Cell surface ATP synthase-released H+ and ATP play key roles in cocoa butter intake-mediated regulation of gut immunity through releases of cytokines in rat.

Proper food intake is important for maintaining good health in humans. Chocolate is known to exert anti-inflammatory effects; however, the mechanisms remain unclear. In this study, we aimed to investigate the effects of cocoa butter intake on gut immunity in rats and rabbits. Cocoa butter intake increased the lymph flow, cell density, and IL-1ß, IL-6 and IL-10 levels in mesenteric lymph. Clodronate, a macrophage depletion compound, significantly enhanced the release of all cytokines. The immunoreactivities of macrophage markers CD68 and F4/80 in the jejunal villi were significantly decreased with clodronate. Piceatannol, a selective cell surface ATP synthase inhibitor significantly reduced the cocoa butter intake-mediated releases of IL-1ß, IL-6 and IL-10. The immunoreactivities of cell surface ATP synthase were observed in rat jejunal villi. Shear stress stimulation on the myofibroblast cells isolated from rat jejunum released ATP and carbon dioxide depended with H+ release. In rabbit in vivo experiments, cocoa butter intake increased the concentrations of ATP and H+ in the portal vein. The in vitro experiments with isolated cells of rat jejunal lamina propria the pH of 3.0 and 5.0 in the medium released significantly IL-1ß and IL-6. ATP selectively released IL-10. These findings suggest that cocoa butter intake regulates the gut immunity through the release and transport of IL-1ß, IL-6, and IL-10 into mesenteric lymph vessels in a negative feedback system. In addition, the H+ and ATP released from cell surface ATP synthase in jejunal villi play key roles in the cocoa butter intake-mediated regulation of gut immunity.

Portal blood flow-dependent NO-mediated lymph formation in rat jejunum.

The higher permeability of the venules in jejunal microcirculation to albumin contributes to the increased mesenteric lymph formation. Recently, we demonstrated that water intake induced serotonin release from enterochromaffin cells in rat jejunum, serotonin of which circulated through the portal vein into blood circulation and then increased the mesenteric lymph formation. The mode of action of serotonin remains unclear. Therefore, we aimed to clarify the mechanisms involved in the regulation of the jejunal lymph formation with permeant albumin in in vivo rat experiments. We investigated the effects of intravenous administration of serotonin or water intake on the jejunal-originated lymph volume and the concentration of albumin in the lymph in the presence or absence of L-NAME. The effects of intravenous administration of L-NAME, nicardipine, A23187, and ML-7 on the lymph formation with permeant albumin were also evaluated. Serotonin or water intake significantly increased the mesenteric lymph volume with permeant albumin in the jejunal microcirculation. The serotonin- and water intake-mediated responses were significantly reduced by the pretreatment with intravenous administration of L-NAME. Intravenous administration of L-NAME itself also decreased significantly the jejunal lymph formation. Administration of A23187 and ML-7 significantly reduced the jejunal lymph formation with permeant albumin. In contrast, administration of nicardipine significantly increased the lymph formation. In conclusion, portal venous blood flow- or serotonin-mediated NO release from venular endothelial cells plays physiologically key roles in the lymph formation in rat jejunum via the extrusion of calcium ions and inactivation of MLCK in endothelial cells.

Water intake releases serotonin from enterochromaffin cells in rat jejunal villi.

The present study aims to investigate the roles of water intake in serotonin production and release in rat jejunum. We evaluated the changes in concentrations of serotonin in the portal vein and mesenteric lymph vessel induced by the intragastric administration of distilled water. The density of granules in enterochromaffin cells and the immunoreactivity of serotonin in the jejunal villi were investigated before and after water intake. The effects of intravenous administration of serotonin and/or ketanserin on mesenteric lymph flow and concentrations of albumin and IL-22 in the lymph were also addressed. Water intake increased serotonin concentration in the portal vein, but not in the mesenteric lymph vessel. The flux of serotonin through the portal vein was significantly larger than that through the mesenteric lymph vessel. Water intake decreased the density of granules in the enterochromaffin cells and increased the immunoreactivity of serotonin in the jejunal villi. The intravenous administration of serotonin increased significantly mesenteric lymph flow and the concentrations of albumin and IL-22; both were significantly reduced by the intravenous pretreatment with ketanserin. We showed that serotonin released from enterochromaffin cells by water intake was mainly transported through the portal vein. Additionally, serotonin in blood was found to increase mesenteric lymph formation with permeant albumin in the jejunal villi via the activation of 5-HT2 receptor.

