Monte Carlo simulation is an essential tool in emission tomography that can assist in the design of new medical imaging devices, the optimization of acquisition protocols, and the development or assessment of image reconstruction algorithms and correction techniques. GATE, the Geant4 Application for Tomographic Emission, encapsulates the Geant4 libraries to achieve a modular, versatile, scripted simulation toolkit adapted to the field of nuclear medicine. In particular, GATE allows the description of time-dependent phenomena such as source or detector movement, and source decay kinetics. This feature makes it possible to simulate time curves under realistic acquisition conditions and to test dynamic reconstruction algorithms. This paper gives a detailed description of the design and development of GATE by the OpenGATE collaboration, whose continuing objective is to improve, document, and validate GATE by simulating commercially available imaging systems for PET and SPECT. Large effort is also invested in the ability and the flexibility to model novel detection systems or systems still under design. A public release of GATE licensed under the GNU Lesser General Public License can be downloaded at the address http://www-lphe.ep.ch/GATE/. Two benchmarks developed for PET and SPECT to test the installation of GATE and to serve as a tutorial for the users are presented. Extensive validation of the GATE simulation platform has been started, comparing simulations and measurements on commercially available acquisition systems. References to those results are listed. The future prospects toward the gridification of GATE and its extension to other domains such as dosimetry are also discussed.
The study was initiated to determine if CD34+ cell selection of small volume bone marrow samples could be effectively performed on the Isolex® 300i Magnetic Cell Selection System® device and if the results obtained from these samples would be comparable to results from large standard volume samples. The impact on the CD34+ recovery using a full versus a half vial of Isolex® CD34 reagent and the effects of shipping a post-selection product were evaluated.
Study Design and Methods
A protocol to evaluate CD34+ cell selection with two ranges of smaller volume bone marrow samples (~50 mL and ~100mL) was developed and instituted at the three PACT facilities. The study was performed in 2 phases.
Mean post-selection CD34+ recoveries from the two sizes of samples were (104.1%) and 103.3% (smallest and largest volumes, respectively), and mean CD34+ recoveries were 115.6% and 88.7% with full and half vials of reagent respectively. Mean CD34+ recoveries for post-shipment smaller volume samples (106.8%) and for larger volume samples (116.4%) and mean CD34+ recoveries of 99.9% and 127.4% for post-shipment samples processed with full and half vials of reagent respectively were obtained.
Mean CD34+ recovery was 76.8% for post-selection samples and 74.0% for post-shipment samples.
Results from this study suggest that smaller volume bone marrow sample processing on the Isolex® system is as efficient or more efficient when compared to standard volume sample processing. Post-processing mean CD34+ recovery results obtained using a full vial or a half vial of CD34 reagent were not significantly different.
CD34+ cell selection; bone marrow; cell therapy; regenerative medicine
More detailed sequence standards that keep up with revolutionary sequencing technologies will aid the research community in evaluating data.
To assess the frequency and clinical characteristics of patients with mutations of major amyotrophic lateral sclerosis (ALS) genes in a prospectively ascertained, population-based epidemiologic series of cases.
The study population includes all ALS cases diagnosed in Piemonte, Italy, from January 2007 to June 2011. Mutations of SOD1, TARDBP, ANG, FUS, OPTN, and C9ORF72 have been assessed.
Out of the 475 patients included in the study, 51 (10.7%) carried a mutation of an ALS-related gene (C9ORF72, 32; SOD1, 10; TARDBP, 7; FUS, 1; OPTN, 1; ANG, none). A positive family history for ALS or frontotemporal dementia (FTD) was found in 46 (9.7%) patients. Thirty-one (67.4%) of the 46 familial cases and 20 (4.7%) of the 429 sporadic cases had a genetic mutation. According to logistic regression modeling, besides a positive family history for ALS or FTD, the chance to carry a genetic mutation was related to the presence of comorbid FTD (odds ratio 3.5; p = 0.001), and age at onset ≤54 years (odds ratio 1.79; p = 0.012).
We have found that ∼11% of patients with ALS carry a genetic mutation, with C9ORF72 being the commonest genetic alteration. Comorbid FTD or a young age at onset are strong indicators of a possible genetic origin of the disease.
