Use Of Different Protocols Research Articles

An affordable method to obtain cultured endothelial cells from peripheral blood

The culture of endothelial progenitor cells (EPC) provides an excellent tool to research on EPC biology and vascular regeneration and vasculogenesis. The use of different protocols to obtain EPC cultures makes it difficult to obtain comparable results in different groups. This work offers a systematic comparison of the main variables of most commonly used protocols for EPC isolation, culture and functional evaluation. Peripheral blood samples from healthy individuals were recovered and mononuclear cells were cultured. Different recovery and culture conditions were tested: blood volume, blood anticoagulant, coating matrix and percentage of foetal bovine serum (FBS) in culture media. The success of culture procedure, first colonies of endothelial cells appearance time, correlation with number of circulating EPC (cEPC) and functional comparison with human umbilical vein endothelial cells (HUVEC) were studied. The use of heparin, a minimum blood volume of 30 ml, fibronectin as a coating matrix and endothelial growing media-2 supplemented with 20% FBS increased the success of obtaining EPC cultures up to 80% of the processed samples while reducing EPC colony appearance mean time to a minimum of 13 days. Blood samples exhibiting higher cEPC numbers resulted in reduced EPC colony appearance mean time. Cells isolated by using this combination were endothelial cell-like EPCs morphological and phenotypically. Functionally, cultured EPC showed decreased growing and vasculogenic capacity when compared to HUVEC. Thus, above-mentioned conditions allow the isolation and culture of EPC with smaller blood volumes and shorter times than currently used protocols.

Gaps in the Traceability Chain of Human Growth Hormone Measurements

Human growth hormone (hGH) is measured for the diagnosis of secretion disorders. These measurements fall under the EU Directive 98/79/EC on in vitro diagnostic medical devices requiring traceability of commercial calibrator values to higher-order reference materials or procedures (Off J Eur Communities 1998 Dec 7;L 331:1-37). External quality assessment schemes show large discrepancies between results from different methods, even though most methods provide results traceable to the recommended International Standard (IS 98/574). The aim of this study was to investigate possible causes for these discrepancies. We investigated the commutability and recovery of hGH in reconstituted IS 98/574. We tested different reconstitution protocols and used 4 different serum matrices for spiking. These IS preparations were measured together with serum samples. We quantified hGH by 5 different methods in 4 different laboratories. Results from the different methods correlated well for the serum samples. Mean discrepancies between results from different methods were ≤20%. None of the IS preparations was commutable for all the method comparisons. The recovery of hGH in preparations of IS 98/574 depended on the reconstitution protocol (>10-fold differences) and background matrix (relative differences ≤17% for different serum matrices). The use of different protocols for reconstitution and spiking of hGH reference preparations affects quantification by immunoassays, potentially leading to a bias between commercial methods, despite the use of calibrators with values claimed to be traceable to the same higher-order reference material.

