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At least 271 records · Page 15Linked to original sources
PubMedDate not supplied

Noncompetitive immunoassays using bifunctional unilamellar vesicles or liposomes.

Small unilamellar vesicles (SUVs) functionalized with an enzyme label and with specific ligands for biological molecules are useful as signal enhancement vehicles in the development of enzyme-linked immunoadsorbent assays and other biosensor applications. Bifunctional vesicles were prepared by covalently attaching horseradish peroxidase (HRP) and an antibody to the outside of the lipid bilayer of an SUV. The reaction conditions were optimized to obtain 7-12 antibody molecules and 100-200 HRP molecules per vesicle. The enzyme retained 70-80% of its specific activity after immobilization, and the presence of immobilized proteins on the vesicle surface apparently increased the vesicle stability. To minimize the background signal and maximize the specific signal, the immunoassay protocol was optimized with respect to (1) the type and concentration of blocking agent, (2) the diluents for HRP-antibody-vesicles and sample, (3) the incubation period, and (4) the incubation temperature. The bifunctional vesicles were used in a noncompetitive immunoassay to detect d-dimer, a fibrin dimer formed at the early stages of thrombosis. A second conjugate, HRP-antibody, was prepared, characterized, and used as a control against which to compare the assay using vesicles. The assay results using vesicles led to a detection limit for d-dimer in human plasma 9 times lower than what was achieved using the conventional enzyme-antibody conjugate assay.

Antibodies↗
PubMed1985

Theoretical considerations for optimizing intensity differences between primary musculoskeletal tumors and normal tissue with spin-echo magnetic resonance imaging.

In the radiographic assessment of primary musculoskeletal tumors, it is important for therapy planning to accurately define the extent of a tumor. Using a double spin-echo pulse sequence, the T1 and T2 relaxation times and relative hydrogen densities of several neoplastic tissues and of several normal tissues in four patients were measured. Neoplasms measured included one fibrosarcoma, two osteosarcomas, and one giant cell tumor. Normal tissues measured included normal muscle, fat, and bone marrow. Using a mathematical model of the double spin-echo pulse sequence, the intensity difference between each tumor and each normal tissue for multiple values of TR and TE was calculated. These calculated intensity differences were then used to plot isodifference contour curves for each tissue pair. These plots enabled us to pick combinations of TR and TE that optimized the signal difference between tumor and normal tissue. When comparing tumor with predominantly fatty tissue such as marrow or subcutaneous fat, optimal signal difference in our imager occurred at a TR of 600 to 800 msec and a very short TE. When comparing tumor with muscle, optimal signal difference occurred with very long TR times, and TE times ranging from 30 to 90 msec. These preliminary results suggest that an optimal scanning protocol for primary musculoskeletal tumors should contain at least two different pulse sequences with widely separated TR values (500 and 2000 msec in our instrument), and short to intermediate values of TE (28 and 56 msec in our instrument). It is believed that analysis of isodifference contour plots is a useful method for optimizing intensity differences between any two tissue types.

Bone Neoplasms↗
PubMed1994

Management of patients with acute asthma: what do we know? What do we need to know?

Management of patients with acute, severe asthma mandates the use of comprehensive monitoring and aggressive therapeutic drug regimens. Global clinical and objective assessment criteria can help determine the severity of the acute episode and will also help determine optimal management protocols. The response to aggressive therapy, rather than the initial severity of the attack, predicts outcome. Beta 2 sympathomimetic agents are the treatment of choice for acute asthmatic episodes. Aminophylline and parasympatholytic agents may be useful adjuvants, but more data are needed before firm conclusions can be reached regarding risk-benefit ratios. The optimal timing of steroid therapy is not yet known, but even this form of treatment is slow in producing results.

Acute Disease↗
PubMed1987

Cryopreservation of in vitro cultured mouse preimplantation embryos.

We investigated two different freezing protocols on mouse embryos that were either grown in vivo or were grown in vitro for a certain period. Our results confirm that an extended in vitro culture period makes the embryo more susceptible for injury due to freezing and thawing. Furthermore when freezing two cell mouse embryos we could observe that this early stage is more dependent on optimal cryopreservation protocol parameters than later stages.

Animals↗
PubMed1982

Determination of half-dose depth in skin for soft x-rays.

