Establishing long-term cultures with self-renewing acute myeloid leukemia stem/progenitor cells

OBJECTIVE: With the emergence of the concept of the leukemia stem cell, assays to study them remain pivotal in understanding (leukemic) stem cell biology. METHODS: We have cultured acute myeloid leukemia CD34(+) cells on bone marrow stroma. Long-term expansion was monitored and self-renewal was addressed by replating of Leukemic-cobblestone area-forming cells (L-CAs). Also, lentiviral vectors were generated that could target L-CAs. RESULTS: A strong expansion was observed in about 75% of the acute myeloid leukemia cases (n = 30) and long-term cultures could be maintained for up to 24 weeks on MS5 bone marrow stromal cells. Cells that were able to initiate leukemic cobblestone areas resided in the CD34(+) population and were absent from the CD34(-) population. Self-renewal within these L-CAs was determined by sequential passaging of these L-CAs onto new MS5 stromal layers, which resulted in the generation of second, third, and fourth L-CAs, which were able to sustain long-term expansion and generated high numbers of immature undifferentiated suspension cells. CD34(+) cells that were able to initiate long-term cultures all coexpressed MEIS1 and HOXA9, and expressed elevated BMI1 levels. CONCLUSION: We present a novel long-term leukemic stem/progenitor assay in which new drugs can be tested and in which genes can be overexpressed or downmodulated using a lentiviral approach in order to obtain more insight into the process of leukemic transformation and self-renewal.     

Dissecting T cell lineage relationships by cellular barcoding

T cells, as well as other cell types, are composed of phenotypically and functionally distinct subsets. However, for many of these populations it is unclear whether they develop from common or separate progenitors. To address such issues, we developed a novel approach, termed cellular barcoding, that allows the dissection of lineage relationships. We demonstrate that the labeling of cells with unique identifiers coupled to a microarray-based detection system can be used to analyze family relationships between the progeny of such cells. To exemplify the potential of this technique, we studied migration patterns of families of antigen-specific CD8(+) T cells in vivo. We demonstrate that progeny of individual T cells rapidly seed independent lymph nodes and that antigen-specific CD8(+) T cells present at different effector sites are largely derived from a common pool of precursors. These data show how locally primed T cells disperse and provide a technology for kinship analysis with wider utility.     

Demographics of extra-articular calcaneal fractures: including a review of the literature on treatment and outcome

Introduction  Extra-articular calcaneal fractures represent 25–40% of all calcaneal fractures and an even higher percentage of up to 60% is seen in children. A disproportionately small part of the literature on calcaneal fractures involves the extra-articular type. The aim of this study was to investigate the incidence of extra-articular calcaneal fractures in a Level 1 trauma centre, define the distribution of the various types of fractures and compare patient demographics between extra- and intra-articular calcaneal fractures. In addition the literature was reviewed for the most common types of extra-articular calcaneal fractures with regard to incidence, treatment and clinical outcome. Methods  The radiological records between 2003 and 2005 were reviewed for intra- and extra-articular calcaneal fractures. Patient gender-distribution and age were compared. A literature search was conducted for the treatment of extra-articular calcaneal fractures. Results  In this 3-year study period a total of 49 patients with 50 extra-articular calcaneal fractures and 91 patients with 101 intra-articular fractures were identified. The median age for the first group was 32.7 years, and for the second group 40.3 years; P = 0.04. Male predominance was significantly less pronounced for extra-articular (63%) compared with intra-articular fractures (79%; P = 0.04). Conclusion  One-third of all calcaneal fractures are extra-articular. Significant differences exist between the intra- and extra-articular groups, in terms of lower age and male–female ratio. The literature study shows inconsistencies in treatment options, but most extra-articular fractures are well manageable conservatively.     

Inflammation-induced changes in rostral ventromedial medulla mu and kappa opioid receptor mediated antinociception

Acute microinjection of mu-, delta-, or kappa-opioid receptor (MOPr, DOPr, KOPr) agonists into the rostral ventromedial medulla (RVM) produces antinociception. Thermal antinociception produced by MOPr and DOPr agonists is potentiated during inflammation [Hurley RW, Hammond DL. The analgesic effects of supraspinal mu and delta opioid receptor agonists are potentiated during persistent inflammation. J Neurosci 2000;20:1249-59]. Whether this potentiation extends to other stimulus modalities or to KOPr agonists is unknown. To examine these issues, rats received a unilateral intraplantar injection of complete Freund's adjuvant (CFA). Antinociception produced by RVM infusion of the KOPr agonist, U69593, and the MOPr agonist, DAMGO, was tested 4h-2 weeks thereafter. Thermal paw withdrawal latencies (PWLs) were assessed using the Hargreaves method. Mechanical thresholds were determined with the Von Frey and Randall-Selitto method. PWLs of the inflamed paw were reduced 4h-2 weeks after CFA injection. Infusion of either U69593 or DAMGO increased PWLs in CFA treated rats. A bilateral enhancement of the response to both agonists was observed 2 weeks relative to 4h post-CFA injection. Mechanical thresholds of the inflamed paw were decreased for >2 weeks post-CFA injection. Infusion of either agonist elevated thresholds of the inflamed and non-inflamed paws of CFA-treated rats. The magnitude of these effects was greater 2 weeks post-CFA injection for DAMGO and increased progressively for U69593. These data demonstrate that RVM infusion of MOPr or KOPr agonists attenuates CFA-evoked thermal and tactile allodynia and that these effects increase during prolonged inflammation. The augmented response of the non-inflamed paw to agonists suggests that inflammation induces centrally-mediated neuroplastic changes which enhance MOPr- and KOPr-mediated antinociception.     

