Abstract: BACKGROUND AND PURPOSE: Astrocytes support neuronal functions by regulating the extracellular ion-homeostasis and levels of neurotransmitters, and by providing fuel such as lactate to the neurons via their astrocytic processes (APs). Whether injured APs are associated with neuronal survival/death is still an unanswered question. We investigated APs in the neuropil, especially those around astrocytes and normal-appearing, degenerating, and dead neurons in cerebral cortical regions peripheral to the cortical infarction (RPI). METHODS: Stroke-positive gerbils were euthanized at various times after the ischemic insult. Ultrathin sections were obtained from the RPI sectioned coronally at the infundibular level. We counted the number of normal-appearing, degenerated, and dead neurons and astrocytes in paraffin sections, the number of cut-ends and mitochondria in APs in the neuropil on electron-microscopic photographs, and determined the percent-volume of APs by Weibel point-counting method. We compared the number of cut-ends and mitochondria and percent-volume of APs around astrocytes at 5 hours and 48 hours, and around normal-appearing, degenerated, and dead neurons at 12 hours. RESULTS: Although the number of astrocytes did not change (average of 12.3+/-0.20%) during 0 to 48 hours, that of the dead neurons increased from 9.71+/-1.34 to 44.39+/-1.40% during 5 to 48 hours postischemia. The number of normal-appearing APs and mitochondria in APs decreased respectively from 13.49+/-0.65 to 1.61+/-0.14/28.20 microm(2) and from 1.86+/-0.18 to 0.61+/-0.07/28.20 microm(2) in the neuropil during 0 to 48 hours. The number of normal-appearing APs around astrocytes decreased from 12.3+/-0.19 to 1.7+/-0.05/38.33 microm(2) with an increase in percent-volume of degenerated APs from 1.17+/-0.04 to 11.45+/-0.23%, from 5 to 48 hours postischemia. The number of normal-appearing APs decreased from 4.36+/-0.52 to 1.56+/-0.17/38.33 microm(2) with an increase in percent-volume of degenerated APs, from 2.41+/-0.52 to 12.55+/-1.0%, from around the normal-appearing to dead neurons, at 12 hours. CONCLUSIONS: In the RPI, heterogeneous degeneration of APs was closely associated with disseminated selective neuronal necrosis and the maturation phenomenon seen in ischemic neuronal injury.

Abstract: Acute lung injury has a range of causes, and occasionally leads to lethal respiratory failure. Despite advances in treatment, acute lung injury continues to have a high mortality rate, and thus a new therapeutic approach is needed. ST2 is an interleukin (IL)-1 receptor-related protein, and its expression is induced by various inflammatory responses. Recently, ST2 has been speculated to exert anti-inflammatory effects; therefore, we investigated the role of the ST2 in the murine model of acute lung injury. To elucidate the function of ST2 in vivo, mice that transiently overexpressed ST2 protein were prepared using the hydrodynamic gene transfer method, and lung injury was induced by intratracheal administration of bleomycin. In bleomycin-treated ST2-overexpressing mice, the increase of neutrophils in the bronchoalveolar lavage fluid (BALF) was markedly suppressed. Additionally, the levels of tumour necrosis factor-alpha and IL-6, as well as the concentration of albumin, in BALF were reduced compared with those of controls. Furthermore, the pulmonary architecture in ST2-overexpressing mice remained almost normal, and the survival rate was significantly improved. From these results, we concluded that ST2 has the potential to suppress the initial stage of acute lung injury, and therefore it may be a useful reagent for the treatment of acute lung injury.

Abstract: SUMMARY BACKGROUND: Hemophilia A is a congenital bleeding disorder caused by a deficiency of coagulation factor VIII. Approximately 30% of hemophilia A patients develop inhibitors against FVIII following replacement therapy. We have reported that neonatal exposure of FVIII antigen can induce antigen-specific immune tolerance by interferon-gamma (IFN-gamma)-dependent T-cell anergy in hemophilia A mice. OBJECTIVE: The thymus plays crucial roles in self-tolerance, with negative selection of self-reactive effector T cells and positive selection of self-reactive regulatory T cells. We investigated the possibility of the induction of antigen-specific immune tolerance by intrathymic injection of FVIII in hemophilia A mice. METHODS: Hemophilia A mice were injected with recombinant FVIII into the thymus under real-time high-resolution image guidance. RESULTS: Anti-FVIII inhibitory antibody titers in mice challenged with intravenous administration of FVIII were significantly lower in mice (n = 22) that had received thymic FVIII injection than in mice (n = 18) without thymic injection (9.4 +/- 2.3 vs. 122.5 +/- 27.6 BU mL(-1), respectively, P = 0.00078). The CD4(+) T cells from thymic-injected mice could not proliferate or produce interleukin (IL)-2, IL-12 and IFN-gamma in response to FVIII. The CD4(+)CD25(+) T cells generated from thymic-treated mice but not from naïve mice efficiently suppressed the in vitro proliferative response of CD4(+) T cells and blocked the in vivo development of anti-FVIII antibodies in the adoptive transfer. CONCLUSION: These data suggest that intrathymic administration of FVIII could result in immune tolerance by induction of FVIII-specific regulatory T cells.

Abstract: We previously created the Alb-DsRed2 transgenic (Tg) rat that specifically expresses the red fluorescent protein, DsRed2, in the liver. Herein, we demonstrate that the DsRed2 expression is sexually dimorphic and exhibits a male-specific pattern. The profiling of sexual dimorphism in DsRed2 expression during pre-pubertal development was investigated using an in vivo fluorescent imaging analysis. The DsRed2 expression decreased gradually in both sexes until 28days after birth. While DsRed2 expression was not persistent in the female liver, the male hepatic expression increased again at 35days. Sexual dimorphic DsRed2 expression did not change in gonadectomized male and female Tg-rats. However, female hepatic DsRed2 was induced 72h after the hypophysectomy. Hepatocytes isolated from the female Tg-rats also revealed DsRed2 induction by 96h in culture. These results suggest that the pituitary hormone suppresses the female hepatic DsRed2 expression causing the sexual dimorphism of DsRed2 expression.

Abstract: Self-renewing, multipotent neural progenitor cells (NPCs) reside in the adult mammalian spinal cord ependymal region. The current study characterized, in vitro, the native differentiation potential of spinal cord NPCs isolated from adult enhanced green fluorescence protein rats. Neurospheres were differentiated, immunocytochemistry (ICC) was performed, and the positive cells were counted as a percentage of Hoescht+ nuclei in 10 random fields. Oligodendrocytes constituted most of the NPC progeny (58.0% of differentiated cells; 23.4% in undifferentiated spheres). ICC and electron microscopy (EM) showed intense myelin production by neurospheres and progeny. The number of differentiated astrocytes was 18.0%, but only 2.8% in undifferentiated spheres. The number of differentiated neurons was 7.4%, but only 0.85% in undifferentiated spheres. The number of differentiated radial glia (RG) was 73.0% and in undifferentiated spheres 80.9%. EM showed an in vitro phagocytic capability of NPCs. The number of undifferentiated NPCs was 32.8% under differentiation conditions and 78.9% in undifferentiated spheres. Compared with ependymal region spheres, the spheres derived from the peripheral white matter of the spinal cord produced glial-restricted precursors. These findings indicate that adult rat spinal cord ependymal NPCs differentiate preferentially into oligodendrocytes and RG, which may support axonal regeneration in future trials of transplant therapy for spinal cord injury.

Abstract: Cell marking is a very important procedure for identifying donor cells after cell and/or organ transplantation in vivo. Transgenic animals expressing marker proteins such as enhanced green fluorescent protein (EGFP) in their tissues are a powerful tool for research in fields of tissue engineering and regenerative medicine. The purpose of this study was to establish transgenic rabbit lines that ubiquitously express EGFP under the control of the cytomegalovirus immediate early enhancer/beta-actin promoter (CAG) to provide a fluorescent transgenic animal as a bioresource. We microinjected the EGFP expression vector into 945 rabbit eggs and 4 independent transgenic candidate pups were obtained. Two of them died before sexual maturation and one was infertile. One transgenic male candidate founder rabbit was obtained and could be bred by artificial insemination. The rabbit transmitted the transgene in a Mendelian manner. Using fluorescence in situ hybridization analysis, we detected the transgene at 7q11 on chromosome 7 as a large centromeric region in two F1 offspring (one female and one male). Eventually, one transgenic line was established. Ubiquitous EGFP fluorescence was confirmed in all examined organs. There were no gender-related differences in fluorescence. The established CAG/EGFP transgenic rabbit will be an important bioresource and a useful tool for various studies in tissue engineering and regenerative medicine.

Abstract: Recent reports have raised concerns over the feasibility of differentiating bone marrow cells (BMCs) into functional hepatocytes. Such augmentation is considered necessary for potential clinical use of these cells in liver diseases. The present investigation was designed to determine the kinetics of transplanted BMCs and evaluate the effects of repeated bone marrow transplantation (BMT) in rat models of CCl(4)-induced liver damage. The early kinetics of transplanted BMCs was evaluated with a charge-coupled-device (CCD) camera using BMCs obtained from green fluorescent protein (GFP) transgenic (Tg) rats and followed up with in vivo imaging system (IVIS) using BMCs obtained from firefly luciferase (luc) Tg rats. We used a portal infusion system for repeated BMT. BMCs were transplanted via a peripheral vein or the portal vein (PV) once or repeatedly using this system. The results revealed that BMCs accumulated more in the damaged liver than in the intact liver. In the experimental group receiving repeated BMT via the PV, the liver fibrosis was milder than that in the group not receiving BMT, and large clusters of albumin-producing cells were detected by albumin staining. The injected BMCs were shown to accumulate in the damaged liver. This strategy of repeated BMT has potential clinical use in enhancing the number of albumin-producing cells and suppressing liver fibrosis. This combination of beneficial effects may contribute to the benefits of cell transplantation therapy. Demonstration of the benefits of BMT in this study may be expected to have great significance for clinical trials.

Abstract: Amyloidogenic transthyretin (ATTR) is the pathogenic protein of familial amyloidotic polyneuropathy (FAP). To establish a tool for analyses of ATTR metabolisms including after liver transplantations, we developed a transgenic rat model expressing human ATTR V30M and confirmed expressions of human ATTR V30M in various tissues. Mass spectrometry for purified TTR revealed that rat intrinsic TTR and human ATTR V30M formed tetramers. Congo red staining and immunohistochemistry revealed that nonfibrillar deposits of human ATTR V30M, but not amyloid deposits, were detected in the gastrointestinal tracts of the transgenic rats. At 24h after liver transplantation, serum human ATTR V30M levels in transgenic rats that received livers from normal rats became lower than detectable levels. These results thus suggest that this transgenic rat may be a useful animal model which analyzes the metabolism of human ATTR V30M including liver transplantation studies.

Abstract: Renal transplantation is a potential treatment for irreversible renal failure in pet cats. Our aim is to reduce warm ischemic time by using nonpenetrating vascular closure staples (VCS), thereby improving graft survival. Experimental cats were divided into the VCS group (n = 4; autotransplantation) or suture group (n = 6; allotransplantation). The renal artery was anastomosed with the external iliac artery in an end-to-end fashion, and the renal vein was attached to the external iliac vein in an end-to-side fashion. Warm ischemic time as well as arterial and venous anastomotic times were measured. Cats in the suture group were administrated cyclosporine and prednisolone orally after transplantation. Ischemic and anastomotic times in the VCS group were significantly reduced compared with the suture group. Two of 6 allografts had a ureteral anastomotic stricture, and 4 allografts were rejected. Histological findings of autografts showed normal structure. In conclusion, VCS staples were useful in feline renal transplantation.

