A double-chamber rotating bioreactor for the development of tissue-engineered hollow organs: From concept to clinical trial

Cell and tissue engineering are now being translated into clinical organ replacement, offering alternatives to fight morbidity, organ shortages and ethico-social problems associated with allotransplantation. Central to the recent first successful use of stem cells to create an organ replacement in man was our development of a bioreactor environment. Critical design features were the abilities to drive the growth of two different cell types, to support 3D maturation, to maintain biomechanical and biological properties and to provide appropriate hydrodynamic stimuli and adequate mass transport. An analytical model was developed and applied to predict oxygen profiles in the bioreactor-cultured organ construct and in the culture media, comparing representative culture configurations and operating conditions. Autologous respiratory epithelial cells and mesenchymal stem cells (BMSCs, then differentiated into chondrocytes) were isolated, characterized and expanded. Both cell types were seeded and cultured onto a decellularized human donor tracheal matrix within the bioreactor. One year post-operatively, graft and patient are healthy, and biopsies confirm angiogenesis, viable epithelial cells and chondrocytes. Our rotating double-chamber bioreactor permits the efficient repopulation of a decellularized human matrix, a concept that can be applied clinically, as demonstrated by the successful tracheal transplantation.     

Are adolescents who moderate their cannabis use at lower risk of later regular and dependent cannabis use?

Aims   To examine whether moderation of cannabis use among adolescent cannabis users is associated with reductions in cannabis use frequency and risk of dependence in young adulthood.
Design   Ten-year representative cohort study with six surveys in adolescence (mean age 14.9–17.4 years) and two in young adulthood (mean age 20.7 and 24.1 years).
Participants   Inception cohort of 1943 Victorian secondary school students (96% response rate), with 1520 (78% of adolescent participants) interviewed in the final wave.
Measurements   Participants were classified into six groups according to the maximum level of adolescent use and the extent of subsequent moderation in such use: non-users, occasional to abstinence, occasional persisting, weekly to abstinence, weekly to occasional and weekly persisting. Outcome measures were weekly+ cannabis use and DSM-IV cannabis dependence at 20 and 24 years.
Findings   Thirty-one per cent reported cannabis use during adolescence. Most adolescent users had moderated their use: from occasional to abstinence (71% of occasional users), weekly to abstinence or weekly to occasional (28% and 48% of weekly+ users, respectively). By age 24, both occasional use groups were at similar, elevated risk of regular and dependent cannabis use compared to non-users. Weekly+ adolescent users were at greatest risk of these outcomes, although the weekly to abstinence group exhibited lower risk than those in the weekly persisting and weekly to occasional groups, who were at similar risk.
Conclusions   While many young people have dynamic cannabis use patterns, a pattern of moderating adolescent cannabis use was associated with less risk of later problematic use than among those persisting, but risks were still elevated substantially compared with never-users.     

A mouse model system to genetically dissect the molecular mechanisms regulating tumorigenesis.

The vast majority of human tumors are of epithelial origin and result from the accumulation of mutations that alter the function of pathways that control critical cellular processes, including proliferation, checkpoint regulation, and apoptosis. Authentically replicating these events in animal models is critical to understanding the biology of cancer and for testing the feasibility of novel therapies. We developed a mouse model that recapitulates the steps of epithelial tumor progression of multiple tissue types (kidney, breast, ovarian surface, and prostate epithelia), which takes advantage of the power of mouse genetics, and that allows for biochemical analysis, genetic selection, and screening. Moreover, this model enables functional interrogation of far more complex tumor genotypes, both of the tumor cells themselves, and of the cells in the tumor microenvironment. This is a crucial advantage, as human tumors result from multiple compound mutations, most of which are difficult to achieve through standard mutant mouse technology. We have applied this model to establish the role of apoptosis in epithelial solid tumor progression and in treatment response, which has provided novel opportunities for cancer therapies in humans.     

Validation of ECG indices of ventricular repolarization heterogeneity: a computer simulation study

INTRODUCTION: Repolarization heterogeneity (RH) is functionally linked to dispersion in refractoriness and to arrhythmogenicity. In the current study, we validate several proposed electrocardiogram (ECG) indices for RH: T-wave amplitude, -area, -complexity, and -symmetry ratio, QT dispersion, and the Tapex-end interval (the latter being an index of transmural dispersion of the repolarization (TDR)). METHODS AND RESULTS: We used ECGSIM, a mathematical simulation model of ECG genesis in a human thorax, and varied global RH by increasing the standard deviation (SD) of the repolarization instants from 20 (default) to 70 msec in steps of 10 msec. T-wave amplitude, -area, -symmetry, and Tapex-end depended linearly on SD. T-wave amplitude increased from 275 +/- 173 to 881 +/- 456 muV, T-wave area from 34 x 10(3)+/- 21 x 10(3) to 141 x 10(3)+/- 58 x 10(3)muV msec, T-wave symmetry decreased from 1.55 +/- 0.11 to 1.06 +/- 0.23, and Tapex-end increased from 84 +/- 17 to 171 +/- 52 msec. T-wave complexity increased initially but saturated at SD = 50 msec. QT dispersion increased modestly until SD = 40 msec and more rapidly for higher values of SD. TDR increased linearly with SD. Tapex-end increased linearly with TDR, but overestimated it. CONCLUSION: T-wave complexity did not discriminate between differences in larger RH values. QT dispersion had low sensitivity in the transitional zone between normal and abnormal RH. In conclusion, T-wave amplitude, -area, -symmetry, and, with some limitations, Tapex-end and T-wave complexity reliably reflect changes in RH.     

