Tatsuya Chikaraishi

Therapeutic Effects of MicroRNA-582-5p and -3p on the Inhibition of Bladder Cancer Progression

Many reports have indicated that the abnormal expression of microRNAs (miRNAs) is associated with the progression of disease and have identified miRNAs as attractive targets for therapeutic intervention. However, the bifunctional mechanisms of miRNA guide and passenger strands in RNA interference (RNAi) therapy have not yet been clarified. Here, we show that miRNA (miR)-582-5p and -3p, which are strongly decreased in high-grade bladder cancer clinical samples, regulate tumor progression in vitro and in vivo. Significantly, the overexpression of miR-582-5p or -3p reduced the proliferation and invasion of UM-UC-3 human bladder cancer cells. Furthermore, transurethral injections of synthetic miR-582 molecule suppressed tumor growth and metastasis in an animal model of bladder cancer. Most interestingly, our study revealed that both strands of miR-582-5p and -3p suppressed the expression of the same set of target genes such as protein geranylgeranyltransferase type I beta subunit (PGGT1B), leucine-rich repeat kinase 2 (LRRK2) and DIX domain containing 1 (DIXDC1). Knockdown of these genes using small interfering RNA (siRNA) resulted in the inhibition of cell growth and invasiveness of UM-UC-3. These findings uncover the unique regulatory pathway involving tumor suppression by both strands of a single miRNA that is a potential therapeutic target in the treatment of invasive bladder cancer.

Suppression of Tregs by anti-glucocorticoid induced TNF receptor antibody enhances the antitumor immunity of interferon-α gene therapy for pancreatic cancer

We have reported that interferon (IFN)‐α can attack cancer cells by multiple antitumor mechanisms including the induction of direct cancer cell death and the enhancement of an immune response in several pancreatic cancer models. However, an immunotolerant microenvironment in the tumors is often responsible for the failure of the cancer immunotherapy. Here we examined whether the suppression of regulatory T cells (Tregs) within tumors can enhance an antitumor immunity induced by an intratumoral IFN‐α gene transfer. First we showed that an intraperitoneal administration of an agonistic anti‐glucocorticoid induced TNF receptor (GITR) monoclonal antibody (mAb), which is reported to suppress the function of Tregs, significantly inhibited subcutaneous tumor growth in a murine pancreatic cancer model. The anti‐GITR mAb was then combined with the intratumoral injection of the IFN‐α‐adenovirus vector. The treatment with the antibody synergistically augmented the antitumor effect of IFN‐α gene therapy not only in the vector‐injected tumors but also in the vector‐uninjected tumors. Immunostaining showed that the anti‐GITR mAb decreased Foxp3+ cells infiltrating in the tumors, while the intratumoral IFN‐α gene transfer increased CD4+ and CD8+ T cells in the tumors. Therefore, the combination therapy strongly inclined the immune balance of the tumor microenvironment in an antitumor direction, leading to a marked systemic antitumor effect. The CCR5 expression on Tregs was downregulated in the antibody‐treated mice, which may explain the decrease of tumor‐infiltrating Tregs. The combination of Treg‐suppression by GITR mAb and the tumor immunity induction by IFN‐α gene therapy could be a promising therapeutic strategy for pancreatic cancer.

ABO Blood Type Incompatible Kidney Transplantation Without Splenectomy Prepared With Plasma Exchange and Rituximab

We have designed a protocol for ABO-incompatible kidney transplantations based on preoperative plasmapheresis with a tacrolimus/mycophenolate mofetil/methylprednisolone/basiliximab protocol using low-dose rituximab (200 mg/body) instead of splenectomy to prevent antibody-mediated acute rejection. Eight patients successfully received transplants with this protocol. The titers of anti-A and -B antibodies as well as the number of CD20(+) cells were readily maintained at a low level posttransplantation. There were no side effects. All patients have renal transplant function with a follow-up of 1-34 months.

Distant metastasis from benign solitary fibrous tumor of the kidney.

Solitary fibrous tumor (SFT) rarely occurs in the kidneys, and only one reported case of renal SFT has shown distant metastasis. We report the second case of renal SFT exhibiting distant metastasis. A 48-year-old man was referred to our hospital because of a right renal mass. An abdominal CT scan detected a large renal tumor, which was suspected to be a renal cell carcinoma. Right radical nephrectomy was performed, and the tumor was found to measure 28 × 18 × 10 cm. The pathological diagnosis was benign solitary fibrous tumor of the kidney. Eight years after the operation, lung and liver metastases developed. Pulmonary segmentectomy and partial hepatectomy were performed. The pathological diagnoses of these resected tissue specimens were compatible with benign SFT.

Induction of Macrophage-Like Immunosuppressive Cells from Mouse ES Cells That Contribute to Prolong Allogeneic Graft Survival

Recent progress in regenerative medicine has enabled the utilization of pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) as a donor resource for transplantation. However, immune suppression is still needed when the donor-recipient combination is allogeneic. Protection of ESCs-derived grafts from host immune response might be achieved thought the utilization of immunosuppressive cells generated from ESCs. In the present study, we show that a certain fraction of immunosuppressive cells can be generated from ESCs and help to suppress immune response against allogeneic grafts. ESCs-derived suppressor cells (ES-SCs) resembled macrophages in terms of cell surface molecule and gene expressions. Furthermore, gene expression analysis including microarray showed that ES-SCs have M1/M2 hybrid phenotype with high expression of genes correlated to immunosuppression of T cell response. Indeed, ES-SCs were effective to block allogeneic T cell proliferation in a nitric oxide-dependent manner, and prolonged the survival of ESCs-derived embryoid bodies or cardiomyocytes grafts transplanted into mouse kidney capsule. Thus, we consider the potential use of these ESCs-derived macrophage-like immunosuppressive cells as cellular therapies to promote long-term graft survival in future therapies.

