The use of quantitative immunofluorescence to measure immunoglobulin levels in mouse myleoma cells during the cell cycle

The technique of quantitative immunofluorescence was applied to a study of immunoglobulin levels in mouse myeloma cells in different phases of the cell cycle.     

Cytokine analysis to predict immunosuppression.

BACKGROUND: Recently, it has been realized that TH1/TH2 cytokine production offer the unique possibility to predict drug efficacy. However, there is still an incessant need to explore assay conditions and techniques of analyzing cytokines, which are specific and reliable for monitoring drug efficacy. METHODS: In this study we used the multiplex bead array technique to detect cytokines of TH1/TH2 cells in whole blood of heart transplanted (HTx) recipients. RESULTS: We found significantly different levels of cytokine expression in HTx recipients compared with cytokine levels in patients prior to HTx. Furthermore, particular cytokine levels were significantly decreased 2 h after drug dosing, compared with cytokine levels before dosing in mitogen-stimulated whole blood. CONCLUSIONS: Cytokine analysis with the multiplex array technique in mitogen-stimulated whole blood provides the possibility to predict immunosuppression.     

Monitoring of Low-Level Virus in Natural Waters

The insoluble polyelectrolyte technique for concentrating virus is extended to extremely low virus levels. The effectiveness of this method employing a coliphage T2 model is a constant 20% over a range of virus levels from 10(3) to 10(-4) plaque-forming units/ml. The efficiency of the method is dependent upon pH control during the concentration phase. Although the study was initiated to develop a method for quantitating the effectiveness of water and wastewater treatment methods for the removal of viruses from waters at low concentrations, the potential of the technique for efficient monitoring of natural waters is apparent.     

Non-invasive determination of coupled motion of the scapula and humerus--an in-vitro validation

Measuring the motion of the scapula and humerus with sub-millimeter levels of accuracy in six-degrees-of-freedom (6-DOF) is a challenging problem. The current methods to measure shoulder joint motion via the skin do not produce clinically significant levels of accuracy. Thus, the purpose of this study was to validate a non-invasive markerless dual fluoroscopic imaging system (DFIS) model-based tracking technique for measuring dynamic in-vivo shoulder kinematics. Our DFIS tracks the positions of bones based on their projected silhouettes to contours on recorded pairs of fluoroscopic images. For this study, we compared markerlessly tracking the bones of the scapula and humerus to track them with implanted titanium spheres using a radiostereometric analysis (RSA) while manually manipulating a cadaver specimen's arms. Additionally, we report the repeatability of the DFIS to track the scapula and humerus during dynamic shoulder motion. The difference between the markerless model-based tracking technique and the RSA was ±0.3 mm in translation and ±0.5° in rotation. Furthermore, the repeatability of the markerless DFIS model-based tracking technique for the scapula and humerus was ±0.2 mm and ±0.4°, respectively. The model-based tracking technique achieves an accuracy that is similar to an invasive RSA tracking technique and is highly suited for non-invasively studying the in-vivo motion of the shoulder. This technique could be used to investigate the scapular and humeral biomechanics in both healthy individuals and in patients with various pathologies under a variety of dynamic shoulder motions encountered during the activities of daily living.     

High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS): Genome-Wide DNA Methylation Analysis of IAP LTR Retrotransposon

In vertebrates, DNA methylation-mediated repression of retrotransposons is essential for the maintenance of genomic integrity. In the current study, we developed a technique termed HT-TREBS (High-Throughput Targeted Repeat Element Bisulfite Sequencing). This technique is designed to measure the DNA methylation levels of individual loci of any repeat families with next-generation sequencing approaches. To test the feasibility of HT-TREBS, we analyzed the DNA methylation levels of the IAP LTR family using a set of 12 different genomic DNA isolated from the brain, liver and kidney of 4 one-week-old littermates of the mouse strain C57BL/6N. This technique has successfully generated the CpG methylation data of 5,233 loci common in all the samples, representing more than 80% of the individual loci of the five targeted subtypes of the IAP LTR family. According to the results, approximately 5% of the IAP LTR loci have less than 80% CpG methylation levels with no genomic position preference. Further analyses of the IAP LTR loci also revealed the presence of extensive DNA methylation variations between different tissues and individuals. Overall, these data demonstrate the efficiency and robustness of the new technique, HT-TREBS, and also provide new insights regarding the genome-wide DNA methylation patterns of the IAP LTR repeat elements.     

