Cell-free tumour DNA testing for early detection of cancer - a potential future tool.

In recent years, detection of cell-free tumour DNA (ctDNA) or liquid biopsy has emerged as an attractive noninvasive methodology to detect cancer-specific genetic aberrations in plasma, and numerous studies have reported on the feasibility of ctDNA in advanced cancer. In particular, ctDNA assays can capture a more 'global' portrait of tumour heterogeneity, monitor therapy response, and lead to early detection of resistance mutations. More recently, ctDNA analysis has also been proposed as a promising future tool for detection of early cancer and/or cancer screening. As the average proportion of mutated DNA in plasma is very low (0.4% even in advanced cancer), exceedingly sensitive techniques need to be developed. In addition, as tumours are genetically heterogeneous, any screening test needs to assay multiple genetic targets in order to increase the chances of detection. Further research on the genetic progression from normal to cancer cells and their release of ctDNA is imperative in order to avoid overtreating benign/indolent lesions, causing more harm than good by early diagnosis. More knowledge on the sources and elimination of cell-free DNA will enable better interpretation in older individuals and those with comorbidities. In addition, as white blood cells are the major source of cell-free DNA in plasma, it is important to distinguish acquired mutations in leukocytes (benign clonal haematopoiesis) from an upcoming haematological malignancy or other cancer. In conclusion, although many studies report encouraging results, further technical development and larger studies are warranted before applying ctDNA analysis for early cancer detection in the clinic.

Genetic testing and surveillance guidelines in hereditary pheochromocytoma and paraganglioma.

Pheochromocytoma and paraganglioma (PPGL) are rare tumours and at least 30% are part of hereditary syndromes. Approximately 20% of hereditary PPGL are caused by pathogenic germ line variants in genes of the succinate dehydrogenase complex (SDHx), TMEM127 or MAX. Herein we present guidelines regarding genetic testing of family members and their surveillance based on a thorough literature review. All cases of PPGL are recommended genetic testing for germ line variants regardless of patient and family characteristics. At minimum, FH, NF1, RET, SDHB, SDHD and VHL should be tested. In addition, testing of MEN1, SDHA, SDHAF2, SDHC, TMEM127 and MAX is recommended. Healthy first-degree relatives (and second-degree relatives in the case of SDHD and SDHAF2 which are maternally imprinted) should be offered carrier testing. Carriers of pathogenic variants should be offered surveillance with annual biochemical measurements of methoxy-catecholamines and bi-annual rapid whole-body magnetic resonance imaging and clinical examination. Surveillance should start 5 years before the earliest age of onset in the family and thus only children eligible for surveillance should be offered pre-symptomatic genetic testing. The surveillance of children younger than 15 years needs to be individually designed. Our guidelines will provide a framework for patient management with the possibility to follow outcome via national registries and/or follow-up studies. Together with improved insights into the disease, this may enable optimisation of the surveillance scheme in order to minimise both anxiety and medical complications while ensuring early disease detection.

Development, Evaluation and Implementation of Chief Complaint Groupings to Activate Data Collection: A Multi-Center Study of Clinical Decision Support for Children with Head Trauma.

BACKGROUND: Overuse of cranial computed tomography scans in children with blunt head trauma unnecessarily exposes them to radiation. The Pediatric Emergency Care Applied Research Network (PECARN) blunt head trauma prediction rules identify children who do not require a computed tomography scan. Electronic health record (EHR) based clinical decision support (CDS) may effectively implement these rules but must only be provided for appropriate patients in order to minimize excessive alerts. OBJECTIVES: To develop, implement and evaluate site-specific groupings of chief complaints (CC) that accurately identify children with head trauma, in order to activate data collection in an EHR. METHODS: As part of a 13 site clinical trial comparing cranial computed tomography use before and after implementation of CDS, four PECARN sites centrally developed and locally implemented CC groupings to trigger a clinical trial alert (CTA) to facilitate the completion of an emergency department head trauma data collection template. We tested and chose CC groupings to attain high sensitivity while maintaining at least moderate specificity. RESULTS: Due to variability in CCs available, identical groupings across sites were not possible. We noted substantial variability in the sensitivity and specificity of seemingly similar CC groupings between sites. The implemented CC groupings had sensitivities greater than 90% with specificities between 75-89%. During the trial, formal testing and provider feedback led to tailoring of the CC groupings at some sites. CONCLUSIONS: CC groupings can be successfully developed and implemented across multiple sites to accurately identify patients who should have a CTA triggered to facilitate EHR data collection. However, CC groupings will necessarily vary in order to attain high sensitivity and moderate-to-high specificity. In future trials, the balance between sensitivity and specificity should be considered based on the nature of the clinical condition, including prevalence and morbidity, in addition to the goals of the intervention being considered.

