H7N9感染与神经细胞的血小板活化因子分泌浓度的相关性研究

H7N9病毒感染除引起患者呼吸道症状外,还可能导致中枢神级系统病症。血小板活化因子(Platelet activating factor,PAF)是一种生物活性磷脂,参与神经系统的部分功能。但尚未有研究讨论PAF是否参与H7N9病毒中枢神经系统疾病致病机制。本研究通过H7N9病毒体外感染小鼠小胶质细胞(BV2)和神经母瘤细胞(N2a)发现,H7N9流感病毒可以感染BV2、N2a细胞,引起细胞明显病变并使细胞活性下降;此外H7N9病毒感染BV2细胞后,PAF浓度水平明显上升,PAF乙酰水解酶(Platelet activating factor acetylhydrolase,PAF-AH)基因pafah1b1和pafah2表达水平明显下降。而N2a细胞染毒后PAF-AH基因表达水平明显上升,染毒48h后胞内PAF浓度明显下降。本研究首次将PAF与流感病毒性脑病联系在一起,提示PAF可能参与流感病毒性脑病的致病过程,可作为治疗的药物靶点进行后续研究。     

血液通路在H5N1高致病性禽流感病毒入侵小鼠中枢神经系统中的作用

【目的】研究血液通路在H5N1高致病性禽流感病毒入侵小鼠中枢神经系统中的作用。【方法】用3株H5N1病毒滴鼻感染BALB/c小鼠,研究小鼠肺、脑、血中的病毒在感染后不同时间点的复制动态及病理进展,通过免疫组化和免疫荧光染色显示病毒在脑部血管内皮细胞及血管周围神经组织的感染情况。【结果】小鼠感染后病毒迅速在肺中高效复制,随即形成病毒血症;感染后第6天病毒在肺中的滴度和在血液样本中的检出率达到峰值,此时小鼠脑部才开始检测到病毒;小鼠脑内血管内皮细胞、脑血管周围神经组织的神经元和神经胶质细胞中可检测到流感病毒NP蛋白。【结论】血液播散可能是高致病性H5N1禽流感病毒进入中枢神经系统的途径之一。     

Dissemination pathways for poliovirus cells to animals models

It is considered there are two main pathways for poliovirus dissemination towards the central nervous system in humans. One is the pathway through the blood brain barrier. The orally ingested virus invades into the blood circulation, and then the virus permeates into the central nervous system through the blood brain barrier. The other is the neural pathway. In this pathway, the intramuscularly-inoculated virus is transported through the axons from the synapse to the cell body in the central nervous system. We have developed the oral infection system using the mouse models. Moreover, we proposed the possibility that PV is transcytosed through the brain capillary epithelia in a specific manner. As for the neural pathway, we have proved that PV is endocytosed into CD155 containing vesicles and the vesicles are retrogradely transported in the axon of rat primary motor neuron. We have also shown that the cytoplasmic dynein takes part in the transport.     

Coronavirus neurovirulence correlates with the ability of the virus to induce proinflammatory cytokine signals from astrocytes and microglia

The molecular and cellular basis of coronavirus neurovirulence is poorly understood. Since neurovirulence may be determined at the early stages of infection of the central nervous system (CNS), we hypothesize that it may depend on the ability of the virus to induce proinflammatory signals from brain cells for the recruitment of blood-derived inflammatory cells. To test this hypothesis, we studied the interaction between coronaviruses (mouse hepatitis virus) of different neurovirulences with primary cell cultures of brain immune cells (astrocytes and microglia) and mouse tissues. We found that the level of neurovirulence of the virus correlates with its differential ability to induce proinflammatory cytokines (interleukin 12 [IL-12] p40, tumor necrosis factor alpha, IL-6, IL-15, and IL-1beta) in astrocytes and microglia and in mouse brains and spinal cords. These findings suggest that coronavirus neurovirulence may depend on a novel discriminatory ability of astrocytes and microglia to induce a proinflammatory response in the CNS.     

