Glycaemic control boosts glucosylated nanocarrier crossing the BBB into the brain.

Recently, nanocarriers that transport bioactive substances to a target site in the body have attracted considerable attention and undergone rapid progression in terms of the state of the art. However, few nanocarriers can enter the brain via a systemic route through the blood-brain barrier (BBB) to efficiently reach neurons. Here we prepare a self-assembled supramolecular nanocarrier with a surface featuring properly configured glucose. The BBB crossing and brain accumulation of this nanocarrier are boosted by the rapid glycaemic increase after fasting and by the putative phenomenon of the highly expressed glucose transporter-1 (GLUT1) in brain capillary endothelial cells migrating from the luminal to the abluminal plasma membrane. The precisely controlled glucose density on the surface of the nanocarrier enables the regulation of its distribution within the brain, and thus is successfully optimized to increase the number of nanocarriers accumulating in neurons.There are only a few examples of nanocarriers that can transport bioactive substances across the blood-brain barrier. Here the authors show that by rapid glycaemic increase the accumulation of a glucosylated nanocarrier in the brain can be controlled.

Identification of ecdysteroidogenic enzyme genes and their expression during pupal diapause in the cabbage armyworm, Mamestra brassicae.

In this study, we identified ecdysteroidogenic enzymes in the cabbage armyworm, Mamestra brassicae, and demonstrated reduced expression of these genes during diapause. Some insects employ a temporary developmental arrest, diapause, to survive in severe environments. The titres of the moulting hormone ecdysteroid were reduced in diapause pupae of M. brassicae; therefore, ecdysteroidogenesis might be suppressed by a diapause-specific mechanism. To clarify expression changes of ecdysteroidogenic enzyme genes during diapause in M. brassicae, we first identified the genes for seven ecdysteroidogenic enzymes: Neverland, Non-molting glossy (Nm-g), CYP307A1 (Spook), CYP306A1 (Phantom), CYP302A1 (Disembodied), CYP315A1 (Shadow) and CYP314A1 (Shade). Enzymatic assays using heterologous expression in Drosophila Schneider 2 (S2) cells and analysis of mRNA distribution indicated that the identified genes were ecdysteroidogenic enzymes of M. brassicae. Expression levels of these ecdysteroidogenic enzyme genes were compared between prothoracic glands in different pupal stages throughout diapause. Immediately after pupation, diapause-destined pupae showed similar expression levels of ecdysteroidogenic enzyme genes to those of nondiapause pupae. All of these genes showed reduced gene expression after diapause initiation. Expression was immediately increased in diapause-destined pupae at the postdiapause quiescence phase. These results indicate that reduced expression of ecdysteroidogenic enzyme genes suppresses ecdysteroidogenesis and maintains developmental arrest during diapause.

Association of the clinical efficacy of vancomycin with the novel pharmacokinetic parameter area under the trough level (AUTL) in elderly patients with hospital-acquired pneumonia.

WHAT IS KNOWN AND OBJECTIVE: The pharmacokinetic-pharmacodynamic parameter that best predicts the efficacy of vancomycin is the ratio of the area under the concentration versus time curve (AUC) to the minimum inhibitory concentration (MIC). A 24-h AUC (AUC24 )/MIC ratio ≥ 400 was recommended in an American consensus review, but vancomycin treatment occasionally fails despite maintenance of AUC24 /MIC ≥ 400. We evaluated the association between clinical efficacy of vancomycin and two novel pharmacokinetic parameters, the 'area under the trough level' (AUTL) and the 'area above the trough level' (AATL), in hospitalized elderly patients with methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. METHODS: The subjects were hospitalized elderly patients who were administered vancomycin for treatment of MRSA pneumonia between 2006 and 2012 at Sasebo Chuo Hospital (Nagasaki, Japan). Pharmacokinetic parameters of vancomycin were estimated for each patient by Bayesian analysis using population pharmacokinetic parameters for Japanese patients. Based on the patient-specific parameters thus obtained, AUC24 values were calculated as the vancomycin dosage divided by vancomycin clearance. AUTL was calculated as the trough serum concentration multiplied by 24 h, whereas AATL was calculated by subtracting AUTL from AUC24 . RESULTS AND DISCUSSION: Logistic regression analysis demonstrated that efficacy of vancomycin was more strongly associated with AUTL than AUC24 . The optimal cut-off value of AUTL was 331 µg∙h/mL, which means that the optimal cut-off value of the trough serum concentration was 13·8 µg/mL. WHAT IS NEW AND CONCLUSION: Efficacy of vancomycin was associated with AUTL, a novel pharmacokinetic parameter. Determining the target AUTL or trough concentration may enhance the efficacy of vancomycin therapy in elderly patients with MRSA pneumonia. Given that nephrotoxicity may increase with a Ctrough in excess of 15 µg/mL, this level should ideally not be exceeded.

