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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↗
PubMed1999

Regional chemotherapy of non-resectable liver metastases from colorectal cancer - literature and institutional review.

BACKGROUND: Cure is possible by resecting colorectal isolated liver metastases. In non-resectable isolated colorectal liver metastases (CRLM), regional chemotherapy has been advocated to optimize the disease control in the liver in order to improve the results of the alternative, systemic chemotherapy. The drugs are delivered by means of hepatic artery infusion (HAI) via ports or pumps; pharmacological modifications of the hepatic arterial blood-flow-like HAI with starch microspheres or stop-flow and perfusion techniques were applied to improve HAI. METHODS: We reviewed the literature and report our progress, up to May 1999, in analyzing the validity of HAI for CRLM therapy. RESULTS: In the majority of phase-II and -III trials, the response rates to HAI were significantly higher than those from systemic chemotherapy, and local disease control could be achieved even when HAI was used second line to systemic chemotherapy. The meta-analysis of randomized trials comparing HAI with either systemic chemotherapy (five trials) or, optionally, either 5-fluorouracil (FU) or symptomatic treatment (two trials) showed a significant advantage of HAI in response (41% vs 14%, P<10(-10)) and median survival time (15 months vs 11 months, P<0.0009). The active anabolite of 5-FU, 5-fluordeoxyuridine (5-FUDR), the drug of choice for HAI in those trials, may cause severe hepatotoxicity. To avoid this toxicity, we developed a HAI protocol using mitoxantrone, 5-FU plus folinic acid (FA) and mitomycin C (MFFM). The response rates of HAI with 5-FU plus FA or MFFM were 45% and 66%, the interim median survival times 19.8 months and 27.4 months. 5-Year survivors were observed in all our protocols. Since no severe hepatotoxicity occurred, 9 of 74 patients were resected after response to HAI with 5-FU plus FA or MFFM, without surgical mortality and with survival times from 2+ months to 58+ months. CONCLUSION: The high response rates, the long survival times, the possibility of achieving 5-year-survival either by HAI alone or by resection after down staging with HAI all sum up to the evidence that HAI could be the primary choice of treatment for CRLM. Phase-III trials are conducted to compare the protocols with optimal regional versus systemic chemotherapy.

Adenocarcinoma↗
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↗
PubMed2001

A practical approach to the first iteration in the optimization of radiation dose and image quality in CT: estimates of the collective dose savings achieved.

CT is a diagnostic imaging modality giving higher patient dose in comparison with other radiological procedures. It contributed an estimated 20% to the collective effective dose to the UK population from medical X-rays in 1990, rising to an estimated 40% in 1999. Tremendous national effort has been expended in reducing patient doses from other radiological procedures with considerable success, but much of the collective dose reduction achieved has been offset by a corresponding increase in the collective dose from CT. Over a period of about 2 years, following the installation of a helical CT scanner, CT scan parameters in this hospital have been adjusted with the aim of working towards optimization of image quality and patient dose. A 33% reduction in annual collective effective dose has been achieved, from about 16.5 manSv to 11 manSv. However, despite this dose reduction, the annual collective effective dose to our sub-population is 2.8 times the value 9 years ago. The increase is almost entirely the result of an increased application of CT; 6149 examinations per annum in 1999 compared with 2210 in 1991. The crucial importance of reducing patient doses from this modality is discussed. Indicative effective doses and image noise values are presented for examination protocols approaching optimization.

Humans↗
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↗
PubMed2000

Distribution of renal lesions in idiopathic systemic vasculitis: A three-dimensional analysis of 87 glomeruli.

