Pharmacological Modulators of Sphingolipid Metabolism for the Treatment of Cystic Fibrosis Lung Inflammation

Cystic Fibrosis (CF) lung disease is characterised by progressive chronic infection and inflammation of the airways. This prolonged airway inflammatory response leads to irreversible lung damage and fibrosis which is believed to be driven by two distinct, coordinated events: a) a defective cystic fibrosis transmembrane regulator (CFTR) causes airway surface dehydration and increased mucus viscosity leading to chronic colonization with Pseudomonas aeruginosa (P.aeruginosa) (Boucher, 2007); b) mutated CFTR triggers the generation of pro-inflammatory and chemotactic cytokines orchestrated by bronchial epithelial cells, independently of infection (Rubin, 2007; Elizur et al., 2008). The chemokine IL-8, abundantly expressed at sites of chronic inflammation, seems to play a major role in driving the formation of neutrophil (PMN)-rich exudates into the lung of CF patients (Khan et al., 1995; Noah et al., 1997; DiMango et al., 1998; Puchelle et al., 2001; Joseph et al., 2005; Perez et al., 2007). Therefore, reduction of the exaggerated production of IL-8 is key therapeutic target in CF. Anti-inflammatory drugs are an attractive therapeutic tool in CF aimed to decrease the rate of decline in lung function. However, the inherent complexity of the inflammatory response combined with the obvious dependency on this response to contain infection and the side effect profiles of common anti-inflammatories, have made identifying the most suitable therapy a major priority. Consensus is growing on sphingolipids (SLs) as novel targets to cure pulmonary disorders including CF, since modulation of cellular ceramide reduces lung inflammation (Lahiri and Futerman, 2007; Uhlig and Gulbins, 2008). The results in the area of ceramide and CF pathophysiology are very interesting, although contradicting due to the animal models used and methods of ceramide detection (Wojewodka , 2011). The accumulation of ceramide has been identified as one of the key regulators of inflammation in CF airways in different CFTR-/- mouse models (Teichgraber, 2008). On the contrary, decreased ceramide levels have been shown in CFTR ko mice (Guibault, 2008). The possible explanation for this discrepancy seems to be the special diet required for CFTR ko mice, that severely affects the concentration of SLs. Other possible causes, such as genetic determinants, could influence individual levels of SLs (Hicks, 2009). In a different study, no significant difference has been found in basal ceramide levels in immortalised CF bronchial epithelial cells and lung homogenate from CFTR ko mice compared to wild type cells and mice (Yu, 2009). Very importantly, ceramide has been demonstrated to accumulate in the lower airways of CF patients and to be positively associated with neutrophilic inflammation (Brodlie, 2010), supporting the hypothesis that reduction of ceramide may be a therapeutic target for CF lung inflammation. Extending our previous study (Dechecchi, 2008), we have recently demonstrated that the iminosugar N-butyldeoxynojirimycin (miglustat), an inhibitor of the first step in glycosphingolipid (GSL) biosynthesis, reducing the P.aeruginosa induced immunoreactive ceramide expression, produces an anti-inflammatory effect in human bronchial epithelial cells in vitro and down-regulates the neutrophil chemotaxis in murine lungs in vivo (Dechecchi, 2011). These findings strengthen the notion that the metabolism of SLs can be manipulated as a therapeutic option for CF lung disease. With regard to new treatments for CF lung pathology, miglustat deserves great attention since it restores CFTR function in respiratory and pancreatic cells in vitro (Norez, 2006; Dechecchi, 2008) and in CF mice (Lubamba, 2009) and produces an anti-inflammatory effect in vitro and in vivo Dechecchi, 2011). Notably, miglustat is a FDA-approved and EMA−designated orally bioavailable orphan drug, used in Europe and USA for the treatment of Gaucher disease and other GSL storage diseases. In this chapter we review the pre-clinical evidence on the anti-inflammatory effect of miglustat in comparative effectiveness studies with the SL inhibitor amitriptyline and the glucocorticoid (GC) dexamethasone. Importance will be placed on the efficacy of each anti-inflammatory molecule to balance between the anti-inflammatory activity and possible impairment of the host defence