TU-E-BRB-01: Similar-Case-Based Optimization of Beam Arrangements in Stereotactic Body Radiation Therapy.

PURPOSE: The quality of a treatment plan for stereotactic body radiotherapy (SBRT) depends on an experience of each treatment planner. Therefore, the treatment plans are subjectively determined by comparison of several treatment plans developed by time consuming iterative manners, while considering the benefit to a tumor and the risk to the surrounding normal tissues. The aim of our study was to develop an automated optimization method for beam arrangements based on similar cases in a database including plans designed by senior experienced treatment planners. METHODS: Our proposed method consists of three steps. First, similar cases were automatically selected based on image features from the treatment planning point of view. We defined four types of image features relevant to planning target volume (PTV) location, PTV shape, lung size, and spinal cord positional features. Second, the beam angles of the similar case were registered to the objective case with respect to lung regions using a linear registration technique. Third, the beam direction of the objective case was locally optimized based on the cost function considering radiation absorption in normal tissues and organs at risk. The proposed method was evaluated with 10 test cases and a treatment planning database including 81 cases by using eight planning evaluation indices such as D95, lung V20, and maximum spinal cord dose. RESULTS: The proposed method may provide usable beam directions, which have no statistically significant differences with the original beam directions (P > 0.05) in terms of the seven planning evaluation indices. Moreover, the mean value of D95 for 10 test cases was improved with a statistically significant difference by using the proposed method, compared with the original beam directions (P = 0.03). CONCLUSIONS: The proposed method could be used as a computer-assisted treatment planning tool for determination of beam directions in SBRT.

WE-G-BRCD-04: Development of Automated Determination Method of Robust Beam Directions against Patient Setup Errors Based on Spatial Distribution of Electron Density in Particle Therapy.

PURPOSE: The three-dimensional (3D) dose distribution covering a tumor region tends to be more breakable if the beam's eye view (BEV) of the 3D electron density (ED) map in a beam direction changes more abruptly with large fluctuations. Our aim of this study was to develop an automated determination method of robust beam directions against the patient setup error based on the ED-based BEV in the beam direction in the particle therapy. METHODS: The basic idea of our proposed method was to find the robust beam directions, whose the ED-based BEV has the spatial fluctuations with low special frequency and small amplitude. For evaluation of the spatial fluctuation in the ED-based BEV in a beam direction, we obtained power spectra of the ED-based BEVs in all directions, i.e., 0 to 355 degree, with an interval of 5 degree. It was assumed that as the average spatial frequency and amplitude of the fluctuation in the ED-based BEV in a beam direction is lower and smaller, respectively, the absolute value of a gradient of the power spectrum becomes larger. Therefore the gradient of the power spectrum was calculated for determination of the robust beam direction. The ED-based BEV was produced by projecting a 3D electron density map derived from the computed tomography (CT) image from a beam source to the distal end of a planning target volume (PTV). Four cases of head and neck cancer patients were selected for evaluation of the proposed method. RESULTS: As a preliminary result, radiation oncologists agreed with most beam directions, which seem to be robust against patient setup errors, suggested by the proposed method. CONCLUSIONS: Our proposed method could be feasible to suggest the robust beam directions against patient setup errors in hadron particle therapy.

