Minocycline promotes remyelination in aggregating rat brain cell cultures after interferon-γ plus lipopolysaccharide-induced demyelination.

Minocycline has been shown to inhibit microglia reactivity, and to decrease the severity and progression of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. It remained to be examined whether minocycline was also able to promote remyelination. In the present study, myelinating aggregating brain cell cultures were used as a model to study the effects of minocycline on microglial reactivity, demyelination, and remyelination. Cultures were treated simultaneously with two inflammatory agents, interferon-γ (IFN-γ) and lipopolysaccharide (LPS), which caused an inflammatory response accompanied by demyelination. The inflammatory response was characterized by microglial reactivity, upregulation of inflammatory cytokines and iNOS, and increased phophorylation of P38 and P44/42 mitogen activated protein (MAP) kinases. Minocycline inhibited microglial reactivity, and attenuated the increased phophorylation of P38 and P44/42 MAP kinases. Demyelination, determined by a decrease in myelin basic protein (MBP) content and immunoreactivity 48 h after the treatment with the inflammatory agents, was not prevented by minocycline. However, 1 week after demyelination was assessed, the MBP content was restored in presence of minocycline, indicating that remyelination was promoted. Concomitantly, in cultures treated with minocycline, the markers of oligodendrocyte precursors cells (OPCs) and immature oligodendrocytes NG2 and O4, respectively, were decreased compared to cultures treated with the inflammatory agents only. These results suggest that minocycline attenuates microglial reactivity and favors remyelination by enhancing the differentiation of OPCs and immature oligodendrocytes.

Bioatividade de ácidos húmicos: efeitos sobre o desenvolvimento radicular e sobre a bomba de prótons da membrana plasmática

A bioatividade de ácidos húmicos (AH) isolados de lodo da estação de tratamento de esgoto (AHL) e de vermicomposto (AHV) foi avaliada pela ação dessas substâncias sobre o transporte de prótons através da membrana plasmática de células de raízes de café e milho e sua relação com o desenvolvimento dessas espécies. Houve estímulo da área superficial radicular em ambas as espécies cultivadas com ambos AH, mostrando uma concentração ótima em torno de 40 mg L-1. Nessa condição, os tratamentos com AHL e AHV estimularam a H+-ATPase de membrana plasmática em plântulas de café e milho. Os AHL foram mais efetivos na promoção desses efeitos do que os AHV. A modificação do perfil cromatográfico dos AH em solução antes e após o cultivo das plântulas revelou que a interação planta-AH promoveu uma redistribuição das massas moleculares dessas substâncias, sugerindo uma dinâmica de mobilização de subunidades funcionais dos AH por exsudatos das raízes. A análise estrutural dos AH detectou a presença de grupamentos de auxina. A análise comparativa da ação desses dois AH sobre as espécies representantes de plantas monocotiledôneas (milho) e dicotiledôneas (café) apontam para a ativação da H+-ATPase de plasmalema como possível marcador metabólico de bioatividade dos ácidos húmicos.

Atratividade de Atta sexdens rubropilosa por plantas de eucalipto atacadas previamente ou não por Thyrinteina arnobia

O sistema de defesa induzido das plantas é ativado após a ocorrência do dano provocado por um herbívoro. O objetivo deste trabalho foi avaliar se plantas de eucalipto (Eucalyptus grandis) atacadas previamente por lagartas de Thyrinteina arnobia tornam-se resistentes ao corte de operárias de Atta sexdens rubropilosa. Não houve diferença significativa no tempo gasto por operárias dessa formiga em iniciar o corte em plantas atacadas ou não. Porém, houve diferença significativa no tamanho de fragmentos cortados. Concluiu-se que plantas previamente atacadas tornam-se menos atrativas para A. sexdens rubropilosa devido à ativação do seu sistema de defesa induzido.

Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon.

