Effects of testosterone replacement therapy on metabolic syndrome among Japanese hypogonadal men: A subanalysis of a prospective randomised controlled trial (EARTH study).

We investigated the effects of testosterone replacement therapy (TRT) on metabolic factors among hypogonadal men with a metabolic syndrome. From the study population of the EARTH study, which was a randomised controlled study in Japan, 65 hypogonadal patients with a metabolic syndrome, comprising the TRT group (n = 32) and controls (n = 33), were included in this study analysis. The TRT group was administered 250 mg of testosterone enanthate as an intramuscular injection every 4 weeks for 12 months. Waist circumference, body mass index, body fat volume and blood pressure were measured in all patients at baseline and at 12 months. In addition, blood biochemical data, including total cholesterol, triglyceride (TG), HDL cholesterol, fasting plasma glucose (FPG) and haemoglobin A1c (HbA1c) levels, were also evaluated. Changes in these categories from baseline to 12 months were compared between the TRT and control groups, with significant differences observed in waist circumference, body fat percentage, FPG, TG and HbA1c levels. No significant differences were observed in other parameters. TRT for 1 year was associated with improvements in some metabolic factors among Japanese men with hypogonadism and metabolic syndrome.

A PLK4 mutation causing azoospermia in a man with Sertoli cell-only syndrome.

About 15% of couples wishing to have children are infertile; approximately half these cases involve a male factor. Polo-like kinase 4 (PLK-4) is a member of the polo protein family and a key regulator of centriole duplication. Male mice with a point mutation in the Plk4 gene show azoospermia associated with germ cell loss. Mutational analysis of 81 patients with azoospermia and Sertoli cell-only syndrome (SCOS) identified one man with a heterozygous 13-bp deletion in the Ser/Thr kinase domain of PLK4. Division of centrioles occurred in wild-type PLK4-transfected cells, but was hampered in PLK-4-mutant transfectants, which also showed abnormal nuclei. Thus, this PLK4 mutation might be a cause of human SCOS and nonobstructive azoospermia.

Y chromosome haplogroup D2a1 is significantly associated with high levels of luteinizing hormone in Japanese men.

The association between the Y chromosome haplogroup D2 and risk of azoospermia and low sperm motility has been previously studied, and it was indicated that haplogroups DE (YAP lineage) are associated with prostate cancer risk in Japanese males. Our assumption had been that Y chromosome haplogroups may be associated with sex hormone levels, because sex hormones have been deemed responsible for spermatogenesis and carcinogenesis. In this study, we assessed the association between Y chromosome haplogroups and sex hormone levels, including those of testosterone, sex hormone-binding globulin (SHBG), follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin-B, and calculated free testosterone (cFT), in 901 young men from the general Japanese population (cohort 1) and 786 Japanese men of proven fertility (cohort 2). We found that the haplogroup D2a1 was significantly associated with high LH levels in a combined analysis involving two cohorts (ß = 0.068, SE = 0.025, p = 0.0075), following correction for multiple testing. To date, this result is the first evidence that implicates Y chromosome haplogroups in an association with sex hormone levels.

SIN3A mutations are rare in men with azoospermia.

A loss of function of the murine Sin3A gene resulted in male infertility with Sertoli cell-only syndrome (SCOS) phenotype in mice. Here, we investigated the relevance of this gene to human male infertility with azoospermia caused by SCOS. Mutation analysis of SIN3A in the coding region was performed on 80 Japanese patients. However, no variants could be detected. This study suggests a lack of association of SIN3A gene sequence variants with azoospermia caused by SCOS in humans.

Effects of bethanechol chloride and distigmine bromide on postvoiding residual volume in patients with underactive bladder.