SU-E-J-117: Computer-Assisted Verification of Accumulated Dose Distribution during the Treatment Time Based on Estimation of Four-Dimensional Dose Distribution Using an Electronic Portal Imaging Device.

PURPOSE: The accumulated dose distributions during the course of radiation treatment are substantially important for verifying whether treatment dose distributions are produced according to planned dose distributions. The purpose of this study was to develop a computer-assisted verification method of accumulated dose distribution during the irradiation of a tumor based on estimation of four-dimensional (4D) dose distribution using an electronic portal imaging device (EPID). METHODS: The 4D 'treatment' computed tomography (CT) images during the irradiation were estimated based on affine transformations including respiratory motions, which were derived by registration between a planning portal dose image and treatment portal dose dynamic image. Planning portal dose images were calculated from planning CT images and an algorithm for calculation of dose spatial distribution. Treatment portal dose images were estimated from EPID dynamic images obtained during a treatment time. The planning portal dose images were registered to the treatment portal dose images to obtain the affine transformation, which could include respiratory motion in a patient body. The CT images at a treatment time were determined by deforming the planning CT images using the affine transformation matrix. 4D dose distributions during a treatment delivery were obtained by applying a dose calculation algorithm to the 4D treatment CT images. Finally, accumulated dose distributions during the course of radiation treatment were verified with planned dose distributions. RESULTS: We applied the proposed method to EPID dynamic images of 2 lung cancer patients, and evaluated the difference in accumulated dose distribution between the plan and treatment using a gamma evaluation (3mm/3%). The average pass rate for 2 cases was 78.2%. CONCLUSIONS: The proposed method can be used for adaptively modifying the plan based on the dose discrepancy between the plan and treatment. This work was partially supported by Grant-in-Aid for Scientific Research (C) (22611011) and Okawa Foundation for Information and Telecommunications.

SU-E-I-91: Development of a Compact Radiographic Simulator Using Microsoft Kinect.

PURPOSE: Radiographic simulator system is useful for learning radiographic techniques and confirmation of positioning before x-ray irradiation. Conventional x-ray simulators have drawbacks in cost and size, and are only applicable to situations in which position of the object does not change. Therefore, we have developed a new radiographic simulator system using an infrared-ray based three-dimensional shape measurement device (Microsoft Kinect). METHODS: We made a computer program using OpenCV and OpenNI for processing of depth image data obtained from Kinect, and calculated the exact distance from Kinect to the object by calibration. Theobject was measured from various directions, and positional relationship between the x-ray tube and the object was obtained. X-ray projection images were calculated by projecting x-rays onto the mathematical three-dimensional CT data of a head phantom with almost the same size. The object was rotated from 0 degree (standard position) through 90 degrees in increments of 10 degrees, and the accuracy of the measured rotation angle values was evaluated. In order to improve the computational time, the projection image size was changed (512*512, 256*256, and 128*128). RESULTS: The x-ray simulation images corresponding to the radiographic images produced by using the x-ray tube were obtained. The three-dimensional position of the object was measured with good precision from 0 to 50 degrees, but above 50 degrees, measured position error increased with the increase of the rotation angle. The computational time and image size were 30, 12, and 7 seconds for 512*512, 256*256, and 128*128, respectively. CONCLUSIONS: We could measure the three-dimensional position of the object using properly calibrated Kinect sensor, and obtained projection images at relatively high-speed using the three-dimensional CTdata. It was suggested that this system can be used for obtaining simulated projection x-ray images before x-ray exposure by attaching this device onto an x-ray tube.

Investigation of the prediction accuracy of vancomycin concentrations determined by patient-specific parameters as estimated by Bayesian analysis.

