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PubMed2002

Experimental colonic phantom for the evaluation of the optimal scanning technique for CT colonography using a multidetector spiral CT equipment.

Our objective was to optimize a scanning protocol for CT colonography using a multidetector scanner with an adaptive-array matrix in a phantom study. A colonic phantom with 27 simulated lesions was examined using a multidetector CT scanner (Somatom Plus 4 Volume Zoom, Siemens, Erlangen, Germany). Three scanning protocols were tested: (a) collimation 1.0 mm, slice thickness 1.0 mm; (b) collimation 2.5 mm, slice thickness 3.0 mm; and (c) collimation 2.5 mm, slice thickness 5.0 mm. Image analysis was performed by two radiologists blinded to the construction of the phantom and to imaging parameters. Sensitivity for lesion detection and image quality was assessed. Computed tomographic colonography detected 27 of 27 lesions with protocol 1 (sensitivity 100%); 26 of 27 lesions with protocol 2 (sensitivity 96.3%); and 23 of 27 lesions with protocol 3 (sensitivity 85.2%). Image quality was graded as optimal for protocol 1, good for protocol 2, and poor for protocol 3. When multidetector CT equipment with an adaptive-array matrix is used, protocols with 1.0- or 3.0-mm slice thickness have a sensitivity for the detection of lesions higher than that obtained with a 5.0-mm slice thickness. Image quality decreases progressively when the slice thickness is increased; therefore, in a clinical setting, we recommend the use of a high-resolution protocol (1.0-mm effective slice thickness) with the highest pitch value in order to reduce scanning time. Dose exposure for such a protocol if combined with 80 mAs is within the range limits recommended by the ICRP for abdominal CT scanning.

Chi-Square Distribution↗
PubMed2001

Optimized 3-D CT scan protocol for longitudinal morphological estimation in craniofacial surgery.

Frequent three-dimensional computed tomography scanning may cause deterioration of the lenses of the eye, which are susceptible to x-rays. The authors performed an experimental study using a phantom head to establish an optimized three-dimensional computed tomography scan protocol for longitudinal morphological estimation in craniofacial surgery. Volume computed tomography scans were performed using a Hi-Speed Advantage SG CT scanner (GE Medical Systems, Milwaukee, WI) in the axial plane with a combination of scan parameters of varied values. The radiation doses induced by each scanning were measured using thermoluminescent dosimeter chips attached to the position of the lenses in the phantom. Two-dimensional images in the coronal plane and three-dimensional images of the osseous surface were generated from each accumulated data set. For each scan parameter, the images generated from data accumulated using different values were compared. The study showed that lens radiation dose increased with tube potential and was almost directly proportional to tube current and 1/pitch. The slice thickness did not affect lens radiation doses significantly. Images with good contrast resolution and low artifact level sufficient for estimating morphological changes were obtained using a low tube potential of 100 kVp and a tube current of 100 mA. In regard to z-axis spatial resolution, a slice thickness of less than 3 mm was required for precisely pointing out bony edges in the two-dimensional reformation images. These results led us to conclude that volume computed tomography for longitudinal examination in craniofacial surgery should be kept to a minimum frequency and performed using a low-dose technique, small slice thickness, and large pitch.

Artifacts↗
PubMed2000

Considerations preliminary to the application of early and immediate loading protocols in dental implantology.

