Kazutoshi Fujita

Transplantation of spermatogonial stem cells isolated from leukemic mice restores fertility without inducing leukemia

More than 70% of patients survive childhood leukemia, but chemotherapy and radiation therapy cause irreversible impairment of spermatogenesis. Although autotransplantation of germ cells holds promise for restoring fertility, contamination by leukemic cells may induce relapse. In this study, we isolated germ cells from leukemic mice by FACS sorting. The cell population in the high forward-scatter and low side-scatter regions of dissociated testicular cells from leukemic mice were analyzed by staining for MHC class I heavy chain (H-2K b / H-2D b ) and for CD45. Cells that did not stain positively for H-2K b /H-2D b and CD45 were sorted as the germ cell-enriched fraction. The sorted germ cell-enriched fractions were transplanted into the testes of recipient mice exposed to alkylating agents. Transplanted germ cells colonized, and recipient mice survived. Normal progeny were produced by intracytoplasmic injection of sperm obtained from recipient testes. When unsorted germ cells from leukemic mice were transplanted into recipient testes, all recipient mice developed leukemia. The successful birth of offspring from recipient mice without transmission of leukemia to the recipients indicates the potential ofautotransplantation of germ cells sorted by FACS to treat infertility secondary to anticancer treatment for childhood leukemia.

Serial Prostate Biopsies are Associated With an Increased Risk of Erectile Dysfunction in Men With Prostate Cancer on Active Surveillance

PURPOSE We determined whether serial prostate needle biopsies predispose men to erectile dysfunction and/or lower urinary tract symptoms over time. MATERIALS AND METHODS Men with prostate cancer on an active surveillance protocol were administered the 5-item Sexual Health Inventory for Men and International Prostate Symptom Score questionnaires on protocol entry, and at a cross-sectional point in 2008. All men had at least 1, 10 to 12-core prostate biopsy at protocol entry and yearly surveillance biopsies thereafter were recommended. RESULTS Of 333 men 231 returned the followup questionnaires. Correlations were found between biopsy number and erectile dysfunction, with increasing biopsy number associated with a decrease in Sexual Health Inventory for Men score (p = 0.04) and a history of 3 or more biopsies associated with a greater decrease in Sexual Health Inventory for Men score than after 2 or fewer biopsies (p = 0.02). Multivariable analysis for biopsy number, age, prostate volume and prostate specific antigen showed that only biopsy number was associated with decreasing Sexual Health Inventory for Men score (p = 0.02). When men were stratified by baseline Sexual Health Inventory for Men, those without preexisting erectile dysfunction (Sexual Health Inventory for Men score 22 to 25) trended toward steeper decreases in Sexual Health Inventory for Men score after 3 or more biopsies (p = 0.06) than did men with baseline mild to moderate erectile dysfunction (Sexual Health Inventory for Men score 8 to 21). No correlation was found between biopsy number and International Prostate Symptom Score. CONCLUSIONS Serial prostate biopsies appear to have an adverse effect on erectile function in men with prostate cancer on active surveillance but do not affect lower urinary tract symptoms.

Isolation of Germ Cells from Leukemia and Lymphoma Cells in a Human In vitro Model: Potential Clinical Application for Restoring Human Fertility after Anticancer Therapy

More than 70% of patients survive childhood cancer, but chemotherapy and radiation therapy may cause irreversible impairment of spermatogenesis. To treat infertility secondary to anticancer treatment for childhood cancer, we have developed a procedure to isolate germ cells from leukemic mice by fluorescence-activated cell sorting with two surface markers, and transplantation of isolated germ cells successfully restored fertility without inducing leukemia. In the present study, we analyzed human germ cells and human malignant cells, including five leukemia cell lines and three lymphoma cell lines, by fluorescence-activated cell sorting with antibodies against MHC class I and CD45. Testicular specimens were obtained from a patient who underwent surgery for testicular rupture. In the high forward scatter and low side scatter region, no malignant cells were found in the MHC class I-negative and CD45-negative fraction (the germ cell fraction), with the exception of K562 cells. A total of 39.2% of the germ cells were found in the germ cell fraction. A total of 1.45% of K562 cells were found in the germ cell fraction. Treatment with IFNγ induced the expression of MHC class I on K562 cells but not on germ cells and made it possible to isolate germ cells from K562 cells. In conclusion, we isolated human germ cells from malignant cells with two surface markers after treatment with IFNγ. Immunophenotyping for each patient will be necessary before isolation and induction of surface marker will be clinically applicable. (Cancer Res 2006; 66(23): 11166-71)

