A Safe Framework for Quantitative In Vivo Human Evaluation of Image Guidance.

Goal: We present a new framework for in vivo image guidance evaluation and provide a case study on robotic partial nephrectomy. Methods: This framework (called the "bystander protocol") involves two surgeons, one who solely performs the therapeutic process without image guidance, and another who solely periodically collects data to evaluate image guidance. This isolates the evaluation from the therapy, so that in-development image guidance systems can be tested without risk of negatively impacting the standard of care. We provide a case study applying this protocol in clinical cases during robotic partial nephrectomy surgery. Results: The bystander protocol was performed successfully in 6 patient cases. We find average lesion centroid localization error with our IGS system to be 6.5 mm in vivo compared to our prior result of 3.0 mm in phantoms. Conclusions: The bystander protocol is a safe, effective method for testing in-development image guidance systems in human subjects.

Targeting Epilepsy Through the Foremen Ovale: How Many Helical Needles are Needed?

Laser ablation of the hippocampus offers medically refractory epilepsy patients an alternative to invasive surgeries. Emerging commercial solutions deliver the ablator through a burr hole in the back of the head. We recently introduced a new access path through the foremen ovale, using a helical needle, which minimizes the amount of healthy brain tissue the needle must pass through on its way to the hippocampus, and also enables the needle to follow the medial axis of the hippocampus more closely. In this paper, we investigate whether helical needles should be designed and fabricated on a patient-specific basis as we had previously proposed, or whether a small collection of pre-defined needle shapes can apply across many patients. We propose a new optimization strategy to determine this needle set using patient data, and investigate the accuracy with which these needles can reach the the medial axis of the hippocampus. We find that three basic tube shapes (mirrored as necessary for left vs. right hippocampi) are all that is required, across 20 patient datasets (obtained from 10 patient CT scans), to reduce worst-case maximum error below 2 mm.

Toward Practical and Accurate Touch-Based Image Guidance for Robotic Partial Nephrectomy.

Partial nephrectomy involves removing a tumor while sparing surrounding healthy kidney tissue. Compared to total kidney removal, partial nephrectomy improves outcomes for patients but is underutilized because it is challenging to accomplish minimally invasively, requiring accurate spatial awareness of unseen subsurface anatomy. Image guidance can enhance spatial awareness by displaying a 3D model of anatomical relationships derived from medical imaging information. It has been qualitatively suggested that the da Vinci robot is well suited to facilitate image guidance through touch-based registration. In this paper we validate and advance this concept toward real-world use in several important ways. First, we contribute the first quantitative accuracy evaluation of touch-based registration with the da Vinci. Next, we demonstrate real-time touch-based registration and display of medical images for the first time. Lastly, we perform the first experiments validating use of touch-based image guidance to improve a surgeon's ability to localize subsurface anatomical features in a geometrically realistic phantom.

Optimization of Curvilinear Needle Trajectories for Transforamenal Hippocampotomy.

BACKGROUND: The recent development of MRI-guided laser-induced thermal therapy (LITT) offers a minimally invasive alternative to craniotomies performed for tumor resection or for amygdalohippocampectomy to control seizure disorders. Current LITT therapies rely on linear stereotactic trajectories that mandate twist-drill entry into the skull and potentially long approaches traversing healthy brain. The use of robotically-driven, telescoping, curved needles has the potential to reduce procedure invasiveness by tailoring trajectories to the curved shape of the ablated structure and by enabling access through natural orifices. OBJECTIVE: To investigate the feasibility of using a concentric tube robot to access the hippocampus through the foramen ovale to deliver thermal therapy and thereby provide a percutaneous treatment for epilepsy without drilling the skull. METHODS: The skull and both hippocampi were segmented from dual CT/MR image volumes for 10 patients. For each of the 20 hippocampi, a concentric tube robot was designed and optimized to traverse a trajectory from the foramen ovale to and through the hippocampus from head to tail. RESULTS: Across all 20 cases, the mean distances (error) between hippocampus medial axis and backbone of the needle were 0.55 mm, 1.11 mm, and 1.66 mm for best, mean, and worst case, respectively. CONCLUSION: These curvilinear trajectories would provide accurate transforamenal delivery of an ablation probe to typical hippocampus volumes. This strategy has the potential to both decrease the invasiveness of the procedure and increase the completeness of hippocampal ablation.

High fidelity, low cost moulage as a valid simulation tool to improve burns education.

