Release of TNF-alpha and IL-1beta from porcine brain endothelium corresponds to the pyrogenic potential of three marketed formulations of amphotericin.

BACKGROUND: Formulations of amphotericin include a deoxycholate suspension (d-Amph), an amphotericin-B lipid complex (Ablc), and a liposomal product (L-Amph). Fever is most frequent with d-Amph, intermediate with Ablc, and lowest with L-Amph. OBJECTIVE: To determine if the release of tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1) from brain endothelium corresponds to the incidence of amphotericin fever. RESULTS: Release of TNF-alpha and IL-1beta after L-Amph treatment was similar to negative controls while after d-Amph treatment release was similar to lipopolysaccharide. Ablc treatment produced intermediate pyrogen release.NF-kappaB expression, a transcriptional regulator for TNF-alpha and IL-1beta genes, corresponded to this secretion pattern. TNF-alpha release was elevated 2 hours (p = 0.0021) after treatment while significant elevations in IL-1beta required 6 hours (p = 0.0009). CONCLUSION: Results from this in vitro study suggest that amphotericin fever may be directly mediated by brain endothelium. These experiments also suggest that amphotericin fever is initially mediated by TNF-alpha.

Organ dysfunction following stem cell transplantation: relationship to plasma cytokine concentrations.

Patients receiving high-dose preparation for stem cell transplantation are at risk for organ dysfunction (OD). Signs of early OD include hypoxia, mental status changes, and liver dysfunction. These early signs have not been correlated with potential cytokine mediators. We compared plasma concentrations of IL-6, TNF-alpha, and IL-10 in OD patients and controls. Cytokines were measured before preparation, 5 days before OD, day of OD, and 5 days after OD. TNF-alpha and IL-10 were not measurable prior to preparation. IL-10 was more likely to be measurable in OD patients than in controls 5 days prior to onset of OD (P = 0.039), on the day of OD (P = 0.023), and 5 days later (P < 0.0001). TNF-alpha was more likely to be measurable only on the day of OD (P = 0.0035). IL-6 was significantly elevated in OD patients at all time points. Patients who had measurable IL-6 on admission were 5.1 times more likely to develop OD (95% CI = 1.4-17.9; P = 0.011). Five days prior to OD for each 100 pg/ml increase in IL-6, patients were 2.75 times more likely to develop OD (95% CI = 1.3-5.8; P = 0.0087). The early elevation of IL-6 in patients who develop OD may help identify a high risk group where preventive therapies can be evaluated.

Stability of suspension formulations of lansoprazole and omeprazole stored in amber-colored plastic oral syringes.

OBJECTIVE: To determine the stability of lansoprazole and omeprazole suspensions at ambient and refrigerated temperatures using HPLC. DESIGN: The contents of lansoprazole and omeprazole capsules were suspended in separate flasks containing sodium bicarbonate 8.4% to concentrations of 3 and 2 mg/mL, respectively. The contents of each flask were drawn into six amber-colored oral syringes, with one-half of the syringes stored at 22 degrees C (ambient) and the other half at 4 degrees C. Lansoprazole and omeprazole concentrations were determined by a stability-indicating HPLC assay at baseline and at 4, 8, 12, and 24 hours, and on days 4, 7, 14, 21, 30, 45, and 60 after mixing. Both omeprazole and lansoprazole were considered stable if they retained > or =90% of the baseline drug concentration. RESULTS: Omeprazole was stable for up to 14 days at 22 degrees C and 45 days at 4 degrees C. Lansoprazole was stable for eight hours at 22 degrees C and for 14 days at 4 degrees C. CONCLUSIONS: When compared with ambient or refrigerated storage conditions, omeprazole was stable for a longer duration than lansoprazole. Pharmacists may use these results to guide compounding and storage of proton-pump inhibitor suspensions.

Antiproliferative activity of shark cartilage with and without tumor necrosis factor-alpha in human umbilical vein endothelium.

