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Biomedical subjects

T Roberts

Publications and source records attributed to T Roberts.

At least 55 records · Page 3Linked to original sources

Observations on centrifugation: application to centrifuge development.

This report outlines the background to the development of an automated, serial, discrete centrifuge, reporting on the criteria considered essential in such an instrument. We established the criteria by examining the detailed logistics of centrifuge operation in a hospital laboratory. The mean sample load per run, using six centrifuges, was 13.6 samples, and the user-selectable cycle time ranged from 00:01:10 to 00:12:33 (hours:minutes:seconds) with a fixed g value of 1050. During the laboratory working window, (0900-1700), only 50% of the centrifuge capacity was utilized and more than one-third of the sample workload was delayed for >5 min because the centrifuges were not emptied promptly. In addition, 35% of the sample workload was centrifuged for less than the time prescribed in the operational specifications. Based on these findings, we designed a new continuous, serial centrifuge to overcome some of the deficiencies noted in the logistics study. The centrifuge operates continuously, nominally treating 150 samples/h, with a cycle time of 5 min at 1,000 g. The cycle time and g value are variable between limits, and their selection governs the throughput rate. Each sample is centrifuged separately in individual rotors mounted in a sturdy carousel with a periphery that traverses a load/unload station. There is no sample delay because of operator absence, and the capacity is fully utilized. The centrifuge can operate in a stand-alone capacity or has the capability of being integrated into a sample preparation system or as a direct front end for high-throughput analyzers.

Centrifugation↗

Behavioral and single chorda tympani taste fiber responses in the common marmoset, Callithrix jacchus jacchus.

Gustatory responses of the common marmoset were studied using single fiber recordings from chorda tympani (CT) nerve and two bottle preference (TBP) tests. Hierarchical cluster analysis of 43 fibers' response profiles revealed 3 major clusters of fibers characterized by predominant sensitivity to sweeteners (S cluster), bitter compounds (Q cluster) or acids (H cluster). NaCl as well as LiCl did not stimulate CT taste fibers. The TBP tests showed relationship between a compound's ability to stimulate the taste fibers and the animals' consumption. Activity in the S cluster was associated with preference, while the activity in the Q cluster was associated with rejection. Marmosets neither preferred nor rejected sweeteners which did not stimulate any CT fibers.

Animals↗

Cytokine transgene expression and promoter usage in primary CD34+ cells using particle-mediated gene delivery.

Induction or short-term transgenic expression of specific cytokines, growth factors, or other candidate therapeutic genes in hematopoietic progenitor or stem cells is potentially applicable to gene therapy for cancer. In this study, we explored the application of a gene gun technique, as an alternative to viral vectors, for ex vivo gene transfer into and transient gene expression in highly enriched CD34+ cells derived from human umbilical cord blood. Twenty-four hours posttransfection, 32.6 to 1500 pg/l x 10(6) CD34+ cells of transient gene expression was routinely obtained for specific cytokine and reporter genes. Transgene expression at the single-cell level was revealed by X-Gal staining of lacZ cDNA-transfected CD34+ cells. Expression of four candidate therapeutic genes, namely human granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, interleukin 2, and interferon gamma, was detectable for 4 to 7 days in CD34+ cells. A human elongation factor 1alpha promoter/intron 1 transcription unit was identified as a strong cellular promoter for CD34+ cells, exhibiting strength similar to that of the commonly employed cytomegalovirus immediate-early promoter. These results suggest that the nonviral, gene gun technique offers an efficient alternative approach for transient transgenic studies of hematopoietic cells and may provide new possibilities for certain cancer gene therapy strategies using CD34+ cells.

Antigens, CD34↗

DNA vaccines expressing either the GP or NP genes of Ebola virus protect mice from lethal challenge.