Signaling pathways activated by the B-cell receptor in chronic lymphocytic leukemia

Over the past decade, several features of the B-cell receptor (BCR) complex have emerged as major markers for prognostic classification of B-cell chronic lymphocytic leukemia (B-CLL). In particular, the absence of somatic mutations within the immunoglobulin variable heavy chain genes (IGHV), the presence of ZAP-70 and a higher ability of the BCR to translate signals within the cell have been associated with an aggressive clinical course. Indeed, the stratification of patients with B-CLL based on BCR features suggests that heterogeneity of B-CLL clinical courses may reflect BCR signaling differences that have arisen during the evolution of leukemia. Therefore, characterizing BCR signaling profiles may help to identify signaling markers useful for patient stratification, disease monitoring and therapeutic targeting in B-CLL

Analysis of B-cell receptor signal transduction in B-cell chronic lymphocytic leukemia by phosphospecific flow-cytometry

B-cell chronic lymphocytic leukemia (B-CLL) is a clonal lymphoproliferative disease characterized by the expansion of mature CD5+ B-cells. Recent studies indicated that variability in clinical outcomes observed in CLL patients is related to differences in the ability to transduce B-cell receptor (BCR)-mediate signals. The BCR signal transduction routes include, in the initial phases, Syk tyrosin kinase and BCR prolonged stimulation has been associated to activation of kinases including mitogen activated protein kinases (MAPK). In this study we used phosphospecific flow cytometry to study single-cell signaling triggered by BCR in peripheral blood B-cells from 21 B-CLL patients. The following proteins were analyzed: Syk, MAPK (Erk, p38, JNK) and NF-kappaB, either in basal condition or after BCR cross-linking achieved by treatment with anti-IgM F(ab\u2019)2. Most cases showed constitutive phosphorylation of Syk and NF-kappaB (86% and 93%, respectively). Erk was constitutively phosphorylated in 81% cases, JNK in 5%, and p38 in 0% (Table 1). The BCR cross-linking increased the constitutive phosphorylation of Syk in 50% cases, of Erk in 27%, and of NF-kappaB in 21%. No cases showed p38 or JNK increased phosphorylation (Table 1). Recent works shows that efficient activation of BCR signaling depends on inactivation of phosphatase proteins (PTPs), and that endogenously generated H2O2 is an important transiently PTPs inhibitor. Therefore, we cross-linked the BCR in the presence of H2O2. In this condition we observed an increase of phosphorylation of all proteins with respect to treatment with anti-IgM alone (Table 1). Furthermore, the treatment of B-CLL cells with H2O2 alone induced increased phosphorylation of signaling proteins with respect to the constitutive level (Syk in 21%, p38 in 56%, Erk in 38%, JNK in 9%, and NF-kappaB in 43% cases, Table1). Conversely, PTP inhibition in normal B cells did not significantly modify the phosphorylation of these proteins. Taken together, these results revealed a clear-cut remodeling of BCR-mediated signaling in B-CLL cells with respect to normal B cells, thus confirming and extending previous results obtained by Western blot analysis. Furthermore, this study provides the first analysis of BCR signaling by phosphospecific flow cytometry in B-CLL cells and indicate that single-cells measurement of phosphoproteins in response to BCR engagement enables to define cell network phenotypes in B-CLL by multidimensional molecular profiles of signaling

