Altered Expression of Natural Cytotoxicity Receptors and NKG2D on Peripheral Blood NK Cell Subsets in Breast Cancer Patients.

Human natural killer (NK) cells are considered professional cytotoxic cells that are integrated into the effector branch of innate immunity during antiviral and antitumoral responses. The purpose of this study was to examine the peripheral distribution and expression of NK cell activation receptors from the fresh peripheral blood mononuclear cells of 30 breast cancer patients prior to any form of treatment (including surgery, chemotherapy, and radiotherapy), 10 benign breast pathology patients, and 24 control individuals. CD3-CD56dimCD16bright NK cells (CD56dim NK) and CD3-CD56brightCD16dim/- NK cells (CD56bright NK) were identified using flow cytometry. The circulating counts of CD56dim and CD56bright NK cells were not significantly different between the groups evaluated, nor were the counts of other leukocyte subsets between the breast cancer patients and benign breast pathology patients. However, in CD56dim NK cells, NKp44 expression was higher in breast cancer patients (P = .0302), whereas NKp30 (P = .0005), NKp46 (P = .0298), and NKG2D (P = .0005) expression was lower with respect to healthy donors. In CD56bright NK cells, NKp30 (P = .0007), NKp46 (P = .0012), and NKG2D (P = .0069) expression was lower in breast cancer patients compared with control group. Only NKG2D in CD56bright NK cells (P = .0208) and CD56dim NK cells (P = .0439) showed difference between benign breast pathology and breast cancer patients. Collectively, the current study showed phenotypic alterations in activation receptors on CD56dim and CD56bright NK cells, suggesting that breast cancer patients have decreased NK cell cytotoxicity.

Casiopeína IIgly induced cytotoxicity to HeLa cells depletes the levels of reduced glutathione and is prevented by dimethyl sulfoxide.

The newly synthesized copper coordination compound Casiopeína IIgly (Cas IIgly) is a promising alternative drug in the treatment of cancer, since it has shown cytotoxicity and genotoxicity in different tumour models. Given its enhanced effects after ascorbic acid-mediated copper reduction, Cas IIgly's activity is thought to be related to oxidative damage. In the present work, oxidized Cas IIgly failed to induce cytosolic oxidative damage in HeLa cells (only 0.9% of the cell population), and in 2.3% of the treated cells when previously reduced, as evaluated through the oxidation of dihydrorhodamine 123 (DHR 123). However, it showed cytotoxicity, since HeLa cells treated with 10-80 microg/mL Cas IIgly proliferated only at 30% of their normal rate, and at 15% when treated with reduced Cas IIgly. This cytotoxicity is strongly abolished in the presence of the hydroxyl scavenger dimethyl sulfoxide. The decrease, from 3994 to 530 nanograms of reduced glutathione (GSH) per million cells after treatment with 80 microg/mL Casiopeína IIgly, indicates that this drug causes the expenditure of this naturally occurring antioxidant. These results altogether suggest that, albeit Cas IIgly induced cytotoxicity is not related to cytosolic DHR 123 oxidation, it may be related to oxidative damage.

Aging-related oxidative stress in healthy humans.

Oxidative stress has been reported to increase with aging; however, the scientific evidence is controversial. We therefore aimed to analyze the relationship between aging and some markers of oxidative stress. A cross-sectional and comparative study was carried out in a sample of 249 healthy subjects: (i) 25-29 years (n = 22); (ii) 30-39 years (24); (iii) 40-49 years (30); (iv) 50-59 years (48); (v) 60-69 years (60), and (vi) >or= 70 years (65). We measured lipoperoxides and total antioxidant status in plasma and superoxide dismutase and glutathione peroxidase activities in erythrocytes. There was an age-related increase in lipoperoxides, which was evident in the comparison of the group of 25-29 years (0.22 0.11 micromol/l) with the group of 60-69 years (0.38 +/- 0.18 micromol/l, p < 0.01) and >or= 70 years (0.42 +/- 0.19, p < 0.001). Conversely, the total antioxidant status showed an age-related decrease (25-29 years, 1.4 +/- 0.31 mmol/l vs 60-69 years, 1.1 +/- 0.21 and >or= 70 years, 1.1 +/- 0.22, p < 0.05 for each). In erythrocytes, glutathione peroxidase activity showed an age-related decrease (25-29 years, 7,966 +/- 1,813 UI/l vs 60-69 years, 6,193 +/- 2,235 and >or= 70 years, 6,547 +/- 2,307, p < 0.001 for each), whereas superoxide dismutase activity was similar in all age groups. Importantly, there was no age-related change in oxidative stress markers in subjects of < 60 years. These findings suggest that age of >or= 60 years may be associated with increased oxidative stress.

