Swine manure injection with low-disturbance applicator and cover crops reduce phosphorus losses.

Injection of liquid swine manure disturbs surface soil so that runoff from treated lands can transport sediment and nutrients to surface waters. We determined the effect of two manure application methods on P fate in a corn (Zea mays L.)-soybean [Glycine max (L.) Merr.] production system, with and without a winter rye (Secale cereale L.)-oat (Avena sativa L.) cover crop. Treatments included: (i) no manure; (ii) knife injection; and (iii) low-disturbance injection, each with and without the cover crop. Simulated rainfall runoff was analyzed for dissolved reactive P (DRP) and total P (TP). Rainfall was applied 8 d after manure application (early November) and again in May after emergence of the corn crop. Manure application increased soil bioavailable P in the 20- to 30-cm layer following knife injection and in the 5- to 20-cm layer following low-disturbance injection. The low-disturbance system caused less damage to the cover crop, so that P uptake was more than threefold greater. Losses of DRP were greater in both fall and spring following low-disturbance injection; however, application method had no effect on TP loads in runoff in either season. The cover crop reduced fall TP losses from plots with manure applied by either method. In spring, DRP losses were significantly higher from plots with the recently killed cover crop, but TP losses were not affected. Low-disturbance injection of swine manure into a standing cover crop can minimize plant damage and P losses in surface runoff while providing optimum P availability to a subsequent agronomic crop.

Phase III trial comparing paclitaxel poliglumex vs docetaxel in the second-line treatment of non-small-cell lung cancer.

Paclitaxel poliglumex (PPX), a macromolecule drug conjugate linking paclitaxel to polyglutamic acid, reduces systemic exposure to peak concentrations of free paclitaxel. Patients with non-small-cell lung cancer (NSCLC) who had received one prior platinum-based chemotherapy received 175 or 210 mg m(-2) PPX or 75 mg m(-2) docetaxel. The study enrolled 849 previously treated NSCLC patients with advanced disease. Median survival (6.9 months in both arms, hazard ratio=1.09, P=0.257), 1-year survival (PPX=25%, docetaxel=29%, P=0.134), and time to progression (PPX=2 months, docetaxel=2.6 months, P=0.075) were similar between treatment arms. Paclitaxel poliglumex was associated with significantly less grade 3 or 4 neutropenia (P<0.001) and febrile neutropenia (P=0.006). Grade 3 or 4 neuropathy (P<0.001) was more common in the PPX arm. Patients receiving PPX had less alopecia and did not receive routine premedications. More patients discontinued due to adverse events in the PPX arm compared to the docetaxel arm (34 vs 16%, P<0.001). Paclitaxel poliglumex and docetaxel produced similar survival results but had different toxicity profiles. Compared with docetaxel, PPX had less febrile neutropenia and less alopecia, shorter infusion times, and elimination of routine use of medications to prevent hypersensitivity reactions. Paclitaxel poliglumex at a dose of 210 mg m(-2) resulted in increased neurotoxicity compared with docetaxel.

Expression of lysophosphatidic acid acyltransferase beta (LPAAT-beta) in ovarian carcinoma: correlation with tumour grading and prognosis.

Lysophosphatidic acid acyltransferase beta (LPAAT-beta) is an enzyme involved in lipid biosynthesis whose role in tumour progression has been of emerging interest in the last few years. We investigated the expression of LPAAT-beta by reverse transcriptase-polymerase chain reaction and immunohistochemistry in 10 ovarian cell lines as well as in a cohort of 106 ovarian tumours and normal ovaries. Lysophosphatidic acid acyltransferase beta mRNA was found in all cell lines and ovarian tumours examined. Expression of LPAAT-beta protein was significantly increased in ovarian carcinomas compared to benign ovarian tissue (chi2 test P-value=0.001, Kruskal-Wallis test P-value <0.0001). Furthermore, LPAAT-beta expression was positively associated with higher tumour grade (P=0.044), higher mitotic index (P<0.0001) and tumour stage (P=0.032). Expression of LPAAT-beta was significantly linked to reduced overall survival time (P=0.024) as well as to shorter progression-free survival time (P=0.012) in patients younger than 60 years. Our study shows that LPAAT-beta is upregulated in ovarian cancer and is more prevalent in poorly differentiated tumours. In addition, LPAAT-beta expression is a predictor of a worse prognosis in patients younger than 60 years. Further studies are needed to investigate if LPAAT-beta may serve as a therapeutic target for certain subgroups of patients.

