ruminant lentivirus (SRLV) transmission to ewes when using infected rams in artificial insemination (AI). Semen from rams, seropositive and PCR-positive in blood but consistently negative for both proviral DNA and viral protein expression in semen, was used to artificially inseminate 19 ewes. Follow-up investigation of these ewes and of two of their offspring indicated that under the study conditions virus transmission through insemination did not occur. These preliminary findings suggest that semen from SRLV-infected rams could be used for AI without the risk of transmitting virus to susceptible ewes or their lambs. Further larger studies will be required to confirm this finding.