Simulation allows the opportunity for repeated practice in controlled, safe conditions. Moulage uses materials such as makeup to simulate clinical presentations. Moulage fidelity can be assessed by face validity (realism) and content validity (appropriateness). The aim of this project is to compare the fidelity of professional moulage to non-professional moulage in the context of a burns management course. Four actors were randomly assigned to a professional make-up artist or a course faculty member for moulage preparation such that two actors were in each group. Participants completed the actor-based burn management scenarios and answered a ten-question Likert-scale questionnaire on face and content validity. Mean scores and a linear mixed effects model were used to compare professional and non-professional moulage. Cronbach's alpha assessed internal consistency. Twenty participants experienced three out of four scenarios and at the end of the course completed a total of 60 questionnaires. Professional moulage had higher average ratings for face (4.30 v 3.80; p=0.11) and content (4.30 v 4.00; p=0.06) validity. Internal consistency of face (α=0.91) and content (α=0.85) validity questions was very good. The fidelity of professionally prepared moulage, as assessed by content validity, was higher than non-professionally prepared moulage. We have shown that using professional techniques and low cost materials we can prepare quality high fidelity moulage simulations.

Expression of hTERT immortalises normal human urothelial cells without inactivation of the p16/Rb pathway.

The CDKN2A locus is frequently inactivated in urothelial cell carcinoma (UCC), yet how this alteration contributes to bladder tumorigenesis is not known. Although most UCC express telomerase, inactivation of the p16/Rb pathway is generally required for in vitro immortalisation. This and the involvement of p16 in senescence of normal human urothelial cells (NHUC) suggest that CDKN2A deletion may aid bypass of senescence and allow immortalisation. CDKN2A encodes p16 and p14ARF and therefore inactivation of this locus can disrupt both the Rb and p53 tumour suppressor pathways. Retrovirus-mediated transduction was used to specifically modulate the p16/Rb and/or p53 tumour suppressor pathways in NHUC and to express human telomerase reverse transcriptase (hTERT). Expression of hTERT bypassed Rb and p53 pathway-dependent barriers to proliferation and immortalised NHUC. TERT-NHUC had normal karyotypes, were non-tumorigenic and unexpectedly retained CDKN2A. Thus, the phenotypic significance of inactivation of CDKN2A in UCC may not be solely related to bypass of senescence. Phenotypic assays in human urothelial cells have relied on cell strains derived from invasive tumours or NHUC immortalised by expression of SV40-large T. The production of genetically normal but immortal NHUC lines now provides a valuable platform for experiments to examine the timing and combination of events necessary for UCC tumorigenesis.

Removal of C-spine protection by A&E triage nurses: a prospective trial of a clinical decision making instrument.

OBJECTIVE: To investigate if triage nurses could safely apply a set of clinical criteria, removing hard collars and spinal boards at initial triage assessment. METHODS: The Nexus clinical decision rules were applied by trained triage nurses to patients who attended the department with cervical collars and/or on spinal boards. Patients were excluded if they were felt to be in need of immediate medical assessment. Data were collected on the time to nursing assessment, time to medical assessment and time spent restrained. Patients were followed up until discharge and their radiological diagnosis confirmed. Hospital records were checked to ensure that no patients re-presented with injuries that had been missed at initial assessment. RESULTS: In total, 112 patients were included in the study. Clinical criteria were met in 59 patients and their collar removed at triage assessment. For low risk patients, this reflects a mean reduction in time spent restrained of 23.3 minutes (p<0.005; 95% confidence interval 20.18 to 26.54). No patient who had a collar removed was found to have a significant injury. CONCLUSIONS: Simple criteria can be applied by accident and emergency triage nurses to allow safe removal of cervical collars and spinal boards. The reduced time patients spent immobilised represents an important improvement in patient care.

DBC1 re-expression alters the expression of multiple components of the plasminogen pathway.

