A 66-year-old female developed chronic inflammatory demyelinating polyneuropathy (CIDP) one year after the diagnosis of aplastic anemia. High-dose intravenous immunoglobulin (IVIg) therapy, followed by IVIg maintenance therapy, rapidly improved her weakness and hyperesthesia in four extremities. In addition, pancytopenia caused by aplastic anemia also improved following IVIg treatment in parallel. This is the first report to show the co-existence of CIDP and aplastic anemia, and a common pathomechanism may be present in these two rare autoimmune disorders.

Background: Recent studies have revealed that the disruption of the blood–brain barrier (BBB) might contribute to the induction of neurodegeneration in the progressive stage of multiple sclerosis (MS). Objective: We investigated a potential target for the serum auto-antibodies responsible for the BBB impairment in patients with secondary progressive MS (SPMS). Methods: We identified undetermined target antigens in human brain microvascular endothelial cells (BMECs) that reacted with auto-antibodies in sera from SPMS patients using a proteomic approach. In addition, we examined how the identified auto-antibodies compromise the BBB integrity. Results: We found that 10 of 11 SPMS sera had auto-antibodies against galectin-3, although the patients with other neurological diseases did not have these antibodies. Downregulation of galectin-3 led to elevated intercellular adhesion molecule-1 (ICAM-1) and phospho-nuclear factor-kappa (NFκ) B p65 expression in the BMECs. Exposure to SPMS patients’ sera also increased the protein levels of ICAM-1 and phospho-NFκB p65 in BMECs, but these effects induced by anti-galectin-3 immunoreactivity were canceled by the downregulation of galectin-3. Conclusion: Galectin-3 is a possible immunological target molecule of the pathogenic auto-antibodies and contributes to the persistent BBB breakdown in patients with SPMS. These antibodies may also serve as a novel biomarker for SPMS.

OBJECTIVE: The clearance of amyloid β (Aß) from the brain could be a novel therapeutic target for Alzheimer's disease (AD). We examined the role of efflux transporters on Aß transport using a new established conditionally immortalized human brain microvascular endothelial cell line. BACKGROUND: Although several reports demonstrate that low-density lipoprotein receptor-related protein-1 (LRP1) is a major Aβ transporter at the blood-brain barrier (BBB), the role of ATP-binding cassette transporters in efflux of Aβ at the human BBB has not yet been elucidated. In addition, conflicting data exist regarding the contribution of ATP-binding cassette transporters to the clearance of Aß through the BBB. DESIGN/METHODS: Uptake of 125I Aß1-40 by a new conditionally immortalized human brain microvascular endothelial cell line, TY09, were examined with or without down-regulation of p-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 1 (MRP4) using siRNAs specific to each molecules. Apical-to-basolateral permeability of TY09 to 125I Aß1-40 was also evaluated. RESULTS: Down-regulation of BCRP increased the uptake of 125I Aß1-40 by TY09 as well as apical-to-basolateral permeability of TY09 to 125I Aß1-40. Down-regulation of P-gp and MRP4 did not influence the uptake and permeability of 125I Aß1-40 with TY09. CONCLUSION: Our results using a new human in vitro BBB model suggest that BCRP act to prevent the blood-borne Aß from entering brain at the human BBB. Study Supported by: Research grant (26461271) from the Japan Society for the Promotion of Science, Tokyo, Japan.