SF1126 is a peptidic pro-drug inhibitor of pan-PI3K/mTORC. A first-in-human study evaluated safety, dose limiting toxicities (DLT), maximum tolerated dose (MTD), pharmacokinetics (PK), pharmacodynamics (PD) and efficacy of SF1126, in patients with advanced solid and B-cell malignancies.
Patients and methods
SF1126 was administered IV days 1 and 4, weekly in 28 day-cycles. Dose escalation utilised modified Fibonacci 3+3. Samples to monitor PK and PD were obtained.
Forty four patients were treated at 9 dose levels (90–1110 mg/m2/day). Most toxicity was grade 1 and 2 with a single DLT at180 mg/m2 (diarrhoea). Exposure measured by peak concentration (Cmax) and area under the time-concentration curve (AUC0-t) was dose proportional. Stable disease (SD) was the best response in 19 of 33 (58%) evaluable patients. MTD was not reached but the maximum administered dose (MAD) was 1110 mg/m2. The protocol was amended to enrol patients with CD20+ B-cell malignancies at 1110 mg/m2. A CLL patient who progressed on rituximab [R] achieved SD after 2 months on SF1126 alone but in combination with R achieved a 55% decrease in absolute lymphocyte count and a lymph node response. PD studies of CLL cells demonstrated SF1126 reduced p-AKT and increased apoptosis indicating inhibition of activated PI3K signalling.
SF1126 is well tolerated with SD as the best response in patients with advanced malignancies.
PI3K/mTORC pathway; SF1126; Pharmacokinetics; Pharmacodynamics; Refractory solid tumours; B-cell malignancies
Hyperglycaemia disproportionately affects African-Americans (AfAs).
We tested the transferability of 18 single-nucleotide polymorphisms (SNPs)
associated with glycaemic traits identified in European ancestry (EuA)
populations in 5,984 non-diabetic AfAs.
We meta-analysed SNP associations with fasting glucose (FG) or
insulin (FI) in AfAs from five cohorts in the Candidate Gene Association
Resource. We: (1) calculated allele frequency differences, variations in
linkage disequilibrium (LD), fixation indices (Fsts) and
integrated haplotype scores (iHSs); (2) tested EuA SNPs in AfAs; and (3)
interrogated within ±250 kb around each EuA SNP in AfAs.
Allele frequency differences ranged from 0.6% to 54%.
Fst exceeded 0.15 at 6/16 loci, indicating modest population
differentiation. All iHSs were <2, suggesting no recent positive
selection. For 18 SNPs, all directions of effect were the same and
95% CIs of association overlapped when comparing EuA with AfA. For
17 of 18 loci, at least one SNP was nominally associated with FG in AfAs.
Four loci were significantly associated with FG (GCK,
p=5.8 × 10-8;
MTNR1B, p=8.5 ×
10-9; and FADS1,
p=2.2 × 10-4) or FI
(GCKR, p=5.9 ×
10-4). At GCK and MTNR1B
the EuA and AfA SNPs represented the same signal, while at
FADS1, and GCKR, the EuA and best AfA
SNPs were weakly correlated (r2<0.2),
suggesting allelic heterogeneity for association with FG at these loci.
Few glycaemic SNPs showed strict evidence of transferability from EuA
to AfAs. Four loci were significantly associated in both AfAs and those with
EuA after accounting for varying LD across ancestral groups, with new
signals emerging to aid fine-mapping.
African ancestry; Genetics; Genome-wide association; LD mapping; Minorities; Type 2 diabetes
To test efficacy and safety of atorvastatin in subjects with clinically isolated syndrome (CIS).
Subjects with CIS were enrolled in a phase II, double-blind, placebo-controlled, 14-center randomized trial testing 80 mg atorvastatin on clinical and brain MRI activity. Brain MRIs were performed quarterly. The primary endpoint (PEP) was development of ≥3 new T2 lesions, or one clinical relapse within 12 months. Subjects meeting the PEP were offered additional weekly interferon β-1a (IFNβ-1a).
