Modeling Acute Myeloid Leukemia Using StarPEG-Heparin Hydrogels.

Biomimetic semi-synthetic hydrogels formed from a combination of star-shaped poly(ethylene glycol) (starPEG) and the glycosaminoglycan, heparin, allows for the three-dimensional (3D) culture of various cells and tissues. In this chapter, we describe methods for the use of starPEG-heparin hydrogels to cultivate primary and immortalized human acute myeloid leukemia (AML) cells. The resulting 3D culture models allow for the study of AML development and response to chemotherapeutic agents.

I22: SAXS/WAXS beamline at Diamond Light Source - an overview of 10 years operation.

Beamline I22 at Diamond Light Source is dedicated to the study of soft-matter systems from both biological and materials science. The beamline can operate in the range 3.7 keV to 22 keV for transmission SAXS and 14 keV to 20 keV for microfocus SAXS with beam sizes of 240 µm × 60 µm [full width half-maximum (FWHM) horizontal (H) × vertical (V)] at the sample for the main beamline, and approximately 10 µm × 10 µm for the dedicated microfocusing platform. There is a versatile sample platform for accommodating a range of facilities and user-developed sample environments. The high brilliance of the insertion device source on I22 allows structural investigation of materials under extreme environments (for example, fluid flow at high pressures and temperatures). I22 provides reliable access to millisecond data acquisition timescales, essential to understanding kinetic processes such as protein folding or structural evolution in polymers and colloids.

Patients with faecal peritonitis admitted to European intensive care units: an epidemiological survey of the GenOSept cohort.

INTRODUCTION: Faecal peritonitis (FP) is a common cause of sepsis and admission to the intensive care unit (ICU). The Genetics of Sepsis and Septic Shock in Europe (GenOSept) project is investigating the influence of genetic variation on the host response and outcomes in a large cohort of patients with sepsis admitted to ICUs across Europe. Here we report an epidemiological survey of the subset of patients with FP. OBJECTIVES: To define the clinical characteristics, outcomes and risk factors for mortality in patients with FP admitted to ICUs across Europe. METHODS: Data was extracted from electronic case report forms. Phenotypic data was recorded using a detailed, quality-assured clinical database. The primary outcome measure was 6-month mortality. Patients were followed for 6 months. Kaplan-Meier analysis was used to determine mortality rates. Cox proportional hazards regression analysis was employed to identify independent risk factors for mortality. RESULTS: Data for 977 FP patients admitted to 102 centres across 16 countries between 29 September 2005 and 5 January 2011 was extracted. The median age was 69.2 years (IQR 58.3-77.1), with a male preponderance (54.3%). The most common causes of FP were perforated diverticular disease (32.1%) and surgical anastomotic breakdown (31.1%). The ICU mortality rate at 28 days was 19.1%, increasing to 31.6% at 6 months. The cause of FP, pre-existing co-morbidities and time from estimated onset of symptoms to surgery did not impact on survival. The strongest independent risk factors associated with an increased rate of death at 6 months included age, higher APACHE II score, acute renal and cardiovascular dysfunction within 1 week of admission to ICU, hypothermia, lower haematocrit and bradycardia on day 1 of ICU stay. CONCLUSIONS: In this large cohort of patients admitted to European ICUs with FP the 6 month mortality was 31.6%. The most consistent predictors of mortality across all time points were increased age, development of acute renal dysfunction during the first week of admission, lower haematocrit and hypothermia on day 1 of ICU admission.

First Report of Tobacco rattle virus in Peony in Alaska.

