bluuebunny/crossref_metadata_2025 huggingface dataset:
Serum Tissue Inhibitor of Metalloproteinases 1 (TIMP-1) Predicts Organ Recovery from Delayed Graft Function after Kidney Transplantation from Donors after Cardiac Death
Authors: Kusaka Mamoru , Kuroyanagi Yoko , Ichino Manabu , Sasaki Hitomi , Maruyama Takahiro , Hayakawa Kunihiro , Shiroki Ryoichi , Sugitani Atsushi , Kurahashi Hiroki , Hoshinaga Kiyotaka
Published: October 2010

Donors after cardiac death (DCD) have recently become an important source of renal transplants to alleviate the shortage of renal grafts in kidney transplantation (KTx), although DCD kidneys often have complications associated with a delayed graft function (DGF). A microarray-based approach using renal biopsy samples obtained at 1 h after KTx from DCD identified the tissue inhibitor of metalloproteinases 1 (TIMP-1) gene as a potential predictive marker for DGF. The current study measured serum TIMP-1 in patients undergoing KTx and analyzed the time course after KTx. The average serum TIMP-1 level before KTx was 240 ± 10 ng/ml ( n = 34). In patients undergoing KTx from a living donor ( n = 23), the serum TIMP-1 levels showed no increase after KTx (POD1: 226 ± 12, POD2: 211 ± 12, and POD3: 195 ± 10 ng/ml), but in one case, the only patient who required post-KTx HD due to DGF, the level on POD1 was the highest among subjects (361 ng/ml). In contrast, patients undergoing KTx from DCDs ( n = 11), the serum TIMP-1 levels increased rapidly after a KTx (POD1: 418 ± 32, POD2: 385 ± 42, and POD3: 278 ± 25 ng/ml). However, two patients who avoided post-KTx HD due to the immediate function of the graft did not show increased levels (<370 ng/ml) on either POD1 or POD2. The peak serum TIMP-1 values appeared to correlate to the post-KTx dialysis period. Furthermore, the increment of serum TIMP-1 on the early POD was found to be predictive of immediate or delayed function of the grafts. These data suggest that monitoring of serum TIMP-1 levels allow the prediction of graft recovery and the need for HD after a KTx from a DCD.
Assessment of Islet Quality following International Shipping of more than 10,000 km
Authors: Ikemoto Tetsuya , Matsumoto Shinichi , Itoh Takeshi , Noguchi Hirofumi , Tamura Yoshiko , Jackson Andrew M. , Shimoda Masayuki , Naziruddin Bashoo , Onaca Nicholas , Yasunami Yohichi , Levy Marlon F.
Published: October 2010

Islet transplantation is an attractive therapy for type 1 diabetes, although some issues remain. One of them is the severe donor shortage in some countries. In this study, we investigated the possibility of international islet shipping beyond 10,000 km to supply islets to countries with donor shortages. Human islets were isolated from six cadaver donors and cultured until shipment. Islets were packed in either gas-permeable bags or in non-gas-permeable bags and shipped from Baylor Research Institute (Dallas, TX, USA) to Fukuoka University (Fukuoka, Japan). Pre- and postshipment islet number, purity, viability, and stimulation index (by glucose stimulation test) were assessed. Shipped 1,500 IE islets were transplanted into streptozotocin-induced diabetic nude mice for in vivo assay. The distance of our shipment was 11,148.4 km, and the mean duration of the shipments was 48.2 ± 8.2 h. The islet number recovery rate (postshipment/preshipment) was significantly higher in gas-permeable bags (56.4 ± 10.1% vs. 20.5 ± 20.6%, p < 0.01). Islet purity was significantly reduced during shipment in non-gas-permeable bags (from 47.7 ± 18.6% to 40.2 ± 28.2 in gas-permeable bags vs. from 50.4 ± 6.4% to 25.9 ± 15.6% in non-gas-permeable bags, p < 0.05). Islet viability and stimulation index did not change significantly between pre- and postshipping, in either gas-permeable bags or in non-gas-permeable bags. One of three diabetic nude mice (33.3%) converted to normoglycemia. It is feasible to ship human islet cells internationally in gas-permeable bags. This strategy would promote basic and preclinical research for countries with donor shortages, even though the research centers are remote (over 10,000 km from the islet isolation center).
Improved Yield and Functional Parameters of Rat Pancreas Islets Isolated under Intramuscular Anesthesia
Authors: Kim Joon Ye , Lee Jeong-Ik , Jeong Jin Ho , Fang Yuhui , Ju Man Ki , Kim Soo Jin , Huh Kyu Ha , Kim Myoung Soo , Kim Yu Seun
Published: October 2010

