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Results for “protocol optimization”

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PubMed2001

The ACVD task force on canine atopic dermatitis (XXIV): allergen-specific immunotherapy.

Allergen-specific immunotherapy (ASIT) has been used for years to treat dogs with atopic dermatitis (AD) and humans with atopic diseases. The efficacy of ASIT has been well documented for humans with respiratory atopic diseases and stinging insect allergy, but its effectiveness seems more controversial for patients with AD. In spite of insufficient evidence derived from randomized controlled trials, multiple open studies and a large body of clinical observations suggest that ASIT is effective in controlling the clinical signs of dogs with AD. As a result of the scarcity of evidence from controlled trials, the true efficacy of ASIT, and the optimal protocols for allergen dose and frequency of injection are currently unknown. Allergen-specific immunotherapy nevertheless may be included in the treatment of canine AD because of its potential advantages and limited disadvantages compared to other forms of therapy. There is no evidence, however, for the preference of any specific treatment protocol. The predictive value of historical, clinical and immunologic features related to the efficacy of ASIT in dogs with AD are discussed in this paper. Adverse reactions, and the requirements for monitoring of patients receiving ASIT, then are reviewed and detailed. Finally, this review highlights aspects of ASIT where further research and controlled studies are needed.

Allergens↗

PubMed1998

Sequential flow cytometry and fluorescence in situ hybridization for the study of formalin-fixed, paraffin-embedded breast cancer cells.

Both flow cytometry and fluorescence in situ hybridization (FISH) are useful techniques in the analysis of cancer tissues. When the two are used in the study of the same specimens, they are usually performed in parallel, separately. This is problematic where there is a scarcity of material, making completion of both studies impossible. Fluorescence in situ hybridization procedures that will utilize excess material discarded from flow cytometry would be advantageous. The present report describes an optimized protocol for performing sequential flow cytometry and FISH using formalin-fixed paraffin-embedded archival material. Although breast cancer tissues were used in this initial study, the protocol is applicable to other cancer tissues as well.

Breast Neoplasms↗

PubMed1999

Trisomy 8 in embryonal rhabdomyosarcoma detected by fluorescence in situ hybridization.

Embryonal rhabdomyosarcoma is the most common malignant soft-tissue tumor in childhood. Cytogenetic studies of this tumor are rare. In one study trisomy 8 was found to be a primary cytogenetic abnormality. In view of the findings of trisomy 8 in a multitude of cancers, we conducted a pilot study to test the hypothesis that a subset of rhabdomyosarcoma also exists with trisomy 8. Accordingly, archival tissues of 12 cases of rhabdomyosarcoma were retrieved and fluorescence in situ hybridization (FISH) using a chromosome 8-specific, alpha-satellite probe was undertaken on formalin-fixed paraffin-embedded tissue sections using the protocol optimized in the Cytogenetics Laboratory at Rhode Island Hospital. The results obtained demonstrated that 6 of 12 tumors showed chromosome 8 trisomy, when a 15% threshold is adopted. In addition, one case was borderline, with 11% of the cells found positive for three fluorescent signals. Future experiments utilizing additional specimens from our centers as well as from other laboratories are needed to confirm and extend the findings of the present study.

Cell Differentiation↗

PubMed1998

Ligand-binding studies: old beliefs and new strategies.

Ligand-binding studies remain a very popular technique among many experimentalists. As far as equilibrium experiments are concerned, saturation and displacement curves are commonly performed for simplicity, convenience or for the sake of tradition. However, alternative protocols, such as 'mixed'-type protocols or multiligand experiments, are also possible. Indeed, there are cases where kinetic experiments, usually considered a 'second-choice' experiment, might have a superior resolving power compared to equilibrium ones. A combination of equilibrium and kinetic experiments might be a powerful solution to overcome limits and shortcomings of each specific technique and is discussed in this issue by G. Enrico Rovati. Thus, a careful choice of the design, a protocol optimization and a computerized analysis of the data can yield a dramatic improvement in the precision of the parameter estimation over more conventional approaches.

Computer Simulation↗

PubMed2002

A new improved method for the concentration of HIV-1 infective particles.