Abstract: BACKGROUND: Transplantation research involving the use of stem cells demands an appropriate in vivo visualization system to monitor cellular fate over an observation period. The new field of in vivo imaging is being developed with fluorescent and luminescent biotechnology, and involves the real-time visualization of complex cellular processes in living animals. METHODS.: Following our recent development of inbred green fluorescent protein (GFP)-transgenic (Tg) rats, we created the establishment of inbred (Lewis) Tg rats with firefly luciferase. The immunogenicity against luciferase was evaluated by the skin grafting test, and the fate of grafts was monitored by in vivo luminescent technique. RESULTS.: The luciferase-Tg rats ubiquitously expressed the marker gene. Conventional skin grafting apparently showed the long-term acceptance of luciferase-Tg rat skin on wild-type rats (>100 days), compared with grafting of GFP-Tg-derived skin (<10 days). This suggests less cellular immune responsiveness against the luciferase protein than GFP. Strikingly, organ transplants with heart and small bowel, and bone marrow cell transplantation showed viability and graft acceptance, demonstrating that cells and organs from luciferase-Tg rats are transplantable and their fate can be tracked for a sufficient time. Taking advantage of less immunogenic luciferase, cellular fate of transplanted mature hepatocytes was also examined. Transplanted hepatocytes proliferated and were monitored selectively in damaged liver, but not in healthy liver, for over 60 days. CONCLUSIONS: We propose on the basis of these findings that the luciferase-Tg rat system with modern optical imaging offers a new platform for a better understanding of stem cell biology and transplantation.

Abstract: BACKGROUND: We demonstrate the long-term effectiveness of KRP-203 treatment in combination with a subtherapeutic dose of cyclosporine A (CsA) on rat renal allografts. METHODS: We tested the effect of KRP-203 in combination with CsA using a rat skin allograft model. The Pharmacokinetic interaction between CsA and KRP-203 was evaluated. The selectivity of KRP-203 for sphingosine-1-phosphate (S1P)1 and S1P3 receptors were investigated in vitro. Heart rate alteration following bolus injection of phosphorylated KRP-203 (KRP-203-P) or FTY720 (FTY720-P) was also monitored in rats. Finally, the long-term effectiveness of KRP-203 in conjunction with a low dose of CsA was investigated in a rat renal transplantation model. RESULTS: Administration of KRP-203 with CsA prolonged skin allograft survival. KRP-203 and CsA had no effect on the pharmacokinetics of the other. While FTY720-P activated both S1P1 and S1P3 receptors, KRP-203-P selectively activated S1P1, but not the S1P3 receptor (EC50:>1000 nM). Compared to FTY720-P, a tenfold higher dose of KRP-203-P was necessary to induce transient bradycardia. With a low dose of CsA (1 mg/kg/day), KRP-203 (0.3 mg/kg/day) significantly prolonged renal allograft survival (P<0.05, survival time: 9.8 days (CsA) vs. >27.4 days (CsA+KRP)). Although a higher dose of CsA (3 mg/kg/day) alone kept recipients alive, this caused severe renal graft dysfunction. Use of KRP-203 (3 mg/kg/day) in conjunction with CsA markedly improved graft function (P<0.05, creatinine clearance: 0.41+/-0.25 ml/min [CsA] vs. 1.15+/-0.16 ml/min [CsA+KRP]). CONCLUSIONS: The selectivity of KRP-203 for S1P1 reduces the risk of bradycardia, and the combination therapy of KRP-203 with CsA represents a safe and effective strategy for use in renal transplantation.

Abstract: BACKGROUND: Replacement of calcineurin inhibitor (CI) with anti-metabolic agents in transplant patients with CI-induced nephrotoxicity is performed clinically and improves renal function, but increases the risk of rejection. We investigated whether the change from cyclosporine (CsA) to a limited dose of mycophenolic acid (MPA) together with a new sphingosine-1-phosphate (S1P) receptor agonist, KRP-203, is sufficient to prevent both transplant vasculopathy and CsA-induced nephrotoxicity. METHODS: Orthotopic aortic transplantation was conducted in a high-responder rat combination of Dark Agouti (DA; major histocompatibility complex [MHC] haplotype RT-1a) to Lewis (RT-1(l)). After CsA administration (15 mg/kg/day) for 2 weeks, the recipients were divided into the following treatment groups for 6 weeks: MPA (10 mg/kg); KRP-203 (KRP; 1 mg/kg); and MPA + KRP. Serum creatinine (Cr), arteriolar hyalinosis and expression of transforming growth factor (TGF)-beta1 in the recipient kidney were examined as parameters indicating nephrotoxicity. Intimal hyperplasia was assessed by vascular occlusion, and graft-infiltrated cells were semi-quantitatively evaluated histologically and then characterized immunohistochemically. RESULTS: Continuous CsA treatment attenuated intimal hyperplasia and cell infiltration (2.9 +/- 0.3% and 0.4 +/- 0.1; p < 0.01 vs vehicle), but increased Cr and hyalinosis (0.43 +/- 0.03 mg/dl and 57.2 +/- 0.4%; p < 0.01) with upregulated TGF-beta1. Replacement of CsA by MPA or KRP treatment alone improved nephrotoxicity, but worsened intimal hyperplasia and cell infiltration. Conversion to MPA + KRP treatment prevented nephrotoxicity (Cr, 0.32 +/- 0.02 mg/dl; hyalinosis, 5.6 +/- 1.3%; p < 0.01 vs CsA) and markedly suppressed intimal hyperplasia and cell infiltration (3.6 +/- 1.2% and 1.0 +/- 0.3; p = not significant vs CsA), with reduced T-cell infiltrates in the graft. CONCLUSIONS: Changing from CsA to a combined therapy of MMF with S1P agonist is a promising strategy in clinical transplantation to overcome CI-induced nephrotoxicity and chronic rejection.

Abstract: AIM: We determined the characteristics of transgene expression of heart grafts following ex vivo gene transfer using an adenovirus vector. Transgene expression was assessed periodically in the same animals by a non-invasive bioimaging system. METHODS: Rat heterotopic heart transplantation was performed in a syngenic combination. We infused 1 x 10(9) plaque-forming units of adenovirus vectors containing firefly luciferase gene into the heart graft via the coronary artery, with preservation at 4 degrees C and transplanted into the cervix of the recipient. Transgene expression was periodically visualized and quantified by a noninvasive bioimaging system without sacrificing experimental animals. RESULTS: Transgene expression in the graft peaked at day 7 and then fell gradually. Transgene expression was also observed in the recipient liver. CONCLUSIONS: We have determined the time course of transgene expression in the heart graft. This constitutes important information about ex vivo gene therapy for heart grafts.

Abstract: The ideal goal of regeneration medicine is to restore form and function to damaged tissues. While stem cell transplantation is considered a promising therapeutic approach, knowing the fate of transplanted cells using appropriate markers is essential. We developed new inbred transgenic rat strains with lacZ and GFP based on the transgenic (Tg) animal technique in rats. These Tg animals expressed most of their marker genes ubiquitously, compared to previous Tg rats. Immunological antigenicity against marker proteins was evaluated using conventional skin grafting, and results suggested lacZ-Tg-derived skin was much less immunogenic than that of GFP-Tg. However, GFP-positive cells from parental transgenic rats were still potential candidates for the study of cellular fate in immune privilege sites, such as the brain. Taking advantage of less immunogenic lacZ, we also examined the role of bone marrow-derived cells (BMDCs) in skin wound healing using an in vivo biological imaging system. Although transplantation of BMDCs enhanced wound healing at the injection site, BMDCs were detected only for a short time, suggesting a transient contribution of autologous BMDC-transplantation in wound healing. Our Tg-rat system may provide great benefits for the elucidation of the cellular process of regenerative medicine, including cell and tissue transplantation.

Abstract: Leptin, the ob gene product, has an important role in the regulation of body weight. Although tamoxifen, a nonsteroidal antiestrogenic agent, is known to have estrogenic effects on fat metabolism, its influence on adipose tissue remains unknown. In the present study, the effect of tamoxifen on the concentration of leptin was investigated in ovariectomized rats treated with tamoxifen or vehicle. The dosage of tamoxifen was extrapolated from the human dosage. Food intake, adipose tissue weight, and plasma insulin were assessed at the end of the experiment. RESULTS: Tamoxifen-treated rats showed a significant reduction of body weight gain, food intake, adipose tissue weight and leptin concentration (p < 0.001). The plasma insulin level was significantly higher after tamoxifen treatment (p = 0.01) in tamoxifen-treated rats than in control rats. We concluded that tamoxifen reduces food intake in the acute phase and a reduction of adipose tissue gain may result in reduced levels of plasma leptin in ovariectomized rats. Furthermore, rats treated with tamoxifen may be resistant to insulin action.

Abstract: To find more effective and less toxic immunosuppressive strategies in long-term treatment for organ transplantation patients, we examined the effects on rat heart allograft survival of a novel sphigosine-1-phosphate receptor agonist, KRP-203, combined with a subtherapeutic dose of cyclosporine (CsA). Rat heart transplantation was performed across a major histocompatibility complex-incompatible (DA to LEW) rat combination. KRP-203 alone showed little or no effect on heart allograft survival. In contrast, KRP-203 combined with a subtherapeutic dose of CsA led to prolonged allograft survival. Histologic analyses showed that the combination completely suppressed acute rejection, as characterized by allograft vasculopathy, mononuclear cell infiltration, and myocardial necrosis in the heart allografts. RT-PCR analysis showed that the allografts treated with CsA or KRP-203 alone showed no suppression of IL-10, IFN-gamma, and TNF-alpha mRNA expression, but when combined with a subtherapeutic dose of CsA it completely suppressed their mRNA expressions. Furthermore, the combination treatment reduced donor-specific antibody production. KRP-203 combined with a subtherapeutic dose of CsA synergistically prolonged rat heart allograft survival. The combination of CsA with KRP-203 may provide an option to prevent allograft rejection and reduce adverse effects.

Abstract: BACKGROUND: A novel immunomodulator, KRP-203, the molecular structure of which has some similarity to FTY720, has been developed for use in organ transplantation. The present study was designed to investigate the potency and safety of KRP-203 on allograft survival against both acute and chronic rejection in rat skin and heart transplantation. METHODS AND RESULTS: KRP-203 significantly prolonged skin or heart allograft survival of a minor histocompatibility complex (mHC)-disparate (LEW to F344) rat combination. Histopathological and immunohistochemical analysis at 100 days after mHC-disparate rat heart transplantation revealed that KRP-203 treatment significantly inhibited infiltration of inflammatory cells, including macrophages and T cells; expression of endothelin-1 and transforming growth factor-beta1; and IgG deposition and eventually attenuated neointimal formation and myocardial fibrosis. KRP-203 also prolonged heart allograft survival in a major histocompatibility complex (MHC)-incompatible (DA to LEW) rat combination, but the efficacy was not as significant. However, KRP-203 combined with a subtherapeutic dose of cyclosporin A synergistically prolonged the heart allograft survival. Flow cytometric analysis demonstrated that KRP-203 reduced the number of peripheral blood mononuclear cells (lymphocytes and monocytes) but not granulocytes and enhanced lymphocyte homing into peripheral lymph nodes. The influence of KRP-203 on heart rate changes in Hartley guinea pigs was examined. KRP-203 had less of a tendency to cause bradycardia than FTY720. CONCLUSIONS: These findings demonstrated that KRP-203 prolonged skin and heart allograft survival and significantly attenuated chronic rejection and bradycardia as an adverse effect. Therefore, KRP-203 offers considerable potential as a novel therapeutic immunosuppressant in patients with organ transplantation.

Abstract: Genetic modification is a promising therapeutic strategy for organ transplantation. In previous studies, we introduced a nonviral method of gene transfer to donor grafts using an organ-selective injection technique to up-regulate gene expression. Based on the attractive methodology of RNA interference for silencing a particular gene expression, we applied our catheter-based injection method to transfer small interference RNA (siRNA)-green fluorescence protein (GFP) into liver and limb grafts. We first quantified the interfering activity after the systemic delivery of siRNA in the liver of Alb-DsRed2 transgenic (Tg) rats using in vivo bioimaging system. Then, using GFP Tg Lewis rats as donors, transient down-regulation of the GFP expression was achieved both in liver- and limb-transplantation models by the preoperative rapid injection of siRNA. Genetic modification by siRNA may provide new therapeutic options for down-regulation of endogenous antigenicity.

