Kazunori Haga

Therapeutic Efficacy of Replication-Competent Retrovirus Vector–Mediated Suicide Gene Therapy in a Multifocal Colorectal Cancer Metastasis Model

Replication-competent retrovirus (RCR) vectors are intrinsically incapable of infecting quiescent cells and have been shown to achieve highly efficient and tumor-restricted replicative spread and gene transfer in vivo after direct intratumoral injection in a variety of primary cancer models. However, i.v. delivery of RCR vectors expressing therapeutic genes has never previously been tested, particularly in an immunocompetent tumor model. Therefore, in the present study, we sought to test the therapeutic effect of an RCR vector (ACE-CD) carrying the yeast cytosine deaminase (CD) gene, which converts the nontoxic prodrug 5-fluorocytosine (5FC) into the chemotoxin 5-fluorouracil, after delivery by infusion into the locoregional circulation in a multifocal hepatic metastasis model of colon cancer. After confirmation of suicide gene cytotoxicity in vitro, multifocal hepatic tumors were established in syngeneic mice with murine CT26 colorectal cancer cells expressing firefly luciferase (CT26-Luc), and the ACE-CD vector was infused via intrasplenic injection into the portal circulation. Fourteen days after locoregional infusion, systemic administration of 5FC resulted in significant inhibition of bioluminescent signals in mice whose tumors had been infected with RCR but not in control mice. Notably, there was no detectable RCR vector spread to normal liver or bone marrow by quantitative PCR analysis. Our results thus show that locoregional delivery of a suicide gene by RCR vectors infused into the portal circulation results in progressive transduction of multiple tumor foci in the liver, without evidence of spread to adjacent normal parenchyma or extrahepatic tissues, and can achieve significant tumor growth inhibition.

Silodosin and its potential for treating premature ejaculation: A preliminary report

Premature ejaculation is a common sexual problem, as is erectile dysfunction. We evaluated silodosin, a highly selective α1A‐adrenoceptor antagonist, as a new treatment option for premature ejaculation. α1‐Adrenoceptor antagonists are widely used for lower urinary tract symptoms, and clinical studies on silodosin have shown excellent clinical efficacy for lower urinary tract symptoms. However, compared with other α1‐adrenoceptor antagonists, silodosin appeared to suppress ejaculation in a relatively higher percent of trial participants. This suppression of ejaculation by silodosin suggested its potential for treating premature ejaculation. Consequently, we evaluated the feasibility of off‐label silodosin as a new treatment option for premature ejaculation. Eight patients suffering premature ejaculation were treated with silodosin. Silodosin (4 mg) was given 2 h before sexual intercourse. Intravaginal ejaculatory latency time, premature ejaculation profile item, clinical global impression change in premature ejaculation and systemic adverse events were recorded. Intravaginal ejaculatory latency time was significantly prolonged (from 3.4 min to 10.1 min, P = 0.003). All patients answered better (much better) or slightly better for their own premature ejaculation problem compared with pretreatment condition in the clinical global impression change. Premature ejaculation profile also significantly improved. Two (25%), three (37.5%) and seven patients (87.5%) experienced anejaculation, reduced semen volume and discomfort during orgasm, respectively. However, these problems were not of major concern for the participants. No systemic adverse effects were reported. The current results support the possible use of silodosin as a new treatment option for premature ejaculation, and suggest that a placebo controlled study assessing its clinical usefulness would be worthwhile.

Adenovirus–Retrovirus Hybrid Vectors Achieve Highly Enhanced Tumor Transduction and Antitumor Efficacy In Vivo