Improvement of the sensitivity of detection systems for human immunodeficiency virus-1 (HIV-1) has been carried out. One approach to improve the sensitivity is purification and/or concentration of the virus from a specimen. In this study, a method for concentrating HIV-1 using polyethylene glycol (PEG) has been re-evaluated and the optimal protocol for concentrating the virus from low-titer specimens was determined. That is, to obtain a virus pellet, a mixture of equal volumes of a specimen and 20% PEG 20,000 solution in saline is incubated at 4 degrees C for 16 h and then centrifuged at 17860 x g in a microcentrifuge for 20 min. HIV-1 in the pellet could be detectable by HIV-1 p24 antigen capture assay for viral protein, reverse transcriptase (RT) assay for viral enzyme, reverse transcriptase polymerase chain reaction (RT-PCR) assay for viral RNA and a virus infectivity assay.

Cell Line↗

PubMed1997

Amplification and cloning of complete enterovirus genomes by long distance PCR.

A method for amplification and cloning of complete enterovirus cDNA genomes is described. Viral RNA was reverse transcribed using an optimized protocol and a reverse transcriptase with reduced RNase H activity. Amplicons corresponding to complete genomes of 14 prototype strains of group B coxsackieviruses and echoviruses were amplified using oligonucleotide primers derived from the Coxsackievirus B3 genomic sequence of the 5' and 3' ends and a mixture of thermostable DNA polymerases. Coxsackievirus B2 amplicon was then cloned and the terminal sequences of the insert were determined. Lipofection of individual clones resulted in productive. Coxsackievirus B2 infection. The method described makes it possible to obtain large amounts of complete enterovirus cDNAs and simplifies the construction of infectious full-length cDNA clones. Successful amplification of all enterovirus prototype strains tested emphasizes the general use of the method described, which provides a rapid and efficient alternative to traditional cloning strategies.

Animals↗

PubMed2000

Efficient plant regeneration through somatic embryogenesis from callus cultures of Oncidium (Orchidaceae).

An efficient method was established for high frequency somatic embryogenesis and plant regeneration from callus cultures of a hybrid of sympodial orchid (Oncidium 'Gower Ramsey'). Compact and yellow-white embryogenic calli formed from root tips and cut ends of stem and leaf segments on 1/2 MS [11] basal medium supplemented with 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ, 0.1-3 mg/l), 2,4-dichlorophenoxyacetic acid (2,4-D, 3-10 mg/l) and peptone (1 g/l) for 4-7 weeks. Embryogenic callus was maintained by subculture on the same medium for callus induction and proliferated 2-4 times (fresh weight) in 1 month. Initiation of somatic embryogenesis and development up to the protocorm-like-bodies (PLBs) from callus cultures was achieved on hormone-free basal medium. Regenerants were recovered from somatic embryos (SEs) after transfer to the same medium and showed normal development. The optimized protocol required about 12-14 weeks from the initiation of callus to the plantlet formation. Generally, the frequency of embryo formation of root-derived callus was higher than stem- and leaf-derived calli. Combinations of naphthaleneacetic acid (NAA) and TDZ significantly promoted embryo formation from callus cultures. The high-frequency (93.8%) somatic embryogenesis and an average of 29.1 SEs per callus (3x3 mm(2)) was found in root-derived callus on a basal medium supplemented with 0.1 mg/l NAA and 3 mg/l TDZ. Almost all the SEs converted and the plantlets grew well with an almost 100% survival rate when potted in sphagnum moss and acclimatized in the greenhouse.

Journal Article↗

PubMedDate not supplied

Fractures of the frontal sinus: classification and its implications for surgical treatment.

PURPOSE: To propose a clinical classification of fractures of the frontal sinus and discuss the implications on treatment. PATIENTS AND METHODS: One hundred patients with fractures of the frontal sinus seen and operated on in two University hospital units (Leuven and Nijmegen). The majority of the patients were surgically treated according to a protocol based on the proposed classification. Fractures were classified according to their location, extent, involvement of the nasofrontal duct, and injury to the dura. Fragments were reduced and fixed, defects were reconstructed with autologous bone grafts, and the sinus cavity was drained, obliterated, or cranialized. In the latter cases, the sinus mucosa was thoroughly removed before obliteration or cranialization. RESULTS: The follow-up ranged from 6 months to 12 years. There were functional complications in 4% of patients. Minor aesthetic sequelae were noticed in 14% of the patients. CONCLUSION: A detailed classification of fractures of the frontal sinus is proposed. It has helped the authors set up a treatment protocol. Optimally, if used by clinicians, it will contribute to a more standardized method of surgical care of patients with such injuries.