In oral implantology, a 3-6 month stress-free healing period is presently accepted as a prerequisite to achieve bone apposition without interposition of a fibrous scar tissue. This protocol was introduced by Brånemark and co-workers in 1977. The aim of the present paper is to review the reasons that led Brånemark and collaborators to require long delayed loading periods. It is shown that the requirement for long delayed loading periods was drawn from the initiation and development periods of their original clinical trial. Demanding conditions were met involving simultaneously: 1) patients with poor bone quality and quantity, 2) non-optimized implant design, 3) short implants, 4) non-optimized surgical placement, 5) non-optimized surgical protocol and 6) biomechanically non-optimized prosthesis. Extrapolation of the requirement for long healing periods from these particular conditions to more standard situations involving refined surgical protocols and careful patient selection might be questioned. Albeit premature loading has been interpreted as inducing fibrous tissue interposition, immediate loading per se is not responsible for fibrous encapsulation. It is the excess of micromotion during the healing phase that interferes with bone repair. A threshold of tolerated micromotion exists, that is somewhere between 50 microns and 150 microns. It is suggested that loading protocols might be shortened through 2 different approaches. The first way would be to decrease stepwise the delayed loading period for free-standing implants below the presently accepted 3-6 months of healing. The second way would be to identify immediate loading protocols that are capable of keeping the amount of micromotion beneath the threshold of deleterious micromotion. Immediate loading protocols for implants-retained overdentures and fixed bridges are reviewed. It is shown that successful premature loading protocols require a careful and strict patient selection aimed to achieve the best primary stability. These various protocols need to be further documented in order to assess their predictability.

Clinical Protocols↗
PubMed1995

Rapid and effective detection of mutations in the p53 gene using nonradioactive single-strand conformation polymorphism (SSCP) technique applied on PhastSystem.

The polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method is a powerful tool for the screening of genetic alterations, including single-base substitutions. In the present study, the conventional SSCP technique was modified on the semiautomated electrophoresis system (PhastSystem) for the detection of mutations in the p53 tumor suppressor gene. The SSCP running conditions were optimized for three PCR-amplified DNA fragments, spanning exons 5 through 9 of the p53 gene, using the PCR-products derived from the CaSki and HaCaT cells as the normal and mutant controls, respectively. The optimized SSCP protocols were tested on nine human vulvar and vaginal carcinoma-derived cell lines. The optimizing experiments indicated that the running temperature and gel density can affect significantly the electrophoretic mobility and resolution of single-stranded DNA molecules. Because the gel temperature is the most important parameter affecting the conformation and thus electrophoretic mobility of single strands, one of the most important advantages of the SSCP technique on the PhastSystem is that the running temperature is controlled precisely. In addition to the fast electrophoretic separation, the PhastSystem also offers the use of a silver staining method allowing direct visualization of DNA with high detection sensitivity. Thus, the important advantage of this modified SSCP technique is the short time required for analysis, including electrophoresis and DNA detection. It is concluded that the SSCP method applied on the PhastSystem has the advantages of simplicity, efficiency, speed and reproducibility, and is suitable for clinical diagnostic purposes.

Base Sequence↗
PubMed2005

Modeling and simulation of cancer immunoprevention vaccine.

UNLABELLED: We present an in silico model that simulates the immune system responses to tumor cells in naive and vaccinated mice. We have demonstrated the ability of this model to accurately reproduce the experimental results. MOTIVATION: In vivo experiments on HER-2/neu mice have shown the effectiveness of Triplex vaccine in the protection of mice from mammary carcinoma. Full protection was conferred using chronic (prophylactic) vaccination protocol while therapeutic vaccination was less efficient. Our in silico model was able to closely reproduce the effects of various vaccination protocols. This model is the first step towards the development of in silico experiments searching for optimal vaccination protocols. RESULTS: In silico experiments carried out on two large statistical samples of virtual mice showed very good agreements with in vivo experiments for all experimental vaccination protocols. They also show, as supported by in vivo experiments, that the humoral response is fundamental in controlling the tumor growth and therefore suggest the selection and timing of experiments for measuring the activity of T cells. CONTACT: francesco@dmi.unict.it SUPPLEMENTARY INFORMATION: http://www.dmi.unict.it/CIG/suppdata_bioinf.html.

Animals↗
PubMed2006

Application of the standard photodynamic treatment protocol for symptomatic circumscribed choroidal hemangioma.