Water intake accelerates ATP release from myofibroblast cells in rats: ATP-mediated podoplanin-dependent control for physiological function and immunity.

We previously demonstrated that water intake increased mesenteric lymph flow and the total flux of IL-22 in rat jejunum. The drained water and the higher permeability of albumin in the jejunal microcirculation contributed to increase the lymph flow and IL-22 transport via the activation of great bulk flow in the jejunal villi. To address the effects of water intake-mediated great bulk flow-dependent mechanical force on jejunal physiological function and immunological regulation of innate lymphoid cells (ILC)-3, we examined the effects of shear stress stimulation on cultured rat myofibroblast cells. Next, we investigated the effects of water intake on podoplanin and IL-22 expressions in cultured human intestinal epithelial cells and rat in vivo jejunal preparations, respectively. Shear stress stimulation of the myofibroblast cells induced ATP release via an activation of cell surface F1/F0 ATP synthase. ATP produced podoplanin expression in the intestinal epithelial cells. Water intake accelerated immunohistochemical expressions of podoplanin and IL-22 in the interepithelial layers and lamina propria of the jejunum. ATP dose-dependently increased IL-22 mRNA expression in ILC-3, which are housed in the lamina propria. Water intake also increased immunohistochemical and mRNA expressions of ecto-nucleoside triphosphate diphosphohydrolases 2 and 5 in jejunal villi. In conclusion, water intake-mediated shear stress stimulation-dependent ATP release from myofibroblast cells maintains higher tissue colloid osmotic pressure in the jejunal microcirculation through podoplanin upregulation in the interepithelial layers. ATP induces IL-22 mRNA expression in ILC-3 in jejunal villi, which may contribute to regulation of mucosal immunity in small intestine.NEW & NOTEWORTHY We investigated effects of shear stress stimulation on cultured myofibroblast cells and water intake on podoplanin and IL-22 expressions in rat jejunal villi. The stimulation induced ATP release from the cells. Water intake accelerated podoplanin and IL-22 expression levels. ATP increased IL-22 mRNA expression in innate lymphoid cells (ILC)-3. Hence, water intake maintains higher osmotic pressure in the jejunal villi through ATP release and podoplanin upregulation. Water intake may regulate the mucosal immunity.

Evaluating Lymph Flow Through the Thoracic Duct Using Urine Osmolarity in Human Participants.

Background: Previous animal studies have shown that intragastric administration of water can accelerate mesenteric lymph flow. Similarly, human studies have shown that abdominal breathing can induce thoracic lymph drainage. In these studies, lymph flow was measured by hemodilution and a corresponding reduction in blood anti-diuretic hormone (ADH) levels, the latter being linked to urine osmolarity. Hence, we questioned if induction of lymph flow through water administration and supine positioning could be measured by monitoring urine osmolarity. Methods and Results: Volunteers were given 250 mL of distilled water and then made to rest for either 10 or 30 minutes in a supine position. Blood samples were taken pre and postrest to monitor changes in plasma ADH, total protein, plasma albumin, red blood cell, and hemoglobin concentrations. Urine was collected to monitor [Na+], [Cl-], and osmolarity. Intake of 250 mL distilled water with 10-minute rest caused a significant reduction in plasma ADH concentration, with decreases in urine [Na+], [Cl-], and osmolarity. We found a linear relationship between the ratio of plasma ADH concentrations after/before rest (between 1.1 and 3.0 pg·mL) and the ratio of urine osmolarity after/before rest (between 180 and 601 mOsm·L). Conclusions: Intake of 250 mL distilled water with 10-minute rest in a supine position caused hemodilution and a reduction in urine osmolarity consistent with thoracic lymph drainage. Urine osmolarity is a simple, safe clinical measure for monitoring lymph flow that could be used to evaluate the technique of lymph edema therapists.