BACKGROUND/OBJECTIVE: There have been many studies of therapeutic drug monitoring (TDM) of vancomycin (VCM) based on Bayesian analysis, but there have been no reports of the accuracy of prediction based on Bayesian-estimated patient-specific parameters. This study was conducted to compare the accuracy of prediction based on the population pharmacokinetic (PPK) method and Bayesian-estimated parameters. METHOD: The subjects were 22 patients who were treated with VCM for MRSA infection and whose blood was sampled twice or more during the administration period. The concentrations between the blood samples were predicted based on the concentrations in the first blood samples based on the PPK method using mean parameters for the Japanese population and Bayesian-estimated patient-specific pharmacokinetic parameters. The mean prediction error (ME), mean absolute error (MAE) and root mean squared error (RMSE) were compared to examine the accuracy of prediction based on Bayesian-estimated patient-specific parameters. RESULTS AND DISCUSSION: The mean measured VCM concentration was 10·43±5·19 µg/mL, whereas the mean concentration predicted based on the PPK method was 8·52±4·34 µg/mL, with an ME of -1·91, MAE of 2·93 and RMSE of 3·21. The mean concentration predicted based on patient-specific parameters was 9·62±4·95 µg/mL with ME of -0·81, MAE of 1·38 and RMSE of 1·74. The ME and MAE for the concentrations predicted using patient-specific parameters were smaller compared with those predicted using the PPK method (P=0·0471 and 0·0003, respectively), indicating superior prediction with a significant difference between approaches. CONCLUSION: Prediction using Bayesian estimates of patient-specific parameters was better than by the PPK method. However, when using patient-specific parameters it is still necessary to fully understand the clinical status of the patient and frequently determine VCM concentrations.

Effect of various estimates of renal function on prediction of vancomycin concentration by the population mean and Bayesian methods.

OBJECTIVE: Renal function was estimated in 129 elderly patients with methicillin-resistant Staphylococcus aureus (MRSA) who were treated with vancomycin (VCM). The estimation was performed by substituting serum creatinine (SCR) measured enzymatically and a value converted using the Jaffe method into the Cockcroft-Gault and Modification of Diet in Renal Disease (MDRD) equations. The serum trough level was predicted from three estimates of renal function by the population mean (PM) and Bayesian methods and the predictability was assessed. METHODS: Two-compartment model-based Japanese population parameters for VCM were used, and the mean prediction error (ME) and root mean squared error (RMSE) were calculated as indices of bias and accuracy, respectively, for predictions by the PM and Bayesian methods. RESULTS: The PM method gave the highest correlation with the measured value using the estimate of renal function obtained by substituting the Jaffe-converted SCR into the Cockcroft-Gault equation. There was no positive or negative bias in the ME and the value was significantly smaller than for other predicted data (P < 0.05). RMSE was also the smallest, indicating that this method increases the predictability of the serum VCM trough level. While, ME showed a negative bias for all values predicted by the Bayesian method, both the ME and RMSE were very small. CONCLUSION: In the application of the PM method for VCM treatment of elderly patients with MRSA, substitution of SCR based on the Jaffe method into the Cockcroft-Gault equation increases the predictability of the serum VCM trough level. The Bayesian method predicted the serum VCM trough level with high accuracy using any of the estimates of renal function.

Bronchoalveolar lymphocytosis correlates with human T lymphotropic virus type I (HTLV-I) proviral DNA load in HTLV-I carriers.

