Despite very similar gene expression profiles, the clinical course of B-cell chronic lymphocytic leukemia (B-CLL) is heterogeneous. Immunoglobulin VH (IgVH) mutational status and expression of B-cell receptor (BCR) signaling mediators have been associated with disease progression. However, the consequences of BCR engagement on cell survival and evolution of the disease remain unclear. We show here that B-CLL cell survival is dependent on the threshold of BCR stimulation induced by immobilized antibody, in contrast to soluble anti-M F(ab) ¶2 antibody, which leads to apoptosis. Measurement of metabolic activity and apoptotic response discriminated two subgroups. ''Nonresponders'' showed low metabolic activity and unmodified apoptotic response upon BCR stimulation. In contrast,''responders'' exhibited increased metabolic activity and inhibition of spontaneous apoptosis. This survival advantage was associated to a BCR-dependent activation profile leading to induction of cyclin D2/cyclindependent kinase 4 (cdk4) expression and G 1 cell cycle progression. The ability to respond to BCR ligation correlated with an unfavorable clinical course and allowed to define an additional group of patients among IgVH-mutated cases exhibiting a risk of progression. Remarkably, we show that Zap70 expression was neither mandatory nor sufficient to generate downstream survival signals and cyclin D2/cdk4 up-regulation. In conclusion, BCR engagement has a significant effect on B-CLL cell survival, activation, and G 1 progression. Furthermore, our results provide new insights in the physiopathology of progressive IgVH-mutated cases.

Progressive cases of B-cell chronic lymphocytic leukemia (CLL) are frequently associated with lymphadenopathy, highlighting a critical role for signals emanating from the tumor environment in the accumulation of malignant B cells. We investigated on CLL cells from 30 untreated patients the consequence of B-cell receptor (BCR) triggering on the membrane expression of CXCR4 and CD62L, two surface molecules involved in trafficking and exit of B-lymphocytes from lymph nodes. BCR stimulation promoted a strictly simultaneous down-regulation of CXCR4 and CD62L membrane expression to a variable extent. The variable BCRdependent decrease of the two proteins was strikingly representative of the heterogeneous capacity of the CLL cells to respond to BCR engagement in a given patient. Functionally, cells down-regulating CXCR4 and CD62L in response to BCR engagement displayed a reduction in both migration toward CXCL12 and adhesion to lymphatic endothelial cells. Remarkably, the ability of CLL cells to respond to BCR ligation was correlated with unfavorable prognostic markers and short progression-free survival. In conclusion, BCR signaling promotes decrease of CXCR4 and CD62L membrane expression in progressive cases only. These results are consistent with the hypothesis that BCR-mediated signaling pathways favor accumulation of a proliferative pool within the lymph nodes of progressive CLL cases. [Cancer Res 2009;69(16):6387-95]

B-cell antigen receptor (BCR)- IntroductionDespite encouraging scientific and therapeutic advances, chronic lymphocytic leukemia (CLL) disease remains incurable with standard therapy, prompting the need for the development of novel therapeutic agents. CLL affects predominantly elderly people and, based on clinical and biologic annotations, the course of the disease can be classified from indolent to more aggressive subtypes. [1][2][3][4][5] CLL is defined as an expansion of monoclonal, slowly dividing CD5 ϩ / CD20 ϩ B lymphocytes. An important prognosis factor for CLL patients is the mutational status of immunoglobulin (Ig) variable heavy chain genes (IGHV) constituting the IgM B-cell antigen receptor (BCR). Considerable amounts of data have shown that antigen-driven signals are involved in the pathogenesis and progression of CLL malignancies. [6][7][8][9] Although CLL cells accumulate and are resistant to cell death in vivo, they rapidly become apoptotic during in vitro culture. Interestingly, several CLL cells evade apoptosis in vitro through an enhanced survival response after BCR stimulation. 6,7,9,10 After BCR triggering, which is mediated by antigen binding to cell surface Igs, prolonged activation of the MEK-ERK and PI3K-AKT pathways, as well as efficient degradation of I]kappa]B and activation of NF-B have been associated with the induction of antiapoptotic and survival signals. [11][12][13] These molecular events lead to important changes in gene transcription and subsequent B-cell fate specification. 7,14 In addition, sustained BCR engagement promotes increased metabolic activity, allowing some restricted G 1 cell-cycle progression. 6,15 Most CLL cells characteristically display lower levels of surface IgM and IgD compared with normal B cells. 1,2,15 Low BCR surface expression is partly explained by an inefficient assembly, trafficking, or both of Igs that are noncovalently bound to CD79a and CD79b. 16 The tyrosine kinase Syk has been shown to function downstream of the BCR complex in CLL B cells. 8,17 Inhibition of Syk expression or activity induces apoptosis of CLL cells both in vitro and in vivo, suggesting a prosurvival role for the kinase. [18][19][20][21][22] Zap70, the second member of the Syk family, is considered a surrogate marker for unmutated IGHV gene expression and probably facilitates BCR signaling in CLL cells independently from its tyrosine kinase activity. 23,24 Effectors downstream of Syk include, among others, phospholipase C␥2 (PLC␥2) that is responsible for mobilization of intracellular pools of calcium. 14,25 In CLL B cells, BCR ligation induces heterogeneous responses in terms of PLC␥2 phosphorylation and intracellular calcium mobilization. 15,26 In turn, sustained calcium uptake activates the serine and threonine phosphatase calcineurin. Once active, calcineurin promotes dephosphorylation and nuclear translocation of the nuclear factor of activated T cells (NFAT) family of transcription factors, which cooperate with other factors to promote transcriptional regulation of numerou...

