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PubMed1987

Cancer therapy by biological response modifiers.

Biological response modifiers (BRMs) are agents or approaches which can modify the biological response of the host to tumors and thereby are expected to augment the resistance to development or progression of cancer. Recent advances in the technology of genetic engineering and monoclonal antibodies have led to rapid progress in this field. There is an increasing number of genetically engineered cytokines, which appear promising for cancer treatment and are becoming available for clinical trials. These include the interferons, leukin-2, tumor necrosis factor, and colony-stimulating factors. For development of optimal therapeutic protocols with these and a variety of immunomodulatory agents, it appears necessary to develop a detailed understanding of the possible mechanisms of their antitumor effects, and to determine the optimal dose and schedule for altering the antitumor effector mechanisms. BRMs would also be expected to be quite useful in the prevention of cancer. Indeed, in a variety of animal tumor model systems, potent protective effects can be demonstrated. However, the strategies for clinical application of such information have yet to be adequately worked out. One more immediate application of this approach is for the prevention of metastatic spread of tumor cells. BRMs, which stimulate natural killer cell activity, have been shown to strongly protect against dissemination of tumors, and clinical strategies for this important aspect of cancer treatment are being developed.

Humans↗

PubMed1999

Extended serial passaging of mammalian neural stem cells in suspension bioreactors.

Neural stem cells (NSCs) are primitive cells that are the "parent" cells of all the cells in the central nervous system (CNS). Their discovery in 1992 opened the door to a multitude of potential therapies and treatments to cure neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, and Huntington's disease, which affect millions of people worldwide and cost billions of dollars in health care each year. This study proposes optimal serial passaging protocols so that mammalian neural stem cells can effectively be grown in suspension culture. We examined stationary culture passaging protocols and developed our own optimal procedure. Also examined was the effect of serially cultivating the neural stem cells in suspension culture for an extended period of time. The cells were grown for over 35 days in suspension with an overall multiplication ratio of over 10(7) with no decrease in growth rate, maximum cell density, or viability. The cells also remained karyotypically normal through 25 doublings and retained their ability to be differentiated into all the major cell types of the CNS-neurons, astrocytes, and oligodendrocytes. For the first time, mammalian neural stem cells were grown on a larger scale in suspension culture and maintained their stem cell characteristics. A semicontinuous scheme for large-scale production is also presented.

Animals↗

PubMed1998

Development and comparison of assays for measuring immunospecific antibody response in serum of chickens and rabbits immunized with human immunoglobulin G.

The antibody response development during polyclonal antibody production is a relevant parameter to monitor during the immunization period to be able to optimize the immunization protocol and to determine the optimal antibody harvest time. Although rabbits and other mammals are most often used for polyclonal antibody production, the chicken is a relevant alternative. There are both scientific reasons, economic reasons, and animal welfare reasons to consider when choosing the chicken instead of a mammal for this purpose, because antibodies in generous quantities can be harvested from the egg yolk. This study compared different assays for measuring antibody response in rabbit and chicken serum. An inhibition liquid phase absorption assay (ILPAA), a rocket immunoelectrophoresis (RIE) assay, and a line immunoelectrophoresis (LIE) assay were compared to ELISAs. The ELISA proved to be the most useful assay for routine use, as it was less time-consuming and because the assay could easily be adapted to both serum antibody types. However, electrophoretic assays were the most useful as combined analytical and quantitative tools and must be considered essential when analyzing specificities of polyclonal antibody preparations.

Animals↗

PubMed2000

Digital analysis of light microscope immunofluorescence: high-resolution co-localization of synaptic proteins in cultured neurons.

A protocol is presented for determining the subcellular distribution of fluorescently labeled proteins in neurons using deconvolved images gathered with a wide-field microscope. The protocol includes optimal settings for the numerical algorithm used to deconvolve the images and an objective method for thresholding the deconvolved images to retain only high-intensity, specific labeling. The effectiveness of the protocol is demonstrated using a fluorescent antibody stain directed towards the alpha1 subunit of the GABA(A) receptor in cultured neurons. We also show, using an antibody against the presynaptic vesicular protein synaptophysin, that the technique can detect presumptive regions of synaptic contact between neurons. Double-labeling with the anti-alpha1 and anti-synaptophysin antibodies in a cultured neuron reveals regions of both synaptic and non-synaptic alpha1 labeling. Thus, numerical postprocessing of wide-field images can be used to efficiently locate receptor proteins in neurons in relation to functionally important structures. This confocal-like functionality is attained without the excessive bleaching and phototoxicity associated with the intense laser excitation light used in confocal techniques.

