Establishment of novel common marmoset embryonic stem cell lines under various conditions.

Pluripotent stem cells (PSCs), embryonic stem cells (ESCs), and induced PSCs (iPSCs) are excellent tools for studying embryonic development in organisms and classified into naïve and primed states. ESC-derived germline chimera individuals can be produced by injecting naïve ESCs/iPSCs into preimplantation embryos, and conversion of primed human ESCs/iPSCs into a naïve state provides insights into epiblast cell features. Non-human ESCs/iPSCs are alternatives to human naïve ESCs/iPSCs, which elicit ethical issues. In this study, we used the common marmoset (Callithrix jacchus) as an animal model. Since 1996, 16 marmoset ESC lines have been established. Because most of these ESC lines are female and were derived >10 years ago, new ESCs, particularly male marmoset ESC lines, are needed. Here, we successfully established 17 novel marmoset ESC lines, including six male ESC lines from in vitro-fertilized (IVF) embryos and 12 ESC lines under feeder-free conditions. This report is the first to establish ESC lines using feeder-free conditions and IVF preimplantation blastocysts in marmosets, and these novel ESC lines could potentially facilitate future non-human primate ESC studies.

Generation of a Nonhuman Primate Model of Severe Combined Immunodeficiency Using Highly Efficient Genome Editing.

Recent advances in genome editing have facilitated the generation of nonhuman primate (NHP) models, with potential to unmask the complex biology of human disease not revealed by rodent models. However, their broader use is hindered by the challenges associated with generation of adult NHP models as well as the cost of their production. Here, we describe the generation of a marmoset model of severe combined immunodeficiency (SCID). This study optimized zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to target interleukin-2 receptor subunit gamma (IL2RG) in pronuclear stage marmoset embryos. Nine of 21 neonates exhibited mutations in the IL2RG gene, concomitant with immunodeficiency, and three neonates have currently survived from 240 days to 1.8 years. Our approach demonstrates highly efficient production of founder NHP with SCID phenotypes, with promises of multiple pre-clinical and translational applications.

Birth of healthy offspring following ICSI in in vitro-matured common marmoset (Callithrix jacchus) oocytes.

Intracytoplasmic sperm injection (ICSI), an important method used to treat male subfertility, is applied in the transgenic technology of sperm-mediated gene transfer. However, no study has described successful generation of offspring using ICSI in the common marmoset, a small non-human primate used as a model for biomedical translational research. In this study, we investigated blastocyst development and the subsequent live offspring stages of marmoset oocytes matured in vitro and fertilized by ICSI. To investigate the optimal timing of performing ICSI, corrected immature oocytes were matured in vitro and ICSI was performed at various time points (1-2 h, 2-4 h, 4-6 h, 6-8 h, and 8-10 h after extrusion of the first polar body (PB)). Matured oocytes were then divided randomly into two groups: one was used for in vitro fertilization (IVF) and the other for ICSI. To investigate in vivo development of embryos followed by ICSI, 6-cell- to 8-cell-stage embryos and blastocysts were nonsurgically transferred into recipient marmosets. Although no significant differences were observed in the fertilization rate of blastocysts among ICSI timing after the first PB extrusion, the blastocyst rate at 1-2 h was lowest among groups at 2-4 h, 4-6 h, 6-8 h, and 8-10 h. Comparing ICSI to IVF, the fertilization rates obtained in ICSI were higher than in IVF (p>0.05). No significant difference was noted in the cleaved blastocyst rate between ICSI and IVF. Following the transfer of 37 ICSI blastocysts, 4 of 20 recipients became pregnant, while with the transfer of 21 6-cell- to 8-cell-stage ICSI embryos, 3 of 8 recipients became pregnant. Four healthy offspring were produced and grew normally. These are the first marmoset offspring produced by ICSI, making it an effective fertilization method for marmosets.

[Prostate cancer of unknown primary origin with multiple lymph nodes metastasis; a case report].

A 62-year-old man were referred our hospital complaining of high prostate specific antigen (PSA) value (32.4 ng/ml) in May 2010. Two sets of biopsies preformed previously at another hospital had not detected any cancers in the prostate. In our hospital, prostate biopsies were performed in July 2010 and February 2011, but cancer was not detected in either occasion. In March 2011, his PSA increased up to 126.7 ng/ml, CT scan showed the swelling of left supraclavicular and para-aortic lymph nodes. Biopsy of the supraclavicular lymph node was performed. Pathology revealed poorly differentiated adenocarcinoma with positive immunohistochemistry for PSA, which was suggestive of metastatic prostate cancer. After 1 year of treatment with androgen deprivation therapy, the patient developed castration resistant prostate cancer and have undergone chemotherapy with docetaxel.

[Diffusion-weighted imaging for prostate cancer localization: comparison of apparent diffusion coefficient values in cancerous and non-cancerous tissues, and the Gleason score of the radical prostatectomy specimens].