Deleted in bladder cancer 1 (DBC1) is a candidate gene for the bladder tumour suppressor locus at 9q33.1. The function of the gene is currently unknown but a cross-species sequence comparison suggests an important role, as it is highly evolutionarily conserved. Here, we transfected a nonexpressing human bladder cancer cell line with a set of human DBC1 cDNA constructs. The effect on global expression patterns was assessed using cDNA microarrays. The cell clone with the lowest level of DBC1 expression showed induced expression of 26 genes including plasminogen activator inhibitor 2 (SERPINB5; 4.6-fold), heparin-binding EGF-like growth factor precursor (DTR; 4.2-fold), small proline-rich protein 2B (SPRR2B; 3.6-fold), metallothionein 1 isoforms (MT1B/MT1A/MT-1F; from 2.9- to 3.2-fold), tissue-type plasminogen activator precursor (PLAT; 2.8-fold) and urokinase-type plasminogen activator precursor (PLAU; 2.7-fold). In clustering analysis, both PLAT and PLAU clustered with the functionally related urokinase plasminogen activator surface receptor (PLAUR; 1.9-fold). Furthermore, 14 human bladder tumours were analysed by real-time quantitative PCR using gene-specific primers for selected (n=20) genes. The expression levels of SERPINB5, PLAU, PLAUR and MT1 correlated with the DBC1 levels, suggesting previously unknown involvement of DBC1 in the urokinase-plasminogen pathway.

Prehospital care in Indonesia.

BACKGROUND: Indonesia is a huge, diverse, and developing country that until recently had no public ambulance service let alone a system of prehospital care. It commonly experiences many natural disasters, manmade conflicts, and violence as well as the daily emergencies seen worldwide. CURRENT SYSTEM: Hospitals of varying standards are widespread but have no system of emergency ambulance or patient retrieval. Indonesia's only public emergency ambulance service, 118, is based in five of the biggest cities and is leading the way in paramedic training and prehospital care. CHALLENGES AND DEVELOPMENTS: There are many challenges faced including the culture of acceptance, vast geographical areas, traffic, inadequate numbers of ambulances, and access to quality training resources. Recently there have been a number of encouraging developments including setting up of a disaster response brigade, better provision of ambulances, and development of paramedic training. CONCLUSIONS: An integrated national regionalised hospital and prehospital system may seem fantastic but with the enthusiasm of those involved and perhaps some help from countries with access to training resources it may not be an unrealistic goal.

A surgical model of composite cystoplasty with cultured urothelial cells: a controlled study of gross outcome and urothelial phenotype.

OBJECTIVES: To study the outcome of composite cystoplasty using cultured urothelial cells combined with de-epithelialized colon or uterus in a porcine surgical model, using appropriate controls, and to characterize the neo-epithelium created by composite cystoplasty. MATERIALS AND METHODS: Urothelial cells were isolated and propagated in vitro from open bladder biopsies taken from nine female minipigs. Cohesive sheets of confluent urothelial cells were transferred to polyglactin carrier meshes and sutured to de-epithelialized autologous colon in four animals and de-epithelialized autologous uterus in five. These composite segments were then used for augmentation cystoplasty. Conventional colocystoplasty, de-epithelialized colocystoplasty and sham operations were carried out in six control animals. After killing the animals at approximately 90 days the bladders were removed for examination and immunohistochemical analysis, using a panel of antibodies against cytokeratins and urothelial differentiation-associated antigens. RESULTS: Macroscopically, the bladders augmented with composite segments derived from uterine muscle had no evidence of shrinkage or contracture. Histological analysis showed that in four of five composite uterocystoplasties, the neo-urothelium was stratified and had a transitional morphology, although in some areas coverage was incomplete. Immunohistochemical analysis showed evidence of squamous differentiation in both native and augmented segments. All composite and de-epithelialized colonic segments showed significant contraction with poor urothelial coverage, reflecting the unsuitability of the thin-walled porcine colon for de-epithelialization. CONCLUSIONS: The functional and macroscopic outcome of bladder augmentation with a composite derived from cultured urothelium and de-epithelialized smooth muscle of uterine origin endorses the feasibility of composite cystoplasty.

Tuberous sclerosis complex (TSC) gene involvement in sporadic tumours.

In tuberous sclerosis patients, inactivation of the tuberous sclerosis complex tumour-suppressor genes TSC1 and TSC2 contributes to the development of a wide range of hamartomatous lesions. These patients do not, however, show an increased risk of the common adult solid cancers. Recent evidence that the TSC genes play a role in the phosphoinositide 3-kinase pathway, a pathway whose dysregulation is implicated in a wide range of human malignancies, raises the possibility that their inactivation could contribute to the development of some sporadic cancers. To date the only evidence for this comes from the finding of mutations of TSC1 in bladder cancer. The mutation spectrum of TSC1 in bladder cancer and functional evidence from TSC1 -gene-replacement studies in bladder tumour cells will be presented. The literature on genetic changes in several other sporadic epithelial cancers reveals relatively common deletions in the region of the TSC genes. In ovarian and gall bladder carcinoma and non-small-cell carcinoma of the lung, deletions in both 16p13 and 9q34 are found at significant frequency. Mutation analyses in such tumours are now merited.

