Regeneration of damaged periodontium is challenging due to its multi-tissue composition. Mesenchymalstem cell-based approaches using adipose-derived stromal cells (ASCs) may contribute toperiodontal reconstruction, particularly when combined with the use of scaffolds to maintain aspace for new tissue growth. The aim of this study was to assess the regenerative potential ofASCs derived from inbred or outbred rats in combination with novel solid scaffolds composed ofPLGA (Poly d,l-lactic-co-glycolic acid) (PLGA-scaffolds). Cultured ASCs seeded onto PLGAscaffolds(ASCs/PLGA) or PLGA-scaffolds (PLGA) alone were transplanted into periodontal fenestrationdefects created in F344 or Sprague Dawley (SD) rats. Micro-CT analysis showed asignificantly higher percentage of bone growth in the ASCs/PLGA groups compared with thePLGA-alone groups at five weeks after surgery. Similarly, histomorphometric analysis demonstratedthicker growth of periodontal ligament and cementum layers in the ASCs/PLGA-groups comparedwith the PLGA-alone groups. In addition, transplanted DiI-labeled ASCs were observed inthe periodontal regenerative sites. The present investigation demonstrated the marked ability ofASCs in combination with PLGA scaffolds to repair periodontal defects.
Of all potential biological therapeutics, monoclonal antibody (mAb)-based therapies are becomingthe dominant focus of clinical research. In particular, smaller recombinant antibody fragments suchas single-chain variable fragments (scFv) have become the subject of intense focus. However, anefficient affinity ligand for antibody fragment purification has not been developed. In the presentstudy, we designed a consensus sequence for the human antibody heavy or light chain-variable regions(Fv) based on the antibody sequences available in the ImMunoGeneTics information system(IMGT), and synthesized these consensus sequences as template Fv antibodies. We then screenedpeptide ligands that specifically bind to the repertoire-derived human Fv consensus antibody usinga 12-mer-peptide library expressed-phage display method. Subsequently, 1 peptide for the VHtemplate and 8 peptides for the VK template were selected as the candidate ligands after 4 roundsof panning the phage display. Using peptide-bead-based immunoprecipitation, the code-4 andcode-13 peptides showed recovery rates of the VH and VK templates that were 20–30% and 40–50%, respectively. Both peptides exhibited better recovery rates for trastuzumab scFv (approximately40%). If it were possible to identify the best combination of VH and VK-binding peptidesamong the ligand peptides suitable for the human mAb Fv sequence, the result could be a promisingpurification tool that might greatly improve the cost efficiencies of the purification process.
(Pro)renin receptor ((P)RR) is a specific receptor for renin and prorenin. The aim of the presentstudy is to clarify expression of (P)RR and pathophysiological roles of (P)RR in human breastcarcinomas. (P)RR expression was studied in 69 clinical cases of breast carcinoma by immunohistochemistry.Effects of (P)RR on cell proliferation were examined in cultured human breast carcinomacells using (P)RR specific small interference RNA. Immunohistochemistry showed that(P)RR immunoreactivity was detected in the breast carcinoma cells in 50 of 69 cases of breastcarcinoma (72%). The analysis on association between (P)RR immunoreactivity and clinicopathologicalparameters showed that the number of (P)RR positive cases was significantly greater inKi-67 (a cell proliferation marker) ≥ 10% group than in Ki-67 < 10% group (P = 0.02). (P)RR wasexpressed in 4 types of human breast carcinoma cell lines. (P)RR specific small interference RNAinhibited proliferation of both MCF-7 (ERα positive) and SK-BR-3 (ERα negative) cells. Thepresent study has shown, for the first time, the expression of (P)RR in human breast carcinomatissues and cultured breast carcinoma cell lines. These findings have raised the possibility that theblockade of the (P)RR signaling may be a novel therapeutic strategy against breast carcinomas.
The purpose of this study was to evaluate trunk stability in seated elderly and young individuals using a new device that inclines a seat while tracking the center of pressure (CoP). We evaluated the locus of CoP, locus length, locus length per second, enveloped area, root mean square area, and locus length per unit area (LNG/AREA). LNG/AREA, which reflects postural adjustments controlled by the spinal proprioceptive reflexes of the lower limbs, was not significantly different between young and elderly individuals. Our device measured trunk stability without influence from the lower extremities, which explains why LNG/AREA did not significantly differ between young and elderly individuals. These findings indicate that the new device can be used to quantify dynamic trunk stability.