Murine leukemia virus (MLV)-based replication-competent retrovirus (RCR) vectors have been shown to mediate efficient, selective, and persistent tumor transduction, thereby achieving significant therapeutic benefit in a wide variety of cancer models. To further augment the efficiency of this strategy, we have developed a delivery method employing a gutted adenovirus encoding an RCR vector (AdRCR); thus, tumor cells transduced with the adenoviral vector transiently become RCR vector producer cells in situ. As expected, high-titer AdRCR achieved significantly higher initial transduction levels in human cancer cells both in vitro and in vivo, as compared to the original RCR vector itself. Notably, even at equivalent initial transduction levels, more secondary RCR progeny were produced from AdRCR-transduced cells as compared to RCR-transduced cells, resulting in further acceleration of subsequent RCR replication kinetics. In pre-established tumor models in vivo, prodrug activator gene therapy with high-titer AdRCR could achieve enhanced efficacy compared to RCR alone, in a dose-dependent manner. Thus, AdRCR hybrid vectors offer the advantages of high production titers characteristic of adenovirus and secondary production of RCR in situ, which not only accelerates subsequent vector spread and progressive tumor transduction, but can also significantly enhance the therapeutic efficacy of RCR-mediated prodrug activator gene therapy.

Effective in vivo and ex vivo gene transfer to intestinal mucosa by VSV-G-pseudotyped lentiviral vectors

BackgroundGene transfer to the gastrointestinal (GI) mucosa is a therapeutic strategy which could prove particularly advantageous for treatment of various hereditary and acquired intestinal disorders, including inflammatory bowel disease (IBD), GI infections, and cancer.MethodsWe evaluated vesicular stomatitis virus glycoprotein envelope (VSV-G)-pseudotyped lentiviral vectors (LV) for efficacy of gene transfer to both murine rectosigmoid colon in vivo and human colon explants ex vivo. LV encoding beta-galactosidase (LV-β-Gal) or firefly-luciferase (LV-fLuc) reporter genes were administered by intrarectal instillation in mice, or applied topically for ex vivo transduction of human colorectal explant tissues from normal individuals. Macroscopic and histological evaluations were performed to assess any tissue damage or inflammation. Transduction efficiency and systemic biodistribution were evaluated by real-time quantitative PCR. LV-fLuc expression was evaluated by ex vivo bioluminescence imaging. LV-β-Gal expression and identity of transduced cell types were examined by histochemical and immunofluorescence staining.ResultsImaging studies showed positive fLuc signals in murine distal colon; β-Gal-positive cells were found in both murine and human intestinal tissue. In the murine model, β-Gal-positive epithelial and lamina propria cells were found to express cytokeratin, CD45, and CD4. LV-transduced β-Gal-positive cells were also seen in human colorectal explants, consisting mainly of CD45, CD4, and CD11c-positive cells confined to the LP.ConclusionsWe have demonstrated the feasibility of LV-mediated gene transfer into colonic mucosa. We also identified differential patterns of mucosal gene transfer dependent on whether murine or human tissue was used. Within the limitations of the study, the LV did not appear to induce mucosal damage and were not distributed beyond the distal colon.

Dissociation between patients and their partners in expectations for sexual life after radical prostatectomy

To analyze expectations for sexual life after radical prostatectomy in patients and their partners, and its influence on sexual motivation and bothers in the postoperative period.

Lymphoepithelioma‐like carcinoma of the renal pelvis

Abstract:  Lymphoepithelioma‐like carcinoma (LELC), best known to occur in the nasopharynx, can arise in a variety of sites, such as the salivary gland, thymus, lung, stomach, skin and uroepithelium. Primary LELC of the uroepithelium is very rare and there is only limited information in the published reports. We managed a case of a 75‐year‐old woman who presented with nausea and gross painless hematuria. She was treated with laparoscopic nephroureterectomy and was diagnosed with a T1N1M0 LELC of the renal pelvis. Unlike nasopharyngeal lymphoepithelioma, immunohistochemical analysis of this urinary LELC was negative for the Epstein‐Barr virus. Herein we report on one more case of primary LELC of the renal pelvis and review of the published reports, particularly those concerning Epstein‐Barr virus expressions. Recognition of this tumor and complete resection are essential for saving patients.

Adult paratesticular rhabdomyosarcoma

Background A 29-year-old male patient presented with a history of painless enlargement of the left hemiscrotum.Investigations Laboratory tests for β-human chorionic gonadotrophin, α-fetoprotein, and lactate dehydrogenase, physical examination, and CT of the chest, abdomen and pelvis. Histologic examination, nerve-sparing retroperitoneal lymph-node dissection.Diagnosis Paratesticular rhabdomyosarcoma with lymph node metastasis.Management Inguinal radical orchiectomy and adjuvant chemotherapy for 48 weeks. Radiotherapy and additional chemotherapy were administered following local recurrence.

