Analysis of radiation-induced apoptosis in human lymphocytes: Flow cytometry using Annexin V and propidium iodide ver

Background: The neutral comet assay was devised to measure double-stranded DNA breaks, but it has also been used to measure apoptosis based on its characteristic DNA fragmentation patterns. There is still uncertainty about the reliability of this method. By comparing the comet assay with a flow cytometry method that uses Annexin V binding to apoptotic cells, we have provided further evidence for evaluating the usefulness of the comet assay for detecting apoptosis. Methods: Apoptosis was induced in human peripheral blood mononuclear cells (PBMC) by ionizing radiation and measured using the comet assay and a flow cytometry method that measures Annexin V and propidium iodide (PI) staining. Results: The Annexin V flow cytometry assay distinguished among early apoptosis, late apoptosis, and an apoptotic or necrotic phase in which the cells were labeled with both Annexin V and PI. The comet assay detected only the latter two phases of apoptosis. Conclusions: The comet assay is a useful tool for measuring the late stages of apoptosis whereas the Annexin V assay measures higher amounts of apoptosis because it can detect cells in an earlier stage of the apoptotic pathway

Differential Effects of Interleukin-7 and Interleukin-15 on NK Cell Anti-Human Immunodeficiency Virus Activity

The ability of interleukin-7 (IL-7) and IL-15 to expand and/or augment effector cell functions may be of therapeutic benefit to human immunodeficiency virus (HIV)-infected patients. The functional effects of these cytokines on innate HIV-specific immunity and their impact on cells harboring HIV are unknown. We demonstrate that both IL-7 and IL-15 augment natural killer (NK) function by using cells (CD3(−) CD16(+) CD56(+)) from both HIV-positive and -negative donors. Whereas IL-7 enhances NK function through upregulation of Fas ligand, the effect of IL-15 is mediated through upregulation of tumor necrosis factor-related apoptosis-inducing ligand. The difference in these effector mechanisms is reflected by the ability of IL-15-treated but not IL-7-treated NK cells to reduce the burden of replication-competent HIV in autologous peripheral blood mononuclear cells (PBMC) (infectious units per million for control NK cells, 6.79; for IL-7-treated NK cells, 236.17; for IL-15-treated cells, 1.01; P = 0.01 versus control). In addition, the treatment of PBMC with IL-15-treated but not IL-7-treated NK cells causes undetectable HIV p24 (five of five cases), HIV RNA (five of five cases), or HIV DNA (three of five cases). These results support the concept of adjuvant immunotherapy of HIV infection with either IL-7 or IL-15 but suggest that the NK-mediated antiviral effect of IL-15 may be superior

Vpr R77Q is associated with long-term nonprogressive HIV infection and impaired induction of apoptosis

The absence of immune defects that occurs in the syndrome of long-term nonprogressive (LTNP) HIV infection offers insights into the pathophysiology of HIV-induced immune disease. The (H[F/S]RIG)(2) domain of viral protein R (Vpr) induces apoptosis and may contribute to HIV-induced T cell depletion. We demonstrate a higher frequency of R77Q Vpr mutations in patients with LTNP than in patients with progressive disease. In addition, T cell infections using vesicular stomatitis virus G (VSV-G) pseudotyped HIV-1 Vpr R77Q result in less (P = 0.01) T cell death than infections using wild-type Vpr, despite similar levels of viral replication. Wild-type Vpr-associated events, including procaspase-8 and -3 cleavage, loss of mitochondrial transmembrane potential (Δψ(m)), and DNA fragmentation factor activation are attenuated by R77Q Vpr. These data highlight the pathophysiologic