RESUMO
Early-life infection has been shown to have profound effects on the brain and behavior across the lifespan, a phenomenon termed "early-life programming". Indeed, many neuropsychiatric disorders begin or have their origins early in life and have been linked to early-life immune activation (e.g. autism, ADHD, and schizophrenia). Furthermore, many of these disorders show a robust sex bias, with males having a higher risk of developing early-onset neurodevelopmental disorders. The concept of early-life programming is now well established, however, it is still unclear how such effects are initiated and then maintained across time to produce such a phenomenon. To begin to address this question, we examined changes in microglia, the immune cells of the brain, and peripheral immune cells in the hours immediately following early-life infection in male and female rats. We found that males showed a significant decrease in BDNF expression and females showed a significant increase in IL-6 expression in the cerebellum following E.coli infection on postnatal day 4; however, for most cytokines examined in the brain and in the periphery we were unable to identify any sex differences in the immune response, at least at the time points examined. Instead, neonatal infection with E.coli increased the expression of a number of cytokines in the brain of both males and females similarly including TNF-α, IL-1ß, and CD11b (a marker of microglia activation) in the hippocampus and, in the spleen, TNF-α and IL-1ß. We also found that protein levels of GRO-KC, MIP-1a, MCP1, IP-10, TNF-α, and IL-10 were elevated 8-hours postinfection, but this response was resolved by 24-hours. Lastly, we found that males have more thin microglia than females on P5, however, neonatal infection had no effect on any of the microglia morphologies we examined. These data show that sex differences in the acute immune response to neonatal infection are likely gene, region, and even time dependent. Future research should consider these factors in order to develop a comprehensive understanding of the immune response in males and females as these changes are likely the initiating agents that lead to the long-term, and often sex-specific, effects of early-life infection.
Assuntos
Cerebelo/imunologia , Infecções por Escherichia coli/imunologia , Hipocampo/imunologia , Microglia/imunologia , Caracteres Sexuais , Animais , Animais Recém-Nascidos , Fator Neurotrófico Derivado do Encéfalo/imunologia , Cerebelo/microbiologia , Feminino , Hipocampo/microbiologia , Inflamação/sangue , Inflamação/imunologia , Inflamação/microbiologia , Mediadores da Inflamação/sangue , Mediadores da Inflamação/imunologia , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Masculino , Microglia/microbiologia , Fenótipo , Ratos Sprague-DawleyRESUMO
In this study, we tested our hypothesis regarding mechanistic cross-talk between gastrointestinal inflammation and memory loss in a mouse model. Intrarectal injection of the colitis inducer 2,4,6-trinitrobenzenesulfonic acid (TNBS) in mice caused colitis via activation of nuclear factor (NF)-κB and increase in membrane permeability. TNBS treatment increased fecal and blood levels of lipopolysaccharide (LPS) and the number of Enterobacteriaceae, particularly Escherichia coli (EC), in the gut microbiota composition, but significantly reduced the number of Lactobacillus johnsonii (LJ). Indeed, we observed that the mice treated with TNBS displayed impaired memory, as assessed using the Y-maze and passive avoidance tasks. Furthermore, treatment with EC, which was isolated from the feces of mice with TNBS-induced colitis, caused memory impairment and colitis, and increased the absorption of orally administered LPS into the blood. Treatment with TNBS or EC induced NF-κB activation and tumor necrosis factor-α expression in the hippocampus of mice, as well as suppressed brain-derived neurotrophic factor expression. However, treatment with LJ restored the disturbed gut microbiota composition, lowered gut microbiota, and blood LPS levels, and attenuated both TNBS- and EC-induced memory impairment and colitis. These results suggest that the gut microbiota disturbance by extrinsic stresses can cause gastrointestinal inflammation, resulting in memory impairment.