AIM: The efficacy of cholinergic drugs for reduction of post-voiding residual volume (PVR) in patients with underactive bladder is still controversial. This study was performed to examine whether cholinergic drugs have such an effect on PVR. METHODS: Patients with underactive bladder treated for more than two months with cholinergic drugs, which were later discontinued, were extracted retrospectively based on their charts. The changes in PVR, cholinesterase activity (ChE), renal function, and voiding function before and after discontinuation of cholinergic drugs were reviewed and analyzed. RESULTS: Twenty-nine patients were included in this study. In multiple linear regression analysis, the discontinuation of distigmine bromide (DB) was indicated as a significant covariate for PVR increase and ChE increase, while bethanechol chloride (BC) was not a significant covariate. The increase in ChE was significantly correlated with both PVR and voided volume after discontinuation of cholinergic drugs. CONCLUSION: DB could reduce PVR via a decrease in ChE. However, BC at doses up to 60 mg did not reduce PVR. DB may be recommended for the reduction of PVR in patients with underactive bladder.

A novel approach inducing transplant tolerance by activated invariant natural killer T cells with costimulatory blockade.

Invariant natural killer T (iNKT) cells are one of the innate lymphocytes that regulate immunity, although it is still elusive how iNKT cells should be manipulated for transplant tolerance. Here, we describe the potential of a novel approach using a ligand for iNKT cells and suboptimal dosage of antibody for CD40-CD40 ligand (L) blockade as a powerful method for mixed chimerism establishment after allogenic bone marrow transplantation in sublethally irradiated fully allo recipients. Mixed-chimera mice accepted subsequent cardiac allografts in a donor-specific manner. High amounts of type 2 helper T cytokines were detected right after iNKT cell activation, while subsequent interferon-gamma production by NK cells was effectively inhibited by CD40/CD40L blockade. Tolerogenic components, such as CD11c(low) mPDCA1(+) plasmacytoid dendritic cells and activated regulatory T cells (Tregs) expressing CD103, KLRG-1 and PD-1, were subsequently augmented. Those activating Tregs seem to be required for the establishment of chimerism because depletion of the Tregs 1 day before allogenic cell transfer resulted in a chimerism brake. These results collectively suggest that our new protocol makes it possible to induce donor-specific tolerance by enhancement of the innate ability for immune tolerance in place of the conventional immunosuppression.

Single-nucleotide polymorphisms in the LRWD1 gene may be a genetic risk factor for Japanese patients with Sertoli cell-only syndrome.

Genetic mechanisms have been implicated as a cause of some cases of male infertility. Recently, ten novel genes involved in human spermatogenesis, including human LRWD1, have been identified by expression microarray analysis of human testictissue. The human LRWD1 protein mediates the origin recognition complex in chromatin, which is critical for the initiation of pre-replication complex assembly in G1 and chromatin organization in post-G1 cells. The Lrwd1 gene expression is specific to the testis in mice. Therefore, we hypothesized that mutation or polymorphisms of LRWD1 participate in male infertility, especially azoospermia. To investigate whether LRWD1 gene defects are associated with azoospermia caused by SCOS and meiotic arrest (MA), mutational analysis was performed in 100 and 30 Japanese patients by direct sequencing of the coding regions, respectively. Statistical analysis was performed for patients with SCOS and MA and in 100 healthy control men. No mutations were found in LRWD1; however, three coding single-nucleotide polymorphisms (SNP1-SNP3) could be detected in the patients. The genotype and allele frequencies in SNP1 and SNP2 were notably higher in the SCOS group than in the control group (P < 0.05). These results suggest the critical role of LRWD1 in human spermatogenesis.

Direct androgen regulation of PDE5 gene or the lack thereof.

