Activation of cellular antioxidative stress and migration activities by purified components from immortalized stem cells from human exfoliated deciduous teeth.

Although stem cell-based regenerative medicine has been extensively studied, it remains difficult to reconstruct three dimensional tissues and organs in combination with vascular systems in vitro. One clinically successful therapy is transplantation of mesenchymal stem cells (MSC) into patients with graft versus host disease. However, transplanted cells are immediately damaged and destroyed because of innate immune reactions provoked by thrombogenic inflammation, and patients need to take immunosuppressive drugs for the immunological regulation of allogeneic cells. This reduces the benefits of stem cell transplantation. Therefore, alternative therapies are more realistic options for clinical use. In this study, we aimed to take advantage of the therapeutic efficacy of MSC and use multiple cytokines released from MSC, that is, stem cells from human exfoliated deciduous teeth (SHEDs). Here, we purified components from conditioned media of immortalized SHED (IM-SHED-CM) and evaluated the activities of intracellular dehydrogenase, cell migration, and antioxidative stress by studying the cells. The immortalization of SHED could make the stable supply of CM possible. We found that the fractionated component of 50-100 kD from IM-SHED-CM had higher efficacy than the original IM-SHED-CM in terms of intracellular dehydrogenase and cell migration in which intracellular signal transduction was activated via receptor tyrosine kinases, and the glutathione peroxidase and reductase system was highly active. Although antioxidative stress activities in the fractionated component of 50-100 kD had slightly lower than that of original IM-SHE-CM, the fraction still had the activity. Thus, the use of fractionated components of 50-100 kD from IM-SHED-CM could be an alternative choice for MSC transplantation because the purified components from CM could maintain the effect of cytokines from SHED.

A stable chimeric fibroblast growth factor (FGF) can successfully replace basic FGF in human pluripotent stem cell culture.

Fibroblast growth factors (FGFs) are essential for maintaining self-renewal in human embryonic stem cells and induced pluripotent stem cells. Recombinant basic FGF (bFGF or FGF2) is conventionally used to culture pluripotent stem cells; however, because of the instability of bFGF, repeated addition of fresh bFGF into the culture medium is required in order to maintain its concentration. In this study, we demonstrate that a heat-stable chimeric variant of FGF, termed FGFC, can be successfully used for maintaining human pluripotent stem cells. FGFC is a chimeric protein composed of human FGF1 and FGF2 domains that exhibits higher thermal stability and protease resistance than do both FGF1 and FGF2. Both human embryonic stem cells and induced pluripotent stem cells were maintained in ordinary culture medium containing FGFC instead of FGF2. Comparison of cells grown in FGFC with those grown in conventional FGF2 media showed no significant differences in terms of the expression of pluripotency markers, global gene expression, karyotype, or differentiation potential in the three germ lineages. We therefore propose that FGFC may be an effective alternative to FGF2, for maintenance of human pluripotent stem cells.

Characterization of Syrian hamster adapted prions derived from L-type and C-type bovine spongiform encephalopathies.

Atypical forms of bovine spongiform encephalopathy (BSE) may be caused by different prions from classical BSE (C-BSE). In this study, we examined the susceptibility of mice overexpressing mouse and hamster chimeric prion protein (PrP) to L-type atypical BSE (L-BSE). None of the transgenic mice showed susceptibility to L-BSE, except mice overexpressing hamster PrP. We also examined the transmission properties of L-BSE in hamsters. The incubation period of hamsters intracerebrally inoculated with L-BSE was 576.8 days, and that of the subsequent passage was decreased to 208 days. Although the lesion and glycoform profiles and relative proteinase K resistant core fragment of the abnormal isoform of PrP (PrPcore) of L-BSE were similar to that of C-BSE, the deposition of the abnormal isoform of PrP (PrPSc) and the molecular weight of PrPcore of L-BSE was different from than that of C-BSE. In hamster models, some prion strain characteristics of L-BSE were indistinguishable from those of C-BSE.

Decreased expression of matrix metalloproteinase-9 and increased expression of tissue inhibitors of matrix metalloproteinase-1 in paratuberculosis-infected cattle in the ELISA-negative subclinical stage.