Due to slow recruitment, enrollment was discontinued after 81 of 152 planned subjects with CIS were randomized and initiated study drug. Median (interquartile range) numbers of T2 and gadolinium-enhancing (Gd) lesions were 15.0 (22.0) and 0.0 (0.0) at baseline. A total of 53.1% of atorvastatin recipients (n = 26/49) met PEP compared to 56.3% of placebo recipients (n = 18/32) (p = 0.82). Eleven atorvastatin subjects (22.4%) and 7 placebo subjects (21.9%) met the PEP by clinical criteria. Proportion of subjects who did not develop new T2 lesions up to month 12 or to starting IFNβ-1a was 55.3% in the atorvastatin and 27.6% in the placebo group (p = 0.03). Likelihood of remaining free of new T2 lesions was significantly greater in the atorvastatin group compared with placebo (odds ratio [OR] = 4.34, p = 0.01). Likelihood of remaining free of Gd lesions tended to be higher in the atorvastatin group (OR = 2.72, p = 0.11). Overall, atorvastatin was well tolerated. No clear antagonistic effect of atorvastatin plus IFNβ-1a was observed on MRI measures.
Atorvastatin treatment significantly decreased development of new brain MRI T2 lesion activity, although it did not achieve the composite clinical and imaging PEP.
Classification of Evidence:
This study provided Class II evidence that atorvastatin did not reduce the proportion of patients with CIS meeting imaging and clinical criteria for starting immunomodulating therapy after 12 months, compared to placebo. In an analysis of a secondary endpoint (Class III), atorvastatin was associated with a reduced risk for developing new T2 lesions.
This report describes the baseline characteristics of patients in the Reduction of Events with Darbepoetin alfa in Heart Failure trial (RED-HF) which is testing the hypothesis that anaemia correction with darbepoetin alfa will reduce the composite endpoint of death from any cause or hospital admission for worsening heart failure, and improve other outcomes.
Methods and results
Key demographic, clinical, and laboratory findings, along with baseline treatment, are reported and compared with those of patients in other recent clinical trials in heart failure. Compared with other recent trials, RED-HF enrolled more elderly [mean age 70 (SD 11.4) years], female (41%), and black (9%) patients. RED-HF patients more often had diabetes (46%) and renal impairment (72% had an estimated glomerular filtration rate <60 mL/min/1.73 m2). Patients in RED-HF had heart failure of longer duration [5.3 (5.4) years], worse NYHA class (35% II, 63% III, and 2% IV), and more signs of congestion. Mean EF was 30% (6.8%). RED-HF patients were well treated at randomization, and pharmacological therapy at baseline was broadly similar to that of other recent trials, taking account of study-specific inclusion/exclusion criteria. Median (interquartile range) haemoglobin at baseline was 112 (106–117) g/L.
The anaemic patients enrolled in RED-HF were older, moderately to markedly symptomatic, and had extensive co-morbidity.
Heart failure; Anaemia
Measurement of prostate specific antigen (PSA) in prostate cancer patients following radical prostatectomy (RP) has been limited by the sensitivity of available assays. Because radical prostatectomy removes the tissue responsible for PSA production, post-surgical PSA is typically undetectable with current assay methods. However, evidence suggests that more sensitive determination of PSA status following RP could improve assessment of patient prognosis, response to treatment, and better target secondary therapy to those who may benefit most. We report the development and validation of an investigational digital immunoassay with two logs greater sensitivity than today’s ultrasensitive third-generation PSA assays.
Reagents were developed for a paramagnetic bead-based ELISA for use with high-density arrays of femtoliter-volume wells. Anti-PSA capture-beads with immunocomplexes and associated enzyme labels were singulated within the wells of the arrays and interrogated for the presence of enzymatic product. Analytical performance of the assay was characterized, its accuracy compared with a commercially available test, and longitudinal serum samples from a pilot study of 33 RP patients were analyzed.
The assay exhibited a functional sensitivity (20% inter-assay CV) of less than 0.00005 ng/mL (0.05 pg/mL), total imprecision of less than 10% from 1 to 50 pg/mL, and excellent agreement with the comparator method. All RP samples were well within the assay measurement capability. PSA values following surgery were examined in the context of five-year biochemical cancer recurrence.