Assuntos
Colite/imunologia , Disbiose/imunologia , Escherichia coli/fisiologia , Microbioma Gastrointestinal/fisiologia , Trato Gastrointestinal/imunologia , Hipocampo/imunologia , Inflamação/imunologia , Lactobacillus johnsonii/fisiologia , Transtornos da Memória/imunologia , Animais , Permeabilidade da Membrana Celular , Colite/induzido quimicamente , Colite/microbiologia , Modelos Animais de Doenças , Disbiose/induzido quimicamente , Disbiose/microbiologia , Fezes/microbiologia , Hipocampo/microbiologia , Humanos , Masculino , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/microbiologia , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Ácido Trinitrobenzenossulfônico , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Bacterial colonisation of the intestine has a major role in the post-natal development and maturation of the immune and endocrine systems. These processes are key factors underpinning central nervous system (CNS) signalling. Regulation of the microbiome-gut-brain axis is essential for maintaining homeostasis, including that of the CNS. However, there is a paucity of data pertaining to the influence of microbiome on the serotonergic system. Germ-free (GF) animals represent an effective preclinical tool to investigate such phenomena. Here we show that male GF animals have a significant elevation in the hippocampal concentration of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid, its main metabolite, compared with conventionally colonised control animals. Moreover, this alteration is sex specific in contrast with the immunological and neuroendocrine effects which are evident in both sexes. Concentrations of tryptophan, the precursor of serotonin, are increased in the plasma of male GF animals, suggesting a humoral route through which the microbiota can influence CNS serotonergic neurotransmission. Interestingly, colonisation of the GF animals post weaning is insufficient to reverse the CNS neurochemical consequences in adulthood of an absent microbiota in early life despite the peripheral availability of tryptophan being restored to baseline values. In addition, reduced anxiety in GF animals is also normalised following restoration of the intestinal microbiota. These results demonstrate that CNS neurotransmission can be profoundly disturbed by the absence of a normal gut microbiota and that this aberrant neurochemical, but not behavioural, profile is resistant to restoration of a normal gut flora in later life.
Assuntos
Trato Gastrointestinal/metabolismo , Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Microbiota , Serotonina/metabolismo , Caracteres Sexuais , Análise de Variância , Animais , Peso Corporal , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Modelos Animais de Doenças , Feminino , Trato Gastrointestinal/microbiologia , Hipocampo/microbiologia , Ácido Hidroxi-Indolacético/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Estresse Psicológico/sangue , Estresse Psicológico/microbiologia , Estresse Psicológico/patologia , Triptofano/metabolismo , Triptofano Hidroxilase/genética , Triptofano Hidroxilase/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pneumococcal meningitis is a life-threatening disease characterized by an acute infection affecting the pia matter, arachnoid and subarachnoid space. The intense inflammatory response is associated with a significant mortality rate and neurologic sequelae, such as, seizures, sensory-motor deficits and impairment of learning and memory. The aim of this study was to evaluate the effects of acute and extended administration of cannabidiol on pro-inflammatory cytokines and behavioral parameters in adult Wistar rats submitted to pneumococcal meningitis. Male Wistar rats underwent a cisterna magna tap and received either 10µl of sterile saline as a placebo or an equivalent volume of S. pneumoniae suspension. Rats subjected to meningitis were treated by intraperitoneal injection with cannabidiol (2.5, 5, or 10mg/kg once or daily for 9 days after meningitis induction) or a placebo. Six hours after meningitis induction, the rats that received one dose were killed and the hippocampus and frontal cortex were obtained to assess cytokines/chemokine and brain-derived neurotrophic factor levels. On the 10th day, the rats were submitted to the inhibitory avoidance task. After the task, the animals were killed and samples from the hippocampus and frontal cortex were obtained. The extended administration of cannabidiol at different doses reduced the TNF-α level in frontal cortex. Prolonged treatment with canabidiol, 10mg/kg, prevented memory impairment in rats with pneumococcal meningitis. Although descriptive, our results demonstrate that cannabidiol has anti-inflammatory effects in pneumococcal meningitis and prevents cognitive sequel.
