RESUMO
Neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS) are traditionally considered strictly neurological disorders. However, clinical presentation is not restricted to neurological systems, and non-central nervous system (CNS) manifestations, particularly gastrointestinal (GI) symptoms, are common. Our objective was to understand the systemic distribution of pathology in archived non-CNS tissues, taken as part of routine clinical practice during life from people with ALS. We examined tissue from 13 people who went on to develop ALS; including sporadic ALS (n = 12) and C9orf72 hexanucleotide repeat expansion (n = 1). The tissue cohort consisted of 68 formalin-fixed paraffin embedded samples from 21 surgical cases (some patients having more than one case over their lifetimes), from 8 organ systems, which we examined for evidence of phosphorylated TDP-43 (pTDP-43) pathology. We identified pTDP-43 aggregates in multiple cell types of the GI tract, including macrophages and dendritic cells within the lamina propria; as well as ganglion/neuronal and glial cells of the myenteric plexus. Aggregates were also noted within lymph node parenchyma, blood vessel endothelial cells, and chondrocytes. We note that in all cases with non-CNS pTDP-43 pathology, aggregates were present prior to ALS diagnosis and in some instances preceded neurological symptom onset by more than 10 years. These data imply that patients with microscopically unexplained non-CNS symptoms could have occult protein aggregation that could be detected many years prior to neurological involvement.
Assuntos
Esclerose Lateral Amiotrófica , Humanos , Esclerose Lateral Amiotrófica/genética , Células EndoteliaisRESUMO
Clinical heterogeneity observed across patients with amyotrophic lateral sclerosis (ALS) is a known complicating factor in identifying potential therapeutics, even within cohorts with the same mutation, such as C9orf72 hexanucleotide repeat expansions (HREs). Thus, further understanding of pathways underlying this heterogeneity is essential for appropriate ALS trial stratification and the meaningful assessment of clinical outcomes. It has been shown that both inflammation and protein misfolding can influence ALS pathogenesis, such as the manifestation or severity of motor or cognitive symptoms. However, there has yet to be a systematic and quantitative assessment of immunohistochemical markers to interrogate the potential relevance of these pathways in an unbiased manner. To investigate this, we extensively characterised features of commonly used glial activation and protein misfolding stains in thousands of images of post-mortem tissue from a heterogeneous cohort of deeply clinically profiled patients with a C9orf72 HRE. Using a random forest model, we show that microglial staining features are the most accurate classifiers of disease status in our panel and that clinicopathological relationships exist between microglial activation status, TDP-43 pathology, and language dysfunction. Furthermore, we detected spatially resolved changes in fused in sarcoma (FUS) staining, suggesting that liquid-liquid phase shift of this aggregation-prone RNA-binding protein may be important in ALS caused by a C9orf72 HRE. Interestingly, no one feature alone significantly impacted the predictiveness of the model, indicating that the collective examination of all features, or a combination of several features, is what allows the model to be predictive. Our findings provide further support to the hypothesis of dysfunctional immune regulation and proteostasis in the pathogenesis of C9-ALS and provide a framework for digital analysis of commonly used neuropathological stains as a tool to enrich our understanding of clinicopathological relationships within and between cohorts. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Proteína C9orf72/genética , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Microglia/patologia , MutaçãoRESUMO
BACKGROUND: The abomasal helminth Teladorsagia circumcincta is one of the most economically important parasites affecting sheep in temperate regions. Infection is particularly detrimental to lambs, in which it can cause pronounced morbidity and severe production losses. Due to the spreading resistance of this parasite to all classes of anthelmintic drugs, teladorsagiosis is having an increasingly severe impact on the sheep industry with significant implications for sheep welfare. Protective immunity develops slowly, wanes rapidly and does not appear to be as effective in young lambs. To investigate the development of immunity to T. circumcincta in sheep and lambs, we used cytokine transcript profiling to examine differences in the abomasal mucosa and gastric lymph node of naïve and previously infected sheep and lambs following challenge. RESULTS: The results of these experiments demonstrated that the abomasal mucosa is a major source of cytokines during abomasal helminth infection. A local Th2-type cytokine response was observed in the abomasal mucosa and gastric lymph node of the previously infected sheep and lambs when compared with those of the naïve during the early stages of infection. In contrast, a pro-inflammatory component more was evident in the abomasal mucosa and gastric lymph node of the naïve sheep when compared with those of the previously infected, which was not observed in the lambs. CONCLUSIONS: The greater levels of Th2-type cytokine transcripts in both the abomasum and gastric lymph node of the previously infected compared with naïve sheep and lambs emphasises the importance of these mechanisms in the immune response to T. circumcincta infection. Younger lambs appear to be able to generate similar Th2-type responses in the abomasum suggesting that the increased morbidity and apparent lack of resistance in younger lambs following continuous or repeated exposure to T. circumcincta is unlikely to be due to a lack of appropriate Th2-type cytokine production.