The assay demonstrated a robust two-log advance in measurement sensitivity relative to current ultrasensitive assays, and the analytical performance for accurate assessment of PSA status after RP.
Holoprosencephaly (HPE) is the most common structural malformation of the human forebrain. There are several important HPE mutational target genes, including the transcription factor SIX3, which encodes an early regulator of Shh, Wnt, Bmp and Nodal signalling expressed in the developing forebrain and eyes of all vertebrates.
To characterise genetic and clinical findings in patients with SIX3 mutations.
Patients with HPE and their family members were tested for mutations in HPE-associated genes and the genetic and clinical findings, including those for additional cases found in the literature, were analysed. The results were correlated with a mutation-specific functional assay in zebrafish.
In a cohort of patients (n = 800) with HPE, SIX3 mutations were found in 4.7% of probands and additional cases were found through testing of relatives. In total, 138 cases of HPE were identified, 59 of whom had not previously been clinically presented. Mutations in SIX3 result in more severe HPE than in other cases of non-chromosomal, non-syndromic HPE. An over-representation of severe HPE was found in patients whose mutations confer greater loss of function, as measured by the functional zebrafish assay. The gender ratio in this combined set of patients was 1.5:1 (F:M) and maternal inheritance was almost twice as common as paternal. About 14% of SIX3 mutations in probands occur de novo. There is a wide intrafamilial clinical range of features and classical penetrance is estimated to be at least 62%.
Our data suggest that SIX3 mutations result in relatively severe HPE and that there is a genotype–phenotype correlation, as shown by functional studies using animal models.
Salmonella Cerro prevalence in US dairy cattle has increased significantly during the past decade. Comparison of 237 Salmonella isolates collected from various human and animal sources between 1986 and 2009 using pulsed- field gel electrophoresis, antimicrobial resistance typing, and spvA screening, showed very limited genetic diversity, indicating clonality of this serotype. Improved subtyping methods are clearly needed to analyze the potential emergence of this serotype. Our results thus emphasize the critical importance of population-based pathogen surveillance for the detection and characterization of potentially emerging pathogens, and caution to critically evaluate the adequacy of diagnostic tests for a given study population and diagnostic application.
Salmonella Cerro; molecular epidemiology; PFGE; emerging clone
The ability to conduct advanced functional genomic studies of the thousands of sequenced bacteria has been hampered by the lack of available tools for making high-throughput chromosomal manipulations in a systematic manner that can be applied across diverse species. In this work, we highlight the use of synthetic biological tools to assemble custom suicide vectors with reusable and interchangeable DNA “parts” to facilitate chromosomal modification at designated loci. These constructs enable an array of downstream applications, including gene replacement and the creation of gene fusions with affinity purification or localization tags. We employed this approach to engineer chromosomal modifications in a bacterium that has previously proven difficult to manipulate genetically, Desulfovibrio vulgaris Hildenborough, to generate a library of over 700 strains. Furthermore, we demonstrate how these modifications can be used for examining metabolic pathways, protein-protein interactions, and protein localization. The ubiquity of suicide constructs in gene replacement throughout biology suggests that this approach can be applied to engineer a broad range of species for a diverse array of systems biological applications and is amenable to high-throughput implementation.
HIV enters the brain soon after infection causing neuronal damage and microglial/astrocyte dysfunction leading to neuropsychological impairment. We examined the impact of HIV on resting cerebral blood flow (rCBF) within the lenticular nuclei (LN) and visual cortex (VC).
