Flavia Regina Souza Lima

The impact of microglial activation on blood-brain barrier in brain diseases

The blood-brain barrier (BBB), constituted by an extensive network of endothelial cells (ECs) together with neurons and glial cells, including microglia, forms the neurovascular unit (NVU). The crosstalk between these cells guarantees a proper environment for brain function. In this context, changes in the endothelium-microglia interactions are associated with a variety of inflammation-related diseases in brain, where BBB permeability is compromised. Increasing evidences indicate that activated microglia modulate expression of tight junctions, which are essential for BBB integrity and function. On the other hand, the endothelium can regulate the state of microglial activation. Here, we review recent advances that provide insights into interactions between the microglia and the vascular system in brain diseases such as infectious/inflammatory diseases, epilepsy, ischemic stroke and neurodegenerative disorders.

Glioblastoma: therapeutic challenges, what lies ahead.

Glioblastoma (GBM) is one of the most aggressive human cancers. Despite current advances in multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome for patients with high grade glioma remains fatal. The knowledge of how glioma cells develop and depend on the tumor environment might open opportunities for new therapies. There is now a growing awareness that the main limitations in understanding and successfully treating GBM might be bypassed by the identification of a distinct cell type that has defining properties of somatic stem cells, as well as cancer-initiating capacity - brain tumor stem cells, which could represent a therapeutic target. In addition, experimental studies have demonstrated that the combination of antiangiogenic therapy, based on the disruption of tumor blood vessels, with conventional chemotherapy generates encouraging results. Emerging reports have also shown that microglial cells can be used as therapeutic vectors to transport genes and/or substances to the tumor site, which opens up new perspectives for the development of GBM therapies targeting microglial cells. Finally, recent studies have shown that natural toxins can be conjugated to drugs that bind to overexpressed receptors in cancer cells, generating targeted-toxins to selectively kill cancer cells. These targeted-toxins are highly effective against radiation- and chemotherapy-resistant cancer cells, making them good candidates for clinical trials in GBM patients. In this review, we discuss recent studies that reveal new possibilities of GBM treatment taking into account cancer stem cells, angiogenesis, microglial cells and drug delivery in the development of new targeted-therapies.

Glioblastoma cells: a heterogeneous and fatal tumor interacting with the parenchyma.

Glioblastomas (GBMs) are considered to be one of the deadliest human cancers, characterized by a high proliferative rate, aggressive invasiveness and insensitivity to radio- and chemotherapy, as well as a short patient survival period. Moreover, GBMs are among the most vascularized and invasive cancers in humans. Angiogenesis in GBMs is correlated with the grade of malignancy and is inversely correlated with patient survival. One of the first steps in tumor invasions is migration. GBM cells have the ability to infiltrate and disrupt physical barriers such as basement membranes, extracellular matrix and cell junctions. The invasion process includes the overexpression of several members of a super-family of zinc-based proteinases, the Metzincin, in particular a sub-group, metalloproteinases. Another interesting aspect is that, inside the GBM tissue, there are up to 30% of microglia or macrophages. However, little is known about the immune performance and interactions of the microglia with GBMs. These singular properties of GBMs will be described here. A sub-population of cells with stem-like properties may be the source of tumors since, apparently, GBM stem cells (GSCs) are highly resistant to current cancer treatments. These cancer therapies, while killing the majority of tumor cells, ultimately fail in GBM treatment because they do not eliminate GSCs, which survive to regenerate new tumors. Finally, GBM patient prognostic has shown little improvement in decades. In this context, we will discuss how the membrane-acting toxins called cytolysins can be a potential new tool for GBM treatment.

Soluble Factors Released by Toxoplasma gondii-Infected Astrocytes Down-Modulate Nitric Oxide Production by Gamma Interferon-Activated Microglia and Prevent Neuronal Degeneration