BONE MARROW-DERIVED STROMAL CELLS INHIBIT SPONTANEOUS APOPTOSIS IN NORMAL AND NEOPLASTIC THYMOCYTES

Normal T cell development depends upon interaction between progenitor cells and the thymic microenvironment. However, the ultimate T cell precursors derive from haematopoietic progenitors in the bone marrow (BM). The malignant counterpart of immature T cells, namely T acute lymphoblastic leukaemia (T-ALL) cells, arise inside the thymus and almost always spread to BM early in the clinical course. Thus, BM and thymic stromal microenvironments play a crucial role in the development of normal and neoplastic immature T cells. In this work we investigated whether the spontaneous apoptosis observed in vitro in normal and neoplastic immature T cells could be regulated by the interaction with BM- and/or thymus-derived stromal cells. To address this issue, we performed lympho-stromal co-cultures between normal human BM stromal or thymic epithelial (TE) cells and normal human unfractionated thymocytes or blasts derived from 5 adult T-ALL patients. The spontaneous apoptosis was completely abolished when normal or leukaemic T cells were co-cultured with BM-derived stromal cells whereas the interaction with TE cells was ineffective. Furthermore, rescued leukaemic -but not normal- T cells were induced to proliferate by the interaction with BM stromal cells. When BM stroma was replaced with established cell lines used as controls, no protective or proliferative effects were observed. Both cell protection and proliferation were dependent upon cell-cell adhesion, as BM-derived supernatant was unable to rescue or induce proliferation in immature T cells. These results point out the role of BM stroma in the regulation of immature T cell survival. On the opposite side, they highlight the preserved ability of T cell precursors to respond to survival stimuli from BM microenvironment. This mechanism, strengthened by the proliferative potential of T blasts, could play a role in the blast spreading to BM during pathogenesis of T-ALL

Single Cell Network Profiling (SCNP) assay reveals B Cell Receptor Signaling heterogeneity in the Context of ZAP-70 Expression in patients with Chronic Lymphocytic Leukemia (CLL)

CLL is a disease with considerably variable clinical course. Among early stage CLL patients that are provided with the \u201cwatch and wait\u201d standard of care, a significant subset of these patients undergo rapid disease progression during which many of them are refractory to currently available therapies. Although several biological indicators including expression level of ZAP-70 have been associated with CLL patient risk stratification, technical challenges related to assay consistency have limited their broad use in the clinical setting. It has been observed that ZAP-70 expression is sufficient to transduce B cell receptor (BCR) signaling and that BCR signaling activation by anti-IgM was enhanced in ZAP-70 positive CLL cells including spleen tyrosine kinase (Syk), a cytosolic protein kinase that is recruited by the activated BCR and forms a protein complex with ZAP-70 that in turn initiates downstream signaling resulting in CLL cell survival. Based on the BCR biology and established prognostic data, we undertook a study designed to interrogate critical signaling pathways associated with BCR while simultaneously assessing the quantity of the ZAP-70 protein in each individual cell. If successful, this approach may glean a better prognostic prediction than existing techniques while building an assay system suitable for translational studies in the varied clinical groups. We used SCNP, a new technology platform which uses multi-parametric flow cytometry to measure biological pathways focusing on intra-cellular signaling and post-translational changes in response to modulators, to examine the relationship between BCR signaling and ZAP-70 expression in patients with CLL. The goal of this investigation is to generate hypotheses that guide development of sensitive and accurate prognostic tests and support translational therapeutic development targeting the BCR pathway for CLL patient management. Methods: SCNP was performed to measure basal and modulated BCR signaling in cryopreserved PBMCs from 10 CLL patients (5 ZAP-70 negative and 5 ZAP-70 positive) who were not currently receiving treatment. Anti-IgM or hydrogen peroxide was used to evoke BCR signaling, which was measured by SCNP using phospho-specific antibodies against signaling proteins including p-Syk, p-ERK, p-p38, p-Lck, p-NF-kB and p-SAPK/JNK. ZAP-70 protein expression was measured simultaneously with the signaling assessment by staining with a ZAP-70 (clone 136F12) antibody. Analysis was performed using WinList\u2122 to gate the CD5+, CD19+ CLL cells separately from the CD5+, CD3+ T cells. Results: The biological heterogeneity among CLL patients was clearly evident when the basal and evoked levels of BCR signaling protein phosphorylation were assessed using SCNP. In comparison to normal B cells, elevated basal levels of p-Syk, p-NF-kB and p-SAPK/JNK were observed in 7 of 10 CLL samples (MFI two fold greater than unstained cells). Following BCR stimulation with anti-IgM, a trend of positive correlation between the nodes (i.e. proteomic readouts in the presence of modulator) p-Syk, p-Lck, p-ERK and p-p38 and increasing amounts of ZAP-70 was observed. Interestingly, p-Syk was found to correlate well with ZAP-70 expression level (Spearman=0.76). However, following hydrogen peroxide modulation, there was no positive correlation observed between ZAP-70 expression and p-Syk, p-ERK, p-p38, and p-Lck, whereas there appeared to be a trend toward elevated p-SAPK/JNK and p-NF-kB in the context of diminished ZAP-70 expression. An SCNP study in a large set of clinical CLL samples is ongoing to further investigate the relationships between BCR signaling and ZAP-70 expression observed in this study in an attempt to understand the impact of ZAP-70 expression on signaling and the relation of both measures to patient outcome. Conclusions: The findings reported here suggest that measurement of BCR signaling pathways using SCNP reveal biological heterogeneity within the known prognostic groups based on the expression level of the ZAP-70 protein. These findings, when validated, may permit identification of high risk individuals at an early stage of disease, as well as guiding selection of suitable treatment strategies in the two differing prognostic groups