Adolescent↗

PubMed2001

Sampling optimization, at site scale, in contamination monitoring with moss, pine and oak.

With the aim of optimizing protocols for sampling moss, pine and oak for biomonitoring of atmospheric contamination and also for inclusion in an Environmental Specimen Bank, 50 sampling units of each species were collected from the study area for individual analysis. Levels of Ca, Cu, Fe, Hg, Ni, and Zn in the plants were determined and the distributions of the concentrations studied. In moss samples, the concentrations of Cu, Ni and Zn, considered to be trace pollutants in this species, showed highly variable long-normal distributions; in pine and oak samples only Ni concentrations were log-normally distributed. In addition to analytical error, the two main source of error found to be associated with making a collective sample were: (1) not carrying out measurements on individual sampling units; and (2) the number of sampling units collected and the corresponding sources of variation (microspatial, age and interindividual). We recommend that a minimum of 30 sampling units are collected when contamination is suspected.

Air Pollutants↗

PubMed2003

Radiation biodosimetry: applications for spaceflight.

The multiparametric dosimetry system that we are developing for medical radiological defense applications could be adapted for spaceflight environments. The system complements the internationally accepted personnel dosimeters and cytogenetic analysis of chromosome aberrations, considered the best means of documenting radiation doses for health records. Our system consists of a portable hematology analyzer, molecular biodosimetry using nucleic acid and antigen-based diagnostic equipment, and a dose assessment management software application. A dry-capillary tube reagent-based centrifuge blood cell counter (QBC Autoread Plus, Becton [correction of Beckon] Dickinson Bioscience) measures peripheral blood lymphocytes and monocytes, which could determine radiation dose based on the kinetics of blood cell depletion. Molecular biomarkers for ionizing radiation exposure (gene expression changes, blood proteins) can be measured in real time using such diagnostic detection technologies as miniaturized nucleic acid sequences and antigen-based biosensors, but they require validation of dose-dependent targets and development of optimized protocols and analysis systems. The Biodosimetry Assessment Tool, a software application, calculates radiation dose based on a patient's physical signs and symptoms and blood cell count analysis. It also annotates location of personnel dosimeters, displays a summary of a patient's dosimetric information to healthcare professionals, and archives the data for further use. These radiation assessment diagnostic technologies can have dual-use applications supporting general medical-related care.

Animals↗

PubMed1998

Disposition and tissue distribution of boron after infusion of borocaptate sodium in patients with malignant brain tumors.

PURPOSE: In the frame of the Czech boron neutron capture therapy (BNCT) project, a clinical Phase I study of borocaptate sodium [Na2B12H11SH (BSH)] as the boron-10 delivery agent was performed to obtain data on disposition and tissue distribution of boron after an infusion of this compound, as well as to establish an optimal protocol for BNCT of malignant cerebral tumors. METHODS AND MATERIALS: The kinetics of boron disposition after an infusion of borocaptate sodium (25 mg/kg body wt over the period of 1 h) was studied in a group of 10 patients with astrocytoma or glioblastoma of cerebral hemispheres using a modification of the Soloway-Messer colorimetric method. The boron content of tissues (tumor, healthy brain, dura mater, muscle, skin, and cranial bone) removed during the operation performed with latencies varying between 3 and 18 h was investigated by atomic emission spectrometry. RESULTS: Compartmental analysis of boron blood concentrations has shown that in the majority of patients (four males and three females), the concentration decline can be adequately described by a two-compartment pharmacokinetic model (i.e., by a biexponential relationship). The calculated half-lives of the initial (fast) phase of the concentration decline varied between 0.85 and 3.65 h, whereas the half-life values for the terminal (slow) phase ranged between 22.2 and 111.8 h. However, in the remaining three patients (all females), the goodness of fit of the boron concentration data was significantly better when a pharmacokinetic model with three compartments was assumed. In these patients, therefore, an additional ultrafast phase with a half-life varying between 17 and 37 min was detected in the beginning of the boron blood concentration decline. On the other hand, in one of these patients, the half-life of the terminal phase was found to be 415 h (i.e., more than 17 days). Such a long persistence in the body is explained by the very high value of the total distribution volume, indicating extensive binding of BSH in peripheral tissues. Another reason may be enterohepatic recycling of BSH. CONCLUSION: Tumor-to-blood ratios higher than 1.5, which are necessary for an effective outcome of BNCT, can be obtained only if the time interval elapsing between the onset of surgery and termination of BSH infusion is at least 12 h.

