An accessible pharmacodynamic transcriptional biomarker for notch target engagement.

γ-Secretase mediates amyloid production in Alzheimer's disease (AD) and oncogenic activity of Notch. γ-Secretase inhibitors (GSIs) are thus of interest for AD and oncology. A peripheral biomarker of Notch activity would aid determination of the therapeutic window and dosing regimen for GSIs, given toxicities associated with chronic Notch inhibition. This study examined the effects of GSI MK-0752 on blood and hair follicle transcriptomes in healthy volunteers. The effects of a structurally diverse GSI on rhesus blood and hair follicles were also compared. Significant dose-related effects of MK-0752 on transcription were observed in hair follicles, but not blood. The GSI biomarker identified in follicles exhibited 100% accuracy in a clinical test cohort, and was regulated in rhesus by a structurally diverse GSI. This study identified a translatable, accessible pharmacodynamic biomarker of GSI target engagement and provides proof of concept of hair follicle RNA as a translatable biomarker source.

Diagnosing common parotid tumours with magnetic resonance imaging including diffusion-weighted imaging vs fine-needle aspiration cytology: a comparative study.

OBJECTIVES: the purpose of this study was to evaluate the accuracy of MRI combined with diffusion-weighted imaging (DWI) vs fine-needle aspiration cytology (FNAC) in diagnosing common parotid masses. METHODS: 25 consecutive patients (mean age 61 years) with parotid masses were included in this study. Informed consent and ethical approval was obtained. 22 patients underwent both MRI combined with DWI and FNAC. From DWI data, apparent diffusion coefficient maps were generated. The MRI study protocol consisted of T(1) weighted spin echo; T(2) weighted and T(2) weighted fat-suppressed turbo spin echo; DWI; and T(1) weighted fat-suppressed post-contrast images. MRI and FNAC diagnoses were compared with histopathology. Youden's index was used to compare the two methods. RESULTS: masses comprised eight Warthin tumours, eight adenomas (six pleomorphic adenomas, two basal cell adenomas), five carcinomas, two lipomas, one haemagioma and one benign lymphadenopathy. Technically, MRI was successful in 24 of the 25 patients (96%), FNAC was successful in 20 of the 23 patients (87.0%). The accuracy, sensitivity and specificity of MRI without DWI were 96%, 80% and 100%, respectively. Diagnostic accuracy did not increase by adding DWI to conventional MRI; however, DWI was helpful for diagnosing benign tumour histology. MRI combined with DWI was successful for determining accurate tumour typing in all benign masses except one lymphadenopathy. When FNAC had adequate material the accuracy, sensitivity and specificity were 95%, 75% and 100%, respectively. Youden's index was 0.80 for MRI and 0.75 for FNAC. CONCLUSIONS: MRI combined with DWI seems to have similar diagnostic potential as FNAC in differentiation of benign vs malignant parotid masses.

Endovascular stent placement in patients with hepatic artery stenoses or thromboses after liver transplant.

Hepatic artery stenosis or thrombosis following liver transplant is a potentially life-threatening complication. Successful liver transplant depends on uncompromised hepatic arterial inflow. Early diagnosis and treatment of complications prolong graft survival. Interventional radiologic techniques are frequently used to treat hepatic artery complications. Twenty patients with hepatic artery stenoses (n = 11) or thromboses (n = 9) were included in this study. Eighteen of the 20 patients were successfully treated by stent placement. In 9 patients, early endovascular interventions were performed 1 to 7 days after surgery. Two patients were operated owing to the effects of dissection and bleeding from the hepatic artery. Repeat endovascular interventions were performed 10 times in 6 patients. Follow-up ranged from 5 months to 4.5 years. Nine patients with patent hepatic arteries died during follow-up owing to reasons unrelated to the hepatic artery interventions. In 3 patients, the stents became occluded at 3, 5, and 9 months after surgery but no clinical symptoms were present.

Nonvascular complications in pediatric liver recipients: multidetector computed tomography evaluation.

