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At least 307 records · Page 17Linked to original sources
PubMed2005

Miniaturized immunoassay microfluidic system with electrokinetic control.

A portable heterogeneous immunoassay system is presented in this paper. It consists of a poly(dimethylsiloxane) (PDMS) based microfluidic chip as the immunoreactor, a miniaturized programmable high voltage sequencer as the power supply and the flow controller, and a laser-optical fiber fluorescence detection module as the signal reader. The operation of this immunoassay system is automatic. The sequential reagent dispensing and washing processes are controlled by the programmable sequencer. The reagent consumption was only 12 microL and the assay time was only 26 min. The detection limit for Escherichia coli O157:H7 bacterial antigen with this miniaturized system was 0.3 ng/muL, lower than that obtained using fluorescence microscope in previous studies.

Antigens, Bacterial↗
PubMed2006

Long-term survival of an allografted submandibular gland in a miniature swine model given immunosuppressant drugs.

We used a model of allografts of submandibular glands in miniature swine to test the requirements of immunosuppressants for the survival of such grafts. Animals in the first group (n=6) were given no immunosuppressant, and the submandibular glands were rejected within 7 days. Those in the low-dose group (n=4) were treated with a low dose of cyclosporine, methylprednisolone, and azathioprine. The allografts were rejected on days 15, 17, 19, and 20. The animals in the high-dose group (n=6) were treated with a high dose of cyclosporin, methylprednisolone, and azathioprine. Three allografts were rejected, on days 35, 48, and 60. One animal died from anaesthetic problems on day 30 after transplantation, and there were no signs of rejection in the allograft. The other two allografts survived for 100 days with secretory function and no signs of rejection. After we discontinued the immunosuppression on day 100, the two remaining allografts were rejected on days 121 and 128. Blood tests and biopsy specimens of the heart, lung, liver, and kidneys were normal in all animals that were given immunosuppressants. To our knowledge, this study is the first to describe long-term survival of allografted submandibular glands with secretory function in miniature swine.

Animals↗
PubMed2004

Grafting miniature DNA binding proteins.

Miniature proteins serve as leads for biological and medicinal applications by positioning all amino acids necessary for biomolecular recognition on a compact protein structure. Protein grafting was recently used to create miniature helical proteins with high DNA binding affinity and specificity.

Amino Acids↗
PubMed2006

On-line hyphenation of flow injection, miniaturized capillary electrophoresis and atomic fluorescence spectrometry for high-throughput speciation analysis.

A hyphenated technique was developed for high-throughput speciation analysis by on-line coupling of flow injection (FI), miniaturized capillary electrophoresis (CE) and atomic fluorescence spectrometry (AFS). Two interfaces were used to couple all three systems: the first to couple FI and CE and the second to couple miniaturized CE and AFS. The first interface was a modified flow through chamber, connected to the FI valve with a piece of PTFE tube (0.1mm i.d.x 20 cm long). The capillary outlet was coupled to the AFS by using the second concentric "tube-in-tube" interface. Split sampling was achieved in the electrokinetic mode. Inorganic mercury (Hg(II)) and methylmercury (MeHg(I)) were taken as model analytes to demonstrate the performance of the developed hyphenated technique. A volatile species generation (VSG) technique was employed to convert the analytes from the CE effluent into their respective volatile species. Baseline separation of Hg(II) and MeHg(I) was achieved by CE in a 50 microm i.d.x 8 cm long capillary at 3.0 kV within 60s. The precisions (RSD, n=12) were in the range of 0.7-0.9% for migration time, 3.8-4.2% for peak area, and 2.1-3.5% for peak height. The detection limits were 0.1 and 0.2 microgmL(-1) (as Hg) for Hg(II) and MeHg(I) with a sample throughput of 60 samples h(-1). The recoveries of both mercury species in the water samples studied were in the range of 93-106%.

Electrophoresis, Capillary↗
PubMed2004

The miniature pig: a unique experimental model for Schistosoma japonicum infection.

