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PubMed2004

Development of miniaturized sample preparation with fibrous extraction media.

Introducing fine polymeric filaments as the extraction medium, a miniaturized sample preparation technique for micro-column liquid chromatography (micro-LC) has been developed along with the investigation of a reproducible preparation scheme of the extraction capillary. The polymeric filaments were packed longitudinally into either a fused-silica capillary or a polyether ether ketone (PEEK) capillary of appropriate dimensions, and the extraction capillary was installed to the injection valve in micro-LC system. The number of packed filaments should be precisely counted before the packing process to make sure the reproducible preparation of the extraction capillary. With conventional stationary phase materials for open-tubular gas chromatography, polymeric coating to the surface of the filaments was also studied in order to further enhance the extraction performance and selectivity. Coated with the polymeric material suitable for the extraction of particular analyte, a dramatic improvement on the extraction power was obtained. The results suggest that the future possibility of novel tailored fibrous extraction medium with an appropriate coating on it, especially for the analysis of complex sample matrices.

Chromatography, Liquid↗
PubMed2005

Development of miniaturized multi-channel high-performance liquid chromatography for high-throughput analysis.

We have developed miniaturized multi-channel high-performance liquid chromatography (HPLC) system. With this system, we can simultaneously separate multiple samples, using a single high-pressure gradient pump, a chip-based sample injection unit, a monolithic silica capillary column array, and a multi-channel UV detection unit based on fiber optics. The injection unit has a simplified structure composed of brass housing and a quartz microchip having microchannels and access ports, which enable a direct injection of sample to multi-channel by commercial multichannel micropipette. Moreover, that possesses a function of microvalve, and on-chip definition of sample injection plugs achieved with a cross channel injection method, providing each column of monolithic silica capillary array. The substances in channels were simultaneously detected with UV having multiple cells. Standard samples were analyzed for characterizing newly developed system, and sharp peaks were obtained with reproducibility data of < 0.9% (R.S.D.). Analysis of tryptic digestion of casein was also employed. These results show that the novel multi-channel HPLC system has the benefits for the high-throughput analysis in the post-genomic analysis/combinatorial chemistry.

Chromatography, High Pressure Liquid↗
PubMed2005

Structural and functional characteristics of irradiation damage to parotid glands in the miniature pig.

PURPOSE: To evaluate the effects of a solitary megadose protocol of ionizing radiation (IR) on the structure and function of the miniature pig (minipig) parotid gland. METHODS AND MATERIALS: Fourteen minipigs were subjected to either 15 or 20 Gy to one parotid gland with a linear accelerator, whereas another four minipigs served as non-IR controls. Salivary flow rates and salivary chemistries were measured pre-IR and 4 and 16 weeks post-IR. A quantitative assessment of gland weight and acinar area and detailed serum chemistry and hematologic analyses were also performed. RESULTS: Parotid flow rates decreased by approximately 50% either with 20 Gy at 4 weeks, or 15 Gy at 16 weeks post-IR. In the 20 Gy group, salivary flow rates were reduced by approximately 80% at 16 weeks post-IR. A significant decrease in salivary calcium and amylase and an increase of salivary potassium levels were found in both IR groups. There were also transient alterations in serum chemistry and hematology parameters post-IR. Parotid gland weights were significantly decreased (-50%) in the 15 and 20 Gy groups at 4 and 16 weeks post-IR. Additionally, the acinar cell area in glands of both IR groups was significantly reduced from that in control glands at both the 4 and 16 weeks time points. CONCLUSION: Structural changes in salivary gland parenchyma occurred relatively early after IR, whereas the alterations in salivary output were relatively delayed. Further, reductions in salivary flow were not proportional to acinar cell area loss. Together, these findings suggest that nonparenchymal IR damage likely contributes to IR-induced salivary hypofunction.

Amylases↗
PubMed2006

Measurement of blast wave by a miniature fiber optic pressure transducer in the rat brain.

