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PubMed1995

Re-engineering drug development: integrating pharmacoeconomic research into the drug development process.

Pharmacoeconomic research will be an increasingly important aspect of drug development as providers, third-party payers, and worldwide government health agencies use cost-effectiveness and quality-of-life data to assist in making decisions on optimal pharmaceutical treatment protocols, formulary listings, and reimbursement. It is in the best interest of pharmaceutical companies to have an established, well-integrated pharmacoeconomic research program that can respond to the dynamic health-care environment and proactively plan a program to optimize patient care. The new paradigm for pharmacoeconomic research will require establishment and successful management of many internal and external customer relationships. This article discusses one company's organization of these relationships and how they are integrated into the drug development process during each stage of the product life cycle.

Drug Evaluation↗
PubMed2006

Optimization of a flow cytometry-based protocol for detection and phenotypic characterization of multipotent mesenchymal stromal cells from human bone marrow.

BACKGROUND: To study the biology of rare bone marrow (BM) multipotent mesenchymal stromal cells (MSCs), recognized protocols are needed. Colony-forming unit-fibroblast (CFU-F) assays have historically been used for the enumeration of MSCs. However, the need to isolate and further analyze MSCs requires new strategies based on cell surface markers. The purpose of this work was to verify the phenotype of BM MSCs in vivo and to develop flow cytometry-based methods for their evaluation. METHODS: Pre-enrichment with D7-FIB-conjugated microbeads, cell sorting for CD45low D7-FIB+ LNGFR+ cells, and CFU-F assay were used to confirm the phenotype of BM MSCs in vivo. Further phenotypic characterization of MSCs was performed using three-color flow cytometry following pre-enrichment or by direct four-color flow cytometry. The sensitivity of direct flow cytometry/rare event analysis for the accurate enumeration of MSCs was validated using 85 samples from patients with neoplastic BM diseases. RESULTS: In normal BM, a significant correlation was found between the frequencies of CFU-Fs and CD45low D7-FIB+ LNGFR+ cells (n = 19, R = 0.719, P = 0.001). Following cell sorting, 15% of these cells were clonogenic. The same cells were enriched using LNGFR-based positive selection, CD45/Glycophorin A-based depletion, or plastic adherence. CD45low D7-FIB+ LNGFR+ cells expressed classic makers of cultured MSCs CD73/SH3 and CD105/SH2 and markers of stromal reticular cells CD106/VCAM and alkaline phosphatase. Novel markers were identified including leukemia inhibitory factor receptor and gp130. CD45low D7-FIB+ LNGFR+ cells were increased fourfold in the floating fat fraction of normal BM aspirates. Their frequency was decreased in chronic lymphocytic leukemia (threefold, n = 13, P = 0.049) and chronic myelogenous leukemia (ninefold, n = 11, P = 0.001) compared with that in age-matched controls (n = 26 and n = 31, respectively). CONCLUSIONS: This study demonstrates the usefulness of flow cytometry-based methods for the detection, enumeration and further phenotypic analysis of BM MSCs. These findings have broad applications for the future evaluation of BM MSCs in health and disease.

Adolescent↗
PubMed2001

TO-PRO-3 is an optimal fluorescent dye for nuclear counterstaining in dual-colour FISH on paraffin sections.