Objective: In our recent in vitro study, sera from chronic inflammatory demyelinating polyneuropathy (CIDP) patients were shown to possess effects to disrupt blood-nerve barrier (BNB). We studied the molecular background of BNB damage in CIDP using patients’ sera and cultured microvascular endothelial cells derived from human peripheral nerve (PnMECs). Methods: We have obtained patient and Institutional Review Board (IRB) approval, as necessary. We evaluated the effects of sera obtained from patients with CIDP on the expression levels of tight junction proteins, intercellular cell adhesion molecule-1 (ICAM-1), actin stress fiber formation and transendothelial electrical resistance (TEER) value in the PnMECs. We then investigated the influence of the CIDP sera on PnMECs in the presence of specific rho-kinase inhibitor (Y-27632) in order to determine whether rho-kinase pathway is involved in BNB alterations induced by patients’ sera. Results: The sera obtained from the patients with CIDP significantly decreased the amount of claudin-5 and ZO-1 protein levels and TEER values in the PnMECs as compared with normal controls. CIDP sera also increased ICAM-1 protein amount and actin stress fiber formation. Treatment with Y-27632 attenuated reduction of TEER values and claudin-5 expression levels induced by CIDP sera. Y-27632 treatment also prevented the increase of ICAM-1protein amount and excessive actin stress fiber formation. Conclusion: Humoral factors in the sera of CIDP patients may disrupt BNB via activation of rho-kinase pathway.

Background: Non-motor symptoms in Parkinson's disease (PD) are considered as an important factor related to the quality of life in PD patients. However, treatment strategies for these symptoms have yet been established. Zonisamide was reported to improve motor functions of PD and have various effects on nervous systems. The present study investigated the efficacy of zonisamide in non-motor symptoms of PD. Methods: 20 patients with PD participated in this study and were treated with zonisamide (25 mg/day) for 12 weeks. When subjects entered in this study, written informed consent was acquired from each subject. General symptoms were evaluated using the Unified PD Rating Scale (UPDRS). Non-motor symptoms were measured by Non Motor Symptom Scale (NMSS), which consists of 9 subdomains. The study protocol got the approval of the Institutional Review Board. Results: Median UPDRS part III score significantly decreased from 25.5 points at baseline to 20.6 points at 12 weeks (p = 0.004). In addition, NMSS total score showed significant improvement from 55.9 +/- 51.6 to 44.3 +/- 50.4 (p = 0.044). Within each NMSS subdomain, significant amelioration was observed in mood/cognition (from 11.9 +/- 18.0 to 7.3 +/- 12.8, p = 0.020) and attention/memory (from 7.3 +/- 8.9 to 4.8 +/- 7.6, p = 0.021) domains. Conclusions: This study suggests that zonisamide improves non-motor symptoms, especially psychical and cognitive symptoms in PD patients.

OBJECTIVE:Effect of fingolimod in multiple sclerosis (MS) is thought to involve the prevention of lymphocyte egress from lymphoid tissues, thereby reducing autoaggressive lymphocyte infiltration into the central nervous system across blood-brain barrier (BBB). However, brain microvascular endothelial cells (BMECs) represent a possible additional target for fingolimod in MS patients by directly repairing the function of BBB, as S1P receptors are also expressed by BMECs. In this study, we evaluated the effects of fingolimod on BMECs and clarified whether fingolimod-phosphate restores the BBB function after exposure to MS sera.METHODS:Changes in tight junction proteins, adhesion molecules and transendothelial electrical resistance (TEER) in BMECs were evaluated following incubation in conditioned medium with or without fingolimod/fingolimod-phosphate. In addition, the effects of sera derived from MS patients, including those in the relapse phase of relapse-remitting (RR) MS, stable phase of RRMS and secondary progressive MS (SPMS), on the function of BBB in the presence of fingolimod-phosphate were assessed.RESULTS:Incubation with fingolimod-phosphate increased the claudin-5 protein levels and TEER values in BMECs, although it did not change the amount of occludin, ICAM-1 or MelCAM proteins. Pretreatment with fingolimod-phosphate restored the changes in the claudin-5 and VCAM-1 protein/mRNA levels and TEER values in BMECs after exposure to MS sera.CONCLUSIONS:Pretreatment with fingolimod-phosphate prevents BBB disruption caused by both RRMS and SPMS sera via the upregulation of claudin-5 and downregulation of VCAM-1 in BMECs, suggesting that fingolimod-phosphate is capable of directly modifying the BBB. BMECs represent a possible therapeutic target for fingolimod in MS patients.

