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Optimizing the exercise protocol for cardiopulmonary assessment.

Twelve normal men performed 1-min incremental exercise tests to exhaustion in approximately 10 min on both treadmill and cycle ergometer. The maximal O2 uptake (VO2 max) and anaerobic threshold (AT) were higher (6 and 13%, respectively) on the treadmill than the cycle; the AT was reached at about 50% of VO2 max on both ergometers. Maximal CO2 output, heart rate, and O2 pulse were also slightly, but significantly higher on the treadmill. Maximal ventilation, gas exchange ratio, and ventilatory equivalents for O2 and CO2 for both forms of exercise were not significantly different. To determine the optimum exercise test for both treadmill and cycle, we exercised five of the subjects at various work rate increments on both ergometers in a randomized design. The treadmill increments were 0.8, 1.7, 2.5, and 4.2%/min at a constant speed of 3.4 mph, and 1.7 and 4.2%/min at 4.5 mph. Cycle increments were 15, 30, and 60 W/min. The VO2 max was significantly higher on tests where the increment magnitude was large enough to induce test durations of 8-17 min, but the AT was independent of test duration. Thus, for evaluating cardiopulmonary function with incremental exercise testing by either treadmill or cycle, we suggest selecting a work rate increment to bring the subject to the limit of his tolerance in about 10 min.

Adult↗

Application of functional genomics to the chimeric mouse model of HCV infection: optimization of microarray protocols and genomics analysis.

BACKGROUND: Many model systems of human viral disease involve human-mouse chimeric tissue. One such system is the recently developed SCID-beige/Alb-uPA mouse model of hepatitis C virus (HCV) infection which involves a human-mouse chimeric liver. The use of functional genomics to study HCV infection in these chimeric tissues is complicated by the potential cross-hybridization of mouse mRNA on human oligonucleotide microarrays. To identify genes affected by mouse liver mRNA hybridization, mRNA from identical human liver samples labeled with either Cy3 or Cy5 was compared in the presence and absence of known amounts of mouse liver mRNA labeled in only one dye. RESULTS: The results indicate that hybridization of mouse mRNA to the corresponding human gene probe on Agilent Human 22 K oligonucleotide microarray does occur. The number of genes affected by such cross-hybridization was subsequently reduced to approximately 300 genes both by increasing the hybridization temperature and using liver samples which contain at least 80% human tissue. In addition, Real Time quantitative RT-PCR using human specific probes was shown to be a valid method to verify the expression level in human cells of known cross-hybridizing genes. CONCLUSION: The identification of genes affected by cross-hybridization of mouse liver RNA on human oligonucleotide microarrays makes it feasible to use functional genomics approaches to study the chimeric SCID-beige/Alb-uPA mouse model of HCV infection. This approach used to study cross-species hybridization on oligonucleotide microarrays can be adapted to other chimeric systems of viral disease to facilitate selective analysis of human gene expression.

Animals↗

Modulating dendritic cells to optimize mucosal immunization protocols.

Oral administration of soluble protein Ag induces tolerance, a phenomenon that has hampered mucosal vaccine design. To provoke active immunity, orally administered Ag must be fed together with a mucosal adjuvant such as cholera toxin (CT). Unfortunately, CT is not suitable for clinical use because of its associated toxicity. There is, therefore, a need to develop alternative mucosal immunization regimens. Here we have attempted to alter the intrinsically tolerogenic nature of the intestine and improve immunization potential by expanding and activating intestinal APC in vivo. Previous studies have indicated that intestinal dendritic cells (DC) present oral Ag, but do so in a tolerogenic manner. In the present study we investigated whether DC can be converted from tolerogenic into immunogenic APC by treating mice with Flt3 ligand (Flt3L), a DC growth factor, and then immunizing with CT. We observed increased local and systemic responses to CT in the presence of elevated numbers of intestinal DC. In parallel, CT induced up-regulation of CD80 and CD86 on these Flt3L-expanded DC. In an attempt to develop a toxin-free adjuvant system, we investigated whether IL-1 could be used as an alternative DC-activating stimulus. Using a combination of Flt3L and IL-1alpha, we observed a potent active response to fed soluble Ag, rather than the tolerogenic response normally observed. These data suggest that Flt3L-expanded DC are well positioned to regulate intestinal responses depending on the presence or the absence of inflammatory signals. Flt3L may therefore be a reagent useful for the design of mucosal immunization strategies.