Fibrocytes mediate intimal hyperplasia post-vascular injury and are regulated by two tissue factor-dependent mechanisms.

BACKGROUND: CD34(+) α-smooth muscle actin (SMA)(+) cells mediate intimal hyperplasia (IH) after mechanical endoluminal injury. We previously found that IH is tissue factor (TF) dependent. The precise phenotype of the CD34(+) cells mediating IH is unknown and the mechanisms of TF are also unknown. OBJECTIVE: To define the phenotype of cells mediating IH and compare the effects of inhibiting TF on different subsets of CD34(+) cells. METHODS: Endoluminal injury was induced in C57BL/6 and two strains of mice expressing a human tissue factor pathway inhibitor (hTFPI) fusion protein on different subsets of CD34(+) cells. Confocal microscopy, immunocytofluorescence and real-time PCR were used to determine phenotype. RESULTS: Neointimal cells in C57BL/6 mice were defined as a subset of fibrocytes (CD34(+) CD45(+) collagen-1(+) ) expressing SMA, CD31, TIE-2, CXCR4 and CXCL12. Similar cells circulated post-injury and were also found in mice expressing hTFPI on CD34(+) CD31(+) cells, though in these mice, hTFPI inhibited CD31(+) fibrocyte hyperplasia, so no IH developed. Mice with hTFPI on all CD34(+) α-SMA(+) cells repaired arteries back to a pre-injured state. No CD31(+) fibrocytes were found in these mice unless an anti-hTFPI antibody was administered. Similar findings in protease activated receptor (PAR)-1-deficient mice suggested hTFPI prevented thrombin signaling through PAR-1. In vitro, thrombin increased the number of CD31(+) fibrocytes. CONCLUSIONS: Inhibition of TF on CD31(+) fibrocytes inhibits IH whereas inhibition on all CD34(+) α-SMA(+) cells (or PAR-1 deficiency) inhibits the appearance of CD31(+) fibrocytes and promotes repair. These data enhance our understanding of IH and suggest novel ways to promote regenerative repair.

Interest in health information exchange in ambulatory care: a statewide survey.

OBJECTIVE: Assess the interest in and preferences of ambulatory practitioners in HIE. BACKGROUND: Health information exchange (HIE) may improve the quality and efficiency of care. Identifying the value proposition for smaller ambulatory practices may help those practices engage in HIE. METHODS: Survey of primary care and specialist practitioners in the State of Colorado. RESULTS: Clinical data were commonly (always [2%], often [29%] or sometimes [49%]) missing during clinic visits. Of 12 data types proposed as available through HIE, ten were considered "extremely useful" by most practitioners. "Clinical notes/consultation reports," "diagnosis or problem lists," and "hospital discharge summaries" were considered the three most useful data types. Interest in EKG reports, diagnosis/problem lists, childhood immunizations, and discharge summaries differed among ambulatory practitioner groups (primary care, obstetrics-gynecology, and internal medicine subspecialties). CONCLUSION: Practitioners express strong interest in most of the data types, but opinions differed by specialties on what types were most important. All providers felt that a system that provided all data types would be useful. These results support the potential benefit of HIE in ambulatory practices.

Anaphylaxis following the ingestion of flour contaminated by house dust mites--a report of two cases from Singapore.

This study presents two patients who developed anaphylaxis after eating mite-contaminated food, and also contains a survey of dust-mites contamination in flour samples from Singapore households. The clinical records of each patient was studied. Patient A developed anaphylaxis twenty minutes following the ingestion of home-made fried fish coated with Japanese flour, while Patient B developed similar life-threatening symptoms one hour after the ingestion of home baked scones. Both patients were NSAID-intolerant and had a history of allergic rhinitis. Skin prick tests showed a strong positive result for dust-mites and for extracts prepared from the ingested flour. Flour samples were also examined microscopically which revealed large numbers of live Dermatophagoides farinae dust-mites. A survey of 57 flour samples showed that 4 samples (7%) were contaminated with dust mites. The findings in the present study confirm that mite-contamination of flour exists in Singaporean households, and it may trigger anaphylaxis in susceptible individuals.