Specific and nonspecific T-cell recruitment in viral meningitis: Possible implications for autoimmunity

Specific and nonspecific T-cell invasion into cerebrospinal fluid has been investigated in the nonfatal viral meningoencephalitis induced by intracerebral inoculation of mice with vaccinia virus. At the peak of the inflammatory process on Day 7 approximately 5 to 10% of the Lyt 2⁺ T cells present are apparently specific for vaccinia virus. Concurrently, in mice primed previously with influenza virus, 0.5 to 1.0% of the appropriate T-cell set located in cerebrospinal fluid is reactive to influenza-infected target cells. This vaccinia virus-induced inflammatory exudate may thus contain as many as 500 influenza-immune memory T cells. These findings are discussed from the aspect that such nonspecific T-cell invasion into the central nervous system during aseptic viral meningitis could result in exposure of potentially brain-reactive T cells to central nervous system components.     

The role of mast cells in virus-induced inflammation in the murine central nervous system

The mononuclear inflammatory response to Sindbis virus infection of the central nervous system is analogous to the cutaneous delayed-type hypersensitivity reaction. It is dependent on sensitized T cells for initiation, but many of the cells present are nonsensitized bone marrow-derived cells. Tissue mast cells have been shown to be important for the development of the delayed-type hypersensitivity reaction in the skin where capillary endothelial cells are joined by tight junctions. To determine whether mast cells are also important for the development of an immune-mediated inflammatory response across the endothelial tight junctions of the blood-brain barrier, the development of mononuclear inflammation in the central nervous system of reserpine-treated mice and mast cell-deficient mice (WBB6F1-W/Wv) was studied after infection with Sindbis virus. Three central nervous system compartments, the cerebrospinal fluid, the meninges, and the brain parenchyma, were evaluated for inflammation by counting the number of cells present, by grading the histopathologic lesions, and by labeling infiltrating cells with 125IUDR. By all parameters inflammation was reduced when mice were treated with reserpine or were deficient in mast cells. Antigen-specific humoral and cellular immune responses were depressed and virus clearance delayed in reserpine-treated mice, but not in mast cell deficient mice. It is concluded that the vasoactive amines released by mast cells in the central nervous system play a facilitating role in the development of the inflammatory response to Sindbis virus.     

Reduced N-acetylaspartate levels in mice lacking aralar, a brain- and muscle-type mitochondrial aspartate-glutamate carrier

Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralar-deficient (Aralar-/-) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar-/- mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar-/- mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar-/- mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.     

Influenza-B-virus-induced eye and brain malformations during early chick embryogenesis and localization of the viral RNA in specific areas

Influenza is prevalent worldwide, and the teratogenic effects of influenza infection have been suspected to occur within the developing central nervous system. We herein report the sequelae of influenza B viral infection during early chick embryogenesis. Chick embryos at Hamburger-Hamilton stage 9 were infected by an in ovo injection under the blastoderm of influenza B virus (B/Taiwan/25/99). At 48 h after infection, gross malformations of the eye and brain, ranging from 25 to 58% of 168 infected embryos, were observed, in contrast to 3–6% among 71 mock-infected controls (p < 0.0001 for both eye and brain malformations). Histological analyses showed extensive tissue degeneration and aggregates of cells in the head mesenchyme, suggesting cell death and heterotopia. Influenza B viral RNA was directly localized by in situ hybridization with probes specific for the HA segment. Viral RNA was extensively detected in the head surface ectoderm and in the lung bud. In the developing brain, viral RNA was specifically located in the anterior neural retina, habenular area, mid-thalamus, and rhombencephalon. Our data show that influenza B virus can be a teratogenic agent in neural and nonneural embryonic tissues, raising concern for transplacental infection during early pregnancy.     

ANALYTICAL DIFFUSION OF INFLUENZA VIRUS AND MOUSE ENCEPHALOMYELITIS VIRUS

Analytical diffusion has been applied to a study of influenza A virus in mouse lung, influenza A virus in the extra-embryonic fluids of the chick, and mouse encephalomyelitis virus in mouse brain. The results from influenza in mouse lung suggested that about 99 per cent of the infectivity was present in particles 200 mµ in diameter, and 1 per cent in particles 6 mµ in diameter. The results from influenza in extra-embryonic fluids indicated that the preparation was inhomogeneous and that the smallest virus units were about 6 mµ in diameter. The results from mouse encephalomyelitis virus indicated that the preparation was also inhomogeneous, with 10 per cent of the infectivity in particles about 15 mµ in diameter. It has been suggested that in virus preparations normal colloidal particles can act as carriers of much smaller virus units.     

