Liposome-induced pulmonary hypertension: properties and mechanism of a complement-mediated pseudoallergic reaction.

Intravenous injection of liposomes can cause significant pulmonary hypertension in pigs, a vasoconstrictive response that provides a sensitive model for the cardiopulmonary distress in humans caused by some liposomal drugs. The reaction was recently shown to be a manifestation of "complement activation-related pseudoallergy" (CARPA; Szebeni J, Fontana JL, Wassef NM, Mongan PD, Morse DS, Dobbins DE, Stahl GL, Bünger R, and Alving CR. Circulation 99: 2302-2309, 1999). In the present study we demonstrate that the composition, size, and administration method of liposomes have significant influence on pulmonary vasoactivity, which varied between instantaneously lethal (following bolus injection of 5 mg lipid) to nondetectable (despite infusion of a 2,000-fold higher dose). Experimental conditions augmenting the pulmonary hypertensive response included the presence of dimyristoyl phosphatidylglycerol, 71 mol% cholesterol, distearoyl phosphatidylcholine, and hemoglobin in liposomes, increased vesicle size and polydispersity, and bolus injection vs. slow infusion. The vasoactivity of large multilamellar liposomes was reproduced with human C3a, C5a, and xenoreactive immunoglobulins, and it correlated with the complement activating and natural antibody binding potential of vesicles. Unilamellar, monodisperse liposomes with 0.19 +/- 0.10 microm mean diameter had no significant vasoactivity. These data indicate that liposome-induced pulmonary hypertension in pigs is multifactorial, it is due to natural antibody-triggered classic pathway complement activation and it can be prevented by appropriate tailoring of the structure and administration method of vesicles.

Hemodynamic changes induced by liposomes and liposome-encapsulated hemoglobin in pigs: a model for pseudoallergic cardiopulmonary reactions to liposomes. Role of complement and inhibition by soluble CR1 and anti-C5a antibody.

BACKGROUND: Intravenous administration of some liposomal drugs can trigger immediate hypersensitivity reactions that include symptoms of cardiopulmonary distress. The mechanism underlying the cardiovascular changes has not been clarified. METHODS AND RESULTS: Anesthetized pigs (n=18) were injected intravenously with 5-mg boluses of large multilamellar liposomes, and the ensuing hemodynamic, hematologic, and laboratory changes were recorded. The significant (P<0.01) alterations included 79+/-9% (mean+/-SEM) rise in pulmonary arterial pressure, 30+/-7% decline in cardiac output, 11+/-2% increase in heart rate, 236+/-54% increase in pulmonary vascular resistance, 71+/-27% increase in systemic vascular resistance, and up to a 100-fold increase in plasma thromboxane B2. These changes peaked between 1 and 5 minutes after injection, subsided within 10 to 20 minutes, were lipid dose-dependent (ED50=4. 5+/-1.4 mg), and were quantitatively reproducible in the same animal several times over 7 hours. The liposome-induced rises of pulmonary arterial pressure showed close quantitative and temporal correlation with elevations of plasma thromboxane B2 and were inhibited by an anti-C5a monoclonal antibody (GS1), by sCR1, or by indomethacin. Liposomes caused C5a production in pig serum in vitro through classic pathway activation and bound IgG and IgM natural antibodies. Zymosan- and hemoglobin-containing liposomes and empty liposomes caused essentially identical pulmonary changes. CONCLUSIONS: The intense, nontachyphylactic, highly reproducible, complement-mediated pulmonary hypertensive effect of minute amounts of intravenous liposomes in pigs represents a unique, unexplored phenomenon in circulation physiology. The model provides highly sensitive detection and study of cardiopulmonary side effects of liposomal drugs and many other pharmaceutical products due to "complement activation-related pseudoallergy" (CARPA).

Effect of sialyl Lewis(x) oligosaccharide on myocardial and cerebral injury in the pig.

