Prospective, controlled study of an intervention to reduce errors in neonatal antibiotic orders.

OBJECTIVE: To evaluate the effectiveness of an interactive computerized order set with decision support (ICOS-DS) in preventing medication errors in neonatal late-onset sepsis (LOS). STUDY DESIGN: Prospective, controlled comparison of error rates in antibiotic orders for neonates admitted to the Medical University of South Carolina neonatal intensive care unit with suspected LOS (after postnatal day of life 3) prior to (n=153) and after (n=146) implementation of the ICOS-DS. Antibiotic orders were independently evaluated by two pharmacists for prescribing errors, potential errors and omissions. Prescribing errors included>10% overdoses or underdoses, inappropriate route, schedule or antibiotic, drug-drug or drug-disease interactions, and incorrect patient demographics. Potential errors included misspelled drugs, leading decimals, trailing zeroes, impractical doses and error-prone abbreviations. Multiple errors and omissions in an order were counted individually. RESULTS: Overall error rate per order decreased from 1.7 to 0.8 (P<0.001) and potential error rate from 1.0 to 0.06 (P<0.001). The reduction in omission error rate per order from 0.2 to 0.1 was not significant (P=0.17). The prescribing error rate per order increased from 0.4 to 0.7 (P=0.03) because of the use of incorrect patient weights (P<0.001). Renal dysfunction was significantly associated with an increased risk of prescribing errors (odds ratio=3.7, P=0.01) which was not significantly different for handwritten versus ICOS-DS orders (P=0.15). CONCLUSIONS: The ICOS-DS significantly improved the quality of neonatal LOS antibiotic orders although the use of incorrect patient weights was increased. In both groups, orders for patients with renal dysfunction were at risk for prescribing errors. Further evaluation of interventions to promote medication safety for this population is needed.

Beta-actin--an unsuitable internal control for RT-PCR.

Despite reports confirming cell-cycle dependent gene expression and a number of studies describing specific circumstances in which beta-actin is also regulated, the mRNA for beta-actin remains a widely used housekeeping gene internal control. Utilizing differential reverse transcriptase-polymerase chain reaction (RT-PCR), we report here the dose-dependent inhibition of beta-actin by matrigel. This was detected by comparison to the very moderate inhibition of the target gene, membrane type-1 matrix metalloproteinase (MT1-MMP), with results independently confirmed by similar findings on MT1-MMP expression using competitive RT-PCR. Furthermore, RT-PCR of the housekeeping gene 18 Svedberg Units (S) rRNA demonstrated excellent consistency, reproducibility and non-regulation by a matrigel treatment. We conclude that beta-actin is highly regulated by matrigel and therefore unsuitable as an internal control in this treatment. Hence, these findings suggest that researchers have a responsibility to ensure that the housekeeping gene of choice is not regulated in their specific application, as such regulation may dramatically affect the accuracy of their results. This study reinforces the necessity for minimally regulated housekeeping genes such as 18S rRNA, and the superiority of competitive templates as internal controls for quantitative applications of RT-PCR.

IS-RT-PCR assay detection of MT-MMP in a human breast cancer cell line.

The in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) is a method that allows the direct localisation of gene expression. The method utilises the dual buffer mediated activity of the enzyme rTth DNA polymerase enabling both reverse transcription and DNA amplification. Labelled nucleoside triphosphates allow the site of expression to be labelled, rather than the PCR primers themselves, giving a more accurate localisation of transcript expression and decreased background than standard in situ hybridisation (ISH) assays. The MDA-MB-231 human breast carcinoma (HBC) cell line was assayed via the IS-RT-PCR technique, using primers encoding MT-MMP (membrane-type matrix metalloproteinase) and human beta-actin. Our results clearly indicate baseline expression of MT-MMP in the relatively invasive MDA-MB-231 cell line at a signal intensity similar to the housekeeping gene beta-actin, and results following induction with Concanavalin A (Con A) are consistent with our previous results obtained via Northern blotting.

Sertoli cells as paracrine modulators of DNA synthesis in rat peritubular myoid cells in culture.

To determine whether Sertoli cells influence DNA synthesis by rat peritubular myoid cells in vitro, the effects of Sertoli cells on [3H]thymidine incorporation by peritubular myoid cells in a coculture situation were examined. Incubation of testicular peritubular myoid cells with Sertoli cells in coculture induced a significant increase in [3H]thymidine incorporation by peritubular myoid cells. This indicates a cell-cell cooperation between Sertoli and peritubular myoid cells in the testis in terms of DNA synthesis. Secreted factors from Sertoli cells, as tested in a parabiotic culture situation, also increased [3H]thymidine incorporation by peritubular myoid cells. Moreover, in terms of total cellular protein, cocultures of Sertoli cells and peritubular myoid cells resulted in a significant increase when compared with the monocultures, and this coculture effect substituted for the stimulatory response of serum on peritubular myoid cell monoculture. This study investigated the cooperative role of Sertoli cells and peritubular myoid cells in paracrine regulation of testicular functions.