BACKGROUND: The breakdown of the blood-brain barrier (BBB) and the blood-nerve barrier (BNB) has been considered a key step in autoimmune diseases of the nervous systems such as neuromyelitis optica and Guillain-Barré syndrome. It had been previously thought that the BNB were leaky compared with the BBB, but recent several reports indicate that the BNB has almost same properties as a barrier system with those with BBB. Although several studies using animals to examine the difference between BBB and BNB have been published, molecular analyses using human microvascular endothelial cells derived from human BBB and BNB have not yet been reported. In this study, we performed detailed comparative analyses for the barrier properties with both of new established endothelial cell lines derived from human BBB and BNB. DESIGN/METHODS: TY10, a conditionally immortalized human brain microvascular endothelial cell line, and FH-BNB, a conditionally immortalized human peripheral nerve microvascular endothelial cell line, were used. Firstly, we measured transendothelial electrical resistance (TEER) values of TY10 cells and FH-BNBs. In addition, Affymetrix chips, Human Transcriptome Array 2.0, were used to elucidate the difference of the expression level of the tight junction molecules on the BBB and the BNB. RESULTS: The TEER values of TY10 cells and FH-BNB cells were 36.3±4.0Ω.cm2 and 38.0±4.4 Ω.cm2, respectively. The expression levels of claudin-3, 4, 6, 15, 16, 17 and 19 mRNA in FH-BNBs were significantly higher than those in TY10 cells. CONCLUSIONS: Our results indicated that barrier functions of the BNB are as tight as those of the BBB, but some differences regarding tight junction molecules participating the maintenance for tight junction were noted between these two barrier systems.

ObjectiveSevere damage to the blood-brain barrier (BBB) allows anti-aquaporin 4 (AQP4) antibodies to access the astrocytic endfeet in neuromyelitis optica (NMO). In the current study, we identified the pathogenic cytokines/chemokines that are responsible for the BBB malfunction induced by NMO sera.MethodsWe measured the levels of 27 cytokines/chemokines in human brain microvascular endothelial cells (BMECs) after exposure to sera obtained from patients with the acute and stable phases of anti-AQP4 antibody-positive NMO spectrum disorder (NMOSD), multiple sclerosis (MS) patients and healthy controls (HC) using a multiplexed fluorescent bead-based immunoassay system.ResultsThe induced protein (IP)-10 level in the cells was markedly increased following exposure to acute phase NMOSD sera. Other cytokines/chemokines including interleukin (IL)-6 and monocyte chemotactic protein (MCP)-1 were also significantly increased in the acute NMOSD group compared to both the MS and HC groups. The up-regulation of the IP-10 levels in the cells after exposure to the acute-phase NMOSD sera was also observed using another specified ELISA, and this effect was significantly decreased during the remission phase in the individual NMOSD patients. Furthermore, the increase in the level of IP-10 after exposure to the sera was significantly correlated with the cerebrospinal fluid/serum albumin ratio.ConclusionsSera from the acute phase of NMO markedly increased the autocrine secretion of IP-10 by BMECs. The over-production of IP-10 in BMECs may play an important role in the pathogenesis of NMO and may therefore help to mediate the trafficking of T cells expressing its receptor across the BBB.

Objective: Neuromyelitis optica (NMO) is an idiopathic inflammatory, necrotizing disease of the central nervous system. NMO-IgGs selectively bound to aquaporin 4 (AQP4) water channel, which is densely expressed in astrocytic foot processes at the blood–brain barrier (BBB). A loss of AQP4 immunostaining in the spinal cord lesions is a hallmark on the histopathological findings with NMO. Although the down-regulation of AQP4 has been thought to be the important phenomenon for the pathophysiology of NMO, the factors which regulate the expression of AQP4 have not yet been elucidated. In the present study, we investigated which factors could regulate the expression of AQP4 in human astrocyte using newly established in vitro human BBB models.