Intraperitoneal (IP) anesthesia is commonly used for laboratory animal experiments including rat islet isolation. However, the direct effects of anesthetics on pancreatic islets have been neglected. This study compared the islet function and recovery yield from rats that were anesthetized using IP and intramuscular (IM) injection. In addition, the lag time required to lose deep pain was measured in the following anesthetics combinations. Lewis rats were anesthetized using ketamine and xylazine (K/X) or zoletil and xylazine (Z/X). A glucose challenge test was performed on each group of prepared islets. The effect of the anesthetic agents (e.g., ketamine, zoletil, xylazine alone, and the combination of K/X and Z/X) on cell lines (rat insulinoma; RIN-5F) was investigated by determining their effect on the cell viability, the amount of insulin, and insulin mRNA expression levels of RIN-5F. The time needed for deep anesthesia in IM anesthesia was significantly shortened in comparison to IP [K/X (IM: 313 ± 66 s, IP: 371 ± 84 s) and Z/X (IM: 206 ± 76 s, IP: 245 ± 92 s)]. In addition, number of isolated islet yield by IM anesthesia was significantly improved [K/X (IM: 1530 ± 242, IP: 1245 ± 149) and Z/X (IM: 1136 ± 226, IP: 511 ± 154)]. The functions of fresh islets, indicated by the stimulation index, acquired under IM anesthesia was better preserved than that of IP. The viability and the insulin secretion of RIN-5F were decreased at 24 and 48 h. Insulin gene expression levels were decreased at 24 h as well. Anesthetics may be absorbed through the pancreas surface to the islets and have a direct effect, resulting in islet exposure and deterioration during isolation. In conclusion, for rodent islet isolation, IM anesthesia is simpler and safer in comparison to IP anesthesia.
Expression of Phosphatidylserine-Specific Phospholipase A1 mRNA in Human THP-1-Derived Macrophages
Authors: Hosono Hiroyuki , Homma Masato , Ogasawara Yoko , Makide Kumiko , Aoki Junken , Niwata Hideaki , Watanabe Machiko , Inoue Keizo , Ohkohchi Nobuhiro , Kohda Yukinao
Published: October 2010

The expression of phosphatidylserine-specific phospholipase A1 (PS-PLA1) is most upregulated in the genes of peripheral blood cells from chronic rejection model rats bearing long-term surviving cardiac allografts. The expression profile of PS-PLA1 in peripheral blood cells responsible for the immune response may indicate a possible biological marker for rejection episodes. In this study, PS-PLA1 mRNA expression was examined in human THP-1-derived macrophages. The effects of several immunosuppressive agents on this expression were also examined in in vitro experiments. A real-time RT-PCR analysis revealed that PS-PLA1 mRNA expression was found in human THP-1-derived macrophages. This expression was enhanced in the cells stimulated with lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. Other TLR ligands (TLR2, 3, 5, 7, and 9) did not show a significant induction of PS-PLA1 mRNA. The time course of the mRNA expression profiles was different between PS-PLA1 and tumor necrosis factor-α (TNF-α), which showed a maximal expression at 12 and 1 h after LPS stimulation, respectively. Among the observed immunosuppressive agents, corticosteroids, prednisolone, 6α-methylprednisolone, dexamethasone, and beclomethasone inhibited PS-PLA1 expression with half-maximal inhibitory concentrations less than 3.0 nM, while methotrexate, cyclosporine A, tacrolimus, 6-mercaptopurine, and mycophenoic acid showed either a weak or moderate inhibition. These results suggest that the expression of PS-PLA1 mRNA in THP-1-derived macrophages is activated via TLR4 and it is inhibited by corticosteroids, which are used at high dosages to suppress chronic allograft rejection.
Prevention of Graft-Versus-Host Diseases by in Vivo supCD28mAb-Expanded Antigen-Specific nTreg Cells
Authors: Kitazawa Yusuke , Li Xiao-Kang , Liu Zhong , Kimura Hiromitsu , Isaka Yoshitaka , Hünig Thomas , Takahara Shiro
Published: October 2010

Naturally occurring CD4+CD25+Treg cells (nTregs) can be exploited to establish an immunologic tolerance to non-self-antigens. The in vivo administration of a single superagonistic CD28-specific monoclonal antibody (supCD28mAb) to naive rat preferentially expanded the nTregs, which induced a potent inhibition of lethality of the graft-versus-host (GvH) diseases. The appearance of increased Foxp3 molecules was accompanied with a polarization towards a Th2 cytokine profile with a decreased production of IFN-γ and increased production of IL-4 and IL-10 in the serum of the antibody-treated rat. The peripheral Foxp3 nTregs are decreased in acute GvHD, while supCD28mAb administration showed that nTregs were preferentially proliferating in vivo, thus resulting in the significant prevention of the GvH disease. Furthermore, antigen-specific nTregs could suppress conventional T-cell proliferation stimulated with alloantigen in vitro. Taken together, our findings demonstrate that the potent regulatory functions of the Tregs for the treatment of GvHD are antigen specific. These data also provide evidence that GvHD is associated with decrease of Tregs in the periphery of the host. The determination of the Foxp3 Tregs can be a helpful tool to discriminate GvHD severity and lethality after allogeneic stem cell transplantation.
Brain Death in Combination with Warm Ischemic Stress during Isolation Procedures Induces the Expression of Crucial Inflammatory Mediators in the Isolated Islets
Authors: Saito Yukihiko , Goto Masafumi , Maya Kozue , Ogawa Norihiko , Fujimori Keisei , Kurokawa Yoshimochi , Satomi Susumu
Published: October 2010

Tissue factor (TF) and monocyte chemoattractant protein-1 (MCP-1) expressed on the islets have been identified as the main trigger of the instant blood-mediated inflammatory reaction (IBMIR) in islet transplantation. Because the key steps that directly induce TF and MCP-1 remain to be determined, we focused on the influence of brain death (BD) on TF and MCP-1 expression in the pancreatic tissues and isolated islets using a rodent model. TF and MCP-1 mRNA levels in the pancreatic tissues were similar between the BD and the control group. However, TF and MCP-1 mRNA in the fresh islets of the BD group were significantly higher than that of the control group ( p < 0.01). BD may thus be suggested to be of great importance as an initiator of TF and MCP-1 induction in the isolated islets. Furthermore, the upregulation of crucial inflammatory mediators induced by BD could be exacerbated by warm ischemic damage during digestion procedures. In the present study, the islet yield and purity were affected by BD. However, almost no influences were observed with respect to islet viability, indicating that the expression of inflammatory mediators rather than islet viability is more susceptible to BD. According to the change in time course of TF and MCP-1 expression in the isolated islets, the selected time point for islet infusion in current clinical islet transplantation was thus shown to be at its worst level, at least with respect to the damage caused by BD and ischemic stress. In conclusion, BD in combination with warm ischemic stress during isolation procedures induces a high expression of TF and MCP-1 in the isolated islets. In order to reduce the expression of crucial inflammatory mediators in the islet grafts, the management of the pancreas from brain-dead donors with early anti-inflammatory treatments is thus warranted.
Human Immune Reactivity against Liver Sinusoidal Endothelial Cells from GalTα(1,3)GalT-Deficient Pigs
Authors: Van Poll Daan , Nahmias Yakoov , Soto-Gutierrez Alejandro , Ghasemi Mitra , Yagi Hiroshi , Kobayashi Naoya , Yarmush Martin L. , Hertl Martin
Published: October 2010

