RESUMO
The exceptional polymorphism observed within genes of the major histocompatibility complex (MHC), a core component of the vertebrate immune system, has long fascinated biologists. The highly polymorphic classical MHC class-I (MHC-I) genes are maintained by pathogen-mediated balancing selection (PMBS), as shown by many sites subject to positive selection, while the more monomorphic non-classical MHC-I genes show signatures of purifying selection. In line with PMBS, at any point in time, rare classical MHC alleles are more likely than common classical MHC alleles to confer a selective advantage in host-pathogen interactions. Combining genomic and expression data from the blood of wild house sparrows Passer domesticus, we found that only rare classical MHC-I alleles were highly expressed, while common classical MHC-I alleles were lowly expressed or not expressed. Moreover, highly expressed rare classical MHC-I alleles had more positively selected sites, indicating exposure to stronger PMBS, compared with lowly expressed classical alleles. As predicted, the level of expression was unrelated to allele frequency in the monomorphic non-classical MHC-I alleles. Going beyond previous studies, we offer a fine-scale view of selection on classical MHC-I genes in a wild population by revealing differences in the strength of PMBS according to allele frequency and expression level.
Assuntos
Complexo Principal de Histocompatibilidade , Pardais , Animais , Alelos , Complexo Principal de Histocompatibilidade/genética , Pardais/genética , Frequência do Gene , Seleção Genética , Variação GenéticaRESUMO
Long-read sequencing offers a great improvement in the assembly of complex genomic regions, such as the major histocompatibility complex (MHC) region, which can contain both tandemly duplicated MHC genes (paralogs) and high repeat content. The MHC genes have expanded in passerine birds, resulting in numerous MHC paralogs, with relatively high sequence similarity, making the assembly of the MHC region challenging even with long-read sequencing. In addition, MHC genes show rather high sequence divergence between alleles, making diploid-aware assemblers incorrectly classify haplotypes from the same locus as sequences originating from different genomic regions. Consequently, the number of MHC paralogs can easily be over- or underestimated in long-read assemblies. We therefore set out to verify the MHC diversity in an original and a haplotype-purged long-read assembly of one great reed warbler Acrocephalus arundinaceus individual (the focal individual) by using Illumina MiSeq amplicon sequencing. Single exons, representing MHC class I (MHC-I) and class IIB (MHC-IIB) alleles, were sequenced in the focal individual and mapped to the annotated MHC alleles in the original long-read genome assembly. Eighty-four percent of the annotated MHC-I alleles in the original long-read genome assembly were detected using 55% of the amplicon alleles and likewise, 78% of the annotated MHC-IIB alleles were detected using 61% of the amplicon alleles, indicating an incomplete annotation of MHC genes. In the haploid genome assembly, each MHC-IIB gene should be represented by one allele. The parental origin of the MHC-IIB amplicon alleles in the focal individual was determined by sequencing MHC-IIB in its parents. Two of five larger scaffolds, containing 6-19 MHC-IIB paralogs, had a maternal and paternal origin, respectively, as well as a high nucleotide similarity, which suggests that these scaffolds had been incorrectly assigned as belonging to different loci in the genome rather than as alternate haplotypes of the same locus. Therefore, the number of MHC-IIB paralogs was overestimated in the haploid genome assembly. Based on our findings we propose amplicon sequencing as a suitable complement to long-read sequencing for independent validation of the number of paralogs in general and for haplotype inference in multigene families in particular.
Assuntos
Complexo Principal de Histocompatibilidade , Passeriformes , Animais , Haplótipos/genética , Complexo Principal de Histocompatibilidade/genética , Antígenos de Histocompatibilidade Classe I/genética , Genoma , Genômica , Passeriformes/genéticaRESUMO
MHC genes are extraordinarily polymorphic in most taxa. Host-pathogen coevolution driven by negative frequency-dependent selection (NFDS) is one of the main hypotheses for the maintenance of such immunogenetic variation. Here, we test a critical but rarely tested assumption of this hypothesis-that MHC alleles affect resistance/susceptibility to a pathogen in a strain-specific way, that is, there is a host genotype-by-pathogen genotype interaction. In a field study of bank voles naturally infected with the tick-transmitted bacterium Borrelia afzelii, we tested for MHC class II (DQB) genotype-by-B. afzelii strain interactions for infection prevalence between 10 DQB alleles and seven strains. One allele (DQB*37) showed an interaction, such that voles carrying DQB*37 had higher prevalence of two strains and lower prevalence of one strain than individuals without the allele. These findings were corroborated by analyses of strain composition of infections, which revealed an effect of DQB*37 in the form of lower ß diversity among infections in voles carrying the allele. Taken together, these results provide rare support at the molecular genetic level for a key assumption of models of antagonistic coevolution through NFDS.
