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Multiple sclerosis (MS) is an immune-mediated central nervous system (CNS) disease characterized by demyelination resulting from oligodendrocyte loss and inflammation. Cuprizone (CPZ) administration experimentally replicates MS pattern-III lesions, generating an inflammatory response through microgliosis and astrogliosis. Potentially remyelinating agents include oligodeoxynucleotides (ODN) with a specific immunomodulatory sequence consisting of the active motif PyNTTTTGT. In this work, the remyelinating effects of ODN IMT504 were evaluated through immunohistochemistry and qPCR analyses in a rat CPZ-induced demyelination model. Subcutaneous IMT504 administration exacerbated the pro-inflammatory response to demyelination and accelerated the transition to an anti-inflammatory state. IMT504 reduced microgliosis in general and the number of phagocytic microglia in particular and expanded the population of oligodendroglial progenitor cells (OPCs), later reflected in an increase in mature oligodendrocytes. The intracranial injection of IMT504 and intravenous inoculation of IMT504-treated B lymphocytes rendered comparable results. Altogether, these findings unveil potentially beneficial properties of IMT504 in the regulation of neuroinflammation and oligodendrogenesis, which may aid the development of therapies for demyelinating diseases such as MS.
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During pregnancy, a decreased availability of zinc to the fetus can disrupt the development of the central nervous system leading to defects ranging from severe malformations to subtle neurological and cognitive effects. We previously found that marginal zinc deficiency down-regulates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and affects neural progenitor cell (NPC) proliferation. This study investigated if marginal zinc deficiency during gestation in rats could disrupt fetal neurogenesis and affect the number and specification of neurons in the adult offspring brain cortex. Rats were fed a marginal zinc deficient or adequate diet throughout gestation and until postnatal day (P) 2, and subsequently the zinc adequate diet until P56. Neurogenesis was evaluated in the offspring at embryonic day (E)14, E19, P2, and P56 measuring parameters of NPC proliferation and differentiation by Western blot and/or immunofluorescence. At E14 and E19, major signals (i.e., ERK1/2, Sox2, and Pax6) that stimulate NPC proliferation and self-renewal were markedly downregulated in the marginal zinc deficient fetal brain. These alterations were associated to a lower number of Ki67 positive cells in the ventricular (VZs) and subventricular zones (SVZs). Following the progression of NPCs into intermediate progenitor cells (IPCs) and into neurons, Pax6, Tbr2 and Tbr1 were affected in the corresponding areas of the brain at E19 and P2. The above signaling alterations led to a lower density of neurons and a selective decrease of glutamatergic neurons in the young adult brain cortex exposed to maternal marginal zinc deficiency from E14 to P2. Current results supports the concept that marginal zinc deficiency during fetal development can disrupt neurogenesis and alter cortical structure potentially leading to irreversible neurobehavioral impairments later in life.
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Adult neural progenitor cells (NPCs) are capable of differentiating into neurons, astrocytes, and oligodendrocytes throughout life. Notch and transforming growth factor ß1 (TGF-ß) signaling pathways play critical roles in controlling these cell fate decisions. TGF-ß has been previously shown to exert pro-neurogenic effects on hippocampal and subventricular zone (SVZ) NPCs in vitro and to interact with Notch in different cellular types. Therefore, the aim of our work was to study the effect of TGF-ß on adult rat brain SVZ NPC glial commitment and its interaction with Notch signaling. Initial cell characterization revealed a large proportion of Olig2+, Nestin+, and glial fibrillary acidic protein (GFAP+) cells, a low percentage of platelet-derived growth factor receptor α (PDGFRα+) or NG2+ cells, and <1% Tuj1+ cells. Immunocytochemical analyses showed a significant increase in the percentage of PDGFRα+, NG2+, and GFAP+ cells upon four-day TGF-ß treatment, which demonstrates the pro-gliogenic effect of this growth factor on adult brain SVZ NPCs. Real-time polymerase chain reaction analyses showed that TGF-ß induced the expression of Notch ligand Jagged1 and downstream gene Hes1. Notch signaling inhibition in cultures treated with TGF-ß produced a decrease in the proportion of PDGFRα+ cells, while TGF-ß receptor II (TßRII) inhibition also rendered a decrease in the proportion of PDGFRα+ cells, concomitantly with a decrease in Jagged1 levels. These findings demonstrate the participation of Notch signaling in TGF-ß effects and illustrate the impact of TGF-ß on glial cell fate decisions of adult brain SVZ NPCs, as well as on oligodendroglial progenitor cell proliferation and maturation.
