PBDE-47 and PBDE mixture (DE-71) toxicities and liver transcriptomic changes at PND 22 after in utero/postnatal exposure in the rat.

Pentabromodiphenyl ethers (PBDE) are found in human tissue, in household dust, and in the environment, and a particular concern is the potential for the induction of cancer pathways from these fat-soluble persistent organic pollutants. Only one PBDE cancer study has been conducted and that was for a PBDE mixture (DE-71). Because it is not feasible to test all PBDE congeners in the environment for cancer potential, it is important to develop a set of biological endpoints that can be used in short-term toxicity studies to predict disease outcome after long-term exposures. In this study, PBDE-47 was selected as the test PBDE congener to evaluate and compare toxicity to that of the carcinogenic PBDE mixture. The toxicities of PBDE-47 and the PBDE mixture were evaluated at PND 22 in Wistar Han rat (Crl: WI (Han)) pups after in utero/postnatal exposure (0, 0.1, 15, or 50 mg/kg; dams, GD6-21; pups, PND 12-PND 21; oral gavage daily dosing). By PND 22, PBDE-47 caused centrilobular hypertrophy and fatty change in liver, and reduced serum thyroxin (T4) levels; similar effects were also observed after PBDE mixture exposure. Transcriptomic changes in the liver included induction of cytochrome p450 transcripts and up-regulation of Nrf2 antioxidant pathway transcripts and ABC membrane transport transcripts. Decreases in other transport transcripts (ABCG5 & 8) provided a plausible mechanism for lipid accumulation, characterized by a treatment-related liver fatty change after PBDE-47 and PBDE mixture exposure. The benchmark dose calculation based on liver transcriptomic data was generally lower for PBDE-47 than for the PBDE mixture. The up-regulation of the Nrf2 antioxidant pathway and changes in metabolic transcripts after PBDE-47 and PBDE mixture exposure suggest that PBDE-47, like the PBDE mixture (NTP 2016, TR 589), could be a liver toxin/carcinogen after long-term exposure.

Tetrabromobisphenol A activates the hepatic interferon pathway in rats.

Tetrabromobisphenol A (TBBPA) is a widely used flame retardant in printed circuit boards, paper, and textiles. In a two-year study, TBBPA showed evidence of uterine tumors in female Wistar-Han rats and liver and colon tumors in B6C3F1 mice. In order to gain further insight into early gene and pathway changes leading to cancer, we exposed female Wistar Han rats to TBBPA at 0, 25, 250, or 1000mg/kg (oral gavage in corn oil, 5×/week) for 13 weeks. Because at the end of the TBBPA exposure period, there were no treatment-related effects on body weights, liver or uterus lesions, and liver and uterine organ weights were within 10% of controls, only the high dose animals were analyzed. Analysis of the hepatic and uterine transcriptomes showed TBBPA-induced changes primarily in the liver (1000mg/kg), with 159 transcripts corresponding to 132 genes differentially expressed compared to controls (FDR=0.05). Pathway analysis showed activation of interferon (IFN) and metabolic networks. TBBPA induced few molecular changes in the uterus. Activation of the interferon pathway in the liver occurred after 13-weeks of TBBPA exposure, and with longer term TBBPA exposure this may lead to immunomodulatory changes that contribute to carcinogenic processes.

Characterization of polybrominated diphenyl ether toxicity in Wistar Han rats and use of liver microarray data for predicting disease susceptibilities.