Water-soluble poly-(L-glutamic acid)-Gly-camptothecin conjugates enhance camptothecin stability and efficacy in vivo.

The therapeutic efficacy of 20(s)-camptothecin (CPT) is limited in humans by the instability of the active lactone form due to preferential binding of the carboxylate to serum albumin and by difficulty in formulation. Formation of an ester bond with an amino acid via the hydroxyl group at carbon 20 of CPT stabilizes the lactone. Linking CPT to a high molecular weight (MW) anionic polymer enhances solubility and improves distribution to the tumor through enhanced permeability and retention (EPR effect). Poly-(L-glutamic acid) (PG) is an anionic homo-polymer that can theoretically bind one molecule of a drug via the gamma carboxylic acid of each monomeric subunit. It has been used to make a water-soluble PG-paclitaxel conjugate currently in Phase II clinical trials that contains 37% paclitaxel by weight and is administered in a 10 min infusion without pre-medication. We evaluated the anti-tumor activity of PG conjugates of CPT after a single intraperitoneal injection using subcutaneous murine B-16 melanoma tumor growth as an indicator. Interposition of a glycine (gly) linker allowed CPT loading up to 50% w/w on the polymer. Increasing the PG MW from 33 to 49 kDa enhanced the efficacy without altering the maximum tolerated dose (MTD). In athymic mice bearing ectopic human colon or lung tumors, efficacy was enhanced compared to free camptothecin. Thus, as with paclitaxel, conjugation of CPT to PG enhanced pharmaceutical properties and preclinical efficacy.

Pharmacological inhibition of phosphatidylcholine biosynthesis is associated with induction of phosphatidylinositol accumulation and cytolysis of neoplastic cell lines.

De novo production of phosphatidic acid (PA) in tumor cells is required for phospholipid biosynthesis and growth of tumor cells. In addition, PA production by phospholipase D has been cited among the effects of certain oncogenes and growth factors. In this report, it has been demonstrated that enhanced phospholipid metabolism through PA in tumor cells can be exploited pharmacologically for development of anticancer agents, such as CT-2584, a cancer chemotherapeutic drug candidate currently in Phase II clinical trials. By inhibiting CTP:choline-phosphate cytidylyltransferase (CT), CT-2584 caused de novo phospholipid biosynthesis via PA to be shunted away from phosphatidylcholine (PC) and into phosphatidylinositol (PI), the latter of which was doubled in a variety of CT-2584-treated tumor cell lines. In contrast, cytotoxic concentrations of cisplatin did not induce accumulation of PI, indicating that PI elevation by CT-2584 was not a general consequence of chemotherapy-induced cell death. Consistent with this mechanism of action, propranolol, an inhibitor of PA phosphohydrolase and phosphatidylcholine biosynthesis, was also cytotoxic to tumor cell lines, induced PI accumulation, and potentiated the activity of CT-2584 in cytotoxicity assays. As expected from biophysical properties of anionic phospholipids on cellular membranes, CT-2584 cytotoxicity was associated with disruption and swelling of endoplasmic reticulum and mitochondria. We conclude that CT-2584 effects a novel mechanism of cytotoxicity to cancer cells, involving a specific modulation of phospholipid metabolism.

Lisofylline suppresses ex vivo release by murine spleen cells of hematopoietic inhibitors induced by cancer chemotherapeutic agents.

