A prospective phase II study of combined androgen blockade in patients with androgen receptor-positive metastatic or locally advanced unresectable salivary gland carcinoma.

Background: There is no standard first-line chemotherapy for recurrent/metastatic (RM) or unresectable locally advanced (LA) salivary gland carcinoma (SGC). Patients and methods: We conducted a single institution, open-label, single arm, phase II trial of combined androgen blockade (CAB) for androgen receptor (AR)-positive SGC. Leuprorelin acetate was administered subcutaneously at a dose of 3.75 mg every 4 weeks. Bicalutamide was administered orally at a daily dose of 80 mg. Patients were treated until progressive disease or unacceptable toxicities. Results: Thirty-six eligible patients were enrolled. Thirty-three patients had RM disease and three patients had LA disease. The pathological diagnoses were salivary duct carcinoma (34 patients, 94%) and adenocarcinoma, NOS (two patients, 6%). The best overall response rate was 41.7% [n = 15, 95% confidence interval (CI), 25.5%-59.2%], the clinical benefit rate was 75.0% (n = 27, 95% CI, 57.8%-87.9%). The median progression-free survival was 8.8 months (95% CI, 6.3-12.3 months) and the median overall survival was 30.5 months (95% CI, 16.8 months to not reached). Additional analyses between treatment outcomes and clinicopathological factors or biomarkers including AR positivity, human epidermal growth factor receptor 2 status, and its complex downstream signaling pathway gene mutations showed no statistically significant differences. Elevated grade 3 liver transaminases and increased serum creatinine were reported in two patients, respectively. Discontinuation of leuprorelin acetate or bicalutamide due to adverse event occurred in one patient. Conclusion: This study suggests that CAB has equivalent efficacy and less toxicity for patients with AR-positive RM or unresectable LA SGC compared with conventional chemotherapy, which warrants further study. Clinical Trial Registration: UMIN-CTR (http://www.umin.ac.jp/ctr/index-j.htm), identification number: UMIN000005703.

Lateral gain is impaired in macular degeneration and can be targeted to restore vision in mice.

Macular degeneration is a leading cause of blindness. Treatments to rescue vision are currently limited. Here, we study how loss of central vision affects lateral feedback to spared areas of the human retina. We identify a cone-driven gain control mechanism that reduces visual function beyond the atrophic area in macular degeneration. This finding provides an insight into the negative effects of geographic atrophy on vision. Therefore, we develop a strategy to restore this feedback mechanism, through activation of laterally projecting cells. This results in improved vision in Cnga3-/- mice, which lack cone function, as well as a mouse model of geographic atrophy. Our work shows that a loss of lateral gain control contributes to the vision deficit in macular degeneration. Furthermore, in mouse models we show that lateral feedback can be harnessed to improve vision following retinal degeneration.

Gut microbiota development of preterm infants hospitalised in intensive care units.

Gut microbiome development affects infant health and postnatal physiology. The gut microbe assemblages of preterm infants have been reported to be different from that of healthy term infants. However, the patterns of ecosystem development and inter-individual differences remain poorly understood. We investigated hospitalised preterm infant gut microbiota development using 16S rRNA gene amplicons and the metabolic profiles of 268 stool samples obtained from 17 intensive care and 42 term infants to elucidate the dynamics and equilibria of the developing microbiota. Infant gut microbiota were predominated by Gram-positive cocci, Enterobacteriaceae or Bifidobacteriaceae, which showed sequential transitions to Bifidobacteriaceae-dominated microbiota. In neonatal intensive care unit preterm infants (NICU preterm infants), Staphylococcaceae abundance was higher immediately after birth than in healthy term infants, and Bifidobacteriaceae colonisation tended to be delayed. No specific NICU-cared infant enterotype-like cluster was observed, suggesting that the constrained environment only affected the pace of transition, but not infant gut microbiota equilibrium. Moreover, infants with Bifidobacteriaceae-dominated microbiota showed higher acetate concentrations and lower pH, which have been associated with host health. Our data provides an in-depth understanding of gut microbiota development in NICU preterm infants and complements earlier studies. Understanding the patterns and inter-individual differences of the preterm infant gut ecosystem is the first step towards controlling the risk of diseases in premature infants by targeting intestinal microbiota.