Abstract: BACKGROUND: The usefulness of bone marrow cells in accelerating wound healing has not been evaluated despite increasing evidence that bone marrow contains mesenchymal stem cells that have multipotentiality to differentiate into various types of cells after they enter the microenvironment of a specific tissue (niche). OBJECTIVES: To determine the effects of bone marrow cells and occlusive dressings in promoting wound healing in rats. METHODS: We investigated by grafting, biopsy and immunohistochemistry whether various types of cells derived from green fluorescent protein (GFP)-transgenic rats would differentiate into wound component cells when administered topically on the wounds of rats. We also investigated whether topical application of bone marrow cells with an occlusive dressing would accelerate the healing of wounds with exposed bones, as measured by planimetry. RESULTS: GFP-labelled bone marrow cells contained multipotent stem cells that sufficiently differentiated into wound myofibroblasts presenting with alpha-smooth muscle actin in granulation tissue. Other types of cells, including myocytes, adipocytes, peripheral blood cells from buffy coat and dermal fibroblasts, did not express myofibroblast characteristics morphologically or immunohistochemically. Application of bone marrow cells and an occlusive dressing accelerated the repair of wounds with exposed bones, compared with an occlusive dressing only or with the topical administration of bone marrow cells plus a semidry to dry dressing. CONCLUSIONS: Our study indicates that bone marrow cells accelerate the healing of wounds at least in part through their differentiation into wound myofibroblasts. Thus, treatment of wounds with bone marrow cells and a supportive occlusive dressing is effective in promoting the formation of healthy granulation tissue and also for the preparation of an ideal wound bed.

Abstract: INTRODUCTION: Recently, human hand transplantation in Europe has shown that motor function may be recovered in some cases. However, little is known about cell trafficking involved the graft nerve. We have succeeded to use green fluorescent protein transgenic (GFP-Tg) rats with various cells strongly expressing GFP in a model a long-term survival of limb graft. In this model, we found retrograde migration of GFP-positive donor cells through the sclatic nerve anastomosis. It is well known that cellular components in the peripheral nerve graft especially Schwann cells, play an important role in the axonal regeneration promoted by nerve grafting. However, it was difficult to distinguish the cellular component of the nerve graft from recipient cells. The purpose of this study was to evaluate the migration of donor origin cells to the recipient's nerve and to examine the contribution of these cells in axonal regeneration using a simplified model of sciatic grafting. METHODS: Nerve defects were created in recipient rats, using three experimental combinations: group 1: wild-type rats from GFP Tg rats; group 2: GFP Tg rats from wild-type rats; group 3: wild-type rats from GFP Tg rats whose nerve grafts had been pretreated by freeze-thawing cycles (representing an acellular graft). The sciatic nerve specimens were examined under excitation light at 1, 2, and 3 weeks after transplantation. RESULTS: GFP-positive area expanded clearly beyond the anastomosis both proximally and distally in group 1 and infiltrated into the middle of the null graft in group 2. On the contrary, freeze-thawing grafts donated GFP Tg rats lost GFP expression completely. Columns of GFP-positive cells were formed in the degenerated graft migrated into the recipient's nerve both ante- and retrograde. The S100-positive GFP-positive cells were considered to be graft-origin Schwann cells. The regenerating axons were accompanied with these double-positive cells in the recipient nerve. In conclusion, we have visualized the contribution of graft cells to axonal regeneration beyond a peripheral nerve anastomosis.

Abstract: We have successfully specified essential sequences of the 5' upstream region for the stage- and tissue-specific expression of mouse Sry by using an in vitro Cre/loxP system. Sry/Cre plasmids carrying Sry 5' sequences of various sizes were transfected into the primary cultured cells from different tissues of CAG/loxP/CAT/loxP/LacZ transgenic fetuses on 11.5-day post coitus (dpc) or 13.5-dpc. Stage- and tissue-specific regulation of Sry expression was disrupted by the deletion of positions 7549-7660 (from -0.4 to -0.5 kb region). In vitro transcription assay also suggested that the region contains element(s) responsible for stage- and tissue-specific expression of mouse Sry. SRY promoter of Shiba goat (Capra hircus var Shiba), a native Japanese miniature goat, showed the tissue-specific activity in the cells from urogenital ridges of the male mouse, but not in the cells from female mice, indicating a possibly different mechanism among species in the regulation of Sry expression.

Abstract: BACKGROUND: The characteristics of adenovirus-mediated gene transfer into the kidney are not well examined. We studied the effects of contact time and temperature on adenovirus-mediated transgene expression in rat kidneys, using catheter-based in vivo gene transfer and a rat renal transplant model ex vivo. METHODS: An adenovirus vector containing the luciferase (Ad-Luc) or beta-galactosidase (Ad-LacZ) gene was introduced in vivo into the kidney via a renal artery catheter. Various contact times and temperatures were evaluated. Ex vivo, the renal graft was injected with Ad-Luc through the renal artery, chilled for 60 min and then transplanted. Luciferase expression was evaluated periodically by a non-invasive bioimaging system or histology. Cells expressing the LacZ gene were identified by immunoelectron microscopy. RESULTS: In in vivo gene transfer, successful transgene expression was achieved; however, its efficiency was independent of contact time or temperature. In ex vivo gene transfer, transgene expression in the renal graft peaked early and gradually decreased. Strong gene expression was observed in the recipients' livers. LacZ expression was detected in fibroblasts, parietal epithelial cells of Bowman's capsule, mesangial cells, podocytes and tubular cells. CONCLUSIONS: This study generated new information about in vivo and ex vivo gene transfer into the kidney, which would be useful for renal gene therapy.

Abstract: Although implantation of multipotent bone marrow-derived stem cells represents an attractive new cell therapy to repair damaged tissues, recent reports have raised serious concerns over the feasibility of using stem cells deriving from the bone marrow to promote cell transdifferentiation. We established transgenic (Tg) rats with reporter genes as specific molecular tags to examine the effect of bone marrow cells (BMCs) on transdifferentiation into tissues/organs. To monitor transdifferentiation events of locally transplanted BMCs into hepatocytes or capillary endothelial cells, a liver injury model and an ischemic hind-limb model were developed in rats. To test the ability of circulating bone marrow-derived cells to give rise to myocytes after skeletal muscle injury, we used a bone marrow cell transplantation model from Tg rats, which showed ubiquitous expression of beta-galactosidase (lacZ), into lethally irradiated non-Tg rats. Our results show that there was little transdifferentiation of BMCs into the targeted cells in these tissue injury models. However, in the ischemic hind-limb model, laser Doppler imaging and histologic analysis showed that both implantation of BMCs and treatment with microspheres incorporating basic fibroblast-like growth factor (bFGF), which enables the release of bFGF at the site of action over a period of time, effectively induced angiogenesis. In conclusion, rat BMCs with specific marker genes could be a useful tool for detecting transdifferentiation events in vivo.

Abstract: Little information currently exists on the repair of muscular tissue at the site of an amputation stump. This study examined the healing process of muscular tissue following composite limb transplantation using transgenic rat models. METHODS: Green fluorescent protein (GFP) transgenic rats were used to study the process of connection of donor muscle with the recipient. A DsRed2/GFP double-reporter transgenic rat and an NCre transgenic rat were used to study cell fusion. These rats have the unique characteristic of changing red fluorescence to green fluorescence by Cre/LoxP recombination when cell-to-cell fusion occurs between the two transgenic strains. Orthotopic hind limb transplantation was performed in two combinations: GFP transgenic rat to Wild Wistar rat and DsRed2/GFP transgenic rat to NCre transgenic rat. RESULTS: We observed extension of donor-derived GFP(+) myofibers into recipient site a few weeks after limb transplantation. A histologic study of the DsRed2/GFP transgenic rat to the NCre transgenic rat combination showed that red myofibers of the DsRed2/GFP rat were partly replaced by green myofibers as a result of Cre-mediated recombination. PCR analysis detected both the recombined transgene (330 bp) and the nonrecombined gene (1420 bp) in muscle around the junction. These findings indicate that the muscles sutured between the amputation stumps fused with each other and that donor-recipient hybrid cells were formed at the muscle junction following limb transplantation. CONCLUSIONS: This basic information shows muscle fusion between donor and recipient at the site of composite tissue transplant using newly established transgenic rats.

Abstract: BACKGROUND: We developed a nonviral gene transfer method using rapid injection of naked DNA targeting the liver and applied it in a rat model of liver transplantation. METHODS: Inbred Dark Agouti and Lewis rats were used. To test the efficacy and adverse effects of systemic or local (catheter-based) injection, different volumes of phosphate-buffered saline containing naked DNA encoding beta-galactosidase (lacZ) were injected. Luciferase expression was followed by non-invasive imaging, and a cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4Ig) protein was tested functionally by allogenic heart transplantation. Gene transfer was then tested in rat auxiliary liver transplantation (ALT) and orthotopic liver transplantation (OLT). The timing of gene transfer was evaluated in the auxiliary liver transplantation model, and OLT was performed using a liver graft to which luciferase or the CTLA4Ig gene was transferred 2 days before. RESULTS: LacZ was expressed extensively in a volume-dependent manner; however, a large volume often induced recipient death. After local delivery of CTLA4Ig cDNA to the liver, survival of Dark Agouti heart grafts lengthened with increased CTLA4Ig serum levels. Liver grafts injected with naked DNA at the time of donation did not survive, but livers grafted 2 days after gene transfer survived. Successful expression of luciferase and production of CTLA4Ig were finally confirmed in the rat that underwent OLT. CONCLUSIONS: We successfully applied a nonviral hydrodynamic gene transfer method to the rat liver and showed its potential in liver grafting. The high incidence of graft failure when this procedure is performed on the day of organ donation is a potential limitation that needs to be overcome in clinical application.

Abstract: Inhibitory antibody formation is the most serious complication of factor (F)VIII replacement therapy in hemophilia A patients. FVIII-deficient mice were used to study new approaches for induction of immune tolerance. Neither antiFVIII inhibitory antibodies nor antiFVIII IgGs were observed in 13 of 14 adult mice that received 0.05 U g(-1) body weight of human FVIII intravenously within 24 h after birth and repeated injections as adults. In contrast, high FVIII antibody titers (>50 Bethesda Units mL(-1)) developed in seven of 13 mice injected on day 3 postpartum and in all adult mice not treated neonatally. One of nine mice and three of 17 mice developed high-titer antiFVIII inhibitory antibody when they were treated initially with 2-fold (0.1 U g(-1) body weight) and 10-fold higher doses (0.5 U g(-1) body weight) FVIII on day 0, respectively. A human FVIII-specific T-cell proliferative response was absent in splenocytes from neonatally treated mice. The tolerance was FVIII specific because antitoxoid antibodies developed after immunization with tetanus toxoid. Splenocytes failed to proliferate or produce interferon (IFN)-gamma in response to FVIII stimulation, yet still secreted interleukin-2. A proliferative response was restored with exogenous IFN-gamma or interleukin-12, suggesting that lack of inhibitor to FVIII was due to IFN-gamma-dependent anergy. Thus, exposure on day 0 to physiological levels of FVIII antigen might be important for induction of immune tolerance. This immune tolerance model may provide a basis for new approaches to prevention of FVIII inhibitors during replacement therapy.

Abstract: BACKGROUND: Donor passenger leukocytes (DPLs) that migrate after organ transplantation stimulate the recipient immune system and normally cause rejection and graft vs. host reaction. However, DPLs also contribute to the unresponsiveness to the donor organ. The quantity and quality of these migrating cells are considered dependent on individual transplanted organs. We compared the DPLs of the liver, which might contain somatic stem cells, with those of intestinal grafts that have highly immunogenetic cells. To study DPLs over a long period, we used green fluorescent protein (GFP) transgenic (Tg) rats developed by us as donors. METHODS: We performed orthotopic liver transplantation (OLT) and small bowel transplantation (SBT) from GFP Tg rats to wild recipients. A short course of tacrolimus (0.64 mg/kg, intramuscularly) was used to prevent antigenicity of the GFP. The fate of the DPLs in the peripheral blood and the recipient bone marrow was monitored by flow cytometry. Using long-surviving recipients, the GFP(+) cells in the graft and various host immunologic organs were measured and characterized by immunohistochemical staining. RESULTS: In both groups, the numbers of the GFP(+) cells in the peripheral blood increased transiently and then gradually decreased to undetectable levels. While no GFP(+) cells were identified in the long-surviving-recipient bone marrow, there were a few GFP(+) cells in the graft liver, graft mesenteric lymph nodes and the recipient spleen. These cells showed major histocompatibility complex (MHC) class II antigen. There was no significant difference in the migration patterns of the GFP(+) cells in the OLT and SBT rats. CONCLUSIONS: In both the OLT and SBT groups, the DPLs migrated transiently in the recipient peripheral blood. A small numbers of MHC class II-positive DPLs were present at the graft site and in the host spleen, but not in the bone marrow. There were no significant differences in the migration patterns of the DPLs between the OLT and SBT rats over the long term.

