An ovine hepatorenal fibrocystic model of a Meckel-like syndrome associated with dysmorphic primary cilia and TMEM67 mutations.

Meckel syndrome (MKS) is an inherited autosomal recessive hepatorenal fibrocystic syndrome, caused by mutations in TMEM67, characterized by occipital encephalocoele, renal cysts, hepatic fibrosis, and polydactyly. Here we describe an ovine model of MKS, with kidney and liver abnormalities, without polydactyly or occipital encephalocoele. Homozygous missense p.(Ile681Asn; Ile687Ser) mutations identified in ovine TMEM67 were pathogenic in zebrafish phenotype rescue assays. Meckelin protein was expressed in affected and unaffected kidney epithelial cells by immunoblotting, and in primary cilia of lamb kidney cyst epithelial cells by immunofluorescence. In contrast to primary cilia of relatively consistent length and morphology in unaffected kidney cells, those of affected cyst-lining cells displayed a range of short and extremely long cilia, as well as abnormal morphologies, such as bulbous regions along the axoneme. Putative cilia fragments were also consistently located within the cyst luminal contents. The abnormal ciliary phenotype was further confirmed in cultured interstitial fibroblasts from affected kidneys. These primary cilia dysmorphologies and length control defects were significantly greater in affected cells compared to unaffected controls. In conclusion, we describe abnormalities involving primary cilia length and morphology in the first reported example of a large animal model of MKS, in which we have identified TMEM67 mutations.

International variations in application of the best-interest standard across the age spectrum.

OBJECTIVE: Ethically and legally, assertions that resuscitation is in a patient's best interest should be inversely correlated with willingness to forego intensive care (and accept comfort care) at the surrogate's request. Previous single country studies have demonstrated a relative devaluation of neonates when compared with other critically ill patients. STUDY DESIGN: In this international study, physicians in Argentina, Australia, Canada, Ireland, The Netherlands, Norway and the United States were presented with eight hypothetical vignettes of incompetent critically ill patients of different ages. They were asked to make assessments about best interest, respect for surrogate autonomy and to rank the patients in a triage scenario. RESULTS: In total, 2237 physicians responded (average response rate 61%). In all countries and scenarios, participants did not accept to withhold resuscitation if they estimated it was in the patient's best interest, except for scenarios involving neonates. Young children (other than neonates) were given high priority for resuscitation, regardless of existing disability. For neonates, surrogate autonomy outweighed assessment of best interest. In all countries, a 2-month-old-infant with meningitis and a multiply disabled 7-year old were resuscitated first in the triage scenario, with more variable ranking of the two neonates, which were ranked below patients with considerably worse prognosis. CONCLUSIONS: The value placed on the life of newborns is less than that expected according to predicted clinical outcomes and current legal and ethical theory relative to best interests. Value assessments on the basis of age, disability and prognosis appear to transcend culture, politics and religion in this domain.

The Δ133p53 isoform and its mouse analogue Δ122p53 promote invasion and metastasis involving pro-inflammatory molecules interleukin-6 and CCL2.

A number of naturally occurring isoforms of the tumour suppressor protein p53 have been discovered, which appear to have differing roles in tumour prevention or promotion. We are investigating the tumour-promoting activities of the Δ133p53 isoform using our mouse model of Δ133p53 (Δ122p53). Here, we report that tumours from Δ122p53 homozygous mice show evidence of invasion and metastasis and that Δ122p53 promotes migration though a 3-dimensional collagen matrix. We also show that Δ122p53 and Δ133p53 promote cell migration in scratch wound and Transwell assays, similar to the 'gain-of-function' phenotypes seen with mutant p53. Using the well-defined B16 mouse melanoma metastatic model, we show that Δ122p53 leads to faster generation of lung metastases. The increased migratory phenotypes are dependent on secreted factors, including the cytokine interleukin-6 and the chemokine CCL2. We propose that Δ122p53 (and Δ133p53) acts in a similar manner to 'gain-of-function' mutant p53 proteins to promote migration, invasion and metastasis, which may contribute to poor survival in patients with Δ133p53-expressing tumours.

Cambridge-Miami score for intestinal transplantation preoperative risk assessment: initial development and validation.