Peonies (Paeonia sp.) are highly valued for their large showy flowers in home gardens and commercially in the cut flower industry. In 2007, scattered peony (Paeonia lactiflora 'Sarah Bernhardt') plants cultivated on small plots at the University of Alaska Experimental Station in Fairbanks displayed distinct leaf ringspot patterns. Symptoms were more severe during the cooler months of the growing season (June and September), with symptom remission in the intervening warmer months. Leaf samples from six symptomatic plants were collected in July and from 20 symptomatic plants in September and assayed for viruses. Leaf samples (1 g) were assayed with a general protocol for plant virus extraction and partial purification with differential centrifugation followed by protein detection on stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1). No distinct proteins indicative of viral coat protein(s) were detected. Tomato spotted wilt virus (TSWV) and Tobacco rattle virus (TRV), known pathogens of peony, were then specifically targeted. Total RNA was extracted from each sample with an RNeasy Plant Mini kit (Qiagen Inc., Valencia, CA) and used as the template for reverse transcription (RT)-PCR with random primers. TSWV was not detected by RT-PCR with tospovirus group-specific primers (Agdia, Inc., Elkhart, IN). A nested set of primers designed from the TRV 16-kDa protein gene on RNA1 (4) amplified an ~600-bp fragment from one of the symptomatic plants. This DNA was directly sequenced (GenBank Accession No. FJ357572) and BLAST searches in GenBank revealed as much as 95% nucleotide (nt) identity with TRV accessions J04347 and X03685. Additional primer pairs specific for TRV (2) amplified overlapping fragments with expected sizes of ~818, ~515, and ~290 bp from the 29- and 16-kDa protein genes on the 3'-end of RNA1 that were directly sequenced. Assembly of these sequences in Sequencher 4.8 (Gene Codes Corp., Ann Arbor, MI) resulted in a 1,422-nt sequence (Accession No. FJ357571) and Clustal X analysis (3) showed 93 to 94% nt identity to TRV isolates, -ORY (AF034622), -PpK20 (AF314165), -Pp085 (AJ586803), and -SYM (D00155). Mechanical inoculation of partially purified virions from the confirmed TRV-infected peony plant to Nicotiana benthamiana gave no symptoms to occasional ringspots, faintly curled leaves, and chlorotic blotches on N. tabacum 'Samsun', and local lesions on Chenopodium amaranticolor. TRV infection of these hosts was confirmed by RT-PCR. With electron microscopy, rod-shaped particles similar to TRV with a distinct central canal characteristic of TRV were seen occasionally only from inoculated N. benthamiana. On the basis of the biological and molecular data, we have determined the virus in the peony to be an isolate of TRV, tentatively named TRV-Peony. TRV was confirmed in only one other peony based on a sequenced 290-nt PCR fragment with 95% identity with the sequence from the other TRV-infected peony. Lack of TRV detection in the other symptomatic peonies was possibly due to low viral concentrations and interfering plant substances. Documentation of TRV in peonies is especially important to help avoid distribution of virus-infected vegetative propagation material. To our knowledge, this is the first report of TRV in this host in Alaska, but also of this virus in Alaska. References: (1) L. C. Lane. Methods Enzymol. 118:687, 1986. (2) D. J. Robinson. J. Phytopathol. 152:286, 2004. (3) J. D. Thompson et al. Nucleic Acids Res. 24:4882, 1997. (4) F. Van Der Wilk et al. Eur. J. Plant Pathol.100:109, 1994.

Resuscitation room blood alcohol concentrations: one-year cohort study.

OBJECTIVE: To clarify the relationship between presenting clinical condition and blood alcohol concentration (BAC) among adult patients admitted to a resuscitation room (RR) of an emergency department (ED) in order to help guide clinical practice. METHOD: Single-site prospective cohort study of all patients admitted to the RR of an inner-city hospital over a one-year period. The study sample comprised all those aged 16 years and over from whom a blood sample was taken, with BAC (results not known to ED staff), pathology by International Classification of Diseases (ICD) version 10 coding, injury severity score for trauma, return visit to hospital and mortality during the subsequent 6-month period, being recorded. RESULTS: 291 (15%) of 1908 presentations had a positive BAC (ie, BAC >10 mg/100 ml) ranging from 11 to 574 mg/100 ml, of which almost 40% were over 240 mg/100 ml (ICD-10 code Y90.8). In addition to collapse from alcohol/drugs, almost half of those presenting following self-harm or assault had a positive BAC. Those with a positive BAC had a higher rate of ED re-attendance in the following 6 months. 10% of all presentations were due to trauma. CONCLUSION: The following five presentations to the RR are associated with a positive BAC: collapse from alcohol/drugs, self-harm, trauma, gastrointestinal bleeding (ICD-10 code K92.2) and non-cardiac chest pain (ICD-10 code R07). Patients with a positive BAC demonstrate a very wide range of pathology, some with severe levels of misuse. This highlights the opportunity for prompt feedback when sober, to ensure all is done to encourage patients to contemplate change in order to reduce re-attendance.