Summary. Umbilical cord blood (UCB) provides immediate access to haemopoietic stem/progenitor cells (HSPC) but low cell number restricts use in full adult bone marrow reconstitution. This study investigated early ex vivo expansion kinetics of UCB AC133 + cells (2-4 · 10 4 /ml), mononuclear cells (MNC, 1-2 · 10 6 /ml) and AC133negative cells (AC133 neg , 2-4 · 10 4 /ml) in stroma-free 8 d liquid culture (fetal bovine serum-supplemented Iscove's-modified Dulbecco's medium (IMDM) with either ÔK36EGÕ [c-Kit ligand, interleukin 3 (IL-3), IL-6, erythropoietin, granulocyte colony-stimulating factor] or ÔTPOFLÕ (thrombopoietin, Flt-3 ligand). Cell enumeration, apoptosis assay and AC133/ CD34/CD38 antigen immunophenotyping were performed at d 0, 3, 5 and 8. All three cell populations went through a proliferation lag phase between d 3 and d 5. AC133 + cells recovered better from lag phase with significantly higher fold increase (FI) when compared with MNC and AC133 neg populations (K36EG FI: 15AE04 ± 5AE46; TPOFL FI: 8AE59 ± 2AE92, P < 0AE05). After 8 d, populations lacking AC133 + cells were significantly more inclined to undergo apoptosis under proliferative conditions (P < 0AE01). Also, when compared with K36EG, 8 d TPOFL-expanded AC133 + cells encompassed a significantly higher percentage of AC133 + and CD34 + early HSPC (K36EG: 20AE50 ± 2AE36; TPOFL: 47AE00 ± 7AE69; P < 0AE05). In conclusion, TPOFL synergism demonstrated the potential for AC133 + HSPC ex vivo expansion inducing self-renewal, early HSPC maintenance and promoting cell survival status.

BackgroundHyaluronic acid (HA), a naturally occurring polysaccharide, is used in the production of dermal fillers for esthetic purposes. As it has a few days of half-life in human tissues, HA-based dermal filler is chemically modified to increase its lifetime in the body. The most common modification used in commercial HA-based filler is the cross-linking of HA chains using 1,4-butanediol diglycidyl ether (BDDE) as cross-linking agent. Residual, or unreacted, BDDE is considered nontoxic when it is <2 parts per million (ppm); therefore, the quantification of residual BDDE in the final dermal filler is mandatory to ensure the safety of the patients.Materials and methodsThe present study describes the detection and characterization of one by-product of the cross-linking reaction between BDDE and HA in alkaline conditions by combining both liquid chromatography and mass spectroscopy (LC–MS).ResultsAfter different analyses, it was found that the alkaline conditions and the high temperatures employed to sterilize the HA–BDDE hydrogel promote the formation of this new by-product, a “propene glicol-like” compound. LC–MS analysis confirmed that this by-product have the same monoisotopic mass as that of BDDE, a different retention time (tR), and also a different UV absorbance (λ=200 nm) pattern. Unlike BDDE, it was observed in the LC–MS analysis that this by-product had a higher detection at 200 nm in the same assay conditions.ConclusionThese results suggest that this new compound does not have an epoxide on its structure. The discussion is open to assess the risk of this new by-product found in the production of HA–BDDE hydrogels (HA dermal fillers) for commercial purposes.

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Mesotherapy/biorevitalization with hyaluronic acid (HA) is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS® (Repairs, Refills, Stimulates) HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15%) and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts.

Alternate splicing of STAT1 produces two isoforms: alpha, known as the active form, and beta, previously shown to act as a dominant-negative factor. Most studies have dealt with STAT1alpha, showing its involvement in cell growth control and cell death. To examine the specific function of either isoform in cell death, a naturally STAT1-deficient human B cell line was transfected to express STAT1alpha or STAT1beta. STAT1alpha, expressed alone, enhanced cell death, potentiated the fludarabine-induced apoptosis, and enhanced the nuclear location, the phosphorylation, and the transcriptional activity of p53. Unexpectedly, STAT1beta, expressed alone, induced cell death through a mechanism that was independent of the nuclear function of p53. Indeed, in STAT1beta-expressing B cells, p53 was strictly cytoplasmic where it formed clusters, and there was no induction of the transcriptional activity of p53. These data reveal a novel role of STAT1beta in programmed cell death, which is independent of p53.