Abstract: BACKGROUND: Multilineage chimerism is a promising strategy to induce donor-specific tolerance. Because the beneficial effect of splenic grafting on tolerance induction is well known, we studied long-term hematopoietic chimerism and the fate of donor-derived cells after allogenic pancreas/spleen transplantation. METHODS: Green fluorescent protein (GFP) transgenic (Tg) Wistar rats were donors and combined pancreas/spleen transplantation (PST) or pancreas transplantation (PT) alone was performed on recipient LEW rats. Graft survival was compared between these two groups and the fate of donor-derived GFP(+) cells was analyzed by flow cytometry. In this system, the donor-derived cells were clearly defined as having lymphocytic or granulocytic lineage by cell size. T-cell subsets of GFP(+) and GFP(-) cells in long graft-surviving rats were also characterized. RESULTS: The survival period of the grafted pancreas in PST rats was significantly longer than that of PT rats (P<0.001). Three of seven PST rats survived >250 days. The chimeric level of donor-derived GFP(+) cells in the recipient peripheral blood was markedly higher in PST rats. In rats with long-surviving grafts, overall peripheral blood chimerism was more than 5%, and both lymphocytes and granulocytes generated from the grafted spleen were stable. T-cell subsets in the recipient LEW rats varied according to the type of cells. CD4(+)CD8(+) subsets decreased in the GFP(+) cells and CD4(-)CD8(+) subsets increased in the GFP(-) (LEW) cells. CONCLUSION: We confirmed the combination effect of the grafted spleen on pancreatic graft survival. Donor lymphocytic and granulocytic lineages were generated in the recipients with long-surviving graft. It suggested that multilineage chimerism was often induced by the spleen graft and protected the pancreatic graft against rejection for a long period.

Abstract: BACKGROUND: The effectiveness and toxicity of many drugs depends on the dosing-time schedule, relative to the circadian rhythms of biochemical, physiological, and behavioural processes. Previous studies have found chronopharmacology of ketamine, which is a N-methyl-d-aspartate (NMDA) receptor antagonist. The in vivo contribution of the NMDA receptor epsilon1 subunit (NR2A) in this effect is unclear. METHODS: In the present study, daily variations in the hypnotic effect of ketamine were determined in wild-type mice and NMDA epsilon1 knockout (KO) mice. RESULTS: The effect of ketamine had a definite daily variation in wild-type mice. No significant difference in blood concentration was observed at different dosing times (10:00 and 22:00). In NMDA receptor epsilon1 KO mice, the hypnotic effect of ketamine was weaker than in wild-type mice and there was no dependence on the time of administration. Significant pharmacokinetic differences were not observed between wild-type and KO mice. CONCLUSIONS: The enhanced hypnotic effect in the active phase of the circadian cycle is likely a result of changes with the time of day in the susceptibility of the central nervous system to ketamine. Knockout of the NMDA receptor epsilon1 subunit gene markedly reduced the effect of ketamine, and eliminated the time-dependent sensitivity to ketamine.

Abstract: BACKGROUND/AIM: Gene transfer into the kidney has great potential as a novel therapeutic approach. However, an efficient method of gene transfer into the kidney has not been established. We explored the transduction efficiency of renal cells in vitro and in vivo using adeno-associated virus (AAV) serotype 1-5 vectors encoding the beta-galactosidase gene. METHODS: In the in vitro study, rat kidney epithelial cell line NRK52E cells were transfected with AAV serotype derived vectors. In the in vivo study, AAV serotype derived vectors were selectively injected into the kidney using a catheter-based gene delivery system in rats and mice mimicking the clinical procedure. The efficiency of gene expression was histologically evaluated on the basis of the beta-galactosidase expression. RESULTS: AAV serotype 1, 2, and 5 vectors transduced in rat kidney epithelial cell line NRK52E cells in vitro, whereas AAV serotype 3 or 4 vectors showed no transduction. In addition, the kidney-specific injection of AAV serotype 2 vectors successfully transduced in tubular epithelial cells, but not in glomerular, blood vessel, or interstitial cells in vivo, whereas the rest of the serotypes showed no transduction. CONCLUSION: Since kidney-specific gene delivery via the renal artery by catheterization is highly feasible in humans, these findings provide useful information for promising strategies in renal gene therapy.

Abstract: BACKGROUND: Recent studies have demonstrated that complete conversion from cyclosporine A (CsA) to mycophenolate mofetil (MMF) prolongs graft survival in patients undergoing clinical organ transplantation. We investigated the effects of conversion from CsA to MMF on recipient kidneys and transplant arteriosclerosis in a rat aortic allograft model as a high responder combination. METHODS: DA (MHC haplotype, RT1a) rat abdominal aortic grafts were orthotopically transplanted into Lewis (RT1l) rats. The recipients were divided into four oral treatment groups: (1) vehicle group, (2) CsA group (15 mg/kg/day), (3) CsA/MMF40 group (conversion from CsA 15 mg/kg/day to MMF 40 mg/kg/day on day 14), and (4) CsA/MMF20 group (conversion from CsA 15 mg/kg/day to MMF 20 mg/kg/day on day 14). On day 28 after transplantation, the rats were sacrificed and the hematoserological parameters were analyzed. The grafted aortas and recipient kidneys also were evaluated histologically and immunohistochemically. RESULTS: The CsA group developed serological renal dysfunction, arteriolar hyalinosis, and apoptosis in the recipient kidneys, whereas the CsA/MMF40 and CsA/MMF20 groups did not. In the vehicle group, we observed remarkable intimal hyperplasia and marked inflammatory cell infiltration including macrophages and T cells. In the CsA group, intimal hyperplasia was evident without infiltration of macrophages or T cells. In the CsA/MMF40 and CsA/MMF20 groups, intimal hyperplasia was abrogated, while adventitial infiltration of and adhesion to the endothelium by macrophages and T cells occurred. CONCLUSIONS: Conversion from CsA to MMF protected recipient kidneys and prevented transplant arteriosclerosis. However, insufficient immunosuppression by MMF might reactivate immune cells. This conversion therapy has preventive potential in transplant patients with CsA-associated nephrotoxicity and transplant arteriosclerosis.

Abstract: The rat has offered an important animal model in biomedical research including surgical procedure. However, advanced genetic manipulation has progressed less far in the rat than in the mouse. Here we report the Cre/LoxP transgenic rat system, demonstrating conditional chromosomal translocation both in the fertilization and adult stage, spatio-temporal gene controlling by catheter-based adenoviral gene transfer, and muscular fusion events in the limb transplant. Taking advantage of the larger body size of the rat than the mouse, this rat system provides a potential value to evaluate biomedical and therapeutic significance for gene therapy and regenerative medicine.

Abstract: Cultured rat keratinocyte sheets form hair follicles in combination with rat vibrissa dermal papillae when they are transplanted subcutaneously in syngeneic rats and athymic mice. In the present study, the histologic details of these induced follicles were analyzed by preparing cultured sheets mixed with normal rat keratinocytes and green fluorescent protein (GFP)-transgenic rat keratinocytes. Histologic examination demonstrated that some induced follicles maintained their size and morphology for at least 18 weeks, whereas others decreased in size and others totally differentiated into cornified structures between 3 and 6 weeks. The percentage of the grafts with GFP-positive cells decreased during the same period. This finding suggests that some GFP-positive cells were transient-amplifying cells that turned into terminally differentiated cells and were lost during this period. Some large follicles and some small follicles maintained their hair-producing ability and the proliferative activity in their hair matrix for 18 weeks. In addition, one 6-week-old follicle contained label-retaining cells in the outer root sheath. Seven of 25 follicles induced from chimera epithelium contained both GFP-positive cells and GFP-negative cells. These results suggest that stem cells are present in the induced follicle and the induced follicle consists of polyclonally derived cells. The presence of early anagen-like large follicles at week 6 and 9 and a telogen-like small follicle at week 18 also suggests that hair-growth cycle phases proceeded in the induced follicles. In conclusion, the follicles induced in the cultured keratinocyte sheets maintained hair-producing ability and proliferative activity for at least 18 weeks. This and the presence of label-retaining cells suggest that there are stem cells in the induced follicles, which seem to have a hair-growth cycle.

Abstract: BACKGROUND AND AIMS: Gastric intestinal metaplasia, which is mainly induced by Helicobacter pylori infection, is thought to be a precancerous lesion of gastric adenocarcinoma. Intestinal metaplastic mucosa expresses intestine specific homeobox genes, Cdx1 and Cdx2, in the human gastric mucosa. We and others have reported that ectopic expression of Cdx2 in the gastric epithelium generates intestinal metaplasia in the transgenic mouse model. METHODS: To clarify the differences in the roles of Cdx1 and Cdx2 in intestinal metaplasia, we generated transgenic mice expressing Cdx1 in the gastric mucosa and compared Cdx1 induced gastric mucosal morphological changes with Cdx2 induced intestinal metaplasia. RESULTS: The gastric mucosa in Cdx1 transgenic mice was completely replaced by intestinal metaplastic mucosa, consisting of all four intestinal epithelial cell types: absorptive enterocytes, goblet, enteroendocrine, and Paneth cells. Paneth cells, which were not recognised in Cdx2 transgenic mice, were in the upper portion of the intestinal metaplastic mucosa. Pseudopyloric gland metaplasia, which was induced in Cdx2 transgenic mice, was not recognised in Cdx1 transgenic mice. Proliferating cell nuclear antigen (PCNA) positive cells were diffusely scattered in Cdx1 induced intestinal metaplastic mucosa while PCNA positive cells in Cdx2 induced intestinal metaplastic mucosa were in the base of the metaplastic mucosa. Intestinal metaplastic mucosa of Cdx1 transgenic mouse stomach was significantly thicker than that of wild-type or Cdx2 transgenic mouse stomach. CONCLUSIONS: We have confirmed that Cdx1 induced gastric intestinal metaplasia but that it differed from Cdx2 induced intestinal metaplasia in differentiation, structure, and proliferation.

Abstract: BACKGROUND: Glomerular crescent formation is a prominent feature of aggressive forms of glomerulonephritis (GN) and is associated with a poor prognosis. We investigated whether the potent immunosuppressive agent mycophenolate mofetil (MMF) could prevent crescent formation in a model of anti-glomerular basement membrane (GBM) GN in the rat. METHODS: GN with glomerular crescents was induced by the injection of anti-GBM antibody to female Wistar-Kyoto (WKY/NCrj) rats. The experimental rats were divided into two groups: rats received vehicle (0.5% carboxymethylcerlose) or MMF (20 mg/kg/day) orally. Body weight was measured and the urine and blood samples were evaluated. The rats were sacrificed at day 14, and histological analysis was performed. The mRNA expression of cytokines and adhesion molecules in the kidney was analysed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Marked proteinuria, glomerular crescent formation and glomerulosclerosis were observed in this model, and these were significantly reduced by MMF treatment. Marked glomerular macrophage and T-cell infiltration was also observed, and MMF treatment significantly inhibited macrophage but not T-cell infiltration. RT-PCR and immunohistochemical analysis revealed that mRNA and protein expression of osteopontin was decreased by the treatment with MMF. In addition, MMF treatment in the early stages of GN could inhibit proteinuria, glomerular crescent formation and glomerulosclerosis. CONCLUSIONS: These findings suggest therapeutic potential for MMF in the inhibition of glomerular crescent formation in GN and provide new insights into the mechanism underlying the amelioration of crescentic GN by MMF treatment.