Galacto-oligosaccharides ameliorate dysbiotic Bifidobacteriaceae decline in Japanese patients with type 2 diabetes.

Gut microbiota affects the host's metabolism, and it is suggested that there are differences in gut microbiota composition between patients with type 2 diabetes and healthy individuals. Additionally, dysbiosis may increase the concentration of lipopolysaccharides (LPS), causing metabolic endotoxemia, which induces impaired glucose tolerance. Several studies have reported relationships between metabolic diseases and the gut microbiota; and prebiotics, such as oligosaccharides, are commonly consumed to regulate gut microbiotas in healthy individuals. Galacto-oligosaccharides (GOS) are a major prebiotic, which specifically increase Bifidobacteriaceae abundance. Recent studies have reported that Bifidobacteriaceae improved metabolic endotoxemia or impaired glucose tolerance. However, there are few studies reporting the effects of GOS on patients with type 2 diabetes. In the current study, we compared clinical parameters, faecal gut microbiota, their associated metabolic products and their components such as LPS, and LPS-binding protein (LBP) produced by the host, between patients with diabetes and healthy controls. We then assessed the effects of GOS on glycaemic control, and gut microbiotas and metabolites in patients with type 2 diabetes in a double-blind controlled manner. LBP levels were significantly higher in patients with diabetes than those of healthy subjects, which was consistent with previous reports. The abundance of Bifidobacteriaceae and the diversity of intestinal microbiota were significantly lower in patients with diabetes than in healthy subjects. Interestingly, Bifidobacteriaceae was markedly restored in patients with diabetes after consumption of GOS, whereas LBP and glucose tolerance did not improve during this short-term trial period. In the present study, we demonstrated that GOS can ameliorate dysbiosis in patients with diabetes, and continuous intake of GOS may be a promising method for managing type 2 diabetes.

The role of TNFalpha and IL-17 in the development of excess IL-1 signaling-induced inflammatory diseases in IL-1 receptor antagonist-deficient mice.

IL-1 receptor antagonist (IL-1Ra)-deficient mice spontaneously develop several inflammatory diseases, resembling rheumatoid arthritis, aortitis, and psoriasis in humans. As adoptive T cell transplantation could induce arthritis and aortitis in recipient mice, it was suggested that an autoimmune process is involved in the development of diseases. In contrast, as dermatitis developed in scid/scid-IL-IRa-deficient mice and could not be induced by T cell transfer, a T cell-independent mechanism was suggested. The expression of proinflammatory cytokines was augmented at the inflammatory sites. The development of arthritis and aortitis was significantly suppressed by the deficiency of TNFalpha or IL-17. The development of dermatitis was also inhibited by the deficiency of TNFalpha. These observations suggest that TNFalpha and IL-17 play a crucial role in the development of autoimmunity downstream of IL-1 signaling, and excess IL-1 signaling-induced TNFalpha also induces skin inflammation in a T cell-independent manner.

Infant formula with galacto-oligosaccharides (OM55N) stimulates the growth of indigenous bifidobacteria in healthy term infants.

The objective of the study was to investigate whether an infant formula supplemented with galacto-oligosaccharides (GOS; OM55N) was able to stimulate the growth of indigenous bifidobacteria and to establish microbiota similar to that of breastfed infants. A randomised, double-blind, placebo-controlled trial was performed using 35 healthy term infants (31-54 days of age; 42±6 days) to determine whether infant formula with 0.3 g/dl GOS (OM55N) stimulated the growth of bifidobacteria in the infants' guts. At the trial onset and 2 weeks after, the infants' faecal samples were examined for microbiota composition (bacterial abundance and α-diversity) and faecal characteristics. Among the 35 infants, 5 were withdrawn and 8 were excluded from the final evaluation before breaking the blinding since the indigenous bifidobacteria were not detected at the trial onset. After 2 weeks, the abundance of Bifidobacteriaceae was significantly increased in the GOS feeding group compared to the control (+11.6±24.1% vs -3.9±13.0%; P=0.043). The Shannon index, which accounts for both abundance and evenness of the present species, was significantly decreased with GOS supplementation (-0.1±0.4 vs +0.4±0.4; P=0.014). Faecal characteristics such as pH and organic acids were similar in both groups, with no statistical differences. No adverse side effects related to the formula consumption were reported. Although the concentration of GOS was relatively low, the infant formula with GOS increased the abundance of bifidobacteria and resulted in a reduced α-diversity of the microbiota.