Elimination of galactose-α( 1 , 3 )galactose (Gal) expression in pig organs has been previously shown to prevent hyperacute xenograft rejection. However, naturally present antibodies to non-Gal epitopes activate endothelial cells, leading to acute humoral xenograft rejection. Still, it is unknown whether xenogeneic pig liver sinusoidal endothelial cells (LSECs) from α( 1 , 3 )galactosyltransferase (GalT)-deficient pigs are damaged by antibody and complement-mediated mechanisms. The present study examined the xeno-antibody response of LSECs from GalT-deficient and wild pigs. Isolated LSEC from wild-type and GalT pigs were expose to human and baboon sera; IgM and IgG binding was analyzed by flow cytometry. Complement activation (C3a and CH50) was quantified in vitro from serum-exposed LSEC cultures using Enzyme-Linked ImmunoSorbent assay (ELISA). Levels of complement-activated cytotoxicity (CAC) were also determined by a fluorescent Live-Dead Assay and by the quantification of LDH release. IgM binding to GalT knockout (KO) LSECs was significantly lower (80% human and 87% baboon) compare to wild-type pig LSEC. IgG binding was low in all groups. Moreover, complement activation (C3a and CH50) levels released following exposure to human or baboon sera were importantly reduced (42% human and 52% baboon), CAC in GalT KO LSECs was reduced by 60% in human serum and by 72% in baboon serum when compared to wild-type LSECs, and LDH release levels were reduced by 37% and 57%, respectively. LSECs from GalT KO pigs exhibit a significant protection to humoral-induced cell damage compared to LSECs from wild pigs when exposed to human serum. Although insufficient to inhibit xenogeneic reactivity completely, transgenic GalT KO expression on pig livers might contribute to a successful application of clinical xenotransplantation in combination with other protective strategies.
Bone Repair Using a Hybrid Scaffold of Self-Assembling Peptide PuraMatrix and Polyetheretherketone Cage in Rats
Authors: Nakahara Hiroyuki , Misawa Haruo , Yoshida Aki , Hayashi Takayuki , Tanaka Masato , Furumatsu Takayuki , Tanaka Noriaki , Kobayashi Naoya , Ozaki Toshifumi
Published: October 2010

Self-assembling peptide scaffold (SAPS) is well known to have very good bone conduction properties. However, the intensity of SAPS is too weak to actually use it for a clinical bone regeneration. Therefore, we have produced a hybrid scaffold system that involves fabricating a cage from polyetheretherketone (PEEK) that has high intensity, filling the interior of this cage with SAPS, and then transplanted this hybrid scaffold to bone defects in rat femurs. After 28 days, soft X-ray radiographs and histological assessment revealed that good new bone formation was clearly observed in the defects transplanted the PEEK cage with SAPS, but not in the PEEK cage only. The PEEK cage maintained a form and osteoconduction ability of internal SAPS, and SAPS promoted bone formation inside the PEEK; therefore, each was in charge of intensity and bone regeneration separately. The present study suggests that hybrid scaffolds made from PEEK cages and SAPS can be useful tools for the regeneration of load-bearing bones, based on the idea that it should be possible to develop ideal bone filler materials by combining the strength of artificial bone with the bone regeneration and bone conduction properties of SAPS.
Treatment of Acute Liver Failure in Mice by Hepatocyte Xenotransplantation
Authors: Yamamoto Tsuyoshi , Navarro-Alvarez Nalú , Soto-Gutierrez Alejandro , Yuasa Takeshi , Iwamuro Masaya , Kubota Yasuhiro , Seita Masayuki , Kawamoto Hironobu , Javed Shahid M. , Kondo Eisaku , Noguchi Hirofumi , Kobayashi Satoru , Nakaji Shuhei , Kobayashi Naoya
Published: October 2010

Liver diseases still have a high mortality even though liver transplantation has become a standard treatment. Currently, hepatocyte transplantation has been proposed as another promising strategy. One limitation is the availability of human livers as a source of hepatocytes. Because of an unlimited supply, the use of porcine hepatocytes might address this problem. Regardless of the source, once isolated hepatocytes lose specific functionality due to the loss of the natural microenvironment. For this reason, we tested the ability of a self-assembling peptide nanofiber (SAPNF) to provide a provisional three-dimensional (3D) support to interact with cells to control their function in vivo. Isolated porcine hepatocytes were embedded in SAPNF, or collagen type I and transplanted by direct injection into the splenic pulp of SCID mice suffering from acute liver failure (ALF) by 90% hepatectomy. SAPNF porcine hepatocyte transplantation produced engraftment that was far superior to that obtained using collagen and prolonged the survival of mice with ALF, in contrast with controls. An ultrastructural evaluation using transmission electron microscopy indicated extensive cell–cell communication and preservation of hepatocyte architecture. The transplanted SAPNF hepatocytes showed higher expression of albumin and PAS and lower apoptotic events assessed by TUNEL staining. Hepatocytes culture in a truly 3D network allows in vivo maintaining of differentiated functions, and once transplanted between widely divergent species can function to correct acute liver failure in mice and prolong their survival.
Engineering Liver Tissues under the Kidney Capsule Site Provides Therapeutic Effects to Hemophilia B Mice
Authors: Ohashi Kazuo , Tatsum Kohei , Utoh Rie , Takagi Soichi , Shima Midori , Okano Teruo
Published: October 2010