Assuntos
Borrelia , Animais , Arvicolinae/genética , Genótipo , Prevalência , RoedoresRESUMO
BACKGROUND: Hosts are often simultaneously infected with several parasite species. These co-infections can lead to within-host interactions of parasites, including mutualism and competition, which may affect both virulence and transmission. Birds are frequently co-infected with different haemosporidian parasites, but very little is known about if and how these parasites interact in natural host populations and what consequences there are for the infected hosts. We therefore set out to study Plasmodium and Haemoproteus parasites in house sparrows Passer domesticus with naturally acquired infections using a protocol where the parasitemia (infection intensity) is quantified by qPCR separately for the two parasites. We analysed infection status (presence/absence of the parasite) and parasitemia of parasites in the blood of both adult and juvenile house sparrows repeatedly over the season. RESULTS: Haemoproteus passeris and Plasmodium relictum were the two dominating parasite species, found in 99% of the analyzed Sanger sequences. All birds were infected with both Plasmodium and Haemoproteus parasites during the study period. Seasonality explained infection status for both parasites in the adults: H. passeris was completely absent in the winter while P. relictum was present all year round. Among adults infected with H. passeris there was a positive effect of P. relictum parasitemia on H. passeris parasitemia and likewise among adults infected with P. relictum there was a positive effect of H. passeris parasitemia on P. relictum parasitemia. No such associations on parasitemia were seen in juvenile house sparrows. CONCLUSIONS: The reciprocal positive relationships in parasitemia between P. relictum and H. passeris in adult house sparrows suggests either mutualistic interactions between these frequently occurring parasites or that there is variation in immune responses among house sparrow individuals, hence some individuals suppress the parasitemia of both parasites whereas other individuals suppress neither. Our detailed screening of haemosporidian parasites over the season shows that co-infections are very frequent in both juvenile and adult house sparrows, and since co-infections often have stronger negative effects on host fitness than the single infection, it is imperative to use screening systems with the ability to detect multiple parasites in ecological studies of host-parasite interactions.
Assuntos
Coinfecção , Haemosporida , Malária Aviária , Parasitos , Plasmodium , Pardais , Animais , Coinfecção/epidemiologia , Humanos , Malária Aviária/epidemiologia , Parasitemia/veterinária , Pardais/parasitologiaRESUMO
The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase-4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.
Assuntos
Cistos , Doenças Renais Policísticas , Cílios/metabolismo , Cistos/metabolismo , Expressão Gênica , Humanos , Rim , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/metabolismoRESUMO
Autoimmune encephalitis (AE) can rarely manifest as a predominantly psychiatric syndrome without overt neurological symptoms. This study's aim was to characterize psychiatric patients with AE; therefore, anonymized data on patients with suspected AE with predominantly or isolated psychiatric syndromes were retrospectively collected. Patients with readily detectable neurological symptoms suggestive of AE (e.g., epileptic seizures) were excluded. Patients were classified as "probable psychiatric AE (pAE)," if well-characterized neuronal IgG autoantibodies were detected or "possible pAE" (e.g., with detection of nonclassical neuronal autoantibodies or compatible cerebrospinal fluid (CSF) changes). Of the 91 patients included, 21 (23%) fulfilled our criteria for probable (autoantibody-defined) pAE and 70 (77%) those for possible pAE. Among patients with probable pAE, 90% had anti-NMDA receptor (NMDA-R) autoantibodies. Overall, most patients suffered from paranoid-hallucinatory syndromes (53%). Patients with probable pAE suffered more often from disorientation (p < 0.001) and impaired memory (p = 0.001) than patients with possible pAE. Immunotherapies were performed in 69% of all cases, mostly with high-dose corticosteroids. Altogether, 93% of the patients with probable pAE and 80% of patients with possible pAE reportedly benefited from immunotherapies (p = 0.251). In summary, this explorative, cross-sectional evaluation confirms that autoantibody-associated AE syndromes can predominantly manifest as psychiatric syndromes, especially in anti-NMDA-R encephalitis. However, in three out of four patients, diagnosis of possible pAE was based on nonspecific findings (e.g., slight CSF pleocytosis), and well-characterized neuronal autoantibodies were absent. As such, the spectrum of psychiatric syndromes potentially responding to immunotherapies seems not to be limited to currently known autoantibody-associated AE. Further trials are needed.