Inhibition of phosphodiesterase-5 (PDE5) is a well-known mechanism for the effective treatment of erectile dysfunction (ED). Androgen supplementation has also been prescribed for treating ED. However, it has been widely accepted that androgen can upregulate PDE5 expression, and thus creating a paradox in which a positive regulator of erectile function (androgen) could possibly increase the level of a negative regulator (PDE5). To solve this paradox, we conducted a systematic search of the PubMed and a non-systematic search of the Internet using PDE5, erectile, penis, testosterone and androgen as keywords. The retrieved papers were analyzed for data concerning the expression and regulation of PDE5 by androgens. Human and rat PDE5A gene sequences were retrieved from GenBank and computer-analyzed. The results showed that a putative androgen-response element (ARE) was reported in a study of human PDE5A gene promoter, and this prompted a separate study on whether androgen regulates PDE5 expression. The positive outcome in the latter study has since been cited in 17 review and editorial articles as the underlying mechanism for androgen's therapeutic effects on ED. In addition, five other research studies also reached the same conclusion. On the other hand, two independent studies on the genome-wide searches for androgen-regulated genes did not find PDE5A as a candidate. Sequence analysis conducted in this study also failed to find ARE in rat PDE5A gene. Two independent studies on Leydig cells also failed to find positive regulation of PDE5 expression by androgen. Two other studies found concomitant reduction of cavernous smooth muscle and PDE5 expression in castrated rats. One of these studies also found no effect of androgen on PDE5 expression in cultured cavernous smooth muscle cells. Thus, it appears that reduced PDE5 expression in castrated animals is due to reduced smooth muscle content and that PDE5A gene is not directly regulated by androgens.

Risedronate prevents persistent bone loss in prostate cancer patients treated with androgen deprivation therapy: results of a 2-year follow-up study.

Androgen deprivation therapy (ADT) for prostate cancer (PCa) causes bone loss. Although we reported previously that risedronate significantly recovers bone mineral density (BMD) for up to 12 months, there have been no reports with longer follow-up periods to date. This study extended our earlier series extending the follow-up period to 24 months. Eligible patients had histologically confirmed PCa without lumbar spine metastasis and underwent ADT. Lumbar spine BMD, urinary deoxypyridinoline (uDPD) and serum bone alkaline phosphatase were measured at 6, 12 and 24 months. Among the total of 96 patients, we analyzed 26 and 18 patients in risedronate administration and control groups, respectively. BMD relative to the young adult mean ratio, uDPD and serum bone alkaline phosphatase of the risedronate administration group recovered significantly after 24 months compared with the control group (P<0.0001, P=0.0001, and P<0.0001, respectively). Transient blurred vision, malaise and vertigo were observed in 1 patient each among the 46 patients treated with risedronate within 28 days after first administration. Oral administration of risedronate is safe and effective for the recovery of ADT-induced bone loss in PCa patients even at 24 months after commencement of treatment.

The relationship of serum and salivary cortisol levels to male sexual dysfunction as measured by the International Index of Erectile Function.

To evaluate the biomarkers of sexual function, we investigated the relationship between questionnaire responses and biological hormones such as testosterone (T) and cortisol (F) in serum and saliva. The study population included 105 men aged 30-72 years (mean: 49+/-4.5, median: 49). Levels of all serum hormones (Total-T, Free-T, Bioavailable-T, Total-F and Bioavailable-F) and salivary hormones (Saliva-T and Saliva-F) were measured directly by liquid chromatography/tandem mass spectrometry. The International Index of Erectile Function (IIEF) was used as a questionnaire to evaluate sexual dysfunction. Free-T and Bioavailable-T showed significant inverse correlations with age (P<0.01). In the group not taking antidepressants, the levels of Bioavailable-F and Saliva-F showed significant inverse correlations with a portion of the IIEF score (P<0.05). However, reductions in Bioavailable-T and Saliva-T showed no association with the IIEF score. In the group taking antidepressants, these hormone levels showed no correlation with IIEF.

Reduced-intensity unrelated cord blood transplantation for treatment of metastatic renal cell carcinoma: first evidence of cord-blood-versus-solid-tumor effect.

We report a 69-year-old man with cytokine-resistant metastatic renal cell carcinoma treated with reduced-intensity unrelated cord blood transplantation. The patient achieved durable donor engraftment with minimal graft-versus-host disease. The patient showed regression of metastatic disease, providing the first evidence of a graft-versus-tumor effect on a solid tumor resulting from cord blood graft.

Role of protein kinase C in central muscarinic inhibitory mechanisms regulating voiding in rats.

