Yusei Kariya

Generation of donor hematolymphoid cells after rat-limb composite grafting.

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.

The Incidence of Venous Thromboembolism Before and After Total Knee Arthroplasty Using 16-Row Multidetector Computed Tomography

We performed a prospective study to determine the incidence of venous thromboembolism (VTE) using 16-row multidetector computed tomography (MDCT). The study included 71 patients who underwent total knee arthroplasty between September 2004 and March 2009. Multidetector computed tomography was performed 4 days before and after surgery. No patient had any presurgical symptoms of VTE. Presurgical and postsurgical incidences of pulmonary thromboembolism plus deep vein thrombosis were 0% and 13%, respectively; pulmonary thromboembolism alone, 1% and 3%, respectively; and deep vein thrombosis alone, 8% and 34%, respectively. Because asymptomatic VTE was noted in 9% of patients before surgery and 51% after surgery, we conclude that performing MDCT before and after total knee arthroplasty may be useful to clarify the incidence of VTE and to develop appropriate strategies for treatment and prevention.

Long-lasting gene expression by particle-mediated intramuscular transfection modified with bupivacaine: combinatorial gene therapy with IL-12 and IL-18 cDNA against rat sarcoma at a distant site.

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-γ 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.

Defective Sorting to Secretory Vesicles in Trans-Golgi Network Is Partly Responsible for Protein C Deficiency. Molecular Mechanisms of Impaired Secretion of Abnormal Protein C R169W, R352W, and G376D

Abstract— Three thrombophilic patients with protein C (PC) deficiency were found to have independent mutations in the PC gene. These mutations resulted in single amino acid substitutions of R169W, R352W, and G376D in the affected PC molecules. These abnormal PC molecules were expressed in CHO-K1 cells in the presence or absence of vitamin K, and their synthesis, posttranslational modification, and secretion were studied. PC G376D was not secreted from the cells and was gradually degraded inside the cells. There was partial secretion of PC R169W and PC R352W, but most of these molecules were not secreted but were degraded intracellularly. On the basis of pulse-chase, immunofluorescence, and endo-&bgr;-N-acetylglucosaminidase H digestion experiments, the majority of wild-type PC molecules localize not in the Golgi apparatus but in the rough endoplasmic reticulum inside the cells. This suggests that wild-type PC molecules are secreted immediately after &ggr;-carboxylation and modification at the Golgi apparatus. In contrast, the mutant PC molecules were retained inside the cells even after modification of oligosaccharides at the trans-Golgi apparatus, which was probably due to impaired conformation of the abnormal molecules. Data suggest that these abnormal PC molecules were not sorted to secretory vesicles in the trans-Golgi network because of conformational defects in addition to the transport defect from the rough endoplasmic reticulum to the Golgi apparatus and were degraded inside the cells, thereby resulting in a PC deficiency in the affected patients.

Predictive blood coagulation markers for early diagnosis of venous thromboembolism after total knee joint replacement

Pulmonary embolism development may be prevented if asymptomatic venous thromboembolism (VTE) can be predicted and treated preoperatively or soon after total knee arthroplasty (TKA). The purpose of this study was to evaluate whether asymptomatic VTE can be predicted by blood coagulation markers preoperatively or early after TKA. This prospective single-centre study enrolled 68 patients (6 men, 62 women; mean age: 71 years) who underwent TKA between September 2004 and August 2009. Sixteen-row multidetector computed tomography was performed 4 days before and after surgery for diagnosis of asymptomatic VTE. Blood samples were taken to measure the plasma levels of soluble fibrin monomer complex (SFMC), D-dimer and cross-linked fibrin degradation products by leukocyte elastase (e-XDP) at 4 days preoperatively, and at 1 hour, 1 day and 4 days postoperatively. The preoperative SFMC, D-dimer and e-XDP levels did not differ significantly between the thrombus (n=36) and no-thrombus (n=32) groups. D-dimer and e-XDP levels showed the most significant increases at days 4 and 1, respectively, after surgery in the thrombus group. With cut-off points of 7.5 μg/ml for D-dimer and 8.2 U/ml for e-XDP, the sensitivities were 75% and 75%, and the specificities were 63% and 59%, respectively. By multiple logistic regression analysis, D-dimer at day 4 and e-XDP at day 1 postoperatively were independent markers for early diagnosis of VTE (odds ratio=1.61 and 1.19, P=0.01 and 0.04, respectively). The postoperative occurrence of new asymptomatic VTE may be predicted by D-dimer at day 4 and e-XDP at day 1 after TKA.

Difficulty of achieving long-term graft survival of MHC-disparate composite graft using CTLA4IG.

Because of recent advances in immunosuppressive drugs and microsurgical techniques, human hand transplantation has been attempted. However, the composite tissue has the strongest antigenicities in the body organs, and then requires heavy immunosuppressant therapy (1, 2). As a strategy for overcoming this aspect, blockade of the co-stimulatory pathway such as B7-CD28 or CD40-CD154 may reduce the need for chronic use of immunosuppressive agents, as shown experimentally (3, 4). We are interested in a recent study by Iwasaki et al. (5). These investigators showed experimentally the immunosuppressive potential of cytotoxic T-lymphocyte antigen-4 immunoglobulin (CTLA4Ig) against the allograft rejection of rat composite tissue. Survival times of composite allograft in a high responder combination of ACI (major histocompatibility complex haplotype; RT1) to LEW (RT1) were significantly prolonged in the rats that received a single dose of CTLA4Ig on day 2 after transplantation (mean SD, 20.5 1.0 days) compared with that of a control with human immunoglobulin G (mean SD, 8.4 0.9 days) (5). We also confirmed the effect of CTLA4Ig on composite tissue allograft in the similar high responder combination of DA (RT1) to LEW using adenovirus-mediated CTLA4Ig gene transfer. However, the grafts were rejected at days 28 and 36 after limb transplantation (unpublished data). The further study of composite tissue allograft transplantation to induce permanent acceptance without chronic use of immunosuppressants should be the goal (6). Here, we tested the combination effect of tacrolimus plus CTLA4Ig on this composite tissue allograft. Nonviral gene transfer to the graft was attempted because of a great advantage for compromised patients with chronic immunosuppression. In the separated issue, we have developed a novel method for enhancing protein expression in the rat muscle by using a nonviral, particle-mediated gene gun (7). Using this procedure, we attempted to induce the CTLA4Ig gene into the muscle and skin of the donor limb. Male adult DA and LEW rats were used as donor and recipient, respectively. Tacrolimus (0.64 mg/kg) was injected intramuscularly for 14 days postoperatively. The composite allograft received combination therapy of tacrolimus plus the CTLA4Ig gene and showed a significant prolongation of graft survival (32.5 2.1 days) compared with that of controls (5.3 1.0 days, P 0.001) or with tacrolimus treatment alone (26.2 1.1 days, P 0.001). A synergistic effect of tacrolimus with CTLA4Ig was observed, but we could not achieve a longsurviving composite tissue allograft. Finally, we would like to ask Iwasaki et al. (5) what the serum CTLA4Ig level in their model was. Our result showed that local gene therapy of CTLA4Ig using a gene gun could permit the prolonged survival of composite allograft, whereas concentrations of CTLA4Ig in these rats was low. We considered that the localized expression of the CTLA4Ig gene in the donor limb might provide a benefit for controlling the local immune response. However, we also felt insufficiency of costimulatory blockade for tolerance induction of major histocompatibility complex-disparate composite graft.

COMPOSITE TISSUE TRANSPLANTATION IN RATS : FUSION OF DONOR MUSCLE TO THE RECIPIENT SITE

UNLABELLED 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.