As part of a search for good animal models for human schistosomiasis, two miniature pigs of the CLAWN strain (C-1, C-2) were inoculated percutaneously with 200 Schistosoma japonicum cercariae of the Chinese strain, and the subsequent infection was monitored parasitologically, pathologically and serologically. Egg excretion into feces began at 5 weeks post-infection (p.i.) and became pronounced from 8 weeks to 17-20 weeks p.i. The average number of eggs in 1 g feces of each pig at the peak period between 8 and 20 weeks were 288 and 277, respectively. C-1 and C-2 were killed and perfused at 27 and 47 weeks p.i. and adult worm numbers recovered were 35 and 15, respectively. C-2 had at least four pairs of viable mature worms but no detectable fecal eggs for a month before perfusion, suggesting that any produced eggs were not excreted into the feces during this period. Egg deposits associated with inflammatory reactions were observed by histological examination of the liver, spleen, pancreas, mesenteric lymph nodes, lung, and small intestine. This suggests that reduced fecal excretion of eggs into the feces did not correlate to reduced parasite numbers in the chronic phase of schistosomiasis. This is the first report showing the miniature pig to be a potential model for human S. japonicum infection.

Animals↗
PubMed2004

NO-1886 inhibits size of adipocytes, suppresses plasma levels of tumor necrosis factor-alpha and free fatty acids, improves glucose metabolism in high-fat/high-sucrose-fed miniature pigs.

The synthetic compound NO-1886 is a lipoprotein lipase activator that has been proven to be highly effective in lowering plasma triglycerides and elevating high-density lipoprotein cholesterol. Recently, we found that NO-1886 also had a plasma glucose-reducing action in high-fat/high-sucrose diet-induced diabetic rabbits. In the current study, we investigated the effects of NO-1886 on the morphology of adipocytes, plasma levels of tumor necrosis factor-alpha (TNF-alpha) and free fatty acids (FFA) in miniature pigs fed a high-fat/high-sucrose diet. Our results showed that feeding a high-fat/high-sucrose diet to miniature pigs increased the size of adipocytes, and the plasma levels of TNF-alpha, FFA, and glucose. This diet also induced insulin resistance and impaired the acute insulin response to glucose loading. Supplementing 1% NO-1886 to the high-fat/high-sucrose diet inhibited adipocyte enlargement, and suppressed plasma levels of TNF-alpha, FFA, and glucose. The decrease in plasma TNF-alpha and FFA was simultaneous with the decrease in plasma glucose. We also found an increased whole body glucose clearance and an increased acute insulin response to intravenous glucose loading by NO-1886 supplementation. These data suggest that NO-1886 improves the glucose metabolism in high-fat/high-sucrose diet-induced diabetic minipigs by decreasing fat deposit, and suppressing plasma TNF-alpha and FFA levels. Therefore, NO-1886 is potentially beneficial for the treatment of insulin-resistant syndrome.

Adipocytes↗
PubMed2001

Cytokine and lymphocyte profiles in miniature swine after oral infection with Toxoplasma gondii oocysts.

Pigs are considered an important source of Toxoplasma gondii infection for humans. A major strategy for immune prophylaxis of toxoplasmosis in swine is the understanding of the immune response against T. gondii infection. The phenotype of peripheral blood mononuclear cells (PBMC) and the kinetics of interferon-gamma (IFN-gamma), interleukin-12 (IL-12) and interleukin-10 (IL-10) transcriptional changes were characterised in miniature swine following infection. A total of 66, 4-9-month-old miniature swine were used for three experiments performed over a period of 2 years. All pigs were fed iota1000 oocysts of the VEG strain of T. gondii and blood samples were obtained on the day of inoculation and at days 3, 6, 10, 17, 25, 32 and 40 after infection. An increase in expression of activation markers CD25 and SLA-DQ was detected in the first week of infection. A significant increase in the percentage of CD8+cells was observed in the second week of infection. Relative competitive RT-PCR analysis indicated an increase in IFN-gamma mRNA as well as a reduction in IL-10 mRNA during the second week post infection. Increase in IL-12 transcription was not observed until the fourth week of infection. The ability of the pigs to respond to T. gondii infection by simultaneously inducing pro-inflammatory cytokines early and anti-inflammatory cytokines later is a likely indication of the requirement to strike a balance between controlling parasite growth and avoiding cytokine toxicity.

Administration, Oral↗
PubMed2001

Miniaturized automated matrix solid-phase dispersion extraction of pesticides in fruit followed by gas chromatographic-mass spectrometric analysis.

In this study a simple and fast miniaturized automated matrix solid-phase dispersion method for the sample preparation and quantitative extraction of pesticides was developed and evaluated. Only 25 mg of sample and 100 microl of organic solvent were used per analysis for this new miniaturized set-up. The extracts were subsequently analysed by GC-MS without any further purification. The method was optimized for oranges and tested for the determination of a variety of organophosphorus pesticides and a pyrethroid at concentration levels below the maximum residue levels set by the European Union and authorities in The Netherlands. The limits of detection were 4-90 microg/kg. The recoveries for pesticides in orange were 83-118% and the relative standard deviations for the total procedure were 10-13% (n=4) at the limit of quantification. The feasibility of the developed method for apple, pear and grapes was also studied. Equally good results were obtained, but for apple the washing step should be omitted.

