Characterisation of the cell suspension harvested from the dermal epidermal junction using a ReCell® kit

Background

The use of non-cultured autologous cells to promote wound healing and in reconstructive procedures is increasing. One common method for preparing these cells is the use of the ReCell^® device. However, despite its current clinical use, no characterisation of the cell suspension produced using a ReCell^® device has been published.

Objective

To characterise the ReCell suspension that is applied to wounds for cell type, viability, yield, stability and proliferative potential.

Methods

The ReCell^® device was used to harvest cells from a 2 cm² piece of split-thickness skin isolated using a dermatome. The resulting cell suspension was analysed for cell yield, cell type, viability over time, proliferative potential and reproducibility.

Results

Average viable cell yield was 1.7 × 10⁶/cm² of tissue, with 75.5% of the total cell isolate viable. Total viable cell number was not significantly reduced after 4 h storage at 22 °C or 4 °C, and was stable for 24 h at 4 °C. Proliferative potential was assessed using a colony forming assay, with 0.3% of viable cells isolated forming keratinocyte colonies. Predominantly the suspension contained keratinocytes (64.3 ± 28.8%) and fibroblasts (30.3 ± 14.0%), with a small population of melanocytes also identified (3.5 ± 0.5%). Finally, the supernatant contained low total protein (0.92 mg/ml) and the supernatant had no significant effects on cell viability or growth when applied ex vivo.

Conclusions

These results suggest the ReCell^® device provides a method for the preparation of a cell suspension with high viability and proliferative potential, containing viable melanocytes and no apparent toxic cell debris. Further work on the sustained viability of these cells in vivo, and in particular after application to the wound, will be important to better understand the potential of the ReCell^® device in the clinic.     

The role of shame in distinguishing perpetrators of intimate partner violence in U.S. veterans

Increasing attention is being paid to the fact that exposure to traumatic stressors in military combat may lead to perpetration of intimate partner violence (IPV). Because shame has been identified as a factor in posttraumatic stress disorder (PTSD), the current cross-sectional study examined the relationship in U.S. veterans between IPV and PTSD, depression, guilt, and shame. We hypothesized that shame would be the strongest correlate of perpetration of IPV and that shame would mediate the relationship between PTSD and IPV. Participants were 264 primarily male and Caucasian mixed-era veterans presenting for psychological treatment at a Veterans Affairs hospital. They completed standard measures of depression, PTSD symptoms, shame, and guilt and a local checklist was used to dichotomize the sample regarding IPV. Discriminant analysis indicated that shame contributed most (standardized canonical discriminant function coefficient = .44) to distinguishing perpetrators of IPV. In addition, the results were consistent with shame as a mediator of the relationship between PTSD and IPV. These results are in line with studies indicating that shame is linked to IPV perpetration in nonveteran samples (Harmon, 2002; Rand, 2004; Schibik, 2002) and suggests that shame may be an important aspect of the relationship between PTSD and IPV.     

Extended PCR conditions to reduce drop-out frequencies in low template STR typing including unequal mixtures

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpF?STR(?) Identifiler(?) amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpF?STR(?) Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency.     

Mitochondrial DNA neutrophil extracellular traps are formed after trauma and subsequent surgery

Introduction

Neutrophil extracellular traps (NETs) have not been demonstrated after trauma and subsequent surgery. Neutrophil extracellular traps are formed from pure mitochondrial DNA (mtDNA) under certain conditions, which is potently proinflammatory. We hypothesized that injury and orthopedic trauma surgery would induce NET production with mtDNA as a structural component.

Methods

Neutrophils were isolated 8 trauma patients requiring orthopedic surgery postinjury and up to 5 days postoperatively. Four healthy volunteers provided positive and negative controls. Total hip replacement patients acted as an uninjured surgical control group. Neutrophil extracellular traps were visualized with DNA (Hoechst 33342TM/Sytox Green/MitoSox/MitoTracker) stains using live cell fluorescence microscopy with downstream quantitative polymerase chain reaction analysis of DNA composition.

Results

Neutrophil extracellular traps were present after injury in all 8 trauma patients. They persisted for 5 days postoperatively. Delayed surgery resulted in NET resolution, but they reformed postoperatively. Total hip replacement patients developed NETs postoperatively, which resolved by day 5. Quantitative polymerase chain reaction analysis of NET-DNA composition revealed that NETs formed after injury and surgery were made of mtDNA with no detectable nuclear DNA component.

Conclusions

Neutrophil extracellular traps formed after major trauma and subsequent surgery contain mtDNA and represent a novel marker of heightened innate immune activation. They could be considered when timing surgery after trauma to prevent systemic NET-induced inflammatory complications.     