Assuntos
Encefalite Antirreceptor de N-Metil-D-Aspartato , Encefalite Antirreceptor de N-Metil-D-Aspartato/terapia , Autoanticorpos , Estudos Transversais , Encefalite , Doença de Hashimoto , Humanos , Estudos Retrospectivos , SíndromeRESUMO
Fast and reliable detection of patients with severe and heterogeneous illnesses is a major goal of precision medicine1,2. Patients with leukaemia can be identified using machine learning on the basis of their blood transcriptomes3. However, there is an increasing divide between what is technically possible and what is allowed, because of privacy legislation4,5. Here, to facilitate the integration of any medical data from any data owner worldwide without violating privacy laws, we introduce Swarm Learning-a decentralized machine-learning approach that unites edge computing, blockchain-based peer-to-peer networking and coordination while maintaining confidentiality without the need for a central coordinator, thereby going beyond federated learning. To illustrate the feasibility of using Swarm Learning to develop disease classifiers using distributed data, we chose four use cases of heterogeneous diseases (COVID-19, tuberculosis, leukaemia and lung pathologies). With more than 16,400 blood transcriptomes derived from 127 clinical studies with non-uniform distributions of cases and controls and substantial study biases, as well as more than 95,000 chest X-ray images, we show that Swarm Learning classifiers outperform those developed at individual sites. In addition, Swarm Learning completely fulfils local confidentiality regulations by design. We believe that this approach will notably accelerate the introduction of precision medicine.
Assuntos
Blockchain , Tomada de Decisão Clínica/métodos , Confidencialidade , Conjuntos de Dados como Assunto , Aprendizado de Máquina , Medicina de Precisão/métodos , COVID-19/diagnóstico , COVID-19/epidemiologia , Surtos de Doenças , Feminino , Humanos , Leucemia/diagnóstico , Leucemia/patologia , Leucócitos/patologia , Pneumopatias/diagnóstico , Aprendizado de Máquina/tendências , Masculino , Software , Tuberculose/diagnósticoRESUMO
The malaria parasite Plasmodium relictum is one of the most widespread species of avian malaria. As in the case of its human counterparts, bird Plasmodium undergoes a complex life cycle infecting two hosts: the arthropod vector and the vertebrate host. In this study, we examined transcriptomes of P. relictum (SGS1) during crucial timepoints within its vector, Culex pipiens quinquefasciatus. Differential gene-expression analyses identified genes linked to the parasites life-stages at: i) a few minutes after the blood meal is ingested, ii) during peak oocyst production phase, iii) during peak sporozoite phase and iv) during the late-stages of the infection. A large amount of genes coding for functions linked to host-immune invasion and multifunctional genes was active throughout the infection cycle. One gene associated with a conserved Plasmodium membrane protein with unknown function was upregulated throughout the parasite development in the vector, suggesting an important role in the successful completion of the sporogonic cycle. Gene expression analysis further identified genes, with unknown functions to be significantly differentially expressed during the infection in the vector as well as upregulation of reticulocyte-binding proteins, which raises the possibility of the multifunctionality of these RBPs. We establish the existence of highly stage-specific pathways being overexpressed during the infection. This first study of gene-expression of a non-human Plasmodium species in its vector provides a comprehensive insight into the molecular mechanisms of the common avian malaria parasite P. relictum and provides essential information on the evolutionary diversity in gene regulation of the Plasmodium's vector stages.