We investigated the gene expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitors of matrix metalloproteinases-1 (TIMP-1) in peripheral blood cells from infected cattle with Mycobacterium avium subsp. paratuberculosis (Map) in the ELISA-negative subclinical stage compared with uninfected control cattle. Significant decreased MMP-9 expression and increased TIMP-1 expression were found in peripheral blood cells from Map-infected cattle after stimulation with Map lysate and Map purified protein derivative (PPD) than in control cattle by real-time RT-PCR analysis. In contrast to the uninfected controls, the activity of MMP-9 was also decreased in peripheral blood cell culture supernatants from Map-infected cattle at 24 hr after Map lysate and MapPPD stimulation by gelatin zymography analysis. As a result, the MMP-9 may play an important role in the development of Mycobacterium avium subsp. paratuberculosis disease.

Intraspecies prion transmission results in selection of sheep scrapie strains.

BACKGROUND: Sheep scrapie is caused by multiple prion strains, which have been classified on the basis of their biological characteristics in inbred mice. The heterogeneity of natural scrapie prions in individual sheep and in sheep flocks has not been clearly defined. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we intravenously injected 2 sheep (Suffolk and Corriedale) with material from a natural case of sheep scrapie (Suffolk breed). These 3 sheep had identical prion protein (PrP) genotypes. The protease-resistant core of PrP (PrPres) in the experimental Suffolk sheep was similar to that in the original Suffolk sheep. In contrast, PrPres in the Corriedale sheep differed from the original PrPres but resembled the unusual scrapie isolate, CH1641. This unusual PrPres was not detected in the original sheep. The PrPres distributions in the brain and peripheral tissues differed between the 2 breeds of challenged sheep. A transmission study in wild-type and TgBoPrP mice, which overexpressing bovine PrP, led to the selection of different prion strains. The pathological features of prion diseases are thought to depend on the dominantly propagated strain. CONCLUSIONS/SIGNIFICANCE: Our results indicate that prion strain selection occurs after both inter- and intraspecies transmission. The unusual scrapie prion was a hidden or an unexpressed component in typical sheep scrapie.

Accumulation of L-type bovine prions in peripheral nerve tissues.

We recently reported the intraspecies transmission of L-type atypical bovine spongiform encephalopathy (BSE). To clarify the peripheral pathogenesis of L-type BSE, we studied prion distribution in nerve and lymphoid tissues obtained from experimentally challenged cattle. As with classical BSE prions, L-type BSE prions accumulated in central and peripheral nerve tissues.

Intraspecies transmission of L-type-like Bovine Spongiform Encephalopathy detected in Japan.

It has been assumed that the agent causing BSE in cattle is a uniform strain (classical BSE); however, different neuropathological and molecular phenotypes of BSE (atypical BSE) have been recently reported. We demonstrated the successful transmission of L-type-like atypical BSE detected in Japan (BSE/JP24 isolate) to cattle. Based on the incubation period, neuropathological hallmarks, and molecular properties of the abnormal host prion protein, the characteristics of BSE/JP24 prion were apparently distinguishable from the classical BSE prion and closely resemble those of bovine amyloidotic spongiform encephalopathy prion detected in Italy.

Isolation of two distinct prion strains from a scrapie-affected sheep.

We performed a transmission study using mice to clarify the characteristics of the most recent case of scrapie in Japan. The mice that were inoculated with the brain homogenate from a scrapie-affected sheep developed progressive neurological disease, and one of the scrapie-affected mice showed unique clinical signs during primary transmission. This mouse developed obesity, polydipsia, and polyuria. In contrast, the other affected mice exhibited weight loss and hypokinesia. In subsequent passages, the mice showed distinct characteristic scrapie phenotypes. This finding may prove that different prion strains coexist in a naturally affected sheep with scrapie.

Biological and biochemical characterization of L-type-like bovine spongiform encephalopathy (BSE) detected in Japanese black beef cattle.

