Stuart L. Bursten

An increase in serum C18 unsaturated free fatty acids as a predictor of the development of acute respiratory distress syndrome.

OBJECTIVE No means exist for predicting the acute respiratory distress syndrome (ARDS), which complicates sepsis, trauma, and a variety of clinical disorders. Because activation of phospholipid-signaling pathways involving the acyl chains oleate and linoleate may initiate and amplify the inflammatory response, and thereby lead to the development of ARDS, we examined whether serum concentrations of these bioactive lipids increase and are predictive of ARDS in at-risk patients. DESIGN Part I: A prospective, single-blind trial. Part II: A prospective, randomized, double-blind trial. SETTING General intensive therapy units in five university teaching hospitals. SUBJECTS Part I: Thirty-nine healthy control patients were studied to determine normal distribution of serum acyl values, followed by 30 patients admitted with onset of sepsis, trauma, or development of ARDS (within 24 hrs of admission) over a 1-yr period. Part II: Eight patients admitted with sepsis syndrome over a 2-month period. INTERVENTIONS Part II: Patients were randomized to receive the substituted methylxanthine, lisofylline (CT1501R), or an identically presented placebo. MEASUREMENTS AND MAIN RESULTS We measured the serum free fatty acid concentrations in the 39 healthy control subjects, and then we prospectively examined the serum free fatty acid concentrations in 30 age-matched patients in samples obtained within 24 hrs from the onset of sepsis, trauma, or development of ARDS. We then prospectively studied eight septic, at-risk patients who were matched for age, Acute Physiology and Chronic Health Evaluation II scores, Multiple Organ Failure index, and Glasgow Coma Score, in a double-blind, placebo-controlled, pilot study. These patients included four patients who received no treatment and four patients who received lisofylline, a compound that decreases serum unsaturated free fatty acids and diminishes acute lung injury in animals caused by sepsis and/or trauma. The calculated ratios of serum free fatty acids (Le., the ratio of C18 unsaturated fatty acids linoleate and oleate to fully saturated palmitate, C16:0) increased and predicted the development of ARDS in at-risk patients. Serum samples from the 30 patients, obtained within 24 hrs from the onset of sepsis, trauma, or development of ARDS, had significantly increased mean acyl chain ratios (1.42 +/- 0.35 [SD]) compared with healthy control subjects (0.86 +/- 0.25; p < .01). Sera from 13 patients with sepsis or trauma who did not develop ARDS (group A [at-risk, non-pre-ARDS]) also had increased acyl ratios (1.23 +/- 0.27) compared with sera from healthy control subjects (0.86 +/- 0.25; p < .01). Sera from seven patients who subsequently developed ARDS (group B [at-risk, pre-ARDS]) had higher acyl ratios (1.70 +/- 0.21) than group A at-risk patients who did not develop ARDS (1.23 +/- 0.27; p < .01) or healthy control subjects (0.86 +/- 0.25; p < .001). Sera from ten group C patients with ARDS at the time of admission to the study had the highest acyl ratios (1.80 +/- 0.75), which exceeded values for healthy control subjects (p < .001) and group A at-risk patients without ARDS (p = .01), but were not significantly different then group B at-risk, pre-ARDS patients (p = .17). Prospective study of eight septic, at-risk patients demonstrated significantly (p < .05) increased serum acyl ratios in the four untreated patients (findings consistent with the first study) but a significantly (p = .02) reduced ratio in the four at-risk patients treated with lisofyline. CONCLUSIONS Increases in unsaturated serum acyl chain ratios differentiate between healthy and seriously iII patients, and identify those patients likely to develop ARDS. Thus, the serum acyl ratio may not only prospectively identify and facilitate the assessment of new treatments in patients at highest risk for developing ARDS, but may also lead to new insights about the pathogenesis of ARDS.

Ct-1501r Selectively Inhibits Induced Inflammatory Monokines In Human Whole Blood ex Vivo

The effect of (R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine (CT-1501R; the nonproprietary name for CT-1501R approved by the United States Name Council is lisofylline), an inhibitor of second messenger signaling through phosphatidic acid, on release of endogenous mediators important in the systemic inflammatory response syndrome (SIRS) was studied using the human whole blood ex vivo assay system. Human blood was stimulated with various endotoxin preparations, zymosan, or protein A, and the levels of secreted monokines were measured by enzyme-linked immunosorbent assay. CT-1501R inhibited tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-6 release in a dose-dependent manner and was active with all stimuli tested including Salmonella and Escherichia coli-derived endotoxin, endotoxin from both rough and smooth E. coli strains, as well as zymosan and protein A. CT-1501R inhibited monokine release by approximately 50% at 200 μM and 30% at 50 μM and was independent of the relative potency of stimulus. CT-1501R also inhibited IL-1α or IL-1β induction of either TNF-α or IL-1β and inhibited the synergistic effects of stimulation with both human IL-1β and murine TNF-α on release of human TNF-α. Inhibition of monokine release following stimulation with monokine) was, in general, greater than that achieved with lipopolysaccharide (LPS) stimulation. Northern blot analysis showed decreased mRNA accumulation of TNF-α and IL-1β in CT-1501R-treated samples following LPS stimulation suggesting that CT-1501R acts at least in part, at the pretranslational level. In contrast, CT-1501R does not inhibit LPS-stimulated IL-8 or IL-1 receptor antagonist (IL-1ra) release in human whole blood or IL-1α-induced release of PGE2 in human foreskin fibroblast cells. These data suggest that CT-1501R may be of use for clinical intervention in SIRS.

