Hyaluronan–CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9

Osteoclast (OC) precursors migrate to putative sites of bone resorption to form functionally active, multinucleated cells. The preOC FLG 29.1 cells, known to be capable of irreversibly differentiating into multinucleated OC-like cells, displayed several features of primary OCs, including expression of specific integrins and the hyaluronan (HA) receptor CD44. OC-like FLG 29.1 cells adhered to and extensively migrated through membranes coated with fibronectin, vitronectin, and laminins, but, although strongly binding to HA, totally failed to move on this substrate. Moreover, soluble HA strongly inhibited OC-like FLG 29.1 cell migration on the permissive matrix substrates, and this behavior was dependent on its engagement with CD44, as it was fully restored by function-blocking anti-CD44 antibodies. HA did not modulate the cell–substrate binding affinity/avidity nor the expression levels of the corresponding integrins. MMP-9 was the major secreted metalloproteinase used by OC-like FLG 29.1 cells for migration, because this process was strongly inhibited by both TIMP-1 and GM6001, as well as by MMP-9–specific antisense oligonucleotides. After HA binding to CD44, a strong down-regulation of MMP-9 mRNA and protein was detected. These findings highlight a novel role of the HA–CD44 interaction in the context of OC-like cell motility, suggesting that it may act as a stop signal for bone-resorbing cells

Surface-antigen expression profiling of B cell chronic lymphocytic leukemia: from the signature of specific disease subsets to the identification of markers with prognostic relevance

Studies of gene expression profiling have been successfully used for the identification of molecules to be employed as potential prognosticators. In analogy with gene expression profiling, we have recently proposed a novel method to identify the immunophenotypic signature of B-cell chronic lymphocytic leukemia subsets with different prognosis, named surface-antigen expression profiling. According to this approach, surface marker expression data can be analysed by data mining tools identical to those employed in gene expression profiling studies, including unsupervised and supervised algorithms, with the aim of identifying the immunophenotypic signature of B-cell chronic lymphocytic leukemia subsets with different prognosis. Here we provide an overview of the overall strategy employed for the development of such an "outcome class-predictor" based on surface-antigen expression signatures. In addition, we will also discuss how to transfer the obtained information into the routine clinical practice by providing a flow-chart indicating how to select the most relevant antigens and build-up a prognostic scoring system by weighing each antigen according to its predictive power. Although referred to B-cell chronic lymphocytic leukemia, the methodology discussed here can be also useful in the study of diseases other than B-cell chronic lymphocytic leukemia, when the purpose is to identify novel prognostic determinants

Loss of MUTYH function in human cells leads to accumulation of oxidative damage and genetic instability

The DNA glycosylase MUTYH (mutY homolog (Escherichia coli)) counteracts the mutagenic effects of 8-oxo-7,8-dihydroguanine (8-oxodG) by removing adenine (A) misincorporated opposite the oxidized purine. Biallelic germline mutations in MUTYH cause the autosomal recessive MUTYH-associated adenomatous polyposis (MAP). Here we designed new tools to investigate the biochemical defects and biological consequences associated with different MUTYH mutations in human cells. To identify phenotype(s) associated with MUTYH mutations, lymphoblastoid cell lines (LCLs) were derived from seven MAP patients harboring missense as well as truncating mutations in MUTYH. These included homozygous p.Arg245His, p.Gly264TrpfsX7 or compound heterozygous variants (p.Gly396Asp/Arg245Cys, p.Gly396Asp/Tyr179Cys, p.Gly396Asp/Glu410GlyfsX43, p.Gly264TrpfsX7/Ala385ProfsX23 and p.Gly264TrpfsX7/Glu480del). DNA glycosylase assays of MAP LCL extracts confirmed that all these variants were defective in removing A from an 8-oxoG:A DNA substrate, but retained wild-type OGG1 activity. As a consequence of this defect, MAP LCLs accumulated DNA 8-oxodG in their genome and exhibited a fourfold increase in spontaneous mutagenesis at the PIG-A gene compared with LCLs from healthy donors. They were also hypermutable by KBrO3--a source of DNA 8-oxodG--indicating that the relatively modest spontaneous mutator phenotype associated with MUTYH loss can be significantly enhanced by conditions of oxidative stress. These observations identify accumulation of DNA 8-oxodG and a mutator phenotype as likely contributors to the pathogenesis of MUTYH variants

Prognostic impact of ZAP-70 expression in chronic lymphocytic leukemia: mean fluorescence intensity T/B ratio versus percentage of positive cells

