Platelet-derived growth factor (PDGF)-AA/AB in human serum are potential indicators of the proliferative capacity of human synovial mesenchymal stem cells.

INTRODUCTION: For expansion of human mesenchymal stem cells (MSCs), autologous human serum is safer than fetal bovine serum in clinical situations. One of the problems with the use of autologous human serum is that its proliferative effect on MSCs varies widely between donors. The threefold goals of this study were: (1) to demonstrate an improved method for preparing human serum; (2) to identify growth factors predictive of proliferative potential; and (3) to identify a cytokine to promote MSC proliferation in human serum. METHODS: Fresh blood was collected using a closed bag system containing glass beads. The bag was shaken at 20 °C for 30 minutes for rapid preparation, or kept stationary at 4 °C for 24 hours for slow preparation. Passage 0 synovial MSCs derived from four donors were cultured with 10 % conventional rapid preparation serum or modified slow preparation serum from four different donors. To perform the colony-forming unit assay, synovial MSCs were cultured in these serums. The protein expression profile in serum was analyzed using cytokine array. The candidate proteins were speculated from the correlation between the colony-forming ability and protein expression. As an evaluation of the candidate proteins, proliferation ability, surface marker phenotype and differentiation capability of synovial MSCs were examined. RESULTS: Compared with rapid preparation serum, slow preparation serum resulted in a significantly higher total colony number and twofold higher expression levels of nine proteins (angiopoietin-1, BDNF, EGF, ENA-78, IGFBP-2, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, RANTES and TfR). Colony number was positively correlated with PDGF-AA/AB concentrations. Exogenous PDGF-AA significantly promoted proliferation of synovial MSCs, whereas PDGF receptor (PDGFR) inhibitor decreased it. Addition of PDGFs or PDGFR inhibitor did not affect surface epitopes of synovial MSCs. Pretreatment with PDGFs or PDGFR inhibitor did not affect chondrogenic, adipogenic, or calcification potentials of synovial MSCs. CONCLUSION: Slow preparation serum contained higher concentrations of PDGF-AA/AB and increased the colony formation number of synovial MSCs. PDGF-AA/AB were indicators of the proliferative potential of human serum. Exogenous PDGF-AA increased proliferation of synovial MSCs without alteration of surface epitopes and differentiation potentials.

Gene expression profiling in peripheral white blood cells in response to the intake of food with different glycemic index using a DNA microarray.

BACKGROUND/AIMS: Transcriptomics technology in human nutrition intervention studies would allow for genome-wide screening of the effects of nutrients. We observed the time course of gene expression changes in peripheral white blood cells (WBC) to elucidate the metabolic changes in the postprandial state that are a reflection and a marker of whole body metabolic changes. METHODS: In a randomized crossover study, 7 healthy subjects consumed test meals of glucose (GL), white rice (WR) and rolled barley (BAR), each containing 75 g of available carbohydrate, and water (WAT). Blood glucose, insulin and nonesterified fatty acid concentrations, as well as the subjective levels of fullness and hunger were measured. Microarray analysis of the WBC and the real-time PCR were examined during 360 min after the intake of the test meals. RESULTS: The number of genes that changed more than 1.5-fold and the expression patterns in the time course were different between the GL, the WR and the BAR groups. Several genes involved in glycolysis and fatty acid ß-oxidation were markedly changed after the intake of the GL, the WR and the BAR; however, these genes did not change at any time point in the WAT. CONCLUSIONS: Gene expression profiling in the WBC can reflect food-related metabolic changes, even in the postprandial state.

An in vitro assay system for antihyperlipidemic agents by evaluating lipoprotein profiles from human intestinal epithelium-like cells.

We developed an in vitro screening system for antihyperlipidemic activity by measuring lipoprotein profiles secreted from human intestinal epithelium-like cells from the colon cancer cell line, Caco-2. Sodium (Na) butyrate at 5 mM differentiated Caco-2 cells into intestinal epithelium-like cells and numerous microvilli on the apical side of cells were observed under transmission electron microscopy. Real-time RT-PCR analysis revealed that Na butyrate stimulated expression levels of intestinal differentiation markers in Caco-2 cells in a dose-dependent manner and 5 mM Na butyrate up-regulated intestinal alkaline phosphatase, sucrase-isomaltase complex, and microsomal triglyceride transfer protein by 8.1-, 1.9-, and 2.1-fold that of non-treated cells, respectively. Lipoprotein secretions from differentiated Caco-2 cells were promoted by lysophosphatidyl choline and Na oleate, which are a stimulator of lipoprotein secretion and a substrate of triglycerides, respectively. We examined the effects of Pluronic L-81, a lipoprotein secretion inhibitor, on lipoprotein profiles of differentiated Caco-2 cells. Pluronic L-81 at 1.0 µg/ml inhibited TG contents in lipoprotein fractions from cells by 25.6 % and secretion was completely suppressed by the agent at 10 µg/ml.

In vitro screening for antihyperlipidemic activities in foodstuffs by evaluating lipoprotein profiles secreted from human hepatoma cells.

We screened the antihyperlipidemic effects of seven edible plants by evaluation of the triglyceride (TG) and cholesterol profiles secreted from HepG2 cells. We found that the water- and ethanol-extracts of Brasenia schreberi at 100 µg/ml exhibited strong inhibitory activities against TG and cholesterol secretions from HepG2 cells stimulated with sodium oleate. Real-time RT-PCR analysis demonstrated that ethanol extract of B. schreberi (BSET) attenuated the expression of the sterol regulatory element binding protein-1c and -2, fatty acid synthase and HMG CoA synthase-1 genes, which are involved in lipid synthesis in hepatocyte/hepatoma cells. Furthermore, we studied the action of BSET on adipose tissue accumulation and serum parameters in mice fed a high-fat diet (HFD). BSET suppressed mesenteric and epididymal adipose tissue accumulation and normalized serum TG and glucose, but not cholesterol levels in HFD-fed mice.