Assuntos
Citocinas/fisiologia , Ostertagia , Ostertagíase/veterinária , Doenças dos Ovinos/parasitologia , Abomaso/parasitologia , Animais , Animais Recém-Nascidos/imunologia , Animais Recém-Nascidos/parasitologia , Animais Recém-Nascidos/fisiologia , Contagem de Células/veterinária , Citocinas/biossíntese , Mastócitos/fisiologia , Ostertagíase/imunologia , Ovinos/parasitologia , Doenças dos Ovinos/imunologia , Transcrição GênicaRESUMO
Continual low-level exposure of sheep to the helminth Teladorsagia circumcincta elicits a temporary protective immunity, where factors in the immune abomasal mucosa prevent penetration of infective larvae, but which is essentially lost within 6 weeks of cessation of parasite challenge. Here, a proteomic approach was used to identify proteins that are differentially regulated in immune compared to naïve sheep, as potential key mediators of immunity. Six naïve sheep and 12 sheep trickle-infected with T. circumcincta were treated with anthelmintic, and the naïve (control) and 6 immune sheep were killed 7 days later. The remaining 6 sheep (immune waning) were killed 42 days after anthelmintic treatment. Abomasal tissue samples were subjected to 2D-gel electrophoresis and densitometric analysis. Selected spots (n=73) were identified by peptide mass fingerprinting and confirmatory Western blotting was carried out for 10 proteins. Spots selectively up-regulated in immune versus control, but not immune waning versus control sheep, included galectin-15 and thioredoxin, which were confirmed by Western blotting. In immune sheep, serum albumin was significantly down-regulated and albumin proteolytic cleavage fragments were increased compared to controls. Unexpectedly, albumin mRNA was relatively highly expressed in control mucosa, down-regulated in immune, and was immunolocalized to mucus-producing epithelial cells. Thus we have identified differential expression of a number of proteins following T. circumcincta trickle infection that may play a role in host protection and inhibition of parasite establishment.
Assuntos
Abomaso/metabolismo , Proteínas de Helminto/metabolismo , Doenças dos Ovinos/metabolismo , Trichostrongyloidea , Tricostrongiloidíase/veterinária , Abomaso/efeitos dos fármacos , Abomaso/imunologia , Imunidade Adaptativa , Animais , Anti-Helmínticos/administração & dosagem , Western Blotting , Eletroforese em Gel Bidimensional , Galectinas/genética , Galectinas/metabolismo , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/imunologia , Mucosa Gástrica/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Helminto/genética , Proteômica , RNA Mensageiro/metabolismo , Albumina Sérica/genética , Albumina Sérica/metabolismo , Ovinos , Doenças dos Ovinos/tratamento farmacológico , Doenças dos Ovinos/imunologia , Carneiro Doméstico , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Tricostrongiloidíase/tratamento farmacológico , Tricostrongiloidíase/imunologia , Tricostrongiloidíase/metabolismoRESUMO
Infection of sheep with the gastric nematode Teladorsagia circumcincta results in distinct Th2-type changes in the mucosa, including mucous neck cell and mast cell hyperplasia, eosinophilia, recruitment of IgA/IgE producing cells and neutrophils, altered T-cell subsets and mucosal hypertrophy. To address the protective mechanisms generated in animals on previous exposure to this parasite, gene expression profiling was carried out using samples of abomasal mucosa collected pre- and post- challenge from animals of differing immune status, using an experimental model of T. circumcincta infection. Recently developed ovine cDNA arrays were used to compare the abomasal responses of sheep immunised by trickle infection with worm-naïve sheep, following a single oral challenge of 50 000 T. circumcincta L3. Key changes were validated using qRT-PCR techniques. Immune animals demonstrated highly significant increases in levels of transcripts normally associated with cytotoxicity such as granulysin and granzymes A, B and H, as well as mucous-cell derived transcripts, predominantly calcium-activated chloride channel 1 (CLCA1). Challenge infection also induced up-regulation of transcripts potentially involved in initiating or modulating the immune response, such as heat shock proteins, complement factors and the chemokine CCL2. In contrast, there was marked infection-associated down-regulation of gene expression of members of the gastric lysozyme family. The changes in gene expression levels described here may reflect roles in direct anti-parasitic effects, immuno-modulation or tissue repair.
