Interaction of glucocorticoid analogues with the human glucocorticoid receptor.

Transient co-transfection of receptor cDNA and suitable reporter genes was used to study human glucocorticoid receptor (hGR) function in a neutral mammalian cell background. A variety of natural and synthetic steroids were analyzed for their ability to activate gene expression through the hGR and to bind to extracts of cells expressing the hGR cDNA. There was very good correlation between these two in vitro parameters for these compounds. Furthermore, correlation of these data with reported in vivo anti-inflammatory potencies was surprisingly close, with two exceptions. The in vitro data suggest an explanation for the discrepant compounds, consistent with published data on their metabolic fate in vivo. The co-transfection assay has utility as a quantitative predictor of in vivo glucocorticoid pharmacology.

Toward effective hospital utilization management. Ten lessons from case studies.

The challenges presented by managed care arrangements and third-party utilization controls have led hospitals to strengthen internal utilization management programs. This article reports findings from a set of case studies on how hospitals have adapted their utilization management programs to their environments. While knowledge acquired from case studies should not be overgeneralized, the findings of these studies suggest some lessons that merit consideration by hospitals advancing their own programs in utilization management.

Molar absorptivities of bilirubin (NIST SRM 916a) and its neutral and alkaline azopigments.

Three laboratories in the U.S. and two in the Netherlands determined molar absorptivities (epsilon) of Standard Reference Material (SRM) 916a Bilirubin from the National Institute of Standards and Technology. In caffeine reagent the average epsilon values were 50,060 and 48, 980 L.mol-1.cm-1 at 432 and 457 nm, respectively. The epsilon value of the blue azopigment, obtained with the Reference Method for total serum bilirubin, was 76,490 L.mol-1.cm-1 at 598 nm. When the addition of alkaline tartrate was omitted, the molar absorptivity of the red azopigment was 56,600 L.mol-1.cm-1 at 530 nm.

Candidate reference method for determination of total bilirubin in serum: development and validation.

This candidate Reference Method for measuring total bilirubin in serum is based on the Jendrassik-Gróf principle (Clin Chem 29: 297-301, 1983). Standard Reference Material no. 916 bilirubin (National Bureau of Standards) is used as the standard. Bilirubin standard solutions may be prepared either in human serum or in 40 g/L albumin solution (human or bovine), because we found the molar absorptivity of the azopigment at 598 nm to be identical in these media. The absorptivities of the unconjugated and conjugated azopigments appear to be identical, but the conjugated azopigment is completely hydrolyzed in the final reaction mixture. Bilirubin added to serum from adults or neonates was quantitatively accounted for. Interference by hemoglobin (up to 2 g/L), ascorbic acid (up to 20 mg/L), or zinc (at physiological concentrations) is negligible. Of the therapeutic drugs we tested, only L-dopa and alpha-methyldopa interfere. We established normal adult reference values for total bilirubin and examined the intraindividual variation in 19 subjects.

Effects of rapid infusion with high pressure and large-bore i.v. tubing on red blood cell lysis and warming.

A prototype large-bore intravenous tubing was developed and tested. Mean flow rates for blood (Hct 45%) and tap water were determined for several catheters at 600 mm Hg, 300 mm Hg, and gravity flow and were statistically analyzed by calculating the 95% confidence intervals. The degree of hemolysis during high pressure and flow was determined by measuring the plasma free hemoglobin using the spectrophotometric method. To determine if cold banked blood can be adequately warmed at high flow rates, thermocouples were used to measure the blood temperature before and after rapid infusion through a blood warmer. Results included maximum flow rates of 1,764 mL/min for tap water, and 1,714 mL/min for blood (Hct 45%) at 600 mm Hg through the large-bore tubing and an 8.5-F catheter. Flow rates for other pressure and catheter combinations were tabulated. The plasma-free hemoglobin increased slightly compared to controls with high pressure (less than or equal to 600 mm Hg) and flow rates. The increase correlated with less than 1% red blood cell lysis in all trials. When 13 C blood was infused through a warmer, blood temperature increased to 25.3 C at the maximum flow rate of 732 mL/min. Slightly higher heat gain resulted with slower infusion rates. We conclude that the prototype large-bore tubing and up to 600 mm Hg pressure provide rapid flow rates without significant hemolysis. Blood warming may be inadequate at higher flow rates.