Rat Sertoli cell extracellular matrix regulates glycosaminoglycan synthesis by peritubular myoid cells in vitro.

We have previously reported metabolic cooperation between Sertoli and peritubular myoid cells in terms of synthesis of one of the main testicular extracellular matrix (ECM) constituents, glycosaminoglycans (GAG). This study concerns Sertoli cell ECM-peritubular myoid cell interactions in terms of GAG synthesis. We have examined the responses of hormones and other regulatory agents such as a combination of follicle-stimulating hormone (FSH), insulin, retinol, and testosterone (FIRT) on peritubular myoid cells, and tested if Sertoli cell ECM or serum factor substitute for the stimulation by FIRT. Testicular peritubular myoid cells cultured on Sertoli cell ECM showed significant increases in the levels of cell- and ECM-associated GAG over that when cultured on uncoated plastic. This indicates a specific cell-substratum interaction between Sertoli cell ECM and peritubular myoid cells in the testis in terms of GAG synthesis. Moreover, in terms of cell-associated GAG synthesis, peritubular myoid cell cultured on Sertoli cell ECM or on plastic in the presence of serum substituted for the stimulatory response of FIRT on peritubular myoid cells cultured on uncoated plastic. The data are discussed in relation to the possible role of cell-substratum interaction in maintaining peritubular myoid cell functions.

Hormonal modulation of the interactions of cultured rat testicular Sertoli and peritubular myoid cells. Effects on glycosaminoglycan synthesis.

Previous investigators have suggested metabolic cooperation between Sertoli and peritubular cells. This study concerns Sertoli cell and peritubular myoid cell interactions in terms of synthesis of one of the main testicular extracellular matrix (ECM) constituents, glycosaminoglycans (GAG). We have tested the effect of hormones and other regulatory agents such as a combination of FSH, insulin, retinol, and testosterone (FIRT) on monocultures of Sertoli and peritubular myoid cells, and have examined whether or not coculture of Sertoli and peritubular myoid cells substitutes for the stimulation by FIRT. Cocultures of Sertoli and testicular peritubular myoid cells showed significant increases in the levels of secreted protein and sulfoprotein, as well as in cell-associated GAG synthesis in untreated cultures. This indicates cell-cell cooperation between Sertoli and peritubular myoid cells in the testis in terms of sulfated protein and GAG synthesis. Addition of the hormone mixture and retinol (FIRT) stimulated cell-associated and ECM-associated GAG in peritubular myoid cells, suggesting a role of circulating hormones in ECM production by peritubular myoid cells in vivo. Cocultures of Sertoli and myoid cells substituted for the stimulatory response of FIRT on peritubular myoid cells, predominantly in terms of cell-associated GAG synthesis, which again emphasizes that the paracrine regulation of testicular ECM synthesis is dependent on Sertoli-myoid cell cooperation.

Collagen biosynthesis in cultured rat testicular Sertoli and peritubular myoid cells.

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.

The effect of iron and ethanol on rat hepatocyte collagen synthesis.

1. Carbonyl iron (2.5% w/w) in rat chow was used to induce iron loading in rat hepatocytes. 2. Acute exposure of cultured hepatocytes from control and iron-loaded rats to ethanol (25-100 mM) resulted in a significant inhibition of protein synthesis. 3. Inhibition of protein synthesis in hepatocytes from iron-loaded rats was primarily due to impaired amino acid uptake by these cells. 4. High concentrations of ethanol stimulated the rate of protein degradation by hepatocytes from iron-loaded rats. 5. Acute administration of ethanol to hepatocytes from control animals did not stimulate the absolute rates of collagen biosynthesis nor induce Type I procollagen mRNA. 6. Acute administration of ethanol did not inhibit procollagen synthesis. 7. Iron overload induced Type I procollagen mRNA and increased the absolute rates of collagen synthesis in hepatocytes. 8. These findings may be relevant for the development of hepatic fibrosis in patients with genetic hemochromatosis who consume excess ethanol.

Effects of iron loading on free radical scavenging enzymes and lipid peroxidation in rat liver.

