The PDE4 Inhibitor Tanimilast Restrains the Tissue-Damaging Properties of Human Neutrophils.

Neutrophils, the most abundant subset of leukocytes in the blood, play a pivotal role in host response against invading pathogens. However, in respiratory diseases, excessive infiltration and activation of neutrophils can lead to tissue damage. Tanimilast-international non-proprietary name of CHF6001-is a novel inhaled phosphodiesterase 4 (PDE4) inhibitor in advanced clinical development for the treatment of chronic obstructive pulmonary disease (COPD), a chronic inflammatory lung disease where neutrophilic inflammation plays a key pathological role. Human neutrophils from healthy donors were exposed to pro-inflammatory stimuli in the presence or absence of tanimilast and budesonide-a typical inhaled corticosteroid drug-to investigate the modulation of effector functions including adherence to endothelial cells, granule protein exocytosis, release of extracellular DNA traps, cytokine secretion, and cell survival. Tanimilast significantly decreased neutrophil-endothelium adhesion, degranulation, extracellular DNA traps casting, and cytokine secretion. In contrast, it promoted neutrophil survival by decreasing both spontaneous apoptosis and cell death in the presence of pro-survival factors. The present work suggests that tanimilast can alleviate the severe tissue damage caused by massive recruitment and activation of neutrophils in inflammatory diseases such as COPD.

Type-1 interferons prolong the lifespan of neutrophils by interfering with members of the apoptotic cascade.

Polymorphonuclear Neutrophils (PMNs) are metabolically highly active phagocytes, present in abundant numbers in the circulation. These active cells take the onus of clearing invading pathogens by crowding at inflammatory sites in huge numbers. Though PMNs are extremely short living and die upon spontaneous apoptosis, extended lifespan has been observed among those cells arrive at the inflammation sites or tackle intracellular infections or face any microbial challenges. The delay/inhibition of spontaneous apoptosis of these short-living cells at the inflammatory core rather helps in combating pathogens. Like many candidates, type-1 interferons (type-1 IFNs) is a group of cytokines predominant at the inflammation site. Although there are some isolated reports, a systematic study is still lacking which addresses the impact of the predominant type of interferon on the spontaneous apoptosis of neutrophils. Here in, we have observed that exposure of these IFNs (IFN-ß, IFN-α & IFN-ω etc) on human neutrophils prevents the degradation of the Bfl1, an important anti-apoptotic partner in the apoptotic cascade. Treatment showed a significant reduction in the release of cytochrome-C in the cytosol, a critical regulator in the intrinsic apoptotic pathway. We also noticed a reduction in the conversion of procaspase -3 to active caspase-3, a crucial executioner caspase towards initiation of apoptosis. Taken together our results show that exposure to interferon interferes with apoptotic pathways of neutrophils and thereby delay its spontaneous apoptosis. These findings would help us further deciphering specific roles if these inflammatory agents are causing any immune-metabolomic changes on PMNs at the inflammatory and infection core.

Low spontaneous apoptosis index at diagnosis predicts a high-risk phenotype in paediatric acute lymphoblastic leukaemia.