Confocal laser scanning microscopy (CLSM) is an extensive but reliable tool for assessing the hybridisation signals in fluorescence in situ hybridisation (FISH). Most CLSMs are equipped with an argon-laser and a helium/neon-laser illumination system with excitation wavelengths of 488, 543 and 633 nm. A protocol for an optimal nuclear counterstaining in combination with dual-colour FISH for these laser illumination systems has not been established so far. Here, we determined the suitability of eleven dimeric and monomeric cyanine nucleic acid stains on paraffin sections of breast carcinoma specimens in combination with dual-colour FISH (Her-2/neu and centromere 17) for CLSM application. Strong staining of cell nuclei was observed for TO-PRO-3 and YO-PRO-3, YOYO-1 and propidium iodide (PI), but only TO-PRO-3 showed specific staining of nuclei without any staining of the cytoplasm. A specific emission in exclusively one distinct fluorescence channel was shown for TO-PRO-3 (633 nm excitation) as well as YOYO-1, BO-PRO-1 and Sytox Green (488 nm excitation), evaluated by a CLSM and confirmed by 3-D fluorescence spectra. High stability of fluorescence intensity was shown for the far-red dyes TO-PRO-3, YO-PRO-3, YOYO-3 and Syto-59 as well as YOYO-1 and PI. Only TO-PRO-3 was due to its high specificity and stability suitable for detection of an amplification of the Her-2/neu gene by dual-colour FISH and CLSM evaluation.

Breast Neoplasms↗
PubMed2003

Analysis and optimization of structure-based virtual screening protocols (1): exploration of ligand conformational sampling techniques.

Ligand conformational flexibility has long been recognized as an important issue in virtual screening (VS). To this end, a number of different methodologies have been adapted to tackle the problem. Many of said techniques were originally designed for ligand derived pharmacophore screens, but have subsequently been fashioned for application within structure-based virtual screening (SVS). A popular adaptation is the pre-calculation of diverse ligand conformations for subsequent docking in target active sites. In this paper, we study a number of the software programs currently being used in conformer generation, analyzing their ability to regenerate known ligand binding conformations. The implications of these studies are discussed, from the perspective of VS in general and SVS in particular.

Computational Biology↗
PubMed2003

Analysis and optimization of structure-based virtual screening protocols. 2. Examination of docked ligand orientation sampling methodology: mapping a pharmacophore for success.

An important element of any structure-based virtual screening (SVS) technique is the method used to orient the ligands in the target active site. This has been a somewhat overlooked issue in recent SVS validation studies, with the assumption being made that the performance of an algorithm for a given set of orientation sampling settings will be representative for the general behavior of said technique. Here, we analyze five different SVS targets using a variety of sampling paradigms within the DOCK, GOLD and PROMETHEUS programs over a data set of approximately 10,000 noise compounds, combined with data sets containing multiple active compounds. These sets have been broken down by chemotype, with chemotype hit rate used to provide a measure of enrichment with a potentially improved relevance to real world SVS experiments. The variability in enrichment results produced by different sampling paradigms is illustrated, as is the utility of using pharmacophores to constrain sampling to regions that reflect known structural biology. The difference in results when comparing chemotype with compound hit rates is also highlighted.

Binding Sites↗
PubMed1997

Implementation of technetium-99m MIBI SPECT imaging guidelines: optimizing the two day stress-rest protocol.

OBJECTIVES: In a previous study in 460 patients, we found that in patients with suspected or known coronary artery disease undergoing stress-rest technetium-99m sestamibi (MIBI) SPECT myocardial perfusion imaging, rest SPECT imaging could be withhold in approximately 20% of patients because of a completely normal stress study. The present study was set up to evaluate the consequences of the implementation of this finding in a subsequent population of patients, and to set standards for the variety of protocols now used for MIBI SPECT imaging. METHODS: Within a period of 4 months, 235 consecutive patients referred for MIBI SPECT scintigraphy were studied. All patients had stable cardiac chest pain and underwent symptom-limited exercise MIBI SPECT perfusion imaging. The stress SPECT images were reconstructed and evaluated immediately after acquisition of the images. In case of a clearly normal stress SPECT study, rest imaging was cancelled. RESULTS: Twenty-six of 235 patients (11%) had a completely normal stress MIBI SPECT study and the rest SPECT imaging procedure could be subsequently cancelled. In 20 patients (9%) the stress SPECT was inconclusive, and in 189 (80%) of patients stress imaging was clearly abnormal. In the first month of the study, the nuclear medicine physicians and cardiologists would interprete only 6% of the stress images as normal, while this number increased to 13% after 9 weeks, with a mean of 11% for the whole investigation period of 4 months. CONCLUSION: In patients undergoing stress MIBI SPECT imaging, it was found justified to cancel rest MIBI SPECT imaging in at least 11% of patients because of a completely normal stress SPECT. As 9% of the images were inconclusive, the number of normal stress images could theoretically increase to 20% if reliable measures are taken to improve reading accuracy. This number is in close agreement with the number of normal stress studies previously reported by our institution and would lead to a considerable reduction of radiation dose, costs, and increased convenience for an important subset of patients.