Recent advances in liver tissue engineering have encouraged further investigation into the evaluation of therapeutic benefits based on animal disease models. In the present study, liver tissues were engineered in coagulation factor IX knockout (FIX-KO) mice, a mouse model of hemophilia B, to determine if the tissue engineering approach would provide therapeutic benefits. Primary hepatocytes were isolated from the liver of wild-type mice and suspended in a mixture of culture medium and extracellular matrix components. The hepatocyte suspension was injected into the space under the bilateral kidney capsules of the FIX-KO mice to engineer liver tissues. The plasma FIX activities (FIX:C) of the untreated FIX-KO mice were undetectable at any time point. In contrast, the liver tissue engineered FIX-KO mice achieved 1.5–2.5% of plasma FIX activities (FIX:C) and this elevated FIX:C level persisted throughout the 90 day experimental period. Significant FIX mRNA expression levels were found in the engineered liver tissues at levels similar to the wild-type livers. The present study demonstrates that liver tissue engineering could provide therapeutic benefits in the treatment of hemophilia B.
Engineering of an Hepatic Organoid to Develop Liver Assist Devices
Authors: Soto-Gutierrez Alejandro , Navarro-Alvarez Nalú , Yagi Hiroshi , Nahmias Yakoov , Yarmush Martin L. , Kobayashi Naoya
Published: October 2010

Cell-based technologies to support/restore liver function represent one of the most promising opportunities in the treatment of acute liver failure. However, the understanding of the constituent cell types that interact to achieve liver-specific structure and function has not been achieved in the development of liver assist devices (LADs). Here we show that hepatocytes migrated toward and adhered and formed sinusoids-like structures in conjunction with liver nonparenchymal cells, and that this liver organoid formed sophisticated tissue after 7 days in an implanted LAD in rodents. Hepatocytes only or in combination with human nonparenchymal liver cell lines (endothelial, cholangiocytes, and stellate cells) were cultured in Matrigel. Ultrastructural analysis showed that the hepatocyte-decorated endothelial vascular structures resemble in vivo sinusoids containing plate-like structures, bile canaliculi, and lumen. The sinusoid-like structures retained albumin secretion and drug metabolism capabilities. In addition, LADs containing cocultures of human liver nonparenchymal cells were transplanted in animals for a week; the liver tissue formed sophisticated structures resembling the liver. These results demonstrate the importance of nonparenchymal cells in the cellular composition of LADs. The novelty of the culture's sinusoid-like organization and function strongly support the integration of liver nonparenchymal units into hepatocyte coculture-based LADs as a potential destination therapy for liver failure.
Bone Marrow Mesenchymal Stromal Cells Attenuate Organ Injury Induced by LPS and Burn
Authors: Yagi Hiroshi , Soto-Gutierrez Alejandro , Kitagawa Yuko , Tilles Arno W. , Tompkins Ronald G. , Yarmush Martin L.
Published: October 2010

Bone marrow mesenchymal stromal cells (MSCs) suppress immune cell responses and have beneficial effects in various inflammatory-related immune disorders. A therapeutic modality for systemic inflammation and its consequences is not available yet. Thus, this work investigates the therapeutic effects of MSCs in injury models induced by lipopolysaccharide (LPS) or burn. Gene expression was analyzed in MSCs when exposed to inflammatory serum from injured animals and it showed remarkable alterations compared to normal culture. In addition, injured animals were transplanted intramuscularly with MSCs. Forty-eight hours after cell transplantation, kidney, lung, and liver were analyzed for infiltration of inflammatory cells and TUNEL-expressing cells. Results showed that MSCs attenuate injury by reducing the infiltration of inflammatory cells in various target organs and by reducing cell death. These data suggest that MSCs emerge as key regulators of immune/inflammatory responses in vivo and as attractive candidates for cell-based treatments for systemic inflammatory-based disorders.
Comparative Analysis of Endoderm Formation Efficiency between Mouse ES Cells and iPS Cells
Authors: Iwamuro Masaya , Komaki Toshiyuki , Kubota Yasuhiro , Seita Masayuki , Kawamoto Hironobu , Yuasa Takeshi , Shahid Javed M. , Hassan Reham A. R. A. , Hassan Wael A. R. A. , Nakaji Shuhei , Nishikawa Yuriko , Kondo Eisaku , Yamamoto Kazuhide , Kobayashi Naoya
Published: October 2010