PURPOSE: We compared magnetic resonance imaging (MRI) with radical prostatectomy specimens to evaluate the diagnostic performance of diffusion-weighted imaging (DWI) and the apparent diffusion coefficient (ADC) value for prostate cancer localization. MATERIALS AND METHODS: We performed a retrospective study of 44 patients who underwent radical prostatectomy. We compared MRI with pathological specimens (74 tumors) to evaluate their diagnostic performance of cancer localization. The ADC value was measured in cancerous and non-cancerous prostate tissues. RESULTS: Of 74 tumors, digital rectal examination, transrectal ultrasonography, T2-weighted imaging (T2WI), DWI, T2WI and DWI detected 9 (12.2%), 9 (12.2%), 26 (35.1%), 30 (40.5%), and 48 (64.9%) tumors, respectively. The mean ADC value was lower in cancerous tissues than in non-cancerous tissues (0.86 +/- 0.15 versus 1.24 +/- 0.16 x 10(-3) mm2/s). The mean ADC values of cancerous and non-cancerous tissues were: 0.85 +/- 0.15 versus 1.28 +/- 0.17 x 10(-3) mm2/s in the peripheral zone; and 0.87 +/- 0.15 versus 1.19 +/- 0.14 x 10(-3) mm2/s in the transition zone. The mean ADC value in patients with a Gleason score of 8 or 9 (0.76 +/- 0.12 x 10(-3) mm2/s) was lower than that in patients with a Gleason score of 6 or 7 (0.86 +/- 0.15 x 10(-3) mm2/s). CONCLUSION: DWI and ADC value were considered to be useful for the diagnosis of prostate cancer localization.

Detecting KRAS mutations in peripheral blood of colorectal cancer patients by peptide nucleic acid clamp PCR.

We investigated the effectiveness of peptide nucleic acid (PNA) clamp PCR for detecting KRAS mutations in peripheral blood samples of colorectal cancer (CRC) patients. We compared KRAS point mutations between tumour tissue and blood samples. Forty-two patients were included in this study. We observed KRAS mutations in formalin-fixed, paraffin-embedded tissues by PCR direct sequencing and in blood samples by PNA clamp PCR. KRAS point mutations were detected in primary tumour tissue samples of 13 patients (31.0%) and in peripheral blood samples of 10 patients (23.8%). KRAS point mutations were detected in both samples for 8 patients (19.0%). The sensitivity, specificity and accuracy for detecting KRAS mutations in peripheral blood and tumour tissue samples were 61.5, 93.1 and 83.3%, respectively. The positive and negative predictive values were 80.0 and 84.4%, respectively. Five patients with mutant KRAS in their plasma preoperatively, did not exhibit KRAS mutations postoperatively. Our method detected KRAS point mutations in peripheral blood samples of CRC patients, which contained extremely small amounts of mutant cells. This method is helpful for identifying metastatic CRC patients in whom metastases will respond to EGFR-targeted monoclonal antibody therapy.

Generation of transgenic non-human primates with germline transmission.

The common marmoset (Callithrix jacchus) is increasingly attractive for use as a non-human primate animal model in biomedical research. It has a relatively high reproduction rate for a primate, making it potentially suitable for transgenic modification. Although several attempts have been made to produce non-human transgenic primates, transgene expression in the somatic tissues of live infants has not been demonstrated by objective analyses such as polymerase chain reaction with reverse transcription or western blots. Here we show that the injection of a self-inactivating lentiviral vector in sucrose solution into marmoset embryos results in transgenic common marmosets that expressed the transgene in several organs. Notably, we achieved germline transmission of the transgene, and the transgenic offspring developed normally. The successful creation of transgenic marmosets provides a new animal model for human disease that has the great advantage of a close genetic relationship with humans. This model will be valuable to many fields of biomedical research.

Naftopidil monotherapy vs naftopidil and an anticholinergic agent combined therapy for storage symptoms associated with benign prostatic hyperplasia: A prospective randomized controlled study.

AIM: The aim of this study was to compare the efficacy and safety of alpha1-adrenoceptor (alpha1-AR) antagonist monotherapy with combination therapy using alpha1-AR antagonist and anticholinergic agent for benign prostatic hyperplasia (BPH) with storage symptoms as the chief complaint. METHODS: In this prospective comparative study, either 25-75 mg/day of naftopidil monotherapy (monotherapy group) or combination therapy using 25-75 mg/day of naftopidil and an anticholinergic agent (10-20 mg/day of propiverine hydrochloride or 2-6 mg/day of oxybutynin hydrochloride; cotherapy group) were administered for 12 weeks to 101 BPH patients with storage symptoms. RESULTS: International prostate symptom score (IPSS) and quality of life (QOL) index improved significantly in both groups, with no marked differences between groups. Maximum flow rate (Qmax) and residual urine volume (RUV) tended to improve in both groups, again with no marked differences between groups. However, median post-therapeutic RUV was significantly worse for the cotherapy group (45.0 mL) than for the monotherapy group (13.5 mL; P = 0.0210). Ratio of patients with increased RUV was also significantly worse for cotherapy (22.9%) than for monotherapy (5.0%; P = 0.038). CONCLUSIONS: Although the anticholinergic dosage was low, the present results suggest that naftopidil monotherapy was as useful as combination therapy of naftopidil and an anticholinergic agent. Therefore, naftopidil is a useful agent as the first choice in BPH patients with storage symptoms.

