Toll-like receptor 4 stimulation with the detoxified ligand monophosphoryl lipid A improves Alzheimer's disease-related pathology.

Alzheimer's disease (AD) is the most common cause of dementia worldwide. The pathogenesis of this neurodegenerative disease, currently without curative treatment, is associated with the accumulation of amyloid ß (Aß) in brain parenchyma and cerebral vasculature. AD patients are unable to clear this toxic peptide, leading to Aß accumulation in their brains and, presumably, the pathology associated with this devastating disease. Compounds that stimulate the immune system to clear Aß may therefore have great therapeutic potential in AD patients. Monophosphoryl lipid A (MPL) is an LPS-derived Toll-like receptor 4 agonist that exhibits unique immunomodulatory properties at doses that are nonpyrogenic. We show here that repeated systemic injections of MPL, but not LPS, significantly improved AD-related pathology in APP(swe)/PS1 mice. MPL treatment led to a significant reduction in Aß load in the brain of these mice, as well as enhanced cognitive function. MPL induced a potent phagocytic response by microglia while triggering a moderate inflammatory reaction. Our data suggest that the Toll-like receptor 4 agonist MPL may be a treatment for AD.

Self-antigen MASH2 combined with the AS15 immunostimulant induces tumor protection in colorectal cancer mouse models.

Human achaete scute homolog 2 (HASH2) and its murine ortholog MASH2 are potential targets for colorectal cancer immunotherapy. We assessed immunogenicity and antitumor potential of recombinant MASH2 protein combined with AS15 immunostimulant (recMASH2+AS15) in CB6F1 and Apc+/Min-FCCC mice. CB6F1 mice received 4 injections of recMASH2+AS15 or AS15 alone before challenge with TC1-MASH2 tumor cells (Tumor Challenge). Apc+/Min-FCCC mice received 9 injections of recMASH2+AS15 or vehicle (phosphate buffer saline [PBS] or AS15 alone), before (two independent Prophylactic Studies) or after (Immunotherapy) colon adenomas were detectable by colonoscopy. CB6F1 mice immunized with recMASH2+AS15 had a significantly smaller mean tumor size and improved survival rate compared to controls (104 mm2 vs. 197 mm2 [p = 0.009] and 67% vs. 7% [p = 0.001], respectively). In Prophylactic Study 1, the mean number of colon adenomas was significantly lower in Apc+/Min-FCCC mice receiving recMASH2+AS15 compared to PBS (1.8 [95% confidence interval 1.0-3.3] vs. 5.2 [3.7-7.4], p = 0.003). Fewer microadenomas were observed in recMASH2+AS15 groups compared to PBS in both Prophylactic Studies (Study 1: mean 0.4 [0.2-1.0] vs. 1.5 [0.9-2.4], p = 0.009; Study 2: 0.4 [0.2-0.6] vs. 1.1 [0.8-1.5], p = 0.001). In the Immunotherapy Study, fewer colon adenomas tended to be observed in recMASH2+AS15-treated mice (4.1 [2.9-6.0]) compared to controls (AS15 4.7 [3.3-6.6]; PBS 4.9 [3.5-6.9]; no significant difference). recMASH2+AS15 induced MASH2-specific antibody and CD4+ responses in both mouse models. recMASH2+AS15 partially protected mice against MASH2-expressing tumors and reduced spontaneous colorectal adenomas in Apc+/Min-FCCC mice, indicating that MASH2/HASH2 antigens are targets for colorectal cancer immunotherapy.

Inhibition of angiogenic properties of brain endothelial cells by platelet-derived sphingosine-1-phosphate.

The platelet-derived lysophospholipid sphingosine-1-phosphate (S1P) is present in blood plasma and is one of the most potent growth factors displaying proangiogenic activity towards endothelial cells (EC) derived from various tissues. The paracrine regulation of brain angiogenesis by platelet-derived growth factors is, however, poorly understood. In the present study, we assessed the role of S1P on brain EC migration and tubulogenesis, using rat brain-derived (RBE4) EC as an in vitro model. We show that S1P inhibits brain EC migration and tubulogenesis, while it displays proangiogenic activity towards noncerebral EC. Overexpression of the S1P receptor S1P-1 in RBE4 cells potentiated all of the S1P-mediated events. We also show that the lack of expression of MT1-MMP, a membrane-bound matrix metalloproteinase that is thought to cooperate with S1P in tubulogenic processes, may explain the antiangiogenic activity of S1P on brain vasculature. Altogether our results support the hypothesis of a tissue-specific, antiangiogenic role of S1P in the brain, which may help to stabilize the cerebral vasculature and thus have crucial impact on the setting and regulation of normal brain vascularization.