Exposure to blast wave that is generated during an explosion may result in brain damage and related neurological impairments. The aim of this study was to investigate pressure changes induced by exposure to blast inside the rat brain. For intracranial pressure measurement we used a miniature optic fiber sensor (o.d. 550 microm) with a computer recording system. The sensor was placed in the third cerebral ventricle of anesthetized rats exposed to 40 kPa blast wave in a pneumatic-pressure driven shock tube. Short pressure waves lasting several ms were detected inside the brain with the magnitude that might result in nervous tissue damage.

Animals↗
PubMed2005

A comparison of synovial fluid pressure after immediate versus gradual mandibular advancement in the miniature pig.

PURPOSE: Mandibular advancement is a commonly performed surgical procedure for the treatment of mandibular hypoplasia. With the increased use of rigid fixation, there has been a decrease in the amount of relapse but an increase in the amount of force transmitted to the condyles. Gradual advancement of the mandible by distraction osteogenesis slowly overcomes the soft-tissue envelope and may decrease the amount of force exerted on the condyles. The purpose of this study was to develop an animal model to measure the magnitude of pressure associated with immediate versus gradual mandibular advancement. MATERIALS AND METHODS: A 2.0-mm pressure transducer was placed in the superior joint space in 2 miniature pigs. In the first animal, immediate advancement of the mandible with rigid fixation was performed. The synovial fluid hydrostatic pressures were measured prior to surgery and postoperatively. A second animal underwent gradual advancement with distraction osteogenesis. The synovial fluid hydrostatic pressures were measured prior to and after each activation of the distraction device. The condyles were examined radiographically and microscopically. RESULTS: The superior joint space fluid pressures increased and remained elevated over a 5-week period after immediate advancement. In the gradually advanced mandible, the pressures were elevated but returned to near baseline prior to the activation the following day. CONCLUSION: This animal model is useful to directly measure the pressure that is exerted on the condyle. This will allow further studies to compare methods for mandibular advancement. It is likely that gradual advancement of the mandible by distraction osteogenesis produces less force and causes less condylar resorption than large mandibular advancement stabilized with rigid fixation.

Animals↗
PubMed2004

Effect of exercise on postprandial lipemia following a higher calorie meal in Yucatan miniature swine.

Exercise has been shown to attenuate the postprandial lipemic (PPL) response to a modest kcal high-fat meal in numerous human studies, but has not been fully examined in swine. In addition, the effects of exercise on a high-fat meal of larger magnitude have not been examined in humans or in swine. Thus, the purpose of this study was to examine the PPL response to a single, high-fat/cholesterol (HFC) meal (approximately 3,000 kcal, 1,300 kcal from fat) and determine if exercise attenuates the PPL response. Sedentary, female Yucatan miniature swine (n = 10) completed 3 PPL trials: (1) pre diet (PRE); (2) post HFC diet (POST); and (3) post HFC diet plus exercise (EX, 45 minutes at 75% heart rate maximum). Blood samples were collected before (0 hour) and at 2, 4, 6, and 8 hours after the single HFC meal for PPL analysis. Postheparin lipoprotein lipase (LPL) activity was assessed at 8 hours. While fasting LPL activity was significantly increased with the HFC diet, the PPL response to the HFC meal did not differ depending on diet. Furthermore, the PPL response was not significantly altered with a single session of exercise, perhaps because of the severity of the HFC meal, the sedentary nature of the swine, or because LPL activity was not elevated after exercise. These findings suggest that administration of a HFC meal of this magnitude (approximately 3,000 kcal, 1,300 kcal from fat) will promote significant elevations in postprandial triglyceride (TG) concentrations, overwhelm the adaptive response to a HFC diet (elevated LPL activity), and attenuate the beneficial effects of a single exercise session on this system.

Animals↗
PubMed2006

Miniature array postdetection-encoded MRI.