Unlike superficial x-rays, the soft x-rays normally used in dermatologic practice spare unaffected underlying organs during treatment of cutaneous malignancies. However, since the dose with depth from soft x-rays varies markedly, it is important to know this relationship for optimal therapeutic results. The peak kilovoltage, and thus the energy of the beam, is generally selected so that the dose to the base of the lesion is one-half the surface dose. An absorbed dose of 3,400 rads to the surface and a dose of about one-half this amount to the base of most malignant lesions is one standard protocol for optimal therapeutic results. An accurate value of half-depth dose in skin is therefore necessary and is readily obtained from ordinary half-value layer measurements using the technic described.

Aluminum↗
PubMed1990

Optimization of multidimensional high-performance liquid chromatography for the determination of drugs in plasma by direct injection, micellar cleanup and photodiode array detection.

Improvements in a multidimensional liquid chromatography system for the direct determination of drug substances in blood plasma are reported. The system employs an on-line micellar chromatographic cleanup followed by a reversed-phase analytical separation. The limit of detection of propranolol is improved by a factor 10 compared to previously reported work. The technique is applied towards the determination of a multicomponent mixture of tricyclic anti-depressants in blood plasma. A protocol for optimization is described.

Chromatography, High Pressure Liquid↗
PubMed1996

Macrophage targeting with technetium-99m labelled J001 acylated poly-galactoside for scintigraphy of inflammation: optimization and assessment of imaging specificity in experimental arthritis.

J001, an acylated poly-(1,3)-galactoside purified from the membrane of Klebsiella pneumoniae, associates selectively with macrophages via the binding to CD11b and CD14 molecules. Inflammatory foci known to recruit macrophages could thus be imaged with technetium-99m labelled J001. This study aims to define the optimal scintigraphic protocol for 99mTc-J001 imaging and to evaluate the specificity of J001 scans. A dose range study was conducted in rabbits with immunological arthritis using six different specific activities ranging from 370 to 11840 MBq·mg-1 while the intravenously injected activity was constant (37 MBq) Radiochemical purity for each preparation was documented together with the in vivo stability of the 99mTc-J001 complex using exclusion-diffusion radioHPLC of serum collected 1 h after radiopharmaceutical administration. Scintigraphic images were recorded at 2, 3 and 4h and analysed using indexes calculated from regions of interest. Specificity of the macrophage imaging was assessed by comparison with scans obtained after administration of 99mTcO4(- )or 99mTc-albumin nanocolloids. A protocol of plasma transfusion was also used to inject 99mTc-J001 after complete removal of radioactive colloids likely to be generated during the labelling. For the higher specific activities (5920 and 11840 MBq.mg-1), radiochemical purity degradation and in vitro 99mTc transchelation were noted. To prevent transchelation and 99mTc bond hydrolysis likely to impair imaging specificity, 1480 MBq.mg-1 corresponding to 25microg injected J001 was found to be the optimal usable specific activity. Results obtained with the various tracers support the hypothesis that macrophage targeting is the main factor involved in the J001 imaging of arthritis.

Animals↗
PubMedDate not supplied

Childhood non-Hodgkin's lymphoma--results of treatment with ALL high-risk protocol.

Between August 1984 and September 1990, 13 children with non-Hodgkin's lymphoma (NHL) were treated with ALL high-risk protocol (5 with TCLSG 842 regimen and 8 with TPOG 882B protocol) at Taichung Veterans General Hospital. Diagnosis of NHL was confirmed by histology. Nine had lymphoblastic lymphoma, three had histiocytic lymphoma and one had small non-cleaved cell lymphoma, according to the Rappaport classification. After thorough clinical evaluation eight children were NHL stage IV; four were stage III; and one was stage II. All these children had received chemotherapy as acute lymphocytic leukemia (ALL) high-risk protocol for a total of three years, including central nervous system (CNS) prophylaxis. Of the 13 patients, 9 (69.2%) gained complete remission. Over-all survival rate was 46.2%, with a median interval of 36 months. The complete remission rate and survival rate for the nine children with lymphoblastic lymphoma was better at 77.7% & 66.6% respectively. Within the nonresponsive group, two failed to remit because of early withdrawal from the protocol; the other two patients were refractory to treatment. Relapse was noted in one patient. As to the side effects, neutropenic fever was the most common problem encountered (9 occurrences in 13 patients). Other major complications included severe mucositis, massive GI bleeding, intracranial thrombosis induced by l-asparaginase and tumor lysis syndrome. Childhood NHL is often of diffuse types in pathology, and most affected children have advanced diseases at diagnosis. Choosing an optimal treatment protocol is important for good treatment results.