Adolescent↗
PubMed2002

Optimizing antibiotics in residents of nursing homes: protocol of a randomized trial.

BACKGROUND: Antibiotics are frequently prescribed for older adults who reside in long-term care facilities. A substantial proportion of antibiotic use in this setting is inappropriate. Antibiotics are often prescribed for asymptomatic bacteriuria, a condition for which randomized trials of antibiotic therapy indicate no benefit and in fact harm. This proposal describes a randomized trial of diagnostic and therapeutic algorithms to reduce the use of antibiotics in residents of long-term care facilities. METHODS: In this on-going study, 22 nursing homes have been randomized to either use of algorithms (11 nursing homes) or to usual practise (11 nursing homes). The algorithms describe signs and symptoms for which it would be appropriate to send urine cultures or to prescribe antibiotics. The algorithms are introduced by inservicing nursing staff and by conducting one-on-one sessions for physicians using case-scenarios. The primary outcome of the study is courses of antibiotics per 1000 resident days. Secondary outcomes include urine cultures sent and antibiotic courses for urinary indications. Focus groups and semi-structured interviews with key informants will be used to assess the process of implementation and to identify key factors for sustainability.

Aged↗
PubMed1995

[Development and use of quality control program in magnetic resonance].

In 1992, two identical 0.5 Magnetic Resonance units were installed in Policlinico S. Orsola-Malpighi in Bologna. A Quality Assurance (QA) protocol was designed by the Medical Physics Department to monitor both systems as of Acceptance Test. Our main goals in drawing up the QA protocol were: 1) Completeness--to check all the most significant physical parameters; 2) Efficiency--to reduce examination time of the QC Protocol; 3) Reliability--to achieve good repeatability of results. The QA protocol consists of two QC programs: 1) Daily check of Variable Echo image SNR of a homogeneous phantom, Eddy Current compensation, laser printer test; 2) Monthly check of SNR of Spin Echo and Fast Scan images, integral uniformity, B0 uniformity, B1 uniformity, T2 stability, ghosts, slice thickness, slice profile, geometrical distortion, resolution power. SNR dependence on FOV, NEX, matrix and slice thickness, and resolution dependence on slice thickness and position were tested as Acceptance test. The daily checks provide continual monitoring of the performance of both systems and laser printer and have shown: 1) strong fluctuations in image reproduction probably due to film emulsion instability; 2) a "warning" of imminent malfunction. The monthly checks were in line with acceptance test data and have shown: 1) different behavior of the two systems that should perform analogously; 2) greater result stability in one system with better results also in terms of diagnostic images. The main aim of our QA protocol is to optimize diagnostic accuracy by checking several physical parameters that act as good "indicators" for possible malfunctioning. We believe this can be done with simple but useful daily QC supporting a routine more complex QC program and can be achieved through continual application of both protocols.

Magnetic Resonance Imaging↗
PubMed2002

Assignment strategy for proteins with known structure.

In protein NMR the assignment of nuclear spin resonances is a prerequisite for all subsequent applications, such as studies of ligand binding, protein-DNA interactions, and dynamics. Resonance assignment is a time consuming step even when the 3D x-ray structure of the protein is available. A new strategy is presented to solve the "inverse" assignment problem, which is the determination of the NMR resonance assignment from a known 3D protein structure. The protocol employs NMR data in the form of residual dipolar couplings and chemical shifts, while it does not require any sequential NMR connectivity information. The assignment problem is mathematically formulated in terms of a weighted matching problem that can be computationally efficiently solved by a combinatorial optimization algorithm. The protocol is applied to ubiquitin using two or three residual dipolar couplings per amino acid measured in Pfl phage medium together with chemical shift information. The algorithm yields for more than 90% of the protein backbone resonances the correct assignment.