A method for performing nuclear magnetic resonance (NMR) measurements simultaneously from more than a single radiofrequency (RF) coil is presented. The method employs the detection of magnetic resonance signals in an array of detectors, where each detector is responsible for detecting a unique frequency bandwidth or a magnetic resonance signal from a unique location in a region in a primary, substantially homogeneous, static magnetic field. The detectors may be separated logically into groups, whereby all the detectors in a given group are essentially RF-decoupled from each other to substantially eliminate cross-talk by switching circuits or by being placed from each other sufficiently remotely. Sampling of detected signals from detectors in this array is done simultaneously over groups of noninteracting detectors. The detected signals from all detectors in a given group are simultaneously transmitted to a single preamplifier, thus increasing significantly the signal-to-noise ratio (SNR) in that preamplifier. Prior to transmitting each detected NMR signal of each detector to the preamplifier, each detected signal is separately and uniquely encoded electronically. This provides a method whereby the signal of each detector is uniquely encoded. Accumulating all these encoded signals, which were simultaneously received in a number of RF detectors into a single amplifier, results in the total signal having a high SNR ratio. This total amplified signal is later decoded into each detector's original signal by a decoding circuitry. Conventional magnetic resonance imaging (MRI) techniques may be thereafter applied to obtain an image. Or else, conventional NMR techniques may be thereafter applied to obtain an improved SNR from a sample, using a single preamplifier with a multitude of detectors. Applying this method to a large number of miniature and closely packed RF detectors placed in an array-like configuration results in an MRI technique with a very fast acquisition time, an increased SNR and a high spatial resolution equivalent to the number of detectors per unit of length. Deblurring and decoupling algorithms allow for images from layers as deep as 6 mm to be acquired.

Algorithms↗
PubMed2005

Temporal characteristics of feeding behavior in the Munich miniature pig.

UNLABELLED: Correlations between meal size and inter-meal intervals as a characteristic of ingestive behavior are generally calculated utilizing Pearson's correlation coefficient. However, this commonly used method may exhibit disadvantages and methodological problems when dealing with dependent time series. Alternatively, a modified version of Pearson's r (r(diff)) or a nonparametric procedure, Pfanzagl's T combined with an expanded sampling theorem offer a statistical alternative for correlational analysis, robust to stochastic interdependencies between time series. In two studies, porcine self-initiated feeding behavior was observed in a closed economy setup, and a comparison of all three correlation coefficients was performed. STUDY 1: Twelve Munich mini-pigs were observed for 2 weeks. The rate of food ingestion was a stable characteristic of each individual pig, feeding and defecation behavior were temporally associated and light/dark cycle differences were seen for feeding. STUDY 2: Eight Munich mini-pigs were studied for 4 weeks. Kendall's Tau was utilized to test the stability of feeding behavior across weeks. Stability increased over time and feeding was more stable during the light period. Both correlation measures revealed a strong association between meal size and inter-meal intervals. CONCLUSION: Munich-miniature pigs exhibited a stable pattern of self-initiated ingestive and excretory behaviors. In both studies, Pfanzagl's T-values as well as r(diff)-values revealed a corresponding and consistent association between meal size and inter-meal intervals. In combination with the conventional Pearson's r, all three coefficients characterize different aspects of feeding behavior.

Animals↗
PubMed2004

Photothermal spectrometry for detection in miniaturized systems: relevant features, strategies and recent applications.

There is a growing interest in using miniaturized analytical devices because they allow to execute the different steps of an analytical process within very short times and with drastic reduction in the amounts of solvents, reagents and samples. As for capillary electrophoresis, these systems require detectors which are sensitive, versatile and adaptable to very small detection volumes. In this respect, photothermal spectrometry which is complementary to fluorescence seems to be a promising detection method. This review describes the basic principle of photothermal spectrometry along with the related methods based on colinear-beam or crossed-beam configuration of the pump and probe lasers. Two experimental set ups especially designed for microfluidic systems as well as for capillary electrophoresis are described. Their characteristics and key features are discussed and the main applications are outlined.

Electrophoresis, Capillary↗
PubMed2006

The role of indirect recognition of MHC class I and II allopeptides in a fully mismatched miniature swine model of lung transplantation.