Cytokine profiling of prostatic fluid from cancerous prostate glands identifies cytokines associated with extent of tumor and inflammation

Cytokines are key mediators of inflammation that may relate to prostate cancer initiation and progression, and that may be useful markers of prostatic neoplasia and related inflammation. In order to better understand the relationship between cytokines and prostate cancer, we profiled cytokines in prostatic fluids obtained from cancerous prostate glands and correlated them to both cancer status and inflammatory grade.

Monocyte chemotactic protein-1 (MCP-1/CCL2) is associated with prostatic growth dysregulation and benign prostatic hyperplasia

Chronic inflammation is commonly observed in benign prostate hyperplasia (BPH), and prostate tissue often contains increased inflammatory infiltrates, including T cells and macrophages. Cytokines are not only key mediators of inflammation but may also play important roles in the initiation and progression of BPH.

Comparative study on evaluation methods for serum testosterone level for PADAM diagnosis

The International Society for the Study of the Aging Male (ISSAM) recommends that a diagnosis be based on a patients total testosterone (TT), calculated free testosterone (cFT), or calculated bioavailable testosterone (cBT) for partial androgen deficiency of the aging male (PADAM). The purpose of this study was to confirm whether hypogonadism of patients with PADAM is related to symptoms and clarify which criteria of testosterone recommended by ISSAM is suitable for Japanese patients. A total of 90 patients with PADAM symptoms were included in this study. Endocrinologic profiles were reviewed as appropriate, and PADAM symptoms were judged by means of several questionnaires. Laboratory values and symptoms were compared between patients with and without hypogonadism. Even when any criterion of testosterone was used for diagnosis of hypogonadism, AMS (total and subscales), IIEF-5, or SDS scores of PADAM symptoms did not differ significantly between patients classified as having and not having hypogonadism. No other endocrinologic variables than testosterone differed significantly between them, either. PADAM symptoms are not related to testosterone level and it is still obscure whether ISSAMs criterion can be adopted for Japanese patients with PADAM. Other pathology needs to be addressed for evaluation and diagnosis of PADAM in Japan.

Endoglin (CD105) as a Urinary and Serum Marker of Prostate Cancer

We have previously shown that endoglin (CD105) is upregulated in prostatic fluid of men with large volume prostate cancer. We chose to assess endoglin levels in urine and serum from men with prostate cancer or at increased risk for the disease: Urine samples were collected after digital rectal examination (DRE) from 99 men whose cancer status was confirmed by biopsy, and serum samples were collected from 20 men without prostate cancer at low risk for the disease and from 69 men diagnosed with prostate cancer that subsequently underwent radical prostatectomy (30 pT2, 39 pT3). Endoglin levels were assessed by ELISA. Urinary endoglin was elevated in men with biopsy‐positive prostate cancer compared to biopsy‐negative men (p = 0.0014). Urinary endoglin levels in men with prostate cancer correlated with radical prostatectomy tumor volume. The area under the receiver operating characteristic (ROC) curve was 0.72 for urinary endoglin and 0.50 for serum prostate‐specific antigen (PSA; sensitivity for cancer detection 73%, specificity 63%). There were no differences in serum endoglin between normal and cancer cases, but there were increases in serum endoglin in non‐organ confined (NOC, pT3+) versus organ‐confined (OC, pT2) cases (p = 0.0004). The area under the ROC curve was 0.75 for serum endoglin and 0.63 for PSA for predicting NOC status, with a sensitivity of 67% and a specificity of 80%. In conclusion, elevations in post‐DRE urinary endoglin suggest there may be value in further studying endoglin as a urinary biomarker of prostate cancer. Endoglin levels in both urine and serum may aid in prostate cancer detection and prognostication.

Differential brain processing of audiovisual sexual stimuli in men: comparative positron emission tomography study of the initiation and maintenance of penile erection during sexual arousal.

