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Novel markers of blood-brain barrier dysfunction in HIV encephalitis
20
P o s t e r A b s t r a c t s / J o u r n a l o f N e u r o i m m u n o l o g y 9 0 (1998) 1 3 - 1 0 5
89
92
Brain M a s t Cell Stimulated by A n t i - l e E or Substance P E n h a n c e s Local T N F - a Production a n d M a y Provide a M e c h a n i s m for Regulated O p e n i n g of the Blood Brain B a r r i e r d u r i n g Early Events of N e u r o i n f l a m m a t i o n IL Cocchiara, A. Bongiovanni, G. Albeggiani, A. Azzolina, D. Geraci, CNR, Ist~tutodi Biologia delloS&iluppo,Italy
I C A M 1 Expression and Fluid Phase Endoeytosis of Brain M i e r o v a s e u l a r E n d o t h d i a l Cells Following I n t e r f e r o n - b e t a - l a a n d TNF-aipha G. Defazin, M. Trnjano, M. CfioreUi, M. Ruggieri, D. Paolicelli, F. Giuliani, P. Livrea~ UniversityofBari
Brain Mast Cell (BMC) are associated whit the cerebral vasculature and neuropeptide containing peripheral nerves and play a rule in inflammation. Turnout necrosis factor-alpha (TNF
We studied the effect of interferon (IF) 13-1a on basal and T N F a induced ICAM 1 expression and fluid phase endocytosis (FPE) of horseradish peroxidase in cultured rat brain microvascular endothelial cells. Pre-treatment of cultures with IFI3-1a for 48 h followed by 24 h coincubation with TNFot (100 U/ml) and IFl3-1a (1000 U/ml) resulted in significant downregulation of TNFct-induced ICAM l expression and FPE. Downregulation was not observed when 24 h coincubation with TNFo~ (100 U/ml) and IF!3-1a (1000 U/ml) was followed by 1000U/ml IFI3-1a for 48 h. These in vitro findings indicate that the blood brain barrier endothelium may be a target o f IFI3-1a, and correlate with the ability of chronic IFI3-1a treatment to prevent clinical exacerbations of RR MS and the appearance on MRI of new lesions enhanced by gadolinium
9O
93
Novel M a r k e r s of Blood-Brain B a r r i e r Dysfunction in H I V Encephalitis L.M. Dallasta, LL. Maguire, L. Pisarov, C.L. Achim, Universityo/Pittsburgh Medical Center,USA
Astroeytes Express CS1 Domain, Involved in L y m p h o c y t e Binding L.J.W. Van der Laan, VrijeUniver~*itei~Dept. CellBiology,Netherlands, C.LA. De Grout, Vroe Universiteit,Dept.Pathology,Netherlands, M. Elites, Cytet, SanDiego, USA,C.D. D O k s ~ VrijeUniversiteit,Dept. CellBiology,Netherlands
The entry of peripheral immune cells into the CNS is restricted by the bloodbrain barrier (BBB), which is formed by continuous, high-resistance tight junctions between the cerebral capillary endothelium. Previous studies assessing BBB pathology in HIV encephalitis (HIVE) have demonstrated serum protein extravasation, however, the route of entry of these proteins and its relationship to trafficking HIV-infected monocytes and CNS viral burden remains unclear. To investigate these issues, a histologic and immunohistochemical analysis of tight junction-associated proteins was performed on post-mortem brains from AIDS patients with and without HIVE, and from HIV-seronegative control subjects. Only brains from patients with HIV encephalitis showed marked alterations in the pattern and level of expression of the tight junction-associated membrane proteins, occludin and ZO- 1. These changes were associated with the perivascular accumulation of CD68-positive mononuclear cells, as well as marked fibrinogen extravasation and severe gliosis. These findings demonstrate that tight junctionassociated alterations in the BBB occur during HIVE and that this may significantly augment the trafficking of HiV-infected monocytes into the CNS. In addition, this proteins may serve as novels markers of BBB dysfunction in neuroinflammatory diseases.