The toxicity of polybrominated diphenyl ethers (PBDEs), flame-retardant components, was characterized in offspring from Wistar Han dams exposed by gavage to a PBDE mixture (DE71) starting at gestation day 6 and continuing to weaning on postnatal day (PND) 21. Offspring from the dams underwent PBDE direct dosing by gavage at the same dose as their dams from PND 12 to PND 21, and then after weaning for another thirteen weeks. Liver samples were collected at PND 22 and week 13 for liver gene expression analysis (Affymetrix Rat Genome 230 2.0 Array). Treatment with PBDE induced 1,066 liver gene transcript changes in females and 1,200 transcriptional changes in males at PND 22 (false discovery rate < 0.01), but only 263 liver transcriptional changes at thirteen weeks in male rats (false discovery rate < 0.05). No significant differences in dose response were found between male and female pups. Transcript changes at PND 22 coded for proteins in xenobiotic, sterol, and lipid metabolism, and cell cycle regulation, and overlapped rodent liver transcript patterns after a high-fat diet or phenobarbital exposure. These findings, along with the observed PBDE-induced liver hypertrophy and vacuolization, suggest that long-term PBDE exposure has the potential to modify cell functions that contribute to metabolic disease and/or cancer susceptibilities.

Release, biological potency, and biochemical integrity of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) combined with Augment(TM) Bone Graft or GEM 21S beta-tricalcium phosphate (beta-TCP).

Over 10 million surgical procedures are performed annually in the United States to treat musculoskeletal injuries, and a significant portion of these involve orthopedic bone grafting. The goals of the study were to evaluate the in vitro and in vivo release kinetics, biological potency and biochemical integrity of rhPDGF-BB combined with large (1000-2000 microm) and small (250-1000 microm) beta-TCP particles. Recombinant human platelet-derived growth factor B homodimer (rhPDGF-BB) is a protein growth factor under development as a therapeutic for accelerating bone healing. Release of the protein was monitored in vitro by ELISA, and in vivo by measurement of radioactive rhPDGF-BB implanted in rat calvarial defects. Biological activity was measured using a cell-based bioassay, and biochemical integrity was determined by SDS-PAGE and high pressure size exclusion chromatography (HPSEC). Release of rhPDGF-BB occurred rapidly from beta-TCP both in vitro and in vivo. Almost 100% of the rhPDGF-BB was recovered from large and small beta-TCP after 90 min in vitro. Approximately 90% of the rhPDGF-BB was depleted from calvarial defect sites within 72 h of implantation. RhPDGF-BB retained 100% of its biological potency compared to reference standard rhPDGF-BB, manifested as a single band at ~30 kDa by SDS-PAGE and a single peak eluted after 13 min by HPSEC following release from beta-TCP. RhPDGF-BB is rapidly released from large and small beta-TCP particles and is biochemically unaltered following release.

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) by rosiglitazone suppresses components of the insulin-like growth factor regulatory system in vitro and in vivo.

Rosiglitazone (Rosi) belongs to the class of thiazolidinediones (TZDs) that are ligands for peroxisome proliferator-activated receptor gamma (PPARgamma). Stimulation of PPARgamma suppresses bone formation and enhances marrow adipogenesis. We hypothesized that activation of PPARgamma down-regulates components of the IGF regulatory system, leading to impaired osteoblast function. Rosi treatment (1 microm) of a marrow stromal cell line (UAMS-33) transfected with empty vector (U-33/c) or with PPARgamma2 (U-33/gamma2) were analyzed by microarray. Rosi reduced IGF-I, IGF-II, IGFBP-4, and the type I and II IGF receptor (IGF1R and IGF2R) expression at 72 h in U-33/gamma2 compared with U-33/c cells (P < 0.01); these findings were confirmed by RT-PCR. Rosi reduced secreted IGF-I from U-33/gamma2 cells by 75% (P < 0.05). Primary marrow stromal cells (MSCs) extracted from adult (8 months) and old (24 months) C57BL/6J (B6) mice were treated with Rosi (1 microm) for 48 h. IGF-I, IGFBP-4, and IGF1R transcripts were reduced in Rosi-treated MSCs compared with vehicle (P < 0.01) and secreted IGF-I was also suppressed (P < 0.05). B6 mice treated with Rosi (20 mg/kg.d) for short duration (i.e. 4 d), and long term (i.e. 7 wk) had reduced serum IGF-I; this was accompanied by markedly suppressed IGF-I transcripts in the liver and peripheral fat of treated animals. To determine whether Rosi affected circulating IGF-I in humans, we measured serum IGF-I, IGFBP-2, and IGFBP-3 at four time points in 50 postmenopausal women randomized to either Rosi (8 mg/d) or placebo. Rosi-treated subjects had significantly lower IGF-I at 8 wk than baseline (-25%, P < 0.05), and at 16 wk their levels were reduced 14% vs. placebo (P = 0.15). We conclude that Rosi suppresses IGF-I expression in bone and liver; these changes could affect skeletal acquisition through endocrine and paracrine pathways.