In pediatric liver transplantation postoperative diagnosis of complications is crucial for graft salvage. Multidetector computed tomography (MDCT) is a technique to evaluate complications. In this study we present nonvascular abdominal complications encountered in pediatric recipients after liver transplantation. We retrospectively examined 113 MDCT examinations in 43 pediatric patients who underwent liver transplantation between 1997 and 2005. Computed tomography (CT) examinations were made by a 16-detector multislice CT scanner. The pathological findings on CT images were: intraperitoneal free fluid, intrahepatic bile duct dilatation, graft liver infarction, perihepatic and intraperitoneal fluid collections (six biloma), colonic and/or intestinal dilatation, splenic infarction, perihepatic hematoma, right adrenal hemorrhage, perihepatic abscess, incisional hernia, intrahepatic biloma and periportal collar. In one patient intestinal hemorrhage was suspected. Intestinal perforation was suspected in three patients. Among these three patients, one patient died before any surgical intervention. In two patients the diagnosis was confirmed at surgery. In pediatric patients, the short examination time, brief sedation duration, and high-resolution images make MDCT an effective radiological method to evaluate nonvascular transplant complications.

PSA-NCAM is up-regulated during optic nerve regeneration in lizard but not in goldfish.

The addition of polysialic acid (PSA) to neural cell adhesion molecule (NCAM) facilitates axon growth. Here we use Western blots and immunohistochemistry to examine expression of PSA-NCAM during optic nerve regeneration. In lizard, retinal ganglion cell axons become transiently PSA-NCAM positive. By contrast, goldfish RGC axons are PSA-NCAM negative both in normal animals and throughout regeneration with the exception of a PSA-NCAM-positive fascicle arising from newly generated RGCs. Transient sialylation of NCAM in lizard may assist regeneration in the nonpermissive reptilian visual pathway and facilitate the reestablishment of a crude topographic map; down-regulation in the long term may contribute to the breakdown in topography. The lack of sialylation in goldfish presumably reflects the permissive nature of the substrate allowing axon regeneration and the successful reestablishment of a topographic map.

Activation of interferon response factor-3 in human cells infected with herpes simplex virus type 1 or human cytomegalovirus.

Activation of cellular interferon-stimulated genes (ISGs) after infection with herpes simplex virus type 1 (HSV-1) or human cytomegalovirus (HCMV) was investigated. The level of ISG54-specific RNA in human fetal lung (HFL) or human foreskin (BJ) fibroblasts increased substantially after infection with either virus in the presence of cycloheximide. HSV-1 particles lacking glycoprotein D or glycoprotein H failed to induce ISG54-specific RNA synthesis, demonstrating that entry of virus particles rather than binding of virions to the cell surface was required for the effect. A DNA-binding complex that recognized an interferon-responsive sequence motif was induced upon infection with HSV-1 or HCMV in the presence of cycloheximide, and the complex was shown to contain the cell proteins interferon response factor 3 (IRF-3) and CREB-binding protein. IRF-3 was modified after infection with HSV-1 or HCMV to a form of lower electrophoretic mobility, consistent with phosphorylation. De novo transcription of viral or cellular genes was not required for the activation of IRF-3, since the effect was not sensitive to inhibition by actinomycin D. Infection of HFL fibroblasts with HSV-1 under conditions in which viral replication proceeded normally resulted in severely reduced levels of the IRF-3-containing complex, defining the activation of IRF-3 as a target for viral interference with ISG induction. In BJ fibroblasts, however, significant activation of IRF-3 was detected even when the viral gene expression program progressed to later stages, demonstrating that the degree of inhibition of the response was dependent on host cell type. As a consequence of IRF-3 activation, endogenous interferon was released from BJ cells and was capable of triggering the appropriate signal transduction pathway in both infected and uninfected cells. Activation of ISG54-specific RNA synthesis was not detected after infection of human U-373MG glioblastoma cells, showing that the induction of the response by infection is cell type dependent.

Sperm transport in the female reproductive tract of the brushtail possum, Trichosurus vulpecula, following superovulation and artificial insemination.

