We investigated the pattern of lipoprotein lipase (LPL) expression in B-cell chronic lymphocytic leukemia (B-CLL) and assessed its prognostic relevance. Expression of LPL mRNA as well as protein was highly restricted to leukemic B cells. The intensity of intracellular immunoreactivity of LPL was higher in samples of patients with unmutated immunoglobulin heavychain variable region genes (IGV H ) compared to those with mutated IGV H genes. LPL mRNA levels in peripheral blood mononuclear cells (PBMNC) from 104 CLL patients differed by 1.5 orders of magnitude between cases with mutated (N ¼ 51) or unmutated (N ¼ 53) IGV H (median: 1.33 vs 45.22 compared to normal PBMNC). LPL expression correlated strongly with IGV H mutational status (R ¼ 0.614; Po0.0001). High LPL expression predicted unmutated IGV H status with an odds ratio of 25.90 (Po0.0001) and discriminated between mutated and unmutated cases in 87 of 104 patients (84%). LPL expression was higher in patients with poor risk cytogenetics. High LPL expression was associated with a shorter treatment-free survival (median 40 vs 96 months, P ¼ 0.001) and a trend for a shorter median overall survival (105 months vs not reached). Our data establish LPL as a prognostic marker and suggest functional consequences of LPL overexpression in patients with B-CLL.

Members of theThe pathomechanisms underlying the disease primarily involve defects that prevent cell turnover because of apoptosis, rather than alterations in cell cycle regulation. 1,2 One of the hallmarks of B-CLL cells is the overexpression of the transmembrane glycoprotein CD23, 1-3 which undergoes spontaneous proteolysis, giving rise to soluble CD23 (sCD23) molecules. 4 The serum concentration of sCD23 can be several hundredfold higher than in healthy individuals and parallels the clinical stage of the disease. 5,6 Two isoforms of CD23 exist, CD23a and CD23b, which are expressed from 2 different promoters. 7 Expression of CD23a is restricted to B lymphocytes, whereas CD23b is found on a number of different hematopoietic cell types, predominantly after interleukin 4 (IL-4) treatment. 8 In B-CLL cells, selective expression of CD23a has been found to be concurrent to a state of cell survival, thereby providing a link between CD23a and the regulation of apoptosis. 9 CD23 is also closely associated with Epstein-Barr virus (EBV)-mediated B-cell immortalization, because a naturally occurring EBV mutant (P3HR1), carrying a deletion of the EBNA2 gene, has lost its ability to induce CD23 expression and to transform primary B cells in vitro. 10-12 EBNA2 activates the CD23a gene through a CBF1 repressor site located in the CD23a proximal promoter. [13][14][15] Several lines of evidence strongly suggest that EBNA2 mimics Notch signaling by acting as a transcriptional activator after binding and masking the repression domain of CBF1. [16][17][18][19] The Notch gene family encodes transmembrane receptors that modulate differentiation, proliferation, and apoptotic programs in response to extracellular ligands expressed on neighboring cells. 20,21 Ligand-mediated stimulation of Notch causes the proteolytic release of the intracellular domain (NotchIC), which then passes into the nucleus where it activates transcription of CBF1 responsive genes. [22][23][24][25] Deregulation of this pathway by overexpression of a constitutively activated form of Notch not only diverts cell fate decisions but is also tumorigenic. For example, truncation of Notch1 found in a subset of human T-cell leukemias leads to the expression of a ligand-independent oncogenic protein lacking the extracellular domain. 26 The truncated Notch proteins consist primarily of the intracellular domain and localize predominantly in the nucleus. Enforced expression of Notch1IC in bone marrow stem cells causes T-cell leukemia in mice, indicating a causative role for Notch1 in T-cell oncogenesis. 27 To elucidate the mechanisms leading to the up-regulation of CD23a in B-CLL cells, we analyzed the CD23a proximal promoter for sequence-specific DNA-protein interactions. By electrophoretic mobility shift assays (EMSAs), we detected a transcription factor For personal use only. on May 10, 2018. by guest www.bloodjournal.org From complex containing Notch2IC that binds to 5 different CBF1 responsive elements. Our data indicate that Notch2 is overexpressed in B-CLL cells, sug...

