© F e r r a t a S t o r t i F o u n d a t i o nderived from co-culture of monocytes and autologous leukemic cells are characterized by a gene expression profile typical of cells with dysregulated immunocompetence which could be relevant in the context of the acquired immunodeficiency commonly found in CLL patients. 12Whether NLC represent CLL-specific tumor-associated macrophages, as recently suggested, 13 is still debated. We have recently reported that hepatocyte growth factor (HGF), together with CXCL12, is produced at high levels by stromal cells and is capable of prolonging the survival of CLL cells which are positive for the HGF receptor, c-MET.14 HGF is a multifunctional cytokine that induces multiple biological responses in target cells, including adhesion, motility, growth, survival and morphogenesis by activating its tyrosine kinase receptor, c-MET. 15,16 In normal individuals, HGF is constitutively produced by fibroblast-like stromal cells in lymphoid tissue and by follicular dendritic cells within the germinal center microenvironment. 15,17,18 NLC are present in the lymph nodes of CLL patients, where they are interspersed with stromal, dendritic and T cells to form proliferation centers. 19 The intriguing finding that HGF levels are higher in sera from CLL patients than from normal controls, 20 together with a still undefined pattern of effects induced by HGF on myelomonocytic cells, prompted us to determine c-MET expression on NLC and on monocytes-macrophages as well as investigate potential downstream events caused by the interaction between HGF and c-MET. MethodsThis study was approved by the review board of the IRCCS AOU San Martino -IST, Genoa, Italy. Full details are provided in the Online Supplementary Methods file. Cell preparationHeparinized blood or bone marrow samples were taken from CLL patients untreated for at least 3 months. The patients' characteristics are summarized in Online Supplementary Table S1. NLC were derived and characterized as described below and in the Online Supplementary Methods. CD14 + monocytes were purified from patients or healthy donors by magnetic beads. The human monocytic cell line THP-1 was utilized in selected experiments. Fluorescence microscopy and flow cytometryNLC or fresh monocytes from CLL or normal donors were challenged with antibodies against c-MET, indoleamine 2,3-dioxygenase (IDO), vimentin, CD68, interleukin (IL)-10, CD14, CXCR4, CD163 and pSTAT3 TYR705 and analyzed by immunofluorescence or flow cytometry. The quantification of pSTAT3 immunopositive cells area is described in detail in the Online Supplementary Methods. Analysis of STAT3 and pSTAT3 activation by western blottingWestern blotting was used to evaluate STAT3, pSTAT3 and bactin in monocytes from normal or CLL donors and in NLC with or without HGF treatment. Immunohistochemistry and immunofluorescence analysis of lymph node and bone marrow biopsiesTissue sections were probed with anti-human c-MET, -IDO, -vimentin, -CD68, and -CD163. 3,3'-Diaminobenzidine (DAB) substrate chromogen...

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To test the ability of dorzolamide hydrochloride and timolol maleate to display antioxidant effects. Methods: Antioxidant activity was tested in whole trabecular meshwork (TM) tissue as collected from corneal donors' biopsy specimens, young (third passage) and old (10th passage) human TM cells, and acellular systems composed of pure DNA and subcellular fractions containing or devoid of mitochondria. Oxidative stress was induced by hydrogen peroxide. Monitored end points included DNA fragmentation as evaluated by the halo test, oxidative DNA damage in terms of 8-hydroxy-2Јdeoxyguanosine, and mitochondrial function as evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide test. Results: The antioxidant effect of dorzolamide and timolol were observed on TM biopsy specimens and human TM cells exposed to hydrogen peroxide. As evaluated in cell subfractions, timolol displays antioxidant activity regardless of mitochondria presence. Conversely, the antioxidant activity of dorzolamide was maximized in the presence of mitochondria-containing subcellular fractions and in young human TM cells with functional mitochondria. Conclusions: The antioxidant effect of timolol was direct. The antioxidant effect of dorzolamide involves mitochondria and is likely to be exerted mainly during the early glaucoma phases when the mitochondrial damage in the TM tissue still occurs at low levels. Clinical Relevance: Timolol has an antioxidant effect on the entire cell, whereas dorzolamide exerts protective activity toward oxidative stress only in the presence of intact mitochondria (ie, in endothelial cells that are younger when the cellular damage is still limited). The important role of mitochondrial damage in primary openangle glaucoma is supported by the finding that mutant myocilin impairs mitochondrial functions in human TM meshwork cells.

Lipoic acid (LA) is a sulfated antioxidant produced physiologically as a coenzyme of the pyruvate dehydrogenase complex; it is currently used for treatment of non-insulin-dependent diabetes to favor the cellular uptake of glucose. We have previously described the angiopreventive potential of molecules sharing common features with LA: N-acetyl cysteine, epigallocatechin-3-gallate and xanthohumol. To expand these studies, we have tested the capacity of LA to modulate angiogenesis in tumor growth using a Kaposi's sarcoma model. Endothelial cells exposed to LA displayed a dose-dependent reduction of cell migration and a time-dependent modulation of the phosphorylation of key signaling molecules. In vivo, LA efficiently repressed angiogenesis in matrigel plugs and KS-Imm tumor growth. We analyzed modulation of gene expression in endothelial cells treated with LA for 5 h (early response), finding a mild anti-apoptotic, antioxidant and anti-inflammatory response. A group of LA-targeted genes was selected to perform real-time polymerase chain reaction time-lapse experiments. The long-term gene regulation (48 h and 4 days) shows higher rates of modulation as compared with the array data, confirming that LA is able to switch the regulation of several genes linked to cell survival, inflammation and oxidative stress. LA induced the production of tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) in KS-Imm and activin-A in KS-Imm and endothelial cells; these factors show anti-angiogenic activity in vivo contributing to explain the inhibitory effect of LA on neovascularization. According to our data, LA has promising anti-angiogenic properties, though its influence on central metabolic pathways should suggest more caution about its widespread and not prescribed use at pharmacological doses.

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