Some splice variants of the fibronectin (Fn) contain the 25 amino acid polypeptide domain termed CSI, which serves as a ligand for an adhesion molecule on lymphocytes and monocytes: u4B1 integrin or VLA-4. VLA-4 is crucial for recruitment of mononuclear cells towards the CNS and induction of experimental allergic encephalomyelitis J. Here we investigated the expression of Fn and of the CSI domain on astrncytes in human brain tissues sections of MS patients and healthy controls, and on human astrncytes obtained from autopsy material and cultured. Binding assays on cultured astrncytes were performed with VLA-4 positive lymphoblastic cell lines. Results showed that astrocytes express Fn in vitro, but not in vivo. In contrast, the CS1 domain was expressed both in vitro, and in rive in MS lesions on astrocytes, indicating that astrocytes express a CSI containing surface protein in rive, different from Fn. Western blot analysis confLrmed this indication. VLA-4 expressing lympboma cell lines bound to CSI expressing cultured astrocytes. This interaction could partly he blocked by an antibody to the CSI dommn as well as by a synthetic CSI peptide. IyedaockT.A. et aL 0992) Nature356:63456
91
94
N o v d In Vivo Model of the H u m a n Blood-brain B a r r i e r L.M, Dallasta, J.L. Maguire, M.G. White, A.P. Mehta, C.A. Wiley, C.L. Achim, UniversityofPi~.sburghMedical Center, USA
T1 andT2 Cytoklnes Regulate CNS M i e r o v a s e u l a r Perieyte Differentiation P. Dore-Duffy. IL Balabanov, WayneState University,USA
To study blood-brain barrier (BBB) perturbations and immune cell trafficking into the human (CNS) during pathologic conditions, we have developed a novel. experimental in vivo model oftbe BBB. This model utilizes human CNS grafts which are injected intracerebrally into SCID mice. The grafts develop from aggregates of isolated human fetal cerebral mierovascular endothelial cells that are combined with human fetal neuroglial ceils. Before implantation, aggregates are cultured in vitro with VEGF and bFGF to promote vasculogenesis. In addition, human endothelial ceils are labeled in vitro with a Dil-labeled tracer to permit cell tracking in rive. Compared to control grafts, human endothelial cellsupplemented grafts demonstrate central blood vessels that are immanureactive for human-specific capillary wall markers as early as two weeks posttransplantation. Furthermore, these graft blood vessels are immunoreactive for the BBB-associated proteins, eecludin and ZO-I. Ultrastmcmrally, photoconversion of Dil demonstrates that the endothelial-lined blood vessels are of human origin. By providing an appropriate CNS milieu and a human microvascalar bed, this in vivo model will permit analysis of neurninfiammatory interactions at the human BBB that may contribute to the development of a variety of CNS inflammatory disorders.
CNS pericytes (PC) are an integral cellular constituent of the blood-brain barrier (BBB). The expression of alpha-smooth muscle actin (ctSMA) have linked the PC with microvascular contractility. However, txSMA is heterogeneously expressed and it is not known whether 0tSMA+ or ctSMA- PC are unique populations or represent states of differentiation/activation. In this study, we examine TI, 'I"2 cytokine regulation of PC aSMA. Primary Lewis rat CNS PC express ¢zSMA (60%) and CDI lb (100%). They are MHC class lI-, class I +and iCAM-I _+. IFNT(500 u/ml) increased ICAM-1, induced MHC class It Ag and significantly reduced ctSMA. PC changed morphology in a polar fashion. oSMA was found at the polar ends coqocalized with the receptor for urokinase piasminogea activator (uPAR), a molecule expressed by migrating cells. TGF[i (10 ng/ml) restored PC quiescence concomitant with an upregulatiun and rearrangement of ctSMA, and downregulation of uPAR, MHC class II and ICAM-I. Results indicate that IF'N'/(TI) activates PC and induces a migrating phenotype, while TGFI~ (T2) restores quiescence. Functional differentiation of PC is likely an important regulatory mechanism at the BBB.
