Chronic lymphocytic leukemia nurse-like cells express hepatocyte growth factor receptor (c-MET) and indoleamine 2,3-dioxygenase and display features of immunosuppressive type 2 skewed macrophages.

Hepatocyte growth factor, produced by stromal and follicular dendritic cells, and present at high concentrations in the sera of patients with chronic lymphocytic leukemia, prolongs the survival of leukemic B cells by interacting with their receptor, c-MET. It is, however, unknown whether hepatocyte growth factor influences microenvironmental cells, such as nurse-like cells, which deliver survival signals to the leukemic clone. We evaluated the expression of c-MET on nurse-like cells and monocytes from patients with chronic lymphocytic leukemia and searched for phenotypic/functional features supposed to be influenced by the hepatocyte growth factor/c-MET interaction. c-MET is expressed at high levels on nurse-like cells and at significantly higher levels than normal on monocytes from patients. Moreover, the hepatocyte growth factor/c-MET interaction activates STAT3(TYR705) phosphorylation in nurse-like cells. Indoleamine 2,3-dioxygenase, an enzyme modulating T-cell proliferation and induced on normal monocytes after hepatocyte growth factor treatment, was detected together with interleukin-10 on nurse-like cells, and on freshly-prepared patients' monocytes. Immunohistochemical/immunostaining analyses demonstrated the presence of c-MET(+) and indoleamine 2,3-dioxygenase(+) cells in lymph node biopsies, co-expressed with CD68 and vimentin. Furthermore nurse-like cells and chronic lymphocytic monocytes significantly inhibited T-cell proliferation, prevented by anti-transforming growth factor beta and interleukin-10 antibodies and indoleamine 2,3-dioxygenase inhibitors, and supported CD4(+)CD25(high+)/FOXP3(+) T regulatory cell expansion. We suggest that nurse-like cells display features of immunosuppressive type 2 macrophages: higher hepatocyte growth factor levels, produced by leukemic or other microenvironmental surrounding cells, may cooperate to induce M2 polarization. Hepatocyte growth factor may thus have a dual pathophysiological role: directly through enhancement of survival of the leukemic clone and indirectly by favoring T-cell immunosuppression.

Ability of dorzolamide hydrochloride and timolol maleate to target mitochondria in glaucoma therapy.

OBJECTIVE: 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-2'-deoxyguanosine, and mitochondrial function as evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium 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 open- angle glaucoma is supported by the finding that mutant myocilin impairs mitochondrial functions in human TM meshwork cells.

Upregulation of stem cell antigen-1 in the lung of neonatal mice exposed to environmental cigarette smoke.

Mice are particularly susceptible to carcinogens when exposure starts early in life. We evaluated the expression of stem cell antigen-1 (Sca-1) gene in the lung of variously aged CD-1 mice, either untreated or exposed to environmental cigarette smoke (ECS) and/or to a light source. Sca-1 expression progressively decreased with age. The expression of Sca-1 gene and the amount of Sca-1 protein, which was exclusively localized in endothelial cells of the pulmonary vasculature, were significantly upregulated in mice exposed either to ECS or ECS plus light throughout the weaning period, starting at birth. These findings may contribute to explain the high vulnerability of mouse lung early in life.

High susceptibility of neonatal mice to molecular, biochemical and cytogenetic alterations induced by environmental cigarette smoke and light.

Our recent studies have shown that both cigarette smoke and UV-containing light, which are the most widespread and ubiquitous mutagens and carcinogens in the world, cause systemic genotoxic damage in hairless mice. Further studies were designed with the aim of evaluating the induction of genotoxic and carcinogenic effects in Swiss albino mice exposed to smoke and/or light since birth. We observed that a 4-month whole-body exposure of mice to mainstream cigarette smoke, starting at birth, caused an early and potent carcinogenic response in the lung and other organs. Our further experiments showed that exposure of mice to environmental cigarette smoke, during the first 5 weeks of life, resulted in a variety of significant alterations of intermediate biomarkers, including cytogenetic damage in bone marrow and peripheral blood, formation of lipid peroxidation products, increase of bulky DNA adduct levels, induction of oxidative DNA damage, and overexpression of OGG1 gene in lung, stimulation of apoptosis, hyperproliferation and loss of Fhit protein in pulmonary alveolar macrophages and/or bronchial epithelial cells, and early histopathological alterations in the respiratory tract. Moreover, exposure of mice to UV-containing light, mimicking solar irradiation, significantly enhanced oxidative DNA damage and bulky DNA adduct levels in lung, and synergized with smoke in inducing molecular alterations in the respiratory tract. The baseline OGG1 expression in lung was particularly high at birth and decreased in post-weanling mice. Oxidative DNA damage and other investigated end-points exhibited differential patterns in post-weanling mice and adult mice. The findings of these studies provide a mechanistic clue to the general concept that the neonatal period and early stages of life are critical in affecting susceptibility to carcinogens.

Biological assays and genomic analysis reveal lipoic acid modulation of endothelial cell behavior and gene expression.

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.