DNA vaccines expressing the envelope glycoprotein (GP) or nucleocapsid protein (NP) genes of Ebola virus were evaluated in adult, immunocompetent mice. The vaccines were delivered into the skin by particle bombardment of DNA-coated gold beads with the Powderject-XR gene gun. Both vaccines elicited antibody responses as measured by ELISA and elicited cytotoxic T cell responses as measured by chromium release assays. From one to four vaccinations with 0.5 microgram of the GP DNA vaccine resulted in a dose-dependent protection from Ebola virus challenge. Maximal protection (78% survival) was achieved after four vaccinations. Mice were completely protected with a priming dose of 0.5 microgram of GP DNA followed by three or four subsequent vaccinations with 1.5 micrograms of DNA. Partial protection could be observed for at least 9 months after three immunizations with 0.5 microgram of the GP DNA vaccine. Comparing the GP and NP vaccines indicated that approximately the same level of protection could be achieved with either vaccine.

Animals↗

Ciao 1 is a novel WD40 protein that interacts with the tumor suppressor protein WT1.

The Wilms tumor suppressor protein, WT1, is a transcription factor capable of activating or repressing transcription of various cellular genes. The mechanisms involved in regulating the transcriptional activities of WT1 are beginning to be unraveled. It appears that physical interactions of other cellular proteins (p53 and par-4) with WT1 can modulate the function of WT1. Here, we report the identification and cloning of a novel WT1-interacting protein termed Ciao 1, a member of the WD40 family of proteins. Ciao 1 specifically interacts with WT1 both in vitro and in vivo. This interaction alters the mobility of a WT1.DNA complex in gel shift assays, and results in a decrease in transcriptional activation mediated by WT1. Ciao 1 does not inhibit binding of WT1 to its consensus nucleotide sequence and does not affect the repression activity of WT1. Thus, Ciao 1 appears to specifically modulate the transactivation activity of WT1 and may function to regulate the physiological functions of WT1 in cell growth and differentiation.

Amino Acid Sequence↗

Molecular characterization of the 1p22 breakpoint region spanning the constitutional translocation breakpoint in a neuroblastoma patient with a t(1;10)(p22;q21).

To characterize the breakpoint in a neuroblastoma patient with a constitutional rearrangement we have constructed a yeast artificial chromosome (YAC) contig extending approximately 6 Mbp in the chromosome 1p22 region that spans the D1S435 and D1S236 loci. This contig has been confirmed by the coincidence of a number of markers in different overlapping YACs. For several of these YACs the overlap was demonstrated following the isolation and sequencing of end clones from which STS markers were generated. The majority of the YACs have been shown not to be chimeric either through the analysis of somatic cell hybrids or fluorescence in situ hybridization. Following the establishment of the contig we have been able to construct a physical map of the region that incorporates six STS and three newly assigned eSTS markers. The generation of this physical map has allowed the reordering of markers in the genetic linkage map for 1p. The physical order is; tel-D1S435-D1S188-D1S424-D1S236-D1D415- D1S420. With the reordering of D1S435 we have been able to join this contig with another reported previously, thereby generating a well characterized 15 Mbp YAC contig in the 1p22-31 region. The 6 Mbp contig described here spans the chromosome 1 constitutional translocation break-point seen in a patient with a t(1;10)(p22;q21) and who had a stage 4S neuroblastoma. YAC fragmentation has been used to define a 200 Kb region within this contig containing the 1p22 breakpoint. Restriction enzyme analysis demonstrates that there are three NotI sites in this region, one of which lies close to the translocation breakpoint site.

Chromosomes, Artificial, Yeast↗

MRI using hyperpolarized noble gases.