Assuntos
Anti-Inflamatórios/farmacologia , Canabidiol/farmacologia , Transtornos Cognitivos/prevenção & controle , Cognição/efeitos dos fármacos , Lobo Frontal/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Meningite Pneumocócica/tratamento farmacológico , Animais , Anti-Inflamatórios/administração & dosagem , Comportamento Animal/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Canabidiol/administração & dosagem , Quimiocina CXCL1/metabolismo , Transtornos Cognitivos/imunologia , Transtornos Cognitivos/microbiologia , Transtornos Cognitivos/fisiopatologia , Transtornos Cognitivos/psicologia , Modelos Animais de Doenças , Regulação para Baixo , Lobo Frontal/imunologia , Lobo Frontal/microbiologia , Lobo Frontal/fisiopatologia , Hipocampo/imunologia , Hipocampo/microbiologia , Hipocampo/fisiopatologia , Injeções Intraperitoneais , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Memória/efeitos dos fármacos , Meningite Pneumocócica/imunologia , Meningite Pneumocócica/microbiologia , Meningite Pneumocócica/fisiopatologia , Meningite Pneumocócica/psicologia , Ratos , Ratos Wistar , Streptococcus pneumoniae/imunologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoAssuntos
Hipocampo/fisiologia , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Processamento de Proteína Pós-Traducional/genética , Sequência de Aminoácidos , Animais , Proteínas Arqueais/biossíntese , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Linhagem Celular , Células Cultivadas , Citomegalovirus/química , Citomegalovirus/genética , Dependovirus/química , Dependovirus/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Viral da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Hipocampo/microbiologia , Hipocampo/virologia , Humanos , Camundongos , Dados de Sequência Molecular , Rede Nervosa/metabolismo , Rede Nervosa/microbiologia , Rede Nervosa/virologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Neurônios/microbiologia , Neurônios/virologia , Ratos , Proteínas Virais/biossíntese , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Inflammation of the subarachnoid and ventricular space contributes to the development of brain damage i.e. cortical necrosis and hippocampal apoptosis in pneumococcal meningitis (PM). Galectin-3 and -9 are known pro-inflammatory mediators and regulators of apoptosis. Here, the gene and protein expression profile for both galectins was assessed in the disease progression of PM. The mRNA of Lgals3 and Lgals9 increased continuously in the cortex and in the hippocampus from 22 h to 44 h after infection. At 44 h after infection, mRNA levels of Lgals9 in the hippocampus were 7-fold and those of Lgals3 were 30-fold higher than in uninfected controls (P<0.01). Galectin-9 protein did not change, but galectin-3 significantly increased in cortex and hippocampus with the duration of PM. Galectin-3 was localized to polymorphonuclear neutrophils, microglia, monocytes and macrophages, suggesting an involvement of galectin-3 in the neuroinflammatory processes leading to brain damage in PM.
Assuntos
Encéfalo/metabolismo , Quimiotaxia de Leucócito/imunologia , Encefalite/metabolismo , Galectina 3/metabolismo , Galectinas/metabolismo , Meningite Pneumocócica/metabolismo , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/imunologia , Antígenos de Diferenciação Mielomonocítica/metabolismo , Biomarcadores/análise , Biomarcadores/metabolismo , Encéfalo/imunologia , Encéfalo/microbiologia , Córtex Cerebral/imunologia , Córtex Cerebral/metabolismo , Córtex Cerebral/microbiologia , Quimiotaxia de Leucócito/genética , Modelos Animais de Doenças , Encefalite/genética , Encefalite/microbiologia , Galectina 3/genética , Galectina 3/imunologia , Galectinas/genética , Galectinas/imunologia , Hipocampo/imunologia , Hipocampo/metabolismo , Hipocampo/microbiologia , Macrófagos/imunologia , Macrófagos/metabolismo , Meningite Pneumocócica/genética , Meningite Pneumocócica/fisiopatologia , Microglia/imunologia , Microglia/metabolismo , Monócitos/imunologia , Monócitos/metabolismo , Degeneração Neural/genética , Degeneração Neural/imunologia , Degeneração Neural/metabolismo , Neutrófilos/imunologia , Neutrófilos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Although the fetal inflammatory response syndrome seems crucial to the association between intrauterine infection and white matter disease in human preterm infants, the underlying mechanisms remain unclear. Using our previously described rabbit model of cerebral cell death in the white matter and hippocampus induced by intrauterine Escherichia coli infection, we investigated inflammatory and astroglial responses in placenta and brain tissues, in correlation with cell death distribution. Brains and placentas were studied 12, 24, or 48 h following intrauterine inoculation of E. coli or saline (groups G12, G24, and G48). Diffuse monocyte-macrophage infiltrates positive for inducible nitric oxide synthase (i-NOS) were significantly more marked in G24 and G48 placentas than in controls. In the G48 fetuses with both diffuse cell death and focal periventricular white matter cysts mimicking cystic periventricular leukomalacia, a strong rabbit macrophage and inducible nitric oxide synthase immunostaining was observed at the border of these cystic lesions. In contrast, in the fetuses with only diffuse and significant cell death, no inflammatory or astroglial responses were detected in the white matter or hippocampus. Cell death was accompanied by i-NOS immunostaining in the hippocampus but not the white matter. Hippocampal cells positive for i-NOS usually displayed a neuronal phenotype. In this model, focal white matter cysts are accompanied by a robust inflammatory response, and diffuse cell death, which may mimic the white matter and hippocampal damage seen in very and extremely pre-term infants, occur in the absence of a detectable brain inflammatory response.