Syngeneic Hematopoietic Stem Cell Transplantation Enhances the Antitumor Immunity of Intratumoral Type I Interferon Gene Transfer for Sarcoma

Sarcoma at advanced stages remains a clinically challenging disease. Interferons (IFNs) can target cancer cells by multiple antitumor activities, including the induction of cancer cell death and enhancement of immune response. However, the development of an effective cancer immunotherapy is often difficult, because cancer generates an immunotolerant microenvironment against the host immune system. An autologous hematopoietic stem cell transplantation (HSCT) is expected to reconstitute a fresh immune system, and expand tumor-specific T cells through the process of homeostatic proliferation. Here we examined whether a combination of autologous HSCT and IFNs could induce an effective tumor-specific immune response against sarcoma. First, we found that a type I IFN gene transfer significantly suppressed the cell growth of various sarcoma cell lines, and that IFN-β gene transfer was more effective in inducing cell death than was IFN-α in sarcoma cells. Then, to examine the antitumor effect in vivo, human sarcoma cells were inoculated in immune-deficient mice, and a lipofection of an IFN-β-expressing plasmid was found to suppress the growth of subcutaneous tumors significantly. Finally, the IFN gene transfer was combined with syngeneic HSCT in murine osteosarcoma models. Intratumoral IFN-β gene transfer markedly suppressed the growth of vector-injected tumors and inhibited formation of spontaneous lung and liver metastases in syngeneic HSCT mice, and an infiltration of many immune cells was recognized in metastatic tumors of the treated mice. The treated mice showed no significant adverse events. A combination of intratumoral IFN gene transfer with autologous HSCT could be a promising therapeutic strategy for patients with sarcoma.

Irreversible immunoexpression of matrix metalloproteinase-9 in proximal tubular epithelium of renal allografts with acute rejection.

Kitajima K, Koike J, Nozawa S, Yoshiike M, Takagi M, Chikaraishi T. Irreversible immunoexpression of matrix metalloproteinase‐9 in proximal tubular epithelium of renal allografts with acute rejection.
Clin Transplant 2011: 25: E336–E344.

Safety of Monitoring Viral and Liver Function Markers in Patients With Prior Resolved Hepatitis B Infection After Kidney Transplantation.

BACKGROUND Hepatitis B virus (HBV) infection is a risk factor of mortality in kidney transplant recipients. However, information on the risk of HBV reactivation in kidney recipients with prior resolved HBV infection is limited. This study aimed to evaluate the safety of simply monitoring viral and liver markers in living donor kidney transplantation (LDKT) recipients with prior resolved HBV infection. METHODS We retrospectively examined the clinical records of LDKT recipients. Changes in the levels of alanine aminotransferase, aspartate aminotransferase, hepatitis B surface antigen (HBs Ag), surface antibody, core antibody, and HBV-DNA after transplantation were evaluated, and the occurrence of de novo HBV-related hepatitis and allograft function were monitored. RESULTS Of 61 consecutive LDKT patients, seven had prior resolved HBV infection. Four patients underwent ABO-compatible LDKT, whereas two underwent ABO-incompatible LDKT. The median age was 64 years (range, 61-69 years), and two patients were women. The causes of end-stage kidney disease were diabetic nephropathy, hypertensive nephrosclerosis, and chronic glomerulonephritis. Five patients were referred to hepatologists. The history of HBV vaccination was not confirmed in all patients. Prophylaxis with entecavir was administered to two patients with ABO-incompatible LDKT before transplantation. All patients tested negative for HBs Ag and HBV-DNA throughout observation, and none developed de novo HBV-related hepatitis or graft loss. CONCLUSIONS Patients with HBV infection without HBV DNA positivity are eligible for kidney transplants without antiviral therapy, even those on rituximab therapy. Monitoring viral and liver markers combined with hepatologist consultations may ensure safe follow-up in LDKT recipients with prior resolved HBV infection.

Synchronous bilateral urothelial cancer in a kidney recipient

Synchronous bilateral urothelial cancer is very rare. We report a 25‐year‐old male kidney recipient with Alport syndrome who developed bilateral synchronous urothelial cancer after transplantation. At the age of 16 this patient was referred to our clinic for a kidney transplantation. A living related donor kidney transplantation was performed with cyclosporin‐based quadruple immunosuppression. He experienced no acute rejection and his graft function was excellent after transplantation. Nine years after transplantation, he complained of asymptomatic gross hematuria and was diagnosed as having a bilateral urothelial cancer in the native upper urinary tracts. A bilateral total nephroureterectomy was undertaken, and the postoperative pathological diagnosis was advanced bilateral urothelial carcinoma. The patient received adjuvant gemcitabine plus cisplatin chemotherapy at a cisplatin dosage reduced by 50%. After 4 years of follow‐up, he is alive with a functioning graft and no evidence of recurrence.

A case report: Primary extragonadal yolk sac tumor of penile shaft in a 2-year-old child

Abstract:  A 2‐year‐old boy, who had the chief complaints of penile swelling and pain, was brought to the hospital by his mother. Penile contusion/trauma was suspected and he was admitted the same day to undergo emergency surgery to eliminate hematoma. The surgery revealed that the origin of the bleeding was not trauma but a tumor lesion of the penile shaft. It was histopathologically identified as a yolk sac tumor and no tumorous lesions were found except that in the penis. Therefore the patient was diagnosed as definitely having a yolk sac tumor originating in the penis. The patient received four cycles of cisplatin, etoposide and bleomycin treatment as adjuvant chemotherapy. Although it was impossible to completely resect the tumor, cisplatin, etoposide and bleomycin chemotherapy was effective and a complete response was achieved. We plan to carefully monitor the patient in the future.