Algorithms↗
PubMed2005

Combination effects of irradiation and irinotecan on cervical squamous cell carcinoma cells in vitro.

Irinotecan HCl (CPT-11) has frequently been used in chemotherapy or concurrent chemoradiotherapy for patients with advanced cervical cancer, although an effective protocol for chemoradiotherapy with CPT-11 has not yet been established. Using the radiosensitive human cervical squamous cell carcinoma cell line ME180 and SN38, a major active metabolite of CPT-11, both the SN38 effects on radiosensitivity and irradiation effects on SN38 sensitivity were investigated to optimize the chemoradiotherapy protocol for CPT-11. SN38 had no effect on radiosensitivity, and irradiation did not affect SN38 sensitivity. Moreover, 3 of 4 post-irradiation surviving subclones obtained from repeatedly irradiated ME180 cells showed no significant changes in their SN38 sensitivities compared with the non-irradiated parent cells. On the other hand, all 7 SN38-resistant subclones established from ME180 cells showed strong reduction in their radiosensitivities. These results suggest that CPT-11 should be administered to advanced cervical cancer patients after, but not before, standard radiotherapy, and concurrent administration of SN38 with radiotherapy should be avoided in order to prevent severe adverse effects, such as watery diarrhea, which is the main adverse effect of pelvic radiotherapy or CPT-11.

Antineoplastic Agents, Phytogenic↗
PubMed2006

Fluorescence quencher improves SCANSYSTEM for rapid bacterial detection.

BACKGROUND AND OBJECTIVES: The optimized scansystem could detect contaminated platelet products within 24 h. However, the system's sensitivity was reduced by a high fluorescence background even in sterile samples, which led to the necessity of a well-trained staff for confirmation of microscope results. MATERIALS AND METHODS: A new protocol of the optimized scansystem with the addition of a fluorescence quencher was evaluated. Pool platelet concentrates contaminated with five transfusion-relevant bacterial strains were tested in a blind study. RESULTS: In conjunction with new analysis software, the new quenching dye was able to reduce significantly unspecific background fluorescence. Sensitivity was best for Bacillus cereus and Escherichia coli (3 CFU/ml). DISCUSSION: The application of a fluorescence quencher enables automated discrimination of positive and negative test results in 60% of all analysed samples.

Bacteriological Techniques↗
PubMed2004

[Prevention of HIV mother to child transmission in Cotonou: child follow-up].

UNLABELLED: The aim of this survey was to analyse the constraints of implementing on a larger scale the protocol of prevention of HIV/AIDS from mother to child by Nevirapine intake. This survey takes stock of the components of the children medical care follow-up. PATIENTS AND METHODOLOGY: Here is a straight line survey on 231 newborns from 222 screened mothers during pregnancy in 36 Health Centres in Cotonou and suburbia. Nevirapine has been administered in a single dose of 200 mg to mothers during labour and a dosage of 7 mg to the infant at birth. These infants have been steadily followed-up from 13 June 2000 to 30 April 2002. Exclusively feeding either on artificial milk or breastfeeding and a premature weaning at four months of age have been recommended. PCR screening has been conducted at one and six months of age or one month after the effective weaning. Cotrimoxazole has been systematically administered at six weeks. RESULTS: HIV/AIDS sero prevalence was globally evaluated at 4% in the screened pregnant women. One hundred and ninety seven mothers and children have effectively benefited from nevirapine intakes and this protocol has been adequately observed in 51% cases. The medical check-up rate was 18.5%. The average age of children under medical care was 10.8 months at the end of the study. The mother to child transmission rate was globally estimated at 20.4% and 7% in an optimal situation (the protocol was observed and the rate of CD4 above 500). In case of breastfeeding exclusively followed by a premature weaning in most cases, the average age of effective weaning was 8.3 months. Twenty-three children died during medical care follow-up basically from dehydration brought about by diarrhea and vomiting during weaning period. CONCLUSION: The survey confirms the potency of nevirapine in preventing HIV transmission from mother to child in non immunosuppressed women and lays emphasis on real problems for which appropriate solutions should be found before the implementation on a larger scale of this protocol.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Conventional cytogenetics in myeloma.