ABSTRACT The maintenance of a benign chronic Toxoplasma gondii infection is mainly dependent on the persistent presence of gamma interferon (IFN-γ) in the central nervous system (CNS). However, IFN-γ-activated microglia are paradoxically involved in parasitism control and in tissue damage during a broad range of CNS pathologies. In this way, nitric oxide (NO), the main toxic metabolite produced by IFN-γ-activated microglia, may cause neuronal injury during T. gondii infection. Despite the potential NO toxicity, neurodegeneration is not a common finding during chronic T. gondii infection. In this work, we describe a significant down-modulation of NO production by IFN-γ-activated microglia in the presence of conditioned medium of T. gondii-infected astrocytes (CMi). The inhibition of NO production was paralleled with recovery of neurite outgrowth when neurons were cocultured with IFN-γ-activated microglia in the presence of CMi. Moreover, the modulation of NO secretion and the neuroprotective effect were shown to be dependent on prostaglandin E2 (PGE2) production by T. gondii-infected astrocytes and autocrine secretion of interleukin-10 (IL-10) by microglia. These events were partially eliminated when infected astrocytes were treated with aspirin and cocultures were treated with anti-IL-10 neutralizing antibodies and RP-8-Br cyclic AMP (cAMP), a protein kinase A inhibitor. Further, the modulatory effects of CMi were mimicked by the presence of exogenous PGE2 and by forskolin, an adenylate cyclase activator. Altogether, these data point to a T. gondii-triggered regulatory mechanism involving PGE2 secretion by astrocytes and cAMP-dependent IL-10 secretion by microglia. This may reduce host tissue inflammation, thus avoiding neuron damage during an established Th1 protective immune response.

STI1 promotes glioma proliferation through MAPK and PI3K pathways

Gliomas are tumors derived from glia or their precursors within the central nervous system. Clinically, gliomas are divided into four grades and the glioblastoma multiforme (GBM), also referred as grade IV astrocytoma, is the most aggressive and the most common glioma in humans. The prognosis for patients with GBM remains dismal, with a median survival of 9–12 months. Despite their striking heterogeneity, common alterations in specific cellular signal transduction pathways occur within most GBMs. Previous work from our group identified the co‐chaperone stress‐inducible protein 1 (STI1) as a cell surface ligand for cellular prion (PrPC), which leads to the activation of several signal transduction pathways, some of which modulate cell survival. In the present work, we used thymidine incorporation assays to investigate the effect of STI1 upon proliferation of the human glioblastoma‐derived cell line A172. Here we report that STI1 is secreted by and induces proliferation in tumor cells, an effect that is modulated by the Erk and PI3K pathways, and that, in contrast to glioma cells, STI1 does not induce proliferation of normal glia. In addition, our data suggest the involvement of PrPC in STI1‐induced proliferation of A172 cells. These results provide initial evidence of a new functional role for STI1 on the physiology of human gliomas, and may lead to the identification of new therapeutic targets in these tumors.

Prion protein and its ligand stress inducible protein 1 regulate astrocyte development

Prion protein (PrPC) interaction with stress inducible protein 1 (STI1) mediates neuronal survival and differentiation. However, the function of PrPC in astrocytes has not been approached. In this study, we show that STI1 prevents cell death in wild‐type astrocytes in a protein kinase A‐dependent manner, whereas PrPC‐null astrocytes were not affected by STI1 treatment. At embryonic day 17, cultured astrocytes and brain extracts derived from PrPC‐null mice showed a reduced expression of glial fibrillary acidic protein (GFAP) and increased vimentin and nestin expression when compared with wild‐type, suggesting a slower rate of astrocyte maturation in PrPC‐null animals. Furthermore, PrPC‐null astrocytes treated with STI1 did not differentiate from a flat to a process‐bearing morphology, as did wild‐type astrocytes. Remarkably, STI1 inhibited proliferation of both wild‐type and PrPC‐null astrocytes in a protein kinase C‐dependent manner. Taken together, our data show that PrPC and STI1 are essential to astrocyte development and act through distinct signaling pathways.

Membrane Elastic Properties and Cell Function

Recent studies indicate that the cell membrane, interacting with its attached cytoskeleton, is an important regulator of cell function, exerting and responding to forces. We investigate this relationship by looking for connections between cell membrane elastic properties, especially surface tension and bending modulus, and cell function. Those properties are measured by pulling tethers from the cell membrane with optical tweezers. Their values are determined for all major cell types of the central nervous system, as well as for macrophage. Astrocytes and glioblastoma cells, which are considerably more dynamic than neurons, have substantially larger surface tensions. Resting microglia, which continually scan their environment through motility and protrusions, have the highest elastic constants, with values similar to those for resting macrophage. For both microglia and macrophage, we find a sharp softening of bending modulus between their resting and activated forms, which is very advantageous for their acquisition of phagocytic functions upon activation. We also determine the elastic constants of pure cell membrane, with no attached cytoskeleton. For all cell types, the presence of F-actin within tethers, contrary to conventional wisdom, is confirmed. Our findings suggest the existence of a close connection between membrane elastic constants and cell function.