The human male psychosexual cycle consists of four phases: excitation, plateau, orgasm, and resolution. Identification of the specific neural substrates of each phase may provide information regarding the brains pathophysiology of sexual dysfunction. We previously analyzed regional cerebral blood flow (rCBF) with H(2)15O-positron emission tomography (PET) during the excitation phase (initiation of penile erection) induced by audiovisual sexual stimuli (AVSS) and identified activation of the cerebellar vermis, the bilateral extrastriate cortex, and right orbitofrontal cortex, suggesting a role of cognition/emotion in the excitement phase. In the present study, we analyzed rCBF of the same six healthy volunteers during the plateau phase (maintenance of penile erection) induced by AVSS and compared the results with those of the excitation phase. Penile rigidity was monitored in real time with RigiScan Plus during PET scanning. Images were analyzed by statistical parametric mapping (SPM) software, and rCBF in the amygdala, hypothalamus, anterior cingulate, and insula was measured. During the plateau phase, primary subcortical activation was noted in the right ventral putamen, indicating motivational factors in the sexual response via the limbic reward circuit. A significant increase in rCBF in the left hypothalamus was also observed during the plateau phase. The right anterior cingulate and left insula were specifically activated during the excitation phase but not during the plateau phase. These results indicate a significant role of the ventral putamen and the hypothalamus in the plateau phase and confirm that paralimbic and limbic components of the human brain differentially coordinate the sexual response in a psychosexual phase-dependent manner.

The miR-130 family promotes cell migration and invasion in bladder cancer through FAK and Akt phosphorylation by regulating PTEN.

Bladder cancer causes an estimated 150,000 deaths per year worldwide. Although 15% of the recurrent bladder cancer becomes an invasive type, currently used targeted therapy for malignant bladder cancer is still not efficient. We focused on the miR-130 family (miR-130b, miR-301a, and miR-301b) that was significantly upregulated in bladder cancer specimens than that of the normal urothelial specimens. We analyzed the functional significance of miR-130 family using a 5637 bladder cancer cell line and revealed that miR-130 family of inhibitors suppressed cell migration and invasion by downregulating focal adhesion kinase (FAK) and Akt phosphorylation. Mechanistic analyses indicate that the miR-130 family directly targets phosphatase and tensin homolog deleted from chromosome 10 (PTEN), resulting in the upregulation of FAK and Akt phosphorylation. In clinical bladder cancer specimens, downregulation of PTEN was found to be closely correlated with miR-130 family expression levels. Overall, the miR-130 family has a crucial role in malignant progression of bladder cancer and thus the miR-130 family could be a promising therapeutic target for invasive bladder cancer.

Prostatic Inflammation Detected in Initial Biopsy Specimens and Urinary Pyuria are Predictors of Negative Repeat Prostate Biopsy

PURPOSE Asymptomatic prostatic inflammation may cause increased prostate specific antigen in some men, leading to unnecessary repeat prostate biopsy. We determined whether histological findings of inflammation in initial biopsy specimens and/or clinical indicators of inflammation could predict the outcome of subsequent biopsy in men with a negative initial biopsy. MATERIALS AND METHODS A total of 105 Japanese men with increased prostate specific antigen underwent repeat prostate biopsy after initial biopsy revealed no evidence of carcinoma. Of the cases 45 (42.8%) were positive for prostate cancer at repeat biopsy. We evaluated initial biopsy specimens for evidence of inflammation by mononuclear and polymorphonuclear leukocytes, serum and urinary white blood count, and C-reactive protein. RESULTS Polymorphonuclear leukocyte infiltrates, urinary white blood count, patient age, prostate specific antigen at repeat biopsy, prostate volume, prostate specific antigen velocity and prostate specific antigen density were associated with the repeat biopsy outcome (p <0.05). Multivariate analysis revealed that age, prostate specific antigen density and urinary white blood count were independent predictors of outcome. On subgroup analysis of 63 men with serum prostate specific antigen less than 10 ng/ml before initial biopsy polymorphonuclear and mononuclear leukocyte inflammation, age, prostate specific antigen at repeat biopsy, prostate volume, prostate specific antigen velocity and prostate specific antigen density were associated with the outcome of repeat biopsy (p <0.05). Multivariate analysis showed that polymorphonuclear leukocyte infiltrate, prostate specific antigen density and age were independent predictors. CONCLUSIONS Age, prostate specific antigen density, polymorphonuclear leukocyte inflammation in initial biopsy specimens and urinary pyuria are indicators of benign repeat biopsy. They help avoid unnecessary repeat biopsy in men with increased prostate specific antigen.