Abstract: BACKGROUND.: Transplant arteriosclerosis is one of the main features of chronic graft failure in organ transplantation. In this article, the authors investigate mechanisms of mycophenolate mofetil (MMF) on prevention of transplant arteriosclerosis in a rat aortic allograft model. METHODS: Orthotopic rat abdominal aortic transplantation was performed from Brown Norway (RT1) to Lewis (RT1) rats. The recipients were divided into three oral treatment groups: (1). vehicle; (2). MMF40 (40 mg/kg); and (3). MMF20 (20 mg/kg). The authors histologically and immunohistochemically evaluated neointima formation; infiltration of macrophages and T cells; and expression of endothelin (ET)-1, platelet-derived growth factor (PDGF)-B, PDGF receptor-beta (Rbeta), transforming growth factor (TGF) beta 1, and osteopontin (OPN). Using cultured rat vascular smooth muscle cells (VSMC), effects of mycophenolic acid (MPA) on ET-1-induced proliferation and ERK1/2 activation were also examined in vitro. RESULTS: In the vehicle group, marked neointima formation was observed, with massive macrophages and T-cell infiltration in neointima, media, and adventitia. Marked expression of ET-1, PDGF-B, PDGFR-beta, TGFbeta1, and OPN were also observed in neointima. In the MMF40 and MMF20 groups, neointima formation was halted, but macrophages and T cells were infiltrated in the adventitia and adhered to the endothelium. In the MMF40 group, medial infiltration by macrophages and T cells and intimal expression of ET-1, PDGF-B, PDGFR-beta, TGFbeta1, and OPN was inhibited compared with the vehicle and MMF20 groups. Furthermore, MPA inhibited ET-1-induced VSMC proliferation but failed to inhibit its ERK1/2 activation. CONCLUSIONS: MMF treatment might have preventive potential in transplant patients with chronic vasculopathy through inhibition of VSMC proliferation.

Abstract: BACKGROUND: Composite tissue allografts are unique because they provide the vascularized bone marrow with stroma, which is the supportive microenvironment. In this study, we investigated the beneficial effect of donor-derived bone marrow cells within the long-surviving recipient rats after limb transplantation. METHODS: Green fluorescent protein (GFP) transgenic rats developed for paramount cell marking were donors, and wild Wistar rats were recipients. Orthotopic hind-limb transplantation was performed using a microsurgical technique. Tacrolimus (1.0 mg/kg) was intramuscularly injected for 14 days postoperatively. The skin graft from GFP donor onto the GFP recipient was performed as a control. Flow cytometric analyses of recipient peripheral blood and bone marrow were carried out at 4 to 6 days, 18 to 21 days, 6 weeks, and 2, 4, 6, 9, and 12 months after transplantation. RESULTS: The rats that received tacrolimus therapy achieved prolonged composite graft acceptance more than 12 months, whereas GFP skin grafts were rejected at 47 days under the same immunosuppressive protocol. Numerous GFP lymphocytes and granulocytes were detected within the recipient bone marrow for the first 6 weeks post limb transplantation. These cells remained relatively stable for more than 12 months. CONCLUSIONS: The results showed that donor-derived hematopoietic stem cells engrafted in recipient bone marrow and differentiated to lymphocytes and granulocytes after limb transplantation. The vascularized bone marrow, transplanted as a part of the hind limb, could have contributed to mixed chimerism and worked as the bone-marrow source in the recipients.

Abstract: Sry expression is essential for initiating male sex differentiation, and the expression occurs only during a restricted period in the developing gonad. It is thought that Sry is part of a pathway of genes that regulate sex determination. Although the interactions of several genes with Sry expression have been suggested, the exact cascade of gene expression regulating Sry transcription is entirely obscure because there is no available cell line expressing Sry and reflecting an in vivo condition. The present study was carried out to investigate the cis-acting element of the mouse Sry that responds stage specifically to its expression, in part, using transgenic mice expressing GFP on the Y chromosome. Ten DNA fragments were generated by digesting the 5' upstream region (positions 5491-8039; 2,549 bp) of mouse Sry with appropriate restriction enzymes. In an electrophoretic mobility assay with these fragments, the region from position 5491 to position 5799 (309 bp) was identified as forming specific protein-DNA complexes with nuclear extracts from 11.5 days post coitus (dpc) gonads, but not from 12.5 and 13.5-dpc gonads. This region also formed specific protein-DNA complexes with the nuclear extracts from adult testicular germ cells that generate only a circular form from Sry. This stage-specific responsive region was narrowed down to positions 5559-5616 by DNase I footprinting analysis. The assay of DNase I hypersensitive (HS) using the nuclear lysates from the 11.5-dpc urogenital ridges demonstrated that the novel HS site was located in the proximity of position 5600. This region DNase I HS was also detected at the same position when the lysates from adult testicular germ cells were applied. The results indicate that the present HS site may be involved in the transcriptional regulation of the linear and/or circular molecule transcripts from mouse Sry gene.

Abstract:

Abstract: Transgenic (Tg) animals with reporter genes are useful models in which to study cell lineage and the process of differentiation into tissues. We developed the green fluorescent protein (GFP)-Tg rat, which is more suitable for transplantation and stem cell research because it is larger than mice. We found that marker gene expression was dependent on each organ and developmental stage. In this study we describe GFP expression in various tissues from embryonic, neonatal, and adult animals. GFP expression in brain, lung, liver, and islet tissues was restricted to early developmental stages, but it was continuously strong in the exocrine pancreas, kidney, and cardiac and skeletal muscles. The CAG promoter that was presumed to induce ubiquitous protein expression might be responsible for the differences in expression.

Abstract:

Abstract: Animals transgenic (Tg) for reporter genes would be useful to following a given cell lineage during differentiation and regeneration processes. Here, we established a beta-galactosidase (lacZ) Tg rat to use as a tool for regenerative research. Strong lacZ expression was observed in the skeletal muscles, myocardium, pancreas, and skin obtained from these lacZ-Tg rats, and moderate lacZ expression was observed in the liver, spleen, kidney, and cartilage. In contrast, brain, vessels, lung, adrenal gland, small intestine, blood leukocytes, bone marrow (BM) cells, and peripheral blood cells showed no lacZ expression. To test whether this lacZ-Tg rat could be used for regenerative research in myocardium, we induced myocardial injury after a lacZ-Tg BM transplant (BMT) into wild-type rats. The results show that lacZ-positive cardiomyocytes were found in the peri-infarct and uninjured myocardium in the BMT recipient rats. These findings suggest that lacZ-Tg rats are useful tool for regenerative research in the myocardium.

Abstract:

Abstract: The immune response is modulated by genetic adjuvants using plasmid vectors expressing cytokines. Skeletal muscle can express a foreign gene intramuscularly administered via a needle injection, and the potential of muscle as a target tissue for somatic gene therapy in treating cancer has been explored. In the present study, we investigated the efficacy of particle-mediated intramuscular transfection modified with a local anesthetic agent, bupivacaine, on luciferase and green fluorescent protein. The results indicate that these proteins are more efficiently expressed and persist longer in muscle modified in this way compared with the needle-injection method. Using an established rat sarcoma model, particle-mediated intramuscular gene-gun therapy with a combination of IL-12 and IL-18 cDNA was conducted. Growth of the distant sarcoma was significantly inhibited by particle-mediated intramuscular combination gene therapy, and the survival rate was also improved. Furthermore, the combination gene-gun therapy maintained significant levels of interferon-gamma and induced a high activity of tumor-specific cytotoxic T lymphocytes. These results suggest that the sustained local delivery of IL-12 and IL-18 cDNA using intramuscular gene-gun therapy modified with bupivacaine can induce long-term antitumor immunity, and can provide the great advantage of inhibiting the disseminated tumor.

Abstract: Suicide gene expression in specific tissue of transgenic animals has been used for cell-specific ablation. To examine the influence of hepatocyte removal, we produced the herpes simplex virus thymidine kinase (HSVtk) transgenic rat, whose gene was regulated by an albumin enhancer promoter. The liver presence of HSVtk was demonstrated in one line of the transgenic rats. We injected ganciclovir (GCV, 50mg/kg) into the rat on alternate days. After 28 days of GCV administration, liver tissues, and blood of the rats were collected. The histological investigation revealed infiltration of T cells, macrophages, granulocytes/neutrophils, and hepatocyte cell death. The biochemistry analysis demonstrated elevated levels of AST, ALT, and total bilirubin in transgenic rat. In conclusion, the transgenic rat with expressed albumin-specific HSVtk developed experimental hepatitis with administration of GCV, and will be a useful model to facilitate the evaluation of drug effects for clinical control of liver disease.

Abstract: When a threshold amount of temporary ischemic insult to induce focal infarction was given to the unilateral cerebral hemisphere of gerbils, a small focal infarct surrounded by a wide penumbra developed in the rostral portion of the cerebral cortex. During the first 5 hours following recirculation, whole astrocytic cell bodies and processes in the ischemic hemisphere were swollen, with an increase in the number of glycogen granules and in number and size of mitochondria. This swelling was an active reaction of astrocytes for neuronal protection, scavenging potassium, glutamate, and other neuronal metabolic products, and for generating fuels for neurons (cyto-reactive edema). This reactive astrocytic swelling continued in the penumbra, but some dead neurons were found disseminated among the surviving neurons. Whereas, at 12 approximately 48 hours, focal infarction developed in which all cell membranes lost their Gibbs-Donnan's equilibrium due to energetic failure of their membranous Na+/K+ ATPase. This is the cytotoxic edema (cyto-necrotic edema). In the infarct focus, when pericapillary astrocytic end-feet were damaged, the capillary BBB was broken; and thus vasogenic edema was superimposed on the cytotoxic edema.

Abstract: Recent studies suggested that conversion from cyclosporine A (CsA) to antimetabolic agents protects allograft vasculopathy. However, the mechanism of this beneficial effect by the conversion therapy is not fully understood. In the present study, we investigated the effects of conversion from CsA to antimetabolic agent mizoribine (MZR) on the formation of transplant arteriosclerosis in rat aortic allografts. Conversion from CsA to MZR significantly prevented intimal hyperplasia and perivascular inflammatory cell infiltration at 28 days after aortic transplantation. These findings suggest that conversion therapy from CsA to antimetabolic agents might have therapeutic potential in transplant patients with chronic vascular rejection.

Abstract: We attempted to cryopreserve spermatozoa from closed colonies (Jcl:SD and Jcl:Wistar), and inbred (BN/Crj, F3441 DuCrj, LEW/Crj, Long-Evans and WKY/NCrj), mutant (Zitter [WTC.ZI-zi] and Tremor [TRM]), transgenic (human A-transferase [A], and green fluorescent protein [GFP]) strains of rats. Rat epididymal spermatozoa suspended in cryopreservation solution (23% egg yolk, 8% lactose monohydrate, and 0.7% Equex Stm, pH 7.4, adjusted with 10% Tris [hydroxymethy] aminomethane) were frozen and stored at -196 degrees C. After thawing at 37 degrees C, the spermatozoa were instilled into the tip of each uterine horn of the recipients. A total of five recipient females for each strain were inseminated with cryopreserved spermatozoa, and normal live offspring of all strains (Jcl:SD: 11, Jcl:Wistar: 13, BN/Crj: 9, F344/DuCrj: 28, LEW/Crj: 4, Long-Evans: 6, WKY/NCrj: 8, TRM: 24, WTC.ZI-zi: 27, A: 30 and GFP: 20) were obtained.

Abstract: BACKGROUND: Direct DNA vaccination of liver allografts before transplantation may provide an effective strategy for inducing protective immunity to infection and malignancy. METHODS: In this study, the authors examined the feasibility of gene gun-mediated vaccination of liver grafts. Using plasmids expressing luciferase and green fluorescent proteins, their expression was tested in a graft liver. RESULTS: Protein expression was observed in the graft liver and significantly enhanced in hepatectomized rats. A short course of tacrolimus (FK506) also evoked the expression of these proteins. Effects of primary immunization to the liver on the humoral response were then tested using an expression plasmid encoding hepatitis B virus surface (HBs) antigen and were compared to that of skin immunization alone. The results showed that local immunization to the liver strongly induced antibody formation. Furthermore, the combination of an immunized partial liver graft with tacrolimus significantly enhanced antibody production against HBs antigen. CONCLUSIONS: A DNA vaccine to the liver may be one strategy for preventing infectious disease associated with liver transplantation under tacrolimus treatment.