Assuntos
Cerebelo/fisiopatologia , Encefalite/metabolismo , Encefalite/fisiopatologia , Efeitos Tardios da Exposição Pré-Natal , Animais , Animais Recém-Nascidos , Morte Celular , Cerebelo/microbiologia , Cerebelo/patologia , Modelos Animais de Doenças , Encefalite/microbiologia , Encefalite/patologia , Infecções por Escherichia coli/patologia , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Hipocampo/microbiologia , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Imunoquímica , Marcação In Situ das Extremidades Cortadas , Inflamação/microbiologia , Complexo Antígeno L1 Leucocitário/metabolismo , Macrófagos/metabolismo , Masculino , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo II , Doenças Placentárias/microbiologia , Doenças Placentárias/patologia , Gravidez , Coelhos , Fatores de TempoRESUMO
In spite of improved antimicrobial therapy, bacterial meningitis still results in brain damage leading to significant long-term neurological sequelae in a substantial number of survivors, as confirmed by several recent studies. Meningitis caused by Streptococcus pneumoniae is associated with a particularly severe outcome. Experimental studies over the past few years have increased our understanding of the molecular mechanisms underlying the events that ultimately lead to brain damage during meningitis. Necrotic damage to the cerebral cortex is at least partly mediated by ischemia and oxygen radicals and therefore offers a promising target for adjunctive therapeutic intervention. Neuronal apoptosis in the hippocampus may represent the major pathological process responsible for cognitive impairment and learning disabilities in survivors. However, the mechanisms involved in causing this damage remain largely unknown. Anti-inflammatory treatment with corticosteroids aggravates hippocampal damage, thus underlining the potential shortcomings of current adjuvant strategies. In contrast, the combined inhibition of matrix metalloproteinase and tumour necrosis factor-alpha converting enzyme protected both the cortex and hippocampus in experimental meningitis, and may represent a promising new approach to adjunctive therapy. It is the hope that a more refined molecular understanding of the pathogenesis of brain damage during bacterial meningitis will lead to new adjunctive therapies.
Assuntos
Dano Encefálico Crônico/microbiologia , Dano Encefálico Crônico/patologia , Meningite Pneumocócica/patologia , Streptococcus pneumoniae/patogenicidade , Dano Encefálico Crônico/enzimologia , Dano Encefálico Crônico/imunologia , Circulação Cerebrovascular , Hipocampo/microbiologia , Hipocampo/patologia , Hipocampo/fisiopatologia , Humanos , Inflamação/enzimologia , Inflamação/imunologia , Inflamação/microbiologia , Inflamação/patologia , Deficiências da Aprendizagem/complicações , Deficiências da Aprendizagem/microbiologia , Deficiências da Aprendizagem/patologia , Deficiências da Aprendizagem/fisiopatologia , Meningite Pneumocócica/complicações , Meningite Pneumocócica/enzimologia , Meningite Pneumocócica/imunologia , Metaloendopeptidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/fisiologiaRESUMO
The purpose of this investigation was to use molecular and immunological techniques to determine whether oral Treponema infected the human brain. Pieces of frontal lobe cortex from 34 subjects were analyzed with species-specific PCR and monoclonal antibodies. PCR detected Treponema in 14/16 Alzheimer's disease (AD) and 4/18 non-AD donors (P < 0.001), and AD specimens had more Treponema species than controls (P < 0.001). PCR also detected Treponema in trigeminal ganglia from three AD and two control donors. Cortex from 15/16 AD subjects and 6/18 controls contained Treponema pectinovorum and/or Treponema socranskii species-specific antigens (P < 0.01). T. pectinovorum and/or T. socranskii antigens were also found in trigeminal ganglia and pons from four embalmed cadavers, and 2/4 cadavers also had Treponema in the hippocampus. These findings suggest that oral Treponema may infect the brain via branches of the trigeminal nerve.
