Differential regulation of sphingosine-1-phosphate- and VEGF-induced endothelial cell chemotaxis. Involvement of G(ialpha2)-linked Rho kinase activity.

We compared stimulus-coupling pathways involved in bovine pulmonary artery (PA) and lung microvascular endothelial cell migration evoked by sphingosine-1-phosphate (S1P), a potent bioactive lipid released from activated platelets, and by vascular endothelial growth factor (VEGF), a well-recognized angiogenic factor. S1P-induced endothelial cell migration was maximum at 1 microM (approximately 8-fold increase with PA endothelium) and surpassed the maximal response evoked by either VEGF (10 ng/ml) (approximately 2.5-fold increase) or hepatocyte growth factor (HGF) (approximately 2.5-fold increase). Migration induced by S1P, but not by VEGF, was significantly inhibited by treatment with antisense oligonucleotides directed to Edg-1 and Edg-3 (endothelial differentiation gene) S1P receptors and by G protein modification. These strategies included pretreatment with pertussis toxin, or transfection with mini-genes encoding a betagamma subunit inhibitory peptide of the beta-adrenergic receptor kinase, or an 11-amino-acid peptide that inhibits G(1alpha2) signaling. Various strategies to interrupt Rho family signaling, including C(3) exotoxin, dominant/negative Rho, or the addition of Y27632, a cell-permeable Rho kinase inhibitor, significantly attenuated S1P- but not VEGF-induced migration. Conversely, pharmacologic inhibition of either myosin light chain kinase, src family tyrosine kinases, or phosphatidylinositol-3' kinase reduced basal endothelial cell migration and abolished VEGF-induced endothelial cell migration but did not inhibit the increase in S1P-induced migration. Whereas VEGF and S1P increased both p42/p44 extracellular regulated kinase and p38 mitogen-activated protein (MAP) kinase activities, only p38 MAP kinase inhibition significantly reduced VEGF- and S1P-stimulated migration. These data confirm S1P as a potent endothelial cell chemoattractant through G(1alpha2)-coupled Edg receptors linked to Rho-associated kinase and p38 MAP kinase activation. The divergence in signaling pathways evoked by S1P and VEGF suggests complex and agonist-specific regulation of endothelial cell angiogenic responses.

Activation of endothelial cell phospholipase D by migrating neutrophils.

BACKGROUND: Vascular endothelium plays a critical role in regulating the integrity of intercellular adhesive and junctional contacts in response to migrating neutrophils during the inflammatory process. Biochemical responses induced in endothelial cells by adherent, migrating neutrophils are poorly understood. This study was undertaken to explore the possibility that endothelial cell phospholipase D (PLD) is activated when neutrophils migrate through endothelial cell monolayers in response to chemotactic stimuli. METHODS: Bovine pulmonary artery endothelial cells (BPAEC) were cultured on polycarbonate filters at the base of inserts in Transwell chambers (Costar, Cambridge, MA) and subsequently labeled with 3H-myristic acid. Human neutrophil migration through BPAEC monolayers was measured in response to a chemoattractant gradient produced by leukotriene B4 (LTB4). After neutrophils were induced to migrate through endothelial monolayers in the presence of 0.1% ethanol, the filters were excised, and cellular lipids were extracted. Levels of labeled phosphatidylethanol (PEt), an index of activation of endothelial cell PLD, were measured in chromatographs of resolved phospholipids. RESULTS: When neutrophils migrated through endothelial monolayers in response to LTB4, endothelial cells accumulated significant levels of PEt, indicating activation of endothelial cell PLD. Kinetic analysis demonstrated that PEt generation closely paralleled chemotactic migration over a 2-hour time period. Direct contact of neutrophils with the endothelium failed to induce PEt formation in the absence of neutrophil chemotaxis, and LTB4 was an ineffective stimulus of endothelial cell PLD activity in the absence of migrating neutrophils. Neutrophil migration-dependent endothelial PLD activation was observed when chemotaxis was induced by an unrelated chemoattractant, serum activated by exposure to Escherichia coli. However, neutrophil migration alone could not account for activation of endothelial cell PLD, since the peptide chemoattractant f-met-leu-phe (FMLP) induced comparable migration of neutrophils through endothelial monolayers but did not induce PEt generation. CONCLUSIONS: Our study indicates that chemotactic migration of neutrophils through endothelial monolayers results in endothelial cell PLD activation. This process may amplify both target and effector cell reactivity during the inflammatory response.