Water intake increases mesenteric lymph flow and the total flux of albumin, long-chain fatty acids, and IL-22 in rats: new concept of absorption in jejunum.

The traditional Japanese health care custom recommends that a suitable volume of water is consumed. However, physiological and immunological mechanisms in support of this practice are unknown. Therefore, we conducted rat and rabbit in vivo experiments to investigate the effects of intragastric administration of distilled water on the jejunal-originated lymph flow and the concentrations and total flux of cells, albumin, long-chain fatty acids, and innate lymphoid cell 3 (ILC-3)-secreted interleukin-22 (IL-22) through mesenteric lymph vessels. The distribution and activity of ILC-3 in rat small intestine by water intake were evaluated using flow cytometry and RT-PCR. The intragastric administration of distilled water caused significant increases in rat mesenteric lymph flow and in the total flux of cells, albumin, long-chain fatty acids, and IL-22 through the lymph vessels. Intravenously injected Evans blue dye was rapidly transported into rabbit mesenteric lymph vessel and cisterna chyli. The distribution of ILC-3 and the expression of IL-22 mRNA were maximal in the lamina propria cells of the rat jejunum. No significant presence of ILC-3 in the lymph was observed in the control and under water intake conditions. In conclusion, the absorbed water in the jejunum is transported through mesenteric lymph vessels. The higher permeability of albumin in the jejunal microcirculation may play key roles in the transport of consumed water and the reservoir and transporter of long-chain fatty acids. Water intake also accelerates the transfer of IL-22 to the mesenteric lymph, which may contribute, in part, to maintaining and promoting the innate immunity in the body. NEW & NOTEWORTHY The higher permeability of albumin-mediated transport of water-soluble substances in mesenteric lymph vessels of the jejunum may have a large impact on the classic concept suggesting that water-soluble small molecules travel to the liver via the portal vein. ILC-3 is mainly housed in the lamina propria of the jejunum, especially its upper part. IL-22 released from the ILC-3 is also transported through mesenteric lymph in collaboration with the albumin-mediated movement of consumed water.

Lymph Drainage from the Chylocyst-Induced Hemodilution in an In Vivo Rabbit Study.

To confirm our previous study that abdominal respiration has induced hemodilution in human subjects, we performed in-vivo experiments involving anesthetized rabbits. Fifteen 6- to 7-month-old male Japanese white rabbits were used in the animal experiments. Anesthesia was maintained with 2.5%-3.0% isoflurane under N2O + 100% O2 inhalation. Ventilation was maintained at 40 mL/breath for 20 breaths/min. Physiological saline solution was administered at rated 18 mL/h during the experiments. First, we attempted to evaluate lymph flow through the thoracic duct using Sonazoid-based contrast-enhanced ultrasound (CEUS)-guided method and then investigated the effects of manual lymph drainage of the chylocyst on the numbers of red blood cells (RBC), hematocrit (Ht) levels, and the blood concentrations of total protein (TP) and hemoglobin (Hb). In this study, we established surgical methods for identifying the left venous angle and chylocyst using Evans blue dye in anesthetized rabbits. We also confirmed that a Sonazoid-based CEUS-guided method was the most useful technique for producing real-time images of lymph flow through the thoracic duct in anesthetized rabbits. In addition, in present experiments involving anesthetized rabbits, we confirmed that manually massaging the chylocyst produced significant hemodilution. Thus, the procedure produced significant reductions of TP, RBC, Hb, and Ht level in the rabbits.

Abdominal Respiration Induces Hemodilution and Related Reduction in ADH Concentration of Blood.