Assuntos
Culex , Malária Aviária , Parasitos , Plasmodium , Animais , Culex/genética , Culex/parasitologia , Malária Aviária/genética , Mosquitos Vetores/parasitologia , Plasmodium/genéticaRESUMO
BACKGROUND: The SARS-CoV-2 pandemic is currently leading to increasing numbers of COVID-19 patients all over the world. Clinical presentations range from asymptomatic, mild respiratory tract infection, to severe cases with acute respiratory distress syndrome, respiratory failure, and death. Reports on a dysregulated immune system in the severe cases call for a better characterization and understanding of the changes in the immune system. METHODS: In order to dissect COVID-19-driven immune host responses, we performed RNA-seq of whole blood cell transcriptomes and granulocyte preparations from mild and severe COVID-19 patients and analyzed the data using a combination of conventional and data-driven co-expression analysis. Additionally, publicly available data was used to show the distinction from COVID-19 to other diseases. Reverse drug target prediction was used to identify known or novel drug candidates based on finding from data-driven findings. RESULTS: Here, we profiled whole blood transcriptomes of 39 COVID-19 patients and 10 control donors enabling a data-driven stratification based on molecular phenotype. Neutrophil activation-associated signatures were prominently enriched in severe patient groups, which was corroborated in whole blood transcriptomes from an independent second cohort of 30 as well as in granulocyte samples from a third cohort of 16 COVID-19 patients (44 samples). Comparison of COVID-19 blood transcriptomes with those of a collection of over 3100 samples derived from 12 different viral infections, inflammatory diseases, and independent control samples revealed highly specific transcriptome signatures for COVID-19. Further, stratified transcriptomes predicted patient subgroup-specific drug candidates targeting the dysregulated systemic immune response of the host. CONCLUSIONS: Our study provides novel insights in the distinct molecular subgroups or phenotypes that are not simply explained by clinical parameters. We show that whole blood transcriptomes are extremely informative for COVID-19 since they capture granulocytes which are major drivers of disease severity.
Assuntos
COVID-19/patologia , Neutrófilos/metabolismo , Transcriptoma , Antivirais/uso terapêutico , COVID-19/virologia , Estudos de Casos e Controles , Regulação para Baixo , Reposicionamento de Medicamentos , Humanos , Neutrófilos/citologia , Neutrófilos/imunologia , Fenótipo , Análise de Componente Principal , RNA/sangue , RNA/química , RNA/metabolismo , Análise de Sequência de RNA , Índice de Gravidade de Doença , Regulação para Cima , Tratamento Farmacológico da COVID-19RESUMO
Coronavirus disease 2019 (COVID-19) is a mild to moderate respiratory tract infection, however, a subset of patients progress to severe disease and respiratory failure. The mechanism of protective immunity in mild forms and the pathogenesis of severe COVID-19 associated with increased neutrophil counts and dysregulated immune responses remain unclear. In a dual-center, two-cohort study, we combined single-cell RNA-sequencing and single-cell proteomics of whole-blood and peripheral-blood mononuclear cells to determine changes in immune cell composition and activation in mild versus severe COVID-19 (242 samples from 109 individuals) over time. HLA-DRhiCD11chi inflammatory monocytes with an interferon-stimulated gene signature were elevated in mild COVID-19. Severe COVID-19 was marked by occurrence of neutrophil precursors, as evidence of emergency myelopoiesis, dysfunctional mature neutrophils, and HLA-DRlo monocytes. Our study provides detailed insights into the systemic immune response to SARS-CoV-2 infection and reveals profound alterations in the myeloid cell compartment associated with severe COVID-19.
Assuntos
Infecções por Coronavirus/imunologia , Células Mieloides/imunologia , Mielopoese , Pneumonia Viral/imunologia , Adulto , Idoso , Antígenos CD11/genética , Antígenos CD11/metabolismo , COVID-19 , Células Cultivadas , Infecções por Coronavirus/sangue , Infecções por Coronavirus/patologia , Feminino , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Células Mieloides/citologia , Pandemias , Pneumonia Viral/sangue , Pneumonia Viral/patologia , Proteoma/genética , Proteoma/metabolismo , Proteômica , Análise de Célula ÚnicaRESUMO
The molecular events causing memory loss and neuronal cell death in Alzheimer's disease (AD) over time are still unknown. Here we found that picomolar concentrations of soluble oligomers of synthetic beta amyloid (Aß42) aggregates incubated with BV2 cells or rat astrocytes caused a sensitised response of Toll-like receptor 4 (TLR4) with time, leading to increased production of TNF-α. Aß aggregates caused long term potentiation (LTP) deficit in hippocampal slices and predominantly neuronal cell death in co-cultures of astrocytes and neurons, which was blocked by TLR4 antagonists. Soluble Aß aggregates cause LTP deficit and neuronal death via an autocrine/paracrine mechanism due to TLR4 signalling. These findings suggest that the TLR4-mediated inflammatory response may be a key pathophysiological process in AD.