Methods to monitor monocytes-mediated amyloid-beta uptake and phagocytosis in the context of adjuvanted immunotherapies.

Antibody-mediated capture of amyloid-beta (Aß) in peripheral blood was identified as an attractive strategy to eliminate cerebral toxic amyloid in Alzheimer's disease (AD) patients and murine models. Alternatively, defective capacity of peripheral monocytes to engulf Aß was reported in individuals with AD. In this report, we developed different approaches to investigate cellular uptake and phagocytosis of Aß, and to examine how two immunological devices--an immunostimulatory Adjuvant System and different amyloid specific antibodies--may affect these biological events. Between one and thirteen months of age, APPswe X PS1.M146V (TASTPM) AD model mice had decreasing concentrations of Aß in their plasma. In contrast, the proportion of blood monocytes containing Aß tended to increase with age. Importantly, the TLR-agonist containing Adjuvant System AS01B primed monocytes to promote de novo Aß uptake capacity, particularly in the presence of anti-Aß antibodies. Biochemical experiments demonstrated that cells achieved Aß uptake and internalization followed by Aß degradation via mechanisms that required effective actin polymerization and proteolytic enzymes such as insulin-degrading enzyme. We further demonstrated that both Aß-specific monoclonal antibodies and plasma from Aß-immunized mice enhanced the phagocytosis of 1 µm Aß-coated particles. Together, our data highlight a new biomarker testing to follow amyloid clearance within the blood and a mechanism of Aß uptake by peripheral monocytes in the context of active or passive immunization, and emphasize on novel approaches to investigate this phenomenon.

Radiation induced-tubulogenesis in endothelial cells is antagonized by the antiangiogenic properties of green tea polyphenol (-) epigallocatechin-3-gallate.

Radiation therapy is a widely-used option for the treatment of a variety of solid tumors. Although effective, ionizing radiation (IR) may give rise to various side effects, including secondary tumors. In agreement with this, recent reports have demonstrated increased invasive potential in different tumor-derived cell lines following radiation treatment. Many of the molecular effects of IR specifically on the endothelial cells involved in tumor neo-vascularization remain unknown. In this study, we found that low sublethal single doses of IR applied to human umbilical vein endothelial cells stimulated cell migration and in vitro tubulogenesis. This correlated with an increase in membrane type-1 matrix metalloproteinase (MT1-MMP) protein expression, a crucial enzyme that promotes endothelial cell migration and tube formation, and of caveolin-1, a protein that regulates tube formation. Cell adhesion was also promoted by IR, reflected in increased gene expression levels of cell surface beta(3) integrin. Pretreatment of the cells with epigallocatechin-3-gallate (EGCg), a green tea catechin that possesses anti-angiogenic properties, prevented most of the IR-induced cellular and molecular events. These observations suggest that current protocols involving radiation therapy for the treatment of cancer can paradoxically promote angiogenesis, but can be improved by combination with anti-angiogenic molecules such as EGCg to target those tumor-derived endothelial cells that escaped IR-induced apoptosis.

Modulation of P-glycoprotein function by sphingosine kinase-1 in brain endothelial cells.

P-glycoprotein (P-gp), an ABC-transporter highly expressed in brain capillaries, protects the brain by extruding xenobiotics. However, its overexpression has also been associated with the multidrug resistance phenotype in tumors. Here, we have investigated the regulation of P-gp transport activity by sphingosine kinase 1 (SphK-1) in brain endothelial cells. We first demonstrated that SphK-1 is overexpressed in endothelial cells (EC) isolated from rat brain tumors compared with EC from normal brain. We also provide evidence that the overexpression of SphK-1 in the cerebral EC line RBE4 leads to the up-regulation of P-gp, both at the gene and protein levels, and that this modulation depends on the catalytic activity of SphK-1. Moreover, we determined the effect of sphingosine-1-phosphate (S1P), the product of SphK-1, on P-gp function. S1P strongly stimulates P-gp transport activity, without modulating its expression. Finally, we found that the S1P-mediated stimulation of P-gp activity is mediated by S1P-1 and S1P-3 receptors at the RBE4 cell surface. Altogether, these results indicate that SphK-1 and its product S1P are involved in the control of P-gp activity in RBE4 cells. Since SphK-1 is overexpressed in EC from brain tumors, these data also suggest that this kinase and its product could contribute to the acquisition and the maintenance of the multidrug resistance phenotype in brain tumor-derived endothelial cells.