Accuracy of interpretation of cranial computed tomography scans in an emergency medicine residency program.

STUDY OBJECTIVES: To determine the concordance of emergency physicians and radiologists in interpreting cranial computed tomography (CT) scans. The study also sought to determine the clinical significance of misinterpretations of cranial CT scans by emergency physicians. DESIGN: Prospective cohort study. SETTING: A county hospital emergency medicine residency program. PARTICIPANTS: Five hundred fifty-five patients undergoing CT scanning during emergency department evaluation. RESULTS: Forty-nine percent (272) of the indications for CT scanning were for trauma, 14.2% (79) were for cerebrovascular accident, 25.1% (139) were for headache, 15.1% (84) were for seizure, and 13.7% (76) were for miscellaneous reasons. The radiologists interpreted 46.1% (256) of the CT scans as abnormal. The most frequent abnormalities were scalp hematoma, 15.2% (39); infarction, 14.1% (36); calcification, 6.3% (16); contusion, 6.3% (16); parenchymal hemorrhage, 5.1% (13); and mass, 5.1% (13). Nonconcordance between radiologists and emergency physicians was found in 38.7% (206) of the cases. Potentially clinically significant misinterpretations were found in 24.1% (131) of the total sample. These misinterpretations included 62 missed major findings (11.4% of total sample): 25 new infarcts, 10 mass lesions, 8 cases of cerebral edema, 8 parenchymal hemorrhages, 5 contusions, 4 subarachnoid hemorrhages, 1 epidural hematoma, and 1 subdural hematoma. However, on chart review, only three patients (0.6%) were found to have been managed inappropriately, and none had an adverse outcome. CONCLUSION: The misinterpretation rate of cranial CT scans by emergency physicians is of potential clinical concern. However, clinical mismanagement is rare. We recommend that more formal education in CT interpretation be included in residency training and continuing medical education programs for emergency physicians.

Screening for sexually transmitted diseases by primary care physicians.

The acquired immunodeficiency syndrome epidemic has drawn attention to screening for sexually transmitted diseases by primary care physicians. A telephone survey of primary care physicians in an area with a high incidence of STDs (Washington, DC) to ascertain the determinants and the extent of screening and counseling for STDs was completed in 1987. Ninety-nine physicians (33 internists, 38 obstetrician/gynecologists, and 28 family/general practitioners), representing 61% of those eligible, completed the interview. One third (39.4%) were screening for gonorrhea, more than one half (57.5%) for syphilis, and almost all (94%) had tested at least one individual for human immunodeficiency virus infection. Analysis suggested that concomitant screening for hepatitis B was significantly and positively associated with screening for gonorrhea and syphilis. Less than half (45.9%) of the physicians asked new patients about their sexual practices. Physicians should take histories of sexual practices and do more preventive counseling.

Characteristics of engraftment after repeated autologous bone marrow transplantation.

The rate of engraftment after autologous bone marrow transplantation (ABMT) is extremely variable and largely unpredictable. To identify factors influencing engraftment, we studied 35 patients with refractory germ cell tumors undergoing high-dose chemotherapy with carboplatin (900-2000 mg/m2) and etoposide (1200 mg/m2) with bone marrow rescue. Prior to the initiation of chemotherapy, bone marrow sufficient for two marrow infusions was harvested (range 0.86-4.82 x 10(8) nucleated cells per kg). All 35 patients received half of the collected bone marrow 3 days after the last dose of chemotherapy; 23 responders received a second round of the same chemotherapy followed by infusion of the second half of the bone marrow. Eighteen patients could be compared for the two transplant episodes. The "rate of engraftment" was defined as the unweighted mean of four parameters: 1) the number of days until the absolute granulocyte count surpassed 0.2 x 10(9)/liter, 2) the number of days until the absolute granulocyte count surpassed 0.5 x 10(9)/liter, 3) the number of days until the last platelet transfusion, and 4) the number of days until the reticulocyte count surpassed 25 x 10(9)/liter. No significant correlation was found between rate of engraftment and such factors as the number of nucleated cells per kg infused, the dose of chemotherapy, extent of prior chemotherapy, tumor response to the high-dose chemotherapy, age of the patient, or the days of granulocytopenic fever (all p greater than 0.20). In contrast, a close correlation was found for the number of units of platelets (p = 0.005) and red blood cells (p = 0.006) transfused following each of the two transplants. There was no significant difference between rate of engraftment after first and second transplantation. Comparison of these data with the results obtained in reported ABMT with separate harvests suggests that the characteristics of the infused marrow determine the rate of engraftment after ABMT. This model of repeated transplantation could provide an important tool for assessing the therapeutic efficacy of hematopoietic growth factors.