This cross-sectional study used arterial spin labeling MRI (ASL-MRI) to measure rCBF within 33 HIV+ and 26 HIV− subjects. Nonparametric Wilcoxon rank sum test assessed rCBF differences due to HIV serostatus. Classification and regression tree (CART) analysis determined optimal rCBF cutoffs for differentiating HIV serostatus. The effects of neuropsychological impairment and infection duration on rCBF were evaluated.
rCBF within the LN and VC were significantly reduced for HIV+ compared to HIV− subjects. A 2-tiered CART approach using either LN rCBF ≤50.09 mL/100 mL/min or LN rCBF >50.09 mL/100 mL/min but VC rCBF ≤37.05 mL/100 mL/min yielded an 88% (29/33) sensitivity and an 88% (23/26) specificity for differentiating by HIV serostatus. HIV+ subjects, including neuropsychologically unimpaired, had reduced rCBF within the LN (p = 0.02) and VC (p = 0.001) compared to HIV− controls. A temporal progression of brain involvement occurred with LN rCBF significantly reduced for both acute/early (<1 year of seroconversion) and chronic HIV-infected subjects, whereas rCBF in the VC was diminished for only chronic HIV-infected subjects.
Resting cerebral blood flow (rCBF) using arterial spin labeling MRI has the potential to be a noninvasive neuroimaging biomarker for assessing HIV in the brain. rCBF reductions that occur soon after seroconversion possibly reflect neuronal or vascular injury among HIV+ individuals not yet expressing neuropsychological impairment.
= acute/early HIV infection;
= analysis of variance;
= arterial spin labeling MRI;
= classification and regression tree;
= cerebral blood flow;
= chronic HIV infection;
= field of view;
= global deficit score;
= highly active antiretroviral therapy;
= HIV-associated neurocognitive disorders;
= lenticular nuclei;
= resting cerebral blood flow;
= echo time;
= inversion time;
= repetition time;
= visual cortex.
To estimate minimally important differences (MIDs) in scores for the modified Rodnan Skin Score (mRSS) and Health Assessment Questionnaire—Disability Index (HAQ‐DI) in a clinical trial on diffuse systemic sclerosis (SSc).
Participants and methods
134 people participated in a 2‐year, double‐blind, randomised clinical trial comparing efficacy of low‐dose and high‐dose d‐penicillamine in diffuse SSc. At 6, 12, 18 and 24 months, the investigator was asked to rate the change in the patient's health since entering the study: markedly worsened, moderately worsened, slightly worsened, unchanged, slightly improved, moderately improved or markedly improved. Patients who were rated as slightly improved were defined as the minimally changed subgroup and compared with patients rated as moderately or markedly improved.
The MID estimates for the mRSS improvement ranged from 3.2 to 5.3 (0.40–0.66 effect size) and for the HAQ‐DI from 0.10 to 0.14 (0.15–0.21 effect size). Patients who were rated to improve more than slightly were found to improve by 6.9–14.2 (0.86–1.77 effect size) on the mRSS and 0.21–0.55 (0.32–0.83 effect size) on the HAQ‐DI score.
MID estimates are provided for improvement in the mRSS and HAQ‐DI scores, which can help in interpreting clinical trials on patients with SSc and be used for sample size calculation for future clinical trials on diffuse SSc.
MECP2 mutations are identifiable in ∼80% of classic Rett syndrome (RTT), but less frequently in atypical RTT. We recruited 110 patients who fulfilled the diagnostic criteria for Rett syndrome and were referred to Cardiff for molecular analysis, but in whom an MECP2 mutation was not identifiable. Dosage analysis of MECP2 was carried out using multiplex ligation dependent probe amplification or quantitative fluorescent PCR. Large deletions were identified in 37.8% (14/37) of classic and 7.5% (4/53) of atypical RTT patients. Most large deletions contained a breakpoint in the deletion prone region of exon 4. The clinical phenotype was ascertained in all 18 of the deleted cases and in four further cases with large deletions identified in Goettingen. Five patients with large deletions had additional congenital anomalies, which was significantly more than in RTT patients with other MECP2 mutations (2/193; p<0.0001). Quantitative analysis should be included in molecular diagnostic strategies in both classic and atypical RTT.
Gross gene deletion; MECP2; Rett syndrome
Rapid release testing reduces the waiting period for administration of time-sensitive cell therapy products. Current assay systems are labor intensive and time consuming. The Endosafe® Portable Test System (PTS™) is a chromogenic Limulus Amebocyte Lysate (LAL) portable endotoxin detection system which provides quantitative results in approximately 15 minutes. To evaluate Endosafe® performance, specifically with cell therapy products, side-by-side testing of traditional LAL systems, and the Endosafe® system was conducted at the Production Assistance for Cellular Therapies (PACT) facilities and the National Institutes of Health's Department of Transfusion Medicine.
