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.

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.

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.

Fatty replacement is more severe in the anterior aspect of the head of the pancreas. The posterior aspect of the head of the pancreas and the area around the CBD tended to be spared.

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The objective of this study was to establish pure blood-nerve barrier (BNB)-derived peripheral nerve pericyte cell lines and to investigate their unique properties as barrier-forming cells. We isolated peripheral nerve, brain, and lung pericytes from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines expressed several pericyte markers such as alpha-smooth muscle actin, NG2, osteopontin, and desmin, whereas they did not express endothelial cell markers such as vWF and PECAM. In addition, these cell lines expressed several tight junction molecules such as occludin, claudin-12, ZO-1, and ZO-2. In particular, the expression of occludin was detected in peripheral nerve and brain pericytes, although it was not detected in lung pericytes by a Western blot analysis. An immunocytochemical analysis confirmed that occludin and ZO-1 were localized at the cell-cell boundaries among the pericytes. Brain and peripheral nerve pericytes also showed significantly higher trans-pericyte electrical resistance values and lower inulin clearances than lung pericytes. We considered that occludin localized at the cell-cell boundaries among the pericytes might mechanically stabilize the microvessels of the BNB and the blood-brain barrier. Furthermore, we also showed that these cell lines expressed many barrier-related transporters. ABCG2, p-gp, MRP-1, and Glut-1 were detected by a Western blot analysis and were observed in the cytoplasm and outer membrane by an immunocytochemical analysis. These transporters on pericytes might facilitate the peripheral nerve-to-blood efflux and blood-to-peripheral nerve influx transport of substrates in cooperation with those on endothelial cells in order to maintain peripheral nerve homeostasis.

Summary 1The variation of floral sex allocation with flower position within inflorescences was investigated in the spring ephemeral, Corydalis ambigua . Investment in female function ( pistil), attraction (corolla) and nectar production decreased from bottom to top flowers, whereas male investment (stamen) did not differ. 2 This self-incompatible species appears to set seeds as a result of visitation by nectar robbing bumblebee queens. The tendency of bees to visit lower flowers first and then move upwards within an inflorescence should result in directional pollen flow from bottom to top flowers. 3 Naturally pollinated upper flowers set fewer seeds than intermediate and lower flowers due to pollen limitation. The lack of differences in seed set and seed mass per pod following artificial outcrossing indicated that resource limitation did not explain the variation in seed production of flowers in different positions. Pollen viability also did not differ significantly between flower positions. 4 A model of pollination was developed that incorporated the visitation pattern of bumblebees and observed variations in nectar distribution between flower positions. This predicted that receipt of outcross pollen would decrease from bottom to top flowers, but that pollen export to other plants would not differ between flower positions provided that the pollen exchange rate of pollinators was either small or positively correlated with nectar content of each flower position. The observed pattern of floral sex allocation would then be parallel to relative success of pollen export and import between flower positions within inflorescences.

ABSTRACT. 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 temperaturesensitive SV40 large T antigen and human telomerase. This cell line, termed FH-BNB, showed a spindle fibershaped 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 FHBNBs had highly specialized barrier properties and they might therefore be a useful tool to analyze the pathophysiology of various neuropathies.