Assuntos
Abomaso/metabolismo , Regulação da Expressão Gênica , Mucosa Intestinal/metabolismo , Doenças dos Ovinos/genética , Trichostrongyloidea/fisiologia , Tricostrongiloidíase/veterinária , Abomaso/parasitologia , Animais , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica/veterinária , Mucosa Intestinal/parasitologia , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos/veterinária , Reação em Cadeia da Polimerase/veterinária , Análise de Sequência de DNA/veterinária , Ovinos , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/parasitologia , Tricostrongiloidíase/imunologia , Tricostrongiloidíase/metabolismo , Tricostrongiloidíase/parasitologiaRESUMO
Sheep intelectin1 and sheep intelectin3 (sITLN1 and sITLN3) were cloned and sequenced. The amino acid sequences of sITLN1 and sITLN3 shared 86% and 91% homology with the previously cloned sheep intelectin2 (sITLN2), respectively. Expression of sITLN1 and sITLN3 transcript was demonstrated in abomasum, lung, colon and gastric lymph node, terminal rectum, skin, jejunum, mesenteric lymph node, ileal peyer's patches, brain, kidney, liver, spleen, skin, ear pinna, heart and ovary in normal sheep tissues. sITLN2 transcript expression was restricted to the abomasal mucosa in normal sheep tissues. Using a non selective chicken anti-intelectin antibody, tissue intelectin protein was demonstrated in mucus neck cells in the abomasum, mucus cells in the colon, free mucus in ileum, goblet cells in the lung, small intestinal epithelium and brush border, epidermal layer of the skin and skin sebaceous glands. The expression of the three sITLN transcripts was examined in two nematode infections in sheep known to induce a Th2 response; a Teladorsagia circumcincta challenge infection model and a Dictyocaulus filaria natural infection. The three sITLN were absent in unchallenged naïve lambs and present in the abomasal mucosa of both naïve and immune lambs following T. circumcincta challenge infection. Upregulation of sITLN2 and sITLN3 was shown in sheep lung following D. filaria natural infection. Intelectins may play an important role in the mucosal response to nematode infections in ruminants.
Assuntos
Regulação da Expressão Gênica/imunologia , Lectinas/metabolismo , Infecções por Nematoides/veterinária , Doenças dos Ovinos/metabolismo , Animais , Clonagem Molecular , Infecções por Nematoides/metabolismo , Ovinos , Doenças dos Ovinos/parasitologiaRESUMO
A novel intelectin molecule designated sheep intelectin 2 (sITLN2) was detected in sheep abomasal mucosa. The full sequence shared 76-83% homology with other mammalian intelectins. Intelectins are mucus-associated proteins that have been shown to be up-regulated in gastrointestinal nematode infections in rodents and in human asthma. Expression of sheep abomasal ITLN2 mRNA was significantly up-regulated on day 10 post-challenge of worm-free sheep with Teladorsagia circumcincta and at day 2 in previously infected, immune sheep. Increased expression of ITLN protein following challenge was confirmed by Western blot and was immunolocalised to the mucous neck cells of the abomasal mucosa. Infection with T. circumcincta was also associated with increased levels of abomasal transcripts encoding sheep mast cell protease-1, ovine galectin-14 and IL4, which collectively suggested a Th2 type response. Intelectin may play an important role in the mucosal response to gastrointestinal nematode infections in ruminants.
Assuntos
Abomaso/imunologia , Galectinas/metabolismo , Enteropatias Parasitárias/imunologia , Infecções por Nematoides/imunologia , Doenças dos Ovinos/parasitologia , Regulação para Cima , Abomaso/parasitologia , Animais , Sequência de Bases , Western Blotting/métodos , Quimases/genética , Quimases/metabolismo , Feminino , Galectinas/genética , Mucosa Gástrica/metabolismo , Mucosa Gástrica/parasitologia , Interações Hospedeiro-Parasita , Interleucina-4/genética , Interleucina-4/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , OvinosRESUMO
Phospholipase A2 (PLA2) enzymes play a central role in the initiation, propagation and resolution of inflammation. Here, we describe de novo expression of group IVC PLA2 (PLA2g4c) within the intestinal epithelium of Trichinella spiralis parasitised mice. This mouse mast cell protease-1 sensitive, calcium-independent PLA2 is not detectable in the jejunal epithelium of uninfected mice but becomes highly expressed within the epithelial compartment within days of nematode establishment. We propose that epithelial PLA2g4c accounts for the increased lysophospholipase activity observed during intestinal nematodiasis and that it plays a major role in the inflammatory response to nematodes.