BACKGROUND: Although administration of the sialyl Lewis(x) oligosaccharide may reduce myocardial injury after ischemia-reperfusion, its effect on coronary and cerebral microvascular regulation and its clinical application during cardiac operation have not been examined. METHODS: Pigs were placed on normothermic cardiopulmonary bypass after 30 minutes of left anterior descending coronary artery occlusion. The hearts were then arrested with cold high potassium cardioplegia. After 1 hour the cross-clamp was removed and the pigs were weaned from cardiopulmonary bypass and perfused for an additional 1 hour. CY-1503 (a sodium salt of the sialyl Lewis(x) oligosaccharide, n = 6) was administered before reperfusion. Six other pigs received saline vehicle. Endothelium-dependent relaxation of precontracted coronary and brain arterioles (70 to 180 microm) to adenosine 5'-diphosphate and endothelium-independent relaxation to sodium nitroprusside were studied in vitro with videomicroscopy. Control values were obtained from uninstrumented pigs. Myeloperoxidase activity in the myocardium and brain was measured to quantify neutrophil infiltration. Cardiac function and perfusion were assessed by left ventricular systolic pressure, maximum rate of increase of left ventricular pressure, left anterior descending coronary artery blood flow and percent segmental shortening, and cerebral vascular resistance, internal carotid artery blood flow, and the constitutively expressed and inducible isoform of nitric oxide synthase mRNA were measured. RESULTS: The impaired myocardial contractile function after ischemia and cardioplegia was not improved by administration of CY-1503. The reduced endothelium-dependent relaxation responses of coronary and brain arterioles during ischemia followed by cardioplegia and cardiopulmonary bypass were improved with CY-1503, but the altered pattern of organ perfusion was not improved. Myeloperoxidase activity was increased in the heart after ischemia-cardioplegia and in the brain after cardiopulmonary bypass. CY-1503 reduced myeloperoxidase activity in both the myocardium and in the brain. Expressions of myocardial inducible isoform or constitutively expressed nitric oxide synthase were not altered in the heart. CONCLUSIONS: Although the sialyl Lewis(x) oligosaccharide does reduce neutrophil infiltration and endothelial injury in the coronary and cerebral microcirculation after cardiopulmonary bypass, it does not have significant beneficial acute effects on organ perfusion or function in the myocardium or brain.

Inhaled nitric oxide does not affect adenosine 5'-diphosphate-dependent platelet activation in infants with persistent pulmonary hypertension of the newborn.

OBJECTIVE: To investigate the effect of inhaled nitric oxide (NO) treatment in newborns with persistent pulmonary hypertension on adenosine 5'-diphosphate (ADP)-dependent platelet activation. METHODS: After parental informed consent, infants with persistent pulmonary hypertension of the newborn were randomly assigned to receive conventional treatment (control group) or treatment with 40 parts per million of inhaled NO. Platelet activation was measured at time of entry and 30 minutes later by surface expression of P-selectin in response to increasing concentrations of the agonist ADP (0, 2, 5, 10, and 20 microM) using fluorescence-activated flow cytometry. RESULTS: We examined 11 infants in the inhaled NO group and 13 in the control group. P-selectin expression, quantified as mean fluorescence, was not significantly different in the two groups of patients at baseline. Median percent change from baseline fluorescence was assessed using the Wilcoxon matched-pairs signed-rank test. At 30 minutes after enrollment there were no statistically significant changes from baseline fluorescence in either group of patients and at all ADP concentrations. CONCLUSION: Thirty minutes of exposure to 40 ppm of inhaled NO does not inhibit ADP-dependent platelet activation as measured by surface expression of P-selectin in infants with persistent pulmonary hypertension of the newborn.

Platelet and neutrophil activation during cardiac surgical procedures: impact of cardiopulmonary bypass.