OBJECTIVE:Pathological breakdown of the blood-brain barrier (BBB) is thought to constitute the beginning of the disease process in neuromyelitis optica (NMO). In the current study, we investigated possible molecular mechanisms responsible for the breakdown of BBB using NMO sera.METHODS:We analysed the effects of sera obtained from anti-aquaporin 4 (AQP4) antibody-positive NMO spectrum disorder (NMOSD) patients, multiple sclerosis (MS) patients and control subjects on the production of claudin-5, matrix-metalloproteinases (MMPs)-2/9, and vascular cell adhesion protein-1 (VCAM-1) in human brain microvascular endothelial cells (BMECs). We also examined whether immunoglobulin G (IgG) purified from NMOSD sera influences the claudin-5 or VCAM-1 protein expression.RESULTS:The disturbance of BBB properties in BMECs following exposure to NMOSD sera was restored after adding the MMP inhibitor, GM6001. The secretion of MMP-2/9 by BMECs significantly increased after applying the NMOSD sera. The sera from NMOSD patients also increased both the MMP-2/9 secretion and the VCAM-1 protein level by BMECs. The IgG purified from NMOSD sera did not influence the BBB properties or the amount of MMP-2/9 proteins, although it did increase the amount of VCAM-1 proteins in BMECs. Reduction in anti-AQP4 antibody titre was not correlated with a reduction in VCAM-1 expression.CONCLUSIONS:The autocrine secretion of MMP-2/9 by BMECs induced by humoral factors, other than IgG, in sera obtained from NMOSD patients potentially increases BBB permeability. IgG obtained from NMOSD sera, apart from anti-AQP4 antibodies, affect the BBB by upregulating VCAM, thereby facilitating the entry of inflammatory cells into the central nervous system.

Diabetic encephalopathy is now accepted as an important complication of diabetes. The breakdown of the blood–brain barrier (BBB) is associated with dementia in patients with type 2 diabetes mellitus (T2DM). The purpose of this study was to identify the possible mechanisms responsible for the disruption of the BBB after exposure to advanced glycation end-products (AGEs). We investigated the effect of AGEs on the basement membrane and the barrier property of the BBB by Western blot analysis, using our newly established lines of human brain microvascular endothelial cell (BMEC), pericytes, and astrocytes. AGEs reduced the expression of claudin-5 in BMECs by increasing the autocrine signaling through vascular endothelial growth factor (VEGF) and matrix metalloproteinase–2 (MMP-2) secreted by the BMECs themselves. Furthermore, AGEs increased the amount of fibronectin in the pericytes through a similar up-regulation of the autocrine transforming growth factor (TGF)–β released by pericytes. These results indicated that AGEs induce basement membrane hypertrophy of the BBB by increasing the degree of autocrine TGF-β signaling by pericytes, and thereby disrupt the BBB through the up-regulation of VEGF and MMP-2 in BMECs under diabetic conditions.

OBJECTIVE:To ascertain the hypothesis that the phenotypic differences between Bickerstaff's brainstem encephalitis (BBE) and Miller Fisher syndrome (MFS) are derived from the differences in the effects of sera on blood-brain barrier (BBB) and blood-nerve barrier.BACKGROUND:Antibodies against GQ1b are frequently detected in BBE and MFS, and these two disorders may share the same pathogenesis, but the clinical phenotypes of BBE and MFS are substantially different.METHODS:The effects of sera obtained from BBE patients, MFS patients and control subjects were evaluated with regard to the expression of tight junction proteins and transendothelial electrical resistance in human brain microvascular endothelial cells (BMECs) and human peripheral nerve microvascular endothelial cells.RESULTS:The sera obtained from BBE patients decreased the transendothelial electrical resistance values and claudin-5 protein expression in BMECs, although the sera obtained from MFS patients had no effect on BMECs or peripheral nerve microvascular endothelial cells. This effect was reversed after the application of matrix metalloproteinase (MMP) inhibitor, GM6001. The presence or absence of anti-GQ1b antibodies did not significantly influence the results. MMP-9 secreted by BMECs was significantly increased after exposure to the sera obtained from BBE patients, whereas it was not changed after exposure to the sera obtained from MFS patients.CONCLUSIONS:Only the sera obtained from BBE patients destroyed BBB and it might explain the phenotypical differences between BBE and MFS. BBE sera disrupted BBB, possibly via the autocrine secretion of MMP-9 from BBB-composing endothelial cells.