Cancer tissues are comprised of various components including tumor cells and the surrounding tumorstroma, which consists of the extracellular matrix and inflammatory cells. Since the tumorstroma plays critical roles in tumor development, investigation of the tumor stroma in addition totumor cells is important to identify useful tumor-associated markers. To discover novel and usefulsero-diagnostic markers, a comparative study of tumor-associated autoantibodies (AAbs) in serafrom lung adenocarcinoma (AC) patients was investigated by two-dimensional immunoblottingwith AC cell lines or each autologous AC tissues. Autoantigens identified from tissue and cell linesamples comprised 58 (45 antigens) and 53 spots (41 antigens), respectively. Thirty-six proteinsincluding Transforming growth factor-beta-induced protein ig-h3 (BIGH3) and Hyaluronan andproteoglycan link protein 1 (HAPLN1) were detected only from tissues, 32 proteins only from celllines, and 9 proteins from both. BIGH3 and HAPLN1 expressions were confirmed in the tumorstroma, but not in AC cell lines by immunostaining and immunoblotting. These data suggest thatautologous tumor tissue and serum are important to coincidently detect AAbs derived from the tumorstroma in addition to tumor cells.
Epigallocatechin gallate (EGCG) is the major polyphenol in green tea and the main bioactivecompound responsible for the health benefits of tea consumption. The molecule exhibits antimicrobialand anti-inflammatory activities. However, little is known about the molecular mechanismsby which EGCG elicits those activities. In this study, we examined the effects of EGCG onlipopolysaccharide (LPS) endocytosis and LPS-induced NO production. We also investigated themechanism with focus on the effect of EGCG on interaction between small GTPase Rab5 and caveolin-1. EGCG suppressed endocytosis of LPS and LPS-induced nitric oxide production inmouse macrophage-like cells (RAW 264). EGCG suppressed the Rab5-caveolin-1 interaction andreduced Rab5 activity. The results suggest that EGCG reduced LPS-induced innate immune responsesthrough suppression of LPS endocytosis by interfering with Rab5-caveolin-1 interactionand by reducing Rab5 activity.
Bone marrow-derived mesenchymal stem cells (BMSCs) are the indispensable component of thebone marrow, being the common precursors for adipocytes and osteoblasts. We show here that adipogenicdifferentiation resulted in increase in the production of adipocyte markers, such as adiponectin,fatty-acid binding proteins (FABP4), peroxisome proliferator-activated receptor γ (PPARγ),as well as the receptor activator of nuclear-κB ligand (RANKL). Co-culture of osteoclast precursors(OCPs) with BMSCs-derived adipocytes significantly enhanced osteoclast differentiation withlow-dose RANKL, whose levels alone could not promote osteoclastogenesis. These results demonstratefor the first time that adipogenic differentiation of BMSCs plays a pivotal role in maintainingbone homeostasis.
Although growing evidence suggests a major role for T cells in the pathogenesis of primary biliarycirrhosis (PBC), the roles of natural killer (NK) and natural killer T (NKT) cells, which predominatein the liver, in the pathogenesis of PBC remain unclear. We investigated the status ofNK and NKT cells in the liver and peripheral blood samples obtained from 11 patients with asymptomaticPBC diagnosed as stage I or II (early PBC) and 7 patients with symptomatic PBCwho underwent liver transplantation (advanced PBC) using flow cytometry and immunohistochemicalstaining. The proportions of NK and NKT cells were significantly decreased in the liver ofpatients with early PBC compared with normal donors. However, the proportion of CD56+ NKTcells was increased in the liver of patients with advanced PBC. Moreover, the proportion of activatedFas ligand (FasL)-positive NKT cells was significantly increased in the liver of patients withadvanced PBC compared with early PBC (P = 0.013). We also found increased expression of FasLon lymphocytes infiltrating around the injured bile duct in advanced PBC using immunohistochemicalstaining. Our results suggest that activated NKT cells may contribute to the biliary epithelialcell death resulting in the progression of PBC.
Here, we investigated which taste ligand induces the CCK (cholecystokinin) release from intestinalSTC-1 cells. We first developed a new assay to measure the release of CCK. The expressionvector for CCK type A receptor (CCKAR) was permanently introduced into HEK293T cells and acell line was established (CCKAR/HEK). Then, STC-1 cells were treated with taste ligands andthe incubated buffer of STC-1 cells containing released CCK was applied to CCKAR/HEK cells.Since CCKAR couples to Gq-signaling, the CCK-induced receptor activation can be monitored bythe method of Ca2+ -imaging. Therefore, when CCK is released from STC-1 cells to culture mediumwith taste stimulation, Ca2+ activation of CCKAR/HEK should be observed. Among five differenttaste ligands (saccharin, Na-glutamate, NaCl, denatonium benzoate, HCl), only denatoniumbenzoate and HCl induced the release of CCK in STC-1 cells. Thus, we found that only specifictaste ligands induce the CCK release, and that other three taste ligands cannot induce the releaseof CCK despite of their ability to elevate the intracellular Ca2+ level in STC-1 cells.