Adjuvants, Immunologic↗

Optimization of RNA isolation from human ocular tissues and analysis of prostanoid receptor mRNA expression using RT-PCR.

PURPOSE: The isolation and analysis of human ocular RNA is problematic due to variables such as rapid degradation, tissue composition, and melanin contamination. The purpose of this work was to optimize an extraction protocol for the isolation of intact total RNA from a variety of diverse human ocular tissues and to employ RT-PCR to assess the expression of mRNA coding for all eight prostanoid receptors. METHODS: Total RNA was extracted from human iris, ciliary body, choroid, and retina using an RNeasy(R) Midi Kit. Total RNA was extracted from human cornea, sclera, and optic nerve using Tri-Pure(R) Isolation Reagent. 1.0 microgram of total RNA was reverse transcribed into cDNA and subsequently amplified by PCR (35 cycles) using primers designed against each of the human prostanoid receptor cDNAs. PCR products were analyzed by gel electrophoresis and endonuclease digestion. RESULTS: The total yield and quality of RNA derived from each tissue varied according to tissue composition and the isolation method employed. RT-PCR analysis revealed that each tissue expressed all prostanoid receptor mRNAs, however, 50 cycles of PCR was required to visualize FP receptor expression in scleral tissue. In all cases, prostanoid receptor mRNA expression was significantly lower than in human nonpregnant myometrium, which was used as the positive control. CONCLUSIONS: The different cellular composition of each ocular tissue ultimately dictated the methodology to be employed for the isolation of total RNA. Thus, two extraction protocols were optimized for the isolation of intact high quality RNA from a variety of human ocular tissues. The identification of all prostanoid receptor mRNAs in a diverse set of human ocular tissues suggests potential mechanisms for prostanoid-based therapeutics aimed at IOP reduction and stimulates speculation as to additional physiological and or pathophysiological roles mediated by prostanoids.

Choroid↗

[Ultrasonography-guided transperineal prostatic biopsy: optimization of sampling protocol after 1,352 biopsies].

We report our experience-based approach in transperineal ultrasound guided biopsy to detect early prostate cancer. We selected patients on the basis of clinical characteristics. We tried to determine correct number and sites of biopsy to reduce patient discomfort and complications without loosing sensibility. Routine TZ biopsy increases detection rate of prostate cancer in T1 patients. Lateral peripheral biopsy resulted in a loss of sensibility of 5% and thus we considered it useless. Literature reported gain of sensibility with lateral peripheral biopsy referred to transrectal technique. Needle angle of incidence is different and therefore the area biopsied differs. Thus, results are not comparable.

Biopsy, Needle↗

[Cryopreservation of mouse embryos in ethylene glycol-based solutions: a search for the optimal and simple protocols].