Asociación molecular y función del agente tensioactivo pulmonar de ternera / Molecular association and function of calf lung surfactant

El agente tensioactivo pulmonar es un material compuesto de fosfolípidos, lípidos neutros y proteínas que se encuentra en la superficie alveolar de los pulmones y facilita la ventilación alveolar. La organización molecular de los componentes del agente tensioactivo aislado de pulmones de ternera fue analizada por calorimetría diferencial de barrido y por dispersión dinámica de luz y posteriormente comparada con los componentes organizados en liposomas uni y multilamelares; además, se probó la actividad de superficie al desarrollar en cobayos el síndrome de dificultad respiratoria. Los estudios de calorimetría mostraron que las interacciones lípido-proteína fueron considerablemente abatidas en el agente tensioactivo nativo, en comparación con las del agente tensioactivo en forma de liposomas uni o multilamelares. Los experimentos de dispersión dinámica de luz indicaron que el agente tensioactivo nativo tiene forma fibrilar con interacciones limitadas entre lípidos y proteínas, lo que sugiere que se encuentra organizado en una estructura en forma de reja formando una película de estructura estable. Los resultados obtenidos resaltan la importancia de la organización molecular del agente tensioactivo. Cuando éste fue usado para tratar a los animales con síndrome de dificultad respiratoria, los valores del pH arterial y de PaCO2 mejoraron casi hasta alcanzar los valores normales; cuando se utilizó el agente tensioactivo reconstituído como liposomas uni o multilamelares, los animales no se recuperaron. Es importante enfatizar que el método seguido en el protocolo de aislamiento del agente tensioactivo pulmonar de ternera permitió obtenerlo en una forma fisiológicamente activa.

Surfactant, a highly surface-active material composed of phospholipids, neutral lipids and proteins, lines the lungs' alveolar surface facilitating alveolar ventilation. The molecular organization of surfactant components isolated from calf-lungs was analyzed by differential-scanning calorimetry and dynamic light-scattering, and subsequently compared to surfactant components organized in uni and multilamellar liposomes. The respiratory distress syndrome developed in adult guinea pigs was used for assessing surfactant activity. Calorimetry studies showed that lipid-protein interactions were considerably abated in native surfactant as compared to those of surfactant in uni or multi-lamellar liposomes. Light-scattering experiments indicated that native surfactant has a fibrillar shape with limited lipid-protein interactions, suggesting that it is organized in a lattice-like structure forming a stable film. These findings underscore the importance of the native molecular organization of surfactant. When surfactant reconstituted as uni- or multilamellar liposomes was administred to animals under respiratory distress, they did not recover. In contrast, when native surfactant was used to treat sick animals, arterial pH and PaCO2 values improved, almost reaching normal values. It is important to emphasize that fewer steps in the protocol for isolation of calf lung surfactant made it possible to obtain it in a physiologically active molecular form.

Induction of oxidative damage by copper-based antineoplastic drugs (Casiopeínas).