UNLABELLED: Considerable evidence suggests that indirect recognition of MHC allopeptides plays an important role in solid-organ rejection. Here, we examine whether immunization with class I or class II allopeptides accelerates rejection in a fully MHC-mismatched lung transplant model in miniature swine. METHODS: Recipients were immunized with either donor-derived class I or class II peptides. Sensitization to the peptides was confirmed by DTH testing and in vitro proliferation assays. Nonimmunized control (n = 6), class I peptide-immunized (n = 3), and class II peptide-immunized (n = 3) swine were transplanted with fully mismatched lungs using only a 12-day course of tacrolimus. RESULTS: One control animal rejected its graft on postoperative day 103, while the others maintained their grafts for over 1 year. In the class I peptide-immunized group, two recipients rejected their grafts (days 14 and 52). The third animal has not rejected the graft (day 120, experiment is ongoing). In contrast, in the class II-peptide immunized group, only one animal rejected its graft on day 52, while the others maintained their grafts over 1 year. Both anti-donor IgM and IgG antibodies were detectable in all acute rejectors, although no alloantibody was detectable in long-term acceptors. Regardless of the fate of the graft, all animals have maintained their proliferative responses to the peptides. However, only acceptors maintained donor-specific hyporesponsiveness in cell-mediated lymphocytotoxity and mixed lymphocyte reaction assays. CONCLUSIONS: Pretransplant sensitization of lung allograft recipients to donor allopeptides accelerates graft rejection. This appears particularly true for class I-derived allopeptides, suggesting that class II molecules may be less antigenic when presented indirectly.

Animals↗
PubMed2006

Thymectomy does not abrogate long-term acceptance of MHC class I-disparate lung allografts in miniature Swine.

UNLABELLED: We have previously reported that tolerance to class I disparate lung allografts in miniature swine could be induced using an intensive 12-day course of tacrolimus and that pretransplant sensitization with immunogenic MHC class I allopeptides failed to block the induction of tolerance. We also have previously reported the importance of the presence of the thymus in the induction of tolerance to isolated heart, kidney, and combined heart-kidney transplants. In this study, we examined the impact of thymectomy on tolerance induction in lung transplantation. METHODS: Orthotopic left lung transplantation was performed using MHC class I-disparate donors. The recipients received a 12-day course of high-dose tacrolimus (n = 6). Total thymectomies were performed in three of the swine 21 days prior to transplantation. Lung grafts were monitored by chest radiography and serial open lung biopsy. RESULTS: All euthymic recipients maintained their grafts for over 1 year. None of the thymectomized recipients has experienced graft loss in the 6 to 10 months following transplantation. Although isolated lesions of obliterative bronchiolitis were occasionally seen in one thymectomized animal on biopsy, donor-specific unresponsiveness has been observed on assays of cell-mediated lymphocytotoxicity in all recipients. Moreover, co-culture assays have shown that recipient lymphocytes can strongly inhibit the normally robust response of naïve recipient-matched lymphocytes to donor antigen. This inhibition was not seen when using stimulators primed with third-party antigens against appropriate targets. CONCLUSIONS: These data suggest that thymus-independent peripheral regulatory mechanisms may be sufficient to induce and maintain long-term acceptance of the lung allografts.

Animals↗
PubMed2006

An MHC class II disparity raises the threshold for tolerance induction in pulmonary allografts in miniature swine.