Adult↗

PubMed1998

How to better define the characteristics of dispersion of results in liquid chromatographic analyses through an interlaboratory study. Example of collaborative studies on ketoprofen and spiramycin.

The aim of this study was to use statistical tools, especially the analysis of variance (ANOVA), to improve knowledge of the characteristics of the dispersion of results in high-performance liquid chromatography (HPLC) methods for quantitative analysis. It is in this regard that two interlaboratory studies have been carried out in collaboration with Rhône-Poulenc Rorer. The first concerned the analysis of a single drug product (ketoprofen) and was typically a "simple analysis". The second one involved a complex mixture of drug products and related substances (spiramycin), requiring far more constraining analysis conditions. Preliminary studies of the analyses were carried out to develop an optimized protocol. Statistical exploitation of the data for ketoprofen showed that there was no significant influence of the factors "laboratory" and "preparation", under the conditions of the study. On the other hand, in the case of spiramycin, a significant influence of the factors "laboratory" and "preparation" was observed under the conditions of the collaborative study, indicating that the latter factor must be taken into account to establish certified assays. Results of these two studies will help to determine the factors that have a significant influence, depending on the product and the chromatographic method used. By completing the statistical data base, interlaboratory studies will also contribute in the near future to the elaboration of more rigorous protocols for analytical transfers.

Chromatography, High Pressure Liquid↗

PubMed2000

Long-circulating gadolinium-loaded liposomes: potential use for magnetic resonance imaging of the blood pool.

In our previous paper, we reported a method of liposome loading with Gadolinium (Gd) via so called polychelating amphiphilic polymer (PAP). A novel Gd-containing polymeric probe, suitable for the incorporation into the liposomal membrane, was prepared from a low-molecular-weight DTPA-polylysine by linking its N-terminus to a lipid anchor, NGPE-PE. When compared with known membranotropic MR probes, such as Gd-DTPA-SA and Gd-DTPA-PE, liposomes containing new membrane-bound polychelator possess enhanced relaxivity for water protons resulting in an increase of tissue signal intensity on MR images. In this study, we developed the optimized protocol to prepare a liposomal MR contrast agent with high relaxivity and narrow size distribution. Gd-containing liposomes were additionally modified with PEG to provide longevity in vivo. We also demonstrated that upon intravenous administration in rabbit and dog, the new preparation causes a prolonged decrease in the blood T(1) value (reflecting the proton relaxation rate in the blood) and may be considered as a potential contrast agent for MRI of the blood pool.

Journal Article↗

PubMed2002

Bioconversion of eucalyptus bark waste into soil conditioner.

An optimized protocol for the bioconversion of eucalyptus bark was devised. It comprised: (i) mechanical reduction in bark size to 0.5-3.0 cm, (ii) moistening to 60-65%, (iii) fortification with ligninase-rich fungus Volvariella sp. (S-1) and 2% urea and (iv) maintenance of this composting mix under aerobic and ambient condition for 14-15 weeks. The resulting bark soil conditioner (BSC) was an easily crumbling, reddish brown biomass, with physico-chemical and microbial properties which would enrich soil fertility/productivity.

Bacteria↗

PubMed1998

Preparation of 211At-labeled humanized anti-Tac using 211At produced in disposable internal and external bismuth targets.

These studies describe the production and purification of 211At as well as the procedure for labeling humanized anti-Tac, the antibody to the alpha-chain of the IL-2 receptor (IL-2R alpha), which has been shown to be a useful target for immunotherapy. The optimized protocol combines the advantages of the two-stage dry distillation procedure with the astatination of trialkylstannyl substances as labeling compounds for proteins. The 211At was produced by bombarding either an external or a recently developed disposable internal bismuth target with alpha-particles from a Cyclotron Corporation CS-30 cyclotron. The 211At was found to contain less than 0.01% 210At. The production rate for the external target was 0.15 mCi +/- 0.056 microA(-1) h(-1) (n = 9) (5.55 MBq mcroA[-1] h[-1]). The production rate for the internal target was 0.44 +/- 0.14 mCi microA(-1) h(-1) (n = 16) (16.28 MBq mcroA[-1] h[-1]).