PURPOSE: The aim of the present study is to determine in HeLa cells and in human lymphocytes, by an easy and fast method, the induction of oxidative damage to plasma membrane lipids and nuclear DNA by Casiopeínas, which are recently synthesized coordination complexes that have been considered as a promising chemotherapeutic alternative for the treatment of cancer, since they have shown cytotoxicity and genotoxicity in several cancer cell lines and xenotransplanted tumours. The presence of an oxidized copper atom in their structure strongly suggests that their mode of action seems to be related to reactive oxygen species (ROS) generation after copper atom reduction through the Fenton and Haber-Weiss system. METHOD: Lipid peroxidation was evaluated as thiobarbituric acid reactive malondialdehyde, cytotoxicity by the fluorescein diacetate/ethidium bromide stain and genotoxicity as DNA fragmentation by the comet assay. Cells were treated with ten different Casiopeínas in a concentration range higher than their IC(50) (10-100 microM), both oxidized and reduced in the presence of ascorbic acid. RESULTS: In almost all the cases, copper reduction enhanced cytotoxicity but, unlike copper nitrate used as positive control, none of them induced appreciable lipid peroxidation. Three Casiopeínas: Cas Igly, Cas-III-H-a and Cas-III-E-a, showed low, moderate and high rates of genotoxicity, respectively, and this effect was enhanced upon addition of ascorbic acid. CONCLUSION: These results suggest that ROS generation might be the cause of cytotoxicity, which seems to be related to initial genetic damage rather than to lipid peroxidation. HeLa cells showed to be more sensitive than normal cells.

Induction of DNA damage by free radicals generated either by organic or inorganic arsenic (AsIII, MMAIII, and DMAIII) in cultures of B and T lymphocytes.

The aim of this work is based in the premise that inorganic arsenic (AsIII) and trivalentmethylated metabolites monomethylarsonous (MMAIII) and dimethylarsinous (DMAIII) participate in DNA damage through the generation of reactive oxygen species (ROS). We have utilized two lymphoblastic lines, Raji (B cells) and Jurkat (T cells), which were treated with the trivalent arsenic species (dose: 0-100 microM) and analyzed by two assays (comet assay and flow cytometry) in the determination of DNA damage and ROS effects in vivo. The results showed that the damage to the DNA and the generation of ROS are different in both cellular lines with respect to the dose of organic arsenic, and the order of damage is MMAIII>DMAIII>AsIII. This fact suggests that the DMAIII is not always the more cytotoxic intermediary xenobiotic, as has already been reported in another study.

Copper increases the damage to DNA and proteins caused by reactive oxygen species.

Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS) and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II) greatly increases in the presence of oxidants. ROS, like hydroxyl (.OH) and superoxide (.O(2)) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT-->GC and GC-->AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus, the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage lambda DNA or proteins using either hydrogen peroxide (H(2)O(2)) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks), spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation temperature [T(d)], denaturation enthalpy [DeltaH], and heat capacity [C(p)] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects were most evident when Cu(II) was present. In summary, these results show that the ROS, .O2 and .OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins. The physiological relevance of these structural effects should be further investigated.

Efficient antioxidant capacity against lipid peroxide levels in healthy elderly of Mexico City.

We evaluated antioxidant activity against lipid peroxide levels (LPO) in healthy elderly and adults of Mexico City in comparison with a population of a rural area. The study included free-living subjects: 38 adults aged <60 years and 129 older subjects aged > or = 60 years of urban Mexico City in addition to 37 adults aged <60 years and 88 older subjects aged > or = 60 years of rural area (Actopan, Hidalgo State, Mexico). LPO were observed as higher in adults and elderly of the urban area than among rural subjects (P<0.01), although LPO levels were similar in rural adults and elderly (P>0.05); conversely, in urban area levels were higher in the elderly than in adults (P<0.01). On the other hand, the superoxide dismutase in urban elderly was higher than that in rural elderly (P<0.05) but similar between urban adults and urban elderly (P>0.05). Total oxidant status in urban elderly was higher than that in rural elderly (P<0.01). Our findings allow us to conclude that the urban elderly (residents of Mexico City) have higher oxidative stress than the rural-dwelling elderly, though the urban elderly have efficient antioxidant capacity as a response to elevated LPO.