Additional file 2. Claudin-12 expression in CNS tissue. Immunohistochemical staining for β-galact... more Additional file 2. Claudin-12 expression in CNS tissue. Immunohistochemical staining for β-galactosidase activity (blue), in frozen sections from the eye of claudin-12lacZ/+ C57BL/6J mice and of a Tie2-lacZ mouse, as a positive control. Images show a similar detection of β-galactosidase activity in cells not associated with the vasculature in the brain cortex and in the retina of claudin-12lacZ/+ C57BL/6J mice. Arrows point to vessel associated staining in the brain of the claudin-12lacZ/+ C57BL/6J mouse and to endothelial cell specific staining in the Tie2-LacZ mouse. * highlights the typical punctate staining pattern for β-galactosidase activity in the brain. Boxed areas are shown in higher magnification. Three independent stainings were performed. Scale bars = 10 and 50 μm, as indicated.
Additional file 4. Lack of reagents allowing to localize expression of claudin-12 protein. (A) Im... more Additional file 4. Lack of reagents allowing to localize expression of claudin-12 protein. (A) Immunofluorescence staining of frozen brain sections from WT and claudin-12lacZ/lacZ C57BL/6J mice with the anti-claudin-12 antibody from IBL represented in green produces indistinguishable vascular and apparently junction associated staining in the brain tissues of both, WT and the claudin-12lacZ/lacZ C57BL/6J mice. Scale bar = 50 μm. (B) Immunofluorescence staining of frozen liver sections from WT and claudin-12lacZ/lacZ C57BL/6J mice, using two different antibodies for claudin-12, represented in red. Notice how the antibody from IBL stains WT and claudin-12lacZ/lacZ tissue, while the anti-claudin-12 antibody from Invitrogen does not recognize claudin-12 in neither of the samples. Nuclei are stained with DAPI (blue). Three independent stainings were done. Scale bar = 100 μm.
The sphingosine 1-phosphate (S1P) receptor 1 (S1P1) has emerged as a novel therapeutic target for... more The sphingosine 1-phosphate (S1P) receptor 1 (S1P1) has emerged as a novel therapeutic target for the treatment of multiple sclerosis (MS). FingolimodR (FTY720) is the first functional antagonist of S1P1 that has been approved for oral treatment of MS. Previously, we have developed novel butterfly derivatives of FTY720 that acted similar to FTY720 in reducing disease symptoms in a mouse model of experimental autoimmune encephalomyelitis (EAE). In this study, we have synthesized a piperidine derivative of the oxazolo-oxazole compounds, denoted ST-1505, and its ring-opened analogue ST-1478, and characterized their in-vitro and in-vivo functions. Notably, the 3-piperidinopropyloxy moiety resembles a structural motif of pitolisant, a drug with histamine H3R antagonistic/inverse agonist activity approved for the treatment of narcolepsy. Both novel compounds exerted H3R affinities, and in addition, ST-1505 was characterised as a dual S1P1+3 agonist, whereas ST-1478 was a dual S1P1+5 agonist. Both multitargeting compounds were also active in mice and reduced the lymphocyte numbers as well as diminished disease symptoms in the mouse model of MS. The effect of ST-1478 was dependent on SK-2 activity suggesting that it is a prodrug like FTY720, but with a more selective S1P receptor activation profile, whereas ST-1505 is a fully active drug even in the absence of SK-2. In summary, these data suggest that the novel well soluble piperidine derivatives ST-1505 and ST-1478 hold promise as novel drugs for the treatment of MS and other autoimmune or inflammatory diseases, and by their H3R antagonist potency, they might additionally improve cognitive impairment during disease.
Brain edema is the main cause of death from brain infarction. The polarized expression of the wat... more Brain edema is the main cause of death from brain infarction. The polarized expression of the water channel protein aquaporin-4 (AQP4) on astroglial endfeet surrounding brain microvessels suggests a role in brain water balance. Loss of astrocyte foot process anchoring to the basement membrane (BM) accompanied by the loss of polarized localization of AQP4 to astrocytic endfeet has been shown to be associated with vasogenic/extracellular edema in neuroinflammation. Here, we asked if loss of astrocyte polarity is also observed in cytotoxic/intracellular edema following focal brain ischemia after transient middle cerebral artery occlusion (tMCAO). Upon mild focal brain ischemia, we observed diminished immunostaining for the BM components laminin α4, laminin α2, and the proteoglycan agrin, in the core of the lesion, but not in BMs in the surrounding penumbra. Staining for the astrocyte endfoot anchorage protein β-dystroglycan (DG) was dramatically reduced in both the lesion core and the penumbra, and AQP4 and Kir4.1 showed a loss of polarized localization to astrocytic endfeet. Interestingly, we observed that mice deficient for agrin expression in the brain lack polarized localization of β-DG and AQP4 at astrocytic endfeet and do not develop early cytotoxic/intracellular edema following tMCAO. Taken together, these data indicate that the binding of DG to agrin embedded in the subjacent BM promotes polarized localization of AQP4 to astrocyte endfeet. Reduced DG protein levels and redistribution of AQP4 as observed upon tMCAO might therefore counteract early edema formation and reflect a beneficial mechanism operating in the brain to minimize damage upon ischemia.
Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (C... more Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) in young adults leading to severe disability. Besides genetic traits, environmental factors contribute to MS pathogenesis. Cigarette smoking increases the risk of MS in an HLA-dependent fashion, but the underlying mechanisms remain unknown. Here, we explored the effect of cigarette smoke exposure on spontaneous and induced models of experimental autoimmune encephalomyelitis (EAE) by evaluating clinical disease and, when relevant, blood leukocytes and histopathology. In the relapsing-remitting (RR) transgenic model in SJL/J mice, we observed very low incidence in both smoke-exposed and control groups. In the optico-spinal encephalomyelitis (OSE) double transgenic model in C57BL/6 mice, the early onset of EAE prevented a meaningful evaluation of the effects of cigarette smoke. In EAE models induced by immunization, daily exposure to cigarette smoke caused a delayed onset of EAE followe...
SUMMARY The chronic neuro-inflammatory character of multiple sclerosis (MS) suggests that the nat... more SUMMARY The chronic neuro-inflammatory character of multiple sclerosis (MS) suggests that the natural process to resolve inflammation is impaired. This protective process is orchestrated by specialized pro-resolving lipid mediators (SPMs), but to date, the role of SPMs in MS remains largely unknown. Here, we provide in vivo evidence that treatment with the SPM lipoxin A4 (LXA4) ameliorates clinical symptoms of experimental autoimmune encephalomyelitis (EAE) and inhibits CD4+ and CD8+ T cell infiltration into the central nervous system (CNS). Moreover, we show that LXA4 potently reduces encephalitogenic Th1 and Th17 effector functions, both in vivo and in isolated human T cells from healthy donors and patients with relapsing-remitting MS. Finally, we demonstrate that LXA4 affects the spinal cord lipidome by significantly reducing the levels of pro-inflammatory lipid mediators during EAE. Collectively, our findings provide mechanistic insight into LXA4-mediated amelioration of neuro-inflammation and highlight the potential clinical application of LXA4 for MS.
Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous sys... more Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous system (CNS) which is associated with lower life expectancy and disability. The experimental antigen-induced encephalomyelitis (EAE) in mice is a useful animal model of MS, which allows exploring the etiopathogenetic mechanisms and testing novel potential therapeutic drugs. A new therapeutic paradigm for the treatment of MS was introduced in 2010 through the sphingosine 1-phosphate (S1P) analogue fingolimod (FTY720, Gilenya®), which acts as a functional S1P1 antagonist on T lymphocytes to deplete these cells from the blood. In this study, we synthesized two novel structures, ST-1893 and ST-1894, which are derived from fingolimod and chemically feature a morpholine ring in the polar head group. These compounds showed a selective S1P1 activation profile and a sustained S1P1 internalization in cultures of S1P1-overexpressing Chinese hamster ovary (CHO)-K1 cells, consistent with a functional an...
SUMMARYClostridium perfringens β-toxin (CPB) is a highly active hemolysin β-pore forming toxin an... more SUMMARYClostridium perfringens β-toxin (CPB) is a highly active hemolysin β-pore forming toxin and the essential virulence factor for a severe, necro-hemorrhagic enteritis in animals and humans. In vivo and in vitro it exerts a remarkable cell type specificity towards endothelial cells, platelets and some leucocytic cell lines. The target cell specificity of CPB is, however, poorly understood and a receptor explaining this selective toxicity has not been identified. This has hampered further research into the pathogenesis of C. perfringens type C induced enteritis. Here we identify Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) as the specific membrane receptor for CPB on endothelial cells. CD31 expression is essential for CPB toxicity in endothelial cells and lethality in mice and sufficient to render previously resistant cells highly susceptible to the toxin. We further demonstrate, that the extracellular membrane proximal Ig6 domain of CD31 is required for the in...