BACKGROUND: Platelet and neutrophil activation plays a crucial role in reperfusion injury. To determine whether platelet and neutrophil activation occurs in the coronary circulation after cold cardioplegic arrest and reperfusion, we studied 22 patients undergoing coronary artery bypass or valve procedures, or both procedures. METHODS: Blood was sampled from the coronary sinus and the radial artery (A) before bypass; (B) immediately after cross-clamp release; and (C) 5 minutes after cross-clamp release; and was analyzed for surface markers of platelet (CD62P) and neutrophil (CD11b) activation. RESULTS: During bypass, platelet activation increased significantly (p < 0.01) over prebypass values, but no difference was seen between arterial and coronary sinus samples. Neutrophil activation also increased significantly (p < 0.001) during bypass, but there was no difference between arterial and coronary sinus samples. CONCLUSIONS: Cellular activation occurs locally in the coronary circulation during bypass, but no more so after cold cardioplegic arrest and reperfusion.

Awkward moments in patient-physician communication about HIV risk.

BACKGROUND: Physicians frequently encounter patients who are at risk for HIV infection, but they often evaluate risk behaviors ineffectively. OBJECTIVE: To describe the barriers to and facilitators of comprehensive HIV risk evaluation in primary care office visits. DESIGN: Qualitative thematic and sequential analysis of videotaped patient-physician discussions about HIV risk. Tapes were reviewed independently by physician and patient and were coded by the research team. SETTING: Physicians' offices. PARTICIPANTS: Convenience sample of 17 family physicians and general internists. Twenty-six consenting patients 18 to 45 years of age who indicated concern about or risks for HIV infection on a 10-item questionnaire administered before the physician visit were included. MEASUREMENTS: A thematic coding scheme and a five-level description of the depth of HIV-related discussion. RESULTS: In 73% of the encounters, physicians did not elicit enough information to characterize patients' HIV risk status. The outcome of HIV-related discussions was substantially influenced by the manner in which the physician introduced the topic, handled awkward moments, and dealt with problematic language and the extent to which the physician sought the patient's perspective. Feelings of ineffectiveness and strong emotions interfered with some physicians' ability to assess HIV risk. Physicians easily recognized problematic communication during reviews of their own videotapes. CONCLUSIONS: Comprehensive HIV risk discussions included providing a rationale for discussion, effectively negotiating awkward moments, repairing problematic language, persevering with the topic, eliciting the patient's perspective, responding to fears and expectations, and being empathic. Educational programs should use videotape review and should concentrate on physicians' personal reactions to discussing emotionally charged topics.

The meaning of symptoms in 10 women with somatization disorder and a history of childhood abuse.

OBJECTIVE: To understand the associated experiences and illness behavior in patients with somatization disorder and a history of childhood abuse. DESIGN: In-depth interviews were conducted with patients who had somatization disorder and a history of childhood abuse; qualitative content analysis was then performed. SETTING: Patients were recruited from 2 primary care teaching practices. PARTICIPANTS: Physicians were asked to refer patients suspected of having both conditions, yielding 21 potential participants. Eight declined, and 3 did not meet standardized screening questionnaire criteria, yielding 10 women who participated in the study. Participants and nonparticipants had a similar range of socioeconomic variables. RESULTS: An analysis of the interviews yielded 22 themes. Seven themes relevant to understanding the link between illness behavior and abuse were the abuse experiences, emotional and behavioral reactions to the abuse, relationship of abuse to somatoform symptoms, relationship of abuse to health care use, attempts to tell about the abuse, relationships with physicians, and physician behavior. Childhood attempts to tell adults about the abuse resulted in threats of punishment, contributing to lifelong patterns of secrecy, even with physicians. Six women reported having childhood physicians who were family members, friends, or the abuser's physician, reinforcing their subsequent secrecy. The women reported that their current physicians denied their physical pain as adults, just as the abusers denied their emotional and physical pain in childhood. Seven women reported decreased health care use once they associated symptoms with abuse experiences. Nine women reported spousal abuse. CONCLUSIONS: Somatization and childhood abuse may involve a paradoxical pattern of hiding feelings and reality, while seeking acknowledgment of suffering. Patient insight may decrease health care use. Therefore, the exploration of patient experiences may be useful for women with somatization disorder and a history of childhood abuse. The risks of spousal abuse and denial and rejection in the physician-patient relationship could also be important.