A three-dimensional cell culture system was used as a model to study the influence of low levels of mercury in the developing brain. Aggregating cell cultures of fetal rat telencephalon were treated for 10 days either during an early developmental period (i.e., between days 5 and 15 in vitro) or during a phase of advanced maturation (i.e., between days 25 and 35) with mercury. An inorganic (HgCl2) and an organic mercury compound (monomethylmercury chloride, MeHgCl) were examined. By monitoring changes in cell type-specific enzymes activities, the concentration-dependent toxicity of the compounds was determined. In immature cultures, a general cytotoxicity was observed at 10(-6) M for both mercury compounds. In these cultures, HgCl2 appeared somewhat more toxic than MeHgCl. However, no appreciable demethylation of MeHgCl could be detected, indicating similar toxic potencies for both mercury compounds. In highly differentiated cultures, by contrast, MeHgCl exhibited a higher toxic potency than HgCl2. In addition, at 10(-6) M, MeHgCl showed pronounced neuron-specific toxicity. Below the cytotoxic concentrations, distinct glia-specific reactions could be observed with both mercury compounds. An increase in the immunoreactivity for glial fibrillary acidic protein, typical for gliosis, could be observed at concentrations between 10(-9) M and 10(-7) M in immature cultures, and between 10(-8) M and 3 x 10(-5) M in highly differentiated cultures. A conspicuous increase in the number and clustering of GSI-B4 lectin-binding cells, indicating a microglial response, was found at concentrations between 10(-10) M and 10(-7) M. These development-dependent and cell type-specific effects may reflect the pathogenic potential of long-term exposure to subclinical doses of mercury.

Microglia, Calcification and Neurodegenerative Diseases

Neurodegeneration is a complex process involving different cell types andneurotransmitters. A common characteristic of neurodegenerative disorders such asAlzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis, Huntington’s disease (HD) and Amyotrophic Lateral Sclerosis (ALS) is the occurrence of a neuroinflammatoryreaction in which cellular processes involving glial cells (mainly microglia and astrocytes) and T cells are activated in response to neuronal death. This inflammatory reaction has recently received attention as an unexpected potential target for the treatment of these diseases.Microglial cells have a mesenchymal origin, invade the central nervous system (CNS)prenatally (Chan et al., 2007b) and are the resident macrophages in the CNS (Ransohoff &Perry, 2009). They comprise approximately 10-20% of adult glia and serve as the CNS innateimmune system. In neurodegenerative diseases, microglia is activated by misfoldedproteins. In the case of AD, amyloid- (A ) peptides accumulate extracellularly and activate the microglia locally. In the case of PD, ALS and HD, the misfolded proteins accumulate intracellularly but are still associated with activation of the microglia (Perry et al., 2010). Reactive microglia in the substantia nigra and striatum of PD brains have been described, and increased levels of proinflammatory cytokines and inducible nitric oxide synthase havebeen detected in these brain regions, providing evidence of a local inflammatory reaction (Hirsch & Hunot, 2009). The injection of lipopolysaccharide (a potent microglia activator) into the substantia nigra produces microglial activation and the death of dopaminergic cells. These findings support the hypothesis that microglial activation and neuroinflammationcontribute to PD pathogenesis (Herrera et al., 2000)...

CCR2/CCL2-mediated inflammation protects photoreceptor cells from amyloid-β-induced apoptosis.

Age-related macular degeneration is characterized by the formation of drusen containing amyloid-β (Aβ) and the degeneration of photoreceptors. To explore the largely unknown role of Aβ in the retina, we investigated the effects on photoreceptors of the oligomeric form of Aβ(1-42). Subretinal injection of the Aβ peptide induced misplaced expression of recoverin and synaptophysin in the photoreceptors, oxidative stress in their inner and outer segments, and finally apoptosis. Aβ did not induce cell death in purified photoreceptor cell cultures, but did so in retinal cell cultures, thereby suggesting that the cellular environment plays a role in Aβ-induced photoreceptor apoptosis. Subretinal injection of Aβ was followed by activation and migration of microglial cells and then by photoreceptor apoptosis. Microglial cells phagocytosed rhodopsin-containing debris and Aβ in the subretinal space. Quantitative RT-PCR allowed us to identify a specific gene expression profile associated with the Aβ-induced progression of retinal degeneration and consistent with oxidative stress, inflammation, and an apoptotic program. The gene most highly upregulated in Aβ-injected retinas was that for the chemokine CCL2, and its absence or that of its cognate receptor CCR2 greatly reduced migration of activated microglial cells to the site of retinal injury and profoundly worsened photoreceptor degeneration and disorganization of the retinal pigment epithelium in Aβ-injected retinas. Our study pinpoints the roles of Aβ and of CCL2/CCR2 axis-dependent inflammation in photoreceptor apoptosis.