DEATH RECEPTOR 3 EXPRESSION AND SERUM LEVEL OF SOLUBLE TL1A CORRELATE WITH INDOLENT, EARLY STAGE B-CELL CHRONIC LYMPHOCYTIC LEUKEMIA

Introduction. B-cell chronic lymphocytic leukemia (B-CLL) accounts for approximately 30% of leukemia diagnosed in the Western coun- tries and shows an increasing incidence with the age of the popula- tion. Analysis of survival times has led to the establishment of stag- ing systems according to various prognostic markers, including Rai stage, IGHV mutational status, expression of CD38 and Zap70. Clin- ically, B-CLL is a heterogeneous disease with variable presentation and evolution. Two major subtypes can be distinguished, indolent and aggressive, which require different treatment strategies. Howev- er, prediction of the clinical course of individual patients with the same stage and risk group remains variable. Receptors of the TNFR superfamily play a fundamental role in promoting the growth of B-cell chronic lymphocytic leukemia. Death receptor (DR) 3 is a TNFR- superfamily member expressed in lymphocyte-enriched tissues. DR3 and its ligand, TNF-like ligand 1A (TL1A), are implicated in regulato- ry mechanisms of adaptive immune response under physiological and pathological settings. Recently, we have demonstrated that DR3 is expressed on the surface of B cell receptor (BCR)-stimulated B cells and interaction of DR3 with TL1A reduces proliferation of suboptimally activated healthy B cells in vitro, without affecting cell survival. These findings prompted us to examine the expression of DR3 and TL1A in B-CLL and their possible role as risk factors for disease progression. Methods. DR3 surface expression of 37 B-CLL samples was measured by flow cytometry at baseline and following stimulation with F(ab\u2019)2 anti-human IgM conjugated to latex microspheres. TL1A serum lev- els of 26 B-CLL samples were measured by ELISA. Correlation analy- sis with clinical and biological parameters were performed using GraphPad Prism software. Results. Here, our preliminary results show that DR3 is expressed on the surface of activated CLL B cells and TL1A is present in the serum of B-CLL patients. Moreover, we show that BCR-induced DR3 expression is more frequently detected in sam- ples with indolent, early-stage disease (Rai 0). The relevance of these findings has been confirmed by serum TL1A measurement showing that higher serum levels of TL1A are more frequently detected in B- CLL patients with favorable prognostic parameters (i.e. absence of CD38 expression) and early-stage disease. Conclusions. Taken togeth- er, these findings suggest that in B-CLL the TL1A/DR3 modulatory function on cell metabolism, in the presence of antigen stimulation, is a feature of indolent, early-stage disease. Thus, we can assume that these factors could be useful in monitoring disease activity and may be of prognostic relevance in B-CLL

The prostate specific membrane antigen regulates the expression of IL-6 and CCL-5 in prostate tumour cells by activating the MAPK pathways

IL-6 and CCL5 are implicated in the development and progression of several forms of tumours incliding PCa. The PSMA expression is augmented in high-grade and metastatic tumours. Some clinical observations suggest that the increased secretion of IL-6 and CCL5 and the higher PSMA expression may be correlated