Assuntos
Peptídeos beta-Amiloides/fisiologia , Neurônios/fisiologia , Agregados Proteicos/fisiologia , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Animais Recém-Nascidos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Mamíferos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/fisiopatologia , Agregação Patológica de Proteínas/psicologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
Passerine birds belong to the most species rich bird order and are found in a wide range of habitats. The extremely polymorphic adaptive immune system of passerines, identified through their major histocompatibility complex class I genes (MHC-I), may explain some of this extreme radiation. Recent work has shown that passerines have higher numbers of MHC-I gene copies than other birds, but little is currently known about expression and function of these gene copies. Non-passerine birds have a single highly expressed MHC-I gene copy, a pattern that seems unlikely in passerines. We used high-throughput sequencing to study MHC-I alleles in siskins (Spinus spinus) and determined gene expression, phylogenetic relationships and sequence divergence. We verified between six and 16 MHC-I alleles per individual and 97% of these were expressed. Strikingly, up to five alleles per individual had high expression. Out of 88 alleles 18 were putatively non-classical with low sequence divergence and expression, and found in a single phylogenetic cluster. The remaining 70 alleles were classical, with high sequence divergence and variable degrees of expression. Our results contradict the suggestion that birds only have a single dominantly expressed MHC-I gene by demonstrating several highly expressed MHC-I gene copies in a passerine.
Assuntos
Genes MHC da Classe II/genética , Seleção Genética/genética , Alelos , Animais , Aves , Evolução Molecular , Éxons/genética , FilogeniaRESUMO
Most diurnal birds have cone-dominated retinae and tetrachromatic colour vision based on ultra-violet/violet-sensitive UV/V cones expressing short wavelength-sensitive opsin 1 (SWS1), S cones expressing short wavelength-sensitive opsin 2 (SWS2), M cones expressing medium wavelength-sensitive opsin (RH2) and L cones expressing long wavelength-sensitive opsin (LWS). Double cones (D) express LWS but do not contribute to colour vision. Each cone is equipped with an oil droplet, transparent in UV/V cones, but pigmented by carotenoids: galloxanthin in S, zeaxanthin in M, astaxanthin in L and a mixture in D cones. Owls (Strigiformes) are crepuscular or nocturnal birds with rod-dominated retinae and optical adaptations for high sensitivity. For eight species, the absence of functional SWS1 opsin has recently been documented, functional RH2 opsin was absent in three of these. Here we confirm the absence of SWS1 transcripts for the Long-eared owl (Asio otus) and demonstrate its absence for the Short-eared owl (Asio flammeus), Tawny owl (Strix aluco) and Boreal owl (Aegolius funereus). All four species had transcripts of RH2, albeit with low expression. All four species express all enzymes needed to produce galloxanthin, but lack CYP2J19 expression required to produce astaxanthin from dietary precursors. We also present ocular media transmittance of the Eurasian eagle owl (Bubo bubo) and Short-eared owl and predict spectral sensitivities of all photoreceptors of the Tawny owl. We conclude that owls, despite lacking UV/V cones, can detect UV light. This increases the sensitivity of their rod vision allowing them, for instance, to see UV-reflecting feathers as brighter signals at night.
Assuntos
Carotenoides/metabolismo , Visão de Cores/fisiologia , Células Fotorreceptoras Retinianas Cones/metabolismo , Opsinas de Bastonetes/genética , Estrigiformes/fisiologia , Transcriptoma/fisiologia , Raios Ultravioleta , Animais , Primers do DNA/química , Expressão Gênica , Visão Noturna/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Visão Ocular/fisiologia , Xantofilas/metabolismoRESUMO
In the original publication of this article, the author Magarida Rodrigues was written incorrectly.
RESUMO
Despite the wealth of genomic and transcriptomic data in Parkinson's disease (PD), the initial molecular events are unknown. Using LD score regression analysis, we show significant enrichment in PD heritability within regulatory sites for LPS-activated monocytes and that TLR4 expression is highest within human substantia nigra, the most affected brain region, suggesting a role for TLR4 inflammatory responses. We then performed extended incubation of cells with physiological concentrations of small alpha-synuclein oligomers observing the development of a TLR4-dependent sensitized inflammatory response with time, including TNF-α production. ROS and cell death in primary neuronal cultures were significantly reduced by TLR4 antagonists revealing that an indirect inflammatory mechanism involving cytokines produced by glial cells makes a major contribution to neuronal death. Prolonged exposure to low levels of alpha-synuclein oligomers sensitizes TLR4 responsiveness in astrocytes and microglial, explaining how they become pro-inflammatory, and may be an early causative event in PD.