Chromosome analysis has become an important diagnostic tool in the assessment of patients with multiple myeloma. Conventional cytogenetic analysis of myeloma cells is complicated by the difficulty in inducing myeloma cells to divide. A method for the culture and harvest of bone marrow samples from patients with myeloma is presented together with a protocol for producing G-banded metaphases for microscopic analysis. It is recommended that two or more cultures be established from each sample. There is extensive literature describing the use of various cocktails of mitogens to assist in obtaining dividing myeloma cells with little consensus as to the optimal method. A protocol is given for stimulating cells to divide using interleukin-4, the method in routine use in the Victorian Cancer Cytogenetics Service.

Bone Marrow Cells↗
PubMed2002

Growth factors, cytokines and dendritic cell development.

Dendritic cells (DC) initiate tumor specific immune responses in animal studies and initial human trials suggest that certain tumor-antigen loaded DC preparations generate clinical responses. DC may be obtained from blood or generated in vitro from precursor cells. In vitro generation of DC from precursor cells, under the influence of cytokines, has been favoured to date as a source because of the greater numbers of DC produced. However, the different cytokine combinations and serum or plasma component(s) used, differentiate precursor cells into DC with different physiological properties and ultimate immunogenicity. Thus, the quality of in vitro cytokine derived DC may have a profound influence on clinical outcomes. The administration of certain growth factors, which increase the number of circulating blood DC, may provide an alternative source of DC for use in clinical trials. Although clinical trials in prostate cancer, melanoma and metastatic renal carcinoma patients are encouraging, some data suggest certain DC preparations and administration protocols are sub optimal, even potentially tumor enhancing. As basic scientific studies establish how to provide DC with stable phenotype, resistance to tumour inhibitory factors and high migratory capacity, the technology for producing cytokine derived DC in vitro using Good Manufacturing Practise (GMP) conditions needs to be developed. Future DC vaccination protocols will require careful control of the DC used for tumor-antigen loading and repetitive long term DC vaccination may be necessary to maintain effective anti-tumor immune responses.

Animals↗
PubMed1994

Protocols for in vivo measurement of the ion transport defects in cystic fibrosis nasal epithelium.

New treatments for cystic fibrosis (CF), including gene therapy, are currently being assessed. These aim to correct the basic defects of increased sodium absorption and decreased chloride secretion in airway epithelia. Assessment of these bioelectric parameters, particularly in the nasal epithelium, is likely to be used as a measure of treatment efficacy. However, the optimal in vivo protocol to discriminate cystic fibrosis from non-cystic fibrosis subjects is unclear. We have, therefore, compared three protocols for measurement of the cystic fibrosis ion transport defects in vivo in the nasal epithelium. Sodium absorption was measured using both the baseline potential difference and the response to the sodium channel blocker, amiloride. Chloride secretion was assessed in the presence of amiloride, using perfusion with isoprenaline, or terbutaline, or a low chloride solution followed by isoprenaline. Baseline potential difference (PD) and the absolute response to amiloride clearly differentiated the increased sodium absorption in the cystic fibrosis subjects. The responses both to terbutaline (delta PD: non-CF: -0.8 (SEM 0.7) mV; CF: -3.6 (0.5) mV) and isoprenaline (non-CF: 1.5 (0.6) mV; CF: -2.9 (0.6) mV) differentiated the two groups of subjects, but there was considerable overlap of values. Perfusion with a low chloride solution (non-CF: 12.6 (1.2) mV; CF: 0.6 (0.4) mV), as well as subsequent perfusion with isoprenaline (non-CF: 10.0 (1.1) mV; CF: -1.4 (0.4) mV) allowed clear separation of the two groups, with no overlap of values. Some CF subjects showed a transient hyperpolarization to these stimuli, which could clearly be differentiated from the sustained responses seen in non-cystic fibrosis subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

PCR detection of group A bovine rotaviruses in feces.