BACKGROUND: A study was undertaken to investigate the pathogenesis of pulmonary involvement in human T lymphotropic virus type I (HTLV-I) carriers. METHODS: The bronchoalveolar lavage (BAL) cell profile of 30 HTLV-I carriers (15 asymptomatic HTLV-I carriers (AHCs) and 15 symptomatic HTLV-I carriers (SHCs)) with chronic inflammatory diseases of respiratory tract and eight patients with HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) was investigated. The HTLV-I proviral deoxyribonucleic acid (DNA) load in peripheral blood mononuclear cells (PBMCs) and BAL fluid from HTLV-I carriers was estimated using the quantitative polymerase chain reaction method and the correlation between the lymphocyte number in BAL fluid and the HTLV-I proviral DNA load in PBMCs and BAL fluid was examined. RESULTS: The percentage of lymphocytes in BAL fluid was increased (>18%) in 11 of 30 HTLV-I carriers although there was no significant difference compared with control subjects. In HTLV-I carriers the lymphocyte number in BAL fluid correlated well with the copy number of HTLV-I proviral DNA in PBMCs. In addition, the copy number of HTLV-I proviral DNA in BAL fluid correlated well with the number of lymphocytes (both CD4+ and CD8+ cells) in BAL fluid. CONCLUSIONS: These findings suggest that pulmonary lymphocytosis can occur in a subset of HTLV-I carriers without HAM/TSP and that the increased HTLV-I proviral DNA load may be implicated in the pathogenesis of pulmonary involvement in HTLV-I carriers.

Paradoxical roles of different nitric oxide synthase isoforms in colonic injury.

Nitric oxide (NO) is a free radical that is largely produced by three isoforms of NO synthase (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). NO regulates numerous processes in the gastrointestinal tract; however, the overall role that NO plays in intestinal inflammation is unclear. NO is upregulated in both ulcerative colitis and Crohn's disease as well as in animal models of colitis. There have been conflicting reports on whether NO protects or exacerbates injury in colitis or is simply a marker of inflammation. To determine whether the site, timing, and level of NO production modulate the effect on the inflammatory responses, the dextran sodium sulfate model of colitis was assessed in murine lines rendered deficient in iNOS, nNOS, eNOS, or e/nNOS by targeted gene disruption. The loss of nNOS resulted in more severe disease and increased mortality, whereas the loss of eNOS or iNOS was protective. Furthermore, concomitant loss of eNOS reversed the susceptibility found in nNOS-/- mice. Deficiencies in specific NOS isoforms led to distinctive alterations of inflammatory responses, including changes in leukocyte recruitment and alterations in colonic lymphocyte populations. The present studies indicate that NO produced by individual NOS isoforms plays different roles in modulating an inflammatory process.

Influence of human T lymphotrophic virus type I on cryptogenic fibrosing alveolitis - HTLV-I associated fibrosing alveolitis: proposal of a new clinical entity.

Human T lymphotrophic virus type-I (HTLV-I), a human retrovirus, infects CD4+ lymphocytes and is thought to modify their function; a possible association with pulmonary diseases has also been suggested. However, little is known about the influence of HTLV-I on cryptogenic fibrosing alveolitis (CFA), a chronic inflammatory interstitial lung disease of unknown aetiology. In order to clarify the influence of HTLV-I infection on CFA, 72 CFA patients with and without HTLV-I infection were examined. HTLV-I positive CFA patients were likely to have larger affected areas and to show traction bronchiectasis with honeycombing change. An imbalance of matrix metalloproteinases and tissue inhibitor of metalloproteinases were also observed in the BALF of HTLV-I positive CFA patients. CD3+/CD25+ lymphocyte percentage was significantly higher in the BALF of HTLV-I positive patients compared to negative patients. MIP-1alpha, IP-10 and sICAM levels in BALF were also significantly higher in HTLV-I positive patients than in negative patients. The levels of MCP-1 and IL-8 were not significantly different. In HTLV-I positive patients, the MIP-1alpha and IP-10 levels showed a significant positive correlation with percentage of CD3+/CD25 lymphocytes. HTLV-I positive CFA patients showed a larger lesion than negative patients and exhibited increased levels of certain cytokines that correlated with activated T cells in the BALF. We suggest that HTLV-I infection may contribute to the development of CFA via activation of T cells. We also propose that these features should be taken into consideration in the treatment of CFA in HTLV-I infected individuals.

The transcription factor T-bet regulates mucosal T cell activation in experimental colitis and Crohn's disease.