Assuntos
Astrócitos/metabolismo , Microglia/metabolismo , Doença de Parkinson/metabolismo , Receptor 4 Toll-Like/metabolismo , alfa-Sinucleína/metabolismo , Animais , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular , Citocinas/metabolismo , Humanos , Inflamação/patologia , Microglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/patologia , Substância Negra/patologiaRESUMO
The pathogenesis of Alzheimer's disease (AD) is thought to involve acute neurotoxic effects exerted by oligomeric forms of amyloid-ß 1-42 (Aß). Application of Aß oligomers in physiological concentrations have been shown to transiently elevate internal Ca2+ in cultured astroglia. While the cellular machinery involved has been extensively explored, to what degree this important signalling cascade occurs in organised brain tissue has remained unclear. Here we adapted two-photon excitation microscopy and calibrated time-resolved imaging (FLIM), coupled with patch-clamp electrophysiology, to monitor Ca2+ concentration ([Ca2+]) inside individual astrocytes and principal neurons in acute brain slices. Inside the slice tissue local micro-ejection of Aß in sub-micromolar concentrations triggered prominent [Ca2+] elevations in an adjacent astrocyte translated as an approximately two-fold increase (averaged over â¼5min) in basal [Ca2+]. This elevation did not spread to neighbouring cells and appeared comparable in amplitude with commonly documented spontaneous [Ca2+] rises in astroglia. Principal nerve cells (pyramidal neurons) also showed Ca2+ sensitivity, albeit to a lesser degree. These observations shed light on the extent and dynamics of the acute physiological effects of Aß on brain cells in situ, in the context of AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Astrócitos/metabolismo , Cálcio/metabolismo , Hipocampo/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos beta-Amiloides/administração & dosagem , Animais , Astrócitos/efeitos dos fármacos , Cátions Bivalentes/metabolismo , Fármacos do Sistema Nervoso Central/administração & dosagem , Hipocampo/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Microscopia de Fluorescência , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/administração & dosagem , Ratos Sprague-Dawley , Análise de Célula Única , Técnicas de Cultura de TecidosRESUMO
One potential therapeutic strategy for Alzheimer's disease (AD) is to use antibodies that bind to small soluble protein aggregates to reduce their toxic effects. However, these therapies are rarely tested in human CSF before clinical trials because of the lack of sensitive methods that enable the measurement of aggregate-induced toxicity at low concentrations. We have developed highly sensitive single vesicle and single-cell-based assays that detect the Ca2+ influx caused by the CSF of individuals affected with AD and healthy controls, and we have found comparable effects for both types of samples. We also show that an extracellular chaperone clusterin; a nanobody specific to the amyloid-ß peptide (Aß); and bapineuzumab, a humanized monoclonal antibody raised against Aß, could all reduce the Ca2+ influx caused by synthetic Aß oligomers but are less effective in CSF. These assays could be used to characterize potential therapeutic agents in CSF before clinical trials.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Bioensaio , Cálcio/metabolismo , Líquido Cefalorraquidiano/química , Vesículas Citoplasmáticas/efeitos dos fármacos , Fragmentos de Peptídeos/antagonistas & inibidores , Agregados Proteicos/efeitos dos fármacos , Idoso , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Animais , Anticorpos Monoclonais Humanizados/farmacologia , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Clusterina/farmacologia , Meios de Cultura/farmacologia , Vesículas Citoplasmáticas/metabolismo , Feminino , Humanos , Transporte de Íons/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Cultura Primária de Células , Ratos , Anticorpos de Domínio Único/farmacologiaRESUMO
BACKGROUND: High-throughput sequencing enables high-resolution genotyping of extremely duplicated genes. 454 amplicon sequencing (454) has become the standard technique for genotyping the major histocompatibility complex (MHC) genes in non-model organisms. However, illumina MiSeq amplicon sequencing (MiSeq), which offers a much higher read depth, is now superseding 454. The aim of this study was to quantitatively and qualitatively evaluate the performance of MiSeq in relation to 454 for genotyping MHC class I alleles using a house sparrow (Passer domesticus) dataset with pedigree information. House sparrows provide a good study system for this comparison as their MHC class I genes have been studied previously and, consequently, we had prior expectations concerning the number of alleles per individual. RESULTS: We found that 454 and MiSeq performed equally well in genotyping amplicons with low diversity, i.e. amplicons from individuals that had fewer than 6 alleles. Although there was a higher rate of failure in the 454 dataset in resolving amplicons with higher diversity (6-9 alleles), the same genotypes were identified by both 454 and MiSeq in 98% of cases. CONCLUSIONS: We conclude that low diversity amplicons are equally well genotyped using either 454 or MiSeq, but the higher coverage afforded by MiSeq can lead to this approach outperforming 454 in amplicons with higher diversity.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Complexo Principal de Histocompatibilidade/genética , Animais , PardaisRESUMO