Current concepts in the treatment of intra-articular calcaneal fractures: results of a nationwide survey

The treatment of intra-articular calcaneal fractures is controversial and randomised clinical trials are scarce. Moreover, the socio-economic cost remains unclear. The aim of this study was to estimate the incidence, treatment preferences and socio-economic cost of this complex fracture in the Netherlands. This data may aid in planning future clinical trials and support education. The method of study was of a cross-sectional survey design. A written survey was sent to one representative of both the traumatology and the orthopaedic staff in each hospital in the Netherlands. Data on incidence, treatment modalities, complications and follow-up strategies were recorded. The socio-economic cost was calculated. The average response rate was 70%. Fracture classifications, mostly by Sanders and Essex-Lopresti, were applied by 29%. Annually, 920 intra-articular calcaneal fractures (0.4% incidence rate) were treated, mainly with ORIF (46%), conservative (39%) and percutaneous (10%) treatment. The average non-weight-bearing mobilisation was 9 weeks (SD 2 weeks). An outcome score, mainly AOFAS, was documented by 7%. A secondary arthrodesis was performed in 21% of patients. The socio-economic cost was estimated to be euro21.5-30.7 million. Dutch intra-articular calcaneal fracture incidence is at least 0.4% of all fractures presenting to hospitals. Better insight into treatment modalities currently employed and costs in the Netherlands was obtained.     

The sinus tarsi approach in displaced intra-articular calcaneal fractures: a systematic review

Purpose  

Although open reduction and internal fixation is currently considered the gold standard in surgical treatment of displaced intra-articular calcaneal fractures, various different approaches exist including the limited lateral approach. The aim of this systematic review was to combine the results of studies using the sinus tarsi approach, which is the most frequently applied limited lateral approach.     

Predicted versus executed surgical orthognathic treatment

ObjectivesThis study aimed to analyse combined surgical-orthodontic treatment plans, compare them with the actual surgery performed, and define factors resulting in changes of the original plan during orthodontic pre-surgical preparation.Study designThe clinical files of 312 orthognathic surgery patients, operated between January 2008 and December 2010, were retrospectively reviewed. Of these 312 patients, 129 had a bimaxillary operation. One hundred sixty patients had osteotomy of the lower jaw only and 23 had osteotomy of the upper jaw only. Factors analysed in the study include Angle Class malocclusion, patient sex, and age. Lip-to-incisor relationship, overjet, overbite and midline deviations of the upper and lower jaw were recorded. Effects of surgical assisted rapid palatal expansion (SARPE) on the eventual surgery were also investigated. Reasons for changing the original treatment plan at the time of the finished pre-surgical-orthodontic alignment were analysed.ResultsThe original treatment plan was changed in 42 of the 312 patients (13.5%). Changes occurred generally in case of a larger interval between set-up of the first treatment plan and the eventual operation (average 22.4 versus 16.4 months for patients with changed versus unchanged treatment plan, respectively). All Class I patients had surgery performed as planned. Class III patients had a significantly higher rate of altered treatment plan (27.3%) than Class II patients (7.6%). More men (52.4%) saw their treatment plan changed, although there were more women than men in the study population (59.6 versus 40.4%).ConclusionOne in seven patients (13.5%) had a different operation than was planned at the start of treatment. Class III patients with small overjet and overbite commonly have a treatment plan for a monomaxillary operation that, after decompensation, needs to be adapted to a bimaxillary operation.     

Validation of anatomical landmarks-based registration for image-guided surgery: An in-vitro study

IntroductionPerioperative navigation is a recent addition to orthognathic surgery. This study aimed to evaluate the accuracy of anatomical landmarks-based registration.Materials and methodsEighty-five holes (1.2 mm diameter) were drilled in the surface of a plastic skull model, which was then scanned using a SkyView cone beam computed tomography scanner. DICOM files were imported into BrainLab ENT 3.0.0 to make a surgical plan. Six anatomical points were selected for registration: the infraorbital foramena, the anterior nasal spine, the crown tips of the upper canines, and the mesial contact point of the upper incisors. Each registration was performed five times by two separate observers (10 times total).ResultsThe mean target registration error (TRE) in the anterior maxillary/zygomatic region was 0.93 ± 0.31 mm (p < 0.001 compared with other anatomical regions). The only statistically significant inter-observer difference of mean TRE was at the zygomatic arch, but was not clinically relevant.ConclusionWith six anatomical landmarks used, the mean TRE was clinically acceptable in the maxillary/zygomatic region. This registration technique may be used to access occlusal changes during bimaxillary surgery, but should be used with caution in other anatomical regions of the skull because of the large TRE observed.