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PubMed2004

A single-platform approach using flow cytometry and microbeads to evaluate immune reconstitution in mice after bone marrow transplantation.

The monitoring of immune reconstitution in murine models of HC transplantation, using accurate and automated methods, is necessary in view of the recent developments of hematopoietic cell (HC) transplantation (including reduced intensity conditioning regimens) as well as emerging immunological concepts (such as the involvement of dendritic cells or regulatory T cells). Here, we describe the use of a single-platform approach based on flow cytometry and tubes that contain a defined number of microbeads to evaluate absolute blood cell counts in mice. This method, previously used in humans to quantify CD34+ stem cells or CD4+ T cells in HIV infected patients, was adapted for mouse blood samples. A CD45 gating strategy in this "lyse no wash" protocol makes it possible to discriminate erythroblasts or red blood cell debris from CD45+ leukocytes, thus avoiding cell loss. Tubes contain a lyophilized brightly fluorescent microbead pellet permitting the acquisition of absolute counts of leukocytes after flow cytometric analysis. We compared this method to determine absolute counts of circulating cells with another method combining Unopette reservoir diluted blood samples, hemocytometer, microscopic examination and flow cytometry. The sensitivity of this single-platform approach was evaluated in different situations encountered in allogeneic HC transplantation, including immune cell depletion after different conditioning regimens, activation status of circulating cells after transplantation, evaluation of in vivo cell depletion and hematopoietic progenitor mobilization in the periphery. This single-platform flow cytometric assay can also be proposed to standardize murine (or other mammalian species) leukocyte count determination for physiological, pharmacological/toxicological and diagnostic applications in veterinary practice.

Animals↗
PubMed2004

Thin film MRI-high resolution depth imaging with a local surface coil and spin echo SPI.

A multiple echo, single point imaging technique, employing a local surface coil probe, is presented for examination of thin film samples. Depth images with a nominal resolution of 5 microm were acquired with acquisition times on the order of 10 min. The method may be used to observe dynamic phenomenon such as polymerization, wetting, and drying in thin film samples. It is readily adapted to spatially resolved diffusion coefficient and T2 relaxation time mapping.

Algorithms↗
PubMed2005

Rapid detection and differentiation of bovine herpesvirus 1 and 5 glycoprotein C gene in clinical specimens by multiplex-PCR.

A multiplex polymerase chain reaction (multiplex-PCR) to detect and differentiate bovine herpesvirus 1 (BoHV-1) and 5 (BoHV-5) was developed using primers for the gene sequence that encodes the glycoprotein C. The technique was assessed against the BoHV-1 and BoHV-5 cell culture adapted strains, and clinical samples collected from animals with clinical signs of BoHV-1 (n = 10) or BoHV-5 (n = 7) infection and with diagnosis confirmed by virus isolation in cell culture and semi-nested PCR. Fifteen clinical samples from asymptomatic animals were included as control group. For the evaluation of the amplifiability of the extracted nucleic acid from clinical specimens was included a bovine internal control that amplified a 626 bp fragment of the ND5 gene present in the bovine mitochondrial DNA. For DNA extraction, a combination of the phenol/chloroform/isoamyl alcohol and silica/guanidine isothiocyanate methods was used. The specificity of the BoHV-1 and BoHV-5 amplicons from standard strains were confirmed by sequence analysis. All the positive clinical samples for BoHV included in this study were characterized as BoHV-1 or BoHV-5 by the difference in length of the amplified product visualized in a agarose gel (354 bp size for BoHV-1, and 159 bp for BoHV-5). The internal control was amplified in all clinical specimens. Non-specific reactions were not observed when the multiplex-PCR was assessed with other viruses (bovine viral diarrhea virus and rabies virus) and BoHV-negative clinical samples from fetuses and adult cattle obtained from a slaughterhouse.

Animals↗
PubMed2004

Improved specificity for Giardia lamblia cyst quantification in wastewater by development of a real-time PCR method.