Decreased expression of VEGF-A in rat experimental autoimmune encephalomyelitis and in cerebrospinal fluid mononuclear cells from patients with multiple sclerosis.

Vascular endothelial growth factor A (VEGF-A) stimulates angiogenesis, but is also pro-inflammatory and plays an important role in the development of neurological disease, where it can have both attenuating and exacerbating effects. VEGF-B, a related molecule, is highly expressed in the central nervous system and seems to be important in neurological injury. A few studies have indicated that VEGF-A may play a role in the pathogenesis of multiple sclerosis (MS), but the role of VEGF-B has not been studied. We have studied the expression of VEGF-A, -B and their receptors by mRNA in situ hybridization, immunohistochemistry and real-time PCR in spinal cord from LEW rats with experimental autoimmune encephalomyelitis (EAE) and in cerebrospinal fluid (CSF) and blood samples from MS patients. Whereas VEGF-A is downregulated in glia in EAE, the infiltrating inflammatory cells are positive for VEGF-A. Expression of VEGF-B and the VEGF receptors is unaltered. In addition, the levels of VEGF-A mRNA in mononuclear cells [corrected] in CSF are lower in MS patients compared with controls. These results demonstrate a complex regulation of VEGF-A during neuroinflammation and suggest that VEGF-B is not involved in the pathogenesis of MS.

Molecular identity, expression and functional analysis of interleukin-1alpha and its isoforms in rat testis.

Interleukin-1alpha (IL-1alpha) is a proinflammatory cytokine that has also been found to act as a paracrine mediator involved in the regulation of testicular functions. The present review provides an overview of the role of IL-1alpha in testicular physiology. Bioactive IL-1alpha isolated from adult rat testis was found to consist of three distinct immunoreactive protein species with apparent sizes of 45, 24 and 19 kDa. These isoforms showed bioactivity in a thymocyte proliferation and steroidogenesis assays with different biopotencies. The background of the molecular heterogeneity and processing, secretion and regulation of the isoforms of testicular IL-1alpha are discussed. All three isoforms have been found to be secreted into the testis tubular lumen and interstitial space. We have provided evidence that IL-1alpha is a paracrine factor that may be of importance in, e.g., the regulation of Leydig cell steroidogenesis. Pathophysiologically, testicular IL-1alpha may contribute to testicular relapse of acute lymphocytic leukemia in boys.

Signaling alterations in activation-induced nonresponsive CD8 T cells.

Costimulation-dependent production and autocrine use of IL-2 by activated CD8 T cells results in initial clonal expansion, but this is transient. The cells quickly become anergic, unable to produce IL-2 in response to Ag and costimulation, irrespective of the form of costimulation. This activation-induced non-responsiveness (AINR) differs from "classical" anergy in that it results despite the cells receiving both signal 1 and signal 2. AINR cells can still proliferate in response to exogenous IL-2, but can no longer produce it. Other TCR-mediated events including cytolytic function and IFN-gamma production are not affected in the AINR state. To characterize the mechanism(s) responsible for lack of IL-2 production in CD8 T cells in the AINR state, microspheres bearing immobilized anti-TCR Abs or peptide-MHC complexes, B7-1, and ICAM-1 were used to provide well-defined stimuli to the cells. Comparison of normal and AINR cells revealed that in AINR cells extracellular signal-regulated kinase (ERK) is upregulated more transiently, Janus kinase activation is substantially reduced, and activation of p38 is eliminated. PMA and ionomycin restored proliferation and IL-2 production in AINR cells, indicating a signaling defect upstream of Ras and protein kinase C. Inhibitors of ERK (PD98059) and of p38 kinase (SB202190) blocked IL-2 mRNA expression and proliferation of both peptide-MHC/B7-1/ICAM-1-stimulated normal cells and PMA/ionomycin-stimulated AINR cells. Together these results demonstrate that activation of at least ERK and p38 is essential for IL-2 production by CD8 T cells and that up-regulation of these mitogen-activated protein kinases, along with Janus kinase, is defective in AINR cells.