Many cytotoxic cancer therapeutic drugs activate stress response signaling pathways that transcriptionally activate a variety of genes. We decided to determine if cytotoxic therapies induce inflammatory cytokines with inhibitory effects on hematopoiesis and if lisofylline (LSF), a novel antiinflammatory compound, suppresses this induction. Mice were treated with cytosine beta-d-arabinofuranoside (AraC), cis-platinum(II)diammine-dichloride (CisP), etoposide (VP-16), or melphalan at clinically relevant doses, with or without LSF. Spleen cell conditioned media (CM) derived from mice treated with cytotoxic agents, but not from control or LSF treated mice, reduced colony formation by murine bone marrow progenitors belonging to the myeloid, erythroid, megakaryocytic, and B-lymphoid lineages. LSF (100 mg/kg), administered either simultaneously with or up to 48 hours before the cytotoxic agents, suppressed the release of this inhibitory activity. Treatment of inhibitory CM with neutralizing antibodies against known growth inhibitory cytokines, including tumor necrosis factor alpha, transforming growth factor beta, and macrophage inflammatory protein-1alpha, resulted in enhanced colony growth. We conclude that treatment of mice with chemotherapeutic drugs induces the ex vivo production of multilineage hematopoietic inhibitors and that induction of these inhibitors could be abrogated by treatment with LSF. These findings suggest a mechanism whereby LSF can accelerate recovery of hematopoiesis following cytotoxic therapies.

A randomized placebo-controlled trial of lisofylline in HLA-identical, sibling-donor, allogeneic bone marrow transplant recipients. The Lisofylline Marrow Transplant Study Group.

The purpose of the study was to evaluate the effect of lisofylline (LSF) on engraftment, regimen-related toxicities (RRT), and mortality in patients undergoing allogeneic bone marrow transplantation (BMT). We performed a multicenter, randomized placebo-controlled trial in 60 patients with hematologic malignancies receiving BMT from HLA-identical sibling donors. Patients were randomized to receive either placebo, 2 mg/kg LSF or 3 mg/kg LSF every 6 h, beginning before conditioning and continuing to day 21 or hospital discharge. Treatment groups were balanced with respect to conditioning regimen and disease stage. However, significantly more patients in the 2 mg/kg LSF group were at high risk for RRT due to performance status >/=1, age >/=40 years, and prior exposure to CMV. Nausea and vomiting were the only adverse events observed in a higher proportion of LSF-treated patients that led to study withdrawal in six of 42 patients (14%). The times to neutrophil recovery to >/=500/microl and platelet recovery (>20 000/microl) were not improved by LSF treatment. Nevertheless, no patient who received treatment with 3 mg/kg LSF developed a documented infection between day 0 and 35 or had a serious or fatal infection between day 0 and 100 (P = 0.003 vs placebo for both). The day-100 survival rate was also significantly improved in the 3 mg/kg LSF group (89%), compared with either the 2 mg/kg LSF (48%) or placebo (61%) groups (log-rank test, 3 mg/kg LSF vs placebo, P = 0. 026). We conclude that treatment with LSF 3 mg/kg reduced the incidence of infections and improved 100-day survival in patients receiving related-donor allogeneic bone marrow transplantation. Bone Marrow Transplantation (2000) 25, 283-291.

Conjugation of camptothecins to poly-(L-glutamic acid).

Conjugation of water-insoluble cancer chemotherapeutic drugs to macromolecular polymers can lead to improved pharmaceutical properties and improved therapeutic ratios due to accumulation of the polymer-drug conjugate in tumor tissue through the enhanced permeability and retention (EPR) to macromolecules associated with tumor vasculature. Pharmaceutical shortcomings of certain active camptothecins including difficulty in formulation and instability of the active lactone form due to interactions with human albumin might be improved by conjugation to polymers. In this report, conjugations of camptothecin (CPT), 10-hydroxy-CPT, and 9-amino-CPT to poly-(L-glutamic acid) (PG) are described; coupling was accomplished either through the 20(S)-hydroxyl or 9 and 10 substituents with and without the use of a glycine linker. Studies using a PG paclitaxel conjugate (PG-TXL), which is currently in Phase I testing, demonstrated that PG enhanced aqueous solubility, prolonged plasma residence time, and greatly increased the distribution of paclitaxel to tumor tissue in a murine model. In this report, we describe the use of similar conjugation technology for CPT derivatives and demonstrate that these difficult to formulate compounds can be rendered water soluble, that their maximum tolerated doses are increased, and that they retain substantial anti-tumor activity in syngeneic and xenogeneic tumor models. Preliminary data suggest that PG with molecular weights between 37 and 50 kDa with CPT loading between 14% and 37% with or without glycine linkers display enhanced efficacy compared with nonconjugated camptothecins administered at their maximum tolerated dose.