PURPOSE: To describe the results of photodynamic therapy (PDT) using a standard protocol, developed for treating choroidal neovascularization (CNV), for the treatment of circumscribed choroidal hemangioma (CCH). METHODS: A prospective, uncontrolled, consecutive case series of patients with symptomatic CCH that were treated using the standard PDT protocol was evaluated periodically with ophthalmic exams and echography. RESULTS: Nine CCH patients were included in the study. Mean tumor height decreased from 2.7 mm before treatment to 0.8 mm at the end of the follow-up period. Mean visual acuity improved from 6/15 to 6/12. Six patients required one PDT session, two patients two sessions, and one patient three sessions. Side effects included transient visual disturbances in two patients. One patient, who concomitantly presented with age-related macular degeneration, developed CNV. CONCLUSIONS: The standard PDT protocol is effective for the treatment of CCH. Complications are uncommon. Further studies should assess the optimal PDT protocol for the treatment of CCH.

Adult↗
PubMed2005

Optimization of a blood pool contrast agent injection protocol for MR angiography.

PURPOSE: To design an ideal first-pass profile for MR angiography (MRA) by optimizing a multiphasic injection protocol based on two experimental animal models. MATERIALS AND METHODS: An equivalent contrast-enhanced (CE) MRA injection protocol was developed with controlled injection modalities (injection rate, volume, and dose) in rabbits and pigs. P792, a blood pool contrast agent, was injected in 17 male New Zealand rabbits and five farm pigs with variable injection schemes (mono- and multiphasic). From the gadolinium (Gd) blood concentration data, a simulation of an MR acquisition was performed to evaluate the impact of such an injection protocol on MR arterial signal and to select the best injection protocol. RESULTS: An empirical relationship between the arterial peak concentration and the injection parameters was found in the rabbits and pigs, allowing precise prediction of the first-pass profile. Of the four injection scheme strategies tested (standard bolus and bi-, tri-, and multiphasic injection protocols), the multiphasic "ramp" injection protocol provided the most optimal contrast agent pharmacokinetics with a durable plateau of concentration. CONCLUSION: Ramp injection protocol provides an optimized first-pass profile for CE-MRA.

Animals↗
PubMedDate not supplied

Comparison of 4 blood storage methods in a protocol for equine pre-operative autologous donation.

OBJECTIVE: To compare viability of equine whole blood stored by 4 different methods, and to establish optimal storage protocols for an equine autologous blood donation program. STUDY DESIGN: In vitro study of stored equine whole blood. Animals- Six healthy adult horses. METHODS: Blood from each horse was collected into 4 different containers: glass bottles containing acid-citrate-dextrose solution (ACD), plastic bags containing ACD, citrate-phosphate-dextrose (CPD), and CPD with supplemental adenine (CPDA-1). Blood was stored for 5 weeks and sampled at 2-day intervals. Standard hematologic and biochemical variables were evaluated, and adenosine-5-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) concentrations were measured and normalized to total hemoglobin content. RESULTS: Plasma hemoglobin, % hemolysis, lactate, potassium, ammonia, and lactate dehydrogenase (LDH) increased, whereas glucose concentration and pH decreased in all stored blood over 5 weeks. There was a temporal increase in hemolysis with all storage methods, but the increase was greatest in glass bottles. Lactate and ammonia were highest in CPD and CPDA-1 samples, indicating more active red blood cell (RBC) metabolism. 2,3-DPG concentrations decreased during storage, but were optimally preserved with CPDA-1. ATP concentrations were significantly higher for blood stored in CPDA-1, and were lowest in glass bottles. CONCLUSIONS: Hematologic and biochemical values measured for blood stored in CPDA-1 are suggestive of improved RBC viability compared with other storage methods. With the exception of ATP, results from stored equine blood were similar to those reported for other species. CLINICAL RELEVANCE: Commercial CPDA-1 bags appear to be the optimal storage method for equine whole blood.

2,3-Diphosphoglycerate↗
PubMed2006

Comparison of the pro-inflammatory potential of monocytes from healthy adults and those with peripheral arterial disease using an in vitro culture model.