The protozoan parasite Giardia lamblia is the most common cause of waterborne disease outbreaks associated with drinking water in the United States. The conventional method used for the enumeration of Giardia cysts in water is based on immunofluorescence with monoclonal antibodies. It is tedious and time-consuming and has the major drawback to be non-specific for the only species infecting humans, G. lamblia. We have developed a real-time polymerase chain reaction (PCR) method using fluorescent TaqMan technology, which improved the specificity of G. lamblia cyst quantification compared to the immunofluorescence assay (IFA). However, this PCR was not totally specific for G. lamblia species and amplified Giardia ardeae target as well. This method showed a sensitivity of 0.45 cysts per reaction and an efficiency of 95% in purified suspensions. We have then applied this quantification method to raw wastewater, a medium containing numerous debris, particles and PCR inhibitors. The adaptation to these environmental samples was realized by a screening of three cyst purification methods and six DNA extraction protocols. Real-time quantification was accomplished by the simultaneous amplification of unknown samples and a tenfold serial dilution of purified G. lamblia cysts. For all samples, the concentrations observed with TaqMan PCR method were compared to the IFA values. Giardia spp. cysts were detected in all non-spiked raw wastewater samples with IFA procedure and the concentrations of Giardia spp. cysts used for the comparison between the two methods ranged between 3.3x10(2)/l and 4.3x10(3)/l. The highest TaqMan PCR/IFA ratios were observed when Percoll/sucrose flotation was combined with DNA extraction protocol optimized for cyst wall lysis, impurities adsorption on a resin, and double step protein digestion and column purification. The concentrations observed with this TaqMan PCR method ranged from 2.5x10(2) to 2.4x10(3) G. lamblia cysts/l and only one sample resulted in a no amplification curve. Thus, we developed a TaqMan PCR method increasing the rapidity and specificity of G. lamblia cyst quantification. The combination of Percoll/sucrose flotation and DNA extraction optimized protocol before TaqMan assay has provided a good indication of the G. lamblia contamination level in raw sewage samples.

Animals↗
PubMed2003

Strategy for identifying protein-protein interactions of gel-separated proteins and complexes by mass spectrometry.

A strategy for identifying and characterizing protein interactions among gel-separated proteins and complexes has been developed and tested. The method involves the efficient recovery of proteins or complexes from native gels without affecting their conformational integrity. The use of limited proteolysis of protein complexes, isolated from the gel or formed from the interaction of gel-recovered proteins with potential binding partners, has enabled local binding domains to be efficiently identified using a combination of microfiltration and mass spectrometric analysis. The application of mass spectrometry affords high detection sensitivities, enabling the strategy to be applied to low levels of protein and protein mixtures. The approach is demonstrated for both antigen-antibody and peptide-protein complexes for which protein-binding regions are characterized among simple peptide mixtures and proteolytic digests. The strategy can be easily adapted to achieve high sample throughput and automation using gel-excision robotics and provides a means to study protein interactions in complex biological mixtures and extracts.

Animals↗
PubMed1990

Modified high-performance liquid chromatographic determination of diamine oxidase activity in plasma.

A previous high-performance liquid chromatographic determination of diamine oxidase activity suitable for tissue homogenates was modified in order to adapt it to plasma samples. Simple additional steps were introduced after the enzyme reaction and before the chromatographic separation, both according to the previous method. In this way the sensitivity and the reproducibility of the overall procedure was suitable for routine plasma diamine oxidase estimations.

Amine Oxidase (Copper-Containing)↗
PubMed1986

Transitional explant reduction assay--a new in vitro testing system for intravesical chemotherapy.

Eighty-five tumors from 49 patients with transitional cell carcinoma were examined in a model for in vitro sensitivity using tumor explant reduction assay. Individual sensitivity patterns were obtained for each tumor tested. Explants were most frequently found to be sensitive to cis-platinum (49 per cent) and least frequently sensitive to thiotepa (20 per cent). Thirteen patients were studied on multiple sequential intervals, with resistance to specific agents noted to develop in 53 per cent of the patients. To date, a prospective cross-over study has been conducted on seven patients with 15 correlates. Overall, clinical correlation of this assay for both resistance and sensitivity is 93 per cent. We report a new assay for transitional cell carcinoma of the bladder that is adaptable to small specimen samples and provides adequate material for testing 73 per cent of the time.

Carcinoma, Transitional Cell↗
PubMed2001

Stability of salivary steroids: the influences of storage, food and dental care.

We studied influences of dental care, food and storage on the reproducibility of salivary steroid levels. Cortisol (F), 17OH-progesterone (17OHP) and Progesterone (P) were measured using adapted commercial radioimmunoassays. Saliva samples of healthy adults (n = 15; m:8; f:7) were collected directly before and after dental care, and directly before and after breakfast with various foodstuffs. A second experiment investigated stability of steroids under different storage conditions. Four series of identical saliva portions (I: Native saliva; II: Centrifuged saliva; III: Saliva with trifluor acetate (TFA); IV: Saliva with 0.5% NaN(3)) were stored at room temperature and at 4 degrees C for up to three weeks. To demonstrate influences of repeated thawing and re-freezing of saliva on steroid values, saliva samples (n = 15) were divided into identical portions. These portions were frozen and re-thawed up to 5 times before measurement. Neither dental care nor intake of bread or milk effected the reproducibility of F, 170HP, and P. Steroid levels decreased significantly in the course of three weeks under different storage conditions (P < 0.001). This decrease was clinically relevant from the second week onward, with exception of NaN(3) treated samples. After repeated freezing and re-thawing 17OHP and P decreased slightly (about 5%). Only F decreased significantly after the third thawing (P < 0.001). The results show the usefulness of standardized handling of saliva samples for improving reproducibility and reliability of salivary steroid measurements.