A slab gel electrophoresis technique for measurement of plasma retinol-binding protein, cellular retinol-binding and retinoic-acid-binding proteins in human skin

At least four different proteins that bind retinoids could be present in a vitamin A target tissue like the skin. In order to separate cellular retinoid-binding proteins (CRBP and CRABP) from serum retinol-binding protein (RBP) and albumin, a one-step procedure was devised. The technique is based on slab polyacrylamide gel electrophoresis (PAGE) of the extracted proteins incubated with tritiated retinoids. The procedure was used to study binding proteins in the skin. The results show that epidermal extracts (the epithelial part of the skin) contain no RBP activities whereas dermal extracts (the mesenchymal part of the skin) contain 1.6 +/- 0.81 pmol/mg protein of RBP. This technique further showed higher levels of CRABP in both epidermal (9.05 +/- 1.16 pmol/mg protein) and dermal (1.5 +/- 0.54 pmol/mg protein) extracts than those previously determined by other less specific techniques. On the other hand CRBP levels were found to be lower in the two tissues (epidermis 0.2 +/- 0.1 pmol/mg and dermis 0.12 +/- 0.05 pmol/mg protein). New conditions to measure specifically CRABP with the charcoal/dextran technique could be developed and analyzed by the PAGE technique; a dissociation constant of 13.7 nM was then calculated for epidermal CRABP. This PAGE technique appears to be the most appropriate method for the study of retinoid-binding proteins including RBP in human skin.     

Validation of a new model-based tracking technique for measuring three-dimensional, in vivo glenohumeral joint kinematics

Shoulder motion is complex and significant research efforts have focused on measuring glenohumeral joint motion. Unfortunately, conventional motion measurement techniques are unable to measure glenohumeral joint kinematics during dynamic shoulder motion to clinically significant levels of accuracy. The purpose of this study was to validate the accuracy of a new model-based tracking technique for measuring three-dimensional, in vivo glenohumeral joint kinematics. We have developed a model-based tracking technique for accurately measuring in vivo joint motion from biplane radiographic images that tracks the position of bones based on their three-dimensional shape and texture. To validate this technique, we implanted tantalum beads into the humerus and scapula of both shoulders from three cadaver specimens and then recorded biplane radiographic images of the shoulder while manually moving each specimen's arm. The position of the humerus and scapula were measured using the model-based tracking system and with a previously validated dynamic radiostereometric analysis (RSA) technique. Accuracy was reported in terms of measurement bias, measurement precision, and overall dynamic accuracy by comparing the model-based tracking results to the dynamic RSA results. The model-based tracking technique produced results that were in excellent agreement with the RSA technique. Measurement bias ranged from -0.126 to 0.199 mm for the scapula and ranged from -0.022 to 0.079 mm for the humerus. Dynamic measurement precision was better than 0.130 mm for the scapula and 0.095 mm for the humerus. Overall dynamic accuracy indicated that rms errors in any one direction were less than 0.385 mm for the scapula and less than 0.374 mm for the humerus. These errors correspond to rotational inaccuracies of approximately 0.25 deg for the scapula and 0.47 deg for the humerus. This new model-based tracking approach represents a non-invasive technique for accurately measuring dynamic glenohumeral joint motion under in vivo conditions. The model-based technique achieves accuracy levels that far surpass all previously reported non-invasive techniques for measuring in vivo glenohumeral joint motion. This technique is supported by a rigorous validation study that provides a realistic simulation of in vivo conditions and we fully expect to achieve these levels of accuracy with in vivo human testing. Future research will use this technique to analyze shoulder motion under a variety of testing conditions and to investigate the effects of conservative and surgical treatment of rotator cuff tears on dynamic joint stability.     