Charles River Laboratories provided each center with a PTS™ reader and two commercially prepared lyophilized Reference Standard Endotoxin (RSE) vials. All samples tested with the Endosafe® system used 0.05 - 5.0 endotoxin units/mL (EU/mL) sensitivity cartridges provided by Charles River. Each vial was reconstituted with LAL water and tested in triplicate using the Endosafe® and in-house LAL methods. Subsequently, each center tested endotoxin content of standard dilutions of cell therapy products thus creating paired test results for each sample. Additionally, fabricated endotoxin-positive samples containing varying concentrations of endotoxin were prepared and shipped to all centers to perform blinded testing.
Valid paired results, based on each center's LAL method and the Endosafe® system criteria were analyzed. Endotoxin detection between paired results was equivalent in most cases.
The Endosafe® system provided reliable results with products typically produced in cell therapy manufacturing facilities, and would be an appropriate test on which to base the release of time-sensitive cell therapy products.
Endotoxin; LAL; Endosafe® PTS™; Comparability
Objectives: To define the incidence of congenital cytomegalovirus (CMV) infection in a defined population in Israel as diagnosed by urine polymerase chain reaction (PCR), and to assess the utility of this method for screening for congenital CMV infection.
Design: A convenient sample of urine specimens from asymptomatic newborns were subjected to CMV PCR. Positive results were validated by urine tube culture and by determination of serum CMV IgM antibodies. Maternal CMV IgG was determined in a representative sample of mothers. Newborns with positive urine specimens underwent full clinical evaluation. Epidemiological characteristics of the mothers were extracted from the medical records.
Settings: Two medical centres in Israel with different population characteristics.
Patients: A total of 2000 newborns (1000 in each medical centre).
Main outcome measure: Presence of CMV DNA in the urine.
Results: Despite significant epidemiological differences between the populations in the two hospitals, the CMV seroprevalence was similar, 80.5% and 85%. Fourteen of the 2000 newborns screened (0.7%) were PCR positive. Urine culture was positive in nine of 10 specimens; IgM was positive in only two of 13 newborns with positive PCR. Eleven newborns underwent full or partial evaluation, and only one (9%) was symptomatic.
Conclusions: The incidence of congenital CMV infection in the study population was 0.7%; over 90% were asymptomatic. Urinary CMV PCR is a reliable, rapid, and convenient method, and thus may serve as a screening tool for the detection of congenital CMV infection.
bladder cancer; gemcitabine; fractionated cisplatin; outpatient; chemotherapy
KIT; testicular germ cell tumours
A variety of human symptoms have been associated with exposure to the dinoflagellate Pfiesteria and have been grouped together into a syndrome termed "possible estuary-associated syndrome." Prospective cohort studies of health effects associated with exposure to estuarine waters that may contain Pfiesteria spp. and related organisms are in progress in North Carolina, Virginia, and Maryland. The three studies recruited cohorts of 118-238 subjects who work or engaged in recreation in estuary waters. Baseline health and neuropsychological evaluations are conducted, and study subjects are followed prospectively for 2-5 years with periodic assessments of health and performance on a battery of neuropsychological tests. Health symptoms and estuary water exposure are recorded by telephone interviews or diaries every 1-2 weeks. Water quality information, including measurements of Pfiesteria spp., is collected in the areas where the subjects are working. Because it is not possible to measure individual exposure to Pfiesteria or a toxin produced by this organism, these studies examine surrogate exposure measures (e.g., time spent in estuary waters, in a fish kill area, or in waters where Pfiesteria DNA was detected by molecular amplification). Preliminary analyses of the first 2 years (1998-2000) of data indicate that none of the three ongoing cohorts have detected adverse health effects. However, there have not been any reported fish kills associated with Pfiesteria since the studies began, so it is possible that none of the study subjects have been exposed to toxin-producing Pfiesteria spp.