PPARgamma2 Regulates a Molecular Signature of Marrow Mesenchymal Stem Cells.

Bone formation and hematopoiesis are anatomically juxtaposed and share common regulatory mechanisms. Bone marrow mesenchymal stromal/stem cells (MSC) contain a compartment that provides progeny with bone forming osteoblasts and fat laden adipocytes as well as fibroblasts, chondrocytes, and muscle cells. In addition, marrow MSC provide an environment for support of hematopoiesis, including the development of bone resorbing osteoclasts. The PPARgamma2 nuclear receptor is an adipocyte-specific transcription factor that controls marrow MSC lineage allocation toward adipocytes and osteoblasts. Increased expression of PPARgamma2 with aging correlates with changes in the MSC status in respect to both their intrinsic differentiation potential and production of signaling molecules that contribute to the formation of a specific marrow micro-environment. Here, we investigated the effect of PPARgamma2 on MSC molecular signature in respect to the expression of gene markers associated exclusively with stem cell phenotype, as well as genes involved in the formation of a stem cell supporting marrow environment. We found that PPARgamma2 is a powerful modulator of stem cell-related gene expression. In general, PPARgamma2 affects the expression of genes specific for the maintenance of stem cell phenotype, including LIF, LIF receptor, Kit ligand, SDF-1, Rex-1/Zfp42, and Oct-4. Moreover, the antidiabetic PPARgamma agonist TZD rosiglitazone specifically affects the expression of "stemness" genes, including ABCG2, Egfr, and CD44. Our data indicate that aging and anti-diabetic TZD therapy may affect mesenchymal stem cell phenotype through modulation of PPARgamma2 activity. These observations may have important therapeutic consequences and indicate a need for more detailed studies of PPARgamma2 role in stem cell biology.

The Thermotoga maritima phenotype is impacted by syntrophic interaction with Methanococcus jannaschii in hyperthermophilic coculture.

Significant growth phase-dependent differences were noted in the transcriptome of the hyperthermophilic bacterium Thermotoga maritima when it was cocultured with the hyperthermophilic archaeon Methanococcus jannaschii. For the mid-log-to-early-stationary-phase transition of a T. maritima monoculture, 24 genes (1.3% of the genome) were differentially expressed twofold or more. In contrast, methanogenic coculture gave rise to 292 genes differentially expressed in T. maritima at this level (15.5% of the genome) for the same growth phase transition. Interspecies H2 transfer resulted in three- to fivefold-higher T. maritima cell densities than in the monoculture, with concomitant formation of exopolysaccharide (EPS)-based cell aggregates. Differential expression of specific sigma factors and genes related to the ppGpp-dependent stringent response suggests involvement in the transition into stationary phase and aggregate formation. Cell aggregation was growth phase dependent, such that it was most prominent during mid-log phase and decayed as cells entered stationary phase. The reduction in cell aggregation was coincidental with down-regulation of genes encoding EPS-forming glycosyltranferases and up-regulation of genes encoding beta-specific glycosyl hydrolases; the latter were presumably involved in hydrolysis of beta-linked EPS to release cells from aggregates. Detachment of aggregates may facilitate colonization of new locations in natural environments where T. maritima coexists with other organisms. Taken together, these results demonstrate that syntrophic interactions can impact the transcriptome of heterotrophs in methanogenic coculture, and this factor should be considered in examining the microbial ecology in anaerobic environments.

Functional genomics-based studies of the microbial ecology of hyperthermophilic micro-organisms.