A case of L-type-like atypical bovine spongiform encephalopathy was detected in 14-year-old Japanese black beef cattle (BSE/JP24). To clarify the biological and biochemical properties of the prion in BSE/JP24, we performed a transmission study with wild-type mice and bovinized transgenic mice (TgBoPrP). The BSE/JP24 prion was transmitted to TgBoPrP mice with the incubation period of 199.7 +/- 3.4 days, which was shorter than that of classical BSE (C-BSE) (223.5 +/- 13.5 days). Further, C-BSE was transmitted to wild-type mice with the incubation period of about 409 days, whereas BSE/JP24 prion inoculated mice showed no clinical signs up to 649 days. Severe vacuolation and a widespread and uniform distribution of PrP(Sc) were pathologically observed in the brain of BSE/JP24 prion affected TgBoPrP mice. The molecular weight and glycoform ratio of PrP(Sc) in BSE/JP24 were different from those in C-BSE, and PrP(Sc) in BSE/JP24 exhibited weaker proteinase K resistance than that in C-BSE. These findings revealed that the BSE/JP24 prion has distinct biological and biochemical properties reported for that of C-BSE. Interestingly, a shorter incubation period was observed at the subsequent passage of the BSE/JP24 prion to TgBoPrP mice (152.2 +/- 3.1 days). This result implies that BSE/JP24 prion has newly emerged and showed the possibility that L-type BSE prion might be classified into multiple strains.

Experimental transmission of two young and one suspended bovine spongiform encephalopathy (BSE) cases to bovinized transgenic mice.

Bovine spongiform encephalopathy (BSE) is caused by a prion that primarily consists of an abnormal isoform of the prion protein (PrP(Sc)). Since PrP(Sc) is partially resistant to proteolytic digestion, the routine diagnosis of BSE is based on the immunological detection of the proteinase K (PK)-resistant moiety of PrP(Sc) (PrP(core)). However, transmission studies are indispensable in order to demonstrate prion infectivity and to analyze prion characteristics. Transmission experiments were accordingly performed on 2 young BSE cases (BSE/JP8, BSE/JP9) and 1 suspected BSE case (Suspended-1) that were detected by the BSE screening program in Japan. In this study, we attempted to transmit the prion from these 3 animals by using transgenic mice overexpressing bovine PrP (TgBoPrP). In spite of the use of BSE-sensitive transgenic mice, none of the mice developed neurological signs nor accumulated PrP(Sc) in their brains for more than 600 days post-inoculation, even with subsequent blind passages. The results of a dilution experiment using the classical BSE prion indicated that prion infectivity in these 3 cattle was below the detection limit of 10(3.0) LD(50)/g.

Corticotropin-releasing hormone and urocortin expression in peripheral blood cells from experimentally infected cattle with Mycobacterium avium subsp. paratuberculosis.

Urocortin (UCN) is a new neuropeptide of the corticotrophin-releasing hormone (CRH) family which plays an important role in immune responses. Mycobacterium avium subspecies paratuberculosis (Map) is the etiological agent of paratuberculosis (Johne's disease). The role of UCN or CRH in the pathogenesis of Map-infection is unknown. In the present study, we first cloned the bovine UCN gene and demonstrated the profile of UCN or CRH expression in peripheral blood cells from Map-infected cattle and uninfected controls by real-time reverse transcription-polymerase chain reaction (RT-PCR) and ELISA analysis. These data are the first observations of the characteristic kinetics of these neuropeptides in Map-infection. UCN or CRH expression in non-stimulated blood samples from infected cattle was higher than that in similarly treated samples from uninfected controls; however, exposure to Map lysate and live Map resulted in down-regulated expression of UCN in infected cattle compared to their counterparts from uninfected controls. These results have provided a direction in understanding the pathogenesis of paratuberculosis and improving diagnostic methods for Map-infection.

Neutralization of interleukin-10 significantly enhances gamma interferon expression in peripheral blood by stimulation with Johnin purified protein derivative and by infection with Mycobacterium avium subsp. paratuberculosis in experimentally infected cattle with paratuberculosis.

Monoclonal antibody neutralization of interleukin-10 (IL-10) increased Johnin purified protein derivative-induced whole-blood gamma interferon (IFN-gamma) secretion 23-fold and also increased IFN-gamma secretion ninefold following in vitro Mycobacterium avium subsp. paratuberculosis infection of peripheral blood mononuclear cells. These results demonstrate the suppressive effect of IL-10 on immune responses to M. avium subsp. paratuberculosis infection in cattle.