Activation of antigen-specific T cells by artificial cell constructs having immobilized multimeric peptide-class I complexes and recombinant B7-Fc proteins.

T cell activation results from the engagement of multiple receptors on T cells by their respective ligands on antigen presenting cells. Studies using artificial cell surface constructs have demonstrated that effective T cell response requires that antigen be presented on a solid surface with dimensions that approximate those of an intact cell. In this report, we describe the cloning and expression of recombinant B7-1-Fc and B7-2-Fc proteins and their incorporation onto 5-microm latex microspheres along with renatured peptide-MHC. These microspheres provide a simple and effective method for the in vitro or in vivo stimulation of antigen-specific T cells under precisely controlled antigen and costimulation conditions.

Inadequate pre-operative evaluation and preparation: a review of 197 reports from the Australian incident monitoring study.

The Australian Incident Monitoring Study database was examined for incidents involving inadequate pre-operative patient preparation and/or evaluation. Of 6271 reports, 727 had appropriate keywords, of which 197 (3.1%) were used for subsequent analysis. All surgical categories were represented. In 10% of reports the patient was not reviewed pre-operatively by an anaesthetist, whilst in 23% the anaesthetist involved in the operating theatre had not performed the pre-operative assessment. Death followed in seven cases, major morbidity in 23 cases, admission to a high-dependency unit or intensive care unit in 17 cases, and surgery was cancelled in nine cases. Poor airway assessment, communication problems and inadequate evaluation were the most common contributing factors. Respondents indicated that the incident was preventable in 57% of cases. Proposed corrective strategies include improved communication, quality assurance activities, development of protocols and additional training. A structured assessment of the airway, along with improvements in information exchange, patient assessment, and use of clearly defined patient management plans and pathways would prevent most of the incidents reported.

Epidural catheter tip cultures: results of a 4-year audit and implications for clinical practice.

BACKGROUND AND OBJECTIVES: The aims of this study were to evaluate the clinical relevance of routine microbiological culture of epidural catheter tips after use in acute pain management, and to identify patterns of culture result with respect to both indications for, and duration of, epidural catheterization. METHODS: The Acute Pain Service (APS) reviews all patients under its care at least daily and keeps detailed records on each. Over a 4-year period, when APS protocol required epidural catheter tips to be sent for microbiological culture on removal, the APS saw 1,810 patients who had received epidural analgesia. The records of these patients were reviewed. RESULTS: Culture results were available for 1,443 (79.7%) patients: 1,027 catheter tips (71.2%) were sterile, while 416 (28.8%) were positive for at least 1 type of microorganism. Clinically, no epidural space infections were identified. The highest positive culture rates were found from epidural catheters used in the treatment of pain from fractured ribs or fractured pelves, while the lowest incidences occurred in elective orthopedic and thoracic surgery. The proportion of epidural catheters with positive culture results steadily increased with the duration of catheterization, but there were no clinically significant differences for catheters left in situ for either 3 or 4 days. CONCLUSIONS: We concluded that a significant proportion of epidural catheter tips may be "culture positive" after removal. It is suggested that this probably represents colonization of the skin at the catheter insertion site and subsequent contamination of the catheter tip on removal of the catheter. The large number of "culture positive" tips in the absence of clinically identifiable epidural space infection suggests that routine culture of epidural catheter tips is clinically irrelevant in the vast majority of cases, and that it is not a good predictor of the presence of an epidural space infection.

Haemolytic-uraemic syndrome outbreak caused by Escherichia coli O111:H-: clinical outcomes.

In South Australia in 1995, we treated 20 children with haemolytic-uraemic syndrome associated with Escherichia coli O111:H-. The source of the outbreak was contaminated locally produced semi-dry fermented sausage (mettwurst). One child died of multiple haemorrhagic cerebral infarcts. Eighteen children required renal dialysis (for a median of 14 days); 12 months after discharge five still had significant impairment of renal function. Other major complications included colonic necrosis (3), cerebral haemorrhage/infarction (3), convulsions (4), and glucose intolerance (2). Although E. coli O111:H- has been associated with sporadic cases of haemolytic-uraemic syndrome, this was the first large outbreak reported in Australia.

Differential scanning calorimetric study of the interaction of cholesterol with the major lipids of the Acholeplasma laidlawii B membrane.