Animals↗

PubMed2000

Heart rate recovery post-exercise as an index of parasympathetic activity.

The time constant (T) obtained by fitting post-exercise heart rate (HR) recovery to a first order exponential decay curve has been promoted as an index of parasympathetic activity. However, acceptance has been limited because reported data are inadequate to assess goodness of fit for the model, determine the best exercise protocol, or optimize the duration of post exercise monitoring. Consequently, we evaluated T for nine healthy volunteers (age 24-46) following treadmill exercise at maximal (max) and two stages sub-max exercise (Bruce protocol). T stabilized only after 3 min of post-exercise monitoring. With max exercise, T varied unacceptably with small changes in onset of monitoring, e.g. -16.7+/-16.6 (-13.2%) in the first 5 s, and residuals of the fitted curve were non-random. In contrast, sub-max exercise produced consistent T values, e.g. -1.9+/-3.2 (-4.2%) in the first 5 s, and residuals were more nearly random. In conclusion, first order decay is an inadequate model for HR recovery following max exercise, but may be reasonable for sub-max levels.

Adult↗

PubMed2001

Use of cryopreserved pronuclear embryos for the production of transgenic mice.

A series of experiments was conducted to test the hypothesis that an improved cryopreservation protocol for pronuclear stage mouse embryos will produce transgenic (Tg) mice by pronuclear gene injection at a rate not significantly different from noncryopreserved embryos. In the first experiment, three cryoprotective agents (CPAs) (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG]) and two cryopreservation protocols, currently used for pronuclear embryos, were compared in regard to their ability to maintain post-thaw morphological integrity and in vitro developmental competence. In the second and third experiments, the optimal cryopreservation protocol determined from the first experiment was used to evaluate in vitro developmental competence of pronuclear embryos following green fluorescence protein gene injection and in vivo developmental competence as well as the gene integration rates. Survival (morphological integrity and development to two cells) of embryos cryopreserved in the presence of DMSO was higher (P < 0.05) than those cryopreserved with either PG or EG. Postinjection developmental competence (development to two cells) of cryopreserved CBA, C57B6/JxCBA-F1 and noncryopreserved (control) embryos was not different (P > 0.05). Postinjection blastocyst formation rate of cryopreserved and noncryopreserved C57B6/JxCBA-F1 embryos was similar (P > 0.05); however, noncryopreserved CBA embryos resulted in a higher blastocyst formation than controls (P < 0.05). While there was no difference in the percentage of transgenic fetuses between cryopreserved and control CBA embryos (P > 0.05), cryopreserved C57B6/JxCBA-F1 embryos resulted in lower transgenic fetuses than control (P < 0.05). These results indicate that the use of cryopreserved mouse pronuclear embryos can be a useful and efficient approach to the production of Tg mice.

Animals↗

PubMed2026

Optimization of protoplast based DNA isolation and genome analysis in a gamma-irradiated Aspergillus niger mutant strain.

Aspergillus niger is an important industrial fungus widely used for citric acid production and a range of biotechnological applications. In this study, a protoplast-based DNA isolation protocol was optimized for a gamma-irradiated A. niger AN-L103_M1 mutant strain, followed by whole-genome sequencing and functional genome analysis. Protoplast yield was strongly influenced by enzyme concentration and the molarity of the osmotic stabilizer. The highest yield was achieved at an enzyme concentration of 50 mg/mL (2.487 ± 0.04 × 10⁸ cells/mL) and 0.8 M KCl (2.550 ± 0.06 × 10⁸ cells/mL), with both factors showing significant effects (p < 0.0001) in GraphPad Prism 11.0.0. Whole-genome sequencing performed using an Illumina NovaSeq 6000 platform yielded a 37.06 Mb draft genome assembled into 537 contigs, with an N50 of 363,084 bp and a GC content of 48.2%. BUSCO 14 analysis showed high completeness (97.95% complete BUSCOs). Functional annotation and KEGG pathway mapping identified genes involved in glycolysis, the tricarboxylic acid cycle, and citrate biosynthesis, while biosynthetic gene cluster analysis revealed diverse potential for secondary metabolite production. These findings provide an optimized workflow for protoplast-based DNA isolation and genome-scale functional analysis in A. niger, proposing a basis for future comparative genomics, transformation studies, and experimentally validated metabolic engineering.