Eicosanoid production from porcine neutrophils and platelets: differential production with various agonists.

Polymorphonuclear neutrophils (PMN) and platelets interact to produce both inflammatory and anti-inflammatory lipid mediators during human disease. Because swine models of human disease are used, it is important to understand the mechanisms involved in the formation of lipid mediators from porcine PMN-platelet interactions. In the present study, we investigated the mechanism of thromboxane (Tx) A2 and lipoxin A4 (LXA4) formation from porcine PMN and platelets, respectively. PMN (10(7)/ml) and platelet (30 x 10(7)/ml) suspensions stimulated with porcine C5a (pC5a), but not recombinant human C5a (rhC5a), significantly enhanced TxB2 formation. After cytochalasin B treatment, pC5a or rhC5a significantly and equally enhanced TxB2 formation from PMN-platelet suspensions. A-23187-induced TxB2 formation from platelets was not significantly augmented by the presence of PMN in these suspensions. A-23187 induced significant LXA4 production from porcine PMN that was not augmented by addition of platelets. Flow cytometric analysis of PMN-platelet suspensions revealed activated platelets adherent to PMN following pC5a stimulation. CV-6209, a platelet-activating factor (PAF) receptor antagonist, dose dependently prevented pC5a-induced platelet adherence to PMN and TxB2 formation. These data demonstrate that 1) porcine PMN alone can biosynthesize LXA4 without the assistance of platelets, which is in sharp contrast to human PMN-platelet interactions, and 2) in the absence of cytochalasin B, pC5a stimulates PAF biosynthesis from porcine PMN, resulting in TxB2 formation from platelets.

Juvenile fibromatosis: hormonal receptors.

Aggressive juvenile fibromatosis, though allegedly a benign process, is as frustrating to manage as it is perplexing to comprehend. The treatment is primarily surgical, with chemotherapy and radiation therapy recently finding support as adjuncts in selected circumstances. Though there is no agreement regarding the etiology of fibromatosis, many have suspected hormonal or traumatic influences. There has been historical, clinical and experimental data demonstrating that fibromatosis seems to be under the influence of estrogen. There have also been anecdotal reports that this tumor has regressed with the use of tamoxifen. To our knowledge, no one has tested these tissues for the presence of estrogen/progesterone receptors. Recently, over a short period of time, we at Children's Memorial Hospital, Chicago, have treated four youngsters with this disorder. All operative specimens were submitted for estrogen and progesterone assays. Although these cases were indistinguishable on histologic examination, two of the four exhibited the presence of estrogen/progesterone receptors. We propose, that from these observations, there potentially may be derived a histochemical classification based upon the presence or absence of estrogen/progesterone receptors. This would serve as an added reference in the definition and treatment of this disease. Should hormonal receptors be present, agents such as tamoxifen conceivably could be employed as part of a post-operative maintenance regimen similar to those protocols applied in the management of hormonally responsive breast cancer.

What is the clock? Translational regulation of circadian bioluminescence.

An oscillation in the cellular level of specific proteins in the unicellular marine alga Gonyaulax is correlated with the prominent circadian rhythm of bioluminescence of living cells and persists under constant conditions. This regulation involves a daily bout of synthesis of a specific protein, which is controlled by the circadian clock at the translational level.

Methodologic factors influencing plasma binding of alpha-1-acid glycoprotein-bound and albumin-bound drugs.