A comparison of the antioxidant protective system and presence of lipid peroxidation was made between rats iron-loaded by two different mechanisms. Superoxide dismutase activity, glutathione peroxidase activity, and reduced glutathione concentrations, together with malondialdehyde production, were measured in the livers of rats chronically iron-overloaded by (a) parenteral iron (primarily Kupffer cell iron deposition) and (b) dietary carbonyl iron (mainly parenchymal iron deposition). In carbonyl iron-treated rats, hepatic superoxide dismutase activity was significantly decreased, whereas hepatocyte lipid peroxidation, as measured by malondialdehyde levels, was significantly increased when compared with control rats at or above iron concentrations of 100 and 185 mumol/g dry wt, respectively. However, no significant decrease in superoxide dismutase activity or significant increase in malondialdehyde levels was observed in iron dextran-treated rats. Glutathione peroxidase activities and reduced glutathione concentrations in rats, iron-loaded by either method, were not significantly different from those of control animals. These results suggest that the deposition of iron in the reticuloendothelial cells of the liver does not lead to lipid peroxidation; however, iron deposited in the parenchymal cells of the liver may lead to an altered free radical antioxidant protective system, resulting in lipid peroxidation in these cells at a similar level of iron loading. We conclude that the cellular site of iron deposition as well as the hepatic iron concentration is important in determining iron-induced liver injury.

The use of in vivo 2H NMR spectroscopy to investigate the effects of obesity and diabetes mellitus upon lipid metabolism in mice.

In vivo deuterium magnetic resonance spectroscopy was used to measure fat utilization rates in diabetic and non-diabetic obese and non-obese mice. Monosodium glutamate-treated mice were used as a model for obesity, and diabetes was induced by administration of streptozotocin. Deuterium levels were enhanced by addition of D2O to drinking water (10% v/v) for a period of 14 days. The deuterium magnetic resonance signals of the body water and adipose tissue were then monitored to measure the rate of deuterium loss from the body. The rates of fat utilization for obese mice were significantly lower (75%, p less than 0.05) (halflife, t1/2 = 113 +/- 13 days) than the rates for non-obese mice (t1/2 = 30.0 +/- 9.0 days). The induction of diabetes caused a large (90%) but proportionally similar increase in fat utilization for both groups of mice (obese, t1/2 = 11.0 +/- 5.2; non-obese, t1/2 = 3.0 +/- 0.9). The results suggest that the induction of diabetes in obese mice does not affect the utilization of fat as a metabolic fuel. These preliminary studies indicate that deuterium magnetic resonance spectroscopy may be a useful technique for non-invasive determination of the rates of fat utilization in vivo.

The 1H NMR visibility of intracellular lactate in Streptococcus faecalis.

1H NMR studies of glycolysis in washed cell suspensions of Streptococcus faecalis indicated that intracellular lactate is not 1H NMR visible. Evidence for this was gained from time course studies of glycolysis at increasing concentrations of glucose. A close correlation existed between the relative increase in the lactate integral and the enzymatically determined extracellular lactate concentration [Lo]. When ionophores which cause the collapse of the positive intracellular/extracellular lactate gradient were added to cell suspensions following fermentation of 5, 10 and 50 mM glucose, the increase in the lactate integral was proportional to the respective increase in [Lo]. A more direct method for determining the origin of the lactate signal involved centrifugation of a cell suspension after fermentation of 50 mM glucose and measurement of lactate in the extracellular and intracellular fluid. 1H spectra of the cell suspension, supernatant and sonicated pellet revealed that the lactate observed in the cell suspension was equivalent to the lactate in the supernatant alone. The intracellular lactate contained in the pellet represented 42% of the total lactate, indicating that only 58% of lactate is detected by in vivo 1H MRS of S. faecalis. This result is in contrast with the high percentage (70-90%) of in vitro lactate which is detected by in vivo 1H MRS of mammalian brain tissue (Williams S. R. et al. Magn. Res. Med. 7, 425-431, 1988). This may be due to a higher proportion of extracellular lactate in mammalian tissue or differences in the intracellular environments of bacterial and mammalian cells.

Water suppression with B0 field gradient homospoil pulses in high-resolution NMR spectroscopy.

The combination of a frequency nonselective excitation suppression method (1331 sequence) with selective excitation followed by gradient-induced dephasing of water transverse magnetization yielded suppression ratios of greater than 10,000:1. The need for gradient preemphasis and correction of B0 field shifts is discussed. The suppression efficiency of this method compared favorably to results obtained using the CPMG spin-echo technique to observe metabolite resonances in a urine sample.

The effect of methotrexate upon tumour ATP as determined by in vivo 31P inversion spin transfer.