Human mesangial cells are resistant to productive infection by multiple strains of human immunodeficiency virus types 1 and 2

Human immunodeficiency virus-associated nephropathy (HIVAN) is a recognized clinical entity of unknown pathogenesis. A role for viral infection of renal cells in the initiation of this process at present is an intriguing but untested hypothesis. Studies in primate models of acquired immunodeficiency syndrome (AIDS) suggest that injury to the mesangial cell may be central to the sclerosing glomerular lesion characteristic of HIVAN. We therefore tested the infectibility of human mesangial cells (HMC) in vitro by a variety of strains of HIV chosen to include a spectrum of tropisms for different cell types. Productive infection of mesangial cells could not be demonstrated using any of the virus strains. Nonetheless, HIV infection of intrinsic renal cells remains an attractive area of inquiry for understanding the natural history of HIVAN.

Acylation of monocyte and glomerular mesangial cell proteins. Myristyl acylation of the interleukin 1 precursors.

Acylation of cellular proteins with the fatty acids myristate or palmitate represents an important mechanism for the co- or posttranslational modification of proteins. Lipid A, the biologically active component of bacterial endotoxin, exerts a number of biochemical effects on responsive cell types. Evidence is presented that lipid A stimulates the synthesis and subsequent myristyl acylation of intracellular monocyte and glomerular mesangial cell proteins. Two of the myristylated monocyte proteins were identified by specific immunoprecipitation as the 33-kD IL 1 alpha and beta precursors; a similar myristylated protein was found in mesangial cells. The 17-kD secretory form of monocyte IL 1 beta did not contain covalently linked myristate. Myristyl acylation of the IL 1 precursor proteins may facilitate the processing or membrane localization of these proteins, which lack characteristic hydrophobic signal sequences. The acylated 33-kD IL 1 alpha may remain preferentially associated with the membrane in an active form, whereas limited proteolysis may convert the biologically inactive IL 1 beta precursor into the extracellular, nonacylated, active 17-kD protein.

Pharmacological inhibition of gelatinase B induction and tumor cell invasion

The 92 kDa matrix metalloproteinase (gelatinase B, MMP‐9) plays a major role in the facilitation of tumor metastasis and in inflammatory disorders characterized by excessive matrix protein destruction. MMP‐9 is transcriptionally induced in multiple cell types by exposure to the inflammatory mediators bacterial endotoxin, interleukin‐1 (IL‐1) or tumor necrosis factor‐α (TNF‐α). CT‐2519, (1‐(5‐isothiocyanatohexyl)‐3,7‐dimethylxanthine), a synthetic small molecule from an anti‐inflammatory compound library, was evaluated for its effect on endotoxin and cytokine‐induced MMP‐9 synthesis by a monocytic leukemic cell line, THP‐1, and a monocyte/macrophage line, RAW 264.7. CT‐2519 dose‐dependently inhibited endotoxin and cytokine‐induced synthesis of MMP‐9 by these cells. Furthermore, both MMP‐9 secretion and matrix invasion by cells of a human fibrosarcoma cell line, HT‐1080, were inhibited by CT‐2519 in a dose‐dependent manner. Northern blot analyses and studies utilizing MMP‐9 promoter constructs indicated that the inhibitory action of CT‐2519 occurs at the level of transcriptional suppression. Given the observation that cellular activation by endotoxin, IL‐1 and TNF‐α may be mediated, at least in part, through induction of certain species of phosphatidic acid (PA), the effect of CT‐2519 on lipid levels was analyzed. CT‐2519 effectively reduced endotoxin‐mediated increases, in particular cellular lipid levels. Pharmacologic modulation of cytokine‐dependent gene products, such as MMP‐9, may offer an important therapeutic approach to the treatment of neoplastic and inflammatory disorders.

Effects of lisofylline on hyperoxia-induced lung injury.