Abstract: We developed a novel protocol for rat orthotopic liver transplantation (OLT), using a suture method to establish hepatic artery flow. After determining that early inferior vena cava (IVC) unclamping maintained better circulation compared with the portal vein (PV) using porto-systemic shunted recipients, we developed a rat OLT model with total vascular reconstruction using a suture method. After connecting the suprahepatic IVC, the infrahepatic IVC was anastomosed, using a running suture method. IVC circulation was established immediately. The PV was anastomosed without intestinal congestion, using porto-systemic shunted recipients. The aortic conduit, including the donor celiac and hepatic artery, was anastomosed to the recipient abdominal aorta end-to-side. Eight of 11 OLT cases (72.7%) survived indefinitely. Biliary connection was achieved using a one-stent method. Three cases died 3-5 days postoperatively. Hepatic angiography showed good patency. The graft liver was histologically normal in long-surviving rats.

Abstract: Because serum albumin is specifically produced by mature hepatocytes, detection system of albumin producing cells could be a valuable tool to visualize liver regeneration or development. We have developed here an albumin enhancer/promoter-driven Alb-DsRed2 Tg rat that expresses DsRed2, having liver-specific reporter gene expression of red fluorescent protein. To study the transdifferentiation of bone marrow cells (BMCs) into albumin producing cells, BMCs from the Alb-DsRed2 Tg rat were injected into rats having acute liver damage caused by 2-acetylaminofluorene plus carbon tetrachloride and chronic liver damage by repeated administration of CCl(4). DsRed2-positive cells were generated in the recipient liver after BMC injection. The number of transdifferentiated DsRed2-positive cells in chronic liver injury model was increased comparing with that in acute injury model. We propose that the Alb-DsRed2 Tg rat is well suited to studying in vivo liver regeneration.

Abstract: BACKGROUND: It is important to develop a nontoxic gene transfer method for immunosuppressed patients. In this study, the authors applied a nonviral gene transfer method using rapid injection of naked DNA into the graft limb in rats. METHODS: Naked DNA (beta-galactosidase, luciferase, or green fluorescent protein expressing plasmid) was used to test an intravascular gene transfer approach in various conditions on the Lewis rat limb. Then, in a rat limb transplantation model, these marker genes were administered preoperatively (day -2) or perioperatively (day 0) to the graft limb by the authors' "venous protocol." The expression level of luciferase was observed over a long period using a noninvasive living image acquisition IVIS system. RESULTS: Effective intravascular delivery of gene to the rat limb was achieved by a rapid bolus injection of naked DNA through the femoral caudal epigastric vein. Using this procedure, the limb graft with the marker gene perioperatively in place was safely transplanted. After limb transplantation, sustained marker gene expression was observed for more than 2 months. CONCLUSIONS: This is the first report showing that the method of rapid injection of naked DNA into the limb can be applied to gene modification for organ transplantation.

Abstract: Although a short course of methotrexate (MTX) has a potential immunoregulatory effect on clinical allograft rejection, little data are available about the drug, and the mechanism of hyporeactivity after withdrawal is still unknown. In previous studies, we achieved permanent graft acceptance through administration of a short course of high-dose MTX during heterotopic ratheart transplantation (HHT) in a combination of DA (MHC haplotype; RT1(a)) to PVG/c (RT1(c)) rats. A 3-week course of MTX (0.25 mg/kg/day) was administered intraperitoneally to the PVG/c recipients of a DA heart graft, and 11 of 16 rats survived longer than 300 days after HHT. The splenic lymphocytes obtained from one recipient showed high reactivity against donor type splenic lymphocytes, but others did not. All serum samples from recipients showed immunosuppressive activity. The serum had anti-donor antibodies. These results showed that tolerance induced by short-course MTX was maintained by a serologic factor believed to be anti-idiotypic antibodies.

Abstract: Gastric intestinal metaplasia occurs as a pathological condition in the gastric mucosa. To clarify how an intestine-specific homeobox gene, Cdx2, affects the morphogenesis of gastric mucosa, we generated transgenic mice expressing Cdx2 in parietal cells. Until Day 18 after birth, the number of parietal cells inthegastric mucosa of transgenic mice was the same as for their normal littermates. However, at Day 19, we detected several glands in which parietal cells disappeared and the proliferating zone moved from the isthmus to the base of the glands. Thereafter, parietal cells decreased gradually and disappeared at Day 37. All of the gastric mucosal cells, except for enterochromaffin-like (ECL) cells, were completely replaced by intestinal metaplasia, consisting of goblet cells, enteroendocrine cells, and absorptive cells expressing alkaline phosphatase. Pseudopyloric gland metaplasia was also formed. The transgenic mouse is a very useful model for clarifying physiological differentiation of gastric and intestinal cell lineages and analyzing the molecular events from intestinal metaplasia to adenocarcinoma.

Abstract: We cloned a rat ABO homologue and established human A- and B-transferase transgenic rats. A DNA fragment corresponding to exon 7 of the human ABO gene was amplified from Wistar rat genomic DNA and sequenced. Using the amplified fragments as a probe for Southern blotting, multiple hybridized bands appeared on both EcoRI- and BamHI-digested genomes of seven rat strains, which showed variations in the band numbers among the strains. Four cDNAs were cloned from a Wistar rat, three of which showed A-transferase activity and one of which showed B-transferase activity. These activities were dependent on the equivalent residues at 266 and 268 of human ABO transferase. Wild Wistar rats expressed A-antigen in salivary gland, intestine, and urinary bladder tissue, but B-antigen was not stained in any organs studied, whereas a transcript from the ABO homologue with B-transferase activity was ubiquitous. Human A-transferase and B-transferase were transferred into Wistar rats. A-transgenic rats expressed A-antigen in ectopic tissue of the brain plexus, type II lung epithelium, pancreas, and epidermis. B-antigen in the B-transgenic rat was expressed in the same organs as A-transgenic rats. These results may shed light on the function and evolution of the ABO gene in primates.

Abstract: Auxiliary partial liver transplantation (APLT) is beneficial for fulminant liver failure when there is potential for recovery of the diseased liver. However, the impact of host hepatectomy on regeneration of the grafted liver is unclear. In this study, we modified a previous rat model of auxiliary whole liver transplantation without portal vein reconstruction, and studied the effect of host hepatectomy on regeneration of the cut liver graft. Thirty percent of the liver was heterotopically transplanted, to connect the recipient's left renal artery and vein with the graft's aortic cuff of the hepatic artery and inferior vena cava, respectively, using a cuff technique; 30% of the recipient liver then was cut. The control group was left intact. The liver grafts were weighed preoperatively and 2 weeks postoperatively. This procedure prevented congestion of the graft liver and achieved a high success rate, even when performed by a surgeon who was relatively inexperienced with the technique. The weight of the grafted liver in the host hepatectomized group significantly increased (P < 0.05) compared with that of the control group. We developed an experimental model of APLT and reviewed the literature on rat heterotopic liver transplantation, and compared the surgical techniques.

Abstract: BACKGROUND: Anastrozole, a new aromatase inhibitor, has been used to treat postmenopausal metastatic breast cancer, and several clinical trials of adjuvant treatment using this agent are ongoing. However, the effects of anastrozole on lipid metabolism are unknown. The aim of this study was to evaluate the effect of anastrozole on lipid metabolism, especially lipoprotein lipase (LPL) activity, compared with tamoxifen in rats. METHODS: Ovariectomized female rats were divided into six groups: C, controls; T, tamoxifen treatment; A, anastrozole treatment; CAT, combined anastrozole/tamoxifen treatment; NAT, no treatment after tamoxifen; and AAT, anastrozole treatment after tamoxifen. The agents were orally administered for 3 weeks. Serum total cholesterol, triglycerides, and LPL activity in postheparin plasma were measured at the end of the experiment. RESULTS: Serum cholesterol levels were significantly lower in the T and CAT groups than in controls (P < 0.001). Serum triglyceride levels were significantly higher in the T group than in the other groups (P < 0.001). LPL activity was significantly lower in T and AAT groups (P < 0.01). There was no significant difference in any parameters in group A. CONCLUSIONS: Anastrozole does not affect lipid metabolism including LPL activity. There was little effect on lipid profiles during combination treatment or following treatment with tamoxifen. In a clinical setting, therefore, anastrozole might be safe for patients with abnormal triglyceride profiles during tamoxifen treatment.

Abstract: There have been few studies on the regulatory elements of the Sry gene, mainly because no Sry-expressing cell lines have yet been established. This paper describes a useful tool for investigating the regulation and upstream region of Sry by means of the in vitro Cre/loxP system. Using plasmids containing the 9.9 kb mouse genomic Sry previously shown to induce testis development in XX transgenic mice, we constructed a Sry/Cre fusion gene plasmid in which Cre expression is controlled by the 5' and 3' untranslated regions of mouse Sry. To distinguish between male and female gonads of 11.5 days post-coitus (d.p.c.) fetuses, double transgenic fetuses carrying both the CAG (cytomegalovirus enhancer and beta-actin promoter)/loxP/lacZ transgene on the autosome and the green fluorescent protein transgene ubiquitously expressed on the Y chromosome were produced by crossing between two transgenic mouse lines. When Sry/Cre plasmids were transfected into the cells that had been prepared from the gonads, brains and livers of double transgenic fetuses, only a small number of X-gal-stained cells were detected among the primary cultured cells from male and female gonads, and none were detected among the cells from the other tissues. The X-gal-positive cells were negative for alkaline phosphatase, indicating that these cells were somatic cells expressing Sry. The Sry/Cre plasmids with a 0.4 kb upstream region of Sry yielded a large number of X-gal-positive cells in the cells from gonads, including various tissues of 11.5 d.p.c. fetuses, indicating the loss of the tissue-specific expression of Sry. The Sry/Cre with a 1.4 kb upstream region maintained tissue-specific activity of Sry. The results indicate that the present in vitro Cre/loxP system using transgenic mice is a simple and useful system for investigating the regulatory element of sex determination-related genes, including Sry.

Abstract: The purpose of this study is to evaluate green fluorescent protein (GFP) transgenic rats for use as a tool for organ transplantation research. The GFP gene construct was designed to express ubiquitously. By flow cytometry, the cells obtained from the bone marrow, spleen, and peripheral blood of the GFP transgenic rats consisted of 77, 91, and 75% GFP-positive cells, respectively. To examine cell migration of GFP-positive cells after organ transplantation, pancreas graft with or without spleen transplantation, heart graft with or without lung transplantation, auxiliary liver and small bowel transplantation were also performed from GFP transgenic rat to LEW (RT1(1)) rats under a 2-week course of 0.64 mg/kg tacrolimus administration. GFP-positive donor cells were detected in the fully allogenic LEW rats after organ transplantation. These results showed that GFP transgenic rat is a useful tool for organ transplantation research such as cell migration study after organ transplantation without donor cell staining.

Abstract: Here we report isolation of an androgen-regulated novel gene from an androgen-dependent mouse mammary Shionogi carcinoma SC-3 cell line. Using a polymerase chain reaction-based subtraction method and Northern blotting analysis, we isolated four androgen-inducible genes from SC-3 cells. Nucleotide sequencings identified three of the genes as cyclin D1, beta-catenin, and fatty acid synthase, respectively, but the fourth, a gene tentatively named as Arg1 (androgen-regulated gene 1), remained undefined. The cloned 2.0-kb sized Arg1 cDNA encoded 414 amino acid sequences. The deduced amino acid sequences, sharing about 30% homology with cathepsin family members at a protein level, had relatively conserved residues around the three proteinase active sites reported earlier. In Northern blotting, Arg1 mRNA was found in kidney, heart, lung, and to a lesser degree, in spleen and liver. Its transcripts were also detected in male reproductive organs on RT-PCR. In addition, its expression levels in prostate were markedly reduced after castration. Unexpectedly, Arg1-expressing COS1 cells showed no significant proteinase activity to various synthesized substrates under neutral or acidic conditions in this study. This might have been due to the replacement of the cysteinyl active site for proteinase to serine residue in the Arg1 amino acid sequences. Given that Arg1 also contains a lipocaline signature known as a binding motif for small hydrophobic molecules at the center of its amino acid sequences, Arg1 is a lipocalin family gene regulated by androgens in prostate and Shionogi carcinoma cells.