Aspergillus niger↗

PubMed1991

Ability, disability, and the functional capacity of patients with cardiovascular disease.

Assessment of functional capacity, of ability and disability among patients with cardiovascular disease raises a number of problems and issues for which there are currently only imperfect or incomplete answers. Emphasis must be placed on the lack of predictable relationship of anatomic abnormality and functional abnormality. For example, the percentage obstruction of the coronary artery documents the anatomic extent of the disease, rather than the limitation of functional capacity; the same lack of predictive value characterizes the decrease in resting ventricular ejection fraction. The response to a challenge of activity or exertion currently appears to offer the optimal method of assessing functional capacity for work, although a brief continuous exercise test may not be the optimal exercise protocol by which to evaluate endurance. As an example, in our laboratory, comparing a low-level continuous exercise test protocol with one with an intermittent exercise design (i.e., periods of exercise alternating with periods at rest), patients typically can perform at least one additional stage of exercise on the discontinuous or intermittent test protocol. This occurred without significant differences in the final heart rate, blood pressure, or rate-pressure product, probably because most patients so tested were limited not by myocardial ischemia but by musculoskeletal problems, fatigue, or dyspnea (8). An unmet need is a comparison of exercise test protocols for the assessment of functional capacity, possibly the development of new test protocols for patients with limited functional capacity, and the evaluation of the relationship of these test data to eight hours of occupational activity in the workplace setting. It appears logical that a diagnostic exercise test should differ from one designed to determine functional capacity, but the results of a variety of exercise test protocols should be compared with the actual physical activity able to be performed in the workplace, as well as with reported symptoms. It should be defined whether testing is to be performed on optimal medical therapy, which I believe should be the case; or whether the technique used for diagnostic exercise testing, that of the minimal medication possible, is to be employed. Next, the time after surgical intervention or following a prolonged hospitalization at which to test should be delineated in that the deconditioning effect of immobilization may substantially decrease effort tolerance, unrelated to the severity of the underlying cardiovascular disease. Finally, should exercise rehabilitation be recommended or required before testing for cardiovascular impairment; major improvement in functional capacity has occurred in previously sedentary patients with a variety of cardiovascular diseases, including those with important manifestations of myocardial ischemia and ventricular dysfunction.(ABSTRACT TRUNCATED AT 400 WORDS)

Cardiovascular Diseases↗

PubMed1993

Biokinetics of technetium-99m-tetrofosmin: myocardial perfusion imaging agent: implications for a one-day imaging protocol.

Tetrofosmin is a 99mTc-labeled myocardial perfusion imaging agent that has shown encouraging results in Phase I and II clinical trials. The purpose of this study was to determine the biokinetics of this agent following administration during exercise and at rest in order to determine an optimal imaging protocol. Twenty patients with suspected coronary artery disease underwent symptom-limited treadmill exercise. Six to 8 mCi of 99mTc-tetrofosmin was injected at peak exercise and 22-24 mCi was injected 4 hr later at rest. Serial 5-min planar images were obtained in the left anterior oblique view at 5, 10, 15, 30, 60, 120 and 180 min after the radiotracer injection. Regions of interest were drawn on the serial images around the entire heart and portions of liver, lung, spleen, gallbladder and gastrointestinal tract. Average decay-corrected counts per pixel in each organ were plotted against time. In addition, heart-to-adjacent organ ratios were also determined. On stress images, the heart had the highest activity at all times, with the exception of gallbladder in the first 15 min. On rest images, the gallbladder, liver and gastrointestinal tract initially had higher activity than the heart; but the activity in these organs cleared rapidly over the subsequent 30-60 min. Heart-to-adjacent organ ratios were > 1.0 at all times in the stress images. Heart-to-organ ratios were < 1.0 in the first 15 min on the rest images for the liver and gastrointestinal tract. However, 30 min later, all ratios on the rest images were > or = 1.0. Technetium-99m-tetrofosmin images were considered to be of good to excellent quality with good myocardial delineation and adequate contrast between the heart and background. These These observations indicate that a convenient one-day tetrofosmin imaging protocol similar in duration to conventional 201Tl imaging is feasible.

