Hemocompatibility evaluation of poly(glycerol-sebacate) in vitro for vascular tissue engineering

Poly(glycerol-sebacate) (PGS) is an elastomeric biodegradable polyester that could potentially be used to engineer blood vessels in vivo. However, its blood-material interactions are unknown. The objectives of this study were to: (a) fabricate PGS-based biphasic tubular scaffolds and (b) assess the blood compatibility of PGS in vitro in order to get some insight into its potential use in vivo. PGS was incorporated into biphasic scaffolds by dip-coating glass rods with PGS pre-polymer. The thrombogenicity (platelet adhesion and aggregation) and inflammatory potential (IL-1beta and TNFalpha expression) of PGS were evaluated using fresh human blood and a human monocyte cell line (THP-1). The activation of the clotting system was assessed via measurement of tissue factor expression on THP-1 cells, plasma recalcification times, and whole blood clotting times. Glass, tissue culture plastic (TCP), poly(l-lactide-co-glycolide) (PLGA), and expanded polytetrafluorethylene (ePTFE) were used as reference materials. Biphasic scaffolds with PGS as the blood-contacting surface were successfully fabricated. Relative to glass (100%), platelet attachment on ePTFE, PLGA and PGS was 61%, 100%, and 28%, respectively. PGS elicited a significantly lower release of IL-1beta and TNFalpha from THP-1 cells than ePTFE and PLGA. Similarly, relative to all reference materials, tissue factor expression by THP-1 cells was decreased when exposed to PGS. Plasma recalcification and whole blood clotting profiles of PGS were comparable to or better than those of the reference polymers tested.     

Region-specific dissociation of neuronal loss and neurofibrillary pathology in a mouse model of tauopathy

Neurofibrillary tangles form in a specific spatial and temporal pattern in Alzheimer's disease. Although tangle formation correlates with dementia and neuronal loss, it remains unknown whether neurofibrillary pathology causes cell death. Recently, a mouse model of tauopathy was developed that reversibly expresses human tau with the dementia-associated P301L mutation. This model (rTg4510) exhibits progressive behavioral deficits that are ameliorated with transgene suppression. Using quantitative analysis of PHF1 immunostaining and neuronal counts, we estimated neuron number and accumulation of neurofibrillary pathology in five brain regions. Accumulation of PHF1-positive tau in neurons appeared between 2.5 and 7 months of age in a region-specific manner and increased with age. Neuron loss was dramatic and region-specific in these mice, reaching over 80% loss in hippocampal area CA1 and dentate gyrus by 8.5 months. We observed regional dissociation of neuronal loss and accumulation of neurofibrillary pathology, because there was loss of neurons before neurofibrillary lesions appeared in the dentate gyrus and, conversely, neurofibrillary pathology appeared without major cell loss in the striatum. Finally, suppressing the transgene prevented further neuronal loss without removing or preventing additional accumulation of neurofibrillary pathology. Together, these results imply that neurofibrillary tangles do not necessarily lead to neuronal death.     

Additional chromosomal abnormalities in patients with a previously detected abnormal karyotype, mental retardation, and dysmorphic features

The detection of chromosomal abnormalities in patients with mental retardation (MR) and dysmorphic features increases with improvements of molecular cytogenetic methods. We report on six patients referred for detailed characterization of chromosomal abnormalities (four translocations, one inversion, one deletion) detected by conventional cytogenetics, in whom metaphase CGH revealed imbalances not involved in the initially detected rearrangements. The detected abnormalities were validated by real-time PCR. Parents were investigated by CGH in four cases. The genomic screening revealed interstitial deletions of 2q33.2-q34, 3p21, 4q12-q13.1, 6q25, 13q22.2-q31.1, and 14q12. The estimated minimum sizes of the deletions ranged from 2.65 to 9.27 Mb. The CGH assay did not reveal imbalances that colocalized with the breakpoints of the inversion or the translocations. The deletion of 6q included ESR1, in which polymorphisms are associated with variation of adult height. FOXG1B, known to be involved in cortical development, was located in the 14q deletion. The results illustrate that whole-genome molecular cytogenetic analysis of phenotypically affected patients with abnormal conventional karyotypes may detect inapparent molecular cytogenetic abnormalities in patients with microscopic chromosomal abnormalities and that these data provide additional information of clinical importance.     