Definitive endoderm (DE) derived from stem cells holds potential to differentiate into hepatocytes. Stem cell therapy using those cells has potential for a treatment of liver disease. To date, various ways of inducing hepatocytes from embryonic stem (ES) cells have been reported by researchers. However, it has not been proved enough that induced pluripotent stem (iPS) cells behave in the same manner as ES cells in endoderm differentiation. The purpose of this study was to establish an efficient method to induce DE from iPS cells, through comparatively analyzing the efficacy of endoderm formation from mouse ES cells. Furthermore, the efficiency of a serum-free medium in the differentiation into DE was investigated. Mouse ES cells and iPS cells were floated in culture medium for 2 or 5 days and embryoid bodies (EB) were formed. Subsequently, DE was induced with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF). RT-PCR and real-time PCR analyses were carried out at each step to determine the gene expression of EB markers. The difference in cellular proliferation between serum-containing and serum-free media was examined by an MTS assay in EB and DE induction. iPS cells showed the paralleled mRNA expression to ES cells in each step of differentiation into EB, but the levels of expression of Sox17 and Foxa2 were relatively higher in ES cell-derived DE, whereas Cxcr4 expression was higher in iPS cell-derived DE. The utilization of serum-free medium for iPS cells showed significantly favorable cellular proliferation during EB formation and subsequent DE induction. Forming EB for 5 days and subsequently DE induction with activin A and bFGF with serum-free medium was an appropriate protocol in iPS cells. This may represent an important step for generating hepatocytes from iPS cells for the development of cell therapy.
Hepatic Differentiation of Mouse iPS Cells in Vitro
Authors: Iwamuro Masaya , Komaki Toshiyuki , Kubota Yasuhiro , Seita Masayuki , Kawamoto Hironobu , Yuasa Takeshi , Shahid Javed M. , Hassan Reham A. R. A. , Hassan Wael A. R. A. , Nakaji Shuhei , Nishikawa Yuriko , Kondo Eisaku , Yamamoto Kazuhide , Fox Ira J. , Kobayashi Naoya
Published: October 2010

Induced pluripotent stem (iPS) cells are pluripotent and are able to unlimitedly proliferate in vitro. This technical breakthrough in creating iPS cells from somatic cells has noteworthy implications for overcoming the immunological rejection and the ethical issues associated with the derivation of embryonic stem cells from embryos. In the current work, we present an efficient hepatic differentiation of mouse iPS cells in vitro. iPS cells were cultured free floating to induce the formation of embryoid bodies (EB) for 5 days. EB were transferred to a gelatin-coated plate and treated with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF) for 3 days to induce definitive endoderm. Cells were further cultured for 8 days with 100 ng/ml hepatocyte growth factor (HGF) to generate hepatocytes. Characterization was performed by RT-PCR assay. Functional analysis for albumin secretion and ammonia removal was also carried out. iPS cell-derived hepatocyte-like cells (iPS-Heps) were obtained at the end of the differentiation program. Expression levels of a gestational hepatocyte gene and lineage-specific hepatic genes intensified in iPS-Heps. The production of albumin increased in a time-dependent manner. iPS-Heps were capable of metabolizing ammonia. We present here instant hepatic differentiation of mouse iPS cells using combined 3-day treatments of activin A and bFGF with subsequent 8-day HGF. Our study will be an important step to generate hepatocytes from human iPS cells as a new source for liver-targeted cell therapies.
Establishment of an Immortalized Porcine Liver Cell Line JSNK-1 with Retroviral Transduction of SV40T
Authors: Shahid Javed M. , Iwamuro Masaya , Sasamoto Hiromi , Kubota Yasuhiro , Seita Masayuki , Kawamoto Hironobu , Nakaji Shuhei , Noguchi Hirofumi , Yamamoto Kazuhide , Kobayashi Naoya
Published: October 2010

Maintenance of freshly isolated porcine liver cells in vitro is limited for a short period of time. Therefore, establishment of easy handling cell lines is extremely important for in vitro study for liver cells and their possible utilization for cell differentiation and growth of stem cells. Porcine liver cells were transduced with a retroviral vector SSR#69 expressing SV40T, one of SSR#69-immortalized porcine liver cell lines, JSNK-1, was established and characterized. Morphology of JSNK-1 cells was spindle shaped. When the cells became confluent, JSNK-1 cells revealed hills-and-valleys pattern. In the presence of vitamin A, JSNK-1 cells showed big droplets inside the cytoplasm, which were positive with PAS staining. JSNK-1 cells showed the gene expression of collagen type 1α1, collagen type 1α2, FLT-1, β-actin, and SV40T. Immunostaining study revealed that JSNK-1 cells produced collagen, vimentin, and α-smooth muscle actin. JSNK-1 cells possessed the characteristics of the liver stellate cells. JSNK-1 cells produced hepatocyte growth factor (HGF) in a time-dependent manner. When cocultured with iPS cells towards the hepatic differentiation, JSNK-1 cells facilitated their hepatic differentiation in terms of albumin production. In conclusion, JSNK-1 cells would be valuable in the study of liver stellate cell pathophysiology and contribute to the optimization of hepatic differentiation of iPS cells.
Characteristics of CD133+ Human Colon Cancer SW620 Cells
Authors: Kawamoto Hironobu , Yuasa Takeshi , Kubota Yasuhiro , Seita Masayuki , Sasamoto Hiromi , Shahid Javed M. , Hayashi Takahiro , Nakahara Hiroyuki , Hassan Reham , Iwamuro Masaya , Kondo Eisaku , Nakaji Shuhei , Tanaka Noriaki , Kobayashi Naoya
Published: October 2010