Assuntos
Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Células-Tronco Neurais/metabolismo , Oligodendroglia/metabolismo , Receptores Notch/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fatores Etários , Animais , Células Cultivadas , Humanos , Ventrículos Laterais/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Galectin-1 (Gal-1), a member of a highly conserved family of animal lectins, binds to the common disaccharide [Galß(1-4)-GlcNAc] on both N- and O-glycans decorating cell surface glycoconjugates. Current evidence supports a role for Gal-1 in the pathophysiology of multiple sclerosis (MS), one of the most prevalent chronic inflammatory diseases. Previous studies showed that Gal-1 exerts neuroprotective effects by promoting microglial deactivation in a model of autoimmune neuroinflammation and induces axonal regeneration in spinal cord injury. Seeking a model that could link demyelination, oligodendrocyte (OLG) responses and microglial activation, here we used a lysolecithin (LPC)-induced demyelination model to evaluate the ability of Gal-1 to preserve myelin without taking part in T-cell modulation. Gal-1 treatment after LPC-induced demyelination promoted a significant decrease in the demyelinated area and fostered more efficient remyelination, concomitantly with an attenuated oligodendroglial progenitor response reflecting less severe myelination damage. These results were accompanied by a decrease in the area of microglial activation with a shift toward an M2-polarized microglial phenotype and diminished astroglial activation. In vitro studies further showed that, mechanistically, Gal-1 targets activated microglia, promoting an increase in their myelin phagocytic capacity and their shift toward an M2 phenotype, and leads to oligodendroglial differentiation. Therefore, this study supports the use of Gal-1 as a potential treatment for demyelinating diseases such as MS.
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Diferenciação Celular/efeitos dos fármacos , Doenças Desmielinizantes , Galectina 1/farmacologia , Galectina 1/uso terapêutico , Microglia/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/genética , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/efeitos dos fármacos , Encéfalo/ultraestrutura , Polaridade Celular/efeitos dos fármacos , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/tratamento farmacológico , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lisofosfatidilcolinas/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/ultraestrutura , Proteínas do Tecido Nervoso/metabolismo , Oligodendroglia/ultraestrutura , Técnicas de Cultura de TecidosRESUMO
Transforming growth factor beta 1 (TGF-beta1), an anti-inflammatory cytokine, has been shown to have pro-neurogenic effects on adult Neural Stem Cells (aNSC) from the dentate gyrus and in vivo models. Here, we expanded the observation of the pro-neurogenic effect of TGF-beta1 on aNSC from the subventricular zone (SVZ) of adult rats and performed a functional genomic analysis to identify candidate genes to mediate its effect. 10 candidate genes were identified by microarray analysis and further validated by qRT-PCR. Of these, Fibulin-2 was increased 477-fold and its inhibition by siRNA blocks TGF-beta1 pro-neurogenic effect. Curiously, Fibulin-2 was not expressed by aNSC but by a GFAP-positive population in the culture, suggesting an indirect mechanism of action. TGF-beta1 also induced Fibulin-2 in the SVZ in vivo. Interestingly, 5 out of the 10 candidate genes identified are known to interact with integrins, paving the way for exploring their functional role in adult neurogenesis. In conclusion, we have identified 10 genes with putative pro-neurogenic effects, 5 of them related to integrins and provided proof that Fibulin-2 is a major mediator of the pro-neurogenic effects of TGF-beta1. These data should contribute to further exploring the molecular mechanism of adult neurogenesis of the genes identified and the involvement of the integrin pathway on adult neurogenesis.