The effect of methotrexate upon tumour growth in the hind leg muscle of rats containing a transplanted mammary adenocarcinoma was investigated by 31P magnetic resonance spectroscopy. Using inversion-transfer techniques, ATP resonances arising from the tumour could be distinguished from those arising from surrounding skeletal muscle. Methotrexate was shown to inhibit, but not prevent, tumour growth as evidenced by the tumour ATP resonances of methotrexate treated animals compared to those of control animals. These findings correlated with decreased tumour volume and enhanced life expectancy.

Association between alcoholism and increased hepatic iron stores.

Although alcoholic liver disease is often associated with some increase in hepatic iron stores, it is now established that when gross iron overload is present, this is due to genetic hemochromatosis. Furthermore, there appears to be a critical iron concentration necessary for the induction of hepatic fibrosis. Lipid peroxidation induced by ethanol and/or iron would appear to play a major role in hepatic damage in both humans and experimental animals. Although the exact mechanism(s) of induction of lipid peroxidation by ethanol and iron remains to be elucidated, both toxins can exert a synergistic effect upon hepatic lipid peroxidation. Iron overload has also been shown to stimulate directly hepatocyte and hepatic procollagen mRNA expression, which is further stimulated by ethanol. The observed synergism between iron and alcohol with respect to both hepatic lipid peroxidation and collagen biosynthesis offers a possible explanation of the apparent early onset of fibrosis and cirrhosis in patients with iron overload who have an excessive alcohol intake.

In vivo determination of ATP in tumors using 31P inversion spin transfer.

The in vivo exchange kinetics of creatine kinase in the hind leg muscle of rats containing a transplanted mammary adenocarcinoma has been investigated using 31P magnetic resonance spectroscopy. Using a solenoid coil, the adenosine triphosphate (ATP) resonances arising from the tumor could be distinguished from ATP resonances arising from the muscle surrounding the tumor by use of inversion spin transfer techniques. This procedure affords a specific method of evaluating ATP metabolism of tumors in vivo.

Water-suppressed volume-selected 1H NMR spectroscopy in vivo: application to study tumor metabolism.

Volume selection using SPACE has been combined with water suppression techniques to provide high-resolution 1H NMR spectra from aqueous solutions. The technique developed was used to obtain spectra from a tumor growing on the hind leg of a rat. Water suppression factors of between 1000 and 2000 were achieved simultaneously with excellent volume selection.

Use of high resolution in vivo volume selected 1H-magnetic resonance spectroscopy to investigate leukemia in humans.

In vivo high resolution volume-selected 1H magnetic resonance spectroscopy of human tibia has been undertaken using spatial coordinates obtained from magnetic resonance images. Adult tibial marrow has a 1H spectrum rich in fatty acid resonances and is readily distinguished from the 1H spectra of surrounding leg muscle. In all four leukemic patients examined, infiltration of fat cells of tibial marrow by proliferating cells rich in mobile H2O protons was evident by magnetic resonance imaging. Selective examination of volumes of tibial marrow (1 cm3) by 1H magnetic resonance spectroscopy confirmed marked differences in the 1H spectra of marrow from these patients. Increases in the H2O peak of the 1H spectra were correlated with infiltration of blast cells and lack of control of the neoplastic disease. These studies are the first to report the use of volume selected magnetic resonance spectroscopy to selectively monitor leukemia in humans.

In vivo determination of body iron stores by natural-abundance deuterium magnetic resonance spectroscopy.

Induction of iron overload in mice using 1% (w/w) dietary carbonyl iron resulted in marked decreases in 1H and 31P NMR relaxation times. Natural-abundance deuterium (2H) NMR spectroscopy has been used to measure 2H T1 values in vivo in the presence of body paramagnetic iron. This procedure offers a method for noninvasive determination of body iron stores.

The utilization of two frequency-shifted sinc pulses for performing volume-selected in vivo NMR spectroscopy.

A new approach to volume-selected in vivo NMR spectroscopy uses two frequency-shifted sinc pulses, in conjunction with pulsed field gradients, to destroy the coherence of the unwanted signals. A hard pi/2 pulse can then be used to read the z magnetization in the region of interest. This method is independent of T2, provides complete volume selection in a single acquisition, and can be readily implemented on most high-field commercial imaging/spectroscopy systems.

Preliminary studies on the potential of in vivo deuterium NMR spectroscopy.

Natural abundance deuterium NMR spectroscopy can be used to characterise in vivo 2H signals arising from water and fat in mice, with acquisition times of less than two minutes. Administration of D(2)0 (10% V/V) in the drinking water enhances these signals so that excellent spectra can be obtained with one scan. Using these procedures the in vivo turnover of 2H in water and fat in mice has been determined. This procedure may be of particular importance in studies of fat turnover in obesity.