A polymerase chain reaction (PCR) protocol has been developed for identification of bovine group A rotavirus infection in feces. Primers (20mers) complementary to 3' ends of double-stranded RNA genome segment 6 of bovine rotavirus NCDV strain were synthesized and used in PCR. Bovine rotavirus RNA from infected cell culture was employed to optimize the PCR protocol. Rotavirus-negative fecal samples were spiked with known quantities of bovine rotavirus, and the sensitivity of the PCR assay was determined. Fecal samples were extracted with phenol and treated to eliminate unidentified PCR inhibitor(s) in feces, and PCR was performed. PCR products were either visualized on ethidium bromide-stained agarose gels or detected by chemiluminescent hybridization. The sensitivity of the assay was 6 x 10(4) viral particles/ml of feces with ethidium bromide-stained agarose gel visualization or 6 x 10(2) viral particles/ml of feces with chemiluminescent hybridization. The PCR assay was applied to 18 fecal specimens from clinical cases. All 16 clinical samples that were positive for rotavirus by enzyme-linked immunosorbent assay (ELISA) or by ELISA and electron microscopy (EM) were positive by PCR. The 2 samples that were rotavirus negative by ELISA or by ELISA and EM were also negative on PCR analysis.

Animals↗
PubMed2001

Optimization of the isotope-coded affinity tag-labeling procedure for quantitative proteome analysis.

The combination of isotope coded affinity tag (ICAT) reagents and tandem mass spectrometry constitutes a new method for quantitative proteomics. It involves the site-specific, covalent labeling of proteins with isotopically normal or heavy ICAT reagents, proteolysis of the combined, labeled protein mixture, followed by the isolation and mass spectrometric analysis of the labeled peptides. The method critically depends on labeling protocols that are specific, quantitative, general, robust, and reproducible. Here we describe the systematic evaluation of important parameters of the labeling protocol and describe optimized labeling conditions. The tested factors include the ICAT reagent concentration, the influence of the protein, SDS, and urea concentrations on the labeling reaction, and the reaction time. We demonstrate that using the optimized conditions specific and quantitative labeling was achieved on standard proteins as well as in complex protein mixtures such as a yeast cell lysate.

Affinity Labels↗
PubMed2003

Kinetic analysis of interactions between bispecific monoclonal antibodies and immobilized antigens using a resonant mirror biosensor.

A resonant mirror biosensor (IAsys) protocol is described for the comparative kinetic analysis of the ability of monoclonal antibodies (Mabs) and bispecific antibodies (Babs) to bind immobilized antigens. The protocol has been optimized and validated using the panel of affinity-purified antibodies, including two parental Mabs, one specific to human immunoglobulin G (hIgG) and another specific to horseradish peroxidase (HRP), and a Bab derived thereof by cell fusion (anti-hIgG/HRP Bab). The real-time kinetic analysis of antigen-antibody interactions using this protocol allows to demonstrate the differences in the avidity of bivalently binding Mabs and monovalent Babs. As shown in our previous study [J. Immunol. Methods 261 (2002) 103], the observed equilibrium association constants (Kass) determined by IAsys using this protocol yield figures almost overlapping with those obtained by solid-phase radioimmunoassay (RIA). The described protocol is suited for the investigation of the effects of valency on the binding properties of antibodies. It also may be applied for the selection of Mabs and Babs with desired features, for different fields of application.

Animals↗