Additional file 2. Claudin-12 expression in CNS tissue. Immunohistochemical staining for β-galact... more Additional file 2. Claudin-12 expression in CNS tissue. Immunohistochemical staining for β-galactosidase activity (blue), in frozen sections from the eye of claudin-12lacZ/+ C57BL/6J mice and of a Tie2-lacZ mouse, as a positive control. Images show a similar detection of β-galactosidase activity in cells not associated with the vasculature in the brain cortex and in the retina of claudin-12lacZ/+ C57BL/6J mice. Arrows point to vessel associated staining in the brain of the claudin-12lacZ/+ C57BL/6J mouse and to endothelial cell specific staining in the Tie2-LacZ mouse. * highlights the typical punctate staining pattern for β-galactosidase activity in the brain. Boxed areas are shown in higher magnification. Three independent stainings were performed. Scale bars = 10 and 50 μm, as indicated.
Additional file 4. Lack of reagents allowing to localize expression of claudin-12 protein. (A) Im... more Additional file 4. Lack of reagents allowing to localize expression of claudin-12 protein. (A) Immunofluorescence staining of frozen brain sections from WT and claudin-12lacZ/lacZ C57BL/6J mice with the anti-claudin-12 antibody from IBL represented in green produces indistinguishable vascular and apparently junction associated staining in the brain tissues of both, WT and the claudin-12lacZ/lacZ C57BL/6J mice. Scale bar = 50 μm. (B) Immunofluorescence staining of frozen liver sections from WT and claudin-12lacZ/lacZ C57BL/6J mice, using two different antibodies for claudin-12, represented in red. Notice how the antibody from IBL stains WT and claudin-12lacZ/lacZ tissue, while the anti-claudin-12 antibody from Invitrogen does not recognize claudin-12 in neither of the samples. Nuclei are stained with DAPI (blue). Three independent stainings were done. Scale bar = 100 μm.
The sphingosine 1-phosphate (S1P) receptor 1 (S1P1) has emerged as a novel therapeutic target for... more The sphingosine 1-phosphate (S1P) receptor 1 (S1P1) has emerged as a novel therapeutic target for the treatment of multiple sclerosis (MS). FingolimodR (FTY720) is the first functional antagonist of S1P1 that has been approved for oral treatment of MS. Previously, we have developed novel butterfly derivatives of FTY720 that acted similar to FTY720 in reducing disease symptoms in a mouse model of experimental autoimmune encephalomyelitis (EAE). In this study, we have synthesized a piperidine derivative of the oxazolo-oxazole compounds, denoted ST-1505, and its ring-opened analogue ST-1478, and characterized their in-vitro and in-vivo functions. Notably, the 3-piperidinopropyloxy moiety resembles a structural motif of pitolisant, a drug with histamine H3R antagonistic/inverse agonist activity approved for the treatment of narcolepsy. Both novel compounds exerted H3R affinities, and in addition, ST-1505 was characterised as a dual S1P1+3 agonist, whereas ST-1478 was a dual S1P1+5 agonist. Both multitargeting compounds were also active in mice and reduced the lymphocyte numbers as well as diminished disease symptoms in the mouse model of MS. The effect of ST-1478 was dependent on SK-2 activity suggesting that it is a prodrug like FTY720, but with a more selective S1P receptor activation profile, whereas ST-1505 is a fully active drug even in the absence of SK-2. In summary, these data suggest that the novel well soluble piperidine derivatives ST-1505 and ST-1478 hold promise as novel drugs for the treatment of MS and other autoimmune or inflammatory diseases, and by their H3R antagonist potency, they might additionally improve cognitive impairment during disease.
Brain edema is the main cause of death from brain infarction. The polarized expression of the wat... more Brain edema is the main cause of death from brain infarction. The polarized expression of the water channel protein aquaporin-4 (AQP4) on astroglial endfeet surrounding brain microvessels suggests a role in brain water balance. Loss of astrocyte foot process anchoring to the basement membrane (BM) accompanied by the loss of polarized localization of AQP4 to astrocytic endfeet has been shown to be associated with vasogenic/extracellular edema in neuroinflammation. Here, we asked if loss of astrocyte polarity is also observed in cytotoxic/intracellular edema following focal brain ischemia after transient middle cerebral artery occlusion (tMCAO). Upon mild focal brain ischemia, we observed diminished immunostaining for the BM components laminin α4, laminin α2, and the proteoglycan agrin, in the core of the lesion, but not in BMs in the surrounding penumbra. Staining for the astrocyte endfoot anchorage protein β-dystroglycan (DG) was dramatically reduced in both the lesion core and the penumbra, and AQP4 and Kir4.1 showed a loss of polarized localization to astrocytic endfeet. Interestingly, we observed that mice deficient for agrin expression in the brain lack polarized localization of β-DG and AQP4 at astrocytic endfeet and do not develop early cytotoxic/intracellular edema following tMCAO. Taken together, these data indicate that the binding of DG to agrin embedded in the subjacent BM promotes polarized localization of AQP4 to astrocyte endfeet. Reduced DG protein levels and redistribution of AQP4 as observed upon tMCAO might therefore counteract early edema formation and reflect a beneficial mechanism operating in the brain to minimize damage upon ischemia.
Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (C... more Multiple sclerosis (MS) is the most common inflammatory disorder of the central nervous system (CNS) in young adults leading to severe disability. Besides genetic traits, environmental factors contribute to MS pathogenesis. Cigarette smoking increases the risk of MS in an HLA-dependent fashion, but the underlying mechanisms remain unknown. Here, we explored the effect of cigarette smoke exposure on spontaneous and induced models of experimental autoimmune encephalomyelitis (EAE) by evaluating clinical disease and, when relevant, blood leukocytes and histopathology. In the relapsing-remitting (RR) transgenic model in SJL/J mice, we observed very low incidence in both smoke-exposed and control groups. In the optico-spinal encephalomyelitis (OSE) double transgenic model in C57BL/6 mice, the early onset of EAE prevented a meaningful evaluation of the effects of cigarette smoke. In EAE models induced by immunization, daily exposure to cigarette smoke caused a delayed onset of EAE followe...
SUMMARY The chronic neuro-inflammatory character of multiple sclerosis (MS) suggests that the nat... more SUMMARY The chronic neuro-inflammatory character of multiple sclerosis (MS) suggests that the natural process to resolve inflammation is impaired. This protective process is orchestrated by specialized pro-resolving lipid mediators (SPMs), but to date, the role of SPMs in MS remains largely unknown. Here, we provide in vivo evidence that treatment with the SPM lipoxin A4 (LXA4) ameliorates clinical symptoms of experimental autoimmune encephalomyelitis (EAE) and inhibits CD4+ and CD8+ T cell infiltration into the central nervous system (CNS). Moreover, we show that LXA4 potently reduces encephalitogenic Th1 and Th17 effector functions, both in vivo and in isolated human T cells from healthy donors and patients with relapsing-remitting MS. Finally, we demonstrate that LXA4 affects the spinal cord lipidome by significantly reducing the levels of pro-inflammatory lipid mediators during EAE. Collectively, our findings provide mechanistic insight into LXA4-mediated amelioration of neuro-inflammation and highlight the potential clinical application of LXA4 for MS.
Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous sys... more Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous system (CNS) which is associated with lower life expectancy and disability. The experimental antigen-induced encephalomyelitis (EAE) in mice is a useful animal model of MS, which allows exploring the etiopathogenetic mechanisms and testing novel potential therapeutic drugs. A new therapeutic paradigm for the treatment of MS was introduced in 2010 through the sphingosine 1-phosphate (S1P) analogue fingolimod (FTY720, Gilenya®), which acts as a functional S1P1 antagonist on T lymphocytes to deplete these cells from the blood. In this study, we synthesized two novel structures, ST-1893 and ST-1894, which are derived from fingolimod and chemically feature a morpholine ring in the polar head group. These compounds showed a selective S1P1 activation profile and a sustained S1P1 internalization in cultures of S1P1-overexpressing Chinese hamster ovary (CHO)-K1 cells, consistent with a functional an...
SUMMARYClostridium perfringens β-toxin (CPB) is a highly active hemolysin β-pore forming toxin an... more SUMMARYClostridium perfringens β-toxin (CPB) is a highly active hemolysin β-pore forming toxin and the essential virulence factor for a severe, necro-hemorrhagic enteritis in animals and humans. In vivo and in vitro it exerts a remarkable cell type specificity towards endothelial cells, platelets and some leucocytic cell lines. The target cell specificity of CPB is, however, poorly understood and a receptor explaining this selective toxicity has not been identified. This has hampered further research into the pathogenesis of C. perfringens type C induced enteritis. Here we identify Platelet Endothelial Cell Adhesion Molecule-1 (CD31 or PECAM-1) as the specific membrane receptor for CPB on endothelial cells. CD31 expression is essential for CPB toxicity in endothelial cells and lethality in mice and sufficient to render previously resistant cells highly susceptible to the toxin. We further demonstrate, that the extracellular membrane proximal Ig6 domain of CD31 is required for the in...
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