An integrative biology approach to understanding keratinocyte collective migration as stimulated by bioglass.

A critical phase of wound healing is the coordinated movement of keratinocytes. To this end, bioglasses show promise in speeding healing in hard tissues and skin wounds. Studies suggest that bioglass materials may promote wound healing by inducing positive cell responses in proliferation, growth factor production, expression of angiogenic factors, and migration. Precise details of how bioglass may stimulate migration are unclear, however, because the common assays for studying migration in wound healing focus on simplified outputs like rate of migration or total change in wound area. These outputs are limited in that they represent the average behavior of the collective, with no connection between the motion of the individual cells and the collective wound healing response. There is a need to apply more refined tools that identify how the motion of the individual cells changes in response to perturbations, such as by bioglass, and in turn affects motion of the cell collective. Here, we apply an integrative biology strategy that combines an in vitro wound healing assay using primary neonatal human keratinocytes with time lapse microscopy and quantitative image analysis. The resulting data set provides the cell velocity field, from which we define key metrics that describe cooperative migration phenotypes. Treatment with growth factors led to faster single-cell speeds compared to control, but the migration was not cooperative, with cells breaking away from their neighbors and migrating as individuals. Treatment with calcium or bioglass led to migration phenotypes that were highly collective, with greater coordination in space compared to control. We discuss the link between bioglass treatment and observed increases in free calcium ions that are hypothesized to promote these distinct coordinated behaviors in primary keratinocytes. These findings have been enabled by the unique descriptors developed through applying image analysis to interpret biological response in migration models. Insight Box/Paragraph Statement: Bioglasses are important materials for tissue engineering and have more recently shown promise in skin and wound healing by mechanisms tied to their unique ionic properties. The precise details, however, of how cell migration may be affected by bioglass are left unclear by traditional cell assay methods. The following describes the integration of migration assays of keratinocytes, cells critical for skin and wound healing, with the tools of time lapse microscopy and image analysis to generate a quantitative description of coordinated, tissue-like migration behavior, stimulated by bioglass, that would not have been accessible without the combination of these analytical tools.

Biomimetic human skin model patterned with rete ridges.

Rete ridges consist of undulations between the epidermis and dermis that enhance the mechanical properties and biological function of human skin. However, most human skin models are fabricated with a flat interface between the epidermal and dermal layers. Here, we report a micro-stamping method for producing human skin models patterned with rete ridges of controlled geometry. To mitigate keratinocyte-induced matrix degradation, telocollagen-fibrin matrices with and without crosslinks enable these micropatterned features to persist during longitudinal culture. Our human skin model exhibits an epidermis that includes the following markers: cytokeratin 14, p63, and Ki67 in the basal layer, cytokeratin 10 in the suprabasal layer, and laminin and collagen IV in the basement membrane. We demonstrated that two keratinocyte cell lines, one from a neonatal donor and another from an adult diabetic donor, are compatible with this model. We tested this model using an irritation test and showed that the epidermis prevents rapid penetration of sodium dodecyl sulfate. Gene expression analysis revealed differences in keratinocytes obtained from the two donors as well as between 2D (control) and 3D culture conditions. Our human skin model may find potential application for drug and cosmetic testing, disease and wound healing modeling, and aging studies.

Coronary Artery Disease and Aspirin Intolerance: Background and Insights on Current Management.

Aspirin is one of the most widely used medications across the global healthcare system and is the foundation in treating ischemic heart disease, as well as secondary prevention for ischemic and valvular heart disease. Challenges arise in treating patients with cardiovascular disease who have concomitant aspirin intolerance. Through an extensive review of the literature, we provide a comprehensive background on the pharmacology of aspirin, the mechanisms behind aspirin intolerance, the importance of aspirin in cardiovascular disease, and the management of aspirin intolerance in both acute coronary syndrome and stable coronary artery disease. Our review includes a multidisciplinary approach from the internist, allergist/immunologist, and cardiologist when evaluating this important patient population.

Impact of Isolated Burns on Major Organs: A Large Animal Model Characterized.