Assuntos
Doença de Alzheimer/microbiologia , Encéfalo/microbiologia , Boca/microbiologia , Treponema/classificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais , Borrelia burgdorferi/isolamento & purificação , Cadáver , Distribuição de Qui-Quadrado , Corantes , DNA Bacteriano/genética , Feminino , Lobo Frontal/microbiologia , Hipocampo/microbiologia , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Ponte/microbiologia , Saliva/microbiologia , Estatísticas não Paramétricas , Treponema/genética , Treponema/imunologia , Gânglio Trigeminal/microbiologia , Nervo Trigêmeo/microbiologiaRESUMO
Drug treatment of late-stage human African Trypanosomiasis (HAT) in which the central nervous system (CNS) is involved may be complicated by a severe post-treatment reactive encephalopathy (PTRE) which can be fatal in up to 10% of cases. In order to understand the immunopathogenesis of this complication, an experimental mouse model has been developed that mirrors many of the pathological features of the PTRE in humans, and which allows various anti-inflammatory therapeutic regimes to be evaluated. Following the development of the PTRE in this model a number of cytokines are increased within the CNS including tumour necrosis factor (TNF) alpha, interleukins 1, 4 and 6, and macrophage inflammatory protein (MIP)-1. These cytokines appear at the same time as astrocyte activation which is an early event occurring before the development of the marked meningoencephalitic inflammatory response. The immunosuppressant drug azathioprine prevents but does not reduce the severity of an established PTRE and has a minimal effect on astrocyte activation. The ornithine decarboxylase inhibitor eflornithine prevents the induction, and ameliorates the severity, of the PTRE, and also reduces the degree of astrocyte activation. The Substance P antagonist RP-67,580 ameliorates the severity of an established PTRE, and also reduces astrocyte activation, indicating an important role of SP in the generation of the inflammatory response. Continued use of this mouse model should lead to further enhancement of our understanding of the pathogenesis of the PTRE and to improved drug regimes to prevent and/or treat it.
Assuntos
Modelos Animais de Doenças , Encefalite/tratamento farmacológico , Encefalite/etiologia , Tripanossomíase Africana/complicações , Analgésicos/uso terapêutico , Animais , Azatioprina/uso terapêutico , Eflornitina/uso terapêutico , Encefalite/induzido quimicamente , Encefalite/imunologia , Hipocampo/efeitos dos fármacos , Hipocampo/microbiologia , Humanos , Imunossupressores/uso terapêutico , Indóis/uso terapêutico , Isoindóis , Camundongos , Tripanossomicidas/uso terapêutico , Tripanossomíase Africana/tratamento farmacológicoRESUMO
The neurotropic murine coronavirus JHM is capable of inducing various forms of neurologic diseases, including demyelination. Neurons have been shown to act as a repository site at the early stages of the disease process (O. Sorensen and S. Dales, J. Virol. 56:434-438, 1985). JHM virus (JHMV) replication and trafficking of viral proteins and virions in cultured rat hippocampal neurons and a neuronal cell line, OBL-21, were examined, with an emphasis placed on the role of the microtubular network. We show here that JHMV spread within the central nervous system occurs transneuronally and that virus protein trafficking was dependent upon microtubules. Viral trafficking occurred asymmetrically, involving both the somatodendritic and the axonal domains. Thus coronavirus can be disseminated from neurons at either the basolateral or the apical domains. A specific interaction between antibodies derived against the microtubule-associated protein tau and JHMV nucleocapsid protein (N) was observed, which can presumably be explained by an overall amino acid similarity of 44% and an identity of 20% between proteins N and tau, with optimal alignment at the microtubule binding domain of tau. Collectively, our data suggest an important role of the microtubule network in viral protein trafficking and distribution. They also draw attention to protein sequence mimicry of a cell component by this coronavirus as one strategy for making use of the host's functions on behalf of the virus.