Clorazepate affects cell surface regulation of delta and kappa opioid receptors, thereby altering buprenorphine-induced adaptation in the rat brain

Concomitant abuse of buprenorphine (BPN) and benzodiazepines (BZD) may relate to a pharmacodynamic interaction between the two. The objective of the present work was to investigate the acute and chronic effects of clorazepate (CRZ) alone or in combination with BPN on selective kappa opiate tritiated ligand [3H]-U69 593 and delta opiate radioligand [3H]-deltorphine II binding in the rat brain. Bmax (maximal receptor density) and Kd (the dissociation constant) were directly determined at different brain regions of interest (ROI) selected for high densities of kappa and/or delta receptors in rats treated with BPN and/or CRZ. The agents were administered either once or for 21 consecutive days. Differences in Bmax and Kd (for both specific ligands) were related to drug treatment and receptor location. Globally, single BPN administration induced no changes in kappa or delta opiate receptor binding, whereas repeated BPN administration up-regulated kappa receptor density and decreased delta affinity. At the kappa receptor level, repeated administration of CRZ acted only on Kd, whereas the delta receptor was up-regulated. Repeated addition of CRZ to BPN had no effect on kappa receptor Bmax versus chronic controls. By significantly decreasing Bmax, CRZ nullified the effect of chronic BPN on the kappa receptor. The modifications were strongest in the nucleus accumbens, where both types of receptor occur. Treatments had region-selective effects in some brain areas, such as the amygdala, periaqueductal gray matter, hypothalamus and caudate putamen. Increased mu and delta receptor densities would be expected to provide reinforcement by enhancing reward, and impairment of kappa receptor availability would be expected to decrease aversion. The effects described are likely to influence addictive behavior among people abusing BZD and BPN.     

Gait adaptation of children with cerebral palsy compared with control children when stepping over an obstacle

This study investigated the adaptive gait pattern in obstacle clearance in 12 normally developing (ND) children (six males, six females; mean age 10y 2mo, SD 10mo) and 12 children who had spastic diplegic cerebral palsy (CP) and who were independent ambulators (10 males, two females; mean age 13y 5mo, SD 2y 7mo). Children in both groups had 8 to 12 years of walking experience. They walked up to and crossed over obstacles of a height of 0%, 10%, and 20% of their leg length. Kinematic aspects of the three-dimensional leg movements were captured with the Vicon system and analyzed with one-way analysis of variance with repeated measure. Both groups increased foot clearance for higher obstacles. Children with CP were slower in approach and crossing speed, and used a wider base of support than the ND children when the obstacle height was increased. Results suggest that motor problems in obstacle clearance in children with CP lie in the implicit process of motor control, i.e. torque dynamics, not the explicit process, i.e. movement-match with the environmental constraints.     

Adrenomedullin gene expression and levels in the cardiovascular system after treatment with lipopolysaccharide

To study the effect of septicaemia, the temporal changes in tissue adrenomedullin (AM) and preproAM mRNA levels were studied in the heart and blood vessels after lipopolysaccharide (LPS) injection. Radioimmunoassay and solution hybridization-RNase protection assays were used to follow the changes in AM and its mRNA levels respectively after intraperitoneal injection of 10 mg/kg LPS in rats. The preproAM mRNA levels increased at 1 h in the right atrium after LPS injection, while the AM contents decreased at 1 h in the left atrium. The preproAM mRNA levels increased at 3 and 6 h in the left ventricle, whereas it increased at 6 h in the right ventricles after LPS injection. There was an increase in preproAM mRNA levels at 1 and 3 h in the mesenteric artery, while AM levels were increased at 1, 3 and 6 h. However, there were no such changes in the thoracic aorta. There were also increases in tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta and IL-6 in the heart, and in the mesenteric artery (TNF-alpha and IL-1beta) and in thoracic aorta (IL-1beta and IL-6). The present results suggest that the biosynthesis and secretion of AM may be increased in cardiovascular tissues of rats injected with LPS, and that AM may play multiple roles in inflammation.     

The VIP-secreting tumor as a differential diagnosis of protracted diarrhea in pediatrics

BACKGROUND: Vasoactive intestinal peptide (VIP) can be produced by mature neurogenic tumors. Pathologically elevated VIP plasma levels cause secretory diarrhea with excessive loss of water and electrolytes. Despite the clinical severity diagnosis of a VIP-secreting tumor is often delayed and subsequently its extirpation as the mainstay of therapy. PATIENTS: We report on two patients with ganglioneuroblastoma and secretory diarrhea. We contrast the case of a 13-month-old boy with advanced symptoms of secretory diarrhea, high VIP plasma levels, and late diagnosis to the case of a 14-month-old boy with mild secretory diarrhea and normal VIP plasma levels but positive proof of VIP in tumor tissue. Reviewing the literature we found 57 cases of pediatric VIP-secreting tumors. RESULTS: The clinical situation is characterized by the typical symptoms of secretory diarrhea with hypokalemia and metabolic acidosis. Histopathology predominantly reveals ganglioneuroblastoma or ganglioneuroma. The symptoms mostly stop after complete resection of the tumor whereas lack of resection is associated with elevated mortality rates. CONCLUSIONS: In case of prolonged therapy-resistant secretory diarrhea the existence of a VIP-secreting tumor should be considered. Diagnostic work-up should include the assessment of VIP plasma levels, catecholamines in urine, and appropriate imaging techniques in order to rule out or confirm the possibility of a VIP producing tumor.