Crucial role of CB(2) cannabinoid receptor in the regulation of central immune responses during neuropathic pain

Neuropathic pain is a clinical manifestation of nerve injury difficult to treat even with potent analgesic compounds. Here, we used different lines of genetically modified mice to clarify the role played by CB2 cannabinoid receptors in the regulation of the central immune responses leading to the development of neuropathic pain. CB2 knock-out mice and wild-type littermates were exposed to sciatic nerve injury, and both genotypes developed a similar hyperalgesia and allodynia in the ipsilateral paw. Most strikingly, knock-outs also developed a contralateral mirror image pain, associated with an enhanced microglial and astrocytic expression in the contralateral spinal horn. In agreement, hyperalgesia, allodynia, and microglial and astrocytic activation induced by sciatic nerve injury were attenuated in transgenic mice overexpressing CB2 receptors. These results demonstrate the crucial role of CB2 cannabinoid receptor in modulating glial activation in response to nerve injury. The enhanced manifestations of neuropathic pain were replicated in irradiated wild-type mice reconstituted with bone marrow cells from CB2 knock-outs, thus demonstrating the implication of the CB2 receptor expressed in hematopoietic cells in the development of neuropathic pain at the spinal cord.

Delta-9-tetrahydrocannabinol (THC) protects partly against demyelination by modulating the inflammatory response: an in vitro study in aggregating brain cell cultures

Delta 9-tetrahydrocannabinol (THC) has been proposed as therapeutic agent in the treatment of multiple sclerosis. In the present study, we examined whether a modulation of brain inflammatory by THC may protect against demyelination. Myelinating aggregating brain cell cultures were subjected to demyelination by a repeated treatment (3x) with the two inflammatory agents interferon-y (IFN-y) and lipopolysaccharide (LPS). The effects of THC on an acute inflammatory reponse were also examined by treating the aggregates with a single application of the two inflammatory agents. THC effects on the demyelinating process and on several mediators of the inflammatory reponse were analyzed. THC treatment partially prevented the decreased immunoreactivity for MBP, and the decrease in MBP content measured by immunoblotting. It prevented IFN-y + LPS -induced microglial reactivity; and decreased the IFN-y + LPS-induced i8ncreased phosphorylation of p44/42 MAP kinase. The other inflammatory markers, I-NOS and TNF-a mRNA expression, and p38 MAP kinase phosphorylation of p44/42 MAP kinase. The other inflammatory markers, I-NOS and TNF-a mRNA expression, and p38 MAP kinase phosphorylation were downregulated by THC treatment following a single application of the inflammatory agents, but not after repeated applications. THC protected partially against the IFN-y + LPS-induced demyelination. The protective effect of THC on IFN-y + LPS-induced demyelination may be due to a decrease of the inflammatory reponse. However, the anti-inflammatory effect of THC on some inflammatory markers is lost when the inflammatory response is more proeminent and of longer duration, suggesting either that the anti-inflammatory effect of a molecule may depend on the properties of the inflammatory response, or that the anti-inflammatory potential of THC decreases in case of repeated exposure.

Modelagem matemática da secagem em camada delgada de bagaço de uva fermentado

O objetivo deste trabalho foi determinar as características da secagem de bagaço de uva fermentado em secador com ar aquecido, avaliar a capacidade descritiva de conhecidos modelos matemáticos de secagem em camada delgada, e obter os valores de difusividade efetiva e a energia de ativação. Os experimentos de secagem foram conduzidos a 50, 60, 70, 80 e 90ºC, com a velocidade do ar de secagem de 1,0 m s-1. Foram comparados dez diferentes modelos matemáticos de secagem em camada delgada, de acordo com os valores do coeficiente de determinação (R²), qui-quadrado (χ²), raiz do quadrado médio residual (RQMR) e erro médio relativo (P), estimados pelas curvas de secagem. Os efeitos da temperatura de secagem nos coeficientes e nas constantes foram preditos pelos modelos de regressão. O modelo de Page modificado foi selecionado para representar o comportamento da secagem em camada delgada de bagaço de uva. Os valores médios da difusividade efetiva variaram de 1,0091 x 10-9 m² s-1 a 3,0421 x 10-9 m² s-1 nas temperaturas avaliadas. A dependência da difusividade efetiva pela temperatura foi descrita pela equação de Arrhenius, com o valor de energia de ativação de 24,512 kJ mol-1.