Abnormal brachial artery flow-mediated vasodilation in young adults with major depression.

Depressive illness in patients without traditional risk factors for coronary artery disease is associated with striking abnormalities of endothelial function and elevation of circulating markers of atherosclerosis propensity. Further studies are needed to define the mechanisms that underlie these observations.

Negative regulation of G(1)/S transition by the candidate bladder tumour suppressor gene DBCCR1.

Deletion of all or part of chromosome 9q is the most common genetic alteration in all stages and grades of bladder cancer. DBCCR1 (deleted in bladder cancer chromosome region candidate 1) maps to the chromosome region 9q32-33, a candidate tumour suppressor locus for bladder cancer. Although no mutations of DBCCR1 have been detected in bladder tumours, expression of DBCCR1 is silenced by promoter hypermethylation in 50% of bladder cancer cell lines analysed. Here we sought to provide functional evidence to authenticate DBCCR1 as a tumour suppressor using gene-transfer methods. Exogenous expression of DBCCR1 protein or an HA epitope-tagged fusion protein, HA-DBCCR1 in NIH3T3 cells and human bladder tumour cell lines resulted in suppression of proliferation. Cell cycle analyses in NIH3T3 cells revealed that DBCCR1-mediated growth inhibition was due to an increase in the number of cells in the G(1) phase of the cell cycle. The levels of apoptosis were not altered. These results demonstrate a role for DBCCR1 in cell cycle control, thereby supporting the hypothesis that this is the tumour suppressor gene targeted by 9q32-33 deletion in bladder cancer.

Role of p53 in the responses of human urothelial cells to genotoxic damage.

Loss of p53 function is a feature of many types of malignancy, including transitional-cell carcinoma (TCC), where it is associated with high-grade lesions and the development of muscle-invasive disease. Genotoxic agents used as part of the treatment strategy may contribute to tumour progression by inducing further non-lethal DNA damage in surviving cells. To determine the role of p53 in cellular responses to genotoxic agents, we used cultured normal human urothelial (NHU) cells and NHU cells with disabled p53 function. Mitomycin C and gamma-radiation caused normal cells to undergo an extended period of cell-cycle arrest, followed by complete recovery of proliferative potential. In contrast, cells with disabled p53 function, whether karyotypically normal (HU-E6 cells) or post-crisis with karyotypic abnormalities (HU-E6P cells), underwent extensive apoptosis. Overall survival was dose-dependent, and surviving HU-E6 cells from low-dose treatments showed clonal karyotypic abnormalities. These findings demonstrate that p53 status is a crucial factor in determining the ability of urothelial cells to survive DNA damage and suggest caution in the use of genotoxic treatments for low-grade tumours as our data imply that malignancies that have not yet lost p53 function will show the same "repair-and-recovery" response as normal cells.

N;-3 and n;-6 polyunsaturated fatty acids induce cytostasis in human urothelial cells independent of p53 gene function.

The role of long-chain polyunsaturated fatty acids (PUFA) in the etiopathology and treatment of cancer is poorly understood. We have studied the effects of n;-3 and n;-6 PUFA on the proliferation and survival of normal human uroepithelial (NHU) cells, cells with disabled p53 function after stable transfection with the human papillomavirus 16 (HPV16) E6 gene (HU-E6), and p53-disabled cells that had passed through crisis and acquired karyotypic abnormalities (HU-E6P). The n;-3 and n;-6 PUFA had distinct reversible antiproliferative and irreversible cytostatic effects according to concentration and exposure time. The reversible antiproliferative effect was partly due to the production of lipoxygenase metabolites. NHU and HU-E6 cells were equally sensitive to n;-3 and n;-6 PUFA, but HU-E6P cells were more resistant to both the antiproliferative and cytostatic effects. Cytostatic concentrations of n;-3 and n;-6 PUFA did not induce apoptosis, but caused permanent growth arrest ("interphase" or "reproductive" cell death) and mRNA levels for genes involved in cell cycle control (p21, p16, p27, cdk1, cdk2, and cdk4) were not altered. Neither n;-3 nor n;-6 PUFA promoted acquisition of karyotypic abnormalities in HU-E6 cells, suggesting that n;-3 and n;-6 PUFA do not cause genotoxic damage. In conclusion, our studies show that the antiproliferative and cytostatic effects of n;-3 and n;-6 PUFA are not dependent on p53 function and, further, that transformation results in a loss of sensitivity to n;-3 and n;-6 PUFA-mediated growth inhibition.