INFLUENCE OF ANTIBIOTICS, SEALING SYSTEM AND GENOTYPE ON SHOOT REGENERATION VIA ORGANOGENESIS IN PASSION FLOWERS

In this work we evaluated the influence of three antibiotics (A) [Meropenem (MER), Cefotaxime (CEF) and Timentin (TIM)], three sealing systems-SS (PVPM, Petri dish, and PVC) and two genotypes (G) (Passiflora edulis ‘FB’ 100 and P. cincinnata) on shoot regeneration from in vitro cultured leaf explants. Explants were grown on MS medium supplemented with 2.0 mg.L-1 6-benzyladenine (BA) plus different concentrations of MER (0, 12.5 and 25.0 mg.L-1), CEF (0, 250 and 500 mg. L-1) or TIM (0, 150 and 300 mg.L-1), for five days in the dark. The flasks were sealed with three types of material (SS): polypropylene folding bag with microbial filter (ESPM), Petri dish with hypoallergenic tape (PDH), and a single layer of polyvinyl chlorine film (PVC). After explant inoculation and sealing of the flasks, these were transferred to a growth room at temperature 27±2°C. The flasks were transferred to a 16/8 h (light/dark) photoperiod, under irradiance of 36 μmol.m-2.s-1, for 30 days. The experimental design was completely randomized in triple factorial scheme. Each treatment was replicated four times, and each replicate consisted of ten leaf explants. Shoot regeneration was evaluated 30 days after inoculation. Data were subjected to variance analysis and means were compared by Tukey’s test at 5% probability. Percentage data were transformed in arcsin before analysis. Significant differences were observed for G, SS, A, and double (G x SS and G x A) and triple (G x SS x A) interactions on the shoot regeneration. In general, the best response with respect to G was obtained with P. edulis ‘FB-100’. For SS ESPM is more indicated, whereas TIM presented the better results among antibiotics evaluated. Regarding to the triple interactions the best responses achieved were P. cincinnata x PDH x R (12.5 mg.L-1), and P. edulis x ESPM x TIM (150 mg.L-1). Based on these results we suggest the use of different protocols for the two species.

Retrospective Study on the Use of Different Protocols for Repeated Transarterial Chemoembolization in the Treatment of Patients with Hepatocellular Carcinoma

To evaluate local tumor control and survival rate after repeated transarterial chemoembolization using two different protocols in hepatocellular carcinoma (HCC) patients. A total of 190 patients (mean, 68 years) with HCC were repeatedly treated with transarterial chemoembolization in 4-week intervals. The chemotherapy protocol consisted of mitomycin C alone (n = 111) and mitomycin C with gemcitabine (n = 79). Embolization was performed with lipiodol and microspheres. Tumor response was evaluated by magnetic resonance imaging using Response Evaluation Criteria In Solid Tumors (RECIST) criteria. Survival rates were calculated using Kaplan-Meier method. In the mitomycin C-only group, we observed partial response in 38.8% (43/111), stable disease in 27% (30/111), and progressive disease in 34.2% (38/111). In the mitomycin C/gemcitabine group (n = 79), partial response was observed in 43% (34/79), stable disease in 16.5% (13/79) and progressive disease in 40.5% (32/79). The overall 1- and 2-year survival rates were 56% and 28%, respectively. The overall median survival time from the start of transarterial chemoembolization treatment was 15 months. The median survival of patients treated with mitomycin C was 16.5 months and it was 12 months for patients treated with a combination of mitomycin C and gemcitabine. No statistically significant difference between the two groups was observed (P = .7). Chemoembolization is an effective minimally invasive therapy option for palliative treatment of HCC patients. Mitomycin C only proves to be effective, the addition of gemcitabine was not advantageous.

Accuracy assessment of the axial images obtained from cone beam computed tomography

The aim of this study was to evaluate accuracy of linear measurements assessed from axial tomograms and the influence of the use of different protocols in two cone beam CT (CBCT) units. A cylinder object consisting of Nylon® (Day Brazil, Sao Paulo, Brazil) with radiopaque markers was radiographically examined applying different protocols from NewTom 3G(TM) (Quantitative Radiology s.r.l, Verona, Veneto, Italy) and i-CAT(TM) (Imaging Sciences International, Hatfield, PA) units. Horizontal (A-B) and vertical (C-D) distances were assessed from axial tomograms and measured using a digital calliper that provided the gold standard for actual values. There were differences when considering acquisition protocols to each CBCT unit. Concerning all analysed protocols from i-CAT(TM) and Newtom 3G(TM), both A-B and C-D distances presented underestimated values. Measurements of the axial images obtained from NewTom 3G(TM) (6 inch 0.16 mm and 9 inch 0.25 mm) were similar to the ones obtained from i-CAT(TM) (13 cm 20 s 0.3 mm, 13 cm 20 s 0.4 mm and 13 cm 40 s 0.25 mm). The use of different protocols from CBCT machines influences linear measurements assessed from axial images. Linear distances were underestimated in both equipments. Our findings suggest that the best protocol for the i-CAT(TM) is 13 cm 20 s 0.3 mm and for the NewTom 3G(TM), the use of 6 inch or 9 inch is recommended.