Abstract: Here, we report characterization of growth factors secreted from androgen-independent mouse mammary Shionogi carcinoma cells. Previous isolation of fibroblast growth factor 8 (FGF8) from androgen-dependent Shionogi carcinoma SC-3 cells prompted us to characterize growth factors secreted from the androgen-independent cells. After several purification procedures, mitogens for NIH3T3 cells from the androgen-independent cells were identified as activins on the grounds that activin betaA- and betaB-subunits are detected in the active fractions by Western blotting and that the growth-promoting effects by the active fractions are specifically inhibited in the presence of follistatin. In addition, exogenous activins, but not inhibin, stimulated the growth of NIH3T3 cells in a dose-dependent manner. Interestingly, transcripts of activin betaB-subunit were predominantly found in the androgen-independent cells while its betaA-subunit was universally expressed in both androgen-dependent and -independent Shionogi carcinoma cells. In concordant with this in vitro finding, transcripts of activin betaB-subunit were enhanced in murine prostates after castration. Therefore, expression of activin betaB-subunit, but not its betaA-subunit, is likely to be related with androgen-depleted cell conditions in prostates, and possibly in androgen-related cancers.

Abstract: Using a previously reported technique for bile collection, we studied the pharmacokinetics of levofloxacin (LVFX) and grepafloxacin (GPFX) in normal rats and in animals with renal failure. Continuous bile drainage was performed using normal and renal-failure Wistar rats. Oral GPFX or LVFX (40 mg/Kg) was administered. The drug concentrations in plasma, urine, and bile were determined by high-performance liquid chromatography. The area under the blood concentration-time curve (AUC) in each renal-failure rat was calculated. There were no significant differences in GPFX concentrations in the serum, urine, and bile between the renal-failure and normal rats, but the LVFX level in the urine of the renal-failure group was statistically significantly lower than in the normal group. The AUC of GPFX had an opposite correlation with the degree of renal failure, but that of LVFX was correlated.

Abstract: Malignant melanoma involving the oral cavity has a highly metastatic potential. Curative surgery is required to resect extensive oral tissues and often results in dysfunction as well as a severe cosmetic deformity in patients with the disease. An alternative technology for the local and sustained delivery of cytokines for cancer immunotherapy has been shown to induce tumor regression, suppression of metastasis, and development of systemic antitumor immunity. However, local immunization of the oral cavity has not previously been studied. In this study, we examined the efficacy of particle-mediated oral gene transfer on luciferase and green fluorescent protein production. The results showed that these proteins were more significantly expressed in oral mucosa than the skin, stomach, liver, and muscle. Using an established oral melanoma model in hamsters, particle-mediated oral gene gun therapy with interleukin (IL) 12 cDNA was then conducted. The results indicated that direct bombardment of mouse IL-12 cDNA suppressed tumor formation and improved the survival rate. The skin tumor model created by inoculation of melanoma cells was also significantly inhibited by the oral bombardment of IL-12 cDNA coupled with an irradiated melanoma vaccine administrated to the oral mucosa, compared to treatment with a percutaneous vaccine. IL-12 gene gun therapy, combined with an oral mucosal vaccine, induced interferon-gamma mRNA expression in the host spleen for a long time. These results suggest that immunization of oral mucosa may induce systemic antitumor immunity more efficiently than immunization of the skin and that oral mucosa may be one of the most suitable tissues for cancer gene therapy by means of particle-mediated gene transfer.

Abstract: Muscle-specific isoform of the mitochondrial ATP synthase gamma subunit (F(1)gamma) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492-8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998) Biochem. Biophys. Res. Commun. 251, 603-608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F(1)gamma gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F(1)gamma wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F(1)gamma Pu-del and F(1)gamma Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F(1)gamma Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F(1)gamma Pu-rich minigene mice revealed that the F(1)gamma Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F(1)gamma pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.

Abstract: The effect of 3-nitropropionic acid (3-NP), a selective inhibitor of succinic dehydrogenase, preconditioning on postischemic neurological deterioration and infarction was examined in gerbils after transient ischemia. The animals were pretreated with 1-80mg/kg of 3-NP 1 day before ischemia induced by two 10-min occlusions of the left common carotid artery. Four milligrams per kilogram 3-NP pretreatment significantly ameliorated postischemic neurological deterioration (stroke index at 7 days postischemia, 1.4+/-1.5 vs. 7.4+/-5.4 in 4 mg/kg-pretreated vs. non-pretreated animals: mean+/-SD) and reduced infarct volume (24+/-4.8 vs. 43+/-12 mm(3)). One and 20 mg/kg 3-NP induced milder neuroprotection, and 80 mg/kg 3-NP aggravated postischemic stroke symptoms and infarction. Thus, appropriate doze of 3-NP preconditioning is effective in ameliorating the postischemic neurological deterioration and reducing infarct volume.

Abstract: We have examined the regional differences in the evolution of energy failure in experimental focal cerebral ischemia. In gerbil brain subjected to repeated unilateral common carotid artery occlusion, the tissue ATP content, pH and succinic dehydrogenase activity decreased at different rates after the circulation had been restored in various cerebral regions. Light microscopical infarction became apparent at different rates following the impairment of the energy metabolism in these regions. In brain cortex with selective neuronal necrosis, only minor alterations in energy metabolism were detectable over a 7-day period following the restoration of the circulation. The present data show that the rate of energy failure is significantly different in various cerebral regions after repeated periods of cerebral ischemia in the gerbil. A slowly evolving impairment of the cerebral energy metabolism after circulation of the brain has been restored appears to be indispensable for the delayed formation of infarction after transient cerebral ischemia.

Abstract: Tamoxifen, a nonsteroidal antiestrogenic antitumor agent, has weak estrogen-like effects on lipid metabolism, however, the mechanism remains unknown. We previously reported that tamoxifen decreases the activity of lipoprotein lipase (LPL), a key enzyme in triglyceride metabolism, in patients with breast cancer. This study evaluated the effect of tamoxifen on LPL activity in vitro and in vivo. In experiment 1, total cholesterol, triglyceride, adipose tissue weight, and LPL activity of post-heparin plasma were measured in ovariectomized female rats with and without tamoxifen treatment. In experiment 2, purified very-low-density lipoprotein (VLDL) and purified LPL were incubated with and without tamoxifen or estrogen, and the triglycerides in VLDL were measured using an enzymatic method. In experiment 1, total cholesterol and adipose tissue weight decreased significantly in tamoxifen-treated rats (p < 0.001 and p < 0.01, respectively). Triglyceride measurements were not significantly different between the two groups, however, the LPL activity was lower in tamoxifen-treated rats (p < 0.005). In experiment 2, triglycerides in VLDL were significantly higher after VLDL and LPL were incubated with tamoxifen and estrogen (p < 0.005). We concluded that tamoxifen inhibits the hydrolytic activity of LPL in vivo and in vitro. This mechanism may explain the elevated serum triglyceride levels in some patients treated with tamoxifen.

Abstract: The development of infarction and/or selective neuronal death in the brain after transient cerebral ischemia depends on the severity of the ischemic episode. After transient cerebral ischemia of the threshold level for the induction of infarction, both changes evolve slowly in various postischemic regions. We examined the relationship of disturbances of energy metabolism to infarction and selective neuronal death in various regions of the postischemic brain subjected to two 10-min occlusions of the unilateral common carotid artery. Our results indicated that in various cerebral regions that developed infarction, the tissue ATP content, in parallel with the succinic dehydrogenase activity, fell to their lowest levels at different times over a 4-day period after circulation had been restored (earliest to latest: dorsolateral thalamus > dorsolateral caudate > chiasmal level cortex > hippocampal CA3 sector > hippocampal CA sector). In the cortex at the infundibular level, disseminated selective neuronal death developed over a 7-day period following restoration of circulation; it was accompanied by only a slight alteration in energy metabolism. The present results indicate that regional differences existed in the rate of energy impairment and evolving infarction in the postischemic gerbil brain. Energy impairment, in association with mitochondrial enzymatic dysfunction, seems to be indispensable for the delayed manifestation of cerebral infarction but not for disseminated selective neuronal death.

Abstract: Renal dysfunction induced by a single injection of cisplatin depends on the timing of the dose. However, the effects of repeated administration of cisplatin on time-dependent toxicity have not been evaluated despite the fact that in clinical practice high doses are repeatedly injected at intervals or low doses are administered daily. We studied chrononephrotoxicity in rats after weekly or daily cisplatin injections. Weekly high doses (5 mg kg(-1)) or daily low doses (1.2 mg kg(-1)) of cisplatin were injected at four time points (3, 9, 15 and 21 h after the light was turned on (HALO)) for 3 weeks. Changes in body weight after weekly cisplatin administration were independent of the timing of the doses. In the group that received daily cisplatin, the loss in body weight in the 3 HALO group was smaller than in animals receiving injections at 15 and 21 HALO (P < 0.05 and 0.001, respectively). The blood urea nitrogen and serum creatinine levels in the control rats showed a significant circadian change (peak at 15 HALO and trough at 9 HALO), but these parameters were markedly elevated in both trials and their circadian variations were disturbed. In conclusion, cisplatin-induced nephrotoxicity was the lowest at 3HALO compared with other time points of both dose regimens.

Abstract: Colocalization of mitochondria is the first step of intermitochondrial interaction or fusion in a cell. Here, we showed colocalization between exogenous mitochondria and endogenous ones or between exogenous proteoliposomes and endogenous mitochondria in mouse fertilized eggs by confocal laser microscopy. Isolated mitochondria from mouse liver and proteoliposomes containing mitochondrial membrane were directly labeled with red fluorescent aliphatic marker, PKH26, which is incorporated into lipid membrane, and then were microinjected into fertilized mouse eggs. Exogenous mitochondria appeared to be almost colocalized with endogenous mitochondria at the 4- and 8-cell stages, when mitochondria were stained with Rhodamine 123 (green fluorescent marker). On the contrary, when liposomes consisted of soy bean phospholipid were microinjected into the eggs as a control, their localization was different from that of endogenous mitochondria. Next, the submitochondrial particles and proteoliposomes were microinjected. Both the proteoliposomes and the submitochondrial particles appeared to colocalize with endogenous mitochondria at the 4-cell stage. These results suggest the existence of a factor that makes liposomes colocalize with mitochondria. Such a proteoliposome would be useful for the development of mitochondrial gene transfer techniques.

Abstract: We investigated in the gerbil model whether the therapeutic effect of a novel Na+/H+ exchange inhibitor SM-20220 on ischemic brain edema could be enhanced by improving the decreased intracellular pH with an alkalizing agent, tris (hydroxymethyl) aminomethane (THAM). The left carotid artery of the animals was occluded twice for 10 min at a 5 hr interval. Ischemia-positive animals were selected and classified into the SM-20220- (0.5 mg/kg, i.p.) THAM- (2.0 ml/kg, i.v., 0.3M-THAM), combination of SM-20220 (0.5 mg/kg, i.p.) and THAM (2.0 ml/kg, i.v.), and vehicle- (0.9% saline, i.p.) treatment groups. Each agent was administered at 0, 6, 12 and 36 hr after recirculation following the 2nd episode of ischemia. The brain water, sodium and potassium contents were measured at 12, 24, and 48 hr after recirculation. The water content of the ischemic hemisphere 12 hr after recirculation was significantly lower in the combination-treated group (79.02%; P < 0.05) than in either the SM-20220- (79.28%) or THAM-treated group (79.32%). At 24 hr after recirculation the water content was significantly lower in the combination-treated group (79.83%, P < 0.05) than in the vehicle group (80.95%). At 48 hr after recirculation there were no significant differences in the water content between the vehicle group and any of the other treatment groups. The changes in brain water (delta H2O) and sodium plus potassium (delta Na + delta K) content in the ischemic hemisphere showed a significant correlation in each group. The combined treatment with the novel Na+/H+ exchange inhibitor SM-20220 and THAM is more effective on ischemic brain edema than treatment with a single agent. The results of this study indicate that improvement of intra- and extracellular acidosis by THAM infusion enhanced the activity of the NHE inhibitor SM-20220.