Automation↗
PubMed1997

A miniature CCD video camera for high-sensitivity light measurements in freely behaving animals.

We developed a miniaturized, high-sensitivity camera that can be placed in areas of difficult access in freely behaving animals for neural tissue imaging. The device consists of a charged coupled device (CCD) chip, a coherent image conduit and miniature light emitting diodes (LEDs). An amplifier circuit is constructed on the camera chip and nine wires are attached for external connections. Placement of LEDs around the image conduit perimeter provides dark-field illumination, which increases detection of cellular-related light scattering changes and doubles the depth-of-view over conventional reflectance imaging procedures. The device has been successfully used to record from several deep brain structures, including the ventral medullary surface of sleeping and waking cats. The procedure allows assessment of light scattering changes that result from neural activity or detection of vital dyes to metabolic or voltage-induced activation.

Analysis of Variance↗
PubMed2002

High-throughput miniaturized immunoassay for human interleukin-13 secreted from NK3.3 cells using homogenous time-resolved fluorescence.

A miniaturized immunoassay for human interleukin-13 (IL-13) using homogeneous time-resolved fluorescence (HTRF) has been developed. In this assay, IL-13 which was secreted from NK3.3 cells stimulated with interleukin-2 (IL-2) was detected by measuring the time-resolved fluorescence after adding a mixture of three reagents, biotinylated anti-IL-13 monoclonal antibody, europium cryptate (fluorescence donor)-labeled different anti-IL-13 monoclonal antibody and crosslinked allophycocyanin (fluorescence acceptor)-conjugated with streptavidin in a 384-well assay plate. The detection limit of IL-13 using this immunoassay was estimated to be less than 600 pg/ml and IL-13 levels measured by this method were very close to those measured by enzyme linked immunosorbent assay (ELISA; the correlation coefficient was 0.9535). The proposed assay requires only a fourth of the quantities of all reagents compared with the assay using a conventional 96-well microtiter plate. Furthermore, there is no need to transfer the culture supernatant to another assay plate and wash the plate. Therefore, this miniaturized immunoassay is economical and efficient and is particularly suitable for high-throughput drug screening.

Antibodies, Monoclonal↗
PubMedDate not supplied

Intraoperative epicardial echocardiography with a miniature high-frequency transducer: imaging techniques and scanning planes.

Intraoperative Doppler echocardiography has the potential to provide anatomic and functional information but is hindered by the large size of standard transducers. We describe new scanning planes accessible through the application of a new 5 MHz miniature transducer with imaging, Doppler, and color-flow capability. Epicardial echocardiography was performed in 15 adults undergoing elective coronary artery bypass grafting. Standard parasternal equivalent, subcostal equivalent, aortopulmonary sulcus and aortosuperior vena caval views were obtained. Previously unobtainable apical four-chamber, five-chamber, and long-axis views were possible by positioning the transducer at the apex. The transducer has a broad bandwidth, allowing high-quality imaging at different depths and could be maneuvered laterally, posteriorly, and over the aorta and pulmonary arteries to provide off-axis views. The proximal-mid coronary arteries were imaged on the beating heart with a standoff medium. Transducer miniaturization should expand the role of epicardial ultrasonography in the surgical management of heart disorders.

Adult↗
PubMed2002

The effect of estrogens and dietary calcium deficiency on the extracellular matrix of articular cartilage in Göttingen miniature pigs.

Clinical observations have suggested that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis (OA). However, positive and negative associations between the incidence of OA and serum estrogen concentrations have been reported. In contrast to this, osteoporosis is regarded as a disease with a strong estrogen-dependent component. Moreover, there is an interaction between estrogen and calcium deficiency: calcium supplementation potentiates the effect of estrogen therapy. The present study was designed to investigate how estrogen deficiency affects the articular cartilage depending on calcium supply. The distribution of different types of glycosaminoglycans and collagens can be used as an indicator for extracellular matrix changes induced by estrogen deficiency. Different levels of dietary calcium were therefore fed to intact and ovariectomized Göttingen miniature pigs for one year before articular cartilage was harvested. The histochemical staining for heavy sulfated glycosaminoglycans in the extracellular matrix of ovariectomized miniature pigs, especially of those fed with a low calcium diet, was stronger in comparison to intact animals. In intact animals type II-collagen was immunodetected in all zones of unmineralized and mineralized articular cartilage, while immunostaining for this protein was negative to weak in the deep radiated fiber zone of ovariectomized minipigs. These results suggest that the synthesis of heavy sulfated glycosaminoglycans and immunohistochemically detectable type II-collagen is possibly influenced by estrogen deficiency. In conclusion, under estrogen deficiency, the extracellular matrix of articular cartilage underwent similar changes to those observed in physiologically aging cartilage where keratan sulfate is increased as a heavy sulfated glycosaminoglycan.