Sympathetically induced myocardial ischaemia causes the heart to release plasma kinin.

A recently developed highly sensitive radioimmunoassay method for detecting plasma kinin was used to re-evaluate the results of previous studies, in which plasma kinin had been measured with a bioassay method. To clarify the mechanism of plasma kinin release in global myocardial ischaemia the left main coronary artery was cannulated using a Griggs type autoperfusing cannula after pentobarbital anaesthesia in open chest dogs. The animals were divided into a non-coronary constricted group (n = 4) and a moderately coronary constricted group (n = 7). Cardiac sympathetic nerve stimulation (10 V, 4 Hz, 2 ms duration) was given to both groups. Haemodynamic recordings and blood samples were taken before and after coronary constriction as well as after sympathetic nerve stimulation. The arterial and coronary sinus plasma kinin concentrations were determined with the new radioimmunoassay method. After sympathetic nerve stimulation apparent myocardial ischaemia occurred and the plasma kinin concentration in coronary sinus blood increased significantly in the constricted group. In the non-constricted group, however, myocardial ischaemia did not appear and no significant change in coronary sinus plasma kinin concentrations was seen. These findings show that there was a pronounced release of plasma kinin from the heart when apparent myocardial ischaemia occurred.

GABAA receptor-mediated input change on orexin neurons following sleep deprivation in mice.

Orexins are bioactive peptides, which have been shown to play a pivotal role in vigilance state transitions: the loss of orexin-producing neurons (orexin neurons) leads to narcolepsy with cataplexy in the human. However, the effect of the need for sleep (i.e., sleep pressure) on orexin neurons remains largely unknown. Here, we found that immunostaining intensities of the α1 subunit of the GABAA receptor and neuroligin 2, which is involved in inhibitory synapse specialization, on orexin neurons of mouse brain were significantly increased by 6-h sleep deprivation. In contrast, we noted that immunostaining intensities of the α2, γ2, and ß2/3 subunits of the GABAA receptor and Huntingtin-associated protein 1, which is involved in GABAAR trafficking, were not changed by 6-h sleep deprivation. Using a slice patch recording, orexin neurons demonstrated increased sensitivity to a GABAA receptor agonist together with synaptic plasticity changes after sleep deprivation when compared with an ad lib sleep condition. In summary, the GABAergic input property of orexin neurons responds rapidly to sleep deprivation. This molecular response of orexin neurons may thus play a role in the changes that accompany the need for sleep following prolonged wakefulness, in particular the decreased probability of a transition to wakefulness once recovery sleep has begun.

Age-related changes of gene expression in mouse kidney: fluorescence differential display--PCR analyses.

We used a fluorescence differential display--PCR (FDD-PCR) technique to analyze the genes expressed in mouse kidneys collected at nine different developmental stages ranging from 3 days to 15 months after birth. We found ten genes that were age-dependent and differentially-expressed in the kidneys during our experimental period. We confirmed by comparative RT-PCR that of the ten cDNAs, seven showed reproducible age-dependent expression. Four of the nucleotide sequences of these cDNA clones, had high homology with known genes (fibronectin, soluble guanylyl cyclase alpha-1 subunit, cytosolic aldehyde dehydrogenase and mitochondrial DNA), and three with expressed sequence tags of unknown genes. The FDD-PCR method was very useful for detecting new age-related genes expressed differentially in the mouse kidney.

Detection of alternatively spliced variant messages of Fas gene and mutational screening of Fas and Fas ligand coding regions in peripheral blood mononuclear cells derived from silicosis patients.

Silicosis is clinically characterized not only by respiratory disorders but by immunological abnormalities such as the appearance of autoantibodies and complications of autoimmune diseases. Dysregulation of apoptosis, particularly in the Fas/Fas ligand (FasL) pathway, has been considered to play a role in the pathogenesis of autoimmune diseases. It has been found that serum soluble Fas (sFas) levels are elevated in silicosis patients (SIL) and the sFas message is dominantly expressed in peripheral blood mononuclear cells (PBMC) derived from these individuals. In the present study, one tried to detect alternatively spliced variant messages including typical sFas message and found four that were highly and frequently expressed, and which possess a signal peptide domain, but not transmembrane and signal transducing domains, in PBMC derived from SIL. Functional mutations were not detected in Fas and FasL genes in silicosis PBMC. Still, alternative spliced variants of the Fas gene including typical sFas message appear to play an important role in the immunological dysregulation in SIL.