To establish effective lymph drainage methods and develop concise and accurate clinical techniques for evaluating lymph drainage in healthy individuals and patients with cancer treatment-related lymph edema, we investigated the numbers of red (RBC) and white (WBC) blood cells, and platelets (PLT) in blood, hematocrit (Ht), and the blood concentrations of total protein (TP), albumin (Alb), and anti-diuretic hormone (ADH) before and after 5 min manual lymph drainage, followed by 30 min rest with or without abdominal respiration in the supine or sitting position in 48 healthy volunteers. The 5 min facial, upper and lower extremities lymph drainage, followed by 30 min rest in the supine position induced significant reductions of the TP and Alb in all subjects, and their RBC and Ht levels in some subjects. The 30 min rest only in the supine position without lymph drainage produced also significant reductions of blood TP and Alb. In addition, abdominal respiration in the supine position without manual lymph drainage caused more significant hemodilution, being significant reductions of TP, Alb, RBC, Ht, and ADH in all volunteers. These findings may be related to effective lymph drainage from the chylocyst. Furthermore, it also resulted in a significantly increased micturition desire. In conclusion, abdominal respiration during 30 min rest in the supine position is effective at inducing lymph drainage, and the associated induction of hemodilution and lowering of the blood ADH concentration (and increased micturition desire in some cases) can be used to accurately assess the extent of lymph drainage.

Pivotal roles of lymphatic endothelial cell layers in the permeability to hydrophilic substances through collecting lymph vessel walls: effects of inflammatory cytokines.

To address physiological and pathophysiological meanings of condensing effect of albumin in lymph through collecting lymph vessel walls, we established human lymphatic endothelial cells (LEC) and evaluated the size-dependent regulation of the permeability of such layers to hydrophilic substances. We also investigated the effects of tumor necrosis factor (TNF)-α or interleukin (IL)-1ß on the permeability and on the morphology of human LEC. Significant amounts of 4 kDa dextran, but not 12 or 66 kDa dextran, passed through the layers. TNF-α or IL-1ß induced significant increases in the permeability to 4 and 12 kDa dextrans. TNF-α or IL-1ß also produced significant redistribution of the cytoskeletal F-actin in the LEC, which resulted in changes in their shape. Pretreatment with Y-27632, a Rho kinase inhibitor, or PD98059, an extracellular signal-regulated kinase (ERK) phosphorylation inhibitor, significantly abolished the TNF-α- or IL-1ß-induced increases in the permeability of the layers to 4 and 12 kDa dextrans. Y-27632 and PD98059 significantly inhibited the changes in the F-actin distribution of the LEC produced by TNF-α or IL-1ß. TNF-α or IL-1ß caused significant increases in ERK 1/2 phosphorylation in the LEC, which were significantly inhibited by Y-27632 or PD98059. These findings suggest that the human LEC layer plays key roles in the transport of hydrophilic substances through collecting lymph vessel walls and that TNF-α or IL-1ß significantly increases the permeability of the layers to 4 and 12 kDa dextrans via Rho kinase activation and the ERK 1/2 phosphorylation-mediated reorganization of F-actin in the LEC.

Cell surface F1/FO ATP synthase contributes to interstitial flow-mediated development of the acidic microenvironment in tumor tissues.