Extracapillary proliferation and fibrinoid necrosis are the main diagnostic glomerular lesions in renal biopsy specimens of patients with idiopathic systemic vasculitis. Neither the incidence nor the correlation between extracapillary proliferation and fibrinoid necrosis in renal biopsy specimens from patients with systemic vasculitis has been systematically evaluated. By means of a three-dimensional analysis, we made a topographic reconstruction of the distribution of extracapillary proliferation and fibrinoid necrosis in affected glomeruli and tested different biopsy-processing protocols to optimize histopathologic analysis in clinical practice. Paraffin blocks of renal biopsy specimens from six patients diagnosed with systemic vasculitis were completely and serially sectioned in 2-microm thick sections and stained with the Gomori trichrome method. Glomeruli were scored per section for the presence of fibrinoid necrosis and extracapillary proliferation. Subsequently, a three-dimensional reconstruction was obtained for 87 glomeruli. In only one glomerulus did fibrinoid necrosis occur without extracapillary proliferation; in 51%, a combination of the two lesions was found; in 22%, extracapillary proliferation occurred in the absence of fibrinoid necrosis; and 26% did not show either lesion. Using the standard protocol from our department (ie, evaluation of 20 consecutive sections in various stainings), the chance of finding extracapillary proliferation was 100% and that of finding fibrinoid necrosis was 73%. If 5 sections stained with the Gomori trichrome were added, the latter percentage increased to 86%. Using skip-serial sections, even better results (87% to 92%) were obtained, with four skips as the best option (92%). In conclusion, our finding that fibrinoid necrosis rarely occurs in the absence of extracapillary proliferation may imply that both lesions are etiologically related. In addition, our observations indicate that the incidence of fibrinoid necrosis may be underestimated in clinical practice, depending on the number of sections evaluated.

Biopsy↗
PubMed2001

A strategy for optimizing quality and quantity of DNA extracted from soil.

The efficiency of a bead beating method was studied in detail with regard to a variety of factors including beating time and speed, volume and temperature of the buffer, as well as amount and type of beads employed. The results presented here reveal that all of these parameters have a significant effect on yield and quality of DNA extracted from soils. Precise adjustment of extraction conditions allows for significantly higher yields of high quality DNA from soils than previously reported. We further evaluated the effect of the extraction conditions on the apparent soil microbial community structures, as observed by polymerase chain reaction (PCR) and RFLP. Differences in the fingerprints of DNA extracted under different conditions suggest that results could be biased when using gentle extraction procedures. Based on multiple subsequent extractions using very harsh extraction conditions, we propose a protocol for the quantification of the total DNA content in soils. Extractions from six soils of different texture and chemical characteristics with selected bead beating protocols revealed that the quality (fragment size and purity) of the extracted DNA was generally very good, but also depended on the soil characteristics. While a single, general protocol for optimal DNA recovery from all soils cannot be given, this study provides detailed guidelines on how to optimize the general method to obtain optimal DNA from individual soils.

Buffers↗
PubMedDate not supplied

The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs.

The short-term storage (at 0 degrees C) and cryopreservation of spermatozoa may be useful for providing gametes for fertilisations performed in programmes for the conservation and management of endangered amphibians. The current study was undertaken to examine the applicability of amphibian spermatozoa storage protocols developed with the cane toad (Bufo marinus) to a wider range of amphibian species, with a view to ultimately using these protocols for endangered species. In Australia, at least 29 species of recently extinct or endangered frogs are from the families the Myobatrachidae and the Hylidae. This study investigated the applicability of short-term storage and cryopreservation protocols developed for cane toad (Bufo marinus) spermatozoa to those of hylid and myobatrachid species. Storage of spermatozoa in intact testes or in suspensions for six days at 0 degrees C showed spermatozoa maintained higher motility in suspensions than those in testes, and hylid spermatozoa maintained greater motility than myobatrachid spermatozoa. However, the protocols for optimal storage at 0 degrees C varied with testis size when spermatozoa were stored in whole testes. Spermatozoa from 13 frog species representing both families were cryopreserved using sucrose as diluent with Me(2)SO or glycerol as cryoprotectants. After cryopreservation hylid spermatozoa showed a greater recovery than myobatrachid spermatozoa and Me(2)SO provided higher recovery than glycerol. The freeze-thaw recovery of spermatozoa was independent of testes weight of the species studied. These results show spermatozoa from the Hylidae and Myobatrachidae may be stored both in the short-term (at 0 degrees C) and long-term by cryopreservation using protocols established for B. marinus.

Animals↗
PubMed2000

[Problems in inducing pulmonary maturation in relation to premature delivery].