Effects of long-chain fatty acids on human urothelial cells in organ culture.

It has been suggested that tumour-derived cells are differentially sensitive to the anti-proliferative and cytotoxic effects of long chain n-3 and n-6 polyunsaturated fatty acids (PuFAs). We have previously shown that PuFAs are also growth suppressive to highly proliferative normal human urinary bladder uro-epithelial (NHU) cells grown in monolayer culture. To determine if the effects on NHU cells are directly related to the proliferative index, we have studied the effects of long chain fatty acids in a bladder organ culture system, where proliferation and differentiation of the urothelium is under homeostatic control. A 50 microM concentration of fatty acids was chosen as this concentration of PuFA was profoundly growth inhibitory to NHU cells in monolayer culture. In organ culture, 50 microM PuFAs had no detectable effect on the proliferation or on the preservation of urothelial differentiated histioarchitecture, as assessed using a panel of phenotypic markers. These results suggest that the effects of PuFA may be modulated by the tissue microenvironment.

The effects of dietary fatty acids on the proliferation of normal human urothelial cells in vitro.

Little is known of the mechanisms by which dietary fatty acids (FAs) may affect normal epithelial cell physiology and thereby directly or indirectly influence tumour incidence and progression. In this study, we have used normal human urothelial cell cultures to investigate whether FAs may modify proliferation of normal human epithelial cells in vitro. FAs were presented as albumin complexes in serum-free medium and the effects on proliferation over a concentration range of 1-100 microM were assayed by thymidine incorporation. Saturated FAs had no effect. At lower concentrations (1-10 microM), mono-unsaturated FAs (MUFAs) and n-3 polyunsaturated FAs (PUFAs) were slightly stimulatory. Concentrations of unsaturated FAs above 10 microM were growth inhibitory in a dose-dependent manner. Oleic acid showed least cytostatic effect, whereas gamma-linolenic acid induced irreversible growth arrest. Although marked morphological effects were observed in irreversibly growth-inhibited cells, the cells remained attached to the substratum and showed no evidence of nuclear pyknosis or apoptosis. The inhibitory effects of single PUFAs could be reduced, but not abolished, by the addition of saturated FAs or MUFAs. Mixtures of different PUFAs were inhibitory in an additive manner. These data suggest that PUFAs have a cytostatic effect on rapidly proliferating epithelial cells which appears unrelated to malignant transformation.

A comparison of four commercial systems used to measure whole-body electrical impedance.

Four commercial whole-body impedance measuring systems (Holtain, RJL, Bodystat and EZcomp) were compared on two separate occasions for a group of normal subjects. The first set of readings in 21 subjects demonstrated a significant difference of approximately 6% between the Holtain measurement and the higher readings from the Bodystat or RJL systems. The differences between the RJL and Bodystat readings were much less (mean difference 0.6%). Similar differences between the Holtain and EZcomp or Bodystat measurements were demonstrated on a second occasion for a group of 11 subjects. Given that these devices operate by supplying a constant current, the differences may be explained by the results from a series of measurements on a whole-body resistance simulator in which it appears that for skin contact resistance > 200 omega the Holtain device is unable to sustain a constant current and therefore records a lower impedance than the true value.

The challenge of Pneumocystis carinii culture.

Published and unpublished data on the cultivation of P. carinii were reviewed by a panel of investigators convened by the National Institutes of Health. Although several cell culture systems allow propagation of P. carinii for a limited time with modest rates of replication, these have not proved adequate for isolation of P. carinii in sufficient quantity to explore important basic biological investigation. Attempts at cell-free culture have yielded only transient proliferation. Because much of the unsuccessful work on cultivation of the organism has been unpublished, the panel agreed that these data may be useful to other investigators in designing experimental strategies for cultivation. Therefore, the purpose of this report is to make available this information to researchers, lest others unknowingly repeat unsuccessful methods. It is hoped that by documenting the history and the complexities of Pneumocystis culture, renewed interest and efforts will be directed toward this fundamental scientific challenge.