BACKGROUND: The Major Histocompatibility Complex (MHC) plays a central role in immunity and has been given considerable attention by evolutionary ecologists due to its associations with fitness-related traits. Songbirds have unusually high numbers of MHC class I (MHC-I) genes, but it is not known whether all are expressed and equally important for immune function. Classical MHC-I genes are highly expressed, polymorphic and present peptides to T-cells whereas non-classical MHC-I genes have lower expression, are more monomorphic and do not present peptides to T-cells. To get a better understanding of the highly duplicated MHC genes in songbirds, we studied gene expression in a phylogenetic framework in three species of sparrows (house sparrow, tree sparrow and Spanish sparrow), using high-throughput sequencing. We hypothesize that sparrows could have classical and non-classical genes, as previously indicated though never tested using gene expression. RESULTS: The phylogenetic analyses reveal two distinct types of MHC-I alleles among the three sparrow species, one with high and one with low level of polymorphism, thus resembling classical and non-classical genes, respectively. All individuals had both types of alleles, but there was copy number variation both within and among the sparrow species. However, the number of highly polymorphic alleles that were expressed did not vary between species, suggesting that the structural genomic variation is counterbalanced by conserved gene expression. Overall, 50% of the MHC-I alleles were expressed in sparrows. Expression of the highly polymorphic alleles was very variable, whereas the alleles with low polymorphism had uniformly low expression. Interestingly, within an individual only one or two alleles from the polymorphic genes were highly expressed, indicating that only a single copy of these is highly expressed. CONCLUSIONS: Taken together, the phylogenetic reconstruction and the analyses of expression suggest that sparrows have both classical and non-classical MHC-I genes, and that the evolutionary origin of these genes predate the split of the three investigated sparrow species 7 million years ago. Because only the classical MHC-I genes are involved in antigen presentation, the function of different MHC-I genes should be considered in future ecological and evolutionary studies of MHC-I in sparrows and other songbirds.
Assuntos
Genes MHC Classe I , Pardais/classificação , Pardais/genética , Animais , Evolução Biológica , Variações do Número de Cópias de DNA , Evolução Molecular , Expressão Gênica , FilogeniaRESUMO
The major histocompatibility complex (MHC) encodes proteins that are central for antigen presentation and pathogen elimination. MHC class I (MHC-I) genes have attracted a great deal of interest among researchers in ecology and evolution and have been partly characterized in a wide range of bird species. So far, the main focus has been on species within the bird orders Galliformes and Passeriformes, while Charadriiformes remain vastly underrepresented with only two species studied to date. These two Charadriiformes species exhibit striking differences in MHC-I characteristics and MHC-I diversity. We therefore set out to study a third species within Charadriiformes, the Icelandic subspecies of black-tailed godwits (Limosa limosa islandica). This subspecies is normally confined to parasite-poor environments, and we hence expected low MHC diversity. MHC-I was partially characterized first using Sanger sequencing and then using high-throughput sequencing (MiSeq) in 84 individuals. We verified 47 nucleotide alleles in open reading frame with classical MHC-I characteristics, and each individual godwit had two to seven putatively classical MHC alleles. However, in contrast to previous MHC-I data within Charadriiformes, we did not find any evidence of alleles with low sequence diversity, believed to represent non-classical MHC genes. The diversity and divergence of the godwits MHC-I genes to a large extent fell between the previous estimates within Charadriiformes. However, the MHC genes of the migratory godwits had few sites subject to positive selection, and one possible explanation could be a low exposure to pathogens.