Mucosal gene therapy using a pseudotyped lentivirus vector encoding murine interleukin-10 (mIL-10) suppresses the development and relapse of experimental murine colitis

BackgroundTherapeutic gene transfer is currently being evaluated as a potential therapy for inflammatory bowel disease. This study investigates the safety and therapeutic benefit of a locally administered lentiviral vector encoding murine interleukin-10 in altering the onset and relapse of dextran sodium sulfate induced murine colitis.MethodsLentiviral vectors encoding the reporter genes firefly-luciferase and murine interleukin-10 were administered by intrarectal instillation, either once or twice following an ethanol enema to facilitate mucosal uptake, on Days 3 and 20 in Balb/c mice with acute and relapsing colitis induced with dextran sulfate sodium (DSS). DSS colitis was characterized using clinical disease activity, macroscopic, and microscopic scores. Bioluminescence optical imaging analysis was employed to examine mucosal lentiviral vector uptake and transgene expression. Levels of tumor necrosis factor-α and interleukin-6 in homogenates of rectal tissue were measured by ELISA. Biodistribution of the lentiviral vector to other organs was evaluated by real time quantitative PCR.ResultsMucosal delivery of lentiviral vector resulted in significant transduction of colorectal mucosa, as shown by bioluminescence imaging analysis. Lentiviral vector-mediated local expression of interleukin-10 resulted in significantly increased levels of this cytokine, as well as reduced levels of tumor necrosis factor-α and interleukin-6, and significantly reduced the clinical disease activity, macroscopic, and microscopic scores of DSS colitis. Systemic biodistribution of locally instilled lentiviral vector to other organs was not detected.ConclusionsTopically-delivered lentiviral vectors encoding interleukin-10 safely penetrated local mucosal tissue and had therapeutic benefit in this DSS model of murine colitis.

Simple and reliable predictor of urinary continence after radical prostatectomy: serial measurement of urine loss ratio after catheter removal.

To evaluate urine loss ratio after catheter removal as a predictive factor of urinary continence after radical prostatectomy.

PTEN Knockout Prostate Cancer as a Model for Experimental Immunotherapy

PURPOSE Testing immunotherapeutic strategies for prostate cancer has been impeded by the lack of relevant tumor models in immunocompetent animals. This opportunity is now provided by the recent development of prostate specific PTEN knockout mice, which show spontaneous development of true adenocarcinoma arising from prostate epithelium and more faithfully recapitulate the human disease than any previous model. We investigated the feasibility of using tumor cells derived from this model to test tumor vaccination and adoptive immunotherapeutic strategies for prostate cancer. MATERIALS AND METHODS PTEN-CaP8 adenocarcinoma cells derived from the biallelic PTEN knockout prostate cancer model were used to vaccinate nontumor bearing litter mates. Tumor specific effector cells were generated from splenocytes of vaccinated mice by mixed lymphocyte-tumor reactions, and antiproliferative effects and cytokine generation were examined in vitro. The effect of vaccination or adoptive immunotherapy on luciferase marked PTEN-CaP8 subcutaneous tumors was monitored by tumor volumetric measurements and noninvasive bioluminescence imaging. RESULTS Vaccination of litter mate mice with irradiated PTEN-CaP8 cells showed a significant prophylactic effect against the subsequent tumor challenge. Effector cells harvested from vaccinated litter mates showed significant interferon-gamma secretion upon co-incubation with PTEN-CaP8 target cells and they were capable of efficient target cell growth inhibition in vitro. Intratumor adoptive transfer of effector cells resulted in significant growth inhibition of preestablished prostate tumors in vivo. CONCLUSIONS The PTEN knockout model serves as a highly useful model in which to investigate tumor cell vaccination and adoptive immunotherapeutic strategies in the context of true adenocarcinoma of the prostate. This model should accelerate efforts to develop effective immunotherapies for human prostate cancer.