Severe burn results in systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction (MOD). Currently, large-animal models of burn-induced SIRS/MOD mostly use secondary insults resulting in a paucity of knowledge on the effect of burn alone on different organ systems. The objective of the current study was to develop and characterize a large animal model of burn-induced SIRS over the course of 2 weeks. Yorkshire swine (n = 16) were randomized to sham controls (n = 4) or 40% total body surface area contact burns (n = 6 at 2 and 14 days post-burn). Blood chemistry and complete blood count analyses were performed at baseline and post-burn days 1, 2, 3, 7, 10, and 14. Upon euthanasia, tissue samples were taken for histopathology. Burns were found to be full thickness and did not re-epithelialize. SIRS was evidenced by increased body temperature, respiration rate, pulse, and white blood cell count for the duration of the experiment. Both acute liver injury and acute kidney injury were induced as determined biochemically and histologically. Histology also revealed atelectasis of the lungs which was associated with increased myeloperoxidase activity. Intestinal structure as well as enterocyte homeostasis was also disrupted. All of these organ abnormalities recovered to varying degrees by 14 days post-burn. We report a unique reproducible large animal model of burn-induced SIRS that can be tailored to specific organ systems for investigation into potential immunomodulatory interventions that prevent organ failure or promote organ recovery after burn injury.

Ontology analysis of global gene expression differences of human bone marrow stromal cells cultured on 3D scaffolds or 2D films.

Differences in gene expression of human bone marrow stromal cells (hBMSCs) during culture in three-dimensional (3D) nanofiber scaffolds or on two-dimensional (2D) films were investigated via pathway analysis of microarray mRNA expression profiles. Previous work has shown that hBMSC culture in nanofiber scaffolds can induce osteogenic differentiation in the absence of osteogenic supplements (OS). Analysis using ontology databases revealed that nanofibers and OS regulated similar pathways and that both were enriched for TGF-ß and cell-adhesion/ECM-receptor pathways. The most notable difference between the two was that nanofibers had stronger enrichment for cell-adhesion/ECM-receptor pathways. Comparison of nanofibers scaffolds with flat films yielded stronger differences in gene expression than comparison of nanofibers made from different polymers, suggesting that substrate structure had stronger effects on cell function than substrate polymer composition. These results demonstrate that physical (nanofibers) and biochemical (OS) signals regulate similar ontological pathways, suggesting that these cues use similar molecular mechanisms to control hBMSC differentiation.

Biopolymer/Calcium phosphate scaffolds for bone tissue engineering.

With nearly 30 years of progress, tissue engineering has shown promise in developing solutions for tissue repair and regeneration. Scaffolds, together with cells and growth factors, are key components of this development. Recently, an increasing number of studies have reported on the design and fabrication of scaffolding materials. In particular, inspired by the nature of bone, polymer/ceramic composite scaffolds have been studied extensively. The purpose of this paper is to review the recent progress of the naturally derived biopolymers and the methods applied to generate biomimetic biopolymer/calcium phosphate composites as well as their biomedical applications in bone tissue engineering.

Using double-stranded DNA probes to promote specificity in target capture.

The most prevalent nucleic acid detection schemes employ single-stranded sequences as probes for capture and detection of oligonucleotide targets in solution. In these systems elevated temperature conditions are generally used to enhance specificity and limit false positives from occurring with mismatched targets. In contrast, the current study uses a strand displacement approach between soluble targets and double-stranded DNA probes (dsProbes) immobilized on microspheres. In our approach the displacement of reporter strands from the dsProbes by the target of interest is promoted by the affinity differences between the reporter strand and the soluble DNA or RNA targets for the immobilized sequences. While displacement activity occurred readily in center mismatched dsProbes with a weaker intrinsic affinity, incorporating a two base-long single-stranded segment at the free end of the immobilized dsProbes resulted in target discrimination not observed for dsProbes possessing only a center mismatch.

Hybridization kinetics between immobilized double-stranded DNA probes and targets containing embedded recognition segments.

We have investigated the time-dependent strand displacement activity of several targets with double-stranded DNA probes (dsProbes) of varying affinity. Here, the relative affinity of various dsProbes is altered through choices in hybridization length (11-15 bases) and the selective inclusion of center mismatches in the duplexes. While the dsProbes are immobilized on microspheres, the soluble, 15 base-long complementary sequence is presented either alone as a short target strand or as a recognition segment embedded within a longer target strand. Compared to the short target, strand displacement activity of the longer targets is slower, but still successful. Additionally, the longer targets exhibit modest differences in the observed displacement rates, depending on the location of recognition segment within the long target. Overall, our study demonstrates that the kinetics of strand displacement activity can be tuned through dsProbe sequence design parameters and is only modestly affected by the location of the complementary segment within a longer target strand.