Microglial stress inducible protein 1 promotes proliferation and migration in human glioblastoma cells.

Microglial activation is a key event in the progression and infiltration of tumors. We have previously demonstrated that the co-chaperone stress inducible protein 1 (STI1), a cellular prion protein (PrP(C)) ligand, promotes glioblastoma (GBM) proliferation. In the present study, we examined the influence of microglial STI1 in the growth and invasion of the human glioblastoma cell line GBM95. We demonstrated that soluble factors secreted by microglia into the culture medium (microglia conditioned medium; MG CM) caused a two-fold increase in the proliferation of GBM95 cells. This effect was reversed when STI1 was removed from the MG CM. In this context, we have shown that microglial cells synthesize and secrete STI1. Interestingly, no difference was observed in proliferation rates when GBM cells were maintained in MG CM or MG CM containing an anti-PrP(C) neutralizing antibody. Moreover, rec STI1 and rec STI1(Δ230-245), which lack the PrP(C) binding site, both promoted similar levels of GBM95 proliferation. In the migration assays, MG CM favored the migration of GBM95 cells, but migration failed when STI1 was removed from the MG CM. We detected metalloproteinase 9 (MMP-9) activity in the MG CM, and when cultured microglia were treated with an anti-STI1 antibody, MMP-9 activity decreased. Our results suggest that STI1 is secreted by microglia and favors tumor growth and invasion through the participation of MMP-9 in a PrP(C)-independent manner.

Thyroid hormone action on astroglial cells fromdistinct brain regions during development

Astrocytes are target to triiodothyronine (T3) hormone action during rat braindevelopment. In this work, we show that astrocytes from distinct developing brain regions aredifferently responsive to thyroid hormone. Distinctly from embryonic or newborn cerebralhemisphere and mesencephalic astrocytes, newborn cerebellar and embryonic hippocampalastrocytes do not change their morphology in response to hormone treatment. We also analysedprotein synthesis and secretion from these T3‐treated astrocytes. The results showed a significantincrease in protein synthesis in astrocytes from older brain regions. Maximum effect, however,was observed in cerebral hemisphere astrocytes from newborn rats. The protein secretion effectwas also more evident in the cerebral hemisphere as well as in cerebellar astrocytes fromnewborn rats. In addition, we examined T3 effects on GFAP/vimentin expression by culturing6‐day old cerebellar astrocytes. In this case T3 seems to induce GFAP expression which might beoccurring as a first step to astrocyte differentiation.

Eradication of bovine brucellosis in the Azores, Portugal - Outcome of a 5-year programme (2002-2007) based on test-and-slaughter and RB51 vaccination.

Bovine brucellosis is an important contagious disease that can cause abortions and infertility in cattle, and can be transmitted to humans. Despite having an eradication programme in place since 1994, in 2000 the situation of bovine brucellosis due to Brucella abortus was not significantly improving in 3 of the 9 islands (Terceira, S. Miguel and S. Jorge) of the archipelago of Azores, an autonomous region of Portugal. Farming on these islands, particularly dairy, is extensive. Therefore, the use of RB51 vaccine, which does not induce antibodies detectable with routine brucellosis diagnostic tests, was implemented. This article reports the results of an eradication programme based on RB51 mass vaccination combined with test-and-slaughter, which was implemented in the Azores during the 2002-2007 period. During the first round of vaccination, both adult cows and heifers were vaccinated. Subsequently, only replacement stock aged 4-12 months, were immunized with RB51.The test-and-slaughter policy based on bulk milk ring test (MRT) and serological surveillance was maintained. During this period, the average brucellosis herd incidence, herd prevalence and individual prevalence decreased 69.26%, 39.26% and 75.41% respectively for the three above-mentioned islands. However, disease reduction approaching eradication was obtained only on the island of Terceira, where a high level of vaccine coverage was rapidly reached and regularly maintained together with strict application of a test-and-slaughter programme. This work shows that the RB51 vaccine could be a useful tool for eradicating bovine brucellosis in well-controlled epidemiological units provided that there is mass vaccine coverage for a sufficiently long period of time and it is combined with an appropriate test-and-slaughter programme.