Hierarchical information system for the cost analysis of clinical laboratory tests.

A microcomputer-based information system that integrates the concepts of text processing, data base processing, and data base analysis has been designed for cost evaluation in our laboratories. This forms a flexible package that is directed by the needs of the user. The package, which has been used to calculate various cost parameters and productivity on the basis of comprehensive data and user-defined rules, serves as a tool for good financial management at various organizational levels in the clinical laboratory.

Pitfalls in the American Monitor kit methods for determination of total and "direct" bilirubin.

We evaluated the American Monitor Corporation kit for total and direct-reacting bilirubin and found that it suffers serious deficiencies, which lead to inaccurate and imprecise results. The main problem with the total-bilirubin procedure is that the short reaction time (2 min) is inadequate for completion of the reaction. The poor precision of the direct-bilirubin method is due to the short reaction time and the inability of the "stabilizer" (hydroxylamine sulfate) to completely destroy the diazo reagent. Depending on when Fehling's reagent is added, the reaction time may vary from 2 min to 7 min. Values for direct bilirubin at 7 min exceed those obtained at 2 min by 17 to 29%. The short reaction time makes color development temperature dependent, an additional source of imprecision. The suboptimal concentration of the diazo reagent results in underestimation of direct-reacting bilirubin. We recommend changes that improve both precision and accuracy of the kit procedures.

Predicting the risk of cancer at the time of breast biopsy. Variation in the benign to malignant ratio.

The benign to malignant ratio (B:M ratio) among breast biopsies (number of benign breast lesions divided by number of breast cancers) is widely believed to be around 4:1 or 5:1. This belief appears to be based upon experience from the 1950's and 1960's. We investigated possible reasons for the current wide variability of the B:M ratio. A straight line relationship between the log B:M ratio and age exists in our data. The B:M ratio is sensitive to racial differences between patient groups but in only minimally affected by varied histologic inclusion criteria. The overall B:M ratio should not be used to counsel women about their breast cancer risk at the time of biopsy. Age-specific B:M ratios provide a more realistic risk assessment.

Fate of the hippocampal mossy fiber projection after destruction of its postsynaptic targets with intraventricular kainic acid.

Intraventricular injections of kainic acid were used to create a model of selective cell death in order to study the fate of afferent projections that are deprived of their postsynaptic targets. This treatment rapidly destroyed hippocampal CA3 pyramidal cells, but not those neurons that give rise to their mossy fiber and entorhinal afferents. Light microscopic studies with the Timm's sulfide silver stain indicated that half or more of the mossy fiber boutons in area CA3b were lost within the first 1-3 days after kainic acid administration. This finding was confirmed by electron microscopy. Electron-dense, usually vacuolated mossy fiber boutons accounted for about 10-20% of the total population present at a 4-hour survival time, but were not encountered in control rats nor at survival times longer than 1 day. Other mossy fiber boutons remained electron lucent, but enlarged, became more rounded in shape, and suffered an apparent loss of synaptic vesicles. It is suggested that degeneration of some mossy fiber boutons and resorption of others into the axon may have accounted for the precipitous decline in their number. The dendritic excrescences contacted by these boutons were nearly all undergoing electron-dense degeneration 4 hours after kainic acid administration. In rats that survived 6-8 weeks mossy fiber boutons remained somewhat scarce, individual boutons appeared relatively small, and only one-third the normal percentage were observed to be engaged in more than one synaptic contact within a single cross section. A qualitative electron microscopic study of the entorhinal projection to area CA3 suggested a response to kainic acid treatment similar to that of the mossy fiber projection, except that no entorhinal boutons were seen to become electron dense. These findings suggest that presynaptic fibers in the mature hippocampus adjust the size of their terminal arborizations and number of synaptic contacts to accommodate a reduction in the target cell population. The rapid loss of mossy fiber boutons may be attributable to an unusual fragility of these structures when they are deprived of the mechanical support normally provided by the pyramidal cell. Finally, the ability of kainic acid administration to alter the number and distribution of presynaptic elements must be taken into account whenever this toxin is used to make selective lesions of postsynaptic cells.

Degeneration of hippocampal CA3 pyramidal cells induced by intraventricular kainic acid.