Five age-dependently expressed genes in mouse brain revealed by the fluorescence differential display-PCR technique.

We used a fluorescence differential display-PCR (FDD-PCR) technique to analyze the genes expressed in mouse brains collected at nine different developmental stages ranging from 3 days to 15 months after birth, and 5 age-dependently expressed genes were found. Age-dependent expression of each of these 5 genes was confirmed by quantitative real-time PCR analysis. Of the 5 genes, 4 (B1-B4) had high homology with the nucleotide sequences of cDNA clones of known mouse genes (myelin proteolipid protein, transferrin, embryo cDNA from the RIKEN full-length enriched library, and protein tyrosine phosphatase), and the rest (B5) with expressed sequence tags of an unknown gene. Sequencing analysis of the full-length cDNA constructed based on the B5 sequence demonstrated that the gene product of B5 was identical to G-substrate, a specific substrate for cGMP-dependent protein kinase. The expression patterns of known genes obtained in our study may provide a further opportunity to investigate the biological and physiological roles of the proteins they encode.

Pulmonary artery connection in the Fontan procedure. Flexible polytetrafluoroethylene conduit for expansion.

From clinical experiences with the Fontan operation in six cases, a few practical contrivances and operative steps are described. We stress that meticulous care should be taken not to cause any stenotic complication in the outflow tract toward the pulmonary artery, particularly by traction of the right atrial appendage only to achieve a direct anastomosis. Conduit repair would be necessitated by cases in order to expand the indicative criteria vertically as well as horizontally. For conduit material, we used consecutively nonvalved polytetrafluoroethylene that was reinforced by handmade stainless steel wire ring (Gore-Tex, Inc) or by spirally built-in stent (IMPRA, Inc). The latter was eventually useful in obtaining natural curving of the conduit without kinking or compression. Some contrivances in anastomosing a conduit were also proposed to achieve an excellent result. We believe these practical contrivances will serve for expansion of the indicative criteria and promise improved operative outcome.

ERP development in the rat in the course of learning two-tone discrimination task.

To clarify some neurophysiological aspects of learning, we investigated the relationship between the course of learning and development of ERP and investigated developmental processes of ERPs. Nine male Sprague-Dawley rats were trained for a two-tone discrimination task and rat P3 and N1 component were longitudinally recorded. Both rat P3 and N1 gradually increased with learning only for target tones. An improvement in the proportion of correct responses preceded the increase in ERPs, and the increase in P3 and N1 proceeded almost simultaneously. These findings suggest that multiple kinds of information processing were acquired with learning the two-tone discrimination task. ERP development could be utilized as an index of establishment of learning.

Rapid identification of human intestinal bifidobacteria by 16S rRNA-targeted species- and group-specific primers.

On the basis of 16S rRNA sequences, species- and group-specific primers for Bifidobacterium adolescentis, B. angulatum, B. bifidum, B. breve, the B. catenulatum group (B. catenulatum and B. pseudocatenulatum), and the B. longum group (B. longum and B. infantis), which are species commonly found in human intestinal tracts, were developed. The specificity of these primers was confirmed through the use of DNA extracted from 46 strains of 31 Bifidobacterium species, as well as 14 non-bifidobacterial species that are the predominant bacteria in the human intestinal tract. The present species-specific primers were applied to the identification of 43 isolated strains, consisting of six strains of B. adolescentis, eight of the B. catenulatum group, seven of B. bifidum, nine of B. breve, and 13 of the B. longum group.

A novel protein interacts with a clock-related protein, rPer1.

Mammalian Per proteins are thought to be important in the mechanism of circadian rhythm. We identified a novel protein PIPS (Per1 interacting protein of the suprachiasmatic nucleus) with the yeast two-hybrid system using PAS domain of rat Per1 (rPer1) as a bait. PIPS is about a 180-kDa protein and expressed mainly in the brain, especially in the hypothalamus including the suprachiasmatic nuclei (SCN). PIPS interacts with mouse Per1 (mPer1) in vitro and in cultured cells transfected with both molecules. Furthermore, it was found that mPer1 translocated PIPS into the nuclei in the cultured cells. Thus, these findings suggest a possibility that PIPS is involved in the feedback loop or output mechanism of circadian rhythm through interacting with Per1 in the SCN.