The aim of this study was to review the physical basis of MRI using hyperpolarized noble gases as well as the present status of preclinical and clinical applications. Non-radioactive noble gases with a nuclear spin 1/2 (He-3, Xe-129) can be hyperpolarized by optical pumping. Polarization is transferred from circularly polarized laser light to the noble-gas atoms via alkali-metal vapors (spin exchange) or metastable atoms (metastability exchange). Hyperpolarization results in a non-equilibrium polarization five orders of magnitude higher than the Boltzmann equilibrium compensating for the several 1000 times lower density of noble gases as compared with liquid state hydrogen concentrations in tissue and allows for short imaging times. Hyperpolarization can be stored sufficiently long (3 h to 6 days) to allow for transport and application. Magnetic resonance systems require a broadband radio-frequency system - which is generally available for MR spectroscopy - and dedicated coils. The hyperpolarized gases are administered as inhalative "contrast agents" allowing for imaging of the airways and airspaces. Besides the known anesthetic effect of xenon, no adverse effects are observed in volunteers or patients. Pulse sequences are optimized to effectively use the non-renewable hyperpolarization before it decays or is destroyed, using fast low-flip-angles strategies to allow for dynamic/breath-hold imaging of highly diffusible (He) or soluble (Xe) gases with in vivo T1-times well below 1 min. Since helium is not absorbed in considerable amounts, its application is restricted to the lung. Xe-129 is also under investigation for imaging of white matter disease and functional studies of cerebral perfusion. Magnetic resonance imaging using hyperpolarized gases is emerging as a technical challenge and opportunity for the MR community. Preliminary experience suggests potential for functional imaging of pulmonary ventilation and cerebral perfusion.

Adult↗

[Acute vascular perforation with shunt formation in the right ventricle after percutaneous transluminal coronary angioplasty. Magnetic resonance tomography and Doppler ultrasound detection of shunt flow].

A coronary artery perforation is a rare complication after percutaneous transluminal coronary angioplasty. The therapy will be determined by the hemodynamic failure of the left or right ventricle. A case of a coronary artery perforation with a shunt from the right coronary artery to the right ventricle after coronary angioplasty is reported. The shunt was detected by coronary angiography and confirmed by magnetic resonance imaging and doppler echocardiography.

Aged↗

[Flow quantification of intracardiac shunt volumes using MR phase contrast technique in the breath holding phase].

PURPOSE: Comparison of a breath-hold, velocity-encoded, phase-difference magnetic resonance (MR) sequence for intracardiac shunt flow measurement with the invasive shunt size determination by oximetry. PATIENTS AND METHODS: 10 patients with different cardiac shunts (6 ASD/3 VSD/1 PDA) and four healthy individuals were investigated using a 1.5 Tesla Siemens Vision system. For flow measurements a breath-hold, velocity-encoded, phase-difference magnetic resonance (MR) sequence was used ('through plane', FLASH 2D-sequence, TR/TE 110/5 ms, 'velocity encoding' 250 cm/s). Mean flow (ml/R-R interval) in the ascending aorta, the pulmonary trunk, and the right and left pulmonary arteries was determined. The ratio of the mean flow in the pulmonary circulation (Qp: sum of the mean flows in the right and left pulmonary arteries) and the systemic circulation (Qs: mean flow in the proximal aorta) was compared with the Qp/Qs ratios determined by the invasive oxymetric technique. Oximetry was performed within 24 hours after MR imaging. RESULTS: In the 4 healthy individuals MR flow measurement yielded a Qp/Qs ratio of 0.96 +/- 0.15. In the 10 patients with the various shunt defects, the non-invasive shunt determination by MR gave a Qp/Qs ratio of 2.09 +/- 0.67. The percentage of the calculated shunt sizes was 47.05 +/- 17.45%. In the comparison with the results determined by the invasive oxymetric technique, the MR data showed a strong correlation of r = 0.87. CONCLUSIONS: Breath-hold, velocity-encoded, phase-difference MR-technique enables a reliable quantification of cardiac shunts within a short acquisition time.

Adult↗

NB4S, a member of the TBC1 domain family of genes, is truncated as a result of a constitutional t(1;10)(p22;q21) chromosome translocation in a patient with stage 4S neuroblastoma.