INTRODUCTION: Preoperative quantification of survival after transplantation would assist in assessing patients. We have developed a preliminary preoperative scoring system, called the Cambridge-Miami (CaMi) score, for transplantation of the small intestine either alone or as a composite graft. METHODS: The score combines putative risk factors for early-, medium-, and long-term survival. Factors included were loss of venous access and impairment of organs or systems not corrected by transplantation. Each factor was scored 0-3. A score of 3 indicated comorbidity approaching a contraindication for transplantation, that which might lead to but was not currently an adverse risk factor scored 1, and that presenting a definite but moderate increase in risk scored 2. The preoperative scores of 20 patients who had received intestinal transplants either isolated or as part of a cluster graft, who had either been followed up postoperatively for at least 10 years, or died within 10 years were compared with their survivals. RESULTS: Postoperative survival and CaMi score inversely correlated when analysed using Spearman test (r(s) = -0.82; P = .0001). A score of <3 associated with survival > or =3 years (12/12 patients) and >3 with survival of <6 months (4/4). Patient Kaplan-Meier (KM) survival curves for patients grouped according to CaMi score became significantly different from group 0 to group 3. Using this as a threshold score patients grouped as either >2 or <3 had significantly different survival rates (log-rank; P = .0001), KM median survival hazard ratio (HR) = 6, and rate of death KM HR = 5. Receiver-operator characteristics indicate a high degree of accuracy for prediction of death with an area under the curve (C statistic) at 3 years of 0.98, at 5 years of 0.82, and at 10 years of 0.65. CONCLUSION: This initial validation suggested that the preoperative CaMi score predicted postoperative survival.

p53 promotes adenoviral replication and increases late viral gene expression.

The tumor suppressor protein, p53, plays a critical role in viro-oncology. However, the role of p53 in adenoviral replication is still poorly understood. In this paper, we have explored further the effect of p53 on adenoviral replicative lysis. Using well-characterized cells expressing a functional p53 (A549, K1neo, RKO) and isogenic derivatives that do not (K1scx, RKOp53.13), we show that virus replication, late virus protein expression and both wtAd5 and ONYX-015 virus-induced cell death are impaired in cells deficient in functional p53. Conversely, by transfecting p53 into these and other cells (IIICF/c, HeLa), we increase late virus protein expression and virus yield. We also show, using reporter assays in IIICF/c, HeLa and K1scx cells, that p53 can cooperate with E1a to enhance transcription from the major late promoter of the virus. Late viral protein production is enhanced by exogenous p53. Taken together, our data suggest that functional p53 can promote the adenovirus (Ad) lytic cycle. These results have implications for the use of Ad mutants that are defective in p53 degradation, such as ONYX-015, as agents for the treatment of cancers.

Accuracy assessment protocols for electromagnetic tracking systems.

Electromagnetic tracking systems have found increasing use in medical applications during the last few years. As with most non-trivial spatial measurement systems, the complex determination of positions and orientations from their underlying raw sensor measurements results in complicated, non-uniform error distributions over the specified measurement volume. This makes it difficult to unambiguously determine accuracy and performance assessments that allow users to judge the suitability of these systems for their particular needs. Various assessment protocols generally emphasize different measurement aspects that typically arise in clinical use. This can easily lead to inconclusive or even contradictory conclusions. We examine some of the major issues involved and discuss three useful calibration protocols. The measurement accuracy of a system can be described in terms of its 'trueness' and its 'precision'. Often, the two are strongly coupled and cannot be easily determined independently. We present a method that allows the two to be disentangled, so that the resultant trueness properly represents the systematic, non-reducible part of the measurement error, and the resultant precision (or repeatability) represents only the statistical, reducible part. Although the discussion is given largely within the context of electromagnetic tracking systems, many of the results are applicable to measurement systems in general.

Aroma therapy for dementia.