We adapted a monocyte:endothelial cell co-culture model to investigate the pro-inflammatory potential of monocytes from patients with peripheral arterial disease (PAD). Isolated monocytes were cultured with human umbilical vein endothelial cells (HUVEC) for 24h, after which the ability of the HUVEC to recruit flowing neutrophils was tested. Development of a usable protocol required comparisons of primary HUVEC with cells that had been passaged and/or frozen and thawed, evaluation of optimal culture media and comparison of monocytes from freshly drawn and stored blood. We found, for instance, that expansion of HUVEC was assisted by inclusion of hydrocortisone, but this agent was withdrawn before the test phase because it reduced responses of HUVEC. Using the optimal practical protocol, we found great variation in the ability of monocytes from different donors to cause neutrophil adhesion. Slightly more ( approximately 20%) monocytes from patients with PAD adhered to HUVEC than monocytes from healthy controls, and the monocytes from PAD patients induced approximately 70% greater subsequent adhesion of neutrophils. Thus, we developed a functional model of inflammatory potential usable in clinically-related studies and found that patients with PAD had circulating monocytes with greater than normal ability to activate endothelial cells.

Cells, Cultured↗
PubMed2004

Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells.

BACKGROUND: Establishing a successful method for testicular stem cell transplantation of frozen-thawed testicular cells would be of immense benefit to boys with childhood cancer undergoing a sterilizing treatment. In this study, we evaluated different cryopreservation protocols in a mouse model by means of testicular germ cell transplantation (TGCT), in order to establish an optimal freezing protocol. METHODS AND RESULTS: In a first series of experiments, we compared an uncontrolled protocol with 1.5 mol/l dimethyl sulphoxide (DMSO) versus a controlled long protocol (cooling to -80 degrees C) and observed a better viability with the latter protocol (36% versus 48%, P < 0.05). We then compared survival after two thawing methods (37 degrees C water versus ice water) in either a DMSO- or an ethylene glycol (EG)-based protocol, and found no difference. In order to evaluate the functional capacity of the cryopreserved testicular suspension, TGCT was performed with both fresh and frozen-thawed suspensions. In 90% of the successfully injected testes, spermatogenesis was reinitiated using fresh suspensions. In contrast, this figure was only 12.5 and 22.7% after cryopreservation, for the short controlled EG protocol and the uncontrolled DMSO protocol, respectively. CONCLUSION: Reinitiation of spermatogenesis is possible after cryopreservation of testicular germ cell suspensions. Although cell survival was acceptable, our results after TGCT show that our protocols need further improvement.

Animals↗
PubMed1995

The use of L5178Y mouse lymphoma cells to assess the mutagenic, clastogenic and aneugenic properties of chemicals.

Guidelines have been proposed to assess the potential of chemicals to affect human health. Written into these guidelines is the requirement that information be submitted on mutagenic activity. Although regulatory agencies accept mutagenicity data from both the hprt and tk loci in mammalian cells, many studies suggest that the L5178Y mouse lymphoma assay at the thymidine kinase locus is likely to detect a greater spectrum of mutagenic lesions. Thus, there is increasing emphasis being placed on this assay in many proposed and published guidelines. The L5178Y mouse lymphoma suspension protocol produces both small and large colonies which are the products of mutants growing at different rates. There is a reduction in the proportion of slowly growing mutants with respect to the total population of cells when expression is carried out in suspension. This potentially leads to quantitatively inaccurate assessments of the mutagenic activity of chemicals. Therefore an in situ procedure was developed that more accurately assesses the mutagenic activity of chemicals by maximizing the detection of small colonies. Many guidelines recommend tests that assess the clastogenic activity of chemicals. Some regulatory agencies accept data from the mouse lymphoma mutation assay to detect clastogens if the protocol is optimized for the detection of small colonies or if colony sizing data are submitted. The conventional suspension assay protocol is not sufficiently validated for this purpose. The in situ protocol has greater potential to meet these requirements.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed2000

Cryopreservation of mouse oocytes using a medium with low sodium content: effect of plunge temperature.