Although much attention has been paid to the genetic, biochemical and physiological aspects of individual hyperthermophiles, how these unique micro-organisms relate to each other and to their natural habitats must be addressed in order to develop a comprehensive understanding of life at high temperatures. Phylogenetic 16 S rRNA-based profiling of samples from various geothermal sites has provided insights into community structure, but this must be complemented with efforts to relate metabolic strategies to biotic and abiotic characteristics in high-temperature habitats. Described here are functional genomics-based approaches, using cDNA microarrays, to gain insight into how ecological features such as biofilm formation, species interaction, and possibly even gene transfer may occur in native environments, as well as to determine what genes or sets of genes may be tied to environmental functionality.

Pain perception during transrectal ultrasound guided prostate needle biopsy: an objective analysis of local anesthesia use.

We assessed the effect of periprostatic nerve blockade during transrectal ultrasound of the prostate prior to obtaining systematic needle biopsies and the discomfort associated with this procedure. A prospective randomized study was performed on 100 men requiring systematic needle biopsy of the prostate. Patients were assigned to two groups: Group 1 received no local anesthesia and Group 2 received a periprostatic injection of 5 ml 1% lidocaine solution (2.5 ml bilaterally) prior to undergoing biopsy of the prostate. The patients were asked to respond to a pre- and post-procedural questionnaire which consisted of four questions designed to evaluate pain perception and pain experienced, respectively, during the entire procedure. Mean pain scores for Group 1 responses vs Group 2 responses were not statistically different for any of the pre-procedural questions. Post-procedural pain scores were significantly lower in Group 2 vs Group 1 (control) for questions 1 and 3: question 1 (2.6+/-1.8 vs 3.8+/-1.8, P<0.05), question 2 (3.0+/-1.9 vs 3.7+/-2.1, P=0.14). Question 3 (2.8+/-2.0 vs 4.3+/-1.9, P<0.05), and question 4 (1.6+/-2.4 vs 2.1+/-2.6, P=0.38). During the study, no patient from Group 2 experienced any adverse reaction from the injection. Our data suggest that periprostatic nerve blockade during transrectal ultrasound of the prostate results in less patient discomfort.

Attunement, calibration, and exploration in fast haptic perceptual learning.

Often, a relatively small number of trials suffices to enhance one's task-specific perceptual capability. In the present experiment, fast perceptual learning was investigated with respect to the perception of the heights or widths of wielded nonvisible rectangular objects. In that haptic perceptual task, inertial differences (mass and moments of inertia) are the basis for perceived size differences. The authors hypothesized that rapid improvement might occur in attunement (attending to the task-relevant inertial variable), calibration (scaling spatial extent to the task-relevant inertial variable), and exploratory behavior (wielding so as to differentiate the task-relevant inertial variable). Twenty-four students performed 25 trials with a set of practice objects; those trials were followed and preceded by 18 trials with a set of test objects. Practice, with knowledge of results (KR), improved both attunement, as measured by regression of perceived spatial extent on the inertial variables, and calibration, as measured by constant and variable error. Of the preceding measures, only variable error improved with practice in the absence of KR. In both KR conditions, however, exploratory behavior decreased in duration and complexity, as measured by recurrence quantification analysis. The present results suggest that the mechanisms involved in fast perceptual learning are more varied and complex than are those encompassed by current accounts.

Disruption of spinal cord white matter and sciatic nerve geometry inhibits axonal growth in vitro in the absence of glial scarring.