Adult↗

PubMed2000

Two-dimensional electrophoretic analysis of Corynebacterium glutamicum membrane fraction and surface proteins.

An improved protocol for the two-dimensional analysis of proteins of the Corynebacterium glutamicum cytoplasmic membrane fraction is described. By use of increased 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) concentrations (2-4%) and an optimized electrophoresis protocol, horizontal streaking of proteins of the cytoplasmic membrane fraction was almost completely avoided. More important, in contrast to a previously published method, both a sample tray and IPG-phor isoelectric focusing unit can be used for the in-gel application of proteins. The described protocol was also found to be suitable for hydrophilic cytoplasmic proteins. Additionally, the preparation and analysis of C. glutamicum cell surface proteins is described. Proteins were extracted with lauroyl sarcosinate and 100-120 spots were separated on two-dimensional (2-D) gels in comparison to 18-20 spots observed previously by standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). C. glutamicum proteins can now be separated into three distinct fractions resembling different functional units of the bacterial cell.

Amino Acid Sequence↗

PubMedDate not supplied

Optimization of techniques in screening CT of the sinuses.

The number of screening examinations of the sinuses performed with CT has markedly increased owing to the widespread and increasing use of endoscopic sinonasal surgery. We reviewed scans from 500 patients who had screening CT examinations of the sinuses for preendoscopic evaluation of inflammatory sinonasal disease to better define an optimal imaging protocol. Three aspects of direct coronal imaging of the paranasal sinuses were investigated: (1) preparation of the patient prior to the examination; (2) technical factors of the CT study, including positioning of the patient, optimal coronal angle, slice thickness, and CT exposure factors; and (3) data display. Our experience indicates that pretreatment of the patient with maximal medical therapy enables the best preendoscopic definition of anatomy, disease pattern, and nonreversible disease component for the treating surgeon. CT technical factors are optimized with scanning in the prone position with thin (3-mm) sections obtained through the anterior paranasal sinuses. This allows optimal visualization of the ostiomeatal unit. The remaining posterior portions of the sinuses are adequately imaged with thicker slices (5 mm). The coronal scan angle used is less critical. Exposure factors (mAs) can be reduced dramatically without image compromise. Data display is optimized when the bone algorithm is used to acquire the data and with image display at intermediate window center and width level. Use of the techniques outlined in this article results in a cost-effective yet diagnostic scan of the sinuses with decreased radiation exposure to the patient.

Cost-Benefit Analysis↗

PubMed2003

Magnetic resonance imaging protocols for examination of the neurocranium at 3 T.

The increasing availability of high-field (3 T) MR scanners requires adapting and optimizing clinical imaging protocols to exploit the theoretically higher signal-to-noise ratio (SNR) of the higher field strength. Our aim was to establish reliable and stable protocols meeting the clinical demands for imaging the neurocranium at 3 T. Two hundred patients with a broad range of indications received an examination of the neurocranium with an appropriate assortment of imaging techniques at 3 T. Several imaging parameters were optimized. Keeping scan times comparable to those at 1.5 T we increased spatial resolution. Contrast-enhanced and non-enhanced T1-weighted imaging was best applying gradient-echo and inversion recovery (rather than spin-echo) techniques, respectively. For fluid-attenuated inversion recovery (FLAIR) imaging a TE of 120 ms yielded optimum contrast-to-noise ratio (CNR). High-resolution isotropic 3D data sets were acquired within reasonable scan times. Some artifacts were pronounced, but generally imaging profited from the higher SNR. We present a set of optimized examination protocols for neuroimaging at 3 T, which proved to be reliable in a clinical routine setting.

Adult↗

PubMed1999

Lower esophageal sphincter dysfunction often precludes safe gastric feeding in stroke patients.