Plasma binding of imipramine and lidocaine, two drugs that bind predominantly to alpha-1 acid glycoprotein (AAG), and of diazepam and phenytoin, albumin-bound drugs, were studied in fresh human plasma. Binding was determined by equilibrium dialysis with 0.1 M pH 7.4 phosphate buffer at 37 degrees C. AAG was determined by immunodiffusion. Lidocaine free fraction (FF) (30-35%) was time-dependent, reaching equilibrium by five hours. However, dialysis time exceeding 8 hours greatly increased lidocaine FF (60%) accompanied by a decrease in AAG concentration. Lidocaine binding and AAG-concentration were not affected by plasma freezing, and were independent of lidocaine concentrations from 0.5-10 micrograms/ml. Imipramine behaved comparably, with FF increasing from 15% at 5 hours of dialysis to 30% at 24 h, concurrent with a drop in AAG concentration. Imipramine binding and AAG concentration were also independent of plasma freezing and drug concentration from 0.25 to 10.0 micrograms/ml. Binding of diazepam (FF = 1.2%) and phenytoin (FF = 17%) were stable at 24 hours, not affected by sample freezing or drug concentration, and were independent of AAG concentration. Therefore, changes in AAG concentration alter plasma protein binding of AAG-bound drugs lidocaine and imipramine. Extended dialysis results in decreased concentrations of immunoreactive AAG and increased FF. In contrast, the binding of albumin-bound drugs diazepam and phenytoin are not affected by these variables.

Interaction of propoxyphene with diazepam, alprazolam and lorazepam.

Healthy volunteers received single doses of three benzodiazepines (diazepam, 10 mg i.v.; alprazolam, 1.0 mg orally; lorazepam, 2 mg i.v.) on two occasions in random sequence. One trial was a control; for the other, subjects ingested propoxyphene, 65 mg every 6 h, for the duration of the benzodiazepine study. The kinetics of each benzodiazepine were determined from multiple plasma concentrations measured following each dose. For diazepam, propoxyphene produced a small and statistically insignificant prolongation of elimination half-life (43 vs 38 h) and reduction of total clearance (0.41 vs 0.47 ml min-1 kg-1). Propoxyphene significantly prolonged alprazolam half-life (18 vs 12 h, P less than 0.005) and reduced total clearance (0.8 vs 1.3 ml min-1 kg-1, P less than 0.005). Propoxyphene had no apparent influence on lorazepam half-life (13.4 vs 13.5 h) or clearance (1.5 vs 1.4 ml min-1 kg-1). Thus propoxyphene significantly impairs the clearance of alprazolam, biotransformed mainly by the oxidative reaction of aliphatic hydroxylation. Propoxyphene has far less effect on the oxidation of diazepam by N-demethylation, and has no apparent influence on lorazepam conjugation.

Clinical importance of the interaction of diazepam and cimetidine.

Cimetidine is known to impair the hepatic microsomal oxidation of diazepam, reducing its clearance and prolonging its half-life. We studied the clinical importance of this effect in 10 patients, who were receiving long-term treatment with diazepam for anxiety, tension, or difficulty in sleeping, in an eight-week double-blind controlled study during which the diazepam dosage remained constant. The study was in four two-week phases: base-line or adaptation, coadministration of cimetidine (300 mg) or matching placebo four times daily, crossover to the opposite treatment (placebo or cimetidine), and recovery treatment with diazepam alone. During the cimetidine phase, plasma concentrations of diazepam plus desmethyldiazepam rose an average of 57 per cent (P less than 0.005), then fell when cimetidine was withdrawn. However, there were no significant changes in scores on the digit-symbol-substitution test, a tracking task, or a reaction-time test. Clinical self-ratings indicated no increases in sedation, fatigue, or drowsiness. Patients experienced shortening of sleep latency (P less than 0.05) and an increase in self-rated depth or soundness of sleep (P less than 0.001) during the cimetidine period, but there were no changes in sleep duration or in the number of nocturnal awakenings. Although coadministration of cimetidine to diazepam-treated patients causes a large increase in plasma diazepam and desmethyldiazepam concentrations, the increase is of minimal clinical importance.