Experimental investigation of tunneling times using Bose-Einstein condensates

The time it takes a quantum system to complete a tunneling event (which in the case of cross-barrier tunneling can be viewed as the time spent in a classically forbidden area) is related to the time required for a state to evolve to an orthogonal state, and an observation, i.e., a quantum mechanical projection on a particular basis, is required to distinguish one state from another. We have performed time-resolved measurements of Landau-Zener tunneling of Bose-Einstein condensates in accelerated optical lattices, clearly resolving the steplike time dependence of the band populations. The use of different protocols enabled us to access the tunneling probability, in two different bases, namely, the adiabatic basis and the diabatic basis. The adiabatic basis corresponds to the eigenstates of the lattice, and the diabatic one to the free-particle momentum eigenstates. Our findings pave the way towards more quantitative studies of the tunneling time for LZ transitions, which are of current interest in the context of optimal quantum control and the quantum speed limit.

Time-resolved measurement of Landau-Zener tunneling in different bases

A comprehensive study of the tunneling dynamics of a Bose--Einstein condensate in a tilted periodic potential is presented. We report numerical and experimental results on time-resolved measurements of the Landau--Zener tunneling of ultracold atoms introduced by the tilt, which experimentally is realized by accelerating the lattice. The use of different protocols enables us to access the tunneling probability, numerically as well as experimentally, in two different bases, namely, the adiabatic basis and the diabatic basis. The adiabatic basis corresponds to the eigenstates of the lattice, and the diabatic one to the free-particle momentum eigenstates. Our numerical and experimental results are compared with existing two-state Landau--Zener models.

Toward the harmonization of immune monitoring in clinical trials: Quo vadis?

A constantly increasing number of cancer immunotherapies are being investigated in clinical trials but no reliable biomarkers to predict clinical benefit currently exist. For some cancer types, biomarkers have proven to be meaningful predictors of patient outcomes (e.g., BCR-ABL in Chronic Myeloid Leukemia or PSA in prostate cancer) and were established as routine tools. As effects of cancer immunotherapy are mediated through the immune system, immune responses may act as natural biomarkers for clinical efficiency if the right factors can be reliably measured. Following this concept, cancer immunotherapy trials over the last decade have often included measures of tumor-specific cellular immune responses as endpoints to identify reliable surrogates for clinical benefit. Substantial efforts were invested throughout the immunotherapy field in setting up suitable cellular immune assays. However, to date, data from clinical trials do not consistently show that immune responses are correlated with clinical endpoints [1]. This lack of correlation is partially interpreted as a consequence of the high variability in assay results, due to the lack of assay standardization, validation and harmonization across laboratories. If harmonization of immune assays can be achieved, assays can be tested as surrogate endpoints in clinical trials, substantially accelerate the development of immunotherapeutic agents and, in addition, offer a rationale to pre-select groups of patients with high probability to benefit from subsequent immunotherapy. A plethora of different immunological assays to monitor antigen-specific T cell responses are available, and some are used by a majority of laboratories. Prime examples are the ELISPOT assay, intracellular cytokine staining (ICS), MHC-peptide multimer staining for detection of antigen-specific CD8+ and increasingly for CD4+ T cells and proliferation assays based on carboxyfluorescein succinimidyl ester (CFSE)-labelling. Additional functional assays that have been introduced more recently are based on CD154 up-regulation on activated antigen-specific CD4+ T cells or the detection of CD107a or cytotoxicity related molecules like perforin or granzyme in CD8+ T cells. For each of these assays, the use of different protocols that have evolved over time translates into a wide range of performances. Differences in the interpretation of the results obtained make comparisons of published data even more complex. This has led to lack of comparability as well as doubts about the integrity of the results that were obtained and published worldwide [2]. Recent developments clearly suggest that “proper” immunomonitoring will have to encompass the parallel use of several tests, which together assess the frequency, function and homing capacity of induced/stimulated T cells present in the circulation and preferentially also locally within the targeted tissue. In view of the influence of both natural and adaptive regulatory T cells on clinical outcome, immunomonitoring should also include screening for these cells. Only the combined use of several techniques may lead to a valid set of surrogate markers for specific immune interventions that allows clinical decisions or optimization of immunotherapeutic strategies. A related proposal was made by a community-wide consensus workshop on clinical trials with cancer vaccines, in which criteria for the use of immune assays were recommended including at least two validated assays to be used in parallel [3]. There is also a surprising disparity between the substantial financial and logistic efforts invested in reagents and man hours to develop immune monitoring techniques and the comparatively minor investment made to date in actually validating and standardizing the available techniques within and between laboratories. This has to be critically re-evaluated as efforts put in the adequate harmonization of assays will support the investments already made. They are also a pre-requisite to meet regulatory requirements on assay standards prior to them being considered as surrogate endpoints for clinical trials. This affects academic institutions and industry partners alike and may reach beyond cancer into the fields of autoimmune and infectious diseases.