Specific and chemoselective multi-alpha-arylation reaction of benzoylformic acid with or without decarbonylation in P(2)O(5)-MsOH and related acidic media

In P(2)O(5)-MsOH, or related acidic media, benzoylformic acid (1) undergoes three types of di- or mono-alpha-arylation reactions with or without decarbonylation ((1) decarbonylative alpha, alpha-diarylation, yielding triarylmethanols 6, (2) decarbonylative alpha-monoarylation, giving benzophenone derivatives 7, and (3) alpha,alpha-diarylation without decarbonylation, affording diarylated carboxylic acids 5) and one simple decarbonylation, without arylation, to form benzoic acid (8), instead of the conventional Friedel-Crafts acylation type reaction. The product ratios are governed by the capability of the acidic medium to form mixed anhydrides with carboxylic acids and the ability of the arenes to accept electrophiles.

Biosynthesis of IL-8 and endothelin-1,2 by immortal simian virus 40 tsA-transformed human endothelial cells.

Immortal human umbilical cord vein endothelial cells (HUVEC-SV40) established in our laboratory by gene transfection of plasmid pSVts58neo were analyzed for their biological activities. The growth and cellular viability of these immortal cells were temperature dependent. Typical bioactive products of endothelial cells, IL-8 and endothelins, were released into the culture medium as in the case of mortal cells. The production of IL-8 was temperature dependent and significantly increased in cells incubated at 37 degrees C as compared with those incubated at 33 degrees C. Such a temperature dependence was not observed in endothelin production. Stimulation of cells with both TNF-alpha and IFN-gamma increased the biosynthesis of IL-8 or endothelins significantly (p<0.05). From these results, it became clear that immortal HUVEC-SV40 cells also secrete IL-8 or endothelins in the same manner as mortal cells, and that they react to the stimulation by cytokines to promote IL-8 or endothelin production.

Man-made mineral fibers induce apoptosis of human peripheral blood mononuclear cells similarly to chrysotile B.

The aim of this study was to investigate whether man-made mineral fibers (MMMF) induce apoptosis of human peripheral blood mononuclear cells (PBMC), as we recently demonstrated for chrysotile B. In vitro cultivation of PBMC with various MMMF as well as chrysotile B clearly produced apoptotic cells. The alteration of the expression for apoptosis related genes at the mRNA level during in vitro cultures of PBMC with various MMMF revealed upregulation of Flice and Apaf-1 genes and down regulation of TNF receptor 1 and Bid genes. These results indicate that MMMF induce apoptosis of PBMC in a similar manner to chrysotile B. However, the process may be mediated not only by the Fas-related apoptotic pathway but also a mitochondrial pathway. Thus, one should be aware that respiratory and immunological abnormalities may occur in workers who are exposed to MMMFs.

Evaluation of cases with silicosis using the parameters related to Fas-mediated apoptosis.

To establish a new clinical index for immunological abnormalities occurring in silicosis, several clinical parameters related to Fas-mediated apoptosis; i.e., membrane Fas expression on peripheral blood lymphocytes (mFas), serum soluble Fas levels (sFas), serum soluble Fas ligand levels (sFasL), and soluble/membrane Fas mRNA expression ratios (s/mFas ExR) in peripheral blood mononuclear cells (PBMC) were investigated. Fifty-eight silicosis patients with no clinical symptoms of autoimmune diseases were the subjects of this study. Factor analysis was performed using 12 clinical parameters including four parameters related to Fas-mediated apoptosis. Two common factors were identified. Factor 1 which consisted of the following parameters; duration of exposure, symptomatic dyspnea, PO2, PCO2, and A-aDO2, should be designated as the respiratory factor for cases with silicosis. The parameters of factor 2 were serum IgG, sFas with high factor loading, titer of ANA, sFasL, and s/mFas ExR. These parameters of factor 2 are indicative of the immunological disorders occurring in silicosis cases. Some cases exhibited abnormalities in parameters of factor 2 but not factor 1. The factor analysis clearly demonstrated that the parameters related to Fas-mediated apoptosis should be the most beneficial for predicting the pre-clinical status of complicated autoimmune diseases in silicosis.