This study investigated sperm transport following superovulation and artificial insemination (AI) in the common brushtail possum, Trichosurus vulpecula. Females were superovulated by treatment with 15 IU pregnant mare serum gonadotrophin (PMSG) then 4 mg luteinizing hormone (LH) 78 h later. Inseminations were performed 27 h after LH (4 million motile spermatozoa/uterus). At 1.5, 3, 6, 9 and 12 h after AI (n=5 per group), females were euthanised and reproductive tracts removed for examination and flushed for sperm. No ovulations had occurred by 1.5 h, but 20% of animals had ovulated by 3 or 6 h, and 80% by 9 or 12 h. The mean numbers of spermatozoa recovered ranged from 249 to 275x10(3) in the uterus; 16-51x10(3) in the isthmus; 8-11x10(3) in the middle segment; and 6-16x10(3) in the ampulla at 1.5, 3 and 6 h after AI. Sperm numbers in all regions decreased at later times (P<0.05) except the isthmus, where 100x10(3) sperm were recovered by 12 h. Highly motile thumbtack sperm (a putative indicator of capacitation in marsupials), were recovered from the isthmus (20%), middle segment (50%) and ampulla (90%) at all sampling times, but not from the uterus. The epithelium of the oviduct segments contained mucus-secreting and ciliated cells and peak secretory activity was observed in the ampulla at 6 h. At 3, 6 and 12 h, many spermatozoa were found in epithelial folds within the isthmus. The present study has provided basic information on sperm transport and storage events within the female reproductive tract of T. vulpecula following superovulation and AI. It is concluded that this model may be useful to better understand pre-fertilization sperm maturation events in the possum, which could facilitate the development of IVF technology.

Retinal structure and visual acuity in a polyprotodont marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata).

The visual system of the fat-tailed dunnart (Sminthopsis crassicaudata), a small polyprotodont marsupial, has been examined both anatomically and behaviourally. The ganglion cell layer was examined in cresyl-violet stained wholemounts and found to contain a mean of 81,400 ganglion cells (SD +/- 3,360); the identification of ganglion cells was supported by a correspondence to optic axon counts. Ganglion cells were distributed as a mid-temporally situated area centralis, embedded in a pronounced visual streak. Localised implants of horseradish peroxidase into retinal wholemounts revealed both A-type and B-type horizontal cells. Sections of the outer retina showed it to be rod-dominated, with a rod-to-cone ratio of 40:1 at the area centralis; cones were found to contain oil droplets but double cones were not a prominent feature. The retinal pigment epithelium consisted of squamous cells. Visual acuity, estimated from counts of peak ganglion cell density (8,300/mm2, SD +/- 1,180) and measurements of posterior nodal distance (2.9 mm), was found to be 2.30 cycles per degree. The value was close to that of 2.36 cycles per degree estimated by behavioural tests using a Mitchell jumping stand; values were similar at low, intermediate and high light levels. Our findings are discussed in relation to the lifestyle of the dunnart.

DNA damage in the nasal passageway: a literature review.

The purpose of this review is to provide a compilation of work examining DNA damage in the nasal cavity. There are numerous methods to identify and quantify damage to DNA and the diversity of methods and toxicologic endpoints is illustrated by the range of studies presented here. There are a large number of independent studies measuring endpoints in the upper respiratory tract; however, with regard to toxicant induced DNA damage in the nasal passageway, the effects of two compounds, 4-(N-Methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and formaldehyde (HCHO), appear to have been extensively characterized. The body of work on NNK and formaldehyde have provided insights into molecular mechanisms of DNA damage and repair and induced cell replication and its relationship to nasal cancer. With new technologies and molecular techniques, the sensitivity to enable evaluations of the minute quantities of nasal tissue available in test species and human biopsy impact the study of the nasal-toxicant interactions. As methods used to characterize DNA damage increase in sensitivity, the importance of both exogenous and endogenous sources of DNA damage, steady-state levels of cellular damage, repair, and resulting mutations, low-dose exposure assessments and inter-species extrapolation will become increasingly complex. Additional studies of DNA damage in the nasal passage will undoubtedly challenge future estimations of risk and impact what are perceived to be acceptable levels of exposure to known and predicted carcinogens. The aim of this paper is to provide to the interested scientist literature relevant to the effects of agents on nasal DNA, so that areas of insufficient information can be identified and used to further develop and expand the knowledge base for nasal DNA toxicant interactions.