Modulation of p-glycoprotein function by caveolin-1 phosphorylation.

p-glycoprotein (p-gp) is an ATP-binding cassette transporter and its overexpression is responsible for the acquisition of the multidrug resistance phenotype in human tumors. p-gp is localized at the blood-brain barrier and is involved in brain cytoprotection. Our previous work used immunoprecipitation to show that caveolin-1 can interact with p-gp. In this study, we provide evidence that caveolin-1 regulates p-gp transport activity in a rat brain endothelial cell line (RBE4). Down-regulation of caveolin-1 by siRNA reduced the interaction between p-gp and caveolin-1, followed by a decrease in [3H]-Taxol and [3H]-Vinblastine accumulation in RBE4 cells. The latter result showed that down-regulation of caveolin-1 enhanced p-gp transport activity. RBE4 cells were also transfected with Sarcoma in order to modulate caveolin-1 phosphorylation. Overexpression of Sarcoma, a protein tyrosine kinase, stimulated caveolin-1 phosphorylation and increased both [3H]-Taxol and [3H]-Vinblastine accumulation as well as Hoechst 33342 accumulation. Transfection of caveolin-1 inhibits p-gp transport activity. Conversely, transfection of the mutant cavY14F decreased the p-gp/caveolin-1 interaction and reduced accumulation of the two p-gp substrates. Thus, our data show that caveolin-1 regulates p-gp function through the phosphorylation state of caveolin-1 in endothelial cells from the blood-brain barrier.

Lebectin, a Macrovipera lebetina venom-derived C-type lectin, inhibits angiogenesis both in vitro and in vivo.

Integrins play an essential role in endothelial cell motility processes during angiogenesis and thus present interesting targets for the development of new anti-angiogenic agents. Snake venoms naturally contain a variety of proteins that can affect integrin-ligand interactions. Recently, the C-type lectin proteins (CLPs) have been characterized as efficient modulators of integrin functions. In this study, we investigated the anti-angiogenic activity of lebectin, a newly discovered CLP from Macrovipera lebetina venom. Human brain microvascular endothelial cells (HBMEC), used as an in vitro model, express alphavbeta3, alphavbeta5, and alpha5beta1 integrins, as well as the alpha2, alpha3, alpha6, and beta4 subunits. Our data show that lebectin acts as a very potent inhibitor (IC(50) approximately 0.5 nM) of HBMEC adhesion and migration on fibronectin by blocking the adhesive functions of both the alpha5beta1 and alphaV integrins. In addition, lebectin strongly inhibits both HBMEC in vitro tubulogenesis on Matrigel trade mark (IC(50) = 0.4 nM) and proliferation. Finally, using both a chicken CAM assay and a Matrigel trade mark Plug assay in nude mice, our results show that lebectin displays potent anti-angiogenic activity in vivo. Lebectin thus represents a new C-type lectin with anti-angiogenic properties with great potential for the treatment of angiogenesis-related diseases.

Medulloblastoma cell invasion is inhibited by green tea (-)epigallocatechin-3-gallate.

Epigallocatechin-3-gallate (EGCG), the major green tea polyphenol, can reach the brain following oral intake and could thus act as an anti-tumoral agent targeting several key steps of brain cancer cells invasive activity. Because integrin-mediated extracellular matrix recognition is crucial during the cell adhesion processes involved in carcinogenesis, we have investigated the effects of EGCG on different cellular integrins of the pediatric brain tumor-derived medulloblastoma cell line DAOY. Using flow cytometry, we report the levels of expression of several cell surface integrins in DAOY. These include high expression of alpha2, alpha3, and beta1 integrins, as well as alphav and beta3 integrins. Moreover, we provide evidence that EGCG can antagonize DAOY cell migration specifically on collagen by increasing cell adhesive ability through specific gene and protein upregulation of the beta1 integrin subunit. Our results suggest that this naturally occurring green tea polyphenol may thus be used as a nutraceutical therapeutic agent in targeting the invasive character of medulloblastomas.