At presents efforts are made to find valid early markers of premature delivery and if it is inevitable to use an optimal therapeutic protocol for induction of pulmonary maturity which may lead to reduction of early neonatal mortality and morbidity (intraventricular-periventricular haemorrhage, necrotizing enterocolitis, bronchopulmonary dysplasia, air-leak syndrome, respiratory distress syndrome--RDS). Hitherto used corticoid monotherapy reduced the incidence of RDS in neonates with a birth weight lower than 1500 g but the incidence is still relatively high and RDS is still one of the main causes of death of immature neonates. Even the use of thyrotropic hormones did not reduce its incidence. Another possible therapeutic approach is ambroxol administration in combination with corticoids. In the presented review the authors discuss the problem of optimal induction of pulmonary maturity in relation to premature delivery.

Ambroxol↗
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↗
PubMed2002

Radiation dose and image quality in diagnostic radiology. Optimization of the dose-image quality relationship with clinical experience from scoliosis radiography, coronary intervention and a flat-panel digital detector.

UNLABELLED: X-rays are known to cause malignancies, skin damage and other side effects and they are thus potentially dangerous. Therefore, it is essential and in fact mandatory to reduce the radiation dose in diagnostic radiology as far as possible. This is also known as the ALARA (as low as reasonably achievable) principle. However, the dose is linked to image quality and the image quality may not be lowered so far that it jeopardizes the diagnostic outcome of a radiographic procedure. The process of reaching this balance between dose and image quality is called optimization. The aim of this thesis was to propose and evaluate methods for optimizing the radiation dose-image quality relationship in diagnostic radiography with a focus on clinical usefulness. The work was performed in three main parts. OPTIMIZATION OF SCOLIOSIS RADIOGRAPHY: In the first part, two recently developed methods for digital scoliosis radiography (digital exposure and pulse fluoroscopy) were evaluated and compared to the standard screen-film method. Radiation dose was measured as kerma area-product (KAP), entrance surface dose (ESD) and effective dose; image quality was assessed with a contrast-detail phantom and through visual grading analysis. Accuracy in angle measurements was also evaluated. The radiation dose for digital exposure was nearly twice as high as the screen-film method at a comparable image quality while the dose for pulsed fluoroscopy was very low but with a considerably lower image quality. The variability in angle measurements was sufficiently low for all methods. Then, the digital exposure protocol was optimized to a considerably lower dose with a slightly lower image quality compared to the baseline. FLAT-PANEL DETECTOR: In the second part, an amorphous-silicon direct digital flat-panel detector was evaluated using a contrast-detail phantom, measuring dose as entrance dose. The flat-panel detector yielded a superior image quality at a lower dose than both storage phosphor plates and screen-film. Equivalent image quality compared to storage phosphor plates was reached at about one-third of the dose. OPTIMIZATION OF PERCUTANEOUS CORONARY INTERVENTION (PCI): In the third part, influence of various settings on radiation dose and image quality in coronary catheterisation and PCI was investigated. Based on these findings, the dose rate for fluoroscopy was reduced to one-third. The dose reduction was evaluated in a clinical series of 154 PCI procedures before and 138 after the optimization. Through this optimization, the total KAP was significantly reduced to two-thirds of the original value. IN SUMMARY: This thesis indicates the possibility of dose reduction in diagnostic radiology through optimization of the radiographic process.

Coronary Angiography↗
PubMed2000

Miniaturization of a functional transcription assay in yeast (human progesterone receptor) in the 384- and 1536-well plate format.

Miniaturization of high throughput screening assays to high-density microplate formats (384 or 1536 wells) is currently the focus of research activity in modern drug discovery facilities. In this article, we describe the adaptation of a fluorescence-based functional transcription assay in yeast for assessing modulators of human progesterone receptor to the 384- and 1536-well microplate format, comparing the experimental results to those obtained in the well-established 96-well format. The experiences gained from the optimization of the liquid-handling procedures and the miniaturization of an enzyme assay (beta-galactosidase) were implemented. Thus optimized pipetting protocols were developed to perform a reporter gene assay in yeast in microplate formats of higher density. In the functional transcription assay in yeast, the reporter gene expression showed the expected dependence on the ligand's dose and affinity in principle in all three microplate formats. For the first time, this assay system has been established in the 1536-well microplate format using CyBi-Well 96/384/1536 as the liquid-handling unit. The comparison of the signal:background ratios showed a lower sensitivity of the assay in the microplate formats of higher density. This study is an example of a successful miniaturization of a yeast cell-based assay to high-density plate formats on the basis of a careful adaptation procedure and optimized liquid-handling conditions.