To address pivotal roles of cell surface F1/FO ATP synthase in the development of acidic microenvironment in tumor tissues, we investigated effects of shear stress stimulation on the cultured human breast cancer cells, MDA-MB-231 and MDA-MB-157, or human melanoma cells, SK-Mel-1. Shear stress stimulation (0.5-5.0 dyn/cm(2)), the levels of which are similar to those produced by the interstitial flow, induced strength-dependent corelease of ATP and H(+) from the cells, which triggered CO2 gas excretion. In contrast, the same level of shear stress stimulation did not induce significant ATP release and CO2 gas excretion from the control human mammary epithelial cells (HMEC). Marked immunocytochemical and mRNA expression of cell surface F1/FO ATP synthase, vacuolar-ATPase (V-ATPase), carbonic anhydrase type IX, and ectonucleoside triphosphate diphosphohydrolase (ENTPDase) 3 were detected in MDA-MB-231 cells, but little or no expression on the HMEC. Pretreatment with cell surface F1/FO ATP synthase inhibitors, but not cell surface V-ATPase inhibitors, caused a significant reduction of the shear stress stimulation-mediated ATP release and CO2 gas excretion from MDA-MB-231 cells. The ENTPDase activity in the shear stress-loaded MDA-MB-231 cell culture medium supernatant increased significantly in a time-dependent manner. In addition, MDA-MB-231 cells displayed strong staining for purinergic 2Y1 (P2Y1) receptors on their surfaces, and the receptors partially colocalized with ENTPDase 3. These findings suggest that cell surface F1/FO ATP synthase, but not V-ATPase, may play key roles in the development of interstitial flow-mediated acidic microenvironment in tumor tissues through the shear stress stimulation-induced ATP and H(+) corelease and CO2 gas production.

Pivotal roles of shear stress in the microenvironmental changes that occur within sentinel lymph nodes.

A sentinel lymph node (SLN) is the first lymph node that receives drainage from a primary tumor. According to their physiological and biomechanical characteristics, we hypothesized that SLN contains lymphatic endothelial cells (LEC) that are constantly loaded with high levels of shear stress, which might contribute to the production of a suitable environment for micrometastasis within them. To test this hypothesis, we investigated the effects of shear stress stimulation on the expression of adhesion molecules on human LEC isolated from the lymph vessels nearest the SLN of breast cancers, and on the release of ATP from human LEC. The study clarified that the shear stress stimulation produced a significant increase of ICAM-1 expression at protein and mRNA levels in human LEC. Next, we examined whether the shear stress-mediated increase of ICAM-1 expression accelerates the attachment of carcinoma cells to human LEC. Finally, in in vivo experiments, we evaluated whether exogenous ATP facilitates the expression of carcinoma cell-ligated adhesion molecules in rat SLN. In conclusion, shear stress stimulation induces ICAM-1 expression on human LEC by activating cell surface F(1) /F(O) ATP synthase, which might contribute to the development of a premetastatic environment within SLN.

Shear stress-mediated F1/FO ATP synthase-dependent CO2 gas excretion from human pulmonary arteriolar endothelial cells.

We studied the physiological role of flow through pulmonary arterioles in CO(2) gas exchange. We established human pulmonary arteriolar endothelial cells (HPAoEC). The cells demonstrated marked immunocytochemical staining of PECAM-1, VEGF R2, ACE-1, and CA type IV on their cell surface. Ten seconds shear stress stimulation caused the co-release of H(+) and ATP via the activation of F(1)/F(O) ATP synthase on the HPAoEC. F(1)/F(O) ATP synthase was immunocytochemically observed on the cell surface of non-permeabilized HPAoEC. In the shear stress-loaded HPAoEC culture media supernatant, ATPase activity increased in a time-dependent manner. The HPAoEC were strongly stained for NTPDase 1, which partially co-localized with purinergic P2Y1. The purinergic P2Y1 receptor agonist UTP (10(-6) M) significantly potentiated the shear stress-induced increase in ATPase activity in the culture medium supernatant. Ten seconds shear stress stimulation also produced stress strength-dependent CO(2) gas excretion from the HPAoEC, which was significantly reduced by the inhibition of F(1)/F(O) ATP synthase or CA IV on the endothelial cell (EC) surface. In conclusion, we have proposed a new concept of CO(2) exchange in the human lung, flow-mediated F(1)/F(O) ATP synthase-dependent H(+) secretion, resulting in the facilitation of a dehydration reaction involving HCO3(-) in plasma and the excretion of CO(2) gas from arteriolar ECs.

Shear stress-induced ATP-mediated endothelial constitutive nitric oxide synthase expression in human lymphatic endothelial cells.