BACKGROUND: Axons within the mature mammalian central nervous system fail to regenerate following injury, usually resulting in long-lasting motor and sensory deficits. Studies involving transplantation of adult neurons into white matter implicate glial scar-associated factors in regeneration failure. However, these studies cannot distinguish between the effects of these factors and disruption of the spatial organization of cells and molecular factors (disrupted geometry). Since white matter can support or inhibit neurite growth depending on the geometry of the fiber tract, the present study sought to determine whether disrupted geometry is sufficient to inhibit neurite growth. RESULTS: Embryonic chick sympathetic neurons were cultured on unfixed longitudinal cryostat sections of mature rat spinal cord or sciatic nerve that had been crushed with forceps ex vivo then immediately frozen to prevent glial scarring. Neurite growth on uncrushed portions of spinal cord white matter or sciatic nerve was extensive and highly parallel with the longitudinal axis of the fiber tract but did not extend onto crushed portions. Moreover, neurite growth from neurons attached directly to crushed white matter or nerve tissue was shorter and less parallel compared with neurite growth on uncrushed tissue. In contrast, neurite growth appeared to be unaffected by crushed spinal cord gray matter. CONCLUSIONS: These observations suggest that glial scar-associated factors are not necessary to block axonal growth at sites of injury. Disruption of fiber tract geometry, perhaps involving myelin-associated neurite-growth inhibitors, may be sufficient to pose a barrier to regenerating axons in spinal cord white matter and peripheral nerves.

Somatosensory attunement to the rigid body laws.

In the most general case, haptic perception of an object's heaviness is most likely the perception of the object's resistance to movement, determined jointly by the object's mass and mass distribution. In two experiments with occluded objects wielded freely in three dimensions, we showed additive effects on perceived heaviness of mass and the inertia tensor. Our manipulations of the inertia tensor were directed specifically at the volume and symmetry of the inertia ellipsoid, quantities that can be understood as important to controlling the level and patterning of muscular forces, respectively. Ellipsoid volume and symmetry were found to have separate effects on perceptual reports of heaviness that were invariant over different tensors. Independent sensitivities to translational inertia and particular characterizations of rotational inertia suggest specialized somatosensory attunement to the rigid body laws.

The critical role of patient follow-up after receiving a diagnosis of prostatic intraepithelial neoplasia.

With the increasing incidence of prostatic intraepithelial neoplasia being found at the time of prostate biopsy and the association of prostatic intraepithelial neoplasia to coexisting prostate cancer and/or the future development of prostate cancer, patient compliance in following post-biopsy follow-up instructions for re-biopsy is becoming more significant in the detection of prostate cancer at an earlier and, therefore, potentially curable stage. During a 3-y period, we reviewed the charts of 130 patients who received an initial diagnosis of prostatic intraepithelial neoplasia after undergoing transrectal ultrasound of the prostate with biopsy. It is our policy to inform the patient of their diagnosis of prostatic intraepithelial neoplasia at the time of the initial biopsy and to recommend a repeat biopsy in 6-12 months. Patients are informed of the diagnosis of prostatic intraepithelial neoplasia verbally and in writing. In addition, a letter is sent to their referring physician with the re-biopsy recommendation. Thirty-nine of 130 patients (30%) were seen for re-biopsy within the specified time. An additional 36 patients (27.69%) were re-biopsied between 12 and 18 months after the initial diagnosis of prostatic intraepithelial neoplasia. An additional 11 patients (7%) were re-biopsied more than 18 months after their initial diagnosis. Forty-four patients failed to return for re-biopsy. Overall, patient follow-up within the desired protocol was poor and must be improved upon to prevent any delays in the diagnosis of prostate cancer. Prostate Cancer and Prostatic Diseases (2001) 4, 63-66

The role of NGF in pregnancy-induced degeneration and regeneration of sympathetic nerves in the guinea pig uterus.

In the guinea pig, pregnancy is associated with a generalised depletion of noradrenaline in uterine sympathetic nerves and, in the areas of the uterus surrounding the foetus, by a complete degeneration of sympathetic nerve fibres. These pregnancy-induced changes have been interpreted as a selective effect of placental hormones on the system of short sympathetic fibres arising from the paracervical ganglia. An alternative explanation is that pregnancy affects the neurotrophic capacity of the uterus. We measured NGF-protein levels in the guinea pig uterine horn, tubal end and cervix at early pregnancy, late pregnancy and early postpartum, using a two-site enzyme-linked immunosorbent assay. For comparative purposes the distribution and relative density of noradrenaline-containing sympathetic nerve fibres were assessed histochemically, and tissue levels of noradrenaline were measured biochemically, using high-performance liquid chromatography with electrochemical detection. In all the uterine regions analysed, NGF-protein levels showed a decline at term pregnancy, but in no case was this change statistically significant. After delivery, NGF-protein levels showed a marked increase in the cervix as well as in both the fertile and empty horns. These results suggest that alterations in NGF-protein do not account for the impairment of uterine sympathetic innervation during pregnancy, but may contribute to their recovery after delivery.