Although ethylene glycol (EG) has been widely used for embryo cryopreservation in domestic animals, few attempts were made to use this molecule to freeze mouse and human embryos. In the few studies that used EG for slow-freezing of mouse and human embryos, complicated protocols for human embryos were used, and the protocols need to be simplified. Besides, freezing mouse morula with EG as a cryoprotectant has not been reported. In this paper, we studied the effects of embryo stages, EG concentration, duration and procedure of equilibration, sucrose supplementation and EG removal after thawing on the development of thawed mouse embryos, using the simple freezing and thawing procedures for bovine embryos. The blastulation and hatching rates (81.92% +/- 2.24% and 68.56% +/- 2.43%, respectively) of the thawed late compact morulae were significantly (P < 0.05) higher than those of embryos frozen-thawed at other stages. When mouse late compact morulae were frozen with different concentrations of EG, the highest rates of blastocyst formation and hatching were obtained with 1.8mol/L EG. The blastulation rate was significantly higher when late morulae were equilibrated in 1.8 mol/L EG for 10 min prior to freezing than when they were equilibrated for 30 min, and the hatching rate of embryos exposed to EG for 10 min was significantly higher than that of embryos exposed for 20 and 30 min. Both rates of blastocyst formation and hatching obtained with two-step equilibration were higher (P < 0.05) than with one-step equilibration in 1.8 mol/L EG. Addition of sucrose to the EG-based solution had no beneficial effects. On the contrary, an increased sucrose level (0.4 mol/L) in the solution impaired the development of the frozen-thawed embryos. In contrast, addition of 0.1 mol/L sucrose to the propylene glycol (PG)-based solution significantly improved the development of the frozen-thawed embryos. Elimination of the cryoprotectant after thawing did not improve the development of the thawed embryos. The cell numbers were less (P < 0.05) in blastocysts developed from the thawed morulae than in the in vivo derived ones. In summary, embryo stage, EG concentration, duration and procedure of equilibration and sucrose supplementation had marked effects on development of the thawed mouse embryos, and a protocol for cryopreservation of mouse embryos is recommended in which the late morulae are frozen in 1.8 mol/L EG using the simple freezing and thawing procedures of bovine embryos after a two-step equilibration and the embryos can be cultured or transferred without EG removal after thawing.

Animals↗

Optimized acquisition and processing protocols for I-123 cardiac SPECT imaging.

BACKGROUND: Deconvolution of septal penetration (DSP) has been developed to improve quantification so as to allow the use of low-energy high-resolution collimators for iodine 123 cardiac single photon emission computed tomography (SPECT) imaging. The purpose of this study is to optimize its acquisition and processing protocols. METHODS AND RESULTS: Planar images of a 9-compartment phantom loaded with variable radioactive concentrations were acquired to derive optimal scatter compensation scaling factors for 20% and 15% photopeak energy window configurations, respectively. A cardiac phantom, loaded with high and low heart-to-calibration ratios (HCRs), respectively, was imaged with both configurations. Repeated acquisitions were done for medium-energy all-purpose collimators for comparison. Critical frequencies for Butterworth filtering were optimized by use of defect contrast and normal short-axis uniformity as selection indices. HCRs were calculated with planar projection and different reconstruction methods, respectively, and then compared with the true HCRs. SPECT produced more accurate HCRs than planar imaging. With the optimized parameters for scatter compensation and filtering, the 2 energy window configurations yielded similar results. Iterative reconstructions with DSP yielded more accurate HCRs than other reconstructions without DSP. CONCLUSION: The optimized protocols based on DSP show promise that quantification of I-123 cardiac SPECT imaging can be achieved with the widely available low-energy high-resolution collimators.

Algorithms↗

Detection protocols for biotinylated probes: optimization using multistep techniques.

Recent studies using biotinylated in situ hybridization (ISH) have utilized a wide range of detection protocols for the biotinylated hybrids, leading to conflicting reports in the literature regarding sensitivity. In this study we compared 11 different detection protocols for biotinylated ISH using a measles virus-specific RNA probe on formalin-fixed, paraffin-embedded central nervous system tissue infected with measles virus. Maximum sensitivity was achieved with five-step detection protocols incorporating the use of a monoclonal antibody to biotin. Single-step detection protocols were found to be insensitive, as shown by their failure to detect viral nucleic acid in infected white-matter cells. Only by increasing the number of steps in the detection protocols were these infected cells demonstrable. Unless pre-hybridization, hybridization, and detection protocols are optimized, the results obtained in pathogenicity studies using ISH could be misinterpreted, leading to false conclusions about nucleic acid distribution. This also applies to the ever-increasing use of ISH for diagnostic purposes.