Molecular cloning of the breakpoints of a t(1;10)(p22q21) constitutional translocation breakpoint in a patient with stage 4S neuroblastoma has identified two genes which are fused in-frame to generate a novel gene. The 1p22 gene, which we have called NB4S , encodes a 7.5 kb transcript with an 810 amino acid open reading frame and is expressed in a wide variety of tissues. NB4S has >88% homology with the mouse EVI -5 gene within the coding region and shows strong homology over a 200 amino acid region with TBC1 box motif genes involved in cell growth and differentiation. The C-teminal end of the protein contains a number of coiled coil domains, indicating a possible protein-protein binding function. The chromosome 10 breakpoint interrupts a novel transcript (TRNG10) which could only be detected in tumor cells. This transcript has no exon/intron structure or significant open reading frame, suggesting that it is a structural RNA which is transcribed but not translated. The chromosome rearrangement creates a fusion gene product which combines the TBC1 motif of NB4S with a polyadenylation signal from TRNG10 , potentially generating a truncated protein with oncogenic properties.

Animals↗

Choosing options for ultrasound screening in pregnancy and comparing cost effectiveness: a decision analysis approach.

OBJECTIVE: To compare the cost effectiveness of different programmes of routine antenatal ultrasound screening to detect four key fetal anomalies: serious cardiac anomalies, spina bifida, Down's syndrome and lethal anomalies, using existing evidence. DESIGN: Decision analysis was used based on the best data currently available, including expert opinion from the Royal College of Obstetricians and Gynaecologists, Working Party and secondary data from the literature, to predict the likely outcomes in terms of malformations detected by each screening programme. SETTING: Results applicable in clinics, hospitals or GP practices delivering antenatal screening. MAIN OUTCOME MEASURE: The number of cases with a 'target' malformation correctly detected antenatally. RESULTS: There was substantial overlap between the cost ranges of each screening programme demonstrating considerable uncertainty about the relative economic efficiency of alternative programmes for ultrasound screening. The cheapest, but not the most effective, screening programme consisted of one second trimester ultrasound scan. The cost per target anomaly detected (cost effectiveness) for this programme was in the range 5,000 pound silver-109,000, pound silver but in any 1000 women it will also fail to detect between 3.6 and 4.7 target anomalies. CONCLUSIONS: The range of uncertainty in the costs did not allow selection of any one programme as a clear choice for NHS purchasers. The results suggested that the overall allocation of resources for routine ultrasound screening in the UK is not currently economically efficient, but that certain scenarios for ultrasound screening are potentially within the range of cost effectiveness reached by other, possibly competing, screening programmes. The model highlighted the weakness of available evidence and demonstrated the need for more information both about current practice and costs.

Cost-Benefit Analysis↗

Topics in microbial risk assessment: dynamic flow tree process.

Microbial risk assessment is emerging as a new discipline in risk assessment. A systematic approach to microbial risk assessment is presented that employs data analysis for developing parsimonious models and accounts formally for the variability and uncertainty of model inputs using analysis of variance and Monte Carlo simulation. The purpose of the paper is to raise and examine issues in conducting microbial risk assessments. The enteric pathogen Escherichia coli O157:H7 was selected as an example for this study due to its significance to public health. The framework for our work is consistent with the risk assessment components described by the National Research Council in 1983 (hazard identification; exposure assessment; dose-response assessment; and risk characterization). Exposure assessment focuses on hamburgers, cooked a range of temperatures from rare to well done, the latter typical for fast food restaurants. Features of the model include predictive microbiology components that account for random stochastic growth and death of organisms in hamburger. For dose-response modeling, Shigella data from human feeding studies were used as a surrogate for E. coli O157:H7. Risks were calculated using a threshold model and an alternative nonthreshold model. The 95% probability intervals for risk of illness for product cooked to a given internal temperature spanned five orders of magnitude for these models. The existence of even a small threshold has a dramatic impact on the estimated risk.