Adolescent↗
PubMed1997

An optimized PCR leads to rapid and highly sensitive detection of Borrelia burgdorferi in patients with Lyme borreliosis.

The present study aimed at developing an optimized PCR protocol fro the sensitive and specific detection of all three Borrelia burgdorferi genospecies pathogenic to humans in Lyme borreliosis patients. A rapid DNA extraction method using alkaline lysis was introduced and was found to be superior to other DNA extraction methods. Nested PCR was performed with primer sets targeting the plasmid-located ospA gene and a chromosomal gene segment encoding a 66-kDa protein (p66). In spiked synovial fluid (SF) fewer than three borreliae/sample were detected. The specificities of the amplicons were confirmed by Southern blot analysis with PCR-derived probes. Urine, cerebrospinal fluid (CSF), and SF specimens from 57 patients with Lyme borreliosis and from 58 controls were examined. In clinical samples the diagnostic sensitivity of PCR was 85% with SF samples, 79% with urine samples, and 91% with paired SF-urine samples from patients with Lyme arthritis and was 79% with CSF samples, 45% with urine samples, and 87% with paired CSF-urine specimens from neuroborreliosis patients. One patient each with neuroborreliosis and with Lyme arthritis had PCR-positive urine samples only. In 17% of all cases both primer sets yielded positive results, while the other patients were positive with only one primer set. Among these, more positive results were obtained with the p66 gene primer than with the ospA primer. The specificity exceeded 99%. We conclude that DNA from B. burgdorferi sensu lato species can sensitively and specifically be detected with the optimized PCR method described. At least two different primer sets should be used, and whenever possible, urine and CSF or SF should be analyzed in parallel to achieve maximum sensitivity of the test. This protocol, therefore, considerably enhances the diagnostic power of PCR in patients with B. burgdorferi infection.

Adolescent↗
PubMed1995

A reliable and sensitive method for non-radioactive northern blot analysis of nerve growth factor mRNA from brain tissues.

This report describes a sensitive, rapid and reproducible protocol for non-isotopic Northern blotting analysis. Church buffer and probes labeled with digoxigenin (DIG) were used for studying the expression of nerve growth factor (NGF) in the rat brain. Using the described method, NGF cRNA probe was hybridized to blotted RNA and the results were compared to Northern blot obtained using the method recommended by the manufacturer (Boehringer Mannheim). Comparison revealed that the blot treated with Church buffer detected at least 10-fold more NGF mRNA as compared to blot hybridized with formamide buffer. In summary, we have developed an optimal hybridization protocol to perform non-radioactive Northern blot analysis using antisense RNA as a probe. This method allowed us to detect the specific low-abundant mRNA and analyze the expression of neurotrophic factors in the rat brain.

Animals↗
PubMed1990

Immune responses of intact and embryonically enucleated frogs to self-lens antigens.

Embryonically enucleated frogs (Xenopus) will tolerate an isogeneic eye implanted during adult life although their immune system has never been previously exposed to eye-specific Ag. We hypothesized that such self-implants survive because they induce tolerance to the eye-specific Ag. We have developed evidence to support this hypothesis by studying the responses of embryonically enucleated or intact frogs, with or without eye implants, to self-lens proteins. After immunization with a low concentration of bovine lens proteins, spleen cells from all intact and all embryonically enucleated frogs displayed significant in vitro proliferation against the Ag. All animals in both groups of frogs also produced high titered xenoantibodies. After immunization with a comparable preparation and concentration of self-lens Ag, splenocytes from only some of the embryonically enucleated and intact frogs showed significant proliferation, and fewer of these frogs produced antibody. When the immunization protocol and Ag concentration were modified to evoke consistent anti-self-lens proliferative and antibody responses from enucleated frogs, intact frogs responded equally well. Because there were no significant differences in the magnitude of the responses, and the percent of enucleated and intact frogs responding did not differ, we conclude that there is little or no immunologic tolerance to self-lens Ag in intact frogs. When either embryonically enucleated or intact frogs were heterotopically implanted with isogeneic eyes and then immunized with self-lens Ag or foreign lens Ag using the optimal immunization protocol, the Ag-specific proliferative responses of their lymphocytes were significantly reduced after immunization with self-, but not foreign Ag. This argues that embryonically enucleated frogs can become tolerant of the organ-specific Ag of the lens, even in adult life.