Assuntos
Hipocampo/microbiologia , Microtúbulos/metabolismo , Vírus da Hepatite Murina/crescimento & desenvolvimento , Neurônios/microbiologia , Sequência de Aminoácidos , Animais , Astrócitos/microbiologia , Astrócitos/ultraestrutura , Transporte Biológico , Capsídeo/genética , Capsídeo/metabolismo , Compartimento Celular , Imunofluorescência , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Dados de Sequência Molecular , Vírus da Hepatite Murina/efeitos dos fármacos , Vírus da Hepatite Murina/metabolismo , Vírus da Hepatite Murina/ultraestrutura , Neuritos/microbiologia , Neuritos/ultraestrutura , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Ratos , Homologia de Sequência de Aminoácidos , Vimblastina/farmacologia , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Proteínas Virais/isolamento & purificação , Vírion/crescimento & desenvolvimento , Vírion/ultraestrutura , Replicação Viral/efeitos dos fármacos , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Competitive quantitative PCRs were used to examine the consequences of stereotactically injecting a highly attenuated herpes simplex virus type 1 mutant into rat brains. This mutant virus, designated RR1CAT/RR2lacZ, was engineered so that coding sequences of the genes UL39 and UL40 specifying the subunits of the viral ribonucleotide reductase were replaced by the chloramphenicol acetyltransferase (CAT) and the lacZ gene coding sequences, respectively. Stereotactic injection of this virus into the hippocampal region of the rat brain resulted in a localized infection. Viral gene products were visualized by immunochemical, cytochemical, or in situ hybridization techniques in the injected hippocampal region at 2 days postinjection. Viral genomes, represented by glycoprotein B (gB), latency-associated transcript (LAT), and lacZ sequences could be amplified by PCR from templates obtained by scraping hippocampal tissue off single 10-microns frozen sections. Both gB message and LAT could be detected by reverse transcriptase (RT)-PCR. At day 7 postinjection, neither CAT message, gB message, nor beta-galactosidase activity could be visualized by the same techniques, although viral DNA was detected by PCR and LAT could be detected by RT-PCR. A similar pattern was seen at 8 weeks, suggesting that latency was established by the mutant virus in cells of the injected hippocampus. By competitive quantitative PCR, hippocampal sections were determined to contain 2.6 x 10(5) genome equivalents (represented by the gB gene) on day 2, 6.2 x 10(4) on day 7, and 8.3 x 10(4) at 8 weeks. By competitive quantitative RT-PCR, the numbers of LAT molecules at the same time points were 3.2 x 10(6), 1.3 x 10(6), and 1.2 x 10(6), respectively. The numbers of LAT molecules per genome equivalent were 12.5, 20.3, and 14.5, respectively, being approximately the same for each of the three time points. The data permit the conclusion that the RR mutant virus establishes latency in the rat brain with the persistence of the viral genome and the production of LAT molecules. Once latency is established, the numbers of viral genomes and LAT RNA molecules remain constant. Thus the competitive quantitative PCR and RT-PCR techniques provide very sensitive and reliable methods to quantitate viral DNA and RNA present in infected tissue.
Assuntos
DNA Viral/análise , Herpes Simples/genética , Herpesvirus Humano 1/patogenicidade , RNA Viral/análise , Latência Viral/genética , Animais , Sequência de Bases , Genoma Viral , Hipocampo/microbiologia , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Ribonucleotídeo Redutases/genética , Células Vero , Proteínas do Envelope Viral/biossíntese , Proteínas do Envelope Viral/genética , VirulênciaRESUMO
We have shown previously using immunofluorescence microscopy that upon infection of polarized hippocampal cells in culture with vesicular stomatitis virus (VSV) and fowl plague virus (FPV) the VSV glycoprotein is delivered to the plasma membrane of the dendrites and of the cell body whereas the FPV hemagglutinin is transported to the axonal surface (Cell, 62 (1990) 63-72). In this work electron microscopy of infected rat hippocampal neurons showed that VSV progeny budded from the plasma membrane of the dendrites and the cell body. The location of the budding virions corresponded to the distribution of the VSV glycoprotein which was detected over the somatodendritic plasma membrane by immunoelectron microscopy. In contrast, no FPV formation was seen in the infected neurons although the FPV hemagglutinin was localized to the axonal surface by immunoelectron microscopy. In Semliki Forest virus (SFV) infected hippocampal cells we observed that the viral glycoproteins were exclusively present in the dendrites and cell body but not in axons.