Degeneration of hippocampal CA3 pyramidal cells was investigated by light and electron microscopy after intraventricular injection of the potent convulsant, kainic acid. Electron microscopy revealed evidence of pyramidal cell degeneration within one hour. The earliest degenerative changes were confined to the cell body and proximal dendritic shafts. These included an increased incidence of lysosomal structures, deformation of the perikaryal and nuclear outlines, some increase in background electron density, and dilation of the cisternae of the endoplasmic reticulum accompanied by detachment of polyribosomes. Within the next few hours the pyramidal cells atrophied and became electron dense. Then these cells became electron lucent once more as ribosomes disappeared and their membranes and organelles broke up and disintegrated. Light microscopic changes correlated with these ultrastructural observations. The dendritic spines and the initial portion of the dendritic shaft became electron dense within four hours and degenerated rapidly, whereas the intermediate segment of the dendrites swelled moderately and became more electron lucent. No degenerative changes were evident in pyramidal cell axons and boutons until one day after kainic acid treatment. Less than one hour after kainic acid administration, astrocytes in the CA3 area swelled, initially in the vicinity of the cell body and mossy fiber layers. It is suggested that the paroxysmal discharges initiated in CA3 pyramidal cells by kainic acid served as the stimulus for this response. Phagocytosis commenced between one and three days after kainic acid administration, but remained incomplete at survival times of 6-8 weeks. Astrocytes, microglia, and probably oligodendroglia phagocytized the degenerating material. These results point to the pyramidal cell body and possibly also the dendritic spines as primary targets of kainic acid neurotoxicity. In conjunction with other data, they support the view that lesions made by intraventricular kainic acid can serve as models of epileptic brain damage.

Loss and reacquisition of hippocampal synapses after selective destruction of CA3-CA4 afferents with kainic acid.

Intraventricular injections of kainic acid were used to destroy the hippocampal CA3-CA4 cells bilaterally in rats, thus denervating the inner third of the molecular layer of the fascia dentata and stratum radiatum of area CA1. Electron microscopic studies showed that this lesion reduced the synaptic density of the CA1 stratum radiatum by an average of 86%. The synaptic density of the inner third of the dorsal dentate molecular layer declined by two-thirds and the corresponding zone of the ventral dentate molecular layer by about half. Within 6-8 weeks the synaptic density of these laminae had been restored to the control value or nearly so. In the CA1 stratum radiatum about 72% of the synaptic contacts destroyed by the lesion were replaced, the inner third of the ventral dentate molecular layer recovered 75% of its lost synapses and the inner third of the dorsal dentate molecular layer apparently recovered virtually all of them. The newly formed synapses did not differ noticeably from those normally present. A kainic acid lesion reduced the synaptic density of the outer two-thirds of the dentate molecular layer by 30% within 3-5 days, despite a virtual absence of presynaptic degeneration in that zone. This result implies a substantial disconnection of perforant path synapses. It did not appear to depend on the extent of denervation of the inner zone. The loss of perforant path synapses was completely reversible. We suggest that the dentate granule cells shed a portion of their synapses in response to a substantial loss of neurons to which they project and regained them when their axons had formed new synaptic connections.

Selective reinnervation of hippocampal area CA1 and the fascia dentata after destruction of CA3-CA4 afferents with kainic acid.

Intraventricular injections of kainic acid were used to destroy the hippocampal CA3-CA4 cells, thus denervating the inner third of the molecular layer of the fascia dentata and stratum radiatum and stratum oriens of area CA1. The responses of intact afferents to such lesions were then examined histologically. The hippocampal mossy fibers densely reinnervated the inner portion of the dentate molecular layer after bilateral destruction of CA4 neurons and to a lesser extent after unilateral destruction. Septohippocampal fibers replaced CA4-derived fibers in the dentate molecular layer only after particularly extensive bilateral CA4 lesions. Medial perforant path fibers showed no anatomical response to any of these lesions. Neither septohippocampal, temporoammonic nor mossy fibers proliferated in or grew into the denervated laminae of area CA1. These results show a preferential ordering in the reinnervation of dentate granule cells which is not readily explained by proximity to the degenerating fibers and also that removal of CA3-CA4-derived innervation more readily elicits translaminar growth in the fascia dentata than in area CA1. These results may be relevant to clinical situations in which neurons of the hippocampal end-blade are lost.