P o s t e r A b s t r a c t s / J o u r n a l o f N e u r o i m m u n o l o g y 9 0 (1998) 1 3 - 1 0 5
89
92
Brain M a s t Cell Stimulated by A n t i - l e E or Substance P E n h a n c e s Local T N F - a Production a n d M a y Provide a M e c h a n i s m for Regulated O p e n i n g of the Blood Brain B a r r i e r d u r i n g Early Events of N e u r o i n f l a m m a t i o n IL Cocchiara, A. Bongiovanni, G. Albeggiani, A. Azzolina, D. Geraci, CNR, Ist~tutodi Biologia delloS&iluppo,Italy
I C A M 1 Expression and Fluid Phase Endoeytosis of Brain M i e r o v a s e u l a r E n d o t h d i a l Cells Following I n t e r f e r o n - b e t a - l a a n d TNF-aipha G. Defazin, M. Trnjano, M. CfioreUi, M. Ruggieri, D. Paolicelli, F. Giuliani, P. Livrea~ UniversityofBari
Brain Mast Cell (BMC) are associated whit the cerebral vasculature and neuropeptide containing peripheral nerves and play a rule in inflammation. Turnout necrosis factor-alpha (TNF
We studied the effect of interferon (IF) 13-1a on basal and T N F a induced ICAM 1 expression and fluid phase endocytosis (FPE) of horseradish peroxidase in cultured rat brain microvascular endothelial cells. Pre-treatment of cultures with IFI3-1a for 48 h followed by 24 h coincubation with TNFot (100 U/ml) and IFl3-1a (1000 U/ml) resulted in significant downregulation of TNFct-induced ICAM l expression and FPE. Downregulation was not observed when 24 h coincubation with TNFo~ (100 U/ml) and IF!3-1a (1000 U/ml) was followed by 1000U/ml IFI3-1a for 48 h. These in vitro findings indicate that the blood brain barrier endothelium may be a target o f IFI3-1a, and correlate with the ability of chronic IFI3-1a treatment to prevent clinical exacerbations of RR MS and the appearance on MRI of new lesions enhanced by gadolinium
9O
93
Novel M a r k e r s of Blood-Brain B a r r i e r Dysfunction in H I V Encephalitis L.M. Dallasta, LL. Maguire, L. Pisarov, C.L. Achim, Universityo/Pittsburgh Medical Center,USA
Astroeytes Express CS1 Domain, Involved in L y m p h o c y t e Binding L.J.W. Van der Laan, VrijeUniver~*itei~Dept. CellBiology,Netherlands, C.LA. De Grout, Vroe Universiteit,Dept.Pathology,Netherlands, M. Elites, Cytet, SanDiego, USA,C.D. D O k s ~ VrijeUniversiteit,Dept. CellBiology,Netherlands
The entry of peripheral immune cells into the CNS is restricted by the bloodbrain barrier (BBB), which is formed by continuous, high-resistance tight junctions between the cerebral capillary endothelium. Previous studies assessing BBB pathology in HIV encephalitis (HIVE) have demonstrated serum protein extravasation, however, the route of entry of these proteins and its relationship to trafficking HIV-infected monocytes and CNS viral burden remains unclear. To investigate these issues, a histologic and immunohistochemical analysis of tight junction-associated proteins was performed on post-mortem brains from AIDS patients with and without HIVE, and from HIV-seronegative control subjects. Only brains from patients with HIV encephalitis showed marked alterations in the pattern and level of expression of the tight junction-associated membrane proteins, occludin and ZO- 1. These changes were associated with the perivascular accumulation of CD68-positive mononuclear cells, as well as marked fibrinogen extravasation and severe gliosis. These findings demonstrate that tight junctionassociated alterations in the BBB occur during HIVE and that this may significantly augment the trafficking of HiV-infected monocytes into the CNS. In addition, this proteins may serve as novels markers of BBB dysfunction in neuroinflammatory diseases.
