James A. Bianco

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.

Preferential radiosensitization of G1 checkpoint-deficient cells by methylxanthines

PURPOSE To develop a checkpoint-based strategy for preferential radiosensitization of human tumors with deficient and/or mutant p53. METHODS AND MATERIALS A549 human lung adenocarcinoma cell lines differing in their expression of the p53 tumor suppressor gene were produced by transduction with the E6 oncogene from human papilloma virus type 16. The cells expressing E6 (E6+) lack a G1 arrest in response to ionizing radiation, are deficient in p53 and p21 expression, and exhibit a fivefold greater clonogenic survival following 10 Gy radiation. RESULTS Postirradiation incubation with millimolar concentrations of the methylxanthine pentoxifylline (PTX) results in preferential radiosensitization of the E6+ cells compared to the LXSN+ vector transduced controls. There is a threefold sensitization of the LXSN+ cells and a 15-fold sensitization of the E6+ cells, which results in equal clonogenic survival of the two lines. Flow cytometry reveals PTX abrogation of the radiation induced G2 arrest for both cell lines. PTX also prolongs G1 transit for both cell lines. Preliminary results are presented using a novel methylxanthine, lisofylline (LSF), which has similar cell cycle effects on G1 and G2 and achieves differential radiosensitization at micromolar concentrations that are sustainable in humans. CONCLUSION This checkpoint-based strategy is a promising approach for achieving preferential radiosensitization of p53- tumors relative to p53+ normal tissues.

Inhibitors of intracellular phosphatidic acid production: novel therapeutics with broad clinical applications

Phosphatidic acids (PAs) are a molecularly diverse group of phospholipids. Certain species of PA are highly active intracellular signaling molecules associated with cellular activation and mitogenesis. Not surprisingly, the PA species active in signaling are not produced by normal cells under homeostatic conditions. Recently the molecular species of PA associated with specific cellular activating events have been identified and compounds that inhibit the formation of specific PA species have been synthesised. We have characterised four classes of PA not derived from phosphatidylinositol hydrolysis that are involved in intracellularsignaling. PA1α is produced following exposure of cells to lipopolysaccharide, IL-1, hypoxia-reoxygenation, IL-8, or platelet activating factor. Lisofylline, a functional inhibitor of PA1a formation, is in Phase II-III trials as a therapeutic to reduce toxicities of anti-cancer therapies and for cytokine-mediated acute systemic inflammatory conditions. PA1α, PA1β and PA2 are pro...

Toxic Epidermal Necrolysis Possibly Linked to Aztreonam in Bone Marrow Transplant Patients

OBJECTIVE AND SETTING: After instituting aztreonam as part of antibiotic prophylaxis in bone marrow transplant (BMT) patients at the Seattle Department of Veterans Affairs Medical Center, the first two cases of toxic epidermal necrolysis (TEN) occurred in more than 250 BMT patients at this center. We have examined the possible cause-and-effect relationship between aztreonam and TEN. PATIENTS: The first patient was a 23-year-old man with acute lymphocytic leukemia receiving a BMT from a related mismatched donor. He experienced profound conjunctivitis and superficial bulla covering 60 percent of his body surface area (BSA). The second patient, a 32-year-old man with lymphoma, received a BMT from a matched unrelated donor. He exhibited lymphocytosis, acute conjunctivitis, and bullous lesions covering 60 percent of his BSA. INTERVENTIONS: Aztreonam was discontinued in both patients. The first patient was treated with pigskin grafting and the second was treated with topical silver sulfadiazine. RESULTS: Despite stabilization of symptoms, both patients eventually died of infectious complications. CONCLUSIONS: Histological data in both patients were more consistent with TEN than graft-versus-host disease (GVHD) in that dermal infiltrates were sparse or absent. The onset of cutaneous symptoms was of more acute onset than acute GVHD, and ocular complaints are uncommon in acute GVHD. Furthermore, the onset of TEN bore a closer temporal relationship to aztreonam than to other drugs administered.

rhGM-CSF After Allogeneic Bone Marrow Transplantation From Unrelated Donors: A Pilot Study of Cyclosporine and Prednisone as Graft-Versus-Host Disease Prophylaxis

Cyclosporine and prednisone were administered as graft-versus-host disease (GVHD) prophylaxis to nine patients undergoing marrow transplant from HLA matched, unrelated donors. RhGM-CSF was administered at a dose of 250 micrograms/m2 daily to all patients. The median day of neutrophil recovery to > or = 500/mm3 was Day 16. Four patients developed Grade II acute GVHD and four developed Grade III acute GVHD. One patient, who survived only 25 days, did not develop GVHD at all. One patient developed systemic infection within the first 28 days after marrow infusion. Comparison of these data to a prior series of patients undergoing bone marrow transplant (BMT) from unrelated donors who were treated with rhGM-CSF along with methotrexate and cyclosporine for GVHD prophylaxis suggests that rhGM-CSF is well-tolerated, neutrophil recovery may be earlier but the severity of GVHD does not appear reduced. Selection of the GVHD prophylaxis regimen may affect the hematopoietic response to cytokine therapy. Further trials with rhGM-CSF in patients undergoing BMT from unrelated donors are required.