Establishment of novel embryonic stem cell lines derived from the common marmoset (Callithrix jacchus).

The successful establishment of human embryonic stem cell (hESC) lines has inaugurated a new era in regenerative medicine by facilitating the transplantation of differentiated ESCs to specific organs. However, problems with the safety and efficacy of hESC therapy in vivo remain to be resolved. Preclinical studies using animal model systems, including nonhuman primates, are essential to evaluate the safety and efficacy of hESC therapies. Previously, we demonstrated that common marmosets are suitable laboratory animal models for preclinical studies of hematopoietic stem cell therapies. As this animal model is also applicable to preclinical trials of ESC therapies, we have established novel common marmoset ESC (CMESC) lines. To obtain marmoset embryos, we developed a new embryo collection system, in which blastocysts can be obtained every 3 weeks from each marmoset pair. The inner cell mass was isolated by immunosurgery and plated on a mouse embryonic feeder layer. Some of the CMESC lines were cultured continuously for more than 1 year. These CMESC lines showed alkaline phosphatase activity and expressed stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, and TRA-1-81. On the other hand, SSEA-1 was not detected. Furthermore, our novel CMESCs are pluripotent, as evidenced by in vivo teratoma formation in immunodeficient mice and in vitro differentiation experiments. Our established CMESC lines and the common marmoset provide an excellent experimental model system for understanding differentiation mechanisms, as well as the development of regenerative therapies using hESCs.

Small cell carcinoma of the prostate with hypercalcemia.

We present a case of small cell prostate carcinoma with hypercalcemia in a 75-year-old man. He was diagnosed as having stage T3bN1M0 adenocarcinoma of the prostate. His serum prostate-specific antigen level was reduced to below the normal range after a combination treatment of a luteinizing hormone-releasing hormone agonist and flutamide for prostate carcinoma. He subsequently experienced increasing fatigue, poor appetite, short time loss of consciousness and pain in his lower abdomen. His serum calcium level and carcinoembryonic antigen were increased. He died 5 months from the start of treatment. The autopsy revealed small cell carcinoma of the prostate and multiple metastasis of the lung, liver, pancreas, lymph nodes and spine.

Phase I study of autologous tumor vaccines transduced with the GM-CSF gene in four patients with stage IV renal cell cancer in Japan: clinical and immunological findings.

We produced lethally irradiated retrovirally GM-CSF-transduced autologous renal tumor cell vaccines (GVAX) from six Japanese patients with stage IV renal cell cancer (RCC). Four patients received GVAX ranging from 1.4 x 10(8) to 3.7 x 10(8) cells on 6-17 occasions. Throughout a total of 48 vaccinations, there were no severe adverse events. After vaccination, DTH skin tests became positive to autologous RCC (auto-RCC) in all patients. The vaccination sites showed significant infiltration by CD4(+) T cells, eosinophils, and HLA-DR-positive cells. The kinetic analyses of cellular immune responses using peripheral blood lymphocytes revealed an enhanced proliferative response against auto-RCC in four patients, and cytotoxicity against auto-RCC was augmented in three patients. T cell receptor beta-chain analysis revealed oligoclonal expansion of T cells in the peripheral blood, skin biopsy specimens from DTH sites, and tumors. Western blot analysis demonstrated the induction of a humoral immune response against auto-RCC. Two of the four patients are currently alive 58 and 40 months after the initial vaccination with low-dose interleukin-2. Our results suggest that GVAX substantially enhanced the antitumor cellular and humoral immune responses, which might have contributed to the relatively long survival times of our patients in the present study.

CA19-9 as a serum marker for poor prognosis in urothelial carcinoma.

INTRODUCTION: To evaluate the role of serum carbohydrate antigen 19-9 (CA19-9) in the prognosis and follow-up evaluation of patients with urothelial carcinoma, the authors studied the association of the serum level and positive rate with clinical features such as infiltration and metastasis in 164 patients admitted to the authors' department. PATIENTS AND METHODS: This study included 164 cases of patients with urohelial carcinoma. The absolute value of the serum CA19-9 level and its positive rate were tested. Simple variant analysis and logistic regression analysis were used for estimation of statistical significance. Kaplan-Meier's test and Cox's proportional hazard model were used for analysis of survival. RESULTS: Significant differences in the serum CA19-9 levels were found with regard to the following parameters: the presence or absence of metastasis, clinical stage, depth of invasion, and degree of differentiation. The positive rate displayed a significant difference only for the presence or absence of metastasis. With regard to the presence of metastasis, serum CA19-9 was a significant risk factor along with depth of invasion in logistic regression analysis. Comparison of the survival rate indicated the prognosis to be significantly poor in the positive group and serum CA19-9 was regarded to be a prognostic risk factor in analysis via the regression model. CONCLUSIONS: Serum CA19-9 is thought to serve as a significant marker for advanced cancer and tumors with highly malignant potential and is useful for predicting prognosis of the disease.