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.

Cloning and expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells.

Lysophosphatidic acid (LPA) and phosphatidic acid (PA) are two phospholipids involved in signal transduction and in lipid biosynthesis in cells. LPA acyltransferase (LPAAT), also known as 1-acyl sn-glycerol-3-phosphate acetyltransferase (EC 2.3.1.51), catalyzes the conversion of LPA to PA. In this study, we describe the isolation and characterization of two human cDNAs that encode proteins possessing LPAAT activities. These two proteins, designated here as LPAAT-alpha and LPAAT-beta, contain extensive sequence sequence similarities to microbial or plant LPAAT sequences. LPAAT-alpha mRNA was detected in all tissues with highest expression in skeletal muscle whereas LPAAT-beta was expressed predominantly in heart and liver tissues. Expression of these two cDNAs in an Escherichia coli strain with a mutated LPAAT gene (plsC) complements its growth defect and shifts the equilibrium of cellular lipid content from LPA to PA and other lipids. Overexpression of these two cDNAs in mammalian cells leads to increased LPAAT activity in cell-free extracts using an in vitro assay that measures the conversion of fluorescently labeled LPA to PA. This increase in LPAAT activity correlates with enhancement of transcription and synthesis of tumor necrosis factor-alpha and interleukin-6 from cells upon stimulation with interleukin-1beta, suggesting LPAAT overexpression may amplify cellular signaling responses from cytokines.

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.

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.

Lisofylline inhibits transforming growth factor beta release and enhances trilineage hematopoietic recovery after 5-fluorouracil treatment in mice.

The effectiveness of endogenous or exogenously administered colony-stimulating factors may be modulated by the presence of hematopoietic inhibitory molecules. Cytotoxic therapy may result in the induction of hematopoietic inhibitors contributing to prolonged myelosuppression, whereas preventing the induction of such inhibitors may accelerate multilineage recovery. Lisofylline [LSF; (R)-1-(5-hydroxyhexyl)-3,7, dimethyl-xanthine], inhibits the signaling and/or release of certain hematopoietic inhibitory molecules such as tumor necrosis factor alpha, macrophage inflammatory protein 1 alpha, transforming growth factor beta, and IFN-gamma. Treatment of murine bone marrow cells with the cytotoxic agent 5-fluorouracil (5-FU) results in the release of a nondialyzable inhibitor of progenitor (colony-forming unit-granulocyte macrophage; CFU-GM) proliferation. When murine bone marrow cells were treated with 5-FU plus LSF, release of this inhibitor of CFU-GM proliferation was blocked. Neutralizing antibody and Western blot analysis indicated that the inhibitor was TGF-beta. We tested the effect of LSF (100 mg/kg i.p., b.i.d.) on multilineage regeneration after high-dose 5-FU or thiotepa treatment in BALB/c mice. In 4 of 5 experiments, LSF significantly accelerated neutrophil recovery (P < or = 0.05, Wilcoxon paired-signed test). In addition, platelet, reticulocyte, and CFU-GM regeneration were significantly accelerated in mice treated with LSF compared to control mice (P < or = 0.05). LSF had no significant effects on the ability of 5-FU to kill hematopoietic progenitor cells, nor did LSF stimulate or inhibit proliferation of CFU-GM. LSF had no effect on chemotherapy-induced killing of tumor cells in vitro, nor on the antitumor activity of 5-FU or thiotepa in BALB/c mice implanted with P388 leukemia cells. Inhibition of hematopoietic inhibitor release may accelerate multilineage recovery after cytotoxic therapy and, as such, may represent an alternative or additional therapy to the use of positively acting lineage specific colony-stimulating factors.