Animals↗
PubMedDate not supplied

Prototype miniature endoluminal MR imaging catheter.

PURPOSE: The feasibility of a miniature endoluminal magnetic resonance (MR) detection coil was investigated for imaging mural and perimural anatomy of small, tubular structures. MATERIALS AND METHODS: To this end, remotely tunable, single-loop, multiturn, receive-only radio-frequency coils, housed in 6-9-F arterial sheaths, were built. A 1.9-T imager was used. Phantom excitation was accomplished with a 62-mm-diameter bird-cage quadrature coil, and ex vivo specimen excitation was accomplished with a single-turn, untuned wire loop. Phantom images obtained with use of a 9-F catheter coil showed a signal-to-noise improvement on the border of 20 dB compared with images obtained with the quadrature coil. An 8-F catheter coil was used to obtain high-resolution (100 microns in-plane pixel size, 500 microns section thickness) spin-echo images (repetition time = 2,400 msec, echo time = 53 msec) of the wall of a fresh ex vivo human popliteal artery. RESULTS: Prospectively, these images were suggestive of the presence of diffuse intimal hyperplasia, medial calcification, and focal atherosclerotic plaque. These findings were confirmed histologically. Three-dimensional restacking of the axial images simplified examination of the normal layers and pathologic changes within the wall. The improved signal-to-noise characteristics of these miniature coils permit fast high-resolution imaging, allowing visualization of microscopic anatomic details. CONCLUSIONS: With further development, this technology may be useful for studying atherosclerosis and for providing imaging guidance during endoluminal MR interventions.

Arteries↗
PubMed2004

Detection of regulatory cells as an assay for allograft tolerance in miniature swine.

BACKGROUND: There is currently a great need for an in vitro assay to assess the presence of tolerance following allotransplantation to determine whether immunosuppressive medications can be discontinued. Our laboratory has recently developed an assay involving coculture inhibition of cell-mediated lympholysis that correlates with tolerance to allografts in swine leukocyte antigen (SLA) Class I-mismatched miniature swine. The potential for clinical application of this assay may depend on 2 important factors: (1) whether the assay can be used in the presence of immunosuppression; and (2) whether frozen-stored naive responder cells can be utilized. METHODS: Long-term tolerant MGH miniature swine that had accepted SLA Class I-mismatched kidney transplants after a 12-day course of cyclosporine or tacrolimus were studied. Two long-term tolerant and 2 naive control animals were treated with a clinically relevant dose of cyclosporine for 2 weeks (trough level 100 to 400 ng/ml) to simulate the ongoing "chronic" immunosuppression used in human recipients of allografts. Cells from tolerant or naive, recipient-matched animals were stimulated for 6 days with donor or third-party SLA. These primed cells were then cocultured with naive unstimulated recipient major histocompatibility complex (MHC)-matched responders and irradiated stimulators. Responder cells were tested both fresh and frozen. RESULTS: Suppression of cytotoxic responses of naive responder cells was observed in all coculture assays using cells from tolerant animals primed against donor antigen in vitro, but not in assays using similarly primed cells from naive animals. Responder cells from tolerant animals receiving immunosuppression had a suppressive activity similar to that from cells of the same animals not receiving immunosuppression. Similar suppression was also observed in coculture assays using either fresh or frozen naive responder cells. CONCLUSIONS: This coculture assay appears to correlate with the presence of tolerance under conditions applicable to the clinical setting. The assay appears to identify peripheral regulatory mechanisms of tolerance in allogeneic transplant recipients, and therefore may provide an approach for determining an appropriate timepoint at which to test withdrawal of immunosuppressive medications.

Animals↗
PubMed2004

One- and two-dimensional miniaturized electrophoresis of proteins with native fluorescence detection.