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Células-Tronco Adultas/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Células-Tronco Neurais/metabolismo , Neurogênese , Fator de Crescimento Transformador beta1/farmacologia , Células-Tronco Adultas/citologia , Células-Tronco Adultas/efeitos dos fármacos , Animais , Astrócitos/metabolismo , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Integrinas/metabolismo , Ventrículos Laterais/citologia , Ventrículos Laterais/crescimento & desenvolvimento , Ventrículos Laterais/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Oligodendroglial damage and demyelination are common pathological features characterizing white matter and neurodegenerative disorders. Identifying the signaling pathways involved in myelin repair through oligodendroglial progenitor maturation is essential for the development of new therapies. This article investigated the role of the Notch signaling pathway in CNS demyelination and apotransferrin-induced remyelination in a focal lysolecithin-induced demyelination model in rats. Notch was found activated in Nestin-expressing neural progenitor cells and in NG2-expressing oligodendroglial precursor cells in the subventricular zone and corpus callosum of lysolecithin-demyelinated rats. Notch activation seemed to be driven by Jagged1, which led to a high expression of downstream gene Hes5 in the subventricular zone of demyelinated rats. Apotransferrin injection induced remyelination, while the injection of the γ-secretase inhibitor reversed this effect. In addition, 24 h after apotransferrin injection, evidence showed Notch activation concomitantly with an increase in F3/contactin levels and the up-regulation of the myelin-associated glycoprotein gene in the subventricular zone and corpus callosum of demyelinated rats. Collected evidence supports the participation of both canonical and non-canonical Notch signaling pathways in demyelination/remyelination. Notch activation was found to trigger Hes5 expression as a consequence of focal demyelination, which might promote oligodendroglial precursor cell proliferation. During apotransferrin-induced remyelination, Notch activation seemed to be mediated by the expression of F3/contactin, which might induce apotransferrin-mediated oligodendroglial maturation. Evidence of the participation of Notch signaling in the demyelination/remyelination process will help further understand demyelinating disorders such as Multiple Sclerosis and the use of aTf should be taken into consideration as a possible therapeutic intervention.
Assuntos
Apoproteínas/farmacologia , Encéfalo/metabolismo , Doenças Desmielinizantes/metabolismo , Bainha de Mielina/metabolismo , Receptores Notch/metabolismo , Transferrina/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Corpo Caloso/efeitos dos fármacos , Corpo Caloso/metabolismo , Corpo Caloso/patologia , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Feminino , Lisofosfatidilcolinas , Masculino , Células-Tronco Neurais/patologia , Oligodendroglia/patologia , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
Peripheral inflammation, both during the prenatal period and in adulthood, impairs adult neurogenesis. We hypothesized that, similar to other programming effects of prenatal treatments, only prenatal inflammation causes long-term consequences in adult neurogenesis and its neurogenic niche. To test this, pregnant Wistar rats were subcutaneously injected with lipopolysaccharide (LPS; 0.5 mg/kg) or saline solution every other day from gestational/embryonic day (GD) 14-20. In addition adult animals were injected with a single intraperitoneal saline or LPS injection (1 mg/kg) and the effects on neurogenesis were assessed 7 days later. Alternatively, to evaluate long-term consequences of adult LPS injections, LPS (1 mg/kg) was administered peripherally to adult rats four times every other day, and the effects on neurogenesis were assessed 60 days later. Prenatal and adult LPS treatments reduced adult neurogenesis and provoked specific microglial (but not astroglial) activation in the dentate gyrus (DG). However, only prenatal inflammation-mediated effects were long-lasting (at least 60 days). Moreover, these effects were specific to the DG since the Subventricular Zone (SVZ) and the Rostral Migratory Stream (RMS) were not affected. In addition, these stimuli caused differential effects on the molecular components of the neurogenic niche; only prenatal LPS treatment reduced the local levels of TGF-ß1 mRNA in the DG. Finally, TGF-ß1 exerted its pro-neurogenic effects via the Smad 2/3 pathway in a neural stem cell culture. Taken together, these data add evidence to the duration, regional specificity and dramatic consequences of prenatal immune programming on CNS physiology, compared with the limited response observed in the adult brain.