Primary care physicians' assessment and prevention of HIV infection.

The degree and depth to which primary care physicians counsel patients at risk for human immunodeficiency virus (HIV) infection is a major concern. To determine which factors influence whether physicians counsel patients at risk for HIV, primary care physicians's clinical experience, knowledge, attitudes, and preventive counseling advice in hypothetical case scenarios were assessed. Ninety-nine adult primary care physicians in the Washington, D.C., metropolitan area were interviewed by telephone from May through November 1987. Ninety-one physicians had tested or referred patients for HIV antibody tests. However, 58% could not name the ELISA or Western blot as the tests. The most frequent HIV prevention recommendations were using condoms (67.7%), abstaining from sexual activity (36.4%), getting tested for HIV (30.3%), and safe sex (23.2%). Naming the HIV antibody tests was the most significant predictor of preventive counseling advice; other significant predictors included physicians' personal comfort with counseling homosexual patients and various physician practice and demographic characteristics. Previous studies showed that homophobia was the main inhibitor of effective AIDS counseling. These results suggest that physicians' lack of knowledge and general discomfort in counseling patients about sexual risk factors, rather than homophobia alone, are important barriers to preventive counseling about HIV infection.

Prevention of release of granulocyte aggregants into sheep lung lymph following endotoxemia by N-acetylcysteine.

Interactions of granulocytes with the lungs are altered by endotoxemia and may be critical in the pathogenesis of endotoxin-induced lung injury. In chronically instrumented unanesthetized sheep, we measured the ability of lung lymph to aggregate normal sheep neutrophils in vitro. We found a marked increase in lung lymph aggregating activity beginning within 1 hour after endotoxin infusion and persisting for several hours. When n-acetylcysteine was administered to the animals before endotoxin infusion, neutrophil aggregating activity in lung lymph after endotoxin infusion was markedly reduced. N-acetylcysteine did not affect neutrophil aggregation, adherence, or leukotriene B4 production in vitro and did not prevent complement activation at concentrations achieved in vivo. It is concluded that endotoxemia causes release from the lungs of substance(s) that activate granulocytes, and that this response is prevented by n-acetylcysteine, possibly as a result of the antioxidant properties of the drug.

Differential inhibition of neutrophil superoxide generation by nonsteroidal antiinflammatory drugs.

In order to further characterize the effects of nonsteroidal antiinflammatory drugs on neutrophil superoxide (O2-) generation, human neutrophils were incubated in the presence of sulfinpyrazone, phenylbutazone, and indomethacin prior to exposure to a variety of oxidative stimuli. Stimuli used included the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP, 5.0 X 10(-7) M), NaF (20 mM), phorbol myristate acetate (PMA, 3.2 X 10(-7) M), and opsonized zymosan (250 micrograms/ml). Superoxide release induced by FMLP was inhibited by all three drugs with half-maximal inhibition (KI50) at 2.5, 30, and 120 microM for sulfinpyrazone, phenylbutazone, and indomethacin, respectively. This inhibition was not due to drug interference with the assay system since comparable inhibition was not observed in a cell-free O2- -generating system. The neutrophil's response to NaF was blunted by sulfinpyrazone (KI50 = 400 microM) and phenylbutazone (KI50 = 65 microM), but was unaffected by indomethacin. A similar inhibitory pattern was observed when zymosan was used as the oxidative stimulus. Sulfinpyrazone and phenylbutazone inhibited the response to zymosan (KI50s of 425 and 32 microM, respectively), whereas indomethacin augmented it. PMA stimulation evoked O2- production which was inhibited by phenylbutazone (KI50 = 350 microM) but not by sulfinpyrazone or indomethacin in concentrations up to 1 mM. The results support the hypothesis that the enzyme system responsible for neutrophil O2- generation can be activated by more than one mechanism. The results also emphasize the need to evaluate pharmacologic modulation of neutrophil responses in light of the stimulus used to evoke the response.