OBJECTIVES: To determine the relationship between stroke and lower esophageal dysfunction with vomiting and to identify an optimal nutrition protocol based on our findings. PATIENTS AND METHODS: The lower and upper esophageal sphincter functions were assessed in 35 patients who had an acute stroke to determine whether gastric or jejunal enteral feeding was the optimal route. Stroke was due to unilateral ischemia in 20 patients, unilateral intracerebral hemorrhage in 8 patients, and global ischemia in 7 patients. Our study consisted of 18 men and 17 women with an average age of 64 years. RESULTS: Using standard esophageal manometric definitions, the lower esophageal sphincter function was below normal in 24 patients: 3 had global anoxia, 5 had unilateral hemorrhage, and 16 had unilateral ischemia. The upper esophageal sphincter function was low in 30 patients: 6 had global anoxia, 7 had unilateral hemorrhage, and 17 had unilateral ischemia. Based on lower esophageal sphincter pressure, 7 patients underwent tube gastrostomy and 13 patients underwent tube jejunostomy placement. All tolerated enteral alimentation well. Prior to lower esophageal sphincter assessment, 4 patients had percutaneous endoscopy gastrostomy feedings that led to aspiration pneumonitis and consultation for tracheostomy; 2 terminally ill patients were referred to the ethics service, and 2 were converted to feeding via jejunostomy tube at the time of tracheostomy and did well. CONCLUSIONS: Vomiting with aspiration due to lower esophageal sphincter dysfunction is common after acute strokes. Esophageal manometry serves as a guide to find the optimal feeding route.

Adult↗

PubMedDate not supplied

Should we consider performing oral glucose tolerance tests more frequently in postmenopausal women for optimal screening of impaired glucose tolerance?

OBJECTIVE: To investigate an optimal screening protocol for impaired glucose tolerance (IGT) and type II or non-insulin-dependent diabetes mellitus (DM) by using fasting plasma glucose (FPG) and oral glucose tolerance test (OGTT) in postmenopausal women. DESIGN: One hundred consecutive postmenopausal women were screened with FPG determination, and then all underwent an OGTT. Basal serum lipid and insulin levels of these women were also determined. Insulin sensitivity was determined by using the homeostasis model assessment. Receiver operating characteristic analysis was performed to determine the efficacy of these variables in detecting women with IGT and DM, and optimal cutoff values were determined. RESULTS: FPG with a cutoff value of 98 mg/dL had the best combination of sensitivity (71%) and specificity (76%) for the detection of IGT and DM. Combined FPG and body mass index screening (with the optimal cutoff value of 26.5 kg/m2) improved the sensitivity to 96% but decreased the specificity to 47%. This combined screening protocol detected 94% of the women with IGT and all diabetic women. CONCLUSIONS: Given that IGT and DM are common among postmenopausal women and DM can be prevented by nonpharmacologic interventions in women with IGT, OGTT may be used more frequently among these women. Our data indicate that for optimal screening of non-insulin-dependent DM and IGT, OGTT should be considered in postmenopausal women, especially when risk factors in addition to age are present. This model may detect most of the women with IGT and almost all diabetic women.

Aged↗

PubMed1995

High-efficiency retroviral-mediated gene transfer into human and nonhuman primate peripheral blood lymphocytes.

Peripheral blood lymphocytes (PBLs) are primary targets for gene therapy of inherited and acquired disorders of the immune system. We describe the development of an optimized transduction system that provides for high-efficiency retrovirus-mediated gene transfer into primary PBLs. This optimized transduction protocol combines centrifugation of the lymphocytes (1000 x g) at the inception of transduction with phosphate depletion, low-temperature incubation (32 degrees C), and the use of the packaging cell line PG13. Gene marking studies of human and primate PBLs using these optimized transduction conditions demonstrated that the transduction efficiency exceeded 50% of the total lymphocyte population. The optimized transduction efficiency of PBLs with amphotropic retroviral vectors was in excess of 25%. The transduction procedure does not alter phenotype, viability, or expansion of the transduced cells. Our data indicate that this optimized transduction system leads to high-efficiency gene transfer into primary human lymphocytes, which obviates the requirement for selection of transduced cells prior to gene-therapy procedures. Thus, large quantities of healthy retrovirally transduced lymphocytes containing a broad immunological repertoire can be generated for use in clinical protocols. Our results represent a significant improvement in the methodology for the transduction of lymphocytes for gene therapy.