A novel DNA microarray for rapid diagnosis of enteropathogenic bacteria in stool specimens of patients with diarrhea

A microarray technique for the detection and identification of enteropathogenic bacteria at the species and subspecies levels was developed in this study, and the target bacteria included pathogenic Escherichia coli, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella enterica, Campylobacter jejuni, Shigellae, Yersinia enterocolitica, and Listeria monocytogenes. The virulence gene of each pathogen was chosen as the amplification target, labeled with a fluorescence dye by multiplex polymerase chain reaction (PCR), and hybridized to the specific virulence gene probes that had been immobilized on a microchip. Stool specimens from 34 patients with diarrhea were tested in this study. Five were positive for multiple genera. Nested PCRs and sequencing were used to amplify and identify the related genes, which were found to share 95.8% to 100% of the nucleotide identity with the corresponding regions in the Genbank database. Real-time PCR was used to determine the number of gene copies to determine the sensitivity of this technique, which was shown to be 58 copies/microl. The results indicated that the microarray technique which targets multiple virulence genes of enteropathogenic bacteria at the species and subspecies levels is an attractive diagnostic tool for rapidly and simultaneously identifying multiple enteropathogenic pathogens in clinical practice, especially in patients with infectious diarrhea.     

Demonstration of a large-conduction ion channel in subcellular fractions of the adrenal medulla

The "tip-dip" technique (formation of a lipid bilayer at the tip of a microelectrode) allows the electrophysiological study of organelle membranes. An ionic channel of large conductance has been found by this technique in membrane preparations from adrenal medulla. This channel is voltage-sensitive and it has 4 levels of conductance. Its subcellular origin is to be determined; it is borne by a structure which sediments between 1,500 and 25,000 X g.     

Hypertension self-control with a portable feedback unit or meditation-relaxation

Thirty borderline essential hypertensives were randomly assigned to a portable constant-cuff blood pressure feedback technique or meditation-relaxation. Each technique was taught in the laboratory, then practiced twice daily at home for four weeks. Subjects mailed daily records of their progress. Seven feedback and ten meditation-relaxation subjects completed the program. Both techniques produced significant systolic and diastolic reductions within practice sessions and diastolic reductions over weeks of training. Neither technique improved reductions nor reduced initial systolic pressure levels over the four weeks. Differences between biofeedback and meditation-relaxation in within-session pressure reductions were not significant.This study was supported by NIMH grant MH27214 to Joanne L. Hager, NIMH grant MH8880-2 to Elliot Mishler, and NIMH grant MH25104 to Richard S. Surwit. The authors are grateful to the Parke-Davis Instrument Co. who provided the instrumentation for this study.     

An approach to practical persistence

 A basic problem in community ecology is determining whether a community of interacting species will survive in the long term. A criterion ensuring this is that of permanence (or uniform persistence), which is based on the idea that species densities for large time are above minimum non-zero levels. There are various mathematical techniques for investigating permanence, but they do not yield an estimate for the minimum levels, and these may lie below minimum viability levels in the biological sense of ‘Practical Persistence’. Here we study a technique for obtaining explicit expressions for the minimum levels when one species is ‘slow’. This is illustrated for a predator–prey problem governed by difference equations, and we note that the technique is applicable even when the dynamics is chaotic.. Received: 7 April 1997 / Revised version: 25 March 1998     

Comparison of effects of in vivo nitrogen dioxide exposure starting from different periods on alveolar macrophage activity, assessed by a chemiluminescence technique in Brown-Norway rats.