Peripheral Nerve Neuropathy is Associated with a Glial Reaction in the Gracile Nucleus Associated with a regulation of the GABA transporter GAT-1

Allodynia (pain in response to normally non painful stimulation) and paresthesia (erroneous sensory experience) are two debilitating symptoms of neuropathic pain. These stem, at least partly, from profound changes in the non-nociceptive sensory pathway that comprises large myelinated neuronal afferents terminating in the gracile and cuneate nuclei. Further than neuronal changes, well admitted evidence indicates that glial cells (especially in the spinal cord) are key actors in neuropathic pain, in particular the possible alteration in astrocytic capacity to reuptake neurotransmitters (glutamate and GABA). Yet, the possibility of such a changed astrocytic scavenging capacity remains unexplored in the dorsal column pathway. The present study was therefore undertaken to assess whether peripheral nerve injury (spared nerve injury model, SNI) could trigger a glial reaction, and especially changes in glutamate and GABA transporters, in the gracile nucleus. SNI surgery was performed on male Sprague-Dawley rats. Seven days after surgery, rats were used for immunofluorescence (fixation and brain slicing), western-blot (fresh brain freezing and protein extraction) or GABA reuptake on synaptosomes. We found that SNI results in a profound glial reaction in the ipsilateral gracile nucleus. This reaction was characterized by an enhanced immunolabelling for microglial marker Iba1 as well as astrocytic protein GFAP (further confirmed by western-blot, p <0.05, n = 7). These changes were not observed in sham animals. Immunofluorescence and western-blot analysis shows that the GABA transporter GAT-1 is upregulated in the ipsilateral gracile nucleus (p <0.001; n = 7), with no detectable change in GAT-3 or glutamate transporters EAAT-1 and EAAT-2. Double immunoflurescence shows that GAT-1 and GFAP colocalize within the same cells. Furthermore, the upregulation of GFAP and GAT-1 were shown to occur all along the rostrocaudal axis of the gracile nucleus. Finally, synaptosomes from ipsilateral gracile nucleus show an increased capacity to reuptake GABA. Together, the data presented herein show that glial cells in the gracile nucleus react to neuropathic lesion, in particular through an upregulation of the GABA transporter GAT-1. Hence, this study points to role of an increased GABA transport in the dorsal column nuclei in neuropathic pain, calling attention to GAT-1 as a putative future pharmacological target to treat allodynia and paresthesia.

Relação entre toxicidade de proteínas Vip3Aa e sua capacidade de ligação a receptores intestinais de lepidópteros-praga

Resumo:O objetivo deste trabalho foi avaliar a toxicidade de novas proteínas Vip3Aa e sua capacidade de ligação a vesículas de membrana da microvilosidade apical (VMMA) do intestino de lagartas neonatas de Spodoptera frugiperda, Anticarsia gemmatalise Heliothis virescens. Proteínas expressas pelos genes vip3Aa42 e vip3Aa43 mostraram-se tóxicas a S. frugiperda (CL50 de 78,2 e 113 ng cm-2, respectivamente) e A. gemmatalis(CL50 de 239,2 e 57,5 ng cm-2, respectivamente), e pouco tóxicas a H. virescens (CL50>5.000 ng cm-2). Os ensaios de ligação às VMMA mostraram que as proteínas unem-se de forma efetiva aos receptores nas vesículas das espécies avaliadas, mas essa capacidade de ligação somente é efetiva na ativação da toxicidade para as populações avaliadas de S. frugiperdae A. gemmatalis.

The blood-brain barrier: cellular basis.

Perfusion experiments with horseradish peroxidase have established that the morphological substrate of the blood-brain barrier is represented by microvascular endothelial cells. They are characterized by complexly arranged tight junctions and a very low rate of transcytotic vesicular transport. They express transport enzymes, carrier systems and brain endothelial cell-specific molecules of unknown function not expressed by any other endothelial cell population. These blood-brain barrier properties are not intrinsic to these cells but are inducible by the surrounding brain tissue. Type I astrocytes injected into the anterior eye chamber of the rat or onto the chick chorioallantoic membrane are able to induce a host-derived angiogenesis and some blood-brain barrier properties in endothelial cells of non-neural origin. Recently we have shown that this cellular interaction is due to the secretion of a soluble astrocyte derived factor(s). Astrocytes are also implicated in the maintenance, functional regulation and the repair of the blood-brain barrier. Complex interactions between other constituents of the microenvironment surrounding the endothelial cells, such as the basement membrane, pericytes, nerve endings, microglial cells and the extracellular fluid, take place and are required for the proper functioning of the blood-brain barrier, which in addition is regionally different as reflected by endothelial cell heterogeneity.