Nontransformed colony-derived stromal cell lines from normal human marrows. I. Growth requirement and myelopoiesis supportive ability.

We report a new method for generating nontransformed human stromal cell lines with a replicative potential of 20 to 25 doublings yielding 10(6) to 3 x 10(7) cells after 4 to 6 weeks. Cells from week-3 to -6 adherent layers of human long-term bone marrow cultures (LTBMC) were plated in methylcellulose in the presence of 20 U/mL interleukin-1 beta (IL-1 beta) and 200 U/mL tumor necrosis factor-alpha (TNF-alpha). After 2 to 3 weeks, we obtained 180 +/- 14 colonies per 10(5) cells seeded. These well-delineated colonies with a dense central core consisted of up to several hundred tightly packed, identical, large refractile cells. Colonies were determined to be clones by sequential examination of the cultures and the linear relationship between the number of colonies counted and cells seeded. Colony-derived cell lines (CDCL) were developed by seeding individual colonies in long-term culture medium (LTCM) supplemented with 20 ng/mL basic fibroblast growth factor (bFGF). The selection of colonies yielding lines with high proliferative capacity was due to the presence of IL-1 beta and TNF-alpha in the semisolid medium. The most effective concentrations for clonal selection were 200 U/mL TNF-alpha and 20 U/mL IL-1 beta. The growth of CDCL in liquid culture depended on the presence of bFGF, with the most effective concentration at 20 ng/mL. CDCL were able to maintain the output of colony-forming units granulocyte/macrophage and burst-forming unit-erythrocyte (CFU-GM and BFU-E) for several weeks from cocultured CD34+ marrow cells. The weekly CFU-GM and BFU-E output from weeks 2-5 was at least the same as observed when using passaged adherent layers. CDCL represent a progenitor cell population for stromal cells that may prove a suitable model for the study of the relationship between marrow stromal cells and hematopoiesis.

Phase III randomized, double-blind placebo-controlled trial of rhGM-CSF following allogeneic bone marrow transplantation.

Preliminary studies in allogeneic BMT suggest that recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) is well tolerated. This is a prospective, multicenter, randomized, double-blind, placebo-controlled trial. Yeast-derived rhGM-CSF 250 micrograms/m2/day or placebo was administered by 4-hour i.v. infusion starting on the day of marrow infusion (day 0) to day 20. All patients received HLA-identical sibling marrow and cyclosporine and prednisone for GVHD prophylaxis. Fifty three patients received rhGM-CSF and 56 received placebo. Comparison of demographics revealed no differences. The time to achieve an absolute neutrophil count of > 0.5 x 10(9) cells/l was shortened in rhGM-CSF treated patients (day 13 vs. 17, P = 0.0001). The incidences of grade III-IV mucositis and infection were significantly reduced (P = 0.005, P = 0.001, respectively) and duration of hospitalization was modestly shortened by 1 day (P = 0.02) in rhGM-CSF treated patients. No differences in platelet recovery, erythrocyte recovery, incidence of veno-occlusive disease, GVHD severity, relapse or survival were observed. In conclusion, rhGM-CSF is well tolerated and reduces post-transplant morbidity in patients undergoing HLA-identical allogeneic BMT.

Phosphatidic acid signaling mediates lung cytokine expression and lung inflammatory injury after hemorrhage in mice.