Nitrogen dioxide (NO2) has been extensively studied for its immune modulating effects on pulmonary cells. Alveolar macrophages (AMs) play an important role in pulmonary immunity. The Brown-Norway (BN) rat has been studied as a high-risk model of allergic diseases. In this study, BN rats were exposed to NO2 from the embryonic or weanling period (EP or WP, respectively). To evaluate the effects of NO2 exposure on pulmonary immunity, the activity levels of rat AMs were assessed as reactive oxygen species-generating capacity, measured by a chemiluminescence (CL) technique, and as cytokine-producing ability. Except for 0.2 ppm of NO2 exposure, the CL responses of AMs obtained from the WP group at 12 weeks old were suppressed significantly. Changes of the cytokine-producing levels suggest that inflammatory reactions are terminated at 12 weeks in the EP group. Correlations between the CL responses and the cytokine levels reveal that NO2 exposure may modulate the direction of AM activation. The CL technique is thought to be useful to evaluate changes in AM activity. In this study, the results suggest that, using the high-risk model of allergic diseases, NO2 exposure from the weanling period has stronger effects on AM activity.     

A method of recording the gastric secretory function in mice

The technique of simultaneous study of stomach functions and the secretory levels of its glands was developed on the CC57W/MvY mice using morphological, bio- and histochemical methods. Protein RNA, DNA contents and levels of gastric tissue SDH activity as well as the composition of the gastric juice were studied. The correlation of gastric secretion with the activity of its tissue SDH was demonstrated.     

The use of fluorescence correlation spectroscopy (FCS) as an alternative biomarker detection technique: a preliminary study

Biomarkers are essential part of daily medical practice. Currently, biomarkers are being used both for diagnostic and prognostic purposes. There are many approaches e.g. ELISA by which biomarker levels are detected from patient samples. However, all these approaches are laborious, time consuming and expensive. There is therefore a general need for exploring new technique which can overcome these drawbacks. Here, we present a preliminary study for detection of serum biomarkers by fluorescence correlation spectroscopy (FCS) based diagnostic technique. FCS is a technique basically used for spatial and temporal analysis of molecular interactions of extremely low-concentration biomolecules in solution. FCS is able to measure diffusion time of the fluorescent molecules passing through the open detection volume and it can also measure the average number of fluorescent molecules passing through the detection volume. Because diffusion speed is correlated with shape and molecular mass of the fluorescent molecule, this property makes it possible to study the complex formation between a small fluorescently labelled and a large unlabelled molecule. In this preliminary study, we utilize this FCS property for detection of serum biomarker. Further studies on various pathological serum samples are warranted to explore further aspects of this technique.     

High-dose combination alkylating agents with autologous bone-marrow support in patients with breast cancer: preliminary assessment of DNA damage in individual peripheral blood lymphocytes using the single cell gel electrophoresis assay.

The single cell gel (SCG) assay is a sensitive electrophoretic technique for detecting the presence of DNA single strand breaks and alkali-labile damage in individual cells. This technique was used to evaluate the levels of DNA damage in cryopreserved peripheral blood lymphocytes (PBLs) from 11 breast cancer patients treated with high doses of cyclophosphamide and cisplatin and provided autologous bone marrow transplantation after treatment. PBL specimens for the SCG study were obtained just prior to treatment, following the administration of cyclophosphamide and cisplatin for 2 days, and upon lymphocytic recovery. Based on a concurrent analysis of DNA damage in cryopreserved and non-cryopreserved PBL samples from three patients, the mean level of DNA migration or the dispersion of damage among cells was not affected by the process of cryopreservation. The pre-treatment samples of several patients contained PBL with increased levels of DNA damage, presumably reflecting persistent DNA damage induced by previous treatment regimens. Chemotherapy resulted in a significant but variable increase in DNA damage in PBL samples from all patients. In this limited study, the level of damage did not correlate with serum levels of cyclophosphamide or with lymphocyte toxicity. Among the post-treatment samples, increased levels of DNA damage were absent in most but not all patients. The presence of damaged cells in the post-treatment samples may be indicative of an inadequate therapy regimen or of DNA damage resulting from non-therapy related processes. Because of its simplicity and short processing time, the SCG assay can be used to evaluate levels of DNA damage during the course of therapy, allowing the dose schedule to be altered to achieve a desired effect level.     