Background & objectives: Significance of apoptosis as a prognostic marker is less well studied in paediatric acute lymphoblastic leukaemia (ALL) cases. Hence, a prospective study, involving 30 paediatric ALL cases, was done to assess the clinical relevance of in vivo apoptosis. Methods: Peripheral blood mononuclear cells from all patients were subjected to annexin V/propidium iodide staining to detect the degree of apoptosis [apoptotic index (AI)] at day 0 and day 35 post-induction chemotherapy. In addition, Bax and Bcl2 apoptotic protein expressions were studied at day 0 and their relative fluorescence mean intensity (RFMI) ratios were calculated. Results: Mean age of patients was 5.1 years. Of the 30 cases, 21 (70%) were at standard-risk, five (17%) at intermediate and four (13%) at high risk. Majority (83%) were B-ALL. Day 8 absolute blast count was >1000/µl in seven (23%) and <1000/µl in 23 of 30 (77%) cases. Day 35 marrow was M1 in 23 (92%) and M2 in two of 25 (8%) cases. AI at day 0 and day 35 ranged from 0.9 to16.6 per cent and 1.4 to 62.8 per cent with a mean of 5.90 and 19.64 per cent, respectively. The Bax/Bcl2 ratio ranged from 0.2 to 3.5 with a mean of 0.83. The ratio was predominantly anti-apoptotic, i.e. <1 (77%). A significant association was noted between low AI at day 0 and high total leucocyte count (P=0.02), T-cell phenotype (P=0.043) and high-risk as per NCI category (P=0.025). Significant increase (>30%) in day 35 AI was seen in only six cases. Interpretation & conclusions: Our study showed that low AI at day 0 was associated with a high-risk clinical phenotype in paediatric ALL. However, studies on larger group, especially with longer follow up or study of relapse cases, will help draw conclusions regarding apoptosis assessment in paediatric ALL.

The Impact of a Long-Acting Oral Sphingosine-1-Phosphate Analogue on Ovarian Aging in a Rat Model.

In animal studies, intravenous continuous infusion or peritoneal injection of sphingosine-1-phosphate (S1P) has been shown to decrease chemotherapy- and radiotherapy-induced apoptosis on primordial follicles. Although a long-acting oral form of an S1P analogue (FTY720, fingolimod) has been recently developed and utilized in women with multiple sclerosis, there are no data exploring its ability to avoid spontaneous follicle apoptosis. Thirty 10-month-old female rats were randomly assigned to 3 groups to investigate whether fingolimod would be able to decrease the spontaneous ovarian follicle apoptosis ratio. An oral analogue form of S1P was administered for 60 days at a dose of 0.1 mg/kg (n = 10) or dose of 1 mg/kg (n = 10) per day. The control group (n = 10) received physiological serum via an orogastric feeding tube. The main outcome measures were anti-Müllerian hormone (AMH) level and nonapoptotic follicle ratio. While low-dose S1P group had comparable AMH levels to high-dose S1P group and controls, high-dose S1P group had higher mean levels of AMH, reaching marginal significance with controls (5.72 ± 0.61 vs 4.81 ± 0.85 ng/mL, P = .050). For the nonapoptotic primordial follicle ratio, both low-dose S1P group (67.0% ± 16.4% vs 29.9% ± 19.5%, P < .001) and high-dose S1P group (51.1% ± 11.5% vs 29.9% ± 19.5%, P = .023) had superior rates when compared with controls. Interestingly, low-dose S1P groups also had a statistically higher nonapoptotic primordial follicle ratio than high-dose S1P group ( P = .047). Our findings suggest that a long-acting oral analogue of S1P might decrease spontaneous follicular apoptosis based on the nonapoptotic primordial follicle ratio and AMH levels when compared with placebo.

STAT3 and NF-κB cooperatively control in vitro spontaneous apoptosis and poor chemo-responsiveness in patients with chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an adult disease characterized by in vivo accumulation of mature CD5/CD19/CD23 triple positive B cells and is currently incurable. CLL cells undergo spontaneous apoptosis in response to in vitro cell culture condition but the underlying mechanism is unclear. We hypothesize that the sensitivity of CLL cells to spontaneous apoptosis may be associated with the constitutive activities of transcription factors STAT3 and/or NF-κB. We now show that the sensitivity of fresh CLL cells to spontaneous apoptosis is highly variable among different patients during 48 hours' cell culture and inversely correlated with in vivo constitutively activated STAT3 and NF-κB (p < 0.001). Both activated STAT3 and NF-κB maintain the levels of anti-apoptotic protein Mcl-1/Bcl-xL and autocrine IL-6 production. CLL cells with higher susceptibility to in vitro spontaneous apoptosis show the greatest chemosensitivity (p < 0.001), which is reflected clinically as achieving a complete response (CR) (p < 0.001), longer lymphocyte doubling times (p < 0.01), time to first treatment (p < 0.01), and progression free survival (p < 0.05). Our data suggest that the sensitivity of CLL cells to in vitro spontaneous apoptosis is co-regulated by constitutively activated STAT3 and NF-κB and reflects the in vivo chemo-responsiveness and clinical outcomes.