Enhancement of antinociception by co-administration of ibuprofen and caffeine in arthritic rats

It has been observed that caffeine improves antinociceptive efficacy of some non-steroidal antiinflammatory drugs (NSAIDs) in several experimental models, however, these effects have been questioned in humans. Controversy in clinical studies may be due to the use of different protocols as well as to high interindividual variability in patient response. In addition, the antinociceptive interaction of ibuprofen + caffeine has not been studied. To assess a possible synergistic antinociceptive interaction, the antinociceptive effects of ibuprofen, and caffeine administered either separately or in combinations were determined in a model of arthritic pain: “Pain-induced functional impairment in the rat (PIFIR model). The antinociceptive efficacies were evaluated using several dose–response curves and time courses. The antinociceptive effects from the combination that produced the greater effect were compared with the maximal antinociceptive effect of either morphine or acetylsalicylic acid alone. The animals were administered with 0.05 ml intra-articular (i.a.) of uric acid to induce nociception. Groups of six rats received either acetylsalicylic acid, morphine, ibuprofen or caffeine, or a combination ibuprofen + caffeine (18 combinations). We report here that caffeine (17.8 and 31.6 mg/kg) is able to potentiate the antinociceptive effect of ibuprofen. This investigation showed that six combinations presented effects of potentiation and twelve combinations only showed antinociceptive effects not different from that of ibuprofen alone. The maximum antinociceptive effect was 270.7 ± 12.7 area units (au), produced by ibuprofen 100 mg/kg + caffeine 17.8 mg/kg; this effect was greater than the maximum produced by morphine 17.8 mg/kg (244.7 ± 22.9 au) in these experimental conditions. The maximum potentiation was 197 % produced with the combination of ibuprofen 17.8 mg/kg + caffeine 17.8 mg/kg. These results suggest that the antinociceptive effect of ibuprofen was significantly potentiated by doses of caffeine that by themselves are ineffective in this model.

Influence of MRI acquisition protocols and image intensity normalization methods on texture classification.

Texture analysis methods quantify the spatial variations in gray level values within an image and thus can provide useful information on the structures observed. However, they are sensitive to acquisition conditions due to the use of different protocols and to intra- and interscanner variations in the case of MRI. The influence was studied of two protocols and four different conditions of normalization of gray levels on the discrimination power of texture analysis methods applied to soft cheeses. Thirty-two samples of soft cheese were chosen at two different ripening periods (16 young and 16 old samples) in order to obtain two different microscopic structures of the protein gel. Proton density and T(2)-weighted MR images were acquired using a spin echo sequence on a 0.2 T scanner. Gray levels were normalized according to four methods: original gray levels, same maximum for all images, same mean for all images, and dynamics limited to micro +/- 3sigma. Regions of interest were automatically defined, and texture descriptors were then computed for the co-occurrence matrix, run length matrix, gradient matrix, autoregressive model, and wavelet transform. The features with the lowest probability of error and average correlation coefficient were selected and used for classification with 1-nearest neighbor (1-NN) classifier. The best results were obtained when using the limitation of dynamics to micro +/- 3sigma, which enhanced the differences between the two classes. The results demonstrated the influence of the normalization method and of the acquisition protocol on the effectiveness of the classification and also on the parameters selected for classification. These results indicate the need to evaluate sensitivity to MR acquisition protocols and to gray level normalization methods when texture analysis is required.