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Giro Denteado/citologia , Lipopolissacarídeos/toxicidade , Neurogênese/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Fatores Etários , Animais , Astrócitos/citologia , Giro Denteado/efeitos dos fármacos , Giro Denteado/embriologia , Giro Denteado/metabolismo , Feminino , Inflamação/patologia , Masculino , Microglia/citologia , Neurogênese/efeitos dos fármacos , Gravidez , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Microglial activation in the substantia nigra (SN) is a ubiquitous feature in PD which could mediate toxic effects. Human mesenchymal stromal cells (hMSCs) possess immunomodulatory properties. We evaluated whether the transplantation of hMSCs obtained from umbilical cord had a neuroprotective effect in a not-immunosuppressed rat Parkinson's disease (PD) model. Rats receiving hMSCs in the SN displayed significant preservation in the number of dopaminergic neurons in the SN at 21 days after lesion and an improved performance in behavioral tests compared to control rats. However, no differences in any inflammatory parameter tested were found. These results suggest that grafted hMSCs exert neuroprotection but not neuromodulatory effects on degenerating dopaminergic neurons.
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Imunocompetência , Mesoderma/citologia , Transtornos Parkinsonianos/imunologia , Transtornos Parkinsonianos/prevenção & controle , Cordão Umbilical/citologia , Animais , Neurônios Dopaminérgicos/imunologia , Neurônios Dopaminérgicos/patologia , Humanos , Mesoderma/imunologia , Mesoderma/transplante , Transtornos Parkinsonianos/patologia , Ratos , Células Estromais/imunologia , Células Estromais/transplante , Substância Negra/imunologia , Substância Negra/patologia , Cordão Umbilical/imunologia , Cordão Umbilical/transplanteRESUMO
Neural stem cells reside in two neurogenic regions of the adult brain: the dentate gyrus of the hippocampus (DG) and the subventricular zone (SVZ). Their proliferation, differentiation, migration and survival are modulated by intrinsic and extrinsic signals, forming a neurogenic niche. Brain cytokines have only been recently regarded as possible components of this neurogenic niche. In particular, we have demonstrated that transforming growth factor-beta (TGF-beta) has a pro-neurogenic effect in the DG in a model of increased neurogenesis by adrenalectomy. We wanted to test whether TGF-beta has a similar effect in another neurogenic region, namely the SVZ. To test this possibility, adult rats were injected with adenoviral vectors expressing TGF-beta (Ad-TGF) or beta-galactosidase (Ad-bgal) in the SVZ and neurogenesis was evaluated 3 weeks later. We have observed that chronic TGF-beta expression increased neurogenesis in the ipsilateral hemisphere of Ad-TGF but not in Ad-bgal-treated rats compared to their contralateral side. In addition, an unspecific effect of the adenoviral vector per se could not be totally discarded. We conclude, under our experimental conditions, that TGF-beta could enhance adult neurogenesis in the SVZ. This data increase the growing evidence supporting a pro-neurogenic role of anti-inflammatory cytokines in the adult brain.