To evaluate the role of protein kinase C in central muscarinic mechanisms regulating voiding, cystometry was performed in conscious rats. Oxotremorine methiodide, a muscarinic agonist was injected i.c.v. in a dose (0.1 microg/rat) shown previously to alter voiding function. Oxotremorine methiodide was also tested after i.c.v. injection of chelerythrine chloride (a protein kinase C inhibitor, 2 microg/rat) or 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7, a protein kinase inhibitor, 5 nmol/rat). In untreated rats, oxotremorine methiodide elicited a bimodal response consisting of an initial increase in bladder capacity, maximal voiding pressure, pressure threshold and post voiding intravesical pressure, but reduced voiding efficiency and bladder compliance. The second response consisted of a decrease in bladder capacity and bladder compliance, increases in maximal voiding pressure and post voiding intravesical pressure, but no change in pressure threshold or voiding efficiency. However, approximately 20 min after pre-treatment with chelerythrine chloride or H-7 in doses that did not alter voiding function, oxotremorine methiodide decreased bladder capacity, increased maximal voiding pressure, but did not change pressure threshold or voiding efficiency. These results indicate that inhibitory and facilitatory muscarinic mechanisms in the brain that control voiding function involve different second messenger systems. Inhibitory mechanisms which are blocked by chelerythrine chloride or H-7 must involve protein kinase C and normally be inactive because the protein kinase inhibitors alone did not alter voiding. On the other hand, facilitatory muscarinic mechanisms which previous studies showed were tonically active are not mediated by chelerythrine chloride or H-7 sensitive signaling pathways.

Multiplex sequence-tagged site PCR for efficient screening of microdeletions in Y chromosome in infertile males with azoospermia or severe oligozoospermia.

The multiplex STS-PCR method was used to detect microdeletions in the long arm of the Y chromosome (Yq) of cytogenetically normal men. One hundred infertile men with azoospermia or oligozoospermia were screened with the multiplex PCR method using 58 STSs, which are specific to Yq for detecting microdeletions on this chromosome. Correlations between the microdeletions on Yq and phenotypes of spermatogenetic disturbance were also examined. Ten patients (10%) had microdeletions on Yq. Seven of the 60 azoospermic patients (11.7%), and 3 of the 40 oligozoospermic patients (7.5%) had microdeletions on Yq. None of the patients showed microdeletions in the AZFa region, but 2 had deletions in the AZFb region, another 2 in the AZFc region, including DAZ, and 1 had deletions in both the AZFb and in the AZFc, including RBM and DAZ. Single microdeletions were found in 4 patients, all of them in the AZFc around DAZ, and 1 patient had 2 microdeletions in the AZFb. The improved multiplex STS-PCR method efficiently detected microdeletions in 10% of azoospermic or severe oligozoospermic men who were cytogenetically normal. All of these microdeletions were presented in the AZFb and/or AZFc regions. This suggests that these regions contain candidate genes for spermatogenesis.

Analysis of SIDS-related civil and criminal court cases in Japan.

Thirty-three sudden infant death syndrome (SIDS)-related civil and criminal lawsuits in Japan were retrieved from judicial precedent databases "Hanrei Masutar (Judicial Decisions Master)" and "Hanrei Taikei (Judicial Decisions System) using "SIDS" as a keyword. Sleeping posture and developmental stage of occurrence were studied in each of the cases retrieved, whether or not a legal autopsy had been performed. The influence exerted on court decisions by Japanese definitions of SIDS as well as the relationship between causes of death and court decisions were studied. Of 33, two were criminal cases (business/professional negligence on the part of the defendants, leading to death), and the rest were civil cases (claims for damages). Because the decision handed down in both criminal cases was "cause of death unknown", these defendants were found innocent. One of these cases was argued in both the court of appeals and the superior court: these courts found SIDS to be the cause of death and consequently the claim for damages was rejected. Both criminal and civil courts dealt with another case: the former found the cause of death to be "unknown" and the defendant innocent, while the latter, finding SIDS the cause of death, declined to review. In cases where the sleeping posture was prone, courts tended to decide the cause of death to be suffocation, especially with neonates. Because diagnosis by exclusion is required in cases of a legal autopsy for SIDS, the diagnosis is difficult without an autopsy. Disagreements between the results of legal autopsy and court decisions occurred in eight cases. With such a discrepancy, a detailed case examination is necessary. In 1983, SIDS was defined in Japan in two different ways; one in a more strict sense and the other being more inclusive. The wider and narrower definitions were unified in 1995 by requiring a survey of the circumstances of death in addition to the narrower definition. Because of this situation, the two cases in the 1980s when legal autopsy was not enforced fell into the category of "SIDS in a wider sense." In no case was a defendant found guilty when the cause of death was judged to be either SIDS/ALTE or unknown. Four cases were rejected when the cause of death was judged to be neither due to suffocation nor SIDS, while seven were accepted either as cases of "joint faults that canceled each other," or as "partial acceptance." In Japan, official views concerning a SIDS diagnosis differ among pediatricians, legal scholars of forensic medicine and pathologists. These differences appeared to influence the legal decisions. Several conferences should be convened as soon as possible to provide an opportunity to resolve the main points of difference between these three professional groups and, thus, attain a unified view.