The brain renin-angiotensin system: a critical analysis.

The concept of a brain renin-angiotensin system originated with the observation that the components necessary for the formation of angiotensin II are present in the central nervous system. This observation has been confirmed and extended, and it is now frequently assumed that there is a functional brain renin-angiotensin system. However, careful analysis of the available evidence has revealed a number of significant problems. It appears that most of the renin-like activity measured in extracts of brain is due to the acid protease cathepsin D; this is unlikely to function as an angiotensin-forming enzyme in vivo. Experiments involving central administration of renin substrate have not provided convincing evidence for a significant renin-renin substrate interaction in vivo. Attempts to demonstrate the presence of angiotensin in the brain have been plagued with problems of specificity and it is still not clear if the peptide is actually present in the central nervous system. These problems do not rule out the possibility that there is a brain renin-angiotensin system, but more definitive evidence is required before it can be concluded that such a tensin system exists.     

Antiviral antibodies stimulate production of reactive oxygen species in cultured canine brain cells infected with canine distemper virus.

Canine distemper is characterized mainly by respiratory, enteric, and nervous symptoms. Infection of the central nervous system results in demyelination, to which inflammation has been shown to contribute significantly. It has been proposed that macrophages play a major role as effector cells in this process. We report that cultured dog brain cells contain a population of macrophages capable of producing reactive oxygen species as measured by luminol-dependent chemiluminescence. In cultures infected with canine distemper virus, a burst of reactive oxygen is triggered by antiviral antibody. This response depends on the presence of viral antigens on the surfaces of infected cells and is mediated by the interaction of antigen-bound antibody with Fc receptors on the macrophages. Since there is no evidence in vitro or in vivo that oligodendrocytes, the cells forming myelin, are infected, our observation supports the hypothesis that "innocent bystander killing" is important in demyelination caused by canine distemper virus. Reactive oxygen species released from macrophages may contribute to destruction of myelin.     

Gamma interferon is a major mediator of antiviral defense in experimental measles virus-induced encephalitis.

Measles virus infection of the central nervous system in the murine model of experimental measles virus-induced encephalitis is successfully controlled by virus-specific T-helper lymphocytes. T cells from BALB/c mice that are resistant to measles virus encephalitis proliferate well against measles virus in vitro, and bulk cultures recognize viral nucleocapsid and hemagglutinin as well as fusion proteins. The measles virus-specific T cells secrete large amounts of interleukin 2 (IL-2), gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha) but no IL-4, IL-6, or IL-10, and hence the cytokine pattern is consistent with that of subtype 1 T-helper lymphocytes. In contrast, cells obtained from measles virus-infected susceptible C3H mice recognize measles virus proteins only weakly and secrete little IFN-gamma and TNF-alpha. Treatment of infected mice with anti-TNF-alpha antibodies has no effect on survival or virus clearance from the brain. Upon neutralization of IFN-gamma in vivo, the phenotype of measles virus-specific T-helper cells isolatable from BALB/c mice is reversed from subtype 1 to subtype 2-like. Anti-IFN-gamma antibody-treated BALB/c mice are susceptible to measles virus encephalitis, and viral clearance from the central nervous system is impaired. These results indicate that IFN-gamma plays a significant role in the control of measles virus infection of the central nervous system.     

In vitro and in vivo infection of neural cells by a recombinant measles virus expressing enhanced green fluorescent protein