Critical factors in assessing risk from exposure to nasal carcinogens.

Anatomical, physiological, biochemical and molecular factors that contribute to chemical-induced nasal carcinogenesis are either largely divergent between test species and humans, or we know very little of them. These factors, let alone the uncertainty associated with our knowledge gap, present a risk assessor with the formidable task of making judgments about risks to human health from exposure to chemicals that have been identified in rodent studies to be nasal carcinogens. This paper summarizes some of the critical attributes of the hazard identification and dose-response aspects of risk assessments for nasal carcinogens that must be accounted for by risk assessors in order to make informed decisions. Data on two example compounds, dimethyl sulfate and hexamethylphosphoramide, are discussed to illustrate the diversity of information that can be used to develop informed hypotheses about mode of action and decisions on appropriate dosimeters for interspecies extrapolation. Default approaches to interspecies dosimetry extrapolation are described briefly and are followed by a discussion of a generalized physiologically based pharmacokinetic model that, unlike default approaches, is flexible and capable of incorporating many of the critical species-specific factors. Recent advancements in interspecies nasal dosimetry modeling are remarkable. However, it is concluded that without the development of research programs aimed at understanding carcinogenic susceptibility factors in human and rodent nasal tissues, development of plausible modes of action will lag behind the advancements made in dosimetry modeling.

Mitogenic responses of rat nasal epithelium to hexamethylphosphoramide inhalation exposure.

Hexamethylphosphoramide (HMPA) is a rat nasal carcinogen that induces squamous cell carcinomas in the anterior portions of the nasal cavity following chronic inhalation exposures as low as 50 ppb. These tumors may arise as a result of P-450-mediated release of formaldehyde (HCHO), a known rat nasal carcinogen. The goal of this research was to investigate early responses of the nasal epithelium to inhaled HMPA. Rats were exposed nose-only to approximately 3 ppm HMPA for 6 h, and killed 18, 48, 96 or 144 h post-exposure. In a separate study, rats were exposed nose-only for 6 h for 1, 2, 3, or 5 consecutive days and killed 18 or 96 h post-exposure. With both single and repeated doses of HMPA, there was no evidence of cytotoxicity in the anterior nose. Olfactory degeneration and necrosis of the dorsal meatus, Bowman's glands and tips of the ethmoid turbinates increased in severity with repeated exposures to HMPA. Cell proliferation was assessed in levels of nasal tissue that included regions of squamous, respiratory, transitional and olfactory epithelium. Regional induction of cell proliferation was measured by BrdU incorporation, and reported as the number of labeled cells/mm basement membrane. At 18 h after a single exposure, there was an increase in cell proliferation in squamous epithelium, which returned to control levels within 48 h. A transitory increase in cell proliferation was observed regions of respiratory and transitional epithelium, although the response of each tissue, in terms of magnitude and peak time of response post-exposure, also differed. Along the dorsal meatus in Level 9, olfactory labeling initially decreased, returned to control levels by 96 h, but again declined at 144 h post-exposure. In repeat dose studies, the squamous epithelium response was variable 18 h post-exposure. For respiratory and transitional epithelium, increased cell proliferation 18 h post-exposure was correlated with increased dose (exposure) of HMPA. Cell proliferation responses following two or more exposures returned to near control levels within 96 h post-exposure. In conclusion, HMPA induced cell proliferation, but not cytotoxicity, in the anterior nose at approximately 3 ppm. These data suggest that HMPA induces proliferative, perhaps mitogenic, responses in the nasal epithelium, and this response may facilitate the fixation of low level genetic damage induced by liberated HCHO.

Acute eosinophilic pneumonia: radiologic and clinical features.