Optical monitoring of synaptic summation along the dendrites of CA1 pyramidal neurons.

The primary function of neurons is to integrate synaptic inputs and to transmit the results to other cells. Recent studies with somatic whole-cell recordings have shown that separate excitatory inputs to hippocampal or cortical pyramidal neurons are summated non-linearly. In the present study, we examined how postsynaptic potentials (PSPs) are summated along the dendrites employing fast optical voltage imaging techniques. Rat hippocampal slices were stained with a fluorescent voltage-sensitive dye (JPW1114) and optical signals were monitored with a 16 x 16 photodiode array system. Two independent input pathways were stimulated individually or in pairs through glass electrodes such that different locations of the dendrites received separate synaptic inputs. We found that (1) the summation of PSPs was sub-linear along the entirety of dendrites, (2) the blockade of GABA(A) receptors suppressed sub-linearity and (3) further blockade of GABA(B) receptors suppressed sub-linearity of the summation of separate inputs on apical dendrites. Our study demonstrates that pyramidal neurons integrate PSPs linearly along the entirety of dendrites; moreover, GABAergic inputs are responsible for maintaining sub-linear summation in CA1 pyramidal neurons.

Immunohistochemical detection of the 150-kDa oxygen-regulated protein in bladder cancer.

OBJECTIVE: To investigate the relationship between the expression of the 150-kDa oxygen-regulated protein (ORP150, which functions as a molecular chaperone in the endoplasmic reticulum for the folding and trafficking of newly synthesized proteins) and the aggressiveness of bladder cancer, and the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs), as the former is a secreting protein through the endoplasmic reticulum and the latter are closely involved in tumour invasion. MATERIALS AND METHODS: Thirty-nine cystectomy specimens, comprising 12 superficial (pT1) and 27 invasive (pT2-pT4) tumours, were immunohistochemically analysed using antibodies against ORP150, VEGF, MMP-1, MMP-2 and MMP-9. Staining was scored from 0 to 3, according to the ratio of positively staining cells. RESULTS: Staining was positive (score 1-3) for ORP150 in 10 of 12 superficial and 25 (93%) of the invasive tumours, with a significantly higher staining score for stage T4 than stage T1 tumours. The trend was the same for the staining score of MMP-2, and there was a significant correlation between ORP150 and MMP-2 expression. CONCLUSIONS: The expression of ORP150 was common in bladder cancer, with a tendency for greater expression in higher stages. The significant correlation between ORP150 and MMP-2 expression suggests that ORP150 acts as a molecular chaperone for MMP-2 secretion and thus tumour invasion.

Inhibition of sperm production in mice by annexin V microinjected into seminiferous tubules: possible etiology of phagocytic clearance of apoptotic spermatogenic cells and male infertility.

