CXCL8 enhances the angiogenic activity of umbilical cord blood-derived outgrowth endothelial cells in vitro.

OECs (outgrowth endothelial cells), also known as late-EPCs (late-endothelial progenitor cells), have a high proliferation potential in addition to in vitro tube formation capability. In ischaemic animal models, injected OECs were integrated into regenerating blood vessels and improved neovascularization. Previous reports have demonstrated the expression of CXCL8 to be up-regulated in ischaemic tissues. It has also been documented that CXCL8 stimulates the angiogenic activity of mature ECs (endothelial cells). Therefore, it has been suggested that CXCL8 plays an important role in neovascularization in ischaemic tissues. However, it is still uncertain whether CXCL8 also stimulates the angiogenic activity of OECs. This study evaluated the effects of CXCL8 on the angiogenic activity of OECs in vitro. OECs were isolated from human UCB (umbilical cord blood)-derived mononuclear cells. Phenotypes of the OECs were assessed by flow cytometry, immunostaining, and real-time RT (reverse transcription)-PCR. The effects of CXCL8 on OECs were investigated by transwell migration assay and capillary tube formation assay on Matrigel. The OEC clones isolated from UCB expressed OEC phenotypes. In addition, CXCL8 receptors (CXCR1 and CXCR2) were expressed on these OEC clones. CXCL8 significantly stimulated the transwell migration and capillary tube formation of OECs. Neutralizing antibody against CXCR2, but not CXCR1, abolished a transwell migration of OECs induced by CXCL8, suggesting the involvement of CXCL8/CXCR2 axis in transwell migration. These results demonstrate that CXCL8 stimulates the angiogenic activity of UCB-derived OECs in vitro.

Cytokine-dependent modification of IL-12p70 and IL-23 balance in dendritic cells by ligand activation of Valpha24 invariant NKT cells.

CD1d-restricted invariant NKT (iNKT) cells play crucial roles in various types of immune responses, including autoimmune diseases, infectious diseases and tumor surveillance. The mechanisms underlying their adjuvant functions are well understood. Nevertheless, although IL-4 and IL-10 production characterize iNKT cells able to prevent or ameliorate some autoimmune diseases and inflammatory conditions, the precise mechanisms by which iNKT cells exert immune regulatory function remain elusive. This study demonstrates that the activation of human iNKT cells by their specific ligand alpha-galactosylceramide enhances IL-12p70 while inhibiting the IL-23 production by monocyte-derived dendritic cells, and in turn down-regulating the IL-17 production by memory CD4(+) Th cells. The ability of the iNKT cells to regulate the differential production of IL-12p70/IL-23 is mainly mediated by a remarkable hallmark of their function to produce both Th1 and Th2 cytokines. In particular, the down-regulation of IL-23 is markedly associated with a production of IL-4 and IL-10 from iNKT cells. Moreover, Th2 cytokines, such as IL-4 and IL-13 play a crucial role in defining the biased production of IL-12p70/IL-23 by enhancement of IL-12p70 in synergy with IFN-gamma, whereas inhibition of the IFN-gamma-promoted IL-23 production. Collectively, the results suggest that iNKT cells modify the IL-12p70/IL-23 balance to enhance the IL-12p70-induced cell-mediated immunity and suppress the IL-23-dependent inflammatory pathologies. These results may account for the long-appreciated contrasting beneficial and adverse consequence of ligand activation of iNKT cells.

Technetium-99m-GSA clearance in mice under long-term dietary restriction.

OBJECTIVE: Dietary restriction (DR) without malnutrition is widely acknowledged to prolong lifespan in laboratory animals. Evidence suggests that DR retards age-related decline in protein turnover of most organs. However, there has been no report about hepatic serum glycoprotein catabolism under DR. In the current study, we evaluate the hepatic uptake of asialoglycoprotein in ICR mice with DR by measuring the plasma clearance of technetium-99m galactosyl human serum albumin (Tc-GSA). METHODS: The amount of food supplied to the restricted mice was 70% of that consumed by the mice fed ad libitum (AL). The regimen was initiated at the age of 7 weeks and terminated after the age of 44 weeks. The plasma clearance of Tc-GSA was measured at the age of 7 weeks, 14 weeks, 28 weeks, and 42 weeks. RESULTS: The restricted animals showed a marked decrease in their body and liver weight, and hepatic uptake of Tc-GSA per liver weight in the restricted mice was greater than that in the mice fed AL. On the other hand, the Tc-GSA plasma clearance in the mice fed AL was stable during the study period, and that in the restricted mice showed no change with age either, and those in the two groups were similar. In addition to the receptor function, there was no difference in the expression of mRNAs of the asialoglycoprotein receptor between the two groups. Serum concentrations of cholinesterase and hepatic mRNAs of glutamine synthetase in the restricted mice were higher than those in the mice fed AL. Serum levels of amino acids in the restricted mice were lower than those in the mice fed AL. CONCLUSIONS: The data presented here show that the DR did not affect the capacity of hepatic serum glycoprotein catabolism, whereas several protein metabolic pathways were affected.

Metabolic response to short-term 4-day energy restriction in a controlled study.