Lisofylline [1-(5 R-hydroxyhexyl)-3,7-dimethylxanthine] decreases lipid peroxidation in vitro and in vivo suppresses proinflammatory cytokine expression in models of lung injury due to sepsis, blood loss, and oxidative damage. In the present experiments, we used a murine hyperoxia model to examine the effects of lisofylline on the activation of nuclear transcriptional regulatory factors [nuclear factor-κB and cAMP response element binding protein (CREB)], the expression of proinflammatory cytokines in the lungs, and the circulating levels of oxidized free fatty acids as well as on hyperoxia-induced lung injury and mortality. Treatment with lisofylline inhibited hyperoxia-associated increases in tumor necrosis factor-α, interleukin-1β, and interleukin-6 in the lungs as well as decreased the levels of hyperoxia-induced serum-oxidized free fatty acids. Although hyperoxic exposure produced activation of both nuclear factor-κB and CREB in lung cell populations, only CREB activation was reduced in the mice treated with lisofylline. Lisofylline diminished hyperoxia-associated increases in lung wet-to-dry weight ratios and improved survival in animals exposed to hyperoxia. These results suggest that lisofylline ameliorates hyperoxia-induced lung injury and mortality through inhibiting CREB activation, membrane oxidation, and proinflammatory cytokine expression in the lungs.Lisofylline [1-(5R-hydroxyhexyl)-3,7-dimethylxanthine] decreases lipid peroxidation in vitro and in vivo suppresses proinflammatory cytokine expression in models of lung injury due to sepsis, blood loss, and oxidative damage. In the present experiments, we used a murine hyperoxia model to examine the effects of lisofylline on the activation of nuclear transcriptional regulatory factors [nuclear factor-kappaB and cAMP response element binding protein (CREB)], the expression of proinflammatory cytokines in the lungs, and the circulating levels of oxidized free fatty acids as well as on hyperoxia-induced lung injury and mortality. Treatment with lisofylline inhibited hyperoxia-associated increases in tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 in the lungs as well as decreased the levels of hyperoxia-induced serum-oxidized free fatty acids. Although hyperoxic exposure produced activation of both nuclear factor-kappaB and CREB in lung cell populations, only CREB activation was reduced in the mice treated with lisofylline. Lisofylline diminished hyperoxia-associated increases in lung wet-to-dry weight ratios and improved survival in animals exposed to hyperoxia. These results suggest that lisofylline ameliorates hyperoxia-induced lung injury and mortality through inhibiting CREB activation, membrane oxidation, and proinflammatory cytokine expression in the lungs.

Oral nifedipine for the treatment of patients with severe hypertension

Ten mg of nifedipine was administered to 19 patients with severe hypertension (mean blood pressure 187 +/- 17/122 +/- 12 mm Hg) without intensive care monitoring. Patients were instructed to bite and swallow the contents of the capsule. Blood pressure declined significantly to a mean of 149 +/- 17/92 +/- 10 mm Hg. No adverse side effects or hypotension occurred. Ten patients required an additional dose 30 to 60 minutes after the initial dose. Mean heart rate increased from 79 to 95 beats per minute without symptomatic consequences. Laboratory parameters measured before and after the four-hour study did not change significantly, although peripheral renin activity rose transiently. Urinary sodium excretion increased 43 percent over four hours after therapy in three patients in whom it was measured. Cardiac output, which was measured noninvasively in seven patients, rose nonsignificantly whereas systemic vascular resistance declined from 2,070 dynes/second/cm-5 to 1,271 dynes/second/cm-5 (statistically significant difference) in 20 minutes. These results indicate that oral nifedipine, when bitten and swallowed, effectively lowers blood pressure in patients with severe hypertension without the occurrence of adverse side effects or hypotension. Oral nifedipine may be used safely in an outpatient setting when urgent intervention is required.

Modulating phosphatidic acid metabolism decreases oxidative injury in rat lungs.

We determined that lisofylline, a potent inhibitor of oleate- and linoleate-containing phosphatidic acid formation (half-maximal inhibitory concentration = 40 nM), prevented oxidant-mediated capillary leak in isolated rat lungs given interleukin-8 (IL-8) intratracheally and perfused with human neutrophils. Lung leak was prevented by lung, but not neutrophil, lisofylline pretreatment. Furthermore, although lisofylline inhibited IL-8-stimulated neutrophil production of phosphatidic acid in vitro, it did not prevent IL-8-stimulated neutrophil adherence, chemotaxis, or intracellular calcium mobilization or N-formyl-Met-Leu-Phe (fMLP)-stimulated oxidant production in vitro. Lisofylline also prevented acute capillary leak in isolated rat lungs perfused only with the oxidant generator purine-xanthine oxidase but did not scavenge O2-(+) or H2O2 in vitro. Finally, lisofylline-mediated protection against lung leak in both models was associated with alterations in lung membrane free fatty acid acyl composition (as reflected by the decreased ratio [linoleate + oleate]/[palmitate]). We conclude that lisofylline prevented both neutrophil-dependent and neutrophil-independent oxidant-induced capillary leak in isolated rat lungs and that protection appears to be mediated by blocking intrinsic lung linoleoyl phosphatidic acid metabolism. We speculate that lisofylline, in addition to our previously reported effects on cytokine signaling by intrapulmonary mononuclear cells, alters intrinsic pulmonary capillary membrane composition and renders this barrier less vulnerable to oxidative damage.