IntroductionMajor progress has been achieved in the past decade in understanding the biology of B-cell chronic lymphocytic leukemia (CLL), leading to new therapeutic concepts and a trend toward improved survival. However, the disease remains incurable, and many patients develop drug resistance. [1][2][3] Despite the long lifespan of CLL cells in vivo, these cells undergo rapid and spontaneous apoptosis in vitro but can be rescued by marrow stromal cells, [4][5][6] nurse-like cells, 7 and follicular dendritic cells. 8 There are also indications that the stromal cells mediate resistance to chemotherapy in CLL cells. 9 This point to the dependence of CLL cells on antiapoptotic stimuli that could be provided in vivo by marrow microenvironment 10,11 and may have a major effect on disease progression and response or resistance to therapy.Bone marrow microenvironment is a complex structure that comprises accessory cells (stromal cells, adipocytes, reticulum cells, endothelial cells, follicular dendritic cells, T cells, and macrophages), matrix proteins, and soluble factors, including growth factors and cytokines. 11,12 Bone marrow stromal cells (BMSCs) represent a major component of the marrow microenvironment. They originate from the mesenchymal stem cells and have hematopoietic supportive properties. They also produce matrix proteins, express integrin ligands, and release several cytokines and growth factors that are involved in the generation and maturation of normal and leukemic B cells. [11][12][13] Therefore, BMSCs could provide a suitable milieu for the development and survival of CLL cells. However, the antiapoptotic cascades and the molecular events that are activated upon the interaction between CLL cells and BMSCs are not fully identified.Several stimuli that are endogenously produced in the microenvironment were shown to activate the antiapoptotic phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. These include cytokines and growth factors, 14 adhesion molecules and matrix proteins, 15 and involve receptors that are expressed on the surface of CLL cells, including the B-cell receptor, CD19, and CD5. [16][17][18] This strongly suggests that the PI3-K/Akt pathway might play a central role in the interaction between CLL cells and the bone marrow microenvironment. The PI3-K/Akt cascade contributes to the regulation of many cellular processes, including motility, proliferation, apoptosis, and tumorigenesis. 14,19 Class I of PI3-K family is the best characterized and comprises p110␣, p110␤, p110␥, and The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 11, 2018. by guest www.bloodjournal.org From p110␦ isotypes. 14,19 The first generation of pan-PI3-K inhibitors (LY294002 and wortmannin) provided substantial information on the molecular mechanism of action and ...

B cell chronic lymphocytic leukemia (B-CLL), the most common type of leukemia in adults, is still essentially incurable despite the development of novel therapeutic strategies. This reflects the incomplete understanding of the pathophysiology of this disease. A comprehensive proteome analysis of primary human B-CLL cells and B cells from younger as well as elderly healthy donors was performed. For comparison, the chronic B cell leukemia cell line JVM-13 was also included. A principal component analysis comprising 6,945 proteins separated these four groups, placing B cells of aged-matched controls between those of young donors and B-CLL patients, while identifying JVM-13 as poorly related cells. Mass spectrometric proteomics data have been made fully accessible via ProteomeXchange with identifier PXD006570-PXD006572, PXD006576, PXD006578, and PXD006589-PXD006591. Remarkably, B cells from aged controls displayed significant regulation of proteins related to stress management in mitochondria and ROS stress such as DLAT, FIS1, and NDUFAB1, and DNA repair, including RAD9A, MGMT, and XPA. ROS levels were indeed found significantly increased in B cells but not in T cells or monocytes from aged individuals. These alterations may be relevant for tumorigenesis and were observed similarly in B-CLL cells. In B-CLL cells, some remarkable unique features like the loss of tumor suppressor molecules PNN and JARID2, the stress-related serotonin transporter SLC6A4, and high expression of ZNF207, CCDC88A, PIGR and ID3, otherwise associated with stem cell phenotype, were determined. Alterations of metabolic enzymes were another outstanding feature in comparison to normal B cells, indicating increased beta-oxidation of fatty acids and increased consumption of glutamine. Targeted metabolomics assays corroborated these results. The present findings identify a potential proteome signature for immune senescence in addition to previously unrecognized features of B-CLL cells and suggest that aging may be accompanied by cellular reprogramming functionally relevant for predisposing B cells to transform to B-CLL cells.