Because phosphatidic acid (PA) pathway signaling may mediate many basic reactions involving cytokine-dependent responses, we investigated the effects of CT1501R, a functional inhibitor of the enzyme lysophosphatidic acid acyltransferase (LPAAT) which converts lysophosphatidic acid (Lyso-PA) to PA. We found that CT1501R treatment not only prevented hypoxia-induced PA increases and lyso-PA consumption in human neutrophils, but also prevented neutrophil chemotaxis and adherence in vitro, and lung injury and lung neutrophil accumulation in mice subjected to hemorrhage and resuscitation. In addition, CT1501R treatment prevented increases in mRNA levels and protein production of a variety of proinflammatory cytokines in multiple lung cell populations after blood loss and resuscitation. Our results indicate the fundamental role of PA metabolism in the development of acute inflammatory lung injury after blood loss.

Potential role for phosphatidic acid in mediating the inflammatory responses to TNF alpha and IL-1 beta.

Tumor necrosis factor alpha (TNF alpha), interleukin 1 beta (IL-1 beta), and endotoxin (LPS) are potent pro-inflammatory mediators which induce multiple and diverse biological responses in a wide variety of cell types. However, these pro-inflammatory mediators also have significant overlap and redundancy in their biological effects. This suggests that there is significant diversity in second messenger signal transduction systems induced by these stimuli to explain the diversity in biological responses, as well as significant redundancy. Here we show that one such second messenger common to several proinflammatory stimuli may be phosphatidic acid (PA). Intracellular PA species, which may have intracellular signaling functions, are rapidly induced in P388 monocytic leukemia cells by TNF alpha, IL-1 beta, or LPS. These PA species vary according to the bond type (i.e., sn-1 ester vs. ether vs. vinyl ether), acyl chain length, and the degree of saturation in the sn-1 and sn-2 positions. Although PA itself may have direct second messenger activities, many of the PA species induced are converted to diacylglycerol species (DG), which are structurally distinct from the DGs generated by phosphatidylcholine-specific phospholipase C (PC-PLC). Lisofylline [(R)-1-(5-hydroxyhexyl)-3,7-dimethylxanthine; LSF] selectively inhibits generation of selected species of PA in P388 cells induced by TNF alpha, IL-1 beta or LPS. TNF alpha-induced sphingomyelin hydrolysis, PLC-mediated PC hydrolysis, and DG kinase-mediated PA formation or TNF alpha-induced NF-kappa B activation and apoptosis are not inhibited by LSF. LSF has a marked protective effect in a variety of acute inflammatory animal models that may be due to inhibition of this shared second messenger pathway involving PA.

Recombinant human granulocyte-macrophage colony-stimulating factor after autologous bone marrow transplantation for lymphoid cancer: an economic analysis of a randomised, double-blind, placebo-controlled trial.

In a blinded retrospective economic evaluation of a double-blind, randomised, placebo-controlled clinical trial, total utilisation and charges for lymphoid cancer patients who received recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) or placebo were compared following autologous bone marrow transplantation. The 40 patients enrolled (22 rhGM-CSF, 18 placebo) could have acute lymphoblastic leukaemia, non-Hodgkins lymphoma or Hodgkin's disease, be of any age, and were undergoing autologous bone marrow transplantation in a metropolitan cancer research centre. Main outcome measures consisted of initial hospital lengths of stay (LOS), total and department charges, rehospitalisation rates and charges, and outpatient charges, all inclusive of the first 100 days following bone marrow infusion. The perspective of the study is that of the third party payer. Initial hospitalisation charges were $US54 100 for patients who received rhGM-CSF and $US68 600 for patients who received placebo (p = 0.05). The difference of $US14 500 was 21% less in patients who received rhGM-CSF, mainly due to lower average LOS with rhGM-CSF (24.2 days) compared with placebo (30.8 days). Outpatient charges were $US9500 (rhGM-CSF) and $US6800 (placebo) {p = 0.18}. Total charges, including readmission (10 per group) were $US12 200 lower in the rhGM-CSF group ($US70 300 vs $US82 500, p = 0.19). The use of rhGM-CSF after autologous bone marrow transplantation was shown to result in substantial cost savings during the initial hospitalisation. When comparing total inpatient and outpatient medical charges within the first 100 days following bone marrow infusion, we found no evidence that these savings were negated.