BACKGROUND: Complementary therapies have become more commonly used over the last decade and have been applied to a range of health problems, including dementia. Of these, aroma therapy is reported to be the most widely used in the British National Health Service (Lundie 1994) and might be of use for people with dementia for whom verbal interaction may be difficult and conventional medicine of only marginal benefit. Aroma therapy has been used for people with dementia to reduce disturbed behaviour (e.g. Brooker 1997), promote sleep (e.g. Wolfe 1996), and stimulate motivational behaviour (e.g. MacMahon 1998). OBJECTIVES: To assess the efficacy of aroma therapy as an intervention for people with dementia. SEARCH STRATEGY: The Cochrane Dementia and Cognitive Improvement Group's Specialized Register was searched on 29 October 2002 to find all relevant trials using the terms: aroma therap*, "aroma therap*", "complementary therap*", "alternative therap*" and "essential oil". The CDCIG Register contains records from all major health care databases and is updated regularly. Additionally, relevant journals were hand searched, and 'experts' in the field of complementary therapies and dementia contacted. SELECTION CRITERIA: All relevant randomized controlled trials (RCTs) were considered. A minimum length of trial and requirements for a follow-up were not included, and participants in included studies had a diagnosis of dementia of any type and severity. The review considered all trials using fragrance from plants defined as aroma therapy as an intervention with people with dementia. Several outcomes were considered in this review, including cognitive function, quality of life, and relaxation. DATA COLLECTION AND ANALYSIS: The titles and abstracts extracted by the searches were screened for their eligibility for potential inclusion in the review, which revealed 2 RCTs of aroma therapy for dementia. Neither of these had published results in a form that we could use. However, individual patient data from one trial were obtained (Ballard 2002) and additional analyses performed. Analysis of co-variance was used for all outcomes, using a random effects model. MAIN RESULTS: The additional analyses conducted revealed a statistically significant treatment effect in favour of the aroma therapy intervention on measures of agitation and neuropsychiatric symptoms. REVIEWER'S CONCLUSIONS: Aroma therapy showed benefit for people with dementia in the only trial that contributed data to this review, but there were several methodological difficulties with this study. More well designed large-scale RCTs are needed before conclusions can be drawn on the effectiveness of aroma therapy. Additionally, several issues need to be addressed, such as whether different aroma therapy interventions are comparable and the possibility that outcomes may vary for different types of dementia.

The sleep of older people in hospital and nursing homes.

Disturbed sleep can affect personal wellbeing and impede the rehabilitation and recovery of older people from illness. This paper reports the findings of a pilot study which included examination of sleep quality and sleep patterns of older people in community hospital and nursing home settings. A marked proportion of older people reported sleeping well in nursing care settings, and those in nursing homes slept better than those in the community hospital. The main causes of sleep disturbance in both settings were: needing to go to the toilet, noise, pain, and discomfort; a similar pattern was seen across the different settings. No discernible difference was found in quality of sleep and whether patients felt rested or not between those patients on hypnotic medication and those who were not. The implications of the findings for practice and future research are discussed.

The human eukaryotic initiation factor 4AI gene (EIF4A1) contains multiple regulatory elements that direct high-level reporter gene expression in mammalian cell lines.

The gene encoding human eukaryotic initiation factor 4A (EIF4A1) is located on chromosome 17p13, 667 bp upstream from the gene encoding the macrophage endosomal protein CD68. The EIF4AI gene contains 10 intervening sequences with the 1397-bp first intron containing a CpG-rich methylation-free island. Sequences capable of enhancing gene expression reside between positions -69 and -371 and positions -504 and -1100 of the EIF4AI 5' flanking sequence and within introns 1, 2, 3, 7, and 9. In macrophage cell lines, EIF4A1 expression vectors give sustained high-level reporter gene expression to levels 10 times higher than that obtained using the human cytomegalovirus immediate-early gene promoter/enhancer. Sequences of the human EIF4AI gene may find application in the development of new vectors for gene therapy and genetic vaccination.

NMR studies of a viral protein that mimics the regulators of complement activation.