OBJECTIVES: The mechanisms and treatment of chronic rejection in pulmonary allotransplantation remain elusive. We have induced robust tolerance to class I-disparate lung allografts in miniature swine using an intensive 12-day course of tacrolimus. Here, we tested whether a tolerant state can be induced in swine receiving fully mismatched lung allografts. METHODS: Orthotopic left lung allografts were performed using MHC class I-disparate (group 1: n = 3) or fully disparate (group 2: n = 6) donors. The recipients received a 12-day postoperative course of tacrolimus (continuous intravenous infusion; target level = 35-50 ng/mL) as their only immunosuppression. RESULTS: All swine in group 1 maintained their grafts long term without developing any lesions of chronic rejection (>497, >432, >451 days). These recipients exhibited donor-specific hyporesponsiveness in cell-mediated lymphocytotoxity (CML) and mixed lymphocyte reaction (MLR) assays. In group 2, five of the six recipients maintained their grafts long term (sacrificed on postoperative days 515, 389, 429, 481, and 438 with viable grafts). Isolated lesions of obliterative bronchiolitis were occasionally seen on biopsy, and donor-specific hyporesponsiveness on assays was consistently observed. The remaining recipient rejected its graft on day 103 with histologic findings of obliterative bronchiolitis. CONCLUSIONS: We report long-term graft acceptance without chronic immunosuppression in five of six recipients across a full MHC disparity, albeit with some evidence of obliterative bronchiolitis. These data suggest that the class II disparity inherent in a fully mismatched transplant increases the requirement for tolerance induction.

Animals↗
PubMed2005

Miniaturized ultrasound arrays for interstitial ablation and imaging.

A potential alternative to extracorporeal, noninvasive HIFU therapy is minimally invasive intense ultrasound ablation that can be performed laparoscopically or percutaneously. An approach to minimally invasive ablation of soft tissue using miniaturized linear ultrasound arrays is presented here. Recently developed 32-element arrays with aperture 2.3 x 49 mm, therapy frequency 3.1 MHz, pulse-echo bandwidths >42% and surface acoustic energy density >80 W/cm2, are described. These arrays are integrated into a probe assembly, including a coupling balloon and piercing tip, suitable for interstitial ablation. An integrated electronic control system allows therapy planning and automated treatment guided by real-time interstitial B-scan imaging. Image quality, challenging because of limited probe dimensions and channel count, is aided by signal processing techniques that improve image definition and contrast, resulting in image quality comparable to typical transabdominal ultrasound imaging. Ablation results from ex vivo and in vivo experiments on mammalian liver tissue show that this approach is capable of ablation rates and volumes relevant to clinical applications of soft tissue ablation such as treatment of liver cancer.

Catheter Ablation↗
PubMed2006

Identification of novel foot-and-mouth disease virus specific T-cell epitopes in c/c and d/d haplotype miniature swine.

To identify foot-and-mouth disease virus (FMDV) specific T-cell epitopes within the non-structural protein 3D in swine, pentadecapeptides were tested in proliferation and Interferon-gamma ELISPOT assays using lymphocytes from two strains of inbred miniature pigs (c/c and d/d haplotype) experimentally infected with FMDV. Lymphocytes of c/c pigs recognized peptides from three different regions in 3D, d/d lymphocytes recognized peptides from two regions, one of them being adjacent to an epitope of c/c pigs and comprising amino acid residues 346-370. Analyses of the response of d/d lymphocytes against peptides representing the structural protein 1A revealed another novel T-cell epitope. Investigation of the phenotype of responding lymphocytes showed a response of CD4(+)CD8(+)MHC-class-II(+) cells, identifying them as activated T-helper cells. This is the first report on FMDV specific T-cell epitopes recognized by swine leukocyte antigen (SLA) inbred swine and provides information useful for the design of novel vaccines against FMDV.

Amino Acid Sequence↗
PubMed2005

Increased fluid secretion after adenoviral-mediated transfer of the human aquaporin-1 cDNA to irradiated miniature pig parotid glands.