Mycobacterium avium subsp. paratuberculosis infection causes suppression of RANTES, monocyte chemoattractant protein 1, and tumor necrosis factor alpha expression in peripheral blood of experimentally infected cattle.

Blood from cattle with subclinical Mycobacterium avium subsp. paratuberculosis infection was stimulated with M. avium subsp. paratuberculosis antigens, and expression of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), RANTES, monocyte chemoattractant protein 1 (MCP-1), and IL-8 was measured. Expression of TNF-alpha, RANTES, and MCP-1 was lower in infected than in uninfected cattle. The reduced response may weaken protective immunity and perpetuate infection.

Variable expression of podocyte-related markers in the glomeruloid bodies in Wilms tumor.

Several podocyte-related markers are organized to express in glomerular differentiation. However, whether expression of them is virtually synchronized and a reliable indicator of the state of differentiation is unknown. The present study investigated, by immunohistochemistry, the divergent expression of several podocyte markers in the improperly differentiated glomeruloid bodies from four cases of Wilms tumors. The glomeruloid bodies were classified into immature (IGB) or mature forms (MGB) based on morphology and epithelial features. Podocytes in IGB expressed WT1, synaptopodin, podocalyxin, and nephrin, and their expression was stronger in MGB. In contrast, Pax2 was strong in IGB and diminished in MGB. p27 was first expressed in MGB. The expression pattern in each molecule mimics normal glomerulogenesis. Podocytes in MGB showed persistent expression of bcl-2 and cytokeratin with synaptopodin, podocalyxin, and nephrin by serial section, a finding unusual for normal glomerulogenesis. Moreover, parietal cells in MGB also occasionally expressed these podocyte markers. The ultrastructure revealed that podocytes in MGB showed tight junctions without foot process formations, which indicated incomplete differentiation. These results suggest that a set of podocyte differentiation markers are occasionally diversely expressed, and raise the possibility that expression of these markers is insufficient to determine the state of terminal differentiation in podocytes.

Localization of intercellular adherens junction protein p120 catenin during podocyte differentiation.

To reveal the role of cadherin complex in podocyte differentiation, the present study describes the localization of the cadherin complex, including p120 catenin (p120ctn), in the developing and in the aminonucleoside nephrosis (PAN nephrosis) rat kidney, by immunofluorescence microscopy and immunogold electron microscopy. p120ctn and beta-catenin were co-localized at the apical part of lateral cell membranes in presumptive podocytes of the S-shaped body, and their localization shifted to the basal margin of lateral cell membranes in the capillary loop stage. There was no expression of the cadherin complex at the slit diaphragm, the intercellular junction of mature podocytes. After the regression of the podocyte junctional structure in PAN nephrosis, the cadherin complex was not re-expressed. The dynamic changes in the localization of the cadherin complex suggest that the it plays an important role during podocyte differentiation, including the rearrangement of the intercellular junction and the formation of the slit diaphragm.

Glomerular differentiation in p27 and p57 double-mutant metanephroi.

The cell cycle inhibitors p27 and p57 are initially concurrently expressed at a critical point of glomerulogenesis and podocyte differentiation. The present study generated mice lacking both p27 and p57, in order to investigate the synergistic roles of these molecules in glomerular differentiation. It appeared that p27 and p57 double-mutant mice died between E16.5 and E18.5, before glomerular differentiation can take place. We harvested E13.5 metanephroi to advance the glomerulogenesis in a metanephric organ culture. Metanephroi of double-mutant and wild-type mice showed no great difference in size and shape at harvest or after 6 days in culture. Histology and morphometry revealed that average glomerular size in metanephroi from double-mutant mice was significantly larger than those in any other mutants. Larger glomeruli in double-mutant metanephroi are composed of an increased number of podocytes. The glomeruli in the double-mutant metanephroi expressed synaptopodin and WT-1 with the same pattern and intensity as those found in wild type. In addition, electron micrography showed the presence of foot processes and slit membrane in podocytes in double mutant. Western blot analysis of metanephroi after 6 days in culture showed an up-regulation of p21 protein in p27 mutant and double-mutant, but not in p57 mutant metanephroi. These findings suggest that p27 and p57 are synergistically involved, in part, to determine the number, but not the differentiation, in podocytes.