Vaccinia virus complement control protein (VCP) is a 243-residue protein that is similar in sequence to the regulators of complement activation; its role is to defend the virus against attack by the host complement system. A fragment of this protein spanning the two complement protein (CP)-modules (residues 126 to 243) which make up the C-terminal half of VCP has been expressed in Pichia pastoris. A 15N-labelled sample was purified for the purposes of structure determination and measurements of dynamics in solution using NMR. Structures were calculated on the basis of 1767 NMR-derived distance and angle restraints, with a longer than normal high-temperature simulated annealing (SA) protocol which improved convergence. The viral CP-modules are structurally very similar to the 15th and 16th CP-modules of human factor H (fH; average r.m.s.d., for invariant Trp and Cys, four pair-wise comparisons,=1.2 A) but less similar to the fifth CP-module of fH (average r.m.s.d.=2.2 A). In the VCP fragment, the orientation of one module with respect to the other is clearly defined by the experimental data, and T1 measurements are consistent with only limited flexibility at the module-module interface. The r.m.s.d. over all of the 118 residues (backbone atoms) is 0.73 A. The intermodular orientation is better defined than, and significantly different from, that observed in a CP-module pair from fH (re-calculated using the extended SA protocol). In VCP the long axis of the second module is tilted by 59(+/-4) degrees with respect to the first module (50(+/-13) degrees in the fH pair), and twisted with respect to the first module by 22(+/-6) degrees (223(+/-17) degrees in fH). The differences between the human and viral proteins may be rationalised in terms of the lack of hydrogen-bond stabilised secondary structure in the N-terminal portion of fH module 16, and the number and type of amino acid side-chains which make up the interface. A similar intermodular interface may be predicted between the third and fourth module of human C4 binding protein and, probably, between the third and fourth modules of the guinea pig acrosomal matrix protein 67; but the formulation of general rules for predicting the structure of interfaces between CP-modules awaits further experimental data.

Solution structure of a pair of complement modules by nuclear magnetic resonance.

A portion of human complement factor H spanning the 15th (H15) and 16th (H16) of its 20 modules, has been expressed in a yeast vector and subjected to structure determination in solution using two-dimensional 1H-NMR. The structure of H15 is very similar to that already established for the fifth module of factor H and H16, consistent with the view that all such complement control (C-) modules share a common overall topology. In addition, the tertiary structures of the component modules of the H15-16 pair are very similar to those of the modules when expressed individually, implying that each folds entirely autonomously within intact factor H. Aromatic residues in the third turn of H15 and the second turn of H16, together with a leucine residue from the linker region, contribute to a small intermodular interface. Comparatively few nuclear Overhauser effects were observable between protons on different modules. Consequently, a wide range of angles of "twist" (131 (+/- 146) degrees, mean value (+/- 1 standard deviation)), i.e. rotation about the long axis of one module with respect to the other, exists in the family of structures generated on the basis of the experimental data. However, much smaller variations occur in the two, orthogonal, angles (175 (+/- 12) degrees and 103 (+/- 6) degrees) that describe the "tilt". These observations may suggest upper limits on the relative flexibility of the two modules. Models were built to assess the outcome of applying such restrictions to all the neighbours within a string of 20 C-modules, and the resulting structures compare well with factor H as visualized by electron microscopy.

Total population study of factors affecting chronic bronchitis prevalence in the coal mining industry of New South Wales, Australia.

The period prevalence of simple chronic bronchitis (SCB) (mucus hypersecretion), defined as chronic cough and sputum production by the MRC respiratory symptom questionnaire administered by occupational physicians and of obstructive chronic bronchitis (OCB) (airflow obstruction) (defined as SCB plus FEV1 less than 80% predicted) have been measured over the period 30 June 1977-30 June 1980 in the entire work force aged between 21 and 60 of the coal industry of New South Wales, Australia (12 357 men). Four dimensional contingency table analysis by a logistic transform method showed highly significant (p less than 0.001) additive affects of age (exposure duration), site of work, smoking, and alcohol consumption on development of overall chronic bronchitis (SCB + OCB). Odds ratios were face work:surface work = 1.78:1, smoker:non-smoker = 4.23:1, alcohol greater than 300 g/wk:alcohol less than 300 g/wk = 2.13:1. There was no evidence for synergistic effects of these factors on the development of mucus hypersecretion. When OCB was analysed separately, the effect of site of work, although in the same direction, was not statistically significant and this was assumed to be due to a "healthy worker" effect or a "swamping" effect of smoking. Age, smoking, and alcohol effects were highly significant (p less than 0.0001) and there was a sharp increase in prevalence of OCB in the age groups 41-50 and 51-60. Odds ratios were face work:surface work = 1.11:1, smoker:non-smoker = 2.66:1, alcohol greater than 300 g/wk:alcohol less than 300 g/wk = 2.91:1. There was no evidence of synergistic effects. These results are consistent with a hypothesis of additive effects of smoking, alcohol, and coal mine dust and fumes on the development of chronic mucus hypersecretion leading to airflow obstruction or a hypothesis of similar additive effects on the development of two separate conditions--mucus hypersecretion with airflow obstruction and mucus hypersecretion without airflow obstruction.