The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-gamma/interleukin (IL)-4 and transforming growth factor (TGF)-beta activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models of chronic intestinal inflammation by taking advantage of mice that lack T-bet and retroviral transduction techniques, respectively. Whereas retroviral transduction of T-bet in CD62L(+) CD4(+) T cells exacerbated colitis in reconstituted SCID mice, T-bet-deficient T cells failed to induce colitis in adoptive transfer experiments suggesting that overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis in vivo. Furthermore, T-bet-deficient CD62L(-) CD4(+) T cells showed enhanced protective functions in Th1-mediated colitis and exhibited increased TGF-beta signaling suggesting that a T-bet driven pathway of T cell activation controls the intestinal balance between IFN-gamma/IL-4 and TGF-beta responses and the development of chronic intestinal inflammation in T cell-mediated colitis. Furthermore, TGF-beta was found to suppress T-bet expression suggesting a reciprocal relationship between TGF-beta and T-bet in mucosal T cells. In summary, our data suggest a key regulatory role of T-bet in the pathogenesis of T cell-mediated colitis. Specific targeting of this pathway may be a promising novel approach for the treatment of patients with Crohn's disease and other autoimmune diseases mediated by Th1 T lymphocytes.

Induction of dauer pupae by fenoxycarb in the silkworm, Bombyx mori.

Topical application of fenoxycarb (1 &mgr;g per animal) at 129 or 132 h of the fifth instar larvae of the silkworm, Bombyx mori, did not induce morphological abnormalities in the pupal stage, but these animals became dauer (permanent) pupae. This condition of B. mori and the endocrine events leading to permanent pupae are discussed in this work. Application of fenoxycarb at 132 h of the fifth instar elicited a high ecdysteroid titre in the pharate pupal stage and a steadily high ecdysteroid titre in the pupal stage. The fenoxycarb-induced permanent pupae had non-degenerating prothoracic glands that secreted low amounts of ecdysteroid and did not respond to recombinant prothoracicotropic hormone (rPTTH) late in the pupal stage. The Bombyx PTTH titre in the haemolymph, determined by a time-resolved fluoroimmunoassay, was lower than that of controls at the time of pupal ecdysis, but higher than controls later in the pupal stage in fenoxycarb-treated animals. After application of fenoxycarb, its haemolymph level, measured by ELISA, reached a peak at pupal ecdysis, then remained low. These results suggest that the fenoxycarb-mediated induction of permanent pupae is only partially a brain-centred phenomenon. It also involves alterations in the hormonal interplay that govern both the initiation of pupal-adult differentiation and changes in the steroidogenic pathway of the prothoracic glands of B. mori.

The membrane-anchored MMP inhibitor RECK is a key regulator of extracellular matrix integrity and angiogenesis.

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.

Distribution of soluble N-ethylmaleimide fusion protein attachment proteins (SNAPs) in the rat nervous system.

Soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) plays an essential role in vesicular transport and the release of neurotransmitters and hormones through associations with NSF and SNAP receptors (SNAREs). Three isoforms (alpha, beta and gamma) of SNAP are expressed in mammals. We have generated isoform-specific antibodies and studied the expression and distribution of these SNAP isoforms in the rat nervous system. Each antibody specifically recognized alpha-, beta- or gamma-SNAP in an isoform-specific manner in immunoblots of brain homogenate. Alpha- and gamma-SNAP were ubiquitously expressed in various tissues, whereas beta-SNAP was expressed only in brain. After subcellular fractionation of brain homogenates, all three isoforms were recovered in both soluble and particulate fractions. Immunohistochemistry revealed that alpha- and beta-SNAP were generally differentially distributed both in synaptic and non-synaptic regions, including brain white matter. The presynaptic location of both alpha- and beta-SNAP was confirmed by immunoelectron microscopy. At the neuromuscular junction, immunoreactive alpha-SNAP was identified in synaptic vesicles, while in the cerebellum, beta-SNAP was present in the presynaptic membranes of basket neuron and mossy fiber terminals. From these results we suggest that both alpha- and beta-SNAP may play an important role in neurotransmitter release as well as in constitutive vesicular transport.