Efeito da temperatura e taxa de cisalhamento nas propriedades de escoamento da polpa de Cupuaçu (T. grandiflorum Schum) integral

O comportamento reológico da polpa de cupuaçu integral foi determinado na faixa de temperatura de 10 a 60ºC. Os efeitos da temperatura e da taxa de deformação foram avaliados por meio de testes em cisalhamento estacionário. As análises reológicas foram conduzidas num reômetro Thermo Haake RheoStress 1. Os reogramas foram descritos pelos modelos reológicos de Ostwald-de-Waelle e o de Herschel-Bulkley. As curvas de escoamento mais bem ajustadas pelo modelo de Ostwald-de-Waelle. O produto apresentou comportamento pseudoplástico, e o índice de comportamento de fluxo (n) decresceu com o aumento da temperatura. O efeito da temperatura sobre a viscosidade aparente foi descrita por uma equação tipo Arrhenius e discutida em termos de energia de ativação. Essa energia aumentou com o aumento da taxa de deformação, sendo obtidos valores na faixa de 1 a 2 kcal/gmol.

The JAK/STAT3 Pathway Is a Common Inducer of Astrocyte Reactivity in Alzheimer's and Huntington's Diseases.

Astrocyte reactivity is a hallmark of neurodegenerative diseases (ND), but its effects on disease outcomes remain highly debated. Elucidation of the signaling cascades inducing reactivity in astrocytes during ND would help characterize the function of these cells and identify novel molecular targets to modulate disease progression. The Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is associated with reactive astrocytes in models of acute injury, but it is unknown whether this pathway is directly responsible for astrocyte reactivity in progressive pathological conditions such as ND. In this study, we examined whether the JAK/STAT3 pathway promotes astrocyte reactivity in several animal models of ND. The JAK/STAT3 pathway was activated in reactive astrocytes in two transgenic mouse models of Alzheimer's disease and in a mouse and a nonhuman primate lentiviral vector-based model of Huntington's disease (HD). To determine whether this cascade was instrumental for astrocyte reactivity, we used a lentiviral vector that specifically targets astrocytes in vivo to overexpress the endogenous inhibitor of the JAK/STAT3 pathway [suppressor of cytokine signaling 3 (SOCS3)]. SOCS3 significantly inhibited this pathway in astrocytes, prevented astrocyte reactivity, and decreased microglial activation in models of both diseases. Inhibition of the JAK/STAT3 pathway within reactive astrocytes also increased the number of huntingtin aggregates, a neuropathological hallmark of HD, but did not influence neuronal death. Our data demonstrate that the JAK/STAT3 pathway is a common mediator of astrocyte reactivity that is highly conserved between disease states, species, and brain regions. This universal signaling cascade represents a potent target to study the role of reactive astrocytes in ND.

Avaliação da influência da temperatura e do tratamento enzimático no comportamento reológico do suco de abacaxi pérola (Ananas Comosus L. merr.)

O objetivo deste trabalho foi determinar o comportamento reológico do suco de abacaxi- pérola natural e tratado com enzimas pectinolíticas. As condições de tratamento enzimático foram otimizadas através de um planejamento experimental do tipo fatorial completo 2k, com três repetições do ponto central. Na avaliação do comportamento reológico foram utilizadas duas amostras submetidas a peneiramento (N e D), analisadas em quatro diferentes temperaturas (10; 25; 50 e 65 °C). As análises reológicas foram realizadas utilizando um viscosímetro de cilindros concêntricos Brookfield e os dados experimentais foram ajustados ao modelo de Mizrahi-Berk. A relação entre temperatura e viscosidade aparente foi descrita por uma equação tipo Arrhenius. A otimização da atividade enzimática indicou, através da análise de variância e da metodologia de superfície de resposta, que as variáveis temperatura e tempo de tratamento exerceram efeito estatisticamente significativo (p<0,05) sobre a concentração de pectina presente na amostra. O modelo utilizado mostrou-se adequado para descrever o comportamento reológico dos sucos de acordo com os parâmetros R², χ² e Bf. Os baixos valores obtidos para o índice de comportamento indicaram um comportamento pseudoplástico (n<1). A temperatura exerceu influência sobre a tensão de cisalhamento e a viscosidade aparente dos sucos analisados, sendo os menores valores observados nas amostras analisadas a 65 °C. A equação tipo Arrhenius descreveu de modo satisfatório o efeito da temperatura sobre a viscosidade aparente. Os valores da energia de ativação (Eat) foram de 4,54 Kcal.g.mol-1 e 4,89 Kcal.g.mol-1, respectivamente, para as amostras do suco de abacaxi natural e despectinizado, aumentando com o tratamento enzimático. A atividade enzimática proporcionou uma redução nos valores dos parâmetros de comportamento reológico das amostras, bem como na viscosidade aparente, em todas as temperaturas utilizadas, sendo o maior percentual de redução observado a 65 °C (41,66%).