Assuntos
Polaridade Celular/fisiologia , Hipocampo/microbiologia , Glicoproteínas de Membrana/análise , Neurônios/microbiologia , Proteínas do Envelope Viral/análise , Animais , Células Cultivadas , Hipocampo/citologia , Vírus da Influenza A/isolamento & purificação , Microscopia , Microscopia Eletrônica , Microscopia Imunoeletrônica , Ratos , Ratos Sprague-Dawley , Vírus da Floresta de Semliki/isolamento & purificação , Vírus da Estomatite Vesicular Indiana/isolamento & purificaçãoRESUMO
Recent studies of human immunodeficiency virus type 1 (HIV-1) encephalitis have shown that in addition to well established white matter damage, the neocortex shows thinning, loss of large neurons and dendritic damage. In order to identify neuronal populations affected in HIV encephalitis and to determine how neuronal damage relates to the severity of HIV infection within the nervous system, we quantified parvalbumin (PV+) and neurofilament (NF+) immunoreactive neurons in the frontal cortex and hippocampus. We found that in the neocortex, the density of NF+ and PV+ neurons was independent of severity of HIV encephalitis, and therefore changes in these neuronal subsets did not account for previously reported neuronal loss. However, neuritic processes of PV+ neurons were fragmented, atrophic and in some cases distended. In contrast to the frontal cortex, there was a trend toward decreased density of PV+ neurons in the hippocampus which only reached significance in the CA3 layer where there was a 50-90% decrease in PV+ neurons. This decrease was closely correlated with the severity of HIV encephalitis. Double-label immunocytochemical analysis confirmed neuritic damage to interneurons. These results suggest that HIV encephalitis differentially involves specific subpopulations of neurons. Since direct HIV infection of neuronal cells was not detected, damage to PV+ cells and fibers may be indirectly mediated by cytokines released by HIV-infected microglia.
Assuntos
Complexo AIDS Demência/patologia , Síndrome da Imunodeficiência Adquirida/patologia , Lobo Frontal/patologia , Hipocampo/patologia , Neurônios/patologia , Lobo Frontal/microbiologia , Proteína Glial Fibrilar Ácida/análise , HIV/isolamento & purificação , Proteína gp41 do Envelope de HIV/análise , Soropositividade para HIV/patologia , Hipocampo/microbiologia , Humanos , Imuno-Histoquímica , Interneurônios/patologia , Tratos Piramidais/patologia , Análise de RegressãoRESUMO
The rabies tissue culture infection test (RTCIT) and rapid rabies enzyme immunodiagnosis (RREID) were compared to the fluorescent-antibody test (FAT) with field specimens. At the French National Reference Center for Rabies, 15,248 specimens were analyzed by FAT and RTCIT, and 2,290 of those specimens were also tested by RREID; 818 other specimens were tested by FAT and RREID in 12 laboratories located in Africa, Asia, and Latin America. The sensitivities and specificities of RREID and RTCIT were comparable. This study showed that both tests can be used as backup procedures to confirm FAT. RREID is also strongly recommended for epidemiological studies and for laboratories which are not equipped for performing FAT.
Assuntos
Vírus da Raiva/isolamento & purificação , Raiva/veterinária , África , Animais , Ásia , Tronco Encefálico/microbiologia , Córtex Cerebral/microbiologia , Técnicas de Cultura , Imunofluorescência , Hipocampo/microbiologia , Técnicas Imunoenzimáticas , América Latina , Valor Preditivo dos Testes , Raiva/diagnóstico , Vírus da Raiva/imunologia , EspectrofotometriaRESUMO
Distribution of viral antigens in the central nervous system of 25 cattle with a persistent bovine viral diarrhea virus (BVDV) infection was studied. Using a polyclonal antiserum produced in pigs and the direct immunofluorescence and immunoperoxidase technique, BVDV antigen was located exclusively in neurons. Predilection sites for viral persistence were cerebral cortex and hippocampus; in other areas of brain and spinal cord, viral antigens were in single neurons or small groups of neurons. There was no morphological evidence of cellular alteration due to viral persistence. Perivascular lymphocytic infiltrations were in affected nervous tissue. It is concluded that the central nervous system is an important location for persistence of BVDV.