The clinical and pathological differences in prevalent round screen-detected and symptomatic invasive breast cancer.

The potential benefits of breast cancer screening include the detection of cancers at a more favourable stage, however, cancers detected during the prevalent round of screening may differ from true screen-detected cancers. These differences are poorly defined. This study prospectively assessed all women between 50 and 64 years of age undergoing curative surgery for breast cancer, both screen-detected and symptomatic, in one screening centre during the prevalent round of the national breast cancer-screening programme. Four hundred and thirty seven patients (364 screen-detected and 73 symptomatic patients) underwent surgery for breast cancer. Symptomatic breast cancers were of a higher grade (p < 0.0001; Chi2) and less likely to be oestrogen receptor positive (49% versus 88%; p < 0.0001; Fisher's exact test); however there was no difference in size of tumour or axillary nodal positivity. This study suggests that tumours detected by screening during the prevalent round of a screening programme are of a more prognostically favourable type than symptomatic breast cancers in the same age group.

TNF and TNFR polymorphisms in severe sepsis and septic shock: a prospective multicentre study.

Tumour necrosis factor (TNF) is an important pro-inflammatory cytokine produced in sepsis. Studies examining the association of individual TNF single nucleotide polymorphisms with sepsis have produced conflicting results. This study investigated whether common polymorphisms of the TNF locus and the two receptor genes, TNFRSF1A and TNFRSF1B, influence circulating levels of encoded proteins, and whether individual polymorphisms or extended haplotypes of these genes are associated with susceptibility, severity of illness or outcome in adult patients with severe sepsis or septic shock. A total of 213 Caucasian patients were recruited from eight intensive care units (ICU) in the UK and Australia. Plasma levels of TNF (P = 0.02), sTNFRSF1A (P = 0.005) and sTNFRSF1B (P = 0.01) were significantly higher in those who died on ICU compared to those who survived. There was a positive correlation between increasing soluble receptor levels and organ dysfunction (increasing SOFA score) (sTNFRSF1A R = 0.51, P < 0.001; sTNFRSF1B R = 0.53, P < 0.001), and in particular with the degree of renal dysfunction. In this study, there were no significant associations between the selected candidate TNF or TNF receptor polymorphisms, or their haplotypes, and susceptibility to sepsis, illness severity or outcome. The influence of polymorphisms of the TNF locus on susceptibility to, and outcome from sepsis remains uncertain.

A cluster randomised, controlled trial of the value of dental health educators in general dental practice.

AIM: To test the effectiveness of dental health educators in general dental practice. OBJECTIVE: To evaluate the effectiveness and cost of primary care trusts seconding dental health educators free of charge to suitable general dental practices to provide dental health counselling to mothers of regularly attending pre-school children at risk to caries. METHOD: Two-cell, parallel group, cluster randomised, controlled clinical trial of two years' duration. CLINICAL SETTING: 30 general dental practices in North-West England. PARTICIPANTS: 269 mothers of 334 pre-school children. INTERVENTIONS: Those in the test group were given visits to a dental health educator over a 2-year period to counsel mothers of at-risk, pre-school children. The rest were held as a control. MAIN OUTCOME MEASURES: Caries prevalence of the children and dental health knowledge, attitudes and toothbrushing skills of the parents. The full costs of the exercise were kept throughout. The statistical analysis controlled for the clustering of children within practices. RESULTS: After 2 years, 271 (81%) children and 248 (92%) mothers remained in the study. There was an 18% difference in mean dmft between the groups in favour of the test group children but this was not statistically significant. At the end of the study there was an 18% difference in mean dmft between the groups in favour of the test group children but this was not statistically significant. No difference in plaque levels was found. The mothers in the test group were more knowledgeable, had better attitudes towards the dental health of their offspring and better toothbrushing skills than those in the control. Each 2-hour session to counsel ten parents cost pound 40. CONCLUSION: Primary care trusts should carefully consider the cost value of seconding dental health educators to counsel parents of regularly attending, at-risk, pre-school children when considering such an option.