It has been proposed that the lower levels of exogenous cholesterol incorporation into the membranes of the sterol-non-requiring as compared to the sterol-requiring mycoplasmas may be due to the much higher glycolipid content of the former and to the reduced ability of glycolipids, as opposed to phospholipids, to incorporate sterols [Efrati et al. (1986) Arch. Biochem. Biophys. 248, 282-288]. In order to test this hypothesis, we have investigated the interaction of cholesterol with the major membrane glyco- and phospholipids of the sterol-non-requiring mycoplasma Acholeplasma laidlawii B, utilizing elaidic acid-homogenous membranes in order to obviate any differences in the nature of cholesterol-lipid interactions due to variations in the fatty acid composition of the different membrane components. Specifically, we have studied the effect of increasing quantities of cholesterol on the thermotropic phase behavior of aqueous dispersions of phosphatidylglycerol, diglucosyl diacylglycerol, and monoglucosyl diacylglycerol, as well as the total membrane polar lipids of this organism, using high-sensitivity differential scanning calorimetry. We find that cholesterol is highly miscible in both the lamellar gel and liquid-crystalline states of phosphatidylglycerol but exhibits limited miscibility in the two neutral glycolipids, particularly in their lamellar gel and crystalline states. We also demonstrate that cholesterol has a limited miscibility in both the lamellar gel and liquid-crystalline states of bilayers composed of the total A. laidlawii B membrane polar lipids. These results demonstrate that the nature of cholesterol-lipid interactions depends markedly on the structure of the glycerolipid polar headgroup and suggests that the incorporation of lower levels of cholesterol into the membranes of the sterol-non-requiring mycoplasmas may indeed be due, at least in part, to their high glycolipid contents. We also show that cholesterol stabilizes the lamellar liquid-crystalline phase of the monoglucosyl diacylglycerol relative to the inverted hexagonal phase at all sterol concentrations, in contrast to the effects of cholesterol on dielaidoylphosphatidylethanolamine, which destabilizes the lamellar liquid-crystalline phase at low concentrations.

Bowel habits of healthy Australian children aged 0-2 years.

OBJECTIVE: To define the bowel habits of healthy Australian children aged up to 2 years. METHODOLOGY: One hundred and forty children were recruited in six age categories with a minimum of 20 children per category. Data on bowel habit was recorded by parents daily for 1 week. RESULTS: Stool frequency decreased with age. Ninety-five per cent of infants 0-14 days passed more than two stools per day compared with 21% at age 13-24 months. Breastfed infants passed more frequent and looser stools than children fed infant formula and solids and also showed a wider variation in frequency. CONCLUSIONS: Mean stool frequency decreased with age as did the variation. This is a combined effect of age and feed type. These data can be used as a guide to the bowel habit of healthy Australian children.

An assessment of prilocaine as a topical anaesthetic agent for fibreoptic bronchoscopy in comparison with lidocaine.

We have evaluated prilocaine as a topical anaesthetic agent for fibreoptic bronchoscopy in comparison with lidocaine in terms of efficacy and safety. Forty patients were included in a randomised double-blind parallel-group study. Efficacy was assessed using visual analogue scales, a patient ranking scale and the number of doses of local anaesthetic and intravenous sedative required. Measures of toxicity included peak plasma concentration of local anaesthetic, whether supplementary oxygen was needed and change in methaemoglobin concentration. For most of the outcome variables, the medians and quartiles were similar for the two local anaesthetics. However, the median peak plasma concentration of prilocaine (0.5 micrograms.ml-1) was less than one-third that of lidocaine (1.76 micrograms.ml-1). The merits and hazards of using multiple-regression modelling to improve the precision of the analysis of the results are considered. We conclude that prilocaine can be used successfully as a topical anaesthetic agent for fibreoptic bronchoscopy and is associated with a lower risk of toxicity.

An investigation of the local anaesthetic effects of pethidine in volunteers.

We have investigated the local anaesthetic effects of 0.1% and 0.2% pethidine compared with 0.5% prilocaine using an intravenous regional anaesthetic technique, in a randomised, double-blind study in volunteers. Both pethidine and prilocaine produced a sensory and motor blockade, although the latter had a more profound effect, with a faster onset and slower recovery. Pethidine in low concentration clearly has a local anaesthetic action on peripheral nerves.