Takashi Ajiki

Gene gun-mediated oral mucosal transfer of interleukin 12 cDNA coupled with an irradiated melanoma vaccine in a hamster model : successful treatment of oral melanoma and distant skin lesion

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-γ 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. Cancer Gene Therapy (2001) 8, 705–712

Bioimaging assessment and effect of skin wound healing using bone-marrow-derived mesenchymal stromal cells with the artificial dermis in diabetic rats

We investigate the relationship between the fate and healing effect of transplanted mesenchymal stromal cells (MSCs) in a rat diabetic skin wound model. Rats are treated with streptozotocin to induce diabetic conditions. A full-thickness skin defect is surgically made on the head of diabetic rats, and covered with an artificial dermis impregnated with either bone marrow cells (BMCs) or bone-marrow-derived MSCs from firefly luciferase (luc) transgenic (Tg) rats. Wound healing is evaluated using planimetry and immunohistochemistry, and the fate of transplanted MSCs is determined using in-vivo luminescent imaging. The diabetic wound treated with MSCs-impregnated artificial dermis is significantly smaller than that treated with artificial dermis alone at 1 week postoperation. Photons of luc+ MSCs are detected at the transplanted site during healing (3 weeks), whereas those of luc+ MSCs are depleted only after 1 week postimplantation. Immunohistochemistry at the healing site treated with MSCs demonstrates that CD31+ vessels increase with expression of vascular endothelial growth factor, suggesting that MSCs accelerate angiogenesis. These findings suggest that transplanted MSCs could be retained at wound sites during the healing process in a diabetic rat model, and subsequently promote wound healing through angiogenesis.

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.

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.

A novel gene therapy to the graft organ by a rapid injection of naked DNA I: long-lasting gene expression in a rat model of limb transplantation.

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 (&bgr;-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.

Early exercise in spinal cord injured rats induces allodynia through TrkB signaling

Rehabilitation is important for the functional recovery of patients with spinal cord injury. However, neurological events associated with rehabilitation remain unclear. Herein, we investigated neuronal regeneration and exercise following spinal cord injury, and found that assisted stepping exercise of spinal cord injured rats in the inflammatory phase causes allodynia. Sprague-Dawley rats with thoracic spinal cord contusion injury were subjected to assisted stepping exercise 7 days following injury. Exercise promoted microscopic recovery of corticospinal tract neurons, but the paw withdrawal threshold decreased and C-fibers had aberrantly sprouted, suggesting a potential cause of the allodynia. Tropomyosin-related kinase B (TrkB) receptor for brain-derived neurotrophic factor (BDNF) was expressed on aberrantly sprouted C-fibers. Blocking of BDNF-TrkB signaling markedly suppressed aberrant sprouting and decreased the paw withdrawal threshold. Thus, early rehabilitation for spinal cord injury may cause allodynia with aberrant sprouting of C-fibers through BDNF-TrkB signaling.

DNA immunization of the grafted liver by particle-mediated gene gun1

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.

Effect of Artificial Nerve Conduit Vascularization on Peripheral Nerve in a Necrotic Bed

Background: Several types of artificial nerve conduit have been used for bridging peripheral nerve gaps as an alternative to autologous nerves. However, their efficacy in repairing nerve injuries accompanied by surrounding tissue damage remains unclear. We fabricated a novel nerve conduit vascularized by superficial inferior epigastric (SIE) vessels and evaluated whether it could promote axonal regeneration in a necrotic bed. Methods: A 15-mm nerve conduit was implanted beneath the SIE vessels in the groin of a rat to supply it with blood vessels 2 weeks before nerve reconstruction. We removed a 13-mm segment of the sciatic nerve and then pressed a heated iron against the dorsal thigh muscle to produce a burn. The defects were immediately repaired with an autograft (n = 10), nerve conduit graft (n = 8), or vascularized nerve conduit graft (n = 8). Recovery of motor function was examined for 18 weeks after surgery. The regenerated nerves were electrophysiologically and histologically evaluated. Results: The vascularity of the nerve conduit implanted beneath the SIE vessels was confirmed histologically 2 weeks after implantation. Between 14 and 18 weeks after surgery, motor function of the vascularized conduit group was significantly better than that of the nonvascularized conduit group. Electrophysiological and histological evaluations revealed that although the improvement did not reach the level of reinnervation achieved by an autograft, the vascularized nerve conduit improved axonal regeneration more than did the conduit alone. Conclusion: Vascularization of artificial nerve conduits accelerated peripheral nerve regeneration, but further research is required to improve the quality of nerve regeneration.

Muscle Is a Target for Preservation in a Rat Limb Replantation Model

Background: Ischemia exceeding 6 hours makes clinical limb replantation difficult and places the patient at risk of functional deficit or limb loss. We investigated the preservation of muscle function and morphology with solutions in rat hindlimb in vivo and in vitro. Methods: Quadriceps femoris muscles from luciferase transgenic rats were preserved for 24 hours at 4°C in extracellular-type trehalose containing Kyoto (ETK), University of Wisconsin (UW), or lactated Ringer’s (LR) solution (control). Muscle luminescence was measured with a bioimaging system. Amputated limbs of Lewis rats preserved with ETK, UW, or LR for 6 or 24 hours at 4°C were transplanted orthotopically. At week 8, terminal latency and amplitude were measured in the tibialis anterior muscle. The muscles were also analyzed histologically. Results: Isolated muscles preserved in ETK or UW had significantly higher luminescence than did muscles immersed in LR (P < 0.05). In the 6-hour-preserved limb transplantation model, although the 3 groups had almost the same terminal latency, electrical amplitude was significantly lower in the LR group. Histologically, muscles preserved with LR showed the most atrophic changes. In the 24-hour-preserved model, the survival rate of the LR group was 37.5% in contrast to 80% in the ETK and UW groups. Electrical signals were not detected in the LR group owing to severe muscle atrophy and fibrosis. The ETK and UW groups showed good muscle function electrophysiologically. Conclusions: Preservation solutions can protect muscle function and morphology in ischemia–reperfusion limbs and improve recipient survival rates after transplantation of long-term-preserved limbs.

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.