Regulation of spontaneous eosinophil apoptosis-a neglected area of importance.

Asthma is characterized by the accumulation of eosinophils in the airways in most phenotypes. Eosinophils are inflammatory cells that require an external survival-prolonging stimulus such as granulocyte macrophage-colony-stimulating factor (GM-CSF), interleukin (IL)-5, or IL-3 for survival. In their absence, eosinophils are programmed to die by spontaneous apoptosis in a few days. Eosinophil apoptosis can be accelerated by Fas ligation or by pharmacological agents such as glucocorticoids. Evidence exists for the relevance of these survival-prolonging and pro-apoptotic agents in the regulation of eosinophilic inflammation in inflamed airways. Much less is known about the physiological significance and mechanisms of spontaneous eosinophil apoptosis even though it forms the basis of regulation of eosinophil longevity by pathophysiological factors and pharmacological agents. This review concentrates on discussing the mechanisms of spontaneous eosinophil apoptosis compared to those of glucocorticoid- and Fas-induced apoptosis. We aim to answer the question whether the external apoptotic stimuli only augment the ongoing pathway of spontaneous apoptosis or truly activate a specific pathway.

Spontaneous in vitro apoptosis of de novo chronic lymphocytic leukemia cells correlates with risk of the disease progression.

Despite significant progress in treatment, chronic lymphocytic leukemia (CLL) still remains an incurable disease. Major advances have been recently made to understand the molecular pathogenesis underlying CLL, but defects in apoptosis are considered to be the most important factors. Although neoplastic cells are resistant to apoptosis in vivo, they show decreased level of spontaneous in vitro apoptosis, with significant differences among CLL patients. This work compares the level of spontaneous CLL cell apoptosis with prognostic factors and clinical course of the disease. In vitro spontaneous apoptosis of peripheral blood lymphocytes was analyzed using Annexin-V assay (confirmed by TUNEL method) in 135 treatment naïve patients with CLL. Levels of apoptosis after 48h culture in patients with stable disease were found to be significantly higher than in the group with progressive course of the disease (p=0.015). Moreover, the level of spontaneous apoptosis after 24h and 48h of incubation correlated inversely with the progression free survival (p=0.026 and 0.009, respectively). These results suggest that in vitro spontaneous apoptosis of CLL cells may be a simple and cheap prognostic test which is relatively quick to use, and can predict the course of the disease and response to treatment. © 2014 Clinical Cytometry Society.

Spontaneous in vitro apoptosis of de novo chronic lymphocytic leukemia cells correlates with risk of the disease progression.

Despite significant progress in treatment, chronic lymphocytic leukemia (CLL) still remains an incurable disease. Major advances have been recently made to understand the molecular pathogenesis underlying CLL, but defects in apoptosis are considered to be the most important factors. Although neoplastic cells are resistant to apoptosis in vivo, they show decreased level of spontaneous in vitro apoptosis, with significant differences among CLL patients. This work compares the level of spontaneous CLL cell apoptosis with prognostic factors and clinical course of the disease. In vitro spontaneous apoptosis of peripheral blood lymphocytes was analyzed using Annexin-V assay (confirmed by TUNEL method) in 135 treatment naïve patients with CLL. Levels of apoptosis after 48 h culture in patients with stable disease were found to be significantly higher than in the group with progressive course of the disease (P = 0.015). Moreover, the level of spontaneous apoptosis after 24 and 48 h of incubation correlated inversely with the progression free survival (P = 0.026 and 0.009, respectively). These results suggest that in vitro spontaneous apoptosis of CLL cells may be a simple and cheap prognostic test which is relatively quick to use, and can predict the course of the disease and response to treatment.