Repeatability and reproducibility of ribotyping and its computer interpretation

Many molecular typing methods are difficult to interpret because their repeatability (within-laboratory variance) and reproducibility (between-laboratory variance) have not been thoroughly studied. In the present work, ribotyping of coryneform bacteria was the basis of a study involving within-gel and between-gel repeatability and between-laboratory reproducibility (two laboratories involved). The effect of different technical protocols, different algorithms, and different software for fragment size determination was studied. Analysis of variance (ANOVA) showed, within a laboratory, that there was no significant added variance between gels. However, between-laboratory variance was significantly higher than within-laboratory variance. This may be due to the use of different protocols. An experimental function was calculated to transform the data and make them compatible (i.e., erase the between-laboratory variance). The use of different interpolation algorithms (spline, Schaffer and Sederoff) was a significant source of variation in one laboratory only. The use of either Taxotron (Institut Pasteur) or GelCompar (Applied Maths) was not a significant source of added variation when the same algorithm (spline) was used. However, the use of Bio-Gene (Vilber Lourmat) dramatically increased the error (within laboratory, within gel) in one laboratory, while decreasing the error in the other laboratory; this might be due to automatic normalization attempts. These results were taken into account for building a database and performing automatic pattern identification using Taxotron. Conversion of the data considerably improved the identification of patterns irrespective of the laboratory in which the data were obtained.

Validation of the human T-lymphocyte cloning assay — ring test report from the EU concerted action on HPRT mutation (EUCAHM)

The T-cell cloning assay, which enables the enumeration and molecular analysis of 6-thioguanine resistant (HPRT-negative) mutant T-cells, has been extensively used for studying human somatic gene mutation in vivo. However, large inter-laboratory variations in the HPRT mutant frequency (MF) call for further investigation of inter-laboratory differences in the experimental methodology, and development of an optimal but easy uniform cloning protocol. As part of the EU Concerted Action on HPRT Mutation (EUCAHM), we have carried out two Ring tests for the T-cell cloning assay. For each test, duplicate and coded samples from three buffy coats were distributed to five laboratories for determination of MF using six different protocols. The results indicated a good agreement between split samples within each laboratory. However, both the cloning efficiencies (CEs) and MFs measured for the same blood donors showed substantial inter-laboratory variations. Also, different medium compositions used in one and the same laboratory resulted in a remarkable difference in the level of MF. A uniform operating protocol (UOP) was proposed and compared with the traditional protocols in the second Ring test. The UOP (preincubation) increased the CE in laboratories traditionally using preincubation, but decreased the CE in laboratories traditionally using priming. Adjusted for donor, use of different protocols contributed significantly to the overall variation in lnCE ( P=0.0004) and lnMF ( P=0.03), but there was no significant laboratory effect on the lnCE ( P=0.38) or lnMF ( P=0.14) produced by the UOP alone. Finally, a simplified version of the UOP using the serum-free medium X-Vivo 10 and PMA was tested in one laboratory, and found to produce a considerable increase in CE. This modified UOP needs to be further evaluated in order to be used for future databases on HPRT MFs in various populations.