PURPOSE: To determine the radiographic and computed tomographic (CT) characteristics of acute eosinophilic pneumonia. MATERIALS AND METHODS: Twelve consecutive patients with acute eosinophilic pneumonia were included in the study. The diagnosis was based on clinical symptoms and results of bronchoalveolar lavage. Plain chest radiographs were obtained in all patients; CT scans were obtained in three patients. Two thoracic radiologists reviewed the radiographs and CT scans. RESULTS: Ten patients had bilateral areas of air-space opacity on images obtained at presentation; in seven of these patients, interstitial areas of opacity were also present. Two patients had bilateral interstitial areas of opacity and no areas of air-space opacity. Interlobular septal thickening and ground-glass attenuation were present on CT scans in two patients; patchy bilateral consolidation was present on CT scans in one patient. Pleural effusion was present on radiographs in seven patients (58%) and was bilateral in five. Pleural effusion was present at some point during the course of disease in all patients. In all patients, air-space disease markedly improved within 3 days of initiation of treatment with corticosteroids. CONCLUSION: Acute eosinophilic pneumonia should be considered as a possible diagnosis when a previously healthy person presents with acute respiratory failure of unknown origin.

Immunosuppressive properties of surfactant and plasma on alveolar macrophages.

Alveolar macrophages have been shown to be major producers of the potent proinflammatory cytokines interleukin-1 beta and tumor necrosis factor-alpha, and of the antiinflammatory cytokine interleukin-1 receptor antagonist. During the adult respiratory distress syndrome the normally surfactant-coated alveolus becomes flooded with plasma proteins, altering the milieu of alveolar cells such as alveolar macrophages. To understand alveolar macrophage function during the adult respiratory distress syndrome, the individual and combined effects of surfactant and plasma on alveolar macrophage cytokine production was examined. A synthetic surfactant (Exosurf) and a bovine-derived surfactant (Survanta) both inhibited production of interleukin-1 beta, pro-interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-1 receptor antagonist in a dose-dependent manner. This inhibition was noted when both endotoxin and heat-killed Staphylococcus aureus were used as stimuli. Autologous plasma also inhibited interleukin-1 beta and tumor necrosis factor-alpha release in a dose-dependent manner, but, unlike surfactant, plasma did not inhibit interleukin-1 receptor antagonist release. Similarly, the combination of plasma and surfactant inhibited interleukin-1 beta and tumor necrosis factor-alpha release but not interleukin-1 receptor antagonist release. In support of these data, interleukin-1 receptor antagonist was detectable in five of six bronchoalveolar lavage fluid samples from patients with adult respiratory distress syndrome at a mean concentration of 465 pg/ml; on the other hand, interleukin-1 beta was not detectable in any of these samples. These results indicate that the relative production of interleukin-1 beta, tumor necrosis factor-alpha, and interleukin-1 receptor antagonist can be altered depending on the local concentration of both surfactant and plasma.

An evaluation of the role of calcium in cell injury.

It has been proposed that a number of chemical-induced cell injuries result from disruption of the ability of the cell to control calcium. Many of the techniques used to develop this theory have relied on indirect measurements of intracellular calcium. The advent of digital imaging fluorescence microscopy has allowed a more direct examination of the relationship between calcium and cell damage. Results indicate that cytosolic calcium does not play a central role in the initiation of oxidative injury in a number of cell types. Changes in calcium homeostasis occur well after the appearance of other indications of cell injury. However, recent studies indicate that a mitochondrial lesion occurs relatively early in the time course of oxidative cell injury. Calcium may play a role in the development of this lesion.

Interactions of cimetidine and ranitidine with aluminum-containing antacids and a clay-containing gastric-protective drug in an "artificial stomach-duodenum" model.