A comprehensive laboratory animal facility pandemic response plan.

The potential of a severe influenza pandemic necessitates the development of an organized, rational plan for continued laboratory animal facility operation without compromise of the welfare of animals. A comprehensive laboratory animal program pandemic response plan was integrated into a university-wide plan. Preparation involved input from all levels of organizational hierarchy including the IACUC. Many contingencies and operational scenarios were considered based on the severity and duration of the influenza pandemic. Trigger points for systematic action steps were based on the World Health Organization's phase alert criteria. One extreme scenario requires hibernation of research operations and maintenance of reduced numbers of laboratory animal colonies for a period of up to 6 mo. This plan includes active recruitment and cross-training of volunteers for essential personnel positions, protective measures for employee and family health, logistical arrangements for delivery and storage of food and bedding, the removal of waste, and the potential for euthanasia. Strategies such as encouraging and subsidizing cryopreservation of unique strains were undertaken to protect valuable research assets and intellectual property. Elements of this plan were put into practice after escalation of the pandemic alerts due to influenza A (H1N1) in April 2009.

Twenty percent of a representative sample of patients taking bupropion have abnormal, asymptomatic electroencephalographic findings.

The risk of dose-dependent seizures is a safety issue with bupropion hydrochloride. To evaluate the presence of specific electroencephalographic (EEG) waveforms, 210 adult subjects taking stable doses of bupropion hydrochloride were recruited to undergo 2 EEGs in a prospective, single-center cohort study. The occurrence of spike waves, sharp waves, and focal slowing was recorded and assessed with a continuation ratio logit model for polytomous responses. This model showed that there was a relationship between sex and the incidence of these waveforms, such that the odds of female subjects having sharp waves was increased by a factor of 2.53 (P = 0.05) when compared with male subjects and controlled for both age and dose. Similarly, female subjects were 2.45 (P = 0.09) times more likely than males to have focal slowing on EEG. Overall, 19.8% (39/197) of this representative population was found to have abnormal, asymptomatic EEG findings. The presence of these waveforms in individuals taking a medication known to lower the seizure threshold may be a risk factor for developing seizures.

DNA density-dependent assembly behavior of colloidal micelles.

A key advantage of DNA-mediated colloidal assembly is the ability to tune the strength of adhesion between particles based on sequence characteristics. In the current study, we have investigated DNA-mediated assembly of polystyrene colloidal particles as a function of sequence length, sequence fidelity, and probe density for DNA sequences patterned from the Salmonella genome. The results of our work indicate that the density of DNA probe strands heavily influences the ability of immobilized sequences to hybridize between surfaces of bidisperse colloidal particles. Incubating suspensions at higher temperatures (to minimize secondary structures that might otherwise compromise duplex formation) was also found to have less effect than duplex density on DNA-mediated particle assembly. We believe these results may add to the understanding and design considerations of directed particle assembly using DNA hybridization, especially in the submicrometer and micrometer size regime.

An innovative design to establish proof of concept of the antidepressant effects of the NR2B subunit selective N-methyl-D-aspartate antagonist, CP-101,606, in patients with treatment-refractory major depressive disorder.

This randomized, placebo-controlled, double-blind study was the first to evaluate the antidepressant efficacy, safety, and tolerability of an NR2B subunit-selective N-methyl-D-aspartate receptor antagonist, CP-101,606. Subjects had major depression, according to Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria and a history of treatment refractoriness to least 1 adequate trial of a selective serotonin reuptake inhibitor. The study had 2 treatment periods. In period 1, subjects first received a 6-week open-label trial of paroxetine and a single-blind, intravenous placebo infusion. Period 1 nonresponders (n = 30) then received a randomized double-blind single infusion of CP-101,606 or placebo plus continued treatment with paroxetine for up to an additional 4 weeks (period 2). Depression severity was assessed using the Montgomery-Asberg Depression Rating Scale and 17-item Hamilton Depression Rating Scale. On the prespecified main outcome measure (change from baseline in the Montgomery-Asberg Depression Rating Scale total score at day 5 of period 2), CP-101,606 produced a greater decrease than did placebo (mean difference, 8.6; 80% confidence interval, -12.3 to -4.5) (P < 0.10). Hamilton Depression Rating Scale response rate was 60% for CP-101,606 versus 20% for placebo. Seventy-eight percent of CP-101,606-treated responders maintained response status for at least 1 week after the infusion. CP-101,606 was safe, generally well tolerated, and capable of producing an antidepressant response without also producing a dissociative reaction. Antagonism of the NR2B subtype of the N-methyl-D-aspartate receptor may be a fruitful target for the development of a new antidepressant with more robust effects and a faster onset compared with those currently available and capable of working when existing antidepressants do not.