Effects of 100% oxygen on lung vascular function in awake sheep.

The purpose of this study was to define the time course of pulmonary toxicity during continuous breathing of 100% oxygen in adult sheep. Sheep were prepared for chronic measurement of vascular pressures, cardiac output, gas exchange, and for collection of lung lymph. Tracheostomies allowed accurate delivery of either 100% oxygen, compressed air, or hypoxic gas. Six sheep breathed 100% oxygen for 72-96 h, and four sheep breathed compressed air for 96 h. In addition to hypoxic challenges [fractional concentration of inspired O2 (FIo2) = 0.12], some sheep received prostaglandin H2 (PGH2)-analogue infusions to further assess pulmonary vascular reactivity. Lung lymph and blood chemotactic activity were measured by granulocyte migration in modified Boyden chambers. Postmortem lung water content and light microscopy were used to measure pulmonary edema. All sheep breathing 100% oxygen died between 72 (n = 2) and 96 h (n = 4). Sheep breathing oxygen demonstrated lung toxicity by measurements of 1) a fourfold increase in flow of protein-rich lung lymph, 2) increased alveolar-arterial difference in Po2 (greater than 20 Torr on FIo2 = 0.21) and hypercapnic acidemia, and 3) a mean extravascular lung water/dry lung weight of 5.7 (controls = 3.3), and histological evidence of interstitial and alveolar edema. Chemotactic activity in lung lymph at base line increased eightfold within 72-96 h of oxygen breathing. In sheep breathing oxygen, the hypoxic pressor response decreased by 50% at 48 h, and by 72-96 h there was a total absence of hypoxic vasoconstriction. There was no increase in lung lymph prostacyclin metabolite, and sodium meclofenamate did not restore the pressor response. In sheep breathing compressed air none of these changes in pulmonary vascular reactivity, lymph flow, or chemotactic activity occurred. We conclude that prolonged breathing of pure oxygen causes pulmonary edema by increasing lung vascular permeability and that respiratory failure results from both edema and loss of pulmonary vascular reactivity.

Free radicals and inflammation: superoxide-dependent activation of a neutrophil chemotactic factor in plasma.

The intravenous administration of superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) to animals with induced inflammation suppresses the inflammatory response and inhibits leukocyte infiltration into the challenged site, suggesting that neutrophil-generated superoxide reacts with an extracellular precursor to generate a substance chemotactic for neutrophils. Plasma exposed to superoxide in vitro becomes potently chemotactic. The appearance of chemotactic activity is inhibited by superoxide dismutase but not by catalase. The chemotactic factor does not stimulate superoxide production or degranulation in neurtrophils. Intradermal injection of superoxide-treated plasma or of a superoxide-generating system causes heavy infiltration of neutrophils to the injection site but does not cause overt signs of inflammation. The chemotactic factor consists of a chloroform-extractable component bound to serum albumin. The superoxide-dependent chemotactic factor appears to play a major role in communication in neutrophil-mediated inflammatory events. Prevention of production of this factor appears to be the major anti-inflammatory action of superoxide dismutase.

Use of the enzyme-linked immunosorbent assay (ELISA) and its microadaptation for the serodiagnosis of toxoplasmosis.

The enzyme-linked immunosorbent assay (ELISA) has proved to be a sensitive and specific quantitative procedure for the serodiagnosis of toxoplasmosis. Using the toxoplasma model, several parameters of the test were investigated. Day-to-day reproducibility was 90% within one twofold dilution and 98% specific when tested against batteries of sera from other diseases. Both the tube method and the microtitration method were used successfully. ELISA results are equivalent to those found in the indirect immunofluorescence test, yet the ELISA procedure is simpler and more rapid to perform.