We evaluated the antiangiogenic activity of shark cartilage, tumor necrosis factor-alpha (TNF-alpha), and a combination of the two using a human umbilical vein endothelial cell proliferation assay. Proliferation of endothelium is a hallmark of angiogenesis, and inhibition of endothelial cell proliferation indicates potential antiangiogenic activity. Shark cartilage produced a concentration-dependent decline in endothelial cell 3H-thymidine incorporation. This activity was heat stable and was found in molecular weight fractions of less than 10 kd. The antiproliferative effect of shark cartilage was specific for vascular endothelium and did not affect the proliferative rate of human astrocytoma cells or human skin fibroblasts. Shark cartilage at a concentration of 500 mu g/ml and TNF-alpha at a concentration of 10 ng/ml reduced endothelial cell proliferation by 32% and 29%, respectively. Treatment of endothelial cells with the combination of shark cartilage and TNF-alpha resulted in a 44% reduction in endothelial cell proliferation. The isolation and identification of the active components of shark cartilage is continuing.

An in vitro model for endothelial permeability: assessment of monolayer integrity.

An essential component of any in vitro model for endothelial permeability is a confluent cell monolayer. The model reported here utilizes primary human umbilical vein endothelial cells (HUVEC) cultured on recently developed polyethylene terephthalate micropore membranes. Using a modification of the Wright-Giemsa stain, confluent HUVEC monolayers grown on micropore membranes were routinely assessed using light microscopy. Determination of confluence using this method was confirmed by scanning electron microscopy. Transendothelial electrical resistance of HUVEC monolayers averaged 27.9 +/- 11.4 omega.cm2, 10 to 21% higher than literature values. Studies characterizing the permeability of the endothelial cell monolayer to 3H-inulin demonstrated a linear relationship between the luminal concentration of 3H-inulin and its flux across HUVEC monolayers. The slope of the flux versus concentration plot, which represents endothelial clearance of 3H-inulin, was 2.01 +/- 0.076 x 10(-4) ml/min (r2 = .9957). The permeability coefficient for the HUVEC monolayer-micropore membrane barrier was 3.17 +/- 0.427 x 10(-6) cm/s with a calculated permeability coefficient of the HUVEC monolayer alone of 4.07 +/- 0.617 x 10(-6) cm/s. The HUVEC monolayer reduced the permeability of the micropore membrane alone to 3H-inulin (1.43 +/- 0.445 x 10(-5) cm/s) by 78%. Evans blue dye-labeled bovine serum albumin could not be detected on the abluminal side without disruption of the HUVEC monolayer. These results demonstrate a model for endothelial permeability that can be extensively assessed for monolayer integrity by direct visualization, transendothelial electrical resistance, and the permeability of indicator macromolecules.

Development of secondary sex characteristics in male rats after fetal and perinatal cimetidine exposure.

Cimetidine has been reported to cause antiandrogenic effects in male pups of female rats receiving cimetidine during gestation. Because of conflicting reports of cimetidine causing permanent antiandrogenic effects in male rats, we studied the sexual development of male rats born to females receiving cimetidine. Water or water and cimetidine (194 mg/kg of body weight per day) were administered to pregnant rats from day 12 of gestation through weaning. A total of 130 male pups were studied. Testicular prostate gland/seminal vesicle weights, anogenital distance, serum testosterone and dihydrotestosterone levels, and seminiferous tubule areas were compared between the two groups. Transfer of cimetidine across the placenta and though breast milk was confirmed by HPLC analysis of serum from female littermates at 0, 10, and 20 days of age. With the exception of a smaller anogenital distance (p < 0.03) and a lower anogenital index (p < 0.05) in the cimetidine-exposed newborn rats, no statistically significant differences were observed in the measured parameters between the cimetidine-exposed and control groups. Cimetidine exposure during the fetal and perinatal periods did not alter the development of secondary sex characteristics in male rat pups.

Effect of interleukin-4 on the synthesis of the third component of complement by pulmonary epithelial cells.