Worldwide, colorectal cancer is the third most common type of cancer affecting both sexes. It has been proposed that a small subset of cancer cells (cancer stem cells) within each tumor is able to initiate tumor growth. In 2007, two research groups simultaneously identified a colon cancer stem cell population in human tumors by the use of CD133 expression. In the present study, we used a human colon cancer cell line, SW620, to analyze the cancer stem cell-like characteristics of CD133+ cells in vitro and in vivo. In vitro, CD133+ SW620 cells had a higher proliferative capacity, were more irradiation- and chemotherapy-resistant, and had a higher expression of β-catenin compared with CD133- cells. Injections of either CD133+ or CD133- cells into the skin or rectal mucosa of NOD/SCID mice led to tumors; however, injection of CD133+ cells resulted in the formation of larger tumors. Tumors derived from injections of CD133- cells did not contain any CD133+ cells, whereas tumors derived from injections of CD133+ cells did contain CD133+ cells, suggesting self-renewing capability. However, the proportion of CD133+ cells in the newly formed tumors in vivo was lower than the proportion of CD133+ cells in vitro. In conclusion, the human colon cancer cell line, SW620, contains both CD133+ and CD133- phenotypes, and the CD133+ phenotype has characteristics consistent with those of cancer stem cells.
Isolation and Propagation of a Human CD133- Colon Tumor-Derived Cell Line with Tumorigenic and Angiogenic Properties
Authors: Navarro-Alvarez Nalú , Kondo Eisaku , Kawamoto Hironobu , Hassan Wael , Yuasa Takeshi , Kubota Yasuhiro , Seita Masayuki , Nakahara Hiroyuki , Hayashi Takahiro , Nishikawa Yuriko , Hassan Reham A. R. A. , Javed Shahid M. , Noguchi Hirofumi , Matsumoto Shinichi , Nakaji Shuhei , Tanaka Noriaki , Kobayashi Naoya , Soto-Gutierrez Alejandro
Published: October 2010

It has been proposed in human colorectal cancers (CRC) a minority subset of cancer cells within tumors able to initiate tumor growth, defined as cancer stem cells (CSC). Solid human primary colonic and its ovarian metastatic cancer tissues were collected from fresh surgical samples and subsequent xenografts were established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. The resulting tumors were disaggregated into single-cell suspensions and a CD133- cell line (NANK) was newly established and analyzed by flow cytometry. Surface markers of progenitor cells were immunophenotypically analyzed, and expression of stem cell and cancer-related genes was characterized. Secreted angiogenesis-associated molecules were investigated by proteomic array technology. Finally, different numbers of NANK were implanted and their tumor-initiating properties were investigated in NOD/SCID mice. Intraperitoneal injection of NANK in NOD/SCID mice induced tumors with developing progressive peritoneal dissemination and ascites. NANK cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Noticeably, NANK lacked the expression of conventional CSC markers CD133 and CD44, self-renewal genes Oct-4 and Nanog, but showed the expression of an important gastrointestinal development marker CDX-2 and BMI-1 that is essential in regulating the proliferative activity of normal and leukemic stem cells. In addition, NANK secreted high amounts of important angiogeneic cytokines. These results provide a novel and extensive model in human CSC for studying the generation and maintenance of phenotypic heterogeneity in CRC.
Characterization of Human Pancreatic Progenitor Cells
Authors: Noguchi Hirofumi , Naziruddin Bashoo , Jackson Andrew , Shimoda Masayuki , Ikemoto Tetsuya , Fujita Yasutaka , Chujo Daisuke , Takita Morihito , Kobayashi Naoya , Onaca Nicholas , Hayashi Shuji , Levy Marlon F. , Matsumoto Shinichi
Published: October 2010

β-Cell replacement therapy via islet transplantation is an effective treatment for diabetes mellitus, but its widespread use is severely limited by the shortage of donor organs. Because pancreatic stem/progenitor cells are abundantly available in the pancreas of these patients and in donor organs, the cells could become a useful target for β-cell replacement therapy. We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we used the techniques to identify and isolate human pancreatic stem/progenitor cells. The cells from a duct-rich population were cultured in 23 kinds of culture media, based on media for mouse pancreatic stem cells or for human embryonic stem cells. The cells in serum-free media formed “cobblestone” morphologies, similar to a mouse pancreatic stem cell line. On the other hand, the cells in serum-containing medium and the medium for human embryonic stem cells formed “fibroblast-like” morphologies. The cells divided actively until day 30, and the population doubling level (PDL) was 6–10. However, the cells stopped dividing after 30 days in any culture conditions. During the cultures, the nucleus/cytoplasm (N/C) ratio decreased, suggesting that the cells entered senescence. Exendin-4 treatment and transduction of PDX-1 and NeuroD proteins by protein transduction technology into the cells induced insulin and pancreas-related gene expression. Although the duplications of these cells were limited, this approach could provide a potential new source of insulin-producing cells for transplantation.
Cell Labeling with a Novel Contrast Agent of Magnetic Resonance Imaging
Authors: Oishi Koichi , Noguchi Hirofumi , Saito Hiroaki , Yukawa Hiroshi , Miyamoto Yoshitaka , Murase Katsutoshi , Hayashi Shuji
Published: October 2010

Cell therapy is a proven and efficient method for treating multiple diseases. For both basic research and clinical practice, the development of noninvasive in vivo imaging methods is essential for monitoring the trafficking or homing of transplanted cells. One attractive approach for the effective imaging of transplanted cells is the efficient labeling of cells with a contrast agent. In this study, we developed a novel contrast agent of magnetic resonance imaging (MRI), TMADM-02. TMADM-02 was efficiently transduced into cells without toxicity. However, the aggregation of TMADM-02 was observed because of its low stability in culture medium. Therefore, TMADM-02 may have led to a false-positive test result. In future studies, we should verify not only the efficiency of labeling cells but also the stability of the contrast agent of MRI for clinical applications.
Efficient Transfection of Sendai Virus Vector to Mouse Pancreatic Stem Cells in the Floating State
Authors: Oishi Koichi , Noguchi Hirofumi , Yukawa Hiroshi , Inoue Makoto , Miyamoto Yoshitaka , Iwata Hisashi , Hasegawa Mamoru , Hayashi Shuji
Published: October 2010