<p>Abstract</p> <p>Background</p> <p>ZAP-70 is an independent negative prognostic marker in chronic lymphocytic leukemia (CLL). Usually, its expression is investigated by flow cytometric protocols in which the percentage of ZAP-70 positive CLL cells is determined in respect to isotypic control (ISO-method) or residual ZAP-70 positive T cells (T-method). These methods, however, beside suffering of an inherent subjectivity in their application, may give discordant results in some cases. The aim of this study was to assess the prognostic significance of these methods in comparison with another in which ZAP-70 expression was evaluated as a Mean-Fluorescence-Intensity Ratio between gated T and CLL cells (T/B Ratio-method).</p> <p>Methods</p> <p>Cytometric files relative to ZAP-70 determination according to the three readouts were retrospectively reviewed on a cohort of 173 patients (test set), all with complete clinical and biological prognostic assessment and time-to-treatment (TTT) available. Findings were then validated in an independent cohort of 341 cases from a different institution (validation set).</p> <p>Results</p> <p>The optimal prognostic cut-offs for ZAP-70 expression were selected at 11% (ISO-method) or 20% of positive cells (T-method), as well as at 3.0 (T/B Ratio-method) in the test set; these cut-offs yielded 66, 60 and 73 ZAP-70⁺cases, respectively. Univariate analyses resulted in a better separation of ZAP-70⁺vs. ZAP-70^-CLL patients utilizing the T/B Ratio, compared to T- or ISO-methods. In multivariate analyses which included the major clinical and biological prognostic markers for CLL, the prognostic impact of ZAP-70 appeared stronger when the T/B-Ratio method was applied. These findings were confirmed in the validation set, in which ZAP-70 expression, evaluated by the T- (cut-off = 20%) or T/B Ratio- (cut-off = 3.0) methods, yielded 180 or 127 ZAP-70⁺cases, respectively. ZAP-70⁺patients according to the T/B Ratio-method had shorter TTT, both if compared to ZAP-70^-CLL, and to cases classified ZAP-70⁺by the T-method only.</p> <p>Conclusions</p> <p>We suggest to evaluate ZAP-70 expression in routine settings using the T/B Ratio-method, given the operator and laboratory independent feature of this approach. We propose the 3.0 T/B Ratio value as optimal cut-off to discriminate ZAP-70⁺(T/B Ratio less than 3.0) from ZAP-70^-(T/B Ratio more/equal than 3.0) cases.</p

Oxidative DNA damage accumulation in gastric carcinogenesis

Abstract: Background-Gastric carcinogenesis is a multifactorial, multistep process, in which chronic inflammation plays a major role. Aims-In order to ascertain whether free radical mediated oxidative DNA damage is involved in such a process, concentrations of 8-hydroxydeoxyguanosine (80HdG), a mutagenic/carcinogenic adduct, and thiobarbituric acid reactive substances (TEARS), as an indirect measure of free radical mediated damage, were determined in biopsy specimens from patients undergoing endoscopy. Patients-Eighty eight patients were divided into histological subgroups as follows: 27 with chronic non-atrophic gastritis, 41 with atrophic gastritis, six with gastric cancer, and 14 unaffected controls. Methods-Intestinal metaplasia, Helicobacter pylori infection, and disease activity were semiquantitatively scored. 80HdG concentrations were assessed by HPLC with electrochemical detection, and TEARS concentrations were fluorimetrically assayed. Results-80HdG concentrations (mean number of adducts/10(5) dG residues) were significantly higher in chronic atrophic gastritis (p=0.0009). Significantly higher concentrations were also detected in the presence of severe disease activity (p=0.02), intestinal metaplasia (p=0.035), and H pylori infection (p=0.001). TEARS concentrations were also higher in atrophic gastritis, though not significantly so. In a multiple logistic regression analysis, 80HdG concentrations correlated best with the presence and severity of H pylori infection (r=0.53, p=0.002). Conclusions-Chronic gastritis is characterised by the accumulation of oxidative DNA damage with mutagenic and carcinogenic potential. H pylori infection is the major determinant for DNA adduct formation

CD30L up-regulates CD30 and IL-4 expression by T cells

AbstractCD30L is frequently expressed on acute myeloid leukemia (AML) blasts. Its presence is associated with the co-expression of interleukin-4 (IL-4) receptor and with the expansion of specific T-helper 2 (Th2) cell subsets producing IL-4 and expressing CD30. Recombinant CD30L-bearing cells up-regulated the expression of surface CD30 and increased the production of IL-4 and soluble (s) CD30 by co-cultured T cells. These findings were confirmed with AML blasts expressing surface CD30L, where blocking anti-CD30 antibodies completely abolished the release of sCD30 and reduced the production of IL-4. Our data indicates a direct role of CD30L+ neoplastic cells in driving the immune response toward a Th2-polarized non-protective state

Laminin-332 (Laminin-5) is the major motility ligand for B cell chronic lymphocytic leukemia

Cell adhesion and motility are central aspects in the pathophysiology of B cell chronic lymphocytic leukemia (B-CLL), but the role of specific extracellular matrix proteins is still to be completely unveiled. Purified peripheral blood neoplastic cells of B-CLL patients migrated poorly on laminins-111,-411,-511, but showed pronounced motility on laminin (LM)-332 in a high percentage of cases. B-CLL cell motility on LM-332 was mediated by the α3β1 integrin and was preferentially observed in cells carrying a mutated IgVH gene profile. Within normal lymph nodes, LM-332 was circumscribed around blood vessels and to areas corresponding to marginal zones, where it was deposited in a pattern reminiscent of reticular fibers. Conversely, in B-CLL involved lymph nodes, a positive LM-332 reticular mesh was diffusely evident, throughout the disrupted nodal architecture. In the present study we identified LM-332 as a crucial motility-promoting factor for B-CLL lymphocytes and as a potential constituent favoring the dissemination of B-CLL lymphocytes through vascular basement membranes and possibly lymph node compartments. © 2007 International Society of Matrix Biology