To clarify the roles of lymphatic endothelial cells (LEC) in the regulation of endothelial constitutive nitric oxide synthase (ecNOS) expression, we examined the effects of shear stress on ecNOS immunohistochemical staining and mRNA and protein expression in human LEC as well as on ATP release from these cells. Shear stress at 0.5 or 1.0 dyn/cm(2) increased ecNOS immunohistochemical staining and ecNOS mRNA and protein expression in cultured LEC. The same strength of shear stress produced a significant release of ATP from the LEC. Exogenous ATP ranging in concentration from 10(-9) to 10(-6) M produced a significant increase in ecNOS immunohistochemical expression in a dose-dependent manner. The increase in ecNOS expression mediated by 10(-6)M ATP was significantly reduced by 10(-5) M suramin. Suramin (10(-5) M) caused a significant reduction in the shear stress-mediated increases in ecNOS immunohistochemical staining and mRNA expression. The shear stress-mediated increases in ecNOS expression were significantly reduced by 3 mM tetraethylammonium, 10(-4) M apamin, 10(-9) M iberiotoxin, 10(-5) M 2-aminoethoxydephenyl borate, or 10(-5)M xestospongin C, but not 10(-5) M glybenclamide or 10(-5) M nifedipine. The shear stress-mediated increases in ecNOS expression were significantly potentiated by pinacidil or NS1619 in a dose-dependent manner. The immunohistochemical expression of small- (SK(Ca)) and big-conductance (BK(Ca)) Ca(2+)-activated K(+) channels was confirmed on the surfaces of human LEC. These findings suggest that shear stress produces a significant release of ATP from LEC, which activates the purinergic P2X/2Y receptor, thereby facilitating ecNOS mRNA and protein expression through inositol 1,4,5-trisphosphate-mediated release of intracellular Ca(2+) ions and the activation of Ca(2+)-activated K(+) channels in LEC.

Chemokine CCL2 facilitates ICAM-1-mediated interactions of cancer cells and lymphatic endothelial cells in sentinel lymph nodes.

We examined the effects of CCL1, CCL2, CCL12 and CCL21 on the expression of adhesion molecules in cultured human lymphatic endothelial cells using immunohistochemical staining or Western blot analysis. In addition, we investigated whether the expressed adhesion molecule was able to facilitate the attachment of carcinoma cells to the lymphatic endothelial cells as an in vitro micrometastatic model. CCL2 caused a selective and significant expression of ICAM-1 on human lymphatic endothelial cells but CCL1, CCL12 and CCL21 did not. By increasing the stimulation time from 4 to 18 and 48 h, the intensity of immunoreactivity for ICAM-1 was significantly increased in a time-dependent manner up to 18 h. The ICAM-1 mRNA levels were also elevated significantly up to 18 h. The CCL2-mediated immunohistochemical expression of ICAM-1 was dose-dependently increased from 10 pg/mL to 1 ng/mL. The CCL2-mediated expression of ICAM-1 was significantly reduced by neutralization of CCL2 using a specific CCL2 antibody. The 18-h treatment with CCL2 caused a significant facilitation of in vitro attachment of MDA-MB-231 and MCF-7 cells to the lymphatic endothelial cells (LECs). The CCL2-mediated response in the attachment assay was also significantly reduced either by the neutralization of CCL2 or by additional treatment with anti-ICAM-1 antibody. Immunohistochemical expression of ICAM-1, but not E-selectin, was strongly observed around and within the metastatic region of sentinel lymph node isolated from breast cancer patients. These findings suggest that CCL2 induces selective and significant expression of ICAM-1 on cultured human lymphatic endothelial cells and then facilitates the attachment of carcinoma cells to the lymphatic endothelial cells, thus providing an in vitro micrometastatic model via the overexpression of ICAM-1.

Recanalization of the collecting lymphatics in rabbit hind leg.