Antigens shared by malignant plasma cells and normal B cells may be involved in graft versus myeloma.

Cytotoxic T cells play an important role in graft-versus-host-disease (GvHD) and graft-versus-leukaemia/myeloma, which may occur in patients treated with an allogeneic stem cell transplantation (ASCT). Here, we describe the selection of a myeloma reactive CD4+ cytotoxic T cell-line (CTL) and two CD4+ clones from this CTL. The CTL was generated from the blood from a patient with multiple myeloma (MM) with graft versus myeloma/GvHD, following an ASCT. The CTL was stimulated using irradiated peripheral blood mononuclear cells and EBV transformed B cells from the myeloma patient (EBVp), both of which were obtained prior to ASCT. Both the CTL and the two T cell clones specifically lysed EBVp and secreted IFN-gamma after coculture with EBVp and autologous myeloma tumour cells in a class II restricted fashion. These results show that myeloma tumour cells and autologous B cells present a common polymorphic peptide that functions as a target for graft derived cytotoxic T cells. Identification of these proteins will give insight into the relationship between graft versus myeloma (GvM) and GvHD and may provide immunotherapeutical targets in the treatment of MM.

The value of blood lactate measurements in ICU: an evaluation of the role in the management of patients on haemofiltration.

In response to clinical demand some point-of-care analysers now provide blood lactate measurements, but recently concern has been expressed about the value and interpretation of these measurements. We undertook this study to evaluate blood lactate measurements in patients with acute renal failure undergoing haemofiltration (HF) with lactate replacement fluid. At baseline, 27 patients had base deficits of >5 mmol/l and 14 (52%) had blood lactates of >3.5 mmol/l. Lactate 'tolerance' was monitored by peak changes in these parameters during the procedure. There was a worsening of base deficit in only three of the patients in whom lactate rises exceeded 10 mmol/l with one survivor. Twelve patients with rises of blood lactate greater than 5 mmol/l improved their base deficit (+1 to +17) with eight (67%) survivors. Of the remaining 12 patients with improved base deficit (+2 to +20), 10 (83%) survived. Lactate tolerance was compromised in patients with co-incidental liver disease, those on inotropic support, and in patients with initial blood lactate measurements of >10 mmol/l and large base deficits. The data suggest that blood lactate and simultaneous acid-base response measurements during HF help to assign correct buffer replacement and should be performed on all patients.

Dental health knowledge and attitudes of regularly attending mothers of high-risk, pre-school children.

AIM: The aims of this study were to discover the knowledge of and attitudes towards dental health of a group of regularly attending mothers of young children at high-risk of caries, and to evaluate their toothbrushing techniques. METHOD: As part of the baseline examination of a randomised controlled trial to test the influence of dental health counselling on the caries increment of at-risk pre-school children, 268 mothers of 334 children completed a questionnaire enquiring about their dental health knowledge and attitudes, and were also observed brushing their children's teeth. RESULTS: Although most mothers (71%) knew that they should brush their children's teeth twice a day using a small toothbrush (94%) only 52% knew that they should use only a small pea-sized amount of paste, and only 3% knew the recommended level of fluoride in toothpaste for these at-risk children. 40% of the children insisted on brushing their own teeth and 40% of the mothers brushed their children's teeth inadequately. Although three-quarters of the mothers knew that sugary foods and drinks should be consumed only at mealtimes, only 7% knew the four foods and drinks supplying most sugar to a child's diet. Although three-quarters of mothers thought that dental decay in milk teeth was very important, only half wanted their children's carious teeth restored. CONCLUSION: Knowledge, attitudes and behaviour about dental health among these regularly attending mothers of at-risk, pre-school children were superficial. Their attitudes to dental health of primary teeth were equivocal and their demonstrated brushing behaviour on the part of their children was inadequate.

The influence of experience and specialisation on the reliability of a common clinical sign.