This study focused on the in vitro infection of mouse and human neuroblastoma cells and the in vivo infection of the murine central nervous system with a recombinant measles virus. An undifferentiated mouse neuroblastoma cell line (TMN) was infected with the vaccine strain of measles virus (MVeGFP), which expresses enhanced green fluorescent protein (EGFP). MVeGFP infected the cells, and cell-to-cell spread was studied by virtue of the resulting EGFP autofluorescence, using real-time confocal microscopy. Cells were differentiated to a neuronal phenotype, and extended processes, which interconnected the cells, were observed. It was also possible to infect the differentiated neuroblastoma cells (dTMN) with MVeGFP. Single autofluorescent EGFP-positive cells were selected at the earliest possible point in the infection, and the spread of EGFP autofluorescence was monitored. In this instance the virus used the interconnecting processes to spread from cell to cell. Human neuroblastoma cells (SH-SY-5Y) were also infected with MVeGFP. The virus infected these cells, and existing processes were used to initiate new foci of infection at distinct regions of the monolayer. Transgenic animals expressing CD46, a measles virus receptor, and lacking interferon type 1 receptor gene were infected intracerebrally with MVeGFP. A productive infection ensued, and the mice exhibited clinical signs of infection, such as ataxia and an awkward gait, identical to those previously observed for the parental virus (Edtag). Mice were sacrificed, and brain sections were examined for EGFP autofluorescence by confocal scanning laser microscopy over a period of 6 h. EGFP was detected in discrete focal regions of the brain and in processes, which extended deep into the parenchyma. Collectively, these results indicate (i) that MVeGFP can be used to monitor virus replication sensitively, in real time, in animal tissues, (ii) that infection of ependymal cells and neuroblasts provides a route by which measles virus can enter the central nervous system in mouse models of encephalitis, and (iii) that upon infection, the virus spreads transneuronally.     

The relationship between QTc interval and cardiac autonomic neuropathy in diabetes mellitus

Methylxanthines are widely consumed because of their stimulating effect primarily on the central nervous system. Their diuretic and respiratory stimulant action is used in clinical medicine. L-Arginine metabolism in the brain is very important for normal brain function. In addition to brain protein synthesis, arginine is a substrate for the production of urea, creatine, nitric oxide, agmatine, glutamic acid, ornithine, proline and polyamines. As known, many of these compounds are very important in brain function. There is no information relating to effects of caffeine on arginine metabolism in the brain, however, there is a lot of new information about arginine metabolism and caffeine action on the central nervous system. So, we have hypothesized the existence of a relationship that may be of interest in understanding mechanisms of caffeine effects on the central nervous system that may have utility in the clinical applications.In our experiment protocol we used male Wistar rats weighing about 200 g. Caffeine was added to the drinking water in gradually increasing amounts, from 2 g/l over the first 3 days, to 4 g/l over the last 7 days. A control group was given drinking water without caffeine. The level of lipid peroxidation, arginase and diamine oxidase (DAO) activity in the brain was measured. The results of our study show that arginase and diamine oxidase were decreased in animals treated with caffeine. The level of lipid peroxidation (MDA) was decreased also.The inhibitory effect of caffeine on arginase activity indicates that caffeine provides more arginine for consumption in other metabolic pathways. Considering the central stimulant effects of caffeine and the decreased lipid peroxidation level, it can be assumed that moderate short-term consumption of caffeine may be beneficial for brain function.     

Semliki Forest virus infects mouse brain endothelial cells and causes blood-brain barrier damage.

Induction of experimental allergic encephalomyelitis is facilitated in a genetically resistant BALB/c mouse strain by a nonpathogenic strain of a neurotropic alphavirus, Semliki Forest virus (SFV-A7). One possible explanation for this enhancement is virus infection of endothelial cells (EC), causing increased permeability of the blood-brain barrier. We have now sought evidence for virus infection of EC in vivo by immunocytochemistry and in situ hybridization. SFV-A7 antigens and RNA were detected in vascular EC and perivascular neurons in cerebellar and spinal cord white matter. Expression of viral antigens was followed by fibrinogen leakage from the blood vessels into brain parenchyma. This was shown by immunoperoxidase staining detecting fibrinogen extravascularly in central nervous system sections of infected mice. Simultaneously, expression of ICAM-1 (intercellular adhesion molecule 1) was induced on brain EC. SFV-A7 replicated in mouse brain microvascular EC in vitro and caused lysis of the cells. SFV-A7 did not induce ICAM-1 expression of mouse brain microvascular EC in vitro, while ICAM-1 was readily induced by gamma interferon and interleukin 1 beta. The observed increase of ICAM-1 expression on EC is immune mediated and not a direct effect of the virus infection. We conclude that SFV-A7 infection causes cerebral microvascular damage which contributes to the facilitation of experimental allergic encephalomyelitis in BALB/c mice.     