OBJECTIVE: This study was designed to examine whether mature collecting lymphatics can regenerate in the adult tissue or not. MATERIALS AND METHODS: The X-ray lymphograms were used to detect network of the collecting lymphatics in rabbit hind leg. Regeneration of the lymphatics was observed after surgical removal of the popliteal lymph node or a part of the popliteal afferent lymphatic. Structure and mechanical properties of the lymphatics were also examined by light and electron microscopes and in vitro functional experiments. RESULTS: One week after removal of the lymph node, only an afferent lymphatic and a deposit of the contrast medium at the popliteal region were observed. Four weeks after the removal, the connection of the afferent and efferent lymphatics at the popliteal region, and collateral lymphatics were present in the leg. Further, 4 weeks after 1-mm excisions of a part of the lymphatic, recanalization was observed between the central and peripheral cut ends of the lymphatic but not after 3- and 10-mm excisions. Endothelial cells and smooth muscle cells could be observed by electron microscope, and contractile proteins, and alpha-smooth muscle actin SM1 and SM2 were immunofluorescently detected in both intact and the regenerated lymphatic walls. In both lymphatics, norepinephrine and acetylcholine induced dose-dependent constriction and dilation of the vessels, respectively. CONCLUSION: The present study demonstrated that mature collecting lymphatics are able to regenerate in the adult tissues.

Establishment of rat lymphatic endothelial cell line.

OBJECTIVE: The objective of the present study was to establish a rat lymphatic endothelial cell line and then to investigate the morphological and immunohistochemical properties of the cells. METHODS: The lymphatic endothelial cells of rat thoracic ducts were isolated enzymatically by trypsin digestion and were cultured in endothelium growth medium (EGM)-2 in an atmosphere of low oxygen (5% O(2), 5% CO(2), and 90% N(2)) or high oxygen (21% O(2), 5% CO(2), and 74% N(2)). RESULTS: The number of the cells cultured in the low-oxygen atmosphere was significantly larger than that obtained in the high-oxygen atmosphere. The cultured cells in the low-oxygen atmosphere showed a monolayer with uniform cobblestone appearance, suggesting the morphological properties of endothelial cells. Factor VIII-related antigen and cell surface carbohydrates (i.e., D-galactose alpha and D-N-acetylgalactosamine alpha) were found on the lymphatic cultured cells. The phagocytosis of 1,1-diocadecyl1-3,3,3',3'-tetramethylindo-carbocyanine perchlorate-labeled acetylated low-density lipoprotein also was observed in the cultured cells. The cytoskeleton protein F-actin was located on the plasma membrane of the cultured cells as circumferential thin bundles and in the cytoplasma as filamentous bundles. CONCLUSIONS: The present study indicates that the choice of EGM-2 as a culture medium and the hypoxic atmosphere ( approximately 5%) enabled us to establish rat lymphatic endothelial cell line.

Comparison of accelerometry and oxymetry for measuring daily physical activity.

To assess the validity of accelerometry in measuring daily physical activity, the energy consumption calculated by accelerometry, with respiratory gas analysis as a reference, was evaluated in 45 non-athletes during various exercise tests. Subjects were required to (1) walk on a treadmill ergometer at various speeds, (2) walk on a treadmill ergometer at a fixed speed and with a stride of 20% more or 20% less than that when walking freely, (3) walk on a treadmill ergometer at a fixed speed wearing either sneakers or leather-soled shoes, and (4) cycle on a bicycle ergometer. There were strong linear relationships between the measurements during the progressively graded treadmill test, with an overall Pearson correlation coefficient of 0.97. The mean estimated difference ranged from -0.77 to 0.27 kcal/min and the coefficients of variation from 13.2% to 22.2%. However, the difference between the methods was not negligible for individual subjects. Accelerometry overestimated energy expenditure during short-step walking, and underestimated it during long-step walking. No significant difference in energy expenditure was found according to the type of shoes worn. Cycling activity was not recorded by accelerometry. Accelerometry is a reasonably accurate and feasible method for evaluating the physical activities of non-athletes, and could be a common tool for epidemiological research and health promotion despite its limitations.