Abstract: This study examined the temporal profile of brain edema in the cerebral cortex associated with selective neuronal death or focal infarction after repeated ischemia at an intensity of ischemic insult just under and above the threshold level to induce infarction. The left carotid artery of adult gerbils was twice occluded for 10 min each time with a 5-hr interval between the blockages. In this model, focal infarction developed in coronal sections examined at the chiasmatic level (face A), whereas only selective neuronal death without infarction was found in the coronal section observed at the infundibular level (face B). In each animal, Evans blue (2%) was intravenously injected 1 hr prior to sacrifice as an indicator of blood-brain barrier (BBB) disruption. Brain edema was assessed by gravimetry in samples taken from both faces at 15 min, 5 hr, 12 hr, 24 hr, and 48 hr after the 2nd 10-min ischemia. Evans blue extravasated only in face A, corresponding to focal infarction at 24 and 48 hr after the 2nd 10-min ischemia. The specific gravities of the ischemic cortex of both faces decreased significantly from control at 15 min (P < 0.05) and had recovered by 5 hr after ischemia. By 12 hr, the specific gravities of both faces had again decreased significantly from the control values (P < 0.05), but did not differ significantly from each other. At 24 and 48 hr, the specific gravities of both faces were significantly lower than the control values (P < 0.01), and the specific gravity of face A was markedly lower than that of face B (P < 0.01). We concluded that in face B, where only selective neuronal death without infarction occurred only cytotoxic edema develops, whereas in face A, where infarction progresses, vasogenic edema develops in addition to cytotoxic edema.

Abstract: A study was carried out of the distribution and density of the neurons remaining in the gerbil cerebral cortex following two 10-min periods of ischemia at either 3-, 5- or 48-h intervals. As the interval between the periods of ischemia increased, the ischemic injury was reduced from severe to milder infarction, and further from more to less intense disseminated selective neuronal necrosis. This model is suitable for studying the mechanisms of transition from selective neuronal necrosis to infarction at the threshold level of infarction.

Abstract: Astrocytic swelling after ischemic insult has been considered a sign of parturbed cell viability. Investigations using cultured astrocytes and C6 glioma cells have revealed that viable astrocytes swell, spatially buffering various metabolites which are increased by the metabolic turmoil following ischemic insults. In the present study, we have studied the temporal profile of ultrastructural changes of astrocytes in the cerebral cortex associated with progressive selective neuronal, death where infarction is not induced. We occluded the left carotid artery of the Mongolian gerbil twice for 10 minutes at a 5 hr interval. In this model, following reperfusion, selective neuronal death progresses in the coronal section cut at the infundibular level. The whole brains of the sham operated control and postischemic animals were fixed by transcardiac perfusion of glutaraldehyde fixatives, at 15 min, 5 and 12 hr after the 2nd 10 min ischemia. Ultrathin sections including the 3rd and 5th cortical layers were prepared from the cut surface at the level of infundibulum. Mild swelling of astrocytic processes and perivascular end-feet was observed in the 15 min group. Glycogen granules were not prominent. In the 5 hr group, we found a few necrotic neurons disseminated in the cortex. All astrocytic cell processes were swollen with increased number of glycogen granules, especially marked in the perivascular end-feet. In the 12 hr group, necrotic neurons increased in number, astrocytic swelling was more extensive, and glycogen granules were evident in astrocytes. No cellular destruction was observed. We conclude: 1. Swelling progresses in astrocytes which however still remain viable and this process is associated with selective progression of neuronal death. 2. Glycogen granules increase in the swollen yet viable astrocytic cell processes.

Abstract: To study morphological changes in the cortex that follow repeated ischemia, one, two, and three 7-min unilateral occlusions of the carotid artery at 6-h intervals, and three, four, and five 7-min similar occlusions at 12-h intervals were produced in gerbils. Animals with one and two 7-min occlusions at 6-h intervals showed selective neuronal necrosis in the cortex; those with three 7-min occlusions at 6-h intervals showed focal infarction in the third layer of the cortex. Animals with three 7-min occlusions at 12-h intervals showed selective neuronal necrosis; those with four 7-min occlusions at 12-h intervals showed focal infarction in the third layer. In animals with five 7-min occlusions at 12 h intervals, infarction affecting all layers of the cortex was seen. Results of the present study indicate that cortical infarction occurred when a brief ischemic insult that does not cause any visible morphological damage in cortical neurons was inflicted repeatedly, and that development of infarction in the cortex following repeated episodes of ischemia depended on both the number of insults and the time intervals between them. This finding suggests that there is a threshold of infarction in repeated ischemia. In our model, various stages of ischemic brain injury could be achieved more easily than in transient ischemia by altering the number of insults or the intervals between them. This model is suitable for studying the pathophysiology on transition from ischemic neuronal necrosis to infarction.

Abstract: BACKGROUND AND PURPOSE: We evaluated the effects of long-term administration of high-colloid oncotic pressure on ischemic brain edema in Mongolian gerbils. METHODS: Animals that exhibited stroke after 35 minutes of unilateral forebrain ischemia were used. The gerbils were divided into albumin- (1 g/kg body wt, 25% albumin; n = 30) and saline-injected (4 mL/kg; n = 30) groups. Both agents were administered intravenously every 12 hours starting immediately after the recirculation. Plasma colloid oncotic pressure, serum sodium and potassium concentrations, and brain water, sodium, and potassium content were measured 24, 48, and 72 hours after recirculation. RESULTS: Plasma colloid oncotic pressure at 24, 48, and 72 hours after recirculation was significantly higher in the albumin- (26.1 +/- 2.3 mm Hg) than in the saline-treated group (18.5 +/- 1.9 mm Hg; P < .01), and brain water content of the ischemic hemisphere was significantly lower in the albumin group (79.5%, 80.2%, and 80.5%, respectively) than in the saline group (80.9%, 81.6%, and 82.1%, respectively; P < .05) at all three time points. Brain sodium content at 24 hours was significantly lower in the albumin than in the saline group (P < .05), while brain potassium content at 24 and 48 hours was significantly higher in the albumin than in the saline group (P < .05). The changes in brain water and sodium plus potassium content, which were calculated from differences between the ischemic and nonischemic hemispheres, showed a significant correlation in both groups (P < .01), but there was no significant difference between the linear regression lines for both groups. CONCLUSIONS: Long-term high-colloid oncotic pressure was effective in treating ischemic brain edema, probably acting by diminishing the bulk flow through the disrupted blood-brain barrier and ameliorating the vasogenic edema.

Abstract: To develop an experimental model which enables quantitative analysis of chronic neuronal loss in the cerebral cortex, repeated ischemic insult was performed using unilateral carotid artery occlusion in Mongolian gerbils. The effect of the time interval between the repeated ischemic insult on the survival rate of the animals and the amount of cortical neuronal loss were examined. The time course of the cortical neuronal damage in repeated ischemic insult was also studied. We repeated the occlusion four times; i.e., one 10-min and three 7-min occlusions (total 31 min of ischemia). The number of animals surviving for 3 weeks after the last ischemic insult was minimum (15.4%) for animals undergoing occlusions at 1-h intervals and maximum (100%) at 24- and 48-h intervals. The number of ischemic neuronal deaths was also dependent upon the time interval, and it was so pronounced as to allow analysis at intervals of 12 hr or 24 hr in the absence of infarction in the cortex. The number of neuronal deaths could not be determined for animals with occlusion at 1-h intervals due to the production of a large infarction, with which the 3-week survival rate was minimum. The temporal profile of cortical neuronal loss in the repeated ischemic insult at 24-h intervals indicated that the number of cortical neurons significantly decreased until 7 days after the start of the ischemic procedure. This model is useful for clarifying the pathophysiology of chronically developing ischemic neuronal death.

Abstract: Transient forebrain ischemia was produced by occluding the common carotid arteries, either unilaterally or bilaterally, in Mongolian gerbils under halothane anesthesia. After 20-min ischemia, the cranial temperature measured in the temporal muscle decreased compared to the preischemic level, the decrease being larger after the bilateral than after the unilateral occlusion. After recirculation, cranial temperature recovered promptly to the preischemic level in the unilateral group, while it was elevated to above the preischemic level in the bilateral group. The rectal temperature also decreased with a similar time course. During 30-min ischemia, the blood pressure of both groups increased to above the preischemic level, the increase being larger in the bilateral group than in the unilateral group. After recirculation, blood pressure of the unilateral group recovered promptly to the preischemic level, while that of the bilateral group decreased to below the preischemic level. When forebrain ischemia was produced immediately after cessation of halothane inhalation, blood pH, PaO2 or PaCO2 did not change significantly from the control level. However, these values showed larger variation in the bilateral group than in the unilateral group. Unilateral occlusion in preselected gerbils provided a good model of transient brain ischemia, giving rise to uniform experimental results.

Abstract: The presence of a basic fibroblast growth factor-like immunoreactive substance was demonstrated in the nuclei of germ cells at stages from spermatocyte to spermatid in adult rat testis by using immunohistochemistry with an antibody raised against a synthetic peptide corresponding to residues 1-10 of bovine basic fibroblast growth factor [1-146]. The fluorescence was very weak in the nuclei and cytoplasm of spermatogonia, Sertoli cells, and most of the interstitial compartments, except for capillary endothelial cells. This is the first study to demonstrate the presence of basic fibroblast growth factor-like immunoreactive material in the nuclei of haploid cells in vivo.

Abstract: The inheritance of a new lethal mutant rate showing congenital osteochondrodysplasia with systemic subcutaneous edema (ocd for the gene symbol) was examined by crossings between the carriers and between F1s derived from the carrier x Long-Evans crossing. The ratio of the affected to phenotypically normal was 276:89 in pups derived from the crossings between the carriers and 83:17 in those at the LE-F2 generation derived from the proven carriers of the LE-F1 animals, thus showing that the ratio is 3:1. The female:male ratio was 47:42 in the affected and 133:143 in the phenotypically normal pups, showing the sex ratio is 1:1, irrespective of the affected or phenotypically normal. The ratio of affected:carrier:noncarrier was 26:58:27 in littermates derived from the crossings between the carriers, showing the ratio is 1:2:1. The results fitted well to the hypothesis that the inheritance is a single autosomal recessive trait. Independence of the gene from linkage group I was also revealed, because the ratio of colored to albino was 14:3 and 65:18 in affected and phenotypically normal pups at the LE-F2 generation, respectively, which fitted to the ratio of 3:1.

Abstract: To determine the etiology of male hypogonadism in a newly found mutant rat (hgn/hgn, with a single autosomal recessive trait), concentrations of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were measured, and the responsiveness of the urogenital organs, hypothalamus, and pituitary gland to testosterone (1 mg/kg s.c. for 7 days), FSH (0.3 AU/kg s.c. for 7 days), human chorionic gonadotropin (hCG) (40 IU/kg s.c. for 7 days), and luteinizing hormone-releasing hormone (LHRH) (0.5 or 5.0 micrograms/kg s.c. for 7 days) were tested. Treatment with testosterone only increased the weights of all of the accessory sex organs, whereas treatment with FSH, hCG, or LHRH did not. Levels of serum FSH and LH were extremely higher and testosterone was lower in hgn/hgn males than in normal males. Serum FSH and LH decreased to levels found in intact animals after treatment with testosterone, suggesting that hypothalamic responsiveness to exogenous testosterone is present in the hgn/hgn males. Thus, the status of the hgn/hgn males was indicated to be due to primary Leydig cell dysfunction.

Abstract: A new rat mutant showing severe male hypogonadism (hgn for the gene symbol) was found and isolated from the hereditary hydronephrosis rat strain originating from the Wistar-Imamichi rat. The affected rats have very tiny testes that weigh 26 mg in the adult, and contain small numbers of seminiferous tubules with degenerated Sertoli cells. It is difficult to identify the gonocyte in the seminiferous tubules in the mutant testis. Very small male accessory sex organs are all present in the mutant. The number of chromosomes is 42 (40A + XY). The structure of each chromosome is normal, showing that the mutant has male sex genes. Thus, it was considered that this status is not due to either lack of the H-Y antigen or Muellerian inhibiting factor or expression of the Tfm. By analyzing the pedigree of the matings producing the mutant, it was concluded that the status was expressed only in the male, but inherited with a single autosomal recessive trait with existence of the phenotypically normal fertile female recessive homozygote. A possible deficiency of certain known or unknown testicular growth or differentiating factor(s) in the mutant is suggested.