Many differentiating spermatogenic cells die by apoptosis during the process of mammalian spermatogenesis. However, very few apoptotic spermatogenic cells are detected by histological examination of the testis, probably due to the rapid elimination of dying cells by phagocytosis. Previous in vitro studies showed that Sertoli cells selectively phagocytose dying spermatogenic cells by recognizing the membrane phospholipid phosphatidylserine (PS), which is exposed to the surface of spermatogenic cells during apoptosis. We examined here whether PS-mediated phagocytosis of apoptotic spermatogenic cells occurs in vivo. For this purpose, the PS-binding protein annexin V was microinjected into the seminiferous tubules of normal live mice, and their testes were examined. The injection of annexin V caused no histological changes in the testis, but significantly increased the number of apoptotic spermatogenic cells as assessed by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay. The number of Sertoli cells did not change in the annexin V-injected testes, and annexin V itself did not induce apoptosis in primary cultured spermatogenic cells. These results indicate that annexin V inhibited the phagocytic clearance of apoptotic spermatogenic cells and suggest that PS-mediated phagocytosis of those cells occurs in vivo. Furthermore, the injection of annexin V into the seminiferous tubules brought about a significant reduction in the number of spermatogenic cells and epididymal sperm in anticancer drug-treated mice. This suggests that the elimination of apoptotic spermatogenic cells is required for the production of sperm.

Antitumor effect of reduction of 150-kDa oxygen-regulated protein expression in human prostate cancer cells.

Heat shock proteins (HSPs)/stress proteins are molecular chaperones that are induced by various environmental and physiological stimuli. Evidence of the relations between the expression of HSPs and the regulation of cell growth or transformation has accumulated. The 150-kDa oxygen-regulated protein (ORP150), a new member of HSP family, functions as a molecular chaperone in the endoplasmic reticulum. We have examined whether transduced antisense ORP150 cDNA reduces tumorigenicity and angiogenicity. Relations between these stress proteins and cancer and possibilities for anticancer gene therapy are described.

[A case of plasmacytoma involving adrenal gland].

We report a case of extramedullary plasmacytoma involving the right adrenal gland. A 52-year-old male was introduced under diagnosis of right adrenal tumor which was found incidentally by computerized tomography in a health check up. Laboratory data showed the presence of M protein and elevation of monoclonal lambda type IgG. It was a hormonally non-active tumor involving the adrenal area. Extramedullary plasmacytoma was confirmed by histological analysis of the resected specimen after laparoscopic right adrenalectomy. Extramedullary plasmacytoma is an uncommon neoplasm and occurs most frequently in the upper respiratory tract and is fairly rare in the adrenal area.

Role of endogenous nitric oxide generation in the regulation of vascular tone and reactivity in small vessels as investigated in transgenic mice using synchrotron radiation microangiography.

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays a central role in regulation of vascular tone and reactivity. The purpose of this study is to clarify the basal tone and microvascular reactivity in eNOS-overexpressing transgenic (Tg) mice in vivo with a microangiography system using monochromatic synchrotron radiation (SR). The mouse femoral artery was cannulated, nonionic contrast media was injected, and microangiography was performed in hindlimbs of mice. Serial images of the small blood vessels (diameter <200 microm) were recorded by the SR microangiography system. At basal conditions, the diameter of tibial arteries in eNOS-Tg mice was larger than that of wild-type mice (179 +/- 8 versus 132 +/- 8 microm; P < 0.01). l-NAME treatment decreased the vessel diameter and canceled the difference in vessel diameters between two genotypes. Acetylcholine- and sodium nitroprusside-induced relaxations of small vessels were significantly reduced in Tg mice compared with wild-type mice (35.0 +/- 9.4 versus 61.6 +/- 6.7%, 85.0 +/- 10.2 versus 97.3 +/- 6.7% of the maximum relaxation, respectively). Our data provide the evidence that overproduced NO from endothelium reduces vascular tone and plays a pivotal role in regulation of vascular tone in small vessels. Furthermore, the reduced NO-mediated relaxation in small vessels of eNOS-Tg mice is demonstrated for the first time in vivo. SR microangiography allows us to evaluate the reactivity in small-sized vessels and appears to be a powerful tool for assessing the microvascular circulation in vivo.