Miniaturized electrophoresis was successfully coupled with native fluorescence detection for direct analysis of proteins in one- and two-dimensional separations. The detection setup was based on direct observation of the UV-induced fluorescence of proteins using a CCD camera and a Hg (Xe) lamp for sample excitation. Protein mixtures were readily separated by size on a 1-cm segment of the one-dimensional gel in 8 min, and a detection limit of 0.04 ng per band was achieved. The dynamic range of the system was larger than 2 orders of magnitude. Miniaturized slab gel electrophoresis was performed on a special holder designed to couple isoelectric focusing with SDS-PAGE. Two-dimensional separation, including rehydration of IEF strip and fluorescence detection was completed in 2.5 h. Approximately 200 protein spots from Escherichia coli were detected on a 1 cm(2) area. A detection limit of 0.1 microg of total protein was achieved. The operation should be amenable to total automation.

Animals↗
PubMed1997

Miniaturized supported liquid membrane device for selective on-line enrichment of basic drugs in plasma combined with capillary zone electrophoresis.

A hollow fiber miniaturized supported liquid membrane (SLM) device for sample preparation is connected on-line with capillary electrophoresis and used for determination of a basic drug, bambuterol, in human plasma. The analyte is extracted from the outside of the hollow fiber (donor) through the liquid membrane (pores of the fiber impregnated with organic solvent) into the acceptor solution in the fiber lumen. The process is driven by differences in pH between the donor and acceptor solution. The whole volume of the acceptor solution can then be injected into the CZE capillary by using the double-stacking procedure for large volume-injection. Very clean extracts of low ionic strength are obtained from the SLM treatment, making this sample pretreatment method compatible with the CZE double-stacking procedure, which in turn makes it possible to inject large volumes of sample onto the separation capillary. Good performance of the whole procedure is demonstrated, and detection limits in the low nanomolar range were obtained in spite of the relatively weak UV absorbance of bambuterol. Extractions through the miniaturized SLM unit can be performed for 5-6 h without regenerating the fiber. The regeneration procedure was tested, and no relevant changes in the performance of the extraction could be found after seven regenerations, allowing the same fiber to be used for a week.

Electrophoresis, Capillary↗
PubMed1997

Miniaturized ion-selective chip electrode for sensor application.

The performance of miniaturized potentiometric cells, with multilayer, planar ion-selective sensors in aqueous electrolyte solutions, human serum, urine, and whole blood, is presented. The basic steps of the fabrication with silicon technology are summarized. The effect of the contact surface between the internal reference system and the ion-sensitive membrane on the analytical characteristics of potassium- and calcium-sensitive sensors is studied. Silicone rubber-, high molecular weight PVC-, carboxylated PVC and aliphatic polyurethane (Tecoflex)-based solvent polymeric membranes were dispensed into anisotropically etched wells on silicon wafers, and the resulted planar sensors were tested in terms of their ion sensitivity (slopes of the cell voltage-pK or pCa calibration curves), long-term stability, and reproducibility. For the assay of potassium in whole blood, the miniaturized potentiometric cell was built in a flow-through manifold. To achieve the required precision, the flow conditions were optimized and the sensors calibrated periodically. The results prove the feasibility of the new sensor design and satisfy the particularly difficult requirements for the analysis of biological samples.

Biosensing Techniques↗
PubMedDate not supplied

New miniaturized hollow-fiber bioreactor for in vivo like cell culture, cell expansion, and production of cell-derived products.

We have developed a miniaturized hollow-fiber bioreactor system for mammalian cell culture with a volume of 1 mL. Cell and medium compartments of the bioreactor are separated by a semipermeable membrane, and oxygenation of the cell compartment is accomplished using an oxygenation membrane. As a result of the geometry of the transparent housing, cells can be observed by microscopy during culture. The leukemic cell lines CCRF-CEM, HL-60, and REH were cultivated up to densities of 3.5 x 10(7)/mL without medium change or manipulation of the cells. As shown using CCRF-CEM cells, growth in the bioreactor was strongly influenced and could be controlled by the medium flow rate. As a consequence, consumption of glucose and generation of lactate varied with flow rate. Depending on the molecular size cutoff of the membranes used, added growth factors such as GM-CSF, as well as factors secreted from the cells, are retained in the cell compartment for up to 1 week. This new miniaturized hollow-fiber bioreactor offers advantages in tissue engineering by continuous nutrient supply for cells in high density, retention of added or autocrine produced factors, and undisturbed long-term culture in a closed system.

Animals↗