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Neurogênese/genética , Neuroimunomodulação/genética , Neurônios/metabolismo , Células-Tronco/metabolismo , Telencéfalo/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fatores Etários , Animais , Proliferação de Células , Citocinas/genética , Citocinas/metabolismo , Vetores Genéticos/genética , Tolerância Imunológica/imunologia , Masculino , Neurônios/citologia , Ratos , Ratos Wistar , Células-Tronco/citologia , Telencéfalo/citologia , Transfecção/métodos , Fator de Crescimento Transformador beta/genética , Regulação para Cima/fisiologiaRESUMO
Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. In this study we present in vivo and in vitro evidence that B. abortus and its lipoproteins activate the innate immunity of the CNS, eliciting an inflammatory response that leads to astrogliosis, a characteristic feature of neurobrucellosis. Intracranial injection of heat-killed B. abortus (HKBA) or outer membrane protein 19 (Omp19), a B. abortus lipoprotein model, induced astrogliosis in mouse striatum. Moreover, infection of astrocytes and microglia with B. abortus induced the secretion of interleukin (IL)-6, IL-1beta, tumor necrosis factor (TNF)-alpha, macrophage chemoattractant protein-1, and KC (CXCL1). HKBA also induced these inflammatory mediators, suggesting the involvement of a structural component of the bacterium. Accordingly, Omp19 induced the same cytokine and chemokine secretion pattern. B. abortus infection induced astrocyte, but not microglia, apoptosis. Indeed, HKBA and Omp19 elicited not only astrocyte apoptosis but also proliferation, two features observed during astrogliosis. Apoptosis induced by HKBA and L-Omp19 was completely suppressed in cells of TNF receptor p55-/- mice or when the general caspase inhibitor Z-VAD-FMK was added to cultures. Hence, TNF-alpha signaling via TNF receptor (TNFR) 1 through the coupling of caspases determines apoptosis. Our results provide proof of the principle that Brucella lipoproteins could be key virulence factors in neurobrucellosis and that astrogliosis might contribute to neurobrucellosis pathogenesis.
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Apoptose , Astrócitos/microbiologia , Astrócitos/patologia , Brucella abortus/fisiologia , Mediadores da Inflamação/metabolismo , Animais , Antígenos de Bactérias/farmacologia , Apoptose/efeitos dos fármacos , Astrócitos/enzimologia , Astrócitos/metabolismo , Proteínas da Membrana Bacteriana Externa/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/microbiologia , Encéfalo/patologia , Brucella abortus/efeitos dos fármacos , Caspases/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocinas/metabolismo , Feminino , Temperatura Alta , Imuno-Histoquímica , Lipopolissacarídeos/farmacologia , Lipoproteínas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Microglia/efeitos dos fármacos , Microglia/microbiologia , Microglia/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The differentiation of neural stem cells toward a neuronal phenotype is determined by the extracellular and intracellular factors that form the neurogenic niche. In this review, we discuss the available data on the functional role of inflammation and in particular, pro- and anti-inflammatory cytokines, on neuronal differentiation from endogenous and transplanted neural stem/progenitor cells. In addition, we discuss the role of microglial cell activation on these processes and the fact that microglial cell activation is not univocally associated with a pro-inflammatory milieu. We conclude that brain cytokines could be regarded as part of the endogenous neurogenic niche. In addition, we propose that accumulating evidence suggests that pro-inflammatory cytokines have a negative effect on neuronal differentiation, while anti-inflammatory cytokines exert an opposite effect. The clarification of the functional role of cytokines on neuronal differentiation will be relevant not only to better understand adult neurogenesis, but also to envisage complementary treatments to modulate cytokine action that could increase the therapeutic benefit of future progenitor/stem cell-based therapies.