Complement activation in the lung is important in a variety of physiological and pathological conditions. The third component of complement, C3, is the pivotal constituent of the complement cascade. C3 is produced in the lung by several cell types including pulmonary epithelial cells. Because pulmonary epithelial cells and T lymphocytes may interact within the lung to regulate local immune responses, we examined the effect of a T lymphocyte-derived cytokine, interleukin-4 (IL-4) on C3 production by A549 human pulmonary epithelial cells. Treatment of A549 cells with IL-4 increased C3 production in a time- and dose-dependent fashion. Concentrations of IL-4 > or = 0.1 ng/ml significantly increased C3 production. Maximal increase in C3 synthesis occurred after stimulation of A549 cells with IL-4 (10 ng/ml) for 3 days. Preincubation of IL-4 with a neutralizing anti-human IL-4 antibody inhibited IL-4's effect on C3 production. The relative abundance of C3 messenger RNA levels in A549 cells increased following IL-4 treatment, indicating that IL-4's effects on C3 production were pretranslational. Intercellular communication between T lymphocytes and pulmonary epithelial cells via cytokines such as IL-4 may be important in the local regulation of C3 gene expression during the inflammatory response.

Effects of injection site and flow rate on the distribution of injected solutions in an extracorporeal membrane oxygenation circuit.

The effects of injection site and flow rates on drug distribution within an extracorporeal membrane oxygenation (ECMO) circuit were studied. Two commercially available reservoirs (30 mL and 50 mL) were connected to a closed ECMO circuit that did not include the membrane oxygenator and heater. The circuit was filled with 150-170 mL of a 9.5% dextran solution in deionized, distilled water with a viscosity approximating that of blood. Flow rates of the circulating solution were set at 75 mL/min and 375 mL/min. Bordeaux red dye was injected into an ECMO circuit at prereservoir, postreservoir, and intrareservoir sites, and samples were obtained from these sites for analysis. Gentamicin was also injected at the prereservoir site, with samples obtained from the reservoir, reservoir tubing, and the prereservoir and postreservoir sites. Postreservoir dye injections resulted in complete mixing at any flow rate. Prereservoir injections at flow rates less than 250 mL/min resulted in incomplete mixing of dye, whereas intrareservoir injections resulted in incomplete mixing at any flow rate. Gentamicin injection was also affected by flow rate, resulting in higher concentrations within the reservoir and reservoir tubing. In patients on ECMO, drug distribution may be substantially altered if drugs are given as a bolus at prereservoir or intrareservoir sites. Efforts should be made to select injection sites that will provide safe and therapeutic drug delivery while minimizing the effects of the ECMO circuit on drug distribution.

Plasma fentanyl levels in infants undergoing extracorporeal membrane oxygenation.

Plasma levels of fentanyl were analyzed in 12 infants undergoing extracorporeal membrane oxygenation who received a fentanyl bolus (5 to 10 micrograms/kg) followed by infusion at 1 to 6.3 micrograms/kg/hr. Fentanyl levels, averaging 11 samples/infant, were measured by radioimmunoassay (mean 19.7 +/- 35.7 ng/ml; n = 140). Eight of the infants, all with a primary diagnosis other than congenital diaphragmatic hernia, survived with relatively short (< 7 days) courses on extracorporeal membrane oxygenation; this group of infants did not develop tolerance to fentanyl and could be maintained on infusion rates of < 5 micrograms/kg/hr throughout. The four infants with congenital diaphragmatic hernia had longer extracorporeal membrane oxygenation runs and three did not survive; their plasma fentanyl levels were consistently higher and while the infusion rates were higher early on extracorporeal membrane oxygenation, they did not exceed 7 micrograms/kg/hr and actually decreased after 5 days on extracorporeal membrane oxygenation. Five infants (42%) received lorazepam in addition to fentanyl for at least one sampling time. The fentanyl infusion dose and plasma level were higher in the congenital diaphragmatic hernia nonsurvivors who did not receive lorazepam (p < 0.001). A decrease in fentanyl clearance correlated with renal dysfunction (p < 0.01). A bolus of fentanyl followed by infusion of relatively low doses (1 to 5 micrograms/kg/hr) provides adequate analgesia for infants on extracorporeal membrane oxygenation, particularly when it is supplemented with intravenous lorazepam whenever needed to control infant movement.