The blood–spinal cord barrier (BSCB) has been recognized as one of the barrier organs in the central nervous system. Unlike the blood–brain barrier (BBB), the function of the BSCB still remains to be fully understood, partly because of the lack of a good in vitro BSCB model. In the present study, we established a microvascular endothelial cell line derived from a rat spinal cord and characterized this new model. A conditionally immortalized cell line, termed rBSCB-1, was established by introducing the temperature-sensitive SV40 large T-antigen gene into primary microvascular endothelial cells isolated from a rat spinal cord. We examined whether this model retains barrier-specific properties. The rBSCB-1 expressed several basic endothelial cell markers and the large T-antigen, and showed temperature-dependent cell growth and barrier function. They also expressed tight junction molecules including claudin-1, 3, 5, 12, occludin, zonula occludens (ZO)-1 and barrier-specific transporters. The rBSCB-1 showed low paracellular permeability to 4 kDa dextran and high transport activity of P-glycoprotein. In addition, the treatment with astrocytic factors increased the barrier properties of rBSCB-1, including their expression levels of tight junctional molecules. The rBSCB-1 cells are the first conditionally immortalized endothelial cell line derived from the BSCB that have been fully characterized as barrier-forming cells. This cell line should be a useful tool to understand the BSCB disruption involved in many neurological disorders including multiple sclerosis and spinal cord injury.

The breakdown of the blood–brain barrier (BBB) has been considered to be a key step in the disease process of a number of neuroimmunological disorders. Although several in vitro BBB models derived from human tissues have been established, no human conditionally immortalized in vitro BBB models using a temperature-sensitive SV40-T antigen ( tsA58 ) and human telomerase reverse transcriptase ( hTERT ) have ever been reported. In the present study, we established a new human brain microvascular endothelial cell line harboring tsA58 and hTERT genes, and extensively characterized this new model. TY08 cells, derived from the human BBB and harboring tsA58 , were infected with retroviruses possessing hTERT genes. We examined whether this new model retains its barrier-specific nature, independent of the passage number. The obtained endothelial cell line, termed TY09, proliferated well under the permissive temperature and stopped growing under the non-permissive temperature, despite the acquisition of hTERT as an additional immortalizing gene. Even with a high-passage number, the cells maintained a spindle-shaped morphology, the expression of the von Willebrand factor, tight junction proteins and transporters. Furthermore, we carried out a transendothelial transport study for TY09 cells and hCMEC/D3 cells, thereby proving that both cell lines have almost the same nature with respect to transcellular permeability of various hydrophilic and hydrophobic substances. The new stable conditionally immortalized TY09 cells, retaining the in vivo BBB functions, should facilitate the performance of future studies for determining the pathophysiology of various neuroimmunological diseases.

The destruction of blood–brain barrier (BBB) and blood-nerve barrier (BNB) has been considered to be a key step in the disease process of a number of neurological disorders including cerebral ischemia, Alzheimer’s disease, multiple sclerosis, and diabetic neuropathy. Although glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) facilitate neuronal or axonal regeneration in the brain or peripheral nerves, their action in the BBB and BNB remains unclear. The purpose of the present study was to elucidate whether these neurotrophic factors secreted from the brain or peripheral nerve pericytes increase the barrier function of the BBB or BNB, using our newly established human brain microvascular endothelial cell (BMEC) line or peripheral nerve microvascular endothelial cell (PnMEC) line. GDNF increased the expression of claudin-5 and the transendothelial electrical resistance (TEER) of BMECs and PnMECs, whereas BDNF did not have this effect. Furthermore, we herein demonstrate that the GDNF secreted from the brain and peripheral nerve pericytes was one of the key molecules responsible for the up-regulation of claudin-5 expression and the TEER value in the BBB and BNB. These results indicate that the regulation of GDNF secreted from pericytes may therefore be a novel therapeutic strategy to modify the BBB or BNB functions and promote brain or peripheral nerve regeneration.