Assessment of undergraduate and graduate programs

Data from an international survey of public relations educators and practitioners raises a number of concerns about how achievement is measured among public relations students. Fewer than one in four academic programs have actually assessed learning outcomes and used the results to enhance their students' educational experiences. Fewer than one in four practitioners have been involved in some way as assessors. The survey suggests that educators place too much stock in grades alone as an appropriate assessment tool. While both educators and practitioners value portfolio reviews as an assessment protocol, practitioners see much greater value in their involvement in this form of assessment than their faculty counterparts. These differences of opinion notwithstanding, current practice and the opinions of educators and practitioners coalesce around the value of capstone experiences such as internships, simulation or case study analysis and surveys of alumni satisfaction as a core around which assessment plans can be built. The Assessment Task Team at the 1998 NCA Summer Conference developed a multidimensional model of assessment that reflects three principles of good practice. First, assessment plans which measure knowledge, behavior and affect at various points in students' academic careers rather than only at the end of a degree program are able to demonstrate change and the value added by the program of study. Second, specific outcomes may be measured by repeated applications of the same assessment protocol or by the use of different protocols over time. Third, appropriate use of a single assessment protocol to measure multiple outcomes, especially when those outcomes are drawn from two or more of the broad categories of knowledge, behavior and affect results in maximizing the information gained while minimizing the resources devoted to assessment.

Phospholipids and their inhibitors

The clinical condition sepsis and its sequelae are caused by numerous mediators that are released by various cell types. The purpose of this review is to describe the results of various studies performed with agents that either inhibited or stimulated the synthesis of, or affected the receptor-binding characteristics of a specific class of these mediators, the phospholipids. Antagonists to platelet-activating factor and thromboxane A2 receptors, monoclonal antibodies to phospholipase A2, agents which increased levels of prostaglandins, and cyclooxygenase, lipoxygenase, and other specific pathway inhibitors, which block the production of leukotrienes and thromboxane A2, were used in the studies described to affect the physiologic correlates of sepsis animal models and human studies. The matters discussed in this paper come from a wide variety of sources, including many broad-based clinical studies of humans with inflammatory disease. Many animal studies are discussed, along with some in vitro cell culture studies and work in molecular genetics. This article reviews a subject that is rapidly evolving, with frequent discoveries. Thus, much of the article discusses research in basic science, particularly the use of experimental drugs in animals. Clinical studies are generally of large numbers of patients showing overt signs of sepsis. Most cited literature was found in reputable, peer review journals, including such major basic science journals as Science, and clinical journals such as the Journal of the American Medical Association, New England Journal of Medicine, and Critical Care Medicine. Occasionally, contradictions do occur in the results of various studies. These contradictions are discussed in this review, and may often be due to the use of different protocols and definitions of the various clinical states. Increases of some mediators, such as platelet-activating factor, thromboxane A2, and the leukotrienes are associated with animal models of sepsis and seem to have a negative effect on the course of sepsis. Antagonists and blockers of these mediators increased survival in these studies. More research is needed to identify how these mediators of inflammation are associated with sepsis, and what the effects of blocking their actions will have on sepsis.

Stimulation of non-specific anti-tumour resistance in the mouse using cell wall preparations from four BCG substrains

The comparative anti-tumour activity of cell walls, deproteinized cell walls and cell wall skeleton (CWS) isolated from four BCG substrains (Canadian, Russian, Glaxo and Swedish) was determined against Ehrlich ascitic carcinoma in CF-1 mice and against L1210 lymphoid leukaemia in B6D2F1/J mice. In Ehrlich carcinoma, the protocol of injection was shown to be of critical importance, since high and low levels of protection and even facilitation of tumour growth were observed according to the protocol used. The oil emulsion used as the vehicle for injection of the fractions (control groups) induced facilitation of tumour growth with some protocols. The highest levels of protection were observed when treatment was performed on day -6 for a single i. p. injection or on days -14 and 0 for two injections. Using the former protocol of immunization, the highest level of protection was detected with cell walls and deproteinized cell walls. CWS isolated from the Canadian and Glaxo substrains were found to be inactive, whereas those isolated from the Swedish and Russian substrains induced significant levels of protection. In L1210 leukaemia, very few preparations induced significant protection. CWS preparations did not induce protection. Canadian and Russian deproteinized cell walls and Canadian cell walls (100 and 250 micrograms, respectively) induced some protection. The use of different protocols of injection did not increase the anti-tumour activity of the Canadian cell walls. The most active preparation in both tumour systems was that of Canadian deproteinized cell walls.