17-alpha-Hydroxyprogesterone↗
PubMed2000

Use of radioimmunoassay as a screen for antibiotics in confined animal feeding operations and confirmation by liquid chromatography/mass spectrometry.

Approximately one-half of the 50,000,000 lb of antibiotics produced in the USA are used in agriculture. Because of the intensive use of antibiotics in the management of confined livestock operations, the potential exists for the transport of these compounds and their metabolites into our nation's water resources. A commercially available radioimmunoassay method, developed as a screen for tetracycline antibiotics in serum, urine, milk, and tissue, was adapted to analyze water samples at a detection level of approximately 1.0 ppb and a semiquantitative analytical range of 1-20 ppb. Liquid waste samples were obtained from 13 hog lagoons in three states and 52 surface- and ground-water samples were obtained primarily from areas associated with intensive swine and poultry production in seven states. These samples were screened for the tetracycline antibiotics by using the modified radioimmunoassay screening method. The radioimmunoassay tests yielded positive results for tetracycline antibiotics in samples from all 13 of the hog lagoons. Dilutions of 10-100-fold of the hog lagoon samples indicated that tetracycline antibiotic concentrations ranged from approximately 5 to several hundred parts per billion in liquid hog lagoon waste. Of the 52 surface- and ground-water samples collected all but two tested negative and these two samples contained tetracycline antibiotic concentrations less than 1 ppb. A new liquid chromatography/mass spectrometry method was used to confirm the radioimmunoassay results in 9 samples and also to identify the tetracycline antibiotics to which the radioimmunoassay test was responding. The new liquid chromatography/mass spectrometry method with online solid-phase extraction and a detection level of 0.5 microg/l confirmed the presence of chlorotetracycline in the hog lagoon samples and in one of the surface-water samples. The concentrations calculated from the radioimmunoassay were a factor of 1-5 times less than those calculated by the liquid chromatography/mass spectrometry concentrations for chlorotetracycline.

Animal Husbandry↗
PubMed1998

Microplate-reverse hybridization method to determine dengue virus serotype.

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3'-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method.

Dengue↗
PubMed1998

Organization of microfibrils in keratin fibers studied by X-ray scattering modelling using the paracrystal concept.

Low-angle X-ray scattering patterns of hard alpha-keratin fibers have been studied for more than 50 years but a completely convincing modelling has never been presented. The models which have been proposed so far are specific to the sample and cannot be adapted to others, mainly because they do not use a parametric analytical expression of the distribution function describing the relative positions of the microfibrils. Our new approach is based on a paracrystal distribution function. In addition, a huge background originating from a non-ordered matrix is taken into account. Various hard alpha-keratins from different origins have been studied using our approach. From the rather good modellings obtained, it appears that the diameter of the microfibril is not origin dependent (7.4 nm) whereas the distances between microfibrils and their electron density profiles are. Hair microfibrils can be reasonably approximated by a solid cylinder but a core and an outer ring are necessary for porcupine. Our method is of course not limited to keratin microfibrils; it can be used for modelling equatorial X-ray scattering profiles of all types of hexagonal fibrillar assemblies, which are in fact widely found in biological tissues.

Animals↗
PubMed1997

Rapid stereospecific high-performance liquid chromatographic determination of levofloxacin in human plasma and urine.

A rapid high-performance liquid chromatographic (HPLC) method for the determination of levofloxacin in human plasma and urine has been validated. A single-step liquid-liquid extraction procedure was used to isolate levofloxacin from the biological matrix prior to quantitative analysis. The compound was separated on an Inertsil C18 reversed-phase HPLC column and quantified by measuring the UV absorbance at 330 nm. The stereospecificity was achieved in the ligand-exchange mode by incorporating chiral reagents directly into the HPLC mobile phase. Ciprofloxacin was used as the internal standard. The method was linear from 0.08 to 5.18 micrograms ml-1 of levofloxacin in plasma and from 23 to 1464 micrograms ml-1 in urine. The overall utility of the method is reflected in its high sample throughput and easy adaptability to robotic automation, thus making the procedure suitable for pharmacological and pharmacokinetic studies of levofloxacin.

Anti-Infective Agents↗
PubMed1996

Formation and decay of the S3 EPR signal species in acetate-inhibited photosystem II.