Genome-wide detection of human copy number variations using high-density DNA oligonucleotide arrays

Recent reports indicate that copy number variations (CNVs) within the human genome contribute to nucleotide diversity to a larger extent than single nucleotide polymorphisms (SNPs). In addition, the contribution of CNVs to human disease susceptibility may be greater than previously expected, although a complete understanding of the phenotypic consequences of CNVs is incomplete. We have recently reported a comprehensive view of CNVs among 270 HapMap samples using high-density SNP genotyping arrays and BAC array CGH. In this report, we describe a novel algorithm using Affymetrix GeneChip Human Mapping 500K Early Access (500K EA) arrays that identified 1203 CNVs ranging in size from 960 bp to 3.4 Mb. The algorithm consists of three steps: (1) Intensity pre-processing to improve the resolution between pairwise comparisons by directly estimating the allele-specific affinity as well as to reduce signal noise by incorporating probe and target sequence characteristics via an improved version of the Genomic Imbalance Map (GIM) algorithm; (2) CNV extraction using an adapted SW-ARRAY procedure to automatically and robustly detect candidate CNV regions; and (3) copy number inference in which all pairwise comparisons are summarized to more precisely define CNV boundaries and accurately estimate CNV copy number. Independent testing of a subset of CNVs by quantitative PCR and mass spectrometry demonstrated a >90% verification rate. The use of high-resolution oligonucleotide arrays relative to other methods may allow more precise boundary information to be extracted, thereby enabling a more accurate analysis of the relationship between CNVs and other genomic features.     

Incidence of delirium and associated mortality in hematopoietic stem cell transplantation patients.

Delirium has been associated with a high risk of mortality in medical patients. Despite the high incidence of delirium in patients who undergo hemapoietic stem cell transplantation (HSCT), delirium as a risk factor for death has not been examined in this population. Thirty adult patients undergoing HSCT who were admitted to the University of Iowa Blood and Marrow Transplantation Program inpatient unit were assessed prospectively from 1 to 2 weeks before transplantation, throughout their inpatient stay, and at 100 days after transplantation. The Delirium Rating Scale and Memorial Delirium Assessment Scale were used twice weekly during the inpatient period to assess delirium severity and occurence. Patients' self-reports of medical history, computerized medical records, and neuropsychological and psychiatric assessments were used to identify pretransplantation risk factors. The incidence of delirium (Delirium Rating Scale score >12 or Memorial Delirium Assessment Scale score >or=8) was 43% and occurred with highest frequency in the first 2 weeks after transplantion. The presence of delirium at any point during hospitalization after transplantation and transplant type (allogeneic) were highly predictive of mortality (p < .0005; odds ratios, 14.0 and 14.4). In conclusion, this study highlights the importance of monitoring for delirium during the acute recovery period after transplantation and suggests that early or even prophylactic treatment for delirium should be studied. Studies to determine the factors that connect delerium soon after transplantation to mortality are highly warranted.     

Neural activation in cognitive motor processes: comparing motor imagery and observation of gymnastic movements

The simulation concept suggested by Jeannerod (Neuroimage 14:S103-S109, 2001) defines the S-states of action observation and mental simulation of action as action-related mental states lacking overt execution. Within this framework, similarities and neural overlap between S-states and overt execution are interpreted as providing the common basis for the motor representations implemented within the motor system. The present brain imaging study compared activation overlap and differential activation during mental simulation (motor imagery) with that while observing gymnastic movements. The fMRI conjunction analysis revealed overlapping activation for both S-states in primary motor cortex, premotor cortex, and the supplementary motor area as well as in the intraparietal sulcus, cerebellar hemispheres, and parts of the basal ganglia. A direct contrast between the motor imagery and observation conditions revealed stronger activation for imagery in the posterior insula and the anterior cingulate gyrus. The hippocampus, the superior parietal lobe, and the cerebellar areas were differentially activated in the observation condition. In general, these data corroborate the concept of action-related S-states because of the high overlap in core motor as well as in motor-related areas. We argue that differential activity between S-states relates to task-specific and modal information processing.     

Within-Breath Control of Genioglossal Muscle Activation in Humans: Effect of Sleep-Wake State

Pharyngeal dilator muscles are clearly important in the pathogenesis of obstructive sleep apnoea syndrome. Substantial data support the role of a local negative pressure reflex in modifying genioglossal activation across inspiration during wakefulness. Using a model of passive negative pressure ventilation, we have previously reported a tight relationship between varying intrapharyngeal negative pressures and genioglossal muscle activation (GGEMG) during wakefulness. In this study, we used this model to examine the slope of the relationship between epiglottic pressure ( P _epi) and GGEMG, during stable NREM sleep and the transition from wakefulness to sleep. We found that there was a constant relationship between negative epiglottic pressure and GGEMG during both basal breathing (BB) and negative pressure ventilation (NPV) during wakefulness (slope GGEMG/ P _epi 1.86 ± 0.3 vs. 1.79 ± 0.3 arbitrary units (a.u.) cmH₂O⁻¹). However, while this relationship remained stable during NREM sleep during BB, it was markedly reduced during NPV during sleep (2.27 ± 0.4 vs. 0.58 ± 0.1 a.u. cmH₂O⁻¹). This was associated with a markedly higher pharyngeal airflow resistance during sleep during NPV. At the transition from wakefulness to sleep there was also a greater reduction in peak GGEMG seen during NPV than during BB. These data suggest that while the negative pressure reflex is able to maintain GGEMG during passive NPV during wakefulness, this reflex is unable to do so during sleep. The loss of this protective mechanism during sleep suggests that an airway dependent upon such mechanisms (as in the patient with sleep apnoea) will be prone to collapse during sleep.     