Roles of nurse practitioners and family physicians in community health centres

Objective

To describe the models of practice used by nurse practitioners (NPs) and FPs in community health centres (CHCs), and to examine the roles of NPs and FPs in these models.

Design

Cross-sectional study using an organizational survey completed by managers of the CHC sites, as well as administrative data on patient sociodemographic characteristics and encounter activities.

Setting

A total of 21 CHCs (13 main sites and 8 satellite sites) operating in eastern Ontario during the period from December 1, 2006, to November 30, 2008.

Participants

A total of 44 849 patients, 53 full-time equivalent FPs, and 41 full-time equivalent NPs.

Main outcome measures

Family physicians’ and NPs’ models of practice, the sociodemographic characteristics and medical profiles of patients who were treated in each model of practice, and FPs’ and NPs’ use of time.

Results

Patients were attributed to 1 of 3 models of practice in CHCs based on the proportion of visits to FPs and NPs: FP care (53% of patients), NP care (29%), and shared care (18%). Patients who received care in the NP model of practice were younger and more likely to be female, be homeless, and not have postsecondary education. Patients who received care in the FP model of practice had more complex medical conditions (cardiovascular disease, mental illness, lung disease, and diabetes) and more annual visits. Patients who received care in the shared care model had intermediate profiles. Nurse practitioners performed more off-site care and walk-in visits. Family physicians and NPs spent a similar proportion of time performing various duties such as direct clinical care and administration tasks.

Conclusion

Although NPs mainly cared for their own patient panels (in the NP care model), they did share some patients with FPs and provide some care to patients under the FP model of practice. Patients who were cared for by FPs and NPs had quite different characteristics.     

Neuropsychological Performance,Brain Imaging,and Driving Violations in Multiple Sclerosis

Objective

To examine the relationship between third ventricular width, a measure of thalamic brain atrophy, and motor vehicle violation type and frequency in a cohort of patients with multiple sclerosis (MS).

Design

Retrospective cohort study.

Setting

Tertiary care university hospital.

Participants

Thirty-five individuals with clinically confirmed relapsing-remitting multiple sclerosis and 35 age-, sex-, and education-matched community-dwelling healthy comparisons (N=70). Participants were aged between 25 and 65 years.

Interventions

Not applicable.

Main Outcome Measures

Data on motor vehicle violations were obtained from an online database (Iowa Courts Online). The violations were categorized as follows: (1) speeding, (2) nonmoving safety, (3) administrative, (4) alcohol-related offense, (5) moving safety, and (6) total violations. Neuropsychological performance in all major cognitive domains was obtained. Thalamic atrophy for the patients with MS was determined via third ventricular width measurement.

Results

The MS group had a greater number of overall violations, administrative violations, and nonmoving safety violations. The groups differed on neuropsychological tasks measuring visuospatial skills, speeded language, learning, and executive functioning, after controlling for affective symptoms. Third ventricular width was associated with total violations as well as moving safety violations. Finally, third ventricular width accounted for a significant variance in driving violation frequency above and beyond demographic variables and neuropsychological factors.

Conclusions

There is an increased frequency of motor vehicle violations among patients with multiple sclerosis, and the number of violations can be predicted by thalamic brain atrophy.     

A nurse developed toolkit for long-term care ombudsmen

Federal regulations provide all nursing home resident access to third party advocates, known as ombudsmen. The ombudsmen are provided unrestricted access to this vulnerable population for complaint investigation and protection of their federally mandated resident's rights. States autonomously administer their ombudsman programs, allowing latitude in hiring and training practices. The majority of state programs rely on a combination of paid and volunteer staff, with most staff lacking formal healthcare training. In an attempt to educate long-term care ombudsmen on common geriatric clinical diagnoses, a clinical toolkit was developed and ombudsmen employed by Alabama Department of Senior Services agreed to pilot test the toolkit. Results of the pilot test did not show ombudsmen with less experience would find the toolkit more useful. Results revealed that all ombudsmen regardless of length of tenure found the toolkit useful.     

Oral Administration of the Broad-Spectrum Antibiofilm Compound Toremifene Inhibits Candida albicans and Staphylococcus aureus Biofilm Formation In Vivo

We here report on the in vitro activity of toremifene to inhibit biofilm formation of different fungal and bacterial pathogens, including Candida albicans, Candida glabrata, Candida dubliniensis, Candida krusei, Pseudomonas aeruginosa, Staphylococcus aureus, and Staphylococcus epidermidis. We validated the in vivo efficacy of orally administered toremifene against C. albicans and S. aureus biofilm formation in a rat subcutaneous catheter model. Combined, our results demonstrate the potential of toremifene as a broad-spectrum oral antibiofilm compound.