Affordance, proper function, and the physical basis of perceived heaviness.

The physical basis of perceived heaviness requires consideration of the haptic perceptual system's role in controlling actions (the system's proper function) and the relation of an object's inertial properties to properties of the human movement system (the object's affordance). We show that the mass of a wielded object and particular scalar variables calculated from the object's inertia tensor combine linearly in determining perceived heaviness. The tensor-derived scalars reflect the symmetry and volume of the corresponding inertia ellipsoid. These measures bear directly on the object's wieldability, that is, on the patterning and level of muscular forces required to move the object in a controlled fashion.

Comparative analysis of prognostic factors in men undergoing radical prostatectomy for adenocarcinoma of the prostate, including DNA ploidy, surgical tumor stage, prostatic specific antigen, Gleason grade, and age.

One hundred consecutive men with adenocarcinoma of the prostate, treated by modified pelvic lymphadenectomy and radical retropubic prostatectomy, were evaluated, comparing DNA ploidy as determined by flow cytometry to surgical tumor stage (pT), preoperative prostatic specific antigen (PSA), Gleason grade, and age at presentation, in an effort to assess the prognostic ability of DNA ploidy. There were 71 (71%) men found to have diploid tumors and 29 (29%) with nondiploid tumors. There was no statistical difference in surgical pathologic stage between these two groups (P = 0.2369). There was no statistical difference when comparing preoperative PSA between these two groups (P = 0.0925). There was no statistical difference when comparing Gleason grade between these two groups (P = 0.5807). Age at presentation was similar in both groups. Based on these findings, it is apparent that longitudinal studies of patient outcome will be necessary to fully assess the prognostic ability of DNA ploidy determined by flow cytometry in men undergoing radical prostatectomy for treatment of adenocarcinoma of the prostate gland.

DNA heterogeneity determined by flow cytometry in prostatic adenocarcinoma--necessitating multiple site analysis.

Although DNA ploidy analysis of prostate cancer is generally associated with grade, stage, clinical outcome, and responsiveness to androgen therapy, one possible reason cited for contrary reports may be tumor heterogeneity. A preliminary report using flow cytometric analysis of punch biopsies demonstrated DNA heterogeneity in five of nine patients. We evaluated 75 patients by cutting whole mounts of formalin fixed prostatectomy tissue every 0.6 cm. All malignant areas and a selected normal area were circumscribed, excised, remounted, and 1-3 50 mu thick sections removed. The nuclei were extracted by a Hedley technique and the DNA stained with propidium iodide. Each whole mount had an average of 1 distinct malignant area (range of 1-6 areas per whole mount block). Nuclei were analyzed on a Becton Dickinson (San Jose, CA) FACScan flow cytometer equipped with RFIT DNA software program. After excluding histograms with CVs > 8.0% and/or "suspicious" diploid histograms having a right "shoulder," 75 or 87 patients still had > or = 2 malignant sites available for analysis (average 4, range 2-9 malignant sites/patient). The 322 histograms had an average CV of 4.4%. Thirty of 75 patients (40%) showed DNA heterogeneity in multiple samples taken from the same prostate. There were 37 prostates with only diploid (D), 1 with only tetraploid (T), 7 with only aneuploid (A), 20 with D plus A, 7 with D plus T, 2 with D plus T plus A, and 1 with a D plus suspected hypodiploid DNA content. Exclusion of the tetraploid and "near diploid aneuploid" cases still resulted in 16% (12/75) of the patients having a diploid versus aneuploid DNA content heterogeneity. Because 40% of the prostates contained a different ploidy depending on which area was sampled, this report suggests multiple sites of malignancy must be analyzed to more accurately assess the ploidy status of prostatic adenocarcinoma.