Myocardial volume and organization are changed by failure of addition of secondary heart field myocardium to the cardiac outflow tract.

Cardiac neural crest ablation results in primary myocardial dysfunction and failure of the secondary heart field to add the definitive myocardium to the cardiac outflow tract. The current study was undertaken to understand the changes in myocardial characteristics in the heart tube, including volume, proliferation, and cell size when the myocardium from the secondary heart field fails to be added to the primary heart tube. We used magnetic resonance and confocal microscopy to determine that the volume of myocardium in the looped heart was dramatically reduced and the compact layer of myocardium was thinner after neural crest ablation, especially in the outflow tract and ventricular regions. Proliferation measured by 5-bromo-2'-deoxyuridine incorporation was elevated at only one stage during looping, cell death was normal and myocardial cell size was increased. Taken together, these results indicate that there are fewer myocytes in the heart. By incubation day 8 when the heart would have normally completed septation, the anterior (ventral) wall of the right ventricle and right ventricular outflow tract was significantly thinner in the neural crest-ablated embryos than normal, but the thickness of the compact myocardium was normal in all other regions of the heart. The decreased volume and number of myocardial cells in the heart tube after neural crest ablation most likely reflects the amount of myocardium added by the secondary heart field.     

The role of nanometer and sub-micron surface features on vascular and bone cell adhesion on titanium

The quantified contribution of pure nanometer (features less than 100 nm in both the lateral and vertical scale) and sub-micron (features larger than 100 nm in the lateral scale) surface structures on the adhesion of vascular (endothelial) and bone (osteoblasts) cells were demonstrated in this study. Compared with flat titanium surfaces, sub-micron surface features led to a 27% increase in surface energy and promoted endothelial cell adhesion density by 200%. In addition, nanometer surface features also led to a 10% increase in surface energy and a 50% increase in endothelial cell adhesion density compared to flat titanium surfaces. Using aligned patterns of such features on titanium, it was clearly identified that both endothelial and bone cells selectively adhered onto sub-micron and nanometer surface features by 400% and 50% more than onto flat regions, respectively. Thus, the surface patterns developed in this study clearly confirmed that sub-micron to nanometer titanium surface features enhanced cytocompatibility properties for both endothelial and bone cells. Although sub-micron features on titanium had the highest surface energy and the greatest cell adhesion densities, nanometer surface features in this study were more efficient surface features increasing both surface energy and cell adhesion more with respect to smaller changes in surface area and surface roughness (compared to sub-micron surface features on titanium which had considerably larger changes in surface area and surface roughness).     

Intragastric infusion of denatonium conditions flavor aversions and delays gastric emptying in rodents

Because most naturally occurring toxins taste bitter to humans, any mechanism that reduces the rate at which bitter substances are ingested and digested should be adaptive. Based on the recent discovery of T2R bitter taste receptors in the gastrointestinal tract of rodents, we asked whether intragastric (IG) infusion of denatonium (a ligand for T2R receptors) would condition a flavor aversion and/or delay gastric emptying. Four experiments tested for post-oral responses to denatonium in rodents. First, Sprague-Dawley rats were trained to associate intake of a flavored solution (the CS+) with IG denatonium infusions, and intake of a different-flavored solution (the CS-) with IG water infusions during 30 min/day sessions. The rats acquired an aversion to the CS+ flavor when it was paired with IG infusions of 10 mM (but not 2.5 mM) denatonium. Intragastric infusions of 10 mM denatonium also delayed gastric emptying of food in the same rats. Second, we asked how long it took for rats to suppress their drinking while being infused IG with 10 mM denatonium. Rats drinking a palatable solution paired with IG infusions of 10 mM denatonium suppressed their licking within 6 min, as compared to rats infused IG with water. Third, we trained C57BL/6J (B6) mice 24 h/day to associate a CS+ flavor paired with IG infusions of 12 mM denatonium (diluted to 6 mM by orally consumed CS+). Like rats, the mice acquired a robust aversion to the CS+ flavor when it was paired with IG infusions of denatonium. A final experiment assessed the potential toxicity of denatonium. To this end, we gave B6 mice a 6 mM denatonium solution as their only source of water for 3 weeks. The mice grew normally and did not display any clinical signs of denatonium toxicosis. This study provides the first evidence that rodents respond to the presence of "bitter" substances in their gastrointestinal tract by generating both behavioral and physiological responses.