The effect of various combinations of plunge temperature and thawing protocol on the survival and viability of mouse oocytes was examined. The oocytes were frozen either in a standard freezing medium (ETFM, embryo transfer freezing medium) or in a low-sodium, choline-based freezing medium (CJ2), with 1.5 M 1,2-propanediol and 0.1 M sucrose, and using a conventional slow cooling method. The criteria used to assess survival were morphological state after thawing (intact or lysed), ability to become fertilized, and ability to develop to the two-cell, morula, and blastocyst stage in vitro. Oocytes frozen in CJ2 and plunged into liquid nitrogen (LN(2)) from -10, -20, or -33 degrees C remained intact and developed to the blastocyst stage at significantly higher rates than oocytes frozen in ETFM. For oocytes plunged into LN(2) from -33 degrees C, very rapid thawing (10 s in 30 degrees C water) was more detrimental than rapid or slow thawing (holding in air at room temperature for 10 or 30 s, respectively, prior to submersion in water at 30 degrees C for 10 s). By contrast, oocytes plunged into LN(2) from -10 or -20 degrees C survived better when thawing was very rapid or rapid than when thawing was slow. With the current protocol CJ2 was very effective over a wide range of plunge temperatures (-20 to -33 degrees C), although the optimal thawing protocol depended on the particular plunge temperature. Over 90% of oocytes surviving after slow cooling in CJ2 to -33 degrees C could be plunged to -196 degrees C with little or no further damage.

Animals↗
PubMed1992

Interlaboratory comparison of microsomal ethoxyresorufin and pentoxyresorufin O-dealkylation determinations: standardization of assay conditions.

Assay conditions and results of cytochrome P-450 dependent 7-ethoxyresorufin (ER) and 7-pentoxyresorufin (PR) O-dealkylation (OD) by rat liver microsomes were compared by four laboratories in the Netherlands. Microsomal mixtures were prepared from control, 3-methylcholanthrene and phenobarbital pretreated animals, resulting in different levels of cytochrome P-450 isozymes. EROD and PROD activities were determined in each laboratory according to their own protocols. Considerable variability was found both between and within laboratories. Further studies demonstrated that protocol differences are important factors causing this interlaboratory variation. Main factors of influence were buffer type, batch of resorufin used for calibration, substrate solvent and substrate concentration. Based on the results obtained, standardized protocols for optimized measurement of microsomal EROD and PROD activities were developed. Additional experiments demonstrated that the use of these standardized protocols reduced intralaboratory variation in both the EROD and the PROD assay, whereas it also reduced the interlaboratory variability for the PROD determinations. The interlaboratory variation for measurement of microsomal EROD activities was only reduced for the laboratories using a Cobas-Bio analyzer. The results of the present study demonstrate clearly that data obtained with EROD and PROD activity measurements are highly sensitive to factors frequently varying from one laboratory to another. In addition, they demonstrate the necessity to be careful with absolute values presented in the literature for these activities, unless well characterized assay conditions are applied.

Animals↗
PubMed2000

Low-temperature recovery strategies for the isolation of bacteria from ancient permafrost sediments.

Permafrost represents a unique ecosystem that has allowed the prolonged survival of certain bacterial lineages at subzero temperatures. To better understand the permafrost microbial community, it is important to identify isolation protocols that optimize the recovery of genetically diverse bacterial lineages. We have investigated the impact of different low-temperature isolation protocols on recovery of aerobic bacteria from northeast Siberian permafrost of variable geologic origin and frozen for 5000 to 3 million years. Low-nutrient media enhanced the quantitative recovery of bacteria, whereas the isolation of diverse morphotypes was maximized on rich media. Cold enrichments done directly in natural, undisturbed permafrost led not only to recovery of increased numbers of bacteria but also to isolation of genotypes not recovered by means of liquid low-temperature enrichments. On the other hand, direct plating and growth at 4 degrees C also led to recovery of diverse genotypes, some of which were not recovered following enrichment. Strains recovered from different permafrost samples were predominantly oligotrophic and non-spore-forming but were otherwise variable from each other in terms of a number of bacteriological characteristics. Our data suggest that a combination of isolation protocols from different permafrost samples should be used to establish a culture-based survey of the different bacterial lineages in permafrost.