Antibodies, Monoclonal↗

Suitability of genomic DNA synthesized by strand displacement amplification (SDA) for AFLP analysis: genotyping single spores of arbuscular mycorrhizal (AM) fungi.

Limited biological samples of microbial origin often yield insufficient amounts of genomic DNA, making application of standard techniques of genetic analysis, like amplified fragment length polymorphism (AFLP), virtually impossible. The Phi29 DNA polymerase based whole genome amplification (WGA) method has the potential to alleviate this technical bottleneck. In the present work, we have sought to investigate the suitability of genomic DNA synthesized using Phi29 based WGA for AFLP analysis. We first used genomic DNA from Saccharomyces cerevisiae to optimize the protocol for the use of SDA-amplified DNA for AFLP analysis. Based on the optimized protocol we obtained AFLP fingerprints which were indistinguishable from the non-amplified genomic DNA. Finally, AFLP analysis was performed using SDA synthesized genomic DNA from single spores of various species of arbuscular mycorrhizal (AM) fungi. Unique and highly reproducible fingerprints for each species were obtained. The present study introduces the application of WGA-mediated AFLP to AM fungal biology; similarly, our protocol could be useful for other microbial genomes currently not amenable to genetic analysis owing to the paucity of starting template.

DNA Replication↗

[MRI of the temporomandibular joint. Technique, results, indications].

An optimized examination protocol for the MRI evaluation of the temporomandibular joint (TMJ) is presented. The MRI protocol is based on an optimized coil technology, sequence design, and a programmed examination protocol for diagnostics of the TMJ. Depending on the clinical findings, MRI provides an all-in-one diagnostic protocol for the diagnosis of degenerative, inflammatory and tumorous lesions of the TMJ. MRI using an optimized examination protocol should become the primary examination protocol for diseases of the TMJ.

Adult↗

Optimizing the time course of brachytherapy and other accelerated radiotherapeutic protocols.

PURPOSE: It is likely that early-responding tissues, such as tumors, repair sublethal damage more rapidly than do late-responding tissues. This difference can be exploited to design protocols with a significantly improved therapeutic advantage for accelerated radiotherapeutic regimens, including brachytherapy. METHODS AND MATERIALS: The time course of potential protocols is computer optimized, maximizing the therapeutic difference between tumor-control probability (TCP), and normal-tissue complication probability (NTCP). These quantities are evaluated with the linear-quadratic model, using clinically derived parameters. The optimization is performed by individually adjusting doses in different parts of the treatment, maximizing the therapeutic advantage. In the main calculations, half times for damage repair were T1/2(late) = 4 h, T1/2(early) = 0.5 h. Two component (fast/slow) repair processes were also investigated. RESULTS: Protocols determined by optimization have significantly greater therapeutic advantage than continuous low-dose rate (CLDR) protocols of the same overall dose and time. The optimized protocols are either (a) acute-dose/gap/CLDR/gap/acute-dose; or (b) a series of acute doses separated by 3-4 h. As a typical example, results are given for 60 Gy/120 h CLDR brachytherapy, which is assumed to give NTCP = 0.2 and TCP = 0.8. Under our assumptions, optimized regimes, with the same overall time and dose, produce an NTCP of approximately 0.11 and TCP of approximately 0.83, a significant therapeutic gain over CLDR. CONCLUSION: Difference in repair rates between early- and late-responding tissues can be exploited to produce clinically practical protocols that are significantly superior to current regimens. Such optimized protocols produce slightly better tumor control than CLDR with the same overall dose and time, significantly less late damage, and similar early normal-tissue sequellae. Temporal optimization, thus, promises to be a powerful tool in designing better treatment protocols.

Animals↗

Improved titers for helper virus-free herpes simplex virus type 1 plasmid vectors by optimization of the packaging protocol and addition of noninfectious herpes simplex virus-related particles (previral DNA replication enveloped particles) to the packaging procedure.