A 230-G-wide EPR signal is induced in acetate-treated photosystem II by 30 s of illumination at 277 K followed by freezing under illumination to 77 K [MacLachlan, D. J., & Nugent, J. H. A. (1993) Biochemistry 32, 9772-9780]. This signal, referred to as the S3 EPR signal, has been interpreted to arise from an S2X+ species where X+ is an amino acid radical. Investigation of the factors responsible for the formation and decay of the S3 EPR signal reveals that the yield of the S3 EPR signal is strongly temperature-dependent and depends on the rate of oxidation of QA-. Quantitation of the number of centers contributing to the S3 EPR signal produced by the optimal continuous illumination times of 3 min at 250 K, 30 s at 273 K, and 5 s at 294 K gave values of 13, 38, and 49 +/- 3%, respectively. By using 5 s of illumination at 294 K to induce the S3 EPR signal, and then illumination at 200 K to reduce QA, both the S3 and QA(-)Fe EPR signals were induced in high yield. This result indicates that the S3 EPR signal does not arise from an acceptor-side species. When saturating laser flashes were used to induce the S3 EPR signal in a dark-prepared, dark-adapted, acetate-treated sample, the yield was small after one flash and close to maximal after two flashes. An EPR signal at g = 4.1 was observed to be formed at intermediate times during the decay of the S3 EPR signal in the dark; the rates of decay of the S3 EPR signal at 273 and 294 K corresponded to the rates of formation of the g = 4.1 EPR signal. These results, together with the flash results, indicate that two steps are involved in both the generation and decay of the S3 EPR signal. The rates of formation and decay of both the S3 and QA(-)Fe EPR signals were measured at 250, 273, and 294 K. A kinetic model is presented that accounts for these kinetic data and the yield of the S3 EPR signal.

Acetates↗
PubMed2005

Finite temperature string method for the study of rare events.

A method is presented for the study of rare events such as conformational changes arising in activated processes whose reaction coordinate is not known beforehand and for which the assumptions of transition state theory are invalid. The method samples the energy landscape adaptively and determines the isoprobability surfaces for the transition: by definition the trajectories initiated anywhere on one of these surfaces has equal probability to reach first one metastable set rather than the other. Upon weighting these surfaces by the equilibrium probability distribution, one obtains an effective transition pathway, i.e., a tube in configuration space inside which conformational changes occur with high probability, and the associated rate. The method is first validated on a simple two-dimensional example; then it is applied to a model of solid-solid transformation of a condensed system.

Journal Article↗
PubMed2005

Adaptation of panic-related psychopathology measures to Russian.

The study reports results of adaptation of panic-related psychopathology measures to Russian, including the Anxiety Sensitivity Index (ASI), the Agoraphobic Cognitions Questionnaire (ACQ), and the Mobility Inventory for Agoraphobia (MIA). Psychometric properties (e.g., reliability, factor structure, endorsement) and external validity of the adaptations were evaluated in a representative sample of Moscow residents (N = 390) and Ukrainian undergraduates (N = 492). The adapted ASI was generally equivalent to the original English-language version. The ACQ showed structural equivalency but did not exhibit expected specificity to panic. Finally, the MIA displayed notable structural discrepancies but had external validity comparable to the original.

Adult↗
PubMed1994

Communal orientation and individualism: factors and correlations with values, social adjustment, and self-esteem.

Contemporary personality structures in the West reportedly are dominated by a problematic self-contained individualism, whereas a community-oriented ensembled individualism is presumed to be more adaptive. With the present sample of American undergraduates, an Individualism scale (Bales & Couch, 1969) was used to measure self-contained individualism, and the Communal Orientation Scale (Clark, Ouellette, Powell, & Milberg, 1987) was used to measure ensembled individualism. Results showed that both instruments predicted a belief in equalitarianism, but individualism was more likely to be associated with interpersonal maladjustment and poor self-esteem. Neither the full scales nor any of their factors displayed significant inverse correlations with each other, thus confirming the existence of negative correlates for self-contained individualism but also demonstrating that the two individualisms are not wholly incommensurable, as has been argued in the past.

Adult↗
PubMed1989

The production and characterisation of monoclonal antibodies against human prolactin and the development of a two-site immunoradiometric assay.

Monoclonal antibodies against human prolactin (PRL) have been produced and characterised and used to develop a sensitive two-site immunoradiometric assay (IRMA). Nine anti-PRL monoclonal antibodies were assessed for reactivity in immunoblotting experiments with PRL, hPL, hGH and pituitary gland extract. There was no detectable crossreactivity with hPL or hGH. In liquid phase radioimmunoassay (RIA) studies using three of the antibodies there was no detectable crossreaction from hPL or hGH. Five antibodies were positive in immunocytochemical studies using sections of human pituitary gland. Using FPLC purified monoclonal antibodies, a two-site IRMA was developed that could assay PRL over the range 17.5-3500 mIU per litre and was readily adapted to assaying serum samples from patients. The two-site IRMA could be performed within one day without loss of sensitivity and has potential as a rapid and simple method for screening clinical samples.

Antibodies, Monoclonal↗