Activation-induced cytidine deaminase (AID) is essential for somatic hypermutation of B-cells. We investigated the expression of AID mRNA by real-time polymerase chain reaction (PCR) in peripheral blood mononuclear cells of 80 patients with B-CLL. AID expression was detected in 45 of 80 patients (56%) at various levels, but was undetectable in 35 patients (44%). AID PCR positivity was associated with unmutated IGV H gene status (22 of 25 patients; P ¼ 0.002) and unfavourable cytogenetics (18 of 23 patients with deletion in 11q or loss of p53; P ¼ 0.040). Using a threshold level of 0.01-fold expression compared to Ramos control cells, even more significant associations were observed (P ¼ 0.001 for IGV H ; P ¼ 0.002 for cytogenetics). A correlation was observed between individual AID levels and the percentage of V H homology (R ¼ 0.41; P ¼ 0.001). AID positivity predicted unmutated IGV H status with an odds ratio of 8.31 (P ¼ 0.003) and poor risk cytogenetics with an odds ratio of 3.46 (P ¼ 0.032). Significance was retained after adjustment for Binet or Rai stages. AID mRNA levels were stable over time. These data suggest a potential role of AID as a prognostic marker in B-CLL.

These results demonstrate a significant increase in circulating TGF-beta1 in patients with AS after the combined spa-exercise therapy in Badgastein. The results also provide evidence for a biological response to speleotherapy and suggest that TGF-beta, through its antiinflammatory function, may play a role in this response.

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Nonstandard abbreviations used: B cell chronic lymphocytic leukemia (B-CLL); bone marrow (BM); BM fibroblast (BMF); BM mononuclear cell (BMMC); BM plasma (BMP); BM stromal cell (BMSC); hairy cell (HC); HC leukemia (HCL); healthy donor (HD); peripheral blood plasma (PBP); procollagen type III aminoterminal propeptide (PIIINP). Conflict of interest:The authors have declared that no conflict of interest exists. IntroductionHairy cell leukemia (HCL) is a chronic lymphoproliferative disorder characterized by the presence of hairy cells (HCs) in peripheral blood, bone marrow (BM), and spleen and is invariably associated with a unique type of BM fibrosis (1-3). Although a rare disease (2% of adult leukemia), HCL represents an excellent model for cancer biotherapy (4) and for understanding the deregulation of cytokines and growth factors in human neoplasia (5-7). The fibrotic process and the associated structural abnormality in BM of HCL patients are mainly due to accumulation of fine argyrophilic reticulin fibers, although collagen fibers can be observed in the advanced stages of the disease (8-11). The composition of reticulin fibers in HCL is not well defined. In normal and fibrotic BM, the distribution of reticulin fibers is identical to that of type III collagen and its precursor, type III procollagen (12,13). Electron microscopic studies of human tissues revealed that reticulin fibers are individual collagen fibrils or small bundles of these fibrils embedded in the interfibrillar matrix of proteoglycans (14-16) and that they are composed mainly of type III collagen surrounding a core of type I collagen fibrils (17,18). In addition to reticulin fibrosis, it has been recently demonstrated that the glycoprotein fibronectin, which is produced and assembled by HCs, contributes to the fibrotic process in BM of HCL patients (19). This process was also found to be particularly enhanced by bFGF, which is endogenously produced by the HCs (20). Since reticulin and fibronectin fibers were found to represent different structures in myelofibrotic BM (21), it appears that BM fibrosis in HCL is a complex process that involves accumulation and assembly of collagenous ECM components (reticulin) (22) and noncollagenous ECM components (fibronectin).In BM, HCs are found in association with randomly dispersed fibroblastoid cells and are surrounded by reticulin fibers (9, 10, 23, 24). These fibroblastoid cells, which have been found in close association with collagen fibers, are the matrix-producing cells and are responsible for the synthesis of reticulin and collagen (9,(25)(26)(27)(28). Other studies confirmed the increase in the collagen fibrils in the intercellular space around the HCs but did not find evidence that HCs give rise to these fibrils (10). These studies also showed that HCs are not argyrophilic (29), suggesting that they may not be the direct source of reticulin fibers. Since no massive increase in fibroblast numbers is observed in the BM of HCL patients (10), the increased production of reticulin and ECM proteins mig...