Animals↗
PubMed1997

Quantification of signal changes in gradient recalled echo FMRI.

Understanding and quantifying the various contributions to functional magnetic resonance imaging (FMRI) signal changes in activated cortical areas is paramount for a clinical application of brain mapping by FMRI. Therefore, all significant contributions to FMRI signal changes, both extra- and intravascular, from macrovessels down to the capillary network, should be taken into account. We present a gradient-recalled-echo FMRI model based on in-flow effects described by the Bloch equations, adding susceptibility effects empirically via T2* differences measured in vitro in human blood samples. Results of these calculations (by systematically varying alpha, echo time (TE), repetition time (TR), as well as blood velocity and T2* upon stimulation) may be used to (a) simulate functional MRI experiments with different measurement protocols and (b) estimate realistic values for important anatomical and physiological details that influence local signal changes in FMRI (i.e., size and distribution of vessels, effective relaxation times of blood, etc.). The excellent agreement between our model calculations and experimental results from conventional gradient recalled echo fMRI in vivo suggests a significant contribution from very slow flow and oxygenation changes, predominantly in small vessels (vblood = 1-4 mm/s). The actual contribution of T1- and T2-related effects is strongly dependent on sequence design and actual sequence parameters used. Thus, the model simulations presented may also be used to optimize measurement protocols for investigating various neurophysiological phenomena.

Adolescent↗
PubMed1998

Experiences with gadodiamide, a non-ionic contrast agent, in MRI of brain metastases.

29 patients suffering from brain metastases were studied with a single (0.1 mmol/kg body weight) and triple dose (0.3 mmol/kg) of gadodiamide, a non-ionic MR contrast agent. The study was performed using an extended imaging protocol with application of T1-weighted images in three orientations. Number of definite and suspected metastases, lesion contrast, edema delineation, and flow artefacts were evaluated. Most of the definite metastases were found in triple dose images. However, the total lesion number could only be determined by looking on all sequences as a whole. Furthermore, the number of suspected metastases could be significantly reduced with triple dose images, but once more all sequences were needed to do so. Flow artefacts were significantly increased by triple dose, whereby lesion contrast and edema delineation was considerably improved. The conclusion is that the combination of high dose application of the contrast agent and an optimized imaging protocol facilitates sufficient imaging of brain metastases. This is important for therapeutical considerations, because patients with more than 1-2 metastases will be treated primarily with radiation therapy instead of neurosurgery.

Brain Neoplasms↗
PubMed1994

Light and electron microscopic in situ hybridization: non-radioactive labeling and detection, double hybridization, and combined hybridization-immunocytochemistry.

We performed light and electron microscopic in situ hybridization, according to the same protocol and without pretreatment of sections, on Lowicryl- and LR Gold-embedded cells. Digoxigenin (DIG)- or biotin-labeled riboprobes were visualized by direct or indirect immunodetection using commercially available gold-antibody conjugates with 0.8-10-nm gold grains. At the ultrastructural level, the main findings were that DIG-labeled probes gave a slightly higher labeling intensity (grains per signal) than biotin. The direct detection method produced a more compact signal, which led to better resolution at medium and high magnifications. Labeling intensities of all gold grain sizes were essentially equal. Grain sizes of 5 nm and larger were highly preferable because available enhancement methods are unsatisfactory for ultrasmall grains. The optimized immunodetection protocols are suitable for double hybridization with two different probes and for combined hybridization and immunocytochemistry.

Antibodies↗
PubMed1997

The utilization of RAPD-PCR for identifying thermophilic and mesophilic Bacillus species.

A random amplified polymorphic DNA fingerprinting assay has been optimized that is able to discriminate between numerous thermophilic and mesophilic bacillus species and strains. Included in the analyses are thermophilic (able to grow at 55 degrees C) strains of Bacillus stearothermophilus, B. kaustophilus, B. coagulans, B. sphaericus, B. thermodenitrificans, B. thermocatenulatus, B. thermoleovorans, B. licheniformis, B. brevis, B. thermoglucosidasius, B. caldolyticus, B. caldotenax, B. caldovelox, B. thermocloacae and B. smithii. Mesophilic strains of B. pumilus, B. subtilis, B. megaterium, B. circulans, B. cereus and B. mycoides can also be used for fingerprinting with the assay. Increasing the concentration of primer from 0.2 to 2.0 microM is shown to have a significant effect on increasing the number of amplification products that can be used for the discrimination or identification of individual strains or species. It is suggested that this may be a general way of improving the resolution of a RAPD protocol. The optimized conditions have been used successfully to trace B. stearothermophilus, B. licheniformis and other bacillus species and strains in an industrial setting.