Cloning, Molecular↗
PubMedDate not supplied

Routine MR imaging protocol with breath-hold fast scans: diagnostic efficacy for focal liver lesions.

PURPOSE: To qualitatively and quantitatively evaluate the diagnostic efficacy of the breath-hold magnetic resonance (MR) imaging sequences in widespread clinical use for detection and characterization of focal hepatic lesions. MATERIALS AND METHODS: Fifty patients with 143 lesions [57 hepatocellular carcinomas (HCC), 10 borderline lesions, 18 metastatic tumors, 21 hemangiomas, and 37 cysts] underwent single-shot fast spin echo (SSFSE), fast spin echo (FSE), and gadolinium-enhanced dynamic fast spoiled gradient-recalled acquisition in steady state (FSPGR) breath-hold MR imaging of the liver. Alternative free receiver operating characteristic (AFROC) analysis was performed to independently and prospectively assess each sequence. RESULTS: For solid lesions, dynamic FSPGR allowed the most sensitive lesion detection and produced the highest lesion conspicuity and lesion-liver contrast-to-noise ratio (CNR). For non-solid benign lesions, SSFSE and FSE produced better results than dynamic FSPGR. SSFSE allowed the most sensitive detection and produced the best lesion conspicuity and lesion-liver CNR. CONCLUSION: SSFSE and dynamic FSPGR comprise the optimal imaging protocol for breath-hold MR assessment of focal hepatic lesions. This combination of sequences allows acquisition of critical diagnostic information about both inherent T2 and T1 lesion contrast and lesion vascularity.

Adult↗
PubMed2002

Heart failure management: multidisciplinary care has intrinsic benefit above the optimization of medical care.

PURPOSE: This work addresses the unanswered question of whether multidisciplinary care (MDC) of heart failure (HF) can reduce readmissions when optimal medical care is applied in both intervention and control groups. METHODS: In a randomized, controlled study, 98 patients (mean age, 70.8 +/- 10.5 years) admitted to hospital with left ventricular failure (New York Heart Association Class IV) were assigned to routine care (RC, n = 47) or MDC (n = 51). All patients received the same components of inpatient, optimal medical care of HF: specialist-led inpatient care; titration to maximum tolerated dose of angiotensin-converting enzyme inhibitor before discharge; attainment of predetermined discharge criteria (weight stable, off all intravenous therapy, and no change in oral regimen for 2 days). Only those in the MDC group received inpatient and outpatient education and close telephone and clinic follow-up. The primary study endpoint was rehospitalization or death for a HF-related issue at 3 months. MAIN FINDINGS: At 3 months, four people had events in the MDC group (7.8% rate over 3 months) compared with 12 people (25.5% rate over 3 months) in the RC group (P = 0.04). CONCLUSION: These data demonstrate for the first time the intrinsic benefit of MDC in the setting of protocol-driven, optimal medical management of HF. Moreover, the event rate of 7.8% at 3 months, as the lowest reported rate for such a high-risk group, underlines the value of this approach to the management of heart failure.

Aged↗
PubMed1999

Evaluation of transmyocardial laser revascularization (TMLR) by gated myocardial perfusion scintigraphy.

TMLR is a novel treatment for patients with coronary artery disease. It comprises the creation of transmyocardial channels thought to improve myocardial perfusion. Gated Tc-99m sestamibi scintigraphy was used to evaluate changes in myocardial perfusion after TMLR. Twelve patients underwent TMLR using a carbon dioxide laser. Sestamibi scans were carried out following a standard protocol prior to and 1, 3, 6, and 12 months after TMLR. Both visual and semi-quantitative assessment showed improvement in 4 patients, deterioration in 2 patients, and no change in the remaining 6 patients each. However, visual and semi-quantitative assessment were concordant in 6 patients and discordant in 6 patients. In 3 of these, semi-quantitative assessment suggested a better outcome, and in 3 patients visual assessment gave better results. Our findings in a small group of patients suggest that about a third benefited from TMLR. Gated myocardial perfusion scintigraphy using technetium-99m sestamibi is suitable for visual evaluation of changes in the lased area over time, but does not allow semi-quantitative evaluation in the patient population typically treated with TMLR. Further investigations using optimized imaging protocols, including positron emission tomography and three dimensional image presentation, are warranted.

Aged↗
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↗