OBJECTIVES: Metabolic rate is affected not solely by diet but also by environmental characteristics such as climate and seasonal changes in day length. In the present study, we conducted a controlled study in which we observed metabolic response to short-term energy restriction (ER). MATERIALS AND METHODS: Thirty-two subjects were divided randomly into a slight ER group and a moderate ER group. The energy intake per day for slight ER vs moderate ER was 1462 kcal vs 1114 kcal. During the 4-day study periods, the same daily timetable, which consists of nutrition, exercise, sleeping and others, was imposed on both groups. The same environment was also provided to both groups. RESULTS: After the 4-day ER, significant decreases in body weight and basal metabolic rate (BMR) were shown in both groups. The decrease in body weight was 2% of the baseline level in both groups, and the decreases in the BMR were 6% of baseline levels in the slight ER group and 13% in the moderate ER group. The decrease in BMR in the moderate ER group was significantly larger than that in the slight ER group. CONCLUSIONS: In a controlled study of short-term ER, we observed a significant decrease in BMR. There was a positive association between the degree of ER and the reduction in BMR. Reductions in BMR were greater than those in body weight. It, thus, appears that the minimization of weight loss is due to dramatic decreases in BMR. This suggests the existence of metabolic resistance against ER.

Effects of dietary restriction on physical performance in mice.

Dietary restriction is known to prolong life in laboratory animals. However, little is known about the effects of dietary restriction on physical performance. To evaluate physical performance, we measured four item indices: time to climb out of obstacles, time to escape restraint by gummed tape, time hanging from a bar, and ability to resist slipping every week. The diets of ICR mice were restricted from the age of 7 weeks through 24 weeks. Body weight of the diet-restricted mice decreased during the 7th to 9th weeks of age. After the 10th week, weight gain resumed. In response to assigned tasks, the diet-restricted mice performed better in all activities: they climbed out of obstacles faster, freed themselves sooner from restraint by gummed tape, hung from a bar longer, and better resisted slipping down a slope. These results suggest that diet-restricted mice have superior physical abilities, such as those required to overcome or avoid risks to life, than do ad-libitum-fed mice.

Ferrochelatase consisting of wild-type and mutated subunits from patients with a dominant-inherited disease, erythropoietic protoporphyria, is an active but unstable dimer.

Erythropoietic protoporphyria (EPP) is an autosomal inherited disease of heme biosynthesis caused by a partial deficiency of the enzyme ferrochelatase. Patients with EPP show only 20-30% normal activity because of mutations in one of the alleles of the ferrochelatase gene. To clarify the molecular mechanisms of this low level of activity, we co-expressed human ferrochelatase carrying His- and HA-tags in a tandem fashion in Escherichia coli. Purification of the His-tag-containing enzyme revealed that the His-enzyme forms an oligomer in association with the HA-enzyme, and analysis by gel-filtration confirmed that the enzyme is a dimer (approximately 80 kDa). Then we expressed homo- and heterodimers composed of the wild-type and engineered mutants of the enzyme (C395Delta, H157A, H263A, H388A) or mutants from EPP patients (I186T, M267I). The levels of homodimeric enzymes produced were low, and the activities of the purified homodimeric mutants were abolished. On the other hand, the heterodimers with wild-type and mutated subunits exhibited potential, but weak, activities without a marked change of Km values for substrates. These results showed that heterodimers containing normal and mutated subunits retain the enzymic activity, which is inconsistent with the hypothesis that ferrochelatase is only active when the dimer contains two normal subunits. Pretreatment at 42 degrees C led to a rapid inactivation of the heterodimeric mutants, indicating instability. Thus, we provide evidence that the instability of the heterodimer containing normal and mutated ferrochelatase as well as the low production levels due to the structural defect of the mutant protein, not the abolishment of the enzymic activity of the heterodimer, causes the weak activity in EPP patients.

Dietary restriction: effects of short-term fasting on protein uptake and cell death/proliferation in the rat liver.

Dietary restriction (DR) is known to prolong life in laboratory animals. Intermittent (alternate-day) fasting or short-term repeated fasting has also been reported to increase the life span of animals. In the present study, we investigated the changes or induction of abnormalities of protein metabolism in rats during fasting, and measured asialoglycoprotein uptake and cell death/proliferation in the liver of rats receiving fasting and refeeding. In the results, liver weight decreased significantly after 48 h of fasting and increased during the refeeding period, returning to the pre-fasting level by 12 h of refeeding. Cell death, determined by single stranded DNA (ssDNA) staining method, increased during the fasting period, and returned to the pre-fasting level during the refeeding period. Cell proliferation, determined using antibodies (Ab) against proliferating cell nuclear antigen, decreased during the fasting period, and increased during the refeeding period. Changes in cell death and cell proliferation were inversely related. However, there was no significant difference in asialoglycoprotein uptake by the whole liver between the ad libitum (AL)-fed rats and 48 h fasted rats. Thus, neither the changes in liver weight nor cell death/proliferation affected asialoglycoprotein uptake on a living body. These results suggest that episodes of 48 h fasting do not induce protein metabolism abnormalities in the liver.