We examined the impact of pretreatment neutrophil count on survival in patients with advanced non-small-cell lung cancer (NSCLC). A total of 388 chemo-naïve patients with stage IIIB or IV NSCLC from a randomised controlled trial were evaluated. The effects of pretreatment peripheral blood neutrophil, lymphocyte and monocyte counts and neutrophil-lymphocyte ratio on survival were examined using the proportional hazards regression model to estimate hazard ratios after adjustment for covariates. The optimal cut-off value was determined by proportional hazards regression analysis with the minimum P-value approach and shrinkage procedure. After adjustment for prognostic factors, the pretreatment elevated neutrophil count was statistically significantly associated with short overall (P=0.0008) and progression-free survival (P=0.024), whereas no association was found between prognosis and lymphocyte or monocyte count. The cut-off value selected for neutrophil count was 4500 mm(-3) (corrected hazard ratio, 1.67; 95% confidence interval (CI), 1.09-2.54). The median survival time was 19.3 months (95%CI, 16.5-21.4) for the low-neutrophil group (4500 mm(-3), n=204) and was 10.2 months (95%CI, 8.0-12.3) for the high-neutrophil group (4500 mm(-3), n=184). We confirmed that pretreatment elevated neutrophil count is an independent prognostic factor in patients with advanced NSCLC receiving modern chemotherapy. Neutrophil count is easily measured at low cost, and it may be a useful indicator of patient prognosis.

Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(؊) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(؊) cells were also highly susceptible to methyl-␤-cyclodextrin (M␤CD) as compared with the WR19L/Fas-SM(؉) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(؊) cells and M␤CD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 (putative molecular mass, 48.6 kDa), which contains a sterile ␣ motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(؊) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against M␤CD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/ Fas-SM(؊) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/Fas-SM(؊) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells.Diverse kinds of phospho-and glycerolipids such as diacylglycerol (DAG), 1 inositol phosphatides, and phosphatidic acid are recognized as bioactive molecules in cell growth and survival (1, 2). Sphingolipid ceramide has recently emerged as a signal mediator of cell functions including apoptosis, differentiation, and secretion (3). Various stresses such as ultraviolet, irradiation, heat shock, hypoxia, and biological factors such as tumor necrosis factor-␣, interferon-␥, and Fas antibody require ceramide generation to execute apoptosis, suggesting the implications of SM as a source of ceramide generation in the induction of cell death (4, 5). It was reported that SM dose-dependently inhibits both deoxycholate-induced apoptosis and subsequent hyper-proliferation in colon epithelial cells (6) and decreases the number of aberrant crypts of colon (7), suggesting the implications of SM in cell death and growth.SM is produced by SM synthase, which is thought to be the only enzyme to synthesize SM in mammalian cells (8). The enzyme catalyzes the reaction in which phosphocholine moiety is transferred from phosphatidylcholine (PC) to ceramide. Thus, the activation of SM synthase subsequently increases the levels of DAG and decreases ceramide at the same time (8). DAG is an important signaling molecule for cell growth through protein kinase C activation (9 -12) and acts competitively against ceramide-induced a...

Accumulating evidences indicate that ceramide is closely involved in apoptotic cell death in neurodegenerative disorders and aging. We examined ceramide levels in the cerebrospinal fluid (CSF) or brain tissues from patients with neurodegenerative disorders and the mechanism of how intra- and extracellular ceramide was regulated during neuronal apoptosis. We screened the ceramide levels in the CSF of patients with neurodegenerative disorders, and found that ceramide was significantly increased in patients with Alzheimer's disease (AD) than in patients with age-matched amyotrophic lateral sclerosis (ALS) and other neurological controls. With immunohistochemistry in AD brains, ceramide was aberrantly expressed in astroglia in the frontal cortices, but not detected in ALS and control brains. To explore for the regulation of ceramide in astroglia in Alzheimer's disease brains, we examined the metabolism of ceramide during neuronal apoptosis. In retinoic acid (RA)-induced neuronal apoptosis, RA slightly increased de novo synthesis of ceramide, but interestingly, RA dramatically inhibited conversion of [14C] ceramide to glucosylceramide (GlcCer), suggesting that the increase of ceramide mass is mainly due to inhibition of the ceramide-metabolizing enzyme GlcCer synthase. In addition, a significant increase of the [14C] ceramide level in the culture medium was detected by chasing and turnover experiments without alteration of extracellular [14C] sphingomyelin levels. A 2.5-fold increase of ceramide mass in the supernatant was also detected after 48 h of treatment with RA. These results suggest a regulatory mechanism of intracellular ceramide through inhibition of GlcCer synthase and a possible role of ceramide as an extracellular/intercellular mediator for neuronal apoptosis. The increased ceramide level in the CSF from AD patients, which may be derived from astroglia, raises a possibility of neuronal apoptosis by the response to intercellular ceramide in AD.