Some splice variants of the fibronectin (Fn) contain the 25 amino acid polypeptide domain termed CSI, which serves as a ligand for an adhesion molecule on lymphocytes and monocytes: u4B1 integrin or VLA-4. VLA-4 is crucial for recruitment of mononuclear cells towards the CNS and induction of experimental allergic encephalomyelitis J. Here we investigated the expression of Fn and of the CSI domain on astrncytes in human brain tissues sections of MS patients and healthy controls, and on human astrncytes obtained from autopsy material and cultured. Binding assays on cultured astrncytes were performed with VLA-4 positive lymphoblastic cell lines. Results showed that astrocytes express Fn in vitro, but not in vivo. In contrast, the CS1 domain was expressed both in vitro, and in rive in MS lesions on astrocytes, indicating that astrocytes express a CSI containing surface protein in rive, different from Fn. Western blot analysis confLrmed this indication. VLA-4 expressing lympboma cell lines bound to CSI expressing cultured astrocytes. This interaction could partly he blocked by an antibody to the CSI dommn as well as by a synthetic CSI peptide. IyedaockT.A. et aL 0992) Nature356:63456
91
94
N o v d In Vivo Model of the H u m a n Blood-brain B a r r i e r L.M, Dallasta, J.L. Maguire, M.G. White, A.P. Mehta, C.A. Wiley, C.L. Achim, UniversityofPi~.sburghMedical Center, USA
T1 andT2 Cytoklnes Regulate CNS M i e r o v a s e u l a r Perieyte Differentiation P. Dore-Duffy. IL Balabanov, WayneState University,USA
To study blood-brain barrier (BBB) perturbations and immune cell trafficking into the human (CNS) during pathologic conditions, we have developed a novel. experimental in vivo model oftbe BBB. This model utilizes human CNS grafts which are injected intracerebrally into SCID mice. The grafts develop from aggregates of isolated human fetal cerebral mierovascular endothelial cells that are combined with human fetal neuroglial ceils. Before implantation, aggregates are cultured in vitro with VEGF and bFGF to promote vasculogenesis. In addition, human endothelial ceils are labeled in vitro with a Dil-labeled tracer to permit cell tracking in rive. Compared to control grafts, human endothelial cellsupplemented grafts demonstrate central blood vessels that are immanureactive for human-specific capillary wall markers as early as two weeks posttransplantation. Furthermore, these graft blood vessels are immunoreactive for the BBB-associated proteins, eecludin and ZO-I. Ultrastmcmrally, photoconversion of Dil demonstrates that the endothelial-lined blood vessels are of human origin. By providing an appropriate CNS milieu and a human microvascalar bed, this in vivo model will permit analysis of neurninfiammatory interactions at the human BBB that may contribute to the development of a variety of CNS inflammatory disorders.
CNS pericytes (PC) are an integral cellular constituent of the blood-brain barrier (BBB). The expression of alpha-smooth muscle actin (ctSMA) have linked the PC with microvascular contractility. However, txSMA is heterogeneously expressed and it is not known whether 0tSMA+ or ctSMA- PC are unique populations or represent states of differentiation/activation. In this study, we examine TI, 'I"2 cytokine regulation of PC aSMA. Primary Lewis rat CNS PC express ¢zSMA (60%) and CDI lb (100%). They are MHC class lI-, class I +and iCAM-I _+. IFNT(500 u/ml) increased ICAM-1, induced MHC class It Ag and significantly reduced ctSMA. PC changed morphology in a polar fashion. oSMA was found at the polar ends coqocalized with the receptor for urokinase piasminogea activator (uPAR), a molecule expressed by migrating cells. TGF[i (10 ng/ml) restored PC quiescence concomitant with an upregulatiun and rearrangement of ctSMA, and downregulation of uPAR, MHC class II and ICAM-I. Results indicate that IF'N'/(TI) activates PC and induces a migrating phenotype, while TGFI~ (T2) restores quiescence. Functional differentiation of PC is likely an important regulatory mechanism at the BBB.