A helper virus-free herpes simplex virus type 1 (HSV-1) plasmid vector system has potential for both gene therapy and physiological studies, but relatively low titers have complicated use of this system. In this article, the packaging efficiency was improved by optimizing the packaging protocol and by adding noninfectious HSV-1-related particles, i.e., previral DNA replication enveloped particles (PREPs), during the packaging procedure. PREPs contain many of the tegument proteins that are thought to enhance an HSV-1 infection. Use of both the optimized packaging protocol and the PREPs resulted in an approximately 50-fold increase in the titer, and five different HSV-1 vectors were packaged using this procedure. A purified vector stock (7.8x10(8) infectious vector particles/ml) was microinjected into the striatum, the rats were sacrificed 4 days after gene transfer, and the brains were found to contain an average of approximately 6740 X-Gal-positive striatal cells. This improved packaging procedure may augment use of this vector system.

Animals↗

[Dose optimization for multislice computed tomography protocols of the midface].

PURPOSE: To optimize multislice computed tomography (MSCT) protocols of the midface for dose reduction and adequate image quality. MATERIALS AND METHODS: MSCT (Somatom Volume Zoom, Siemens) of the midface was performed on 3 cadavers within 24 hours of death with successive reduction of the tube current, applying 150, 100, 70 and 30 mAs at 120 kV as well as 40 and 21 mAs at 80 kV. At 120 kV, a pitch of 0.875 and collimation of 4x1 mm were used, and at 80 kV, a pitch of 0.7 and collimation of 2x0.5 mm. Images were reconstructed in transverse and coronal orientation. Qualitative image analysis was separately performed by two radiologists using a five-point scale (1 = excellent; 5 = poor) applying the following parameters: image quality, demarcation and sharpness of lamellar bone, overall image quality, and image noise (1 = minor; 5 = strong). The effective body dose [mSv] and organ dose [mSv] of the ocular lens (using the dosimetry system "WINdose") were calculated, and the interobserver agreement (kappa coefficient) was determined. RESULTS: For the evaluation of the lamellar bone, adequate sharpness, demarcation and image quality was demonstrated at 120 kV/30 mAs, and for the overall image quality and noise, 120 kV/40 mAs was acceptable. With regard to image quality, the effective body dose could be reduced from 1.89 mSv to 0.34 mSv and the organ dose of the ocular lens from 27.2 mSv to 4.8 mSv. Interobserver agreement was moderate (kappa = 0.39). CONCLUSION: Adequate image quality was achieved for MSCT protocols of the midface with 30 mAs at 120 kV, resulting in a dose reduction of 70 % in comparison to standard protocols.

Face↗

Development of an immunomagnetic bead-immunoliposome fluorescence assay for rapid detection of Escherichia coli O157:H7 in aqueous samples and comparison of the assay with a standard microbiological method.

The objective of this study was to develop and optimize a protocol for the rapid detection of Escherichia coli O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-E. coli O157:H7 immunomagnetic beads in growth medium specific for E. coli O157:H7. The resulting E. coli O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for E. coli O157:H7; the final immunomagnetic bead-E. coli O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a n-octyl-beta-d-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of E. coli O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without E. coli O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify E. coli O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish E. coli O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of E. coli O157:H7 in aqueous samples.

Beverages↗

Principles of low temperature cell preservation.

Cell transplantation is becoming an important technique for treatment of heart failure. Preservation is an integral step in any procedure using cells. There are two primary modes of cell preservation at low temperature, hypothermic preservation at temperatures above freezing and cryogenic preservation at temperatures below freezing. Optimal preservation protocols require a fundamental understanding of the principles involved. This review briefly describes the basic mechanisms of damage during hypothermic and cryogenic preservation and the basic principles for developing optimal protocols for preservation of cells.

Animals↗