OBJECTIVES: To explore the influence of experience and specialisation on clinical judgement by comparing accuracy in diagnosing anaemia between a consultant general surgeon, a consultant ophthalmologist and their registrars. PATIENTS AND METHODS: Conjunctival inspection of 101 patients, subsequent correlation with haemoglobin concentration. MAIN OUTCOME MEASURES: Number of correct and incorrect diagnoses of anaemia. RESULTS: 54 patients were anaemic and 47 were not. Overall accuracy in diagnosing anaemia ranged from 0.61-0.69, sensitivity 0.52-0.65 and specificity 0.62-0.83. Agreements between pairs of examiners were 0.68-0.81, with kappa values of 0.36-0.60 when adjusted for chance agreement. CONCLUSIONS: Neither experience nor specialisation significantly influenced our ability to diagnose anaemia, based on conjunctival inspection. Without critical analysis of clinical signs, we are unaware of their diagnostic limitations.

Progression of skeletal muscle damage during treatment of severe falciparum malaria.

To assess the relationship between severity of malaria and progression of skeletal muscle damage during initial treatment, we studied 28 Thai adults with slide-positive falciparum malaria. Six had uncomplicated malaria (Group 1), 12 had severe non-cerebral malaria (Group 2) and ten had cerebral malaria (Group 3). There were no significant differences between baseline serum creatine kinase (CK) levels in the three groups (P=0.071). There was no change in serum CK during the first 48 h of treatment in Group 1 cases. In Group 2 patients, the median peak serum CK was nine times that at baseline while in Group 3, serum CK peaked at a median concentration 20 times that at presentation. In Groups 2 and 3, the peak serum CK occurred at least 24 h after presentation in more than half the patients, and was independent of intramuscular injections and convulsions during initial therapy. These longitudinal data suggest that: (i) severe falciparum malaria is associated with skeletal muscle damage that increases during initial therapy especially in patients with coma; (ii) the effect of other major treatment or infection-specific factors that are associated with muscle damage does not diminish this relationship; and (iii) cerebral malaria in combination with a high baseline and rising serum CK should pre-empt monitoring and management strategies aimed at preserving renal function including renal dialysis.

The pathophysiologic and prognostic significance of acidosis in severe adult malaria.

OBJECTIVE: To investigate the pathophysiology and prognostic significance of acidosis in severe adult malaria. DESIGN: Cohort study. SETTING: The intensive care unit of an infectious diseases hospital in southern Vietnam. PATIENTS: Three hundred forty-six consecutive adult patients with severe falciparum malaria. INTERVENTIONS: Measurements of baseline venous lactate and pyruvate concentrations and an extensive range of clinical and laboratory variables were made, and patients were followed up carefully until death or discharge from the hospital. Admission arterial blood pH and gas tensions were recorded in 296 patients, and hepatic venous sampling was done in 12 patients. MEASUREMENTS AND MAIN RESULTS: Overall, 198 (67%) patients were acidotic (standard base deficit [SBD], >3.3 mmol/L [n = 196], or arterial Pco2, >45 torr [6 kPa] [n = 3]). Hyperlactatemia (plasma lactate, >4 mmol/L) occurred in 120 (35%) of the 346 patients and was associated significantly with acidosis (p < .0001). The hepatosplanchnic lactate extraction ratio was negatively correlated with mixed venous plasma lactate (r2 = .50; p = .006). Hyperlactatemia, metabolic acidosis (SBD, >3.3), and acidemia (pH <7.35) were strongly positively associated with a fatal outcome (relative risks [95% confidence interval], 4.3 [range, 1.8-10.6], 5.0 [range, 3.0-8.1], and 2.7 [range, 1.8-4.1], respectively). The SBD was the single best clinical or laboratory predictor of fatal outcome. The overall median lactate/pyruvate ratio was raised at 30.6 (range, 20.6-62.3; normal range, <15), suggesting hypoxia and anaerobic glycolysis, and was significantly higher in fatal cases (p < .0001). In an additive multivariate model, the two main independent contributors to metabolic acidosis were plasma creatinine, as a measure of renal dysfunction, and venous plasma lactate, together accounting for 63% of the variance in SBD. In univariate analyses, they contributed 29% and 38%, respectively. CONCLUSIONS: These results confirm the importance of acidosis in the pathophysiology of severe adult malaria and suggest a multifactorial origin involving tissue hypoxia, liver dysfunction, and impaired renal handling of bicarbonate.