Analysis of Variance↗

Dedicated extremity MR imaging: An emerging technology

Dedicated extremity MR imaging represents a radical departure from conventional whole-body scanning. Extremity MR imaging offers such advantages as reduced cost, more convenient and inexpensive setting, greater patient comfort and safety, and high diagnostic power. This article examines some of the features of extremity MR imaging and how this technology is affecting musculoskeletal imaging in today's environment of cost containment and health care reform.

Journal Article↗

Chronic orofacial muscle pain: a new approach to diagnosis and management.

The initial data from this study indicate that there are clearly identifiable chronic muscle pain conditions in the form of localized pain; myofascial pain or regional pain conditions; and fibromyalgia or generalized pain conditions. A clear difference exists between the prevalence of these conditions in male and female patients, with a higher percentage of female patients suffering generalized pain problems and temporomandibular problems. Generalized or localized pain appears to be an individual variant of a similar problem and pain patients may have a genetically determined vulnerability associated with bacterial toxins, particularly within the genitourinary tract. It appears that in fibromyalgia there is an underlying genetic factor that causes abnormalities in the muscle metabolic cycle, and preliminary data suggest that lipid anomalies predispose to fibromyalgia and possibly chronic fatigue syndrome. Patients report infectious events at/or around onset in more than 60 percent of cases. Seventy percent of fibromyalgic cases report orofacial pain.

Chronic Disease↗

The incidence of breast cancer following mantle field radiation therapy as a function of dose and technique.

BACKGROUND: There is an increased incidence of breast cancer following mantle field radiation therapy for Hodgkin's disease (HD). We reviewed the experience at the Mallinckrodt Institute of Radiology (MIR) for radiation factors related to the development of breast cancer after mantle field radiation therapy for HD. METHODS: The radiation therapy records of 152 women treated with mantle field irradiation for HD at MIR between 1966-1985 were reviewed for the development of breast cancer and treatment-related factors. All patients had a minimum of 5 years of follow-up. The treatment era (1966-1974 vs. 1975-1985), stage of HD, mediastinal dose, axillary dose, maximum dose from the anterior field (anterior d(max) dose), the anterior-posterior:posterior-anterior (AP:PA) ratio, age at the time of treatment, length of follow-up, and history of splenectomy were analyzed as possible contributing factors for the development of breast cancer. The observed number of breast cancers was compared to the expected number based on age-adjusted incidences from the Connecticut Tumor Registry. RESULTS: Ten breast cancers occurred in the population. Eight involved an upper outer quadrant. In a multivariate analysis, the development of breast cancer was significantly associated with axillary dose. Patients in the early treatment era were at an increased risk for the development of breast cancer due to high anterior d(max) and breast doses from weighting the fields anteriorly on a low energy linear accelerator. The use of current radiation therapy techniques was not related to an increased risk of breast cancer with a median follow-up of 13 years. CONCLUSIONS: A high dose to the axilla and the anterior d(max) point is significantly associated with the development of breast cancer after mantle field irradiation for HD. Efforts to protect the breast from high doses will likely lessen the increased risk of breast cancer in women treated with radiation therapy for HD.

Adolescent↗

Cloning of the human Gfi-1 gene and its mapping to chromosome region 1p22.

Recently the rat and mouse Growth Factor Independence (Gfi-1) genes have been cloned (Gilks et al., 1993; Zoring et al; 1996). This gene allows cells in culture to overcome the depletion of growth factors in the culture medium and maintain their proliferative potential. As part of a cloning strategy to isolated genes from human chromosome 1p22 which are associated with a constitutional chromosome translocation from a patient with stage 4S neuroblastoma, we have identified the human homologue of the Gfi gene and defined a 50 Kb map position within a well characterised YAC contig from the region. The full length cDNA sequence is 81% homologous with the rodent counterparts and, at the protein level, is even more highly conserved.

Amino Acid Sequence↗