Assuntos
Fosfolipases A2 do Grupo IV/genética , Enteropatias Parasitárias/enzimologia , Mucosa Intestinal/enzimologia , Trichinella spiralis/fisiologia , Triquinelose/enzimologia , Animais , Quimases/metabolismo , Expressão Gênica , Fosfolipases A2 do Grupo IV/metabolismo , Inflamação , Jejuno , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Infection of mice with the nematode Trichinella spiralis triggers recruitment and differentiation of intraepithelial intestinal mucosal mast cells expressing mouse mast cell protease 1 (Mcpt-1), which contributes to expulsion of the parasite. Expression of Mcpt-1 is transforming growth factor (TGF)-beta1-dependent in vitro. TGF-beta1, which is secreted within tissues as a biologically inactive complex with latency-associated peptide, requires extracellular modification to become functionally active. The integrin-alpha(nu)beta(6) mediates local activation of TGF-beta(1) in association with epithelia. Using T. spiralis-infected beta(6)(-/-) mice, we show accumulation of mucosal mast cells in the lamina propria of the small intestine with minimal recruitment into the epithelial compartment. This was accompanied by a coordinate reduction in expression of both Mcpt-1 and -2 in the jejunum and increased tryptase expression, whereas Mcpt-9 became completely undetectable. In contrast, the cytokine stem cell factor, a regulator of mast cell differentiation and survival, was significantly up-regulated in T. spiralis-infected beta(6)(-/-) mice compared with infected beta(6)(+/+) controls. Despite these changes, beta(6)(-/-) mice still appeared to expel the worms normally. We postulate that compromised TGF-beta(1) activation within the gastrointestinal epithelial compartment is a major, but not the only, contributing factor to the observed changes in mucosal mast cell protease and epithelial cytokine expression in beta(6)(-/-) mice.
Assuntos
Antígenos de Neoplasias/genética , Quimases/metabolismo , Integrinas/genética , Mucosa Intestinal/imunologia , Mastócitos/enzimologia , Fator de Crescimento Transformador beta1/metabolismo , Trichinella spiralis , Triquinelose/imunologia , Animais , Medula Óssea/imunologia , Quimases/análise , Quimases/genética , Colo/imunologia , Citocinas/genética , Citocinas/metabolismo , Orelha , Jejuno/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Mutantes , Estômago/imunologiaRESUMO
Evolved functions of integrin-alpha(v)beta(6) include roles in epithelial cell-extracellular matrix protein interactions and in the binding and activation of latent TGF-beta(1). Integrin-alpha(v)beta(6) is also exploited as a receptor by foot-and-mouth disease virus (FMDV) and may play a significant role in its transmission and pathogenesis. The ovine beta(6) integrin subunit was cloned and sequenced (EMBL accession no. AJ439062). Screening of normal ovine tissues by RT-PCR and immunocytochemistry confirmed that integrin-alphavbeta6 is restricted to sheep epithelial cells. Integrin-alphavbeta6 expression was detected in epithelia of the airways, oral cavity, gastrointestinal tract, kidney, sweat glands, hair follicle sheaths, and the epidermis of pedal coronary band (PB) but not of normal skin. Consistent with FMDV tropism, integrin-alphavbeta6 was detected within the basal layers of the stratified squamous epithelium of the oral mucosa and PB. In addition, integrin-alphavbeta6 appears to be constitutively expressed in the normal airways of both cattle and sheep. The latter finding suggests that ruminant airway epithelium presents a highly accessible target for initiation of infection with FMDV by inhalation.
Assuntos
Antígenos de Neoplasias/biossíntese , Vírus da Febre Aftosa/metabolismo , Integrinas/biossíntese , Receptores Virais/biossíntese , Sistema Respiratório/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/genética , Sequência de Bases , Bovinos , Clonagem Molecular , Dimerização , Feminino , Imuno-Histoquímica , Integrinas/genética , Pulmão/metabolismo , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Receptores Virais/genética , Mucosa Respiratória/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , OvinosRESUMO
Peak intestinal mucosal mast cell (MMC) recruitment coincides with expulsion of Trichinella spiralis, at a time when the majority of the MMCs are located within the epithelium in BALB/c mice. Although expression of integrin-alpha(E)beta(7) by MMCs has not been formally demonstrated, it has been proposed as a potential mechanism to account for the predominantly intraepithelial location of MMCs during nematode infection. Co-expression of integrin-alpha(E)beta(7) and the MMC chymase mouse mast cell protease-1, by mouse bone marrow-derived mast cells, is strictly regulated by transforming growth factor (TGF)-beta(1). However, TGF-beta(1) is secreted as part of a latent complex in vivo and subsequent extracellular modification is required to render it biologically active. We now show, for the first time, that intraepithelial MMCs express integrin-alpha(E)beta(7) in Trichinella-infected BALB/c and S129 mice. In S129 mice that lack the gene for the integrin-beta(6) subunit and, as consequence, do not express the epithelial integrin-alpha(v)beta(6), integrin-alpha(E) expression is virtually abolished and recruitment of MMCs into the intestinal epithelium is dramatically reduced despite significant overall augmentation of the MMC population. Because a major function of integrin-alpha(v)beta(6) is to activate latent TGF-beta(1,) these findings strongly support a role for TGF-beta(1) in both the recruitment and differentiation of murine MMCs during nematode infection.