BAFF aids generation of IgG anti-ganglioside antibodies in response to Campylobacter jejuni lipo-oligosaccharide.

Ganglioside mimicry of Campylobacter jejuni lipo-oligosaccharide (LOS) can induce the production of IgG anti-ganglioside antibodies, but the generation mechanism has yet to be clarified. B-cell activating factor belonging to the TNF family (BAFF) helped murine B cells produce anti-ganglioside antibodies against C. jejuni LOS. In splenocyte culture, however, anti-ganglioside antibodies were produced in the presence of a soluble transmembrane activator and calcium-modulating and cyclophilin ligand interactor immunoadhesin (TACI-Ig), a receptor for BAFF. TACI-Ig adenoviral vectors failed to decrease production of anti-ganglioside antibodies in mice sensitized with C. jejuni LOS and did not alter IgG subclasses, evidence that BAFF aids but is not essential for the generation of IgG anti-ganglioside antibodies in response to C. jejuni LOS.

[Usefulness of sharing medication-related reviews based on questionnaire survey, and the cooperation between hospital and community pharmacist at conference prior to discharge].

Owing to the Japanese healthcare system reform, the place where patients receive medical care is beginning to shift from hospital to home. This study aims to evaluate the usefulness of sharing medication-related reviews through a questionnaire survey and hospital pharmacist intervention survey. It appears that almost all community pharmacists have inadequate information at the time of dispensing medication to, monitoring adverse effects of, and counseling cancer patients. There is no doubt that hospital pharmacists should provide more information to community pharmacists at conference prior to discharge. Fortunately, at the Tokyo Women's Medical University Hospital, hospital pharmacists and community pharmacists work closely to contribute to an effective patient care at discharge. Thus, there is an urgent need to ensure greater cooperation between hospital pharmacists and community pharmacists in Japan.

Cutting edge: Guillain-Barre syndrome-associated IgG responses to gangliosides are generated independently of CD1 function in mice.

CD1 molecules present a variety of microbial glycolipids and self-glycolipids to T cells, but their potential role in humoral responses to glycolipid Ags remains to be established. To address this issue directly, we used GM1/GD1a-deficient mice, which, upon immunization with heat-killed Campylobacter jejuni, develop Guillain-Barré syndrome-associated IgG Abs against the GM1/GD1a sugar chain epitopes of bacterial lipo-oligosaccharides (LOS). Our results showed that anti-ganglioside Abs of the IgG1, IgG2b, and IgG3 isotypes were produced in the absence of group 2 CD1 (CD1d) expression. Unlike mouse and human group 2 CD1 molecules that specifically bound LOS, none of the group 1 CD1 molecules (CD1a, CD1b, and CD1c in humans) were capable of interacting with LOS. Thus, these results indicate CD1-independent pathways for anti-ganglioside Ab production.

Antagonistic effect of CCAAT enhancer-binding protein-alpha and Pax5 in myeloid or lymphoid lineage choice in common lymphoid progenitors.

Lymphoid lineage-committed progenitors, such as common lymphoid progenitors (CLPs), maintain a latent myeloid differentiation potential, which can be initiated by stimulation through exogenously expressed cytokine receptors, including IL-2 receptors. Here we show that the transcription factor CCAAT enhancer-binding protein-alpha (C/EBPalpha) is promptly up-regulated in CLPs upon ectopic IL-2 stimulation. Enforced C/EBPalpha expression is sufficient to initiate myeloid differentiation from CLPs, as well as from proT and proB cells, even though proB cells do not give rise to myeloid cells after ectopic IL-2 stimulation. Expression of Pax5, a B lymphoid-affiliated transcription factor, is completely suppressed by enforced C/EBPalpha but not by ectopic IL-2 stimulation in proB cells. Introduction of Pax5 blocks ectopic IL-2 receptor-mediated myeloid lineage conversion in CLPs. These data suggest that C/EBPalpha is a proximal target of cytokine-induced lineage conversion in lymphoid progenitors. Furthermore, complete loss of Pax5 expression triggered by up-regulation of C/EBPalpha is a critical event for lineage conversion from lymphoid to myeloid lineage in CLPs and proB cells.

Accumulation of CD16+ cells with secretion of Ksp37 in decidua at the end of pregnancy.

PROBLEM: Maternal cellular immunity is thought to be in a state of tolerance during pregnancy, but the precise mechanism of immunomodulation is not yet known. We investigated a novel serum protein, killer-specific secretory protein of 37 kDa (Ksp37), produced by cytotoxic lymphocytes, during pregnancy. METHOD OF STUDY: The level of Ksp37 was determined by enzyme linked immunosorbent assay (ELISA) in the sera of healthy pregnant women. Intracellular Ksp37 expression in mononuclear cells, isolated from peripheral blood and decidua at parturition, was examined with a flow cytometer. RESULTS: Serum Ksp37 levels significantly increased at late pregnancy, compared with non-pregnant controls and the first trimester of pregnancy. The flow cytometric analysis exhibited that Ksp37 was mainly expressed in CD16+ natural killer (NK) cells in decidua of term placenta. CONCLUSIONS: Serum Ksp37 level was elevated at late gestational period. CD16+ NK cells in decidua seem to be a main maternal source of Ksp37. Innate immunity, with CD16+ NK cells, may play important roles near parturition.