The blood-nerve barrier (BNB) is a highly specialized unit that maintains the microenvironments of the peripheral nervous system. Since the breakdown of the BNB has been considered a key step in autoimmune neuropathies such as Guillain-Barré syndrome and chronic inflammatory demyelinating polyraduculoneuropathy, it is important to understand the cellular properties of the peripheral nerve microvascular endothelial cells (PnMECs) which constitute the BNB. For this purpose, we established an immortalized cell line derived from human PnMECs. The human PnMECs were transduced with retroviral vectors encoding the temperature-sensitive SV40 large T antigen and human telomerase. This cell line, termed FH-BNB, showed a spindle fiber-shaped morphology, expression of von Willebrand factor and uptake of acetylated low density lipoprotein. These cells expressed tight junction proteins including occludin, claudin-5, ZO-1 and ZO-2 at the cell-cell boundaries. P-glycoprotein and GLUT-1 were also detected by a Western blot analysis and the cells exhibited the functional expression of p-glycoprotein. In addition, transendothelial electrical resistance experiments and paracellular permeabilities of sodium fluorescein and fluorescein isothiocyanate-labeled dextran of molecular weight 4 kDa across these cells demonstrated that FH-BNBs had functional tight junctions. These results indicated that FH-BNBs had highly specialized barrier properties and they might therefore be a useful tool to analyze the pathophysiology of various neuropathies.

Objective: Antibody against GQ1b is frequently detected in Bickerstaff brainstem encephalitis (BBE) and Miller Fisher syndrome (MFS) and has been considered to be pathogenically important in the development of these two disorders; however, the clinical manifestations of there two disorders are obviously different. We hypothesized that there phenotypic differences may be derived from the difference of blood-brain barrier (BBB)/ blood-nerve barrier(BNB) breakdown. Background We demonstrated the effects of sera from patients with BBE and MFS on the impairment of BBB or BNB function and clarified the roles of humoral factor such as Matrix metalloproteinases(MMP) in the destruction of BBB or BNB. Design/Methods: Both human BBB and BNB-derived endothelial cell lines were recently developed in Yamaguchi University, named TY10 and PnMECs. We evaluated transendothelial electrical resistance (TEER), the amount of tight junction proteins including claudin-5 and occludin, and the amount of MMP including MMP-9 and MMP-2 by western blotting in TY10 and PnMECs after the exposure of the patients9 sera. Results: The amount of claudin-5 protein in TY10, not in PnMECs, decreased after exposure of BBE sera. TEER of TY10 also decreased after application of BBE sera. Sera from MFS patients had no effect in these experiments. The amount of MMP-9 and MMP-2 protein in TY10 increased after exposure of BBE sera. Conclusions: Only sera from BBE patients disrupt BBB. This may partially explain the phenotypic defferences between BBE and MFS. BBE sera break BBB, possibly via autocrine secretion of MMP-9 and MMP-2 from BBB-composing endothelial cells. Disclosure: Dr. Saito has nothing to disclose. Dr. Shimizu has nothing to disclose. Dr. Koga has nothing to disclose. Dr. Sano has nothing to disclose. Dr. Haruki has nothing to disclose. Dr. Maeda has nothing to disclose. Dr. Abe has nothing to disclose. Dr. Tasaki has nothing to disclose. Dr. Suzuki has nothing to disclose. Dr. Kusunoki has nothing to disclose. Dr. Mizusawa has received personal compensation for activities with Tanabemitsubishi Co and Sanofi-Aventis Co. Dr. Mizusawa has received personal compensation in an editorial capacity for No to Shinkei and Clinical Neuroscience. Dr. Kanda has nothing to disclose.