High-dose combination alkylating agents with autologous bone-marrow support in patients with breast cancer: preliminary assessment of DNA damage in individual peripheral blood lymphocytes using the single cell gel electrophoresis assay

The single cell gel (SCG) assay is a sensitive electrophoretic technique for detecting the presence of DNA single strand breaks and alkali-labile damage in individual cells. This technique was used to evaluate the levels of DNA damage in cryopreserved peripheral blood lymphocytes (PBLs) from 11 breast cancer patients treated with high doses of cyclophosphamide and cisplatin and provided autologous bone marrow transplantation after treatment. PBL specimens for the SCG study were obtained just prior to treatment, following the administration of cyclophosphamide and cisplatin for 2 days, and upon lymphocytic recovery. Based on a concurrent analysis of DNA damage in cryopreserved and noncryopreserved PBL samples from three patients, the mean level of DNA migration or the dispersion of damage among cells was not affected by the process of cryopreservation. The pre-treatment samples of several patients contained PBL with increased levels of DNA damage, presumably reflecting persistent DNA damage induced by previous treatment regimens. Chemotherapy resulted in a significant but variable increase in DNA damage in PBL samples from all patients. In this limited study, the level of damage did not correlate with serum levels of cyclophosphamide or with lymphocyte toxicity. Among the post-treatment samples, increased levels of DNA damage were absent in most but not all patients. The presence of damaged cells in the post-treatment samples may be indicative of an inadequate therapy regimen or of DNA damage resulting from non-therapy related processes. Because of its simplicity and short processing time, the SCG assay can be used to evaluate levels of DNA damage during the course of therapy, allowing the dose schedule to be altered to achieve a desired effect level.     

Measurement of salivary resistin, visfatin and adiponectin levels

Hormonal determination in saliva offers several advantages. Peptides enter the salivary glands either by active transport mechanisms or are expressed and secreted by the salivary glands themselves. The collection of saliva is a noninvasive, easily repeatable and less stressful technique than blood withdrawal. The purpose of the present study was to introduce a method for measuring salivary resistin, visfatin and adiponectin levels and to evaluate their associations with serum levels. Resistin, visfatin and adiponectin levels were measured in serum and saliva of 50 healthy adult volunteers (17 male and 33 female) using commercial enzyme immunoassay kits for serum with minor modifications. The present study documented the determination of resistin and adiponectin levels in saliva and the significant correlation of salivary levels with serum levels (r=0.441, p<0.01 and r=0.347, p<0.05, respectively). Moreover, the identification of visfatin in saliva was achieved, but no significant correlation with serum visfatin levels was observed. To our knowledge, this is the first study to report the determination of resistin and visfatin in saliva and the significant correlation of salivary resistin with serum levels, while it confirmed the significant association between salivary and serum adiponectin. The introduction of salivary determinations of adipokines could contribute to the elucidation of the physiology and the role of the specific adipokines in various clinical conditions (obesity, insulin resistance, inflammation, reproduction, energy imbalance and stress response).     

Phosphorus-31 NMR of neuroblastoma clonal lines

Phosphorus nuclear magnetic resonance is used to study changes in the levels of the major phosphate-containing intermediary metabolites concomitant with induced cellular differentiation in the N-18 and C-46 neuroblastoma clonal lines. The study reveals differences between the³¹P-NMR profiles of the two clonal lines and also striking differences attendant to dibutyryl cAMP-mediated morphological differentiation in the N-18 clone. Phosphorus-31 NMR would appear to provide a new technique with which to study genetic differentiation.     

Analysis of Age-Related Global DNA Methylation in Chicken

DNA methylation is an epigenetic modification that plays an important role in the normal development and function of organisms. The level of DNA methylation is species-, tissue-, and organelle-specific, and the methylation pattern is determined during embryogenesis. DNA methylation has also been correlated with age. The aim of this study was to determine the global DNA methylation levels and their correlation with age in the chicken, using a Polish autosexing chicken breed, Polbar. A quantitative technique based on an immunoenzymatic assay was used for global DNA methylation analysis. The results show increased global DNA methylation levels with older Polbar embryos. Global DNA methylation levels decrease with the age of hens in the postembryonic stage. This study expands the current knowledge of the Polbar epigenome and the general knowledge of the function of epigenetic mechanisms in birds.