Interactions of cimetidine and ranitidine with aluminum-containing antacids and clay-containing gastric-protective drugs were analyzed in vitro by using an artificial stomach-duodenum model. The model reproduced near-physiologic conditions, taking into account gastric and duodenal flux variations and interactions between gastric mucosa and drugs added to the gastric content. Clay bound cimetidine in acid medium, but the drug was released when the pH increased, resulting in cimetidine amounts in the duodenal site close to those in controls. In contrast, clay bound ranitidine in acid medium and did not release it in the duodenal site. Aluminum-containing antacids did not significantly modify the amount of cimetidine or ranitidine available for absorption. Several factors play a role in the interactions of cimetidine and ranitidine with aluminum-containing antacids and clay-containing gastric-protective drugs: the structure of the antisecretory drugs, gastroduodenal pH, interactions of the antacid and clay with the gastric mucosa, and release of aluminum that could adsorb the drugs or prevent their adsorption by the mucosa. These phenomena are intricate and difficult to analyze without using a physicochemical approach.

Oxidative stress in cultured hepatocytes exposed to acetaminophen.

The effect of acetaminophen (APAP) exposure on the formation of oxidized glutathione (GSSG) was investigated in cultured mouse hepatocytes to determine if oxidative damage is involved in the toxicity of this drug. Incubations of hepatocytes for 24 hr with 1 mM APAP produced a time-dependent loss of cell viability which was preceded by depletion of reduced glutathione (GSH) and an increase in GSSG formation. Pretreatment with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) (0.1 mM) for 30 min, which irreversibly inhibited glutathione reductase (GSSG-Rd) activity, increased the extent of GSSG formation produced by APAP exposure and potentiated its cell killing. Pretreatment of hepatocytes with 20 mM deferoxamine (DFO) for 1 hr to chelate ferric iron decreased GSSG formation and cell killing produced by APAP. Pretreatment with BCNU or DFO did not affect APAP oxidation as determined by the formation of the APAP-GSH conjugate or the covalent binding of APAP metabolites to cellular protein. Hence, increasing the susceptibility of hepatocytes to an oxidative stress with BCNU increased both the formation of GSSG and cell killing produced by APAP. Conversely, decreasing their susceptibility to an oxidative stress by chelating iron with DFO decreased GSSG formation and cell injury. It follows that APAP toxicity involves oxidative processes that occur early in the poisoning process and are a major factor contributing to injury in these cells.

Protection from oxidative damage in mouse liver cells.

Paracetamol toxicity in mouse hepatocytes involved oxidative stress initiated by the formation of NAPQI. This oxidative component of paracetamol injury is associated with the latter stages of the poisoning process. Ebselen, a drug with GSH-peroxidase activity, was effective in ameliorating these oxidative events.

Postnatal mice have low susceptibility to paracetamol toxicity.

The hepatotoxicity of paracetamol in mice of 2, 3, 8-10, 24-26, 32-34, and 52-54 wk of age was determined by lethality data, histopathologic examination of the liver, and appearance of glutamate-pyruvate transaminase and glutamate-oxaloacetate transaminase activities in the plasma over an 8-h exposure period. At a dose of 300 mg/kg, there was evidence of hepatocytic necrosis and transaminase leakage in the 32- to 34- and 52- to 54-wk-old mice, but lethality was only recorded in the oldest age group. At 500 mg/kg, paracetamol produced 30% lethality in 3-wk-old mice and between 50 and 90% lethality in the adult age groups. There was histologic evidence of hepatocytic necrosis at all of these ages and its extent increased with age. Similarly, there were increases in plasma transaminases in each of these age groups. However, in 2-wk-old mice there was no lethality, no hepatocytic necrosis, and no increase in plasma transaminases. The lack of susceptibility of 2-wk-old mice to paracetamol toxicity was not due to immaturity of the cytochrome P-450 enzymes responsible for metabolism of paracetamol to its reactive metabolite (N-acetyl-p-benzoquinone imine). In fact, the activity of this enzyme pathway in 2-wk-old mice was greater than that in adults. The partial clearance of the glutathione-derived metabolites of paracetamol after a nontoxic (50 mg/kg) dose was 80% greater in 2-wk-old mice than in 8- to 10-wk-old mice.(ABSTRACT TRUNCATED AT 250 WORDS)