Role of cell cycle molecules in the pathophysiology of glomerular epithelial cells.

The postmitotic characteristics of podocytes are the basis for the structural assembly and central function (filtration) of the glomeruli. Persistent cell cycle quiescence is required for stability in these cells. In cells of the podocyte lineage, transdifferentiation from the metanephric mesenchyme to mature podocytes is closely associated with tight regulation of the expression of cell cycle molecules. This cell cycle control in podocytes acts as a safeguard for the glomeruli; however, deregulation of this system might result in structural deterioration. This article focuses on the expression of cell cycle molecules in podocyte differentiation and pathology.

Phenotypic changes and cell cycle activation in early tubulointerstitial injury of rat adriamycin nephrosis.

Tubular response, including phenotypic changes against a variety of injuries, is an initial event that promotes tubulointerstitial injuries. Using the progressive kidney disease model of rat adriamycin (ADR) nephrosis, the present study focused on the cell cycle activation and phenotypic changes that occur in the tubuli in early tubulointerstitial injury in ADR nephrosis. At 12 weeks, experimental animals developed overt nephrosis with tubulointerstitial injury, which correlated well with the degree of proteinuria and incidence of glomerulosclerosis. Initial pathology of the tubuli showed a slight dilatation of tubuli, which tended to occur in individual nephrons. Immunohistochemistry demonstrated that vimentin-positive tubuli and osteopontin (OPN)-positive tubuli were associated mostly with proliferating cell nuclear antigen expression. Protein levels of OPN in the renal cortex were correlated with the level of proteinuria by western blotting. Vimentin- and OPN-expressing tubuli were tightly associated with a peritubular influx of alpha-smooth muscle actin (SMA)-positive cells or ED-1-positive cells. In addition, we found thrombomodulin+/ TUNEL+ (dUTP-biotin nick-end labeling) peritubular endothelial cells and ED-1+/alpha-SMA+ cells at an early stage among interstitial inflammatory cells. These results suggest that cell cycle activation in tubular cells forms the background for the phenotypic tubular changes that are involved in chronic tubulointerstitial injury in ADR nephrosis.

Podocyte injury promotes progressive nephropathy in zucker diabetic fatty rats.

The zucker diabetic fatty (ZDF-fa/fa) rat is one of the attractive models for type II diabetes based on impaired glucose tolerance caused by the inherited insulin-resistance gene fa. Characterization of nephropathy in this model may provide useful insights into the mechanism of the progression of diabetic nephropathy. The present study analyzed the pathophysiology of diabetes and nephropathy, including the process of glomerulosclerosis in this model by biochemical and morphometric analyses. In addition, we conducted studies in podocytes in culture to examine the direct effects of high glucose on podocytes. ZDF-fa/fa rats showed overt diabetes despite hyperinsulinemia as early as 3 months of age. Blood glucose levels increased further with a considerable decrease of insulin levels at 5 months. Glomerular filtration rate (GFR) was significantly elevated until 3 months, but fell to the level seen in lean rats by 7 months. Proteinuria started to rise during the period of increased GFR, and increased further after GFR had fallen to within the normal range. Renal fibronectin, collagen iv, and vascular endothelial growth factor mRNA levels were increased at 7 months. Glomerulosclerosis commenced as early as 5 months of age, and was associated with glomerular hypertrophy and mild mesangial expansion with evidence of accentuated podocyte injury, as revealed by increased expression of desmin. Electron microscopy suggested that degeneration of podocytes and the development of tuft adhesions were responsible for the glomerular sclerosis in this model. In addition, glomeruli from the diabetic rats showed up-regulation of the cyclin kinase inhibitors, p21 and p27. Further studies suggested that the increase in p27 expression was predominantly caused by podocytes, because predominant immunolocalization of p27 in podocytes in diabetic rats and high glucose medium induced cell hypertrophy accompanied by p27 up-regulation in differentiated podocyte cell lines. In conclusion, progressive diabetic nephropathy in ZDF-fa/fa rats is associated with evidence of podocyte injury. High concentrations of ambient glucose induced podocyte hypertrophy and stress in vitro, suggesting that the podocyte is a likely target of the diabetic milieu.