Sendai virus (SeV) vectors can efficiently introduce foreign genes without toxicity into various organs and are expected to be clinically applicable. We previously compared the transfectional efficiency of SeV and adenovirus (AdV) vectors by assessing the transfer of the green fluorescent protein (GFP) gene to pancreatic stem cells. Although the gene transfer efficiency was similar between these vectors, SeV vector had a lower toxicity in comparison to the AdV vector. In this study, we assessed the gene transfer efficiency of SeV vector in the floating state to pancreatic stem cells. The efficiency of gene transfer was much higher at all time points and at all concentrations in the floating state versus in the adhesion state. In addition, the pancreatic stem cells transfected with SeV in the floating state maintained their differentiation ability. These data suggest that SeV transfection to pancreatic stem cells in the floating state may be useful in gene transfer technology.
Transduction of Cell-Penetrating Peptides into Induced Pluripotent Stem Cells
Authors: Yukawa Hiroshi , Noguchi Hirofumi , Nakase Ikuhiko , Miyamoto Yoshitaka , Oishi Koichi , Hamajima Nobuyuki , Futaki Shiroh , Hayashi Shuji
Published: October 2010

Induced pluripotent stem (iPS) cells have recently been generated by Yamanaka's group, and then followed by others. iPS cells are expected to have clinical applications including an important role in regenerative medicine. This study focused on the cell-penetrating peptides (CPPs) for differentiation or functional application of iPS cells, because several transduction domains can deliver a large size-independent variety of molecules into cells. Two CPPs, Texas Red-R8 and Rhodamine-TAT, were generated as representative CPPs and these CPPs were tested to determine their ability to penetrate the membrane of iPS cells. Both CPPs were transduced in iPS cells through macropinocytosis classified in endocytosis within 2 h in a manner consistent with many other cells, and no cytotoxicity and influence on their undifferentiated state was observed. In conclusion, CPPs can be utilized for their differentiation or functional application in iPS cells.
Morphometric architecture of the most taxon‐rich species in the U.S. flora: Astragalus lentiginosus (Fabaceae)
Authors: Knaus Brian J.
Published: October 2010

• Premise of the study: The study of infra‐taxa has historically been considered the study of incipient species. Astragalus lentiginosus Douglas ex Hooker (Fabaceae) is the most taxonomically diverse species in the U. S. flora. To gain insight into the nature of infra‐taxa, I ask whether an optimal number of groups exists within A. lentiginosus, whether the varieties represent discrete entities, and whether morphological diversity within A. lentiginosus can be explained by climate.

• Methods: Morphometric measurement was made on herbarium specimens. Seasonality was inferred by fitting sine waves to climatic data. Statistical clustering, ordination, and multiple regression analyses were employed to test hypotheses.

• Key results: K‐means clustering resulted in no clear optimal number of groups within the species. Principal components analysis demonstrated that taxa occupy contiguous regions of morphospace but do not form distinct clusters. Multiple regression demonstrated significant relations among morphology and climatic parameters, which are generally stronger than geographic relations.

• Conclusions: The bewildering amount of diversity contained within A. lentiginosus begs for decomposition, yet its clinal nature precludes it from division into discrete groups. As a species, A. lentiginosus exists as an array of populations in a delicate balance between homogenizing forces, such as gene flow and retention of ancestral characters, and diversifying forces, such as local adaptation and drift. The effect of these forces has become evident in the phenotype of A. lentiginosus, resulting in more infra‐taxa than any other species in the U. S. flora, but has failed to fracture this group into distinct entities.
Is Physical Activity a Risk Factor for Primary Knee or Hip Replacement Due to Osteoarthritis? A Prospective Cohort Study
Authors: WANG YUANYUAN , SIMPSON JULIE ANNE , WLUKA ANITA E. , TEICHTAHL ANDREW J. , ENGLISH DALLAS R. , GILES GRAHAM G. , GRAVES STEPHEN , CICUTTINI FLAVIA M.
Published: October 2010

Objective.

To estimate prospectively any association between measures of physical activity and the risk of either primary knee or hip replacement due to osteoarthritis (OA).

Methods.

Eligible subjects (n = 39,023) were selected from participants in a prospective cohort study recruited 1990–1994. Primary knee and hip replacement for OA during 2001–2005 was determined by linking the cohort records to the National Joint Replacement Registry. A total physical activity level was computed, incorporating both intensity and frequency for different forms of physical activity obtained by questionnaire at baseline attendance.

Results.

There was a dose-response relationship between total physical activity level and the risk of primary knee replacement [hazards ratio (HR) 1.04, 95% CI 1.01–1.07 for an increase of 1 level in total physical activity]. Although vigorous activity frequency was associated with an increased risk of primary knee replacement (HR 1.42, 95% CI 1.08–1.86) for 1–2 times/week and HR 1.24 (95% CI 0.90–1.71) for ≥ 3 times/week), the p for trend was marginal (continuous HR 1.08, 95% CI 1.00–1.16, p = 0.05). The frequency of less vigorous activity or walking was not associated with the risk of primary knee replacement, nor was any measure of physical activity associated with the risk of primary hip replacement.

Conclusion.

Increasing levels of total physical activity are positively associated with the risk of primary knee but not hip replacement due to OA. Physical activity might affect the knee and hip joints differently depending on the preexisting health status and anatomy of the joint, as well as the sort of physical activity performed.
Mycophenolate Mofetil for Induction Treatment of Lupus Nephritis: A Systematic Review and Metaanalysis
Authors: TOUMA ZAHI , GLADMAN DAFNA D. , UROWITZ MURRAY B. , BEYENE JOSEPH , ULERYK ELIZABETH M. , SHAH PRAKESH S.
Published: October 2010

Objective.