Transplantation-associated thrombotic microangiopathy (TA-TMA) is a devastating complication of hematopoietic stem cell transplantation. TA-TMA likely represents the final stage of vascular endothelial injury; however, its pathophysiology is largely unknown, making clinical management difficult. Recently, the association of neutrophil extracellular traps (NETs) with the development of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome has been reported. Thus, we explored whether NETs are also relevant to the occurrence of TA-TMA. We retrospectively analyzed post-transplant trends of serum NET levels in 90 patients, 11 of whom developed TA-TMA. Relative to baseline (before the conditioning regimen), elevated serum NET levels either at 4 weeks after transplantation or as early as the day of transplantation were associated with significantly increased risk of TA-TMA. In contrast, thrombomodulin, a potential marker for TA-TMA, was not helpful to predict the occurrence of TA-TMA in our study. In addition, we directly detected glomerular deposition of NETs in 2 TA-TMA patients. Increased NET levels are a significant risk factor for TA-TMA, suggesting that NET level is a useful biomarker for TA-TMA.

Our data demonstrate that patients in the EBVMCU, a specific clinical subgroup of MTX-LPD, had a better clinical outcome when MTX was withdrawn than did other patients with MTX-LPD.

No abstract

Regardless of the existence of ceramide-related molecules, such as sphingomyelin (SM), neutral sphingomyelinase (nSMase), and SM synthase, in the nucleus, the regulation of ceramide in the nucleus is poorly understood in stress-induced apoptosis. In Fas-induced Jurkat T-cell apoptosis, we found a time-and dose-dependent increase of ceramide content in the nuclear and microsomal fractions. Fas-induced increase of ceramide content in the nucleus also was detected by confocal microscopy using anticeramide antibody. Activation of nSMase and inhibition of SM synthase were evident in the nuclear fraction after Fas cross-linking, whereas nSMase was activated, but SM synthase was not affected, in the microsomal fraction. Pretreatment with D-609, a putative SM synthase inhibitor, enhanced Fas-induced increase of ceramide in the nucleus and induction of apoptosis along with increase of Fas-induced inhibition of nuclear SM synthase. Fas-induced activation of caspase-3 was detected in the nuclear fraction and in whole cell lysate. A caspase-3 inhibitor, acetyl-Asp-GluVal-Asp-chloromethyl ketone, blocked not only Fas-induced increases of apoptosis and ceramide content but also Fas-induced activation of nSMase and inhibition of SM synthase in the nuclear fraction. Taken together, it is suggested that the nucleus is a site for ceramide increase and caspase-3 activation in Fas-induced Jurkat T-cell apoptosis and that caspase-3-dependent regulation of the "SM cycle" consisting of nSMase and SM synthase plays a role in Fas-induced ceramide increase in the nucleus.

Ceramide has emerged as a lipid mediator in apoptosis induced by a variety of stresses. As we previously showed that the activation of AP-1, a nuclear transcription factor was indispensable to ceramide-induced apoptosis in human leukemia HL-60 cells (Sawai, H., Okazaki, T., Yamamoto, H., Okano, H., Takeda, Y., Tashima, M., Sawada, H., Okuma, M., Ishikura, H., Umehara, H.,