Crash compatibility between cars and light trucks: benefits of lowering front-end energy-absorbing structure in SUVs and pickups.

Passenger vehicles are designed to absorb crash energy in frontal crashes through deformation or crush of energy-absorbing structures forward of the occupant compartment. In collisions between cars and light trucks (i.e., pickups and SUVs), however, the capacity of energy-absorption structures may not be fully utilized because mismatches often exist between the heights of these structures in the colliding vehicles. In 2003 automakers voluntarily committed to new design standards aimed at reducing the height mismatches between cars and light trucks. By September 2009 all new light trucks will have either the primary front structure (typically the frame rails) or a secondary structure connected to the primary structure low enough to interact with the primary structures in cars, which for most cars is about the height of the front bumper. To estimate the overall benefit of the voluntary commitment, the real-world crash experience of light trucks already meeting the height-matching criteria was compared with that of light trucks not meeting the criteria for 2000-2003 model light trucks in collisions with passenger cars during calendar years 2001-2004. The estimated benefits of lower front energy-absorbing structure were a 19 percent reduction (p<0.05) in fatality risk to belted car drivers in front-to-front crashes with light trucks and a 19 percent reduction (p<0.05) in fatality risk to car drivers in front-to-driver-side crashes with light trucks.

Comparison of duloxetine, escitalopram, and sertraline effects on cytochrome P450 2D6 function in healthy volunteers.

This study is the first to directly compare the relative effects of duloxetine, escitalopram, and sertraline on the functional activity of the drug-metabolizing cytochrome P450 2D6 enzyme as assessed by changes in the pharmacokinetics of the cytochrome P450 2D6 model substrate drug, metoprolol. Single-dose pharmacokinetics of metoprolol were measured before and after 17 days of treatment with escitalopram 20 mg/d, duloxetine 60 mg/d, or sertraline 100 mg/d in young healthy male and female participants. The outcome measures were changes in metoprolol peak plasma levels, area under the plasma concentration-time curve, and clearance. The results were tested using paired t tests and independent t tests. The addition of each drug produced statistically significant changes in metoprolol pharmacokinetics. The rank order for the change in metoprolol area under the plasma concentration-time curve was duloxetine (180%) > escitalopram (89%) > sertraline (48% and 67%). Compared with sertraline, duloxetine produced statistically significantly larger changes in metoprolol pharmacokinetic parameters. The changes produced by escitalopram and sertraline were not statistically different.

Effect of study criteria on recruitment and generalizability of the results.

OBJECTIVE: Clinical trials are indispensable to drug approval process. This research examined the effect of a specific study criteria on recruitment and generalizability of the results. METHODS: The following were reviewed: (a) the usual inclusion and exclusion criteria for the antipsychotic trials performed at the Institute; (b) epidemiologic data, to determine the effect of study criteria on the target population; and (c) the recruitment procedures/strategies used to identify potential candidates. A survey was conducted to determine the percentage of schizophrenic patients in a conventional outpatient psychiatric clinic conforming to the usual enrollment criteria for antipsychotic trials. RESULTS: Intensive recruitment efforts in a general population of 3.6 million would have been expected to yield only 632 eligible subjects out of 36,000 suffering from schizophrenia. Out of 632, only 50 contacted the research site after an intensive recruitment effort. From those 50, 30 were excluded during a telephone interview. Of the 20 remaining, 6 were excluded for a variety of reasons during a face-to-face interview. Thus, only 14 subjects out of a population of 3.6 million met the study criteria. CONCLUSIONS: These results emphasize the rarified nature of patients-volunteers who enter a clinical trial. Inclusion and exclusion study criteria can severely restrict the number of eligible subjects, dictate recruitment strategies, and in turn affect generalizability of the results.