Dominant localization of prostaglandin D receptors on arachnoid trabecular cells in mouse basal forebrain and their involvement in the regulation of non-rapid eye movement sleep.

Infusion of prostaglandin (PG) D(2) into the lateral ventricle of the brain induced an increase in the amount of non-rapid eye movement sleep in wild-type (WT) mice but not in mice deficient in the PGD receptor (DP). Immunofluorescence staining of WT mouse brain revealed that DP immunoreactivity was dominantly localized in the leptomeninges (LM) of the basal forebrain but that PGD synthase immunoreactivity was widely distributed in the LM of the entire brain. Electron microscopic observation indicated that DP-immunoreactive particles were predominantly located on the plasma membranes of arachnoid trabecular cells of the LM. The region with the highest DP immunoreactivity was clearly defined as bilateral wings in the LM of the basal forebrain located lateral to the optic chiasm in the proximity of the ventrolateral preoptic area, one of the putative sleep centers, and the tuberomammillary nucleus, one of the putative wake centers. The LM of this region contained DP mRNA 70-fold higher than that in the cortex as judged from the results of quantitative reverse transcription-PCR. PGD(2) infusion into the subarachnoid space of this region increased the extracellular adenosine level more than 2-fold in WT mice but not in the DP-deficient mice. These results indicate that DPs in the arachnoid trabecular cells of the basal forebrain mediate an increase in the extracellular adenosine level and sleep induction by PGD(2).

Altered expression of occludin and tight junction formation in psoriasis.

In simple epithelia, tight junctions are well developed and have barrier and fence functions. On the other hand, tight junctions are less developed in stratified epithelia. In the rodent epidermis, only maculae occludentes (i.e. focal strands or spot tight junctions) are observed in the most superficial zone of the granular cell layer. Occludin is an integral membrane protein, and is localized at tight junctions in simple epithelia. In normal epidermis, occludin is expressed at the maculae occludentes in the granular cell layer, indicating that it is associated with keratinocyte differentiation. Thus, we examined occludin expression in psoriasis, in which differentiation of keratinocytes is impaired. In psoriasis, occludin was expressed more broadly in the upper epidermis than in normal epidermis. In addition, immunoelectron microscopy showed occludin to be concentrated on the maculae occludentes in the spinous layer of psoriatic skin. These findings indicate that occludin and the formation of tight junctions are related to the proliferation and differentiation of keratinocytes, and to the pathogenesis of psoriasis.

Effects of juvenile hormone on the secretion of prothoracicotropic hormone in the last- and penultimate-instar larvae of the silkworm Bombyx mori.

The effect of juvenile hormone (JH) on the secretion of the prothoracicotropic hormone (PTTH) was investigated, by examining the changes in hemolymph PTTH titer after the topical application of JH-I on the larvae of the silkworm, Bombyx mori. The titer of PTTH was determined by the time-resolved fluoroimmunoassay. JH-I application at very early stages of development in the fifth (last) instar resulted in a significant increase in the PTTH titer, but this effect became less evident thereafter. After the onset of wandering (day 6 of the fifth instar), JH-I did not affect the hemolymph PTTH titer. JH-I application on day 5 resulted in the delay of spinneret pigmentation on day 6, which is induced by an increase in the ecdysteroid titer on day 5 and is the first visible indication of larval-pupal transformation. However, the JH-I application did not suppress the increase in either PTTH or ecdysteroid titer on day 5, suggesting that JH-I acts on the spinneret to inhibit the response of the tissue to ecdysteroids. JH-I also exhibited a PTTH titer-elevating effect in the fourth instar. These results suggest that JH has a role as a potent stimulator of PTTH secretion in both the penultimate and last instar of the silkworm.