Bacillus↗
PubMed1998

The treatment of late radiation effects with hyperbaric oxygenation (HBO).

BACKGROUND: Late radiation injuries may impose a negative influence on the quality of life in the affected patients. In several entities, standardized treatment protocols are lacking. Hyperbaric oxygenation (HBO) has been shown to have beneficial effects in the treatment of late radiation sequelae. MATERIAL AND METHODS: The basic principles of HBO are reviewed as well as clinical issues. Current study protocols are presented. RESULTS: During HBO-therapy the patient breathes pure oxygen at pressures above 100 kPa. The oxygen solubility within the fluid phase of the blood is largely increased. Biological effects include an increased oxygen diffusibility, improved collagen synthesis and neoangiogenesis as well as an enhancement of antimicrobial defenses. By decreasing the capillary filtration pressure a reduction of edema becomes possible. HBO has been shown to prevent complications following surgery in irradiated tissues. Its efficacy as an adjunct in the treatment of osteonecroses in radiation patients could be demonstrated. In addition, the loss of osseointegrated implants in the maxillofacial bones of these patients could be significantly reduced. Further indications include soft tissue necroses, hemorrhagic cystitis and proctitis in tumor patients that have been treated by radiotherapy as part of a multimodality approach. CONCLUSIONS: HBO in the treatment of late radiation effects is still subject of investigation, but remarkable results have been reported. Optimized treatment protocols need to be determined in various entities. The rate of side effects is acceptable low.

Humans↗
PubMedDate not supplied

Virtual bronchoscopy for three--dimensional pulmonary image assessment: state of the art and future needs.

Virtual bronchoscopy is emerging as a useful approach for assessment of three-dimensional (3D) computed tomographic (CT) pulmonary images. A protocol for virtual bronchoscopic assessment of a 3D CT pulmonary image would have two main stages: (a) preprocessing of image data, which involves extracting objects of interest, defining paths through major airways, and preparing the extracted objects for 3D rendering; and (b) interactive image assessment, which involves use of graphics-based software tools such as surface-rendered views, projection images, virtual endoscopic views, tube views, oblique section images, measurement data, global two-dimensional section images, and cross-sectional views. Although a virtual bronchoscope offers a unique opportunity for exploration and quantitation, it cannot replace a real bronchoscope. Limitations of current virtual endoscopy systems include high cost, lack of visual aids beyond simulated endoscopic views, difficulty in performing interactive anatomic exploration, lack of quantitative information, use of surface rendering instead of volume rendering, and need for substantial off-line display computation. Future needs include development of fully integrated user-friendly virtual bronchoscopes, development of optimal CT protocols for generating artifact-free data sets, and improvements in automated preprocessing of 3D CT images.

Bronchoscopy↗
PubMed1986

Current risks and protocols for operations for double-outlet right ventricle. Derivation from an 18 year experience.

Recent follow-up of 127 patients having repair of double-outlet right ventricle during an 18 year experience (1967 to July, 1984) indicated an overall actuarial survival rate at 12 years of 38%. However, multivariate analysis supported by contingency tables indicated that currently the early (2 week) survival rate after the intraventricular tunnel repair for double-outlet right ventricle with subaortic ventricular septal defect in 6-month-old infants is 99% and the 10 year survival rate 97%. Reoperation of the tunnel repair was rarely required (one of 56 patients), and the functional results were excellent. Results were similar in patients with doubly committed ventricular septal defect, except that two of 10 patients developed typical discrete localized subaortic stenosis late postoperatively. Early and late results in patients with double-outlet right ventricle and subpulmonary ventricular septal defect were poor when an atrial switch operation was part of the repair; when an intraventricular tunnel repair between the left ventricle and aorta was combined with a right ventricular-pulmonary arterial conduit, no early deaths occurred, but three patients died late postoperatively. Several techniques of repair of double-outlet right ventricle with noncommitted ventricular septal defect have provided only a 22% overall 10 year survival rate. These data are used to derive inferences as to optimal treatment protocols.

Actuarial Analysis↗