The treatment of most head and neck cancer patients includes ionizing radiation (IR). Salivary glands in the IR field suffer irreversible damage. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat submandibular glands following IR restored salivary flow to near normal levels. It is unclear if this strategy is useful in larger animals. Herein, we evaluated AdhAQP1-mediated gene transfer after parotid gland IR (20 Gy) in the miniature pig. Sixteen weeks following IR, salivation from the targeted gland was decreased by >80%. AdhAQP1 administration resulted in a dose-dependent increase in parotid salivary flow to approximately 80% of pre-IR levels on day 3. A control Ad vector was without significant effect. The effective AdhAQP1 dose was 2.5 x 10(5) pfu/microl infusate, a dose that leads to comparable transgene expression in murine and minipig salivary glands. Three days after Ad vector administration little change was observed in clinical chemistry and hematology values. These findings demonstrate that localized delivery of AdhAQP1 to IR-damaged salivary glands increases salivary secretion, without significant general adverse events, in a large animal model.

Adenoviridae↗
PubMed1996

Use of miniature biplane transesophageal echocardiography during pediatric atrial catheter interventional procedures.

We reported the use of a new miniature biplane TEE probe during pediatric cardiac interventional catheterization procedures. Use of this imaging modality provided significant advantages during dilation of obstructed venous pathways and closure of interatrial defects. Procedural characteristics and specific congenital heart lesion-related advantages were discussed.

Cardiac Catheterization↗
PubMed2002

Movement of temporomandibular joint tissues during mastication and passive manipulation in miniature pigs.

Movement is an important aspect of the biomechanics of the temporomandibular joint (TMJ). To track the relative movements of TMJ components, radio-opaque markers were implanted in the left squamosal bone, mandible and retrodiscal tissue of miniature pigs. Medial-lateral (ML) and dorsal-ventral (DV) fluoroscopic records were made 8-10 weeks later during chewing and passive manipulation. Marker movements were digitized from the videotapes. During passive manipulation, the deformation of the lateral capsule was also measured with a differential variable-reluctance transducer. The results provide new details about porcine chewing pattern, which is distinguished by a regularly alternating chewing side. During masticatory opening, the mandible had a centre of rotation (CR) well inferior to the condyle and close to the angle. In contrast, the passive opening movement showed a higher CR location close to the condylar neck, indicating a different motion from masticatory opening. The retrodiscal tissue followed the movements of the mandibular condyle during both mastication and passive manipulation. The lateral capsule elongated during ipsilateral shifts and retrusion, implying a possible role in limiting such movements. These movement characteristics provide a useful reference for studies on the TMJ using pigs.

Animals↗
PubMed2000

Bone surface strains and internal bony pressures at the jaw joint of the miniature pig during masticatory muscle contraction.

The long-standing debate on whether the jaw joint is loaded is due in part to the complexity of the factors involved, including a number of different muscles, each with a potentially unique role. This study sought to elucidate how two major jaw muscles, the masseter and the lateral pterygoid, influence jaw-joint loading. Twenty-five 10-month-old miniature pigs were divided into two groups, controls and pigs with the lateral capsular ligament of the jaw joint stripped surgically; this was expected to affect loading by destabilizing the joint. Rosette strain gauges were bonded to: (1) the lateral surface of the squamosal bone (equivalent to the squamosal portion of the temporal bone in humans) at the level of the articular eminence; (2) the lateral surface of the condylar neck; and (3) the lateral surface of the mandibular corpus below the molar region. Semiconductor pressure transducers were placed underneath the surfaces of the anterior slope of the condyle and the posterior slope of the articular eminence through drilled holes. Strains and internal bony pressures were recorded during stimulated tetanic contractions of the masseter or lateral pterygoid muscles. Masseter contraction, either alone or with the contralateral muscle, caused net tension in the squamosal bone and net compression in the condylar neck. The orientations were approximately vertical to the occlusal plane. Masseter contraction elevated both the condylar and eminence pressures from their resting values. The strains caused by lateral pterygoid contractions were much smaller than for the masseter with the exception of the condylar location. Ipsilateral lateral pterygoid contraction decreased both the condylar and eminence pressures from their resting values, perhaps because condylar movement altered the contact between the joint surfaces. Surgical disruption enhanced both pressure changes and bone strains under either muscle contraction but their overall patterns were not altered. In conclusion, both strains and pressures in the jaw joint varied according to specific muscle activity.

Analysis of Variance↗