Studies on haemagglutination by the EMC-MM-Columbia SK-group of viruses

Summary When MM and Columbia SK virus were propagated in the chick embryo, the virus disappeared from the allantoic fluid and from the embryonic brain tissue after 6 to 9 chorioallantoic passages. Amniotic passages, however, resulted in a gradual increase of the mouse infectivity titre of the amniotic fluid and of the fluid of the regularly observed subcutaneous edema of the embryos, though after 22 passages the ID50 titre was still 3 logs lower than that of the original mouse brain suspension. The virus was present not only in amniotic fluid, but also in allantoic fluid and throughout the whole embryo. In spite of the pretty high mouse infectivity titres, the extra-embryonic fluids failed to produce haemagglutination of sheep red cells, whereas low haemagglutination titres could be obtained with suspensions of embryonic brain tissue. No evidence was obtained, that a non-specific inhibitor was responsible for the inability to produce haemagglutination. The virus present in egg fluids is only partially adsorbed, which is in contrast with the almost complete adsorption of virus in mouse brain suspension. The adsorption of a relatively small amount of virus might be one of the factors responsible for lack of haemagglutinating ability. It is suggested, that another coinciding factor, which is necessary for the initiation of the haemagglutination might be present in the central nervous system lesion.Third publication: Antonie van Leeuwenhoek17, 137, 1951.     

Subclinical brain injury caused by H5N1 influenza virus infection

Although H5N1 influenza A viruses can cause systemic infection, their neurotropism and long-term effects on the central nervous system (CNS) are not fully understood. We assessed H5N1viral invasion of the CNS and its long-term effects in a ferret model. An H5N1 virus caused nonsuppurative encephalitis, which lasted for 3 months without neurologic signs. Further, another H5N1 virus caused nonsuppurative vasculitis with brain hemorrhage. Three-dimensional analysis of viral distribution in the brain identified the olfactory system as a major route for brain invasion. The efficient growth of virus in the upper respiratory tract may thus facilitate viral brain invasion.     

Effects of Clinacanthus siamensis leaf extract on influenza virus infection

Ethanolic extracts of 20 medicinal plants were screened for influenza virus NA inhibition and in vitro antiviral activities using MDCK cells in an MTT assay. The vaccine proteins of influenza virus A/New Caledonia/20/99 (H1N1), mouse-adapted influenza virus A/Guizhou/54/89 (A/G)(H3N2) and mouse-adapted influenza virus B/Ibaraki/2/85 (B/I) were used in the NA inhibition assay, and mouse-adapted influenza viruses A/PR/8/34 (H1N1), A/G and B/I were used in the in vitro antiviral assay. The results of the in vitro antiviral assay indicated that the A/G virus was the most susceptible and an extract of the leaf of CS possessed the highest in vitro anti-A/G virus activity (41.98%). Therefore, the A/G virus and the CS extract were selected for studying in vivo anti-influenza virus activity. BALB/c mice were treated with CS extract (100 mg/kg per day, 5 times) orally from 4 hr before to 4 days after infection. CS extract elicited significant production of anti-influenza virus IgG₁ antibody in BAW and increased mouse weight compared to oseltamivir (0.1 mg/kg per day) on day 19 or water on days 17–19 of infection. Moreover, CS extract produced a higher anti-influenza virus IgA antibody level in BAW compared to oseltamivir, and a tendency towards an increase in anti-influenza virus IgA compared to water was shown. The results suggest that CS extract has a protective effect against influenza virus infection.     

Expression of the mouse hepatitis virus receptor by central nervous system microglia

Detection of the mouse hepatitis virus receptor within the central nervous system (CNS) has been elusive. Receptor expression on microglia was reduced during acute infection and restored following immune-mediated virus control. Receptor down regulation was independent of neutrophils, NK cells, gamma interferon, or perforin. Infection of mice devoid of distinct inflammatory cells revealed CD4(+) T cells as the major cell type influencing receptor expression by microglia. In addition to demonstrating receptor expression on CNS resident cells, these data suggest that transient receptor down regulation on microglia aids in establishing persistence in the CNS by assisting virus infection of other glial cell types.