Astatine↗

PubMed2003

Genome-wide RNAi screening in Caenorhabditis elegans.

In Caenorhabditis elegans, introduction of double-stranded RNA (dsRNA) results in the specific inactivation of an endogenous gene with corresponding sequence; this technique is known as RNA interference (RNAi). It has previously been shown that RNAi can be performed by direct microinjection of dsRNA into adult hermaphrodite worms, by soaking worms in a solution of dsRNA, or by feeding worms Escherichia coli expressing target-gene dsRNA. We have developed a simple optimized protocol exploiting this third mode of dsRNA introduction, RNAi by feeding, which allows rapid and effective analysis of gene function in C. elegans. Furthermore, we have constructed a library of bacterial strains corresponding to roughly 86% of the estimated 19,000 predicted genes in C. elegans, and we have used it to perform genome-wide analyses of gene function. This library is publicly available, reusable resource allowing for rapid large-scale RNAi experiments. We have used this library to perform genome-wide analyses of gene function in C. elegans. Here, we describe the protocols used for bacterial library construction and for high-throughput screening in C. elegans using RNAi by feeding.

Animals↗

PubMed2003

Overexpression and purification of isotopically labeled Escherichia coli MutH for NMR studies.

MutH is one of the enzymes involved in the methyl directed -GATC-based DNA repair system. We report a significantly optimized protocol to prepare isotopically (15N and/or 13C) labeled MutH in minimal medium with high yields for NMR studies. Under the various conditions that we have standardized for the affinity purification of His(6) MutH, the yield of the purified MutH has been estimated to be 35-40 mg of protein from 1liter of M9 minimal media. The yield, thus, obtained by this method is significantly higher than those of previously reported methods. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectroscopy analysis revealed that the protein was pure and existed essentially in a monomeric form. Uniformly 15N-labeled protein, thus, produced has been characterized by recording a sensitivity enhanced 2D [15N]-[1H] HSQC spectrum. The dispersion seen in 15N-1H cross-peaks indicates the presence of a well-ordered structure for MutH and proper folding of the purified protein. The spectrum confirms further the existence of MutH as a monomer.

Amino Acid Sequence↗

PubMed1999

Cyclosporine inhibits long-term survival in cardiac allografts treated with monoclonal antibody against CD45RB.

BACKGROUND: We have previously reported that a monoclonal antibody to CD45RB is a novel immunosuppressive agent; however, the optimal regimen in cardiac allografts remains unknown. The present study was undertaken to determine the optimal protocol of this therapy and its interaction with cyclosporine. METHODS: A heterotopic heart allograft model was used in C57BL/6 to BALB/c mice. The following studies were conducted: 1) dose response study (low, intermediate, and high doses at 1, 3, and 9 mg/kg/day respectively), 2) short course (2 days) therapy vs. long course (9 days) therapy, 3) pretreatment (starting on day -1) vs no pretreatment, 4) daily therapy vs. alternative day therapy, and 5) monoclonal antibody treatment with and without cyclosporine. RESULTS: The efficacy of the CD45RB monoclonal antibody was dose and duration dependent (p<0.01). Pretreatment significantly improved the efficacy of this therapy (74.5+/-13.4 days vs. 30.6+/-1.5 days, p<0.01). Daily therapy was superior to alternate day therapy (74.5+/-13.4 days vs. 30.4+/-1.5 days, p<0.03). Interestingly, we found that administration of cyclosporine prior to, at the same time as, or after administration of the CD45RB monoclonal antibody had a detrimental effect on graft survival compared to mAb treated alone (16.6+/-0.4 days, 25+/-2.3 days, and 35.3+/-0.9 days respectively vs. 74.5 days, p<0.01). CONCLUSIONS: Immunosuppression with CD45RB monoclonal antibody is dose and duration dependent. Pretreatment and daily therapy improves results. Addition of cyclosporine inhibits long-term graft survival achieved by the monoclonal antibody alone.

Animals↗