Bacteria↗
PubMed1990

The biophysical profile. A literature review and reassessments of its usefulness in the evaluation of fetal well-being.

Antenatal monitoring studies to assess fetal well-being and to identify the compromised fetus are in widespread use. This literature review analyzes the theoretical basis for fetal monitoring and the clinical research that has defined its utility, and it discusses the variations in protocols and scoring systems for the biophysical profile. While recognizing that there, is as yet, no consensus on the optimal monitoring protocol, the authors espouse one approach in which the cardiac nonstress test and the amniotic fluid volume are the initial tests, with full biophysical profile reserved for abnormal test outcomes. This approach to monitoring should be expeditious without diminishing its predictive value.

Embryonic and Fetal Development↗
PubMed2005

Stem cell transplantation after middle cerebral artery occlusion.

Stem cell lines have been and are being developed to treat damage in the central nervous system after stroke. Stem cells are able to migrate to areas of damage and to differentiate into neurons and glia. Grafts of murine stem cells have been shown to promote recovery from behavioral dysfunction after stroke. We have developed protocols to optimize behavioral testing, animal recovery, and stem cell delivery after middle cerebral artery occlusion. In this chapter we discuss study protocols aimed at integrating in vitro preparation of cells, small animal surgery, behavioral testing batteries, and histological analysis.

Animals↗
PubMed2027

Protocol for Detecting and Sequencing Chikungunya Virus from Field-Collected Mosquitoes.

Arboviral diseases represent a major public health challenge, especially in tropical regions where environmental conditions may favor the proliferation and spread of mosquito vectors. Thus, early and accurate detection of chikungunya virus (CHIKV) in mosquito populations can be a valuable tool for effective surveillance of circulating variants and for identifying new viral introductions. Given the challenges of detecting arboviruses in field-captured mosquitoes, we describe an integrated workflow for CHIKV molecular detection and whole-genome sequencing. This protocol includes mosquito homogenization using a bead-based mechanical disruptor, RNA extraction using TRIzol reagent with minor modifications, molecular screening using CHIKV-specific RT-qPCR, and whole-genome amplification followed by sequencing on Illumina platforms. Despite the protocol being optimized for individual mosquitoes, it results in high-quality RNA suitable for both entomological surveillance and genomic analysis. As this protocol allows recovery of complete CHIKV genomes from mosquito specimens, it can serve as a basis for genomic epidemiology studies, enabling monitoring of viral diversity and lineage dynamics, and facilitating early detection of emerging variants to support timely and targeted public health interventions in endemic and at-risk regions.

Animals↗
PubMed2003

Recombinant expression and purification of Ssa1p (Hsp70) from Saccharomyces cerevisiae using Pichia pastoris.

Heat shock proteins with a molecular mass of 70000 (Hsp70s) are a ubiquitous class of ATP-dependent molecular chaperones involved in the folding of cellular proteins. Sequencing the entire genome of Saccharomyces cerevisiae revealed 14 different genes for Hsp70 proteins in different cellular compartments. Among these 14 Hsp70s, the subclass of Ssa (Ssa1p-Ssa4p) is abundant and essential in the cytosol. Since high yield expression of cytoplasmic Ssa1p is inefficient in Saccharomyces cerevisiae and recombinant expression in E. coli yields low protein levels, we chose Pichia pastoris as the recombinant expression system. In Pichia pastoris, expression levels of Ssa1p are high and Ssa1p is soluble and correctly folded. Also, we present a new protocol for purification of Ssa1p. Previously described purifications include ATP-agarose chromatography leading to Ssa1p partially complexed with ATP. Our optimized purification protocol follows the CiPP strategy (capture, intermediate purification, polishing) avoiding ATP-agarose chromatography, which allows detailed studies on the ATP-dependent Hsp70 functions. We obtained Ssa1p in high purity and 400 times higher quantity compared to previous studies.

Chromatography, Ion Exchange↗