In autoimmune disorders of the peripheral nervous system (PNS), including Guillain–Barré syndrome and chronic inflammatory demyelinating polyradiculoneuropathy, breakdown of the blood-nerve barrier (BNB) has been considered to be a key step in the disease process. Although glucocorticoids (GCs) have been shown to effectively restore the blood–brain barrier (BBB) in some inflammatory central nervous system diseases such as multiple sclerosis, their action against the BNB has not yet been examined. To elucidate the role of GCs on the BNB, we established a novel human immortalized endothelial cell lines derived from the BNB. The established cell line termed “DH-BNBs” expresses two important tight junction proteins, claudin-5 and occludin. Using DH-BNBs, we analyzed how GCs affect BNB function. We herein report that GCs up-regulate the expression of claudin-5 and increase the barrier properties of the BNB. This is the first report which indicates how GCs affect the blood-nerve barrier.

The objectives of this study were to establish pure blood–nerve barrier (BNB) and blood–brain barrier (BBB)‐derived pericyte cell lines of human origin and to investigate their unique properties as barrier‐forming cells. Brain and peripheral nerve pericyte cell lines were established via transfection with retrovirus vectors incorporating human temperature‐sensitive SV40 T antigen ( tsA58 ) and telomerase. These cell lines expressed several pericyte markers such as α‐smooth muscle actin, NG2, platelet‐derived growth factor receptor β, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, the inulin clearance was significantly lowered in peripheral nerve microvascular endothelial cells (PnMECs) through the up‐regulation of claudin‐5 by soluble factors released from brain or peripheral nerve pericytes. In particular, bFGF secreted from peripheral nerve pericytes strengthened the barrier function of the BNB by increasing the expression of claudin‐5. Peripheral nerve pericytes may regulate the barrier function of the BNB, because the BNB does not contain cells equivalent to astrocytes which regulate the BBB function. Furthermore, these cell lines expressed several neurotrophic factors such as NGF, BDNF, and GDNF. The secretion of these growth factors from peripheral nerve pericytes might facilitate axonal regeneration in peripheral neuropathy. Investigation of the characteristics of peripheral nerve pericytes may provide novel strategies for modifying BNB functions and promoting peripheral nerve regeneration. J. Cell. Physiol. 226: 255–266, 2010. © 2010 Wiley‐Liss, Inc.

The breakdown of the blood–brain barrier (BBB) has been considered to be a key step in the disease process of a number of neurological disorders such as cerebral ischemia and Alzheimer's disease. Many in vitro BBB models derived from animal tissues have been established to elucidate the mechanism of BBB insufficiency. However, only a few human immortalized in vitro BBB models have been reported. In the present study, a temperature‐sensitive SV40‐T antigen was introduced to immortalize cells using a retrovirus to obtain a better human in vitro BBB model which sustains physiological properties. This endothelial cell (EC) line, termed TY08, showed a spindle‐shaped morphology. The cells expressed all key tight junctional proteins, such as occludin, claudin‐5, zonula occludens (ZO)‐1 and ZO‐2 at their cell‐to‐cell boundaries, and had low permeability to inulin across its monolayer. The cells also expressed various influx and efflux transporters and exhibited the functional expression of p‐glycoprotein. Furthermore, the TY08 cells grew and proliferated well under the permissive temperature and stopped growing under the non‐permissive temperature to serve as physiological ECs forming the BBB. Thus, conditionally immortalized TY08 cells retaining the in vivo BBB functions should facilitate analyses for determining the pathophysiology of various neurological diseases. J. Cell. Physiol. 225: 519–528, 2010. © 2010 Wiley‐Liss, Inc.