Animals↗

PubMed2026

Optimizing focal vibration therapy for balance and gait: A systematic review.

OBJECTIVE: This systematic review evaluated the efficacy of focal (localized) vibration therapy (FVT) applied to muscles/tendons on balance, gait, and mobility, with a specific focus on defining optimal vibration protocols (frequency, amplitude, dosing) and muscle-targeting strategies to maximize sensorimotor recovery. METHODS: A systematic review was conducted across six databases (CINHAL, Embase, Medline, Web of Science, Scopus, CENTRAL) from January 2000 to May 2025. Studies were included if they involved human participants, applied FVT therapeutically, and reported balance, gait, or mobility outcomes. Data extraction included study characteristics, intervention protocols, and outcomes. Methodological quality was assessed using the PEDro scale. RESULTS: Sixty-two studies (n = 2090 participants) were included. Methodological quality assessment (PEDro scale) indicated 44% of studies met high-quality standards. Biomechanical analysis identified the quadriceps, gastrocnemius/soleus, and plantar muscles as the most effective vibration sites, given their critical roles in gait propulsion and postural stability. The synthesis of protocol data indicated a promising therapeutic window characterized by a vibration frequency of 80-120 Hz (primarily fixed sinusoidal waveforms at a single frequency) and an amplitude of 0.2-0.5 mm (reported only in 12 studies; amplitude was not reported in 23 studies), applied bilaterally for a minimum of 3 sessions per week over 4-12 weeks, which could lead to improved balance and gait performance with benefits sustained for up to 5 months. CONCLUSION: FVT shows potential to improve gait and balance, particularly when targeting lower-extremity muscles with optimized vibration parameters. To advance the field, future research must prioritize the development of standardized protocols and investigate neurophysiological mechanisms to refine FVT as a precision bioengineering solution for mobility deficits.

Humans↗

PubMed2000

[Virtual endoscopy of the upper airway with spiral CT].

PURPOSE: The technical conditions, the optimized scanning protocols and the facilities of virtual endoscopy (VE) are presented in an overview. METHODS: Phantom studies showed that, for a single-row-detector helical CT, collimation of 3.0 mm combined with pitch of 1.5 provided an optimal compromise between image quality and the scan duration per breath-hold. A multi-row-detector helical CT requires only a fraction of the scanning time. This is especially important for patients with dyspnea. RESULTS: The threshold-dependent virtual endoscopic surface rendering is a reliable and rapidly practicable reconstruction algorithm for the imaging of the upper airway. CONCLUSIONS: The VE technique is suitable for the imaging of space-occupying tumors and restricted stenoses. With VE follow-up examinations can be performed non-invasively and interventional procedures can be prepared in an optimal way. Because of the computed tomographic data acquisition the structure of the mucosa is not assessable and there is no opportunity to obtain a biopsy specimen.

Bronchoscopes↗

PubMed1986

MR imaging of the lumbar spine: anatomic correlations and the effects of technical variations.

A correlative anatomic study, a retrospective review of MR images performed in 35 patients, and a series of tests of the effectiveness of various MR scanning techniques were performed in order to improve comprehension of lumbar spine anatomy depicted on MR images, and thereby facilitate development of an optimal scanning protocol. Correlation of MR images with cryomicrotomed cadaver specimens enhanced understanding of the MR depiction of the intervertebral disks, ligamentum flavum, nerve roots, epidural fat, and epidural veins. Experiments were performed to assess the efficacy of a surface coil applied to the back, a solenoidal surface coil, a standard body coil, and an abdominal compression device in optimizing image quality. Experiments were also performed to determine the effect of alterations in the pulse sequence and variations of the phase-encoding axis. Based on these results, a protocol is proposed for routine imaging of the lumbar spine that yields high-resolution sagittal and oblique images and that does not require a surface coil. The recommended protocol employs heavily T1-weighted images with phase encoding along the z axis for sagittal images and along the x axis for axial images. This protocol yields multiple sagittal and oblique axial images through each of the lumbar disks, a larger field of view than obtained with surface coils, and a reduction of total imaging time to as little as 10 min.

Cadaver↗