To systematically review the efficacy and safety of mycophenolic acid and mycophenolate mofetil (MMF) compared to cyclophosphamide (CYC) for the induction treatment of lupus nephritis (LN).

Methods.

Medline, Embase, the Cochrane Center Register of Controlled Trials, and abstracts presented in major international conferences were searched for randomized controlled trials. The primary outcome was renal remission (complete, partial, and overall) and secondary outcomes were adverse events during study period and longterm followup data. Data were compared between groups and relative risk (RR) and 95% CI were calculated.

Results.

Four trials of a total of 618 patients were included. MMF was not superior to CYC for renal remission (partial RR 0.94, 95% CI 0.80 to 1.12; complete RR 0.67, 95% CI 0.35 to 1.28, and overall RR 0.89, 95% CI 0.71 to 1.10). There was a significant reduction in alopecia (RR 5.77, 95% CI 1.56 to 21.38) and amenorrhea (RR 6.64, 95% CI 2.00 to 22.07) with the use of MMF compared to CYC. These results should be interpreted with caution given the width of the CI. There was no significant difference for infections, leukopenia, gastrointestinal symptoms, herpes zoster, endstage renal disease, and death among groups during study period and longterm followup data.

Conclusion.

We could not show that MMF is superior to CYC for the induction treatment of LN. Patients treated with MMF showed reduced risk of certain side effects. MMF can be used as an alternative to CYC for the induction treatment of LN.
Systemic Vasculitis in Patients with Hepatitis C Virus Infection with and without Detectable Mixed Cryoglobulinemia
Authors: TERRIER BENJAMIN , SÈNE DAMIEN , DECHARTRES AGNÈS , SAADOUN DAVID , ORTONNE NICOLAS , ROUVIER PHILIPPE , MUSSET LUCILE , RIGON MATTHIEU RESCHE , MAISONOBE THIERRY , CACOUB PATRICE
Published: October 2010

Objective.

To describe hepatitis C virus (HCV)-related systemic vasculitis in patients without detectable mixed cryoglobulinemia (MC) and to compare them to typical cases of HCV-MC vasculitis.

Methods.

Twelve HCV RNA+ patients with histologically proven vasculitis in the absence of detectable MC (cases) were retrospectively compared with 48 HCV RNA+ patients with MC vasculitis (controls). Each case was matched with 4 controls for age and sex.

Results.

The main epidemiological and virologic features were similar between cases and controls. No clinical difference was found, except for lower rates of arthralgias (33% vs 71%; p = 0.02) and purpura (50% vs 83%; p = 0.03) in cases. Cases showed higher mean serum C3 (1.17 ± 0.21 vs 0.93 ± 0.23 g/l; p = 0.01) and median C4 levels (0.25 vs 0.04 g/l; p < 0.001), lower median serum IgM levels (0.6 vs 1.9 g/l; p < 0.001), and lower rates of rheumatoid factor positivity (8% vs 82%; p < 0.001) than controls. The main histologic features were similar between cases and controls. Immunofluorescence analysis of skin biopsy from 1 case revealed perivascular deposits of C3 and IgA. After treatment, overall clinical response of vasculitis (75% vs 83%) and sustained virological response (40% vs 64%; p = 0.3) were similar between cases and controls, except for higher complete clinical response (42% vs 73%; p = 0.05) in controls.

Conclusion.

HCV-related systemic vasculitis may occur in the absence of detectable MC. Our findings suggest that such vasculitis probably results from immune complex-mediated mechanisms, and that the therapeutic management of such vasculitis should be similar to that of HCV-MC vasculitis.
Adalimumab Improves Sleep and Sleep Quality in Patients with Active Ankylosing Spondylitis
Authors: RUDWALEIT MARTIN , GOOCH KATHERINE , MICHEL BEAT , HEROLD MANFRED , THÖRNER ÅKE , WONG ROBERT , KRON MARTINA , CHEN NAIJUN , KUPPER HARTMUT
Published: October 2010

Objective.

Fatigue and sleep problems are significant in patients with ankylosing spondylitis (AS). This subanalysis of the RHAPSODY study (Review of Safety and Effectiveness with Adalimumab in Patients with Active Ankylosing Spondylitis) was conducted to evaluate the effect of adalimumab on sleep in patients with active AS.

Methods.

All patients (n = 1250) had active AS and received adalimumab 40 mg every other week during the 12-week open-label treatment period. Sleep was assessed by the Medical Outcomes Study Sleep Scale (MOS-SS) at screening and Weeks 6, 12, and 20 (optional continuation period). Effect sizes were calculated to determine clinical significance. Paired Student t tests compared the change in the MOS-SS domains from Week 12 to baseline. Correlation coefficients were calculated to determine the relationship between change in sleep domains and other Week 12 clinical and patient-reported outcomes (Bath AS Disease Activity Index, C-reactive protein, nocturnal pain, total back pain, Bath AS Functional Index, patient’s global assessment of disease activity, morning stiffness, Short Form-36 Health Survey, and Work Productivity and Activity Impairment questionnaire components).

Results.

At Week 12, adalimumab significantly improved sleep in each of the MOS-SS domains (p < 0.001). Effect sizes for 3 of the 6 domains (disturbance, −0.69; adequacy, 0.55; somnolence, −0.52) and both sleep problems indices (Index I, −0.68; Index II, −0.77) were moderate, suggesting clinical significance. Change in the MOS-SS Sleep Problem Index II was moderately correlated with change in most clinical and patient-reported outcomes. Sleep improvements were similar in patients with and without radiographically advanced AS.

Conclusion.

Adalimumab improves overall sleep and sleep quality in patients with active AS.