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Encéfalo/cirurgia , Diferenciação Celular/fisiologia , Inflamação/fisiopatologia , Neurônios/fisiologia , Transplante de Células-Tronco/métodos , Animais , Encéfalo/citologia , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Microglia/fisiologiaRESUMO
Synthetic decapeptides (N=206) covering the entire sequence of mouse liver fumarylacetoacetate hydrolase (FAH) were used to analyze the specificities of the autoantibodies (autoAb) elicited towards this enzyme in mice infected with mouse hepatitis virus (MHV). These autoAb bound mainly to N- and C-terminal FAH peptides, the most reactive sequences being 1-50 and 390-420, respectively. Surprisingly, although FAH sequence 1-50 shares a high degree of homology with various MHV proteins, the C-terminal portion does not. Moreover, whereas the autoAb reacted with homologous peptides surrounding residues 70, 160 and 360, non-similar sequences around residues 130, 210, 240, 250, and 300 were also recognized, indicating that autoAb were not restricted to epitopes with sequence homologies. There was also a lack of correlation between the amount of anti-MHV or anti-FAH antibodies produced and the reactivity towards the peptides. Moreover, the spectrum of peptides recognized by the autoAb of a given mouse did not change significantly with time, which suggests that the MHV-elicited autoimmune response does not induce an epitope recognition spreading. Finally, anti-FAH Ab produced after immunization with rat liver FAH recognized essentially the same mouse FAH regions than autoAb from MHV-infected mice. Results indicated that the induction of the autoAb is not only related to molecular or structural mimicry, but rather supports the Danger model, in which any aggression, in this case the MHV infection, is susceptible to trigger the production of autoAb.
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Especificidade de Anticorpos/imunologia , Autoanticorpos/imunologia , Infecções por Coronavirus/imunologia , Hidrolases/imunologia , Mimetismo Molecular/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Animais , Autoantígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Hidrolases/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Vírus da Hepatite Murina/imunologia , Peptídeos/imunologia , RatosRESUMO
The features of autoantibodies (autoAb) to liver fumarylacetoacetate hydrolase (FAH) elicited in mice infected with mouse hepatitis virus (MHV) were studied by ELISA and western-blot competition assays. All sera tested contained Ab to cryptic FAH epitopes according with results from western-blot tests, whereas ELISA data indicated that some of these same sera did recognize native epitopes of the autoantigen (autoAg). Such differences were detected in individual sera from various mouse strains, and were ascribed to the fact that proteins insolubilized on solid supports expose a variety of conformational and cryptic antigenic determinants. On the other hand, whereas results from both experimental protocols showed that anti-MHV Ab did not cross-react with the soluble autoAg, the opposite situation did not show analogous results. Thus, binding of autoAb to insolubilized FAH could be inhibited by MHV depending on the mouse serum or the experimental protocol used. Additionally, a set of synthetic homologous peptides from mouse FAH and various viral proteins was employed to analyze the Ab repertoire of MHV-infected mice. Results indicated that two homologous peptides were recognized by most Ab: the N-terminal sequences (1-10) from FAH and the nucleocapside, both sharing 50% of identity, and sequence 2317-2326 of the RNA polymerase, a peptide showing 30% of identity with FAH 11-20. Results indicated that MHV-infection triggers at least three distinct Ab populations: anti-MHV, anti-FAH and cross-reacting Ab. This cross-reaction implies either sequential or conformational epitopes from both the viral proteins and the autoAg and may differ between individuals.
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Antígenos Virais/química , Autoimunidade , Vírus da Hepatite Murina/imunologia , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Autoanticorpos/sangue , Autoanticorpos/imunologia , Autoantígenos/imunologia , Doenças Autoimunes/etiologia , Doenças Autoimunes/virologia , Ligação Competitiva , Reações Cruzadas/imunologia , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/imunologia , Feminino , Hidrolases/química , Hidrolases/imunologia , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaRESUMO
The observation that mice infected with mouse hepatitis virus (MHV) develop autoantibodies directed mainly to liver fumarylacetoacetate hydrolase (FAH) enabled the development of an ELISA applicable to the detection of MHV-infection. The method, based on the titration of antibodies to semipurified FAH from rat liver, is easy, economical, and does not require the isolation of viral proteins from large MHV stocks. Furthermore, since sera from mice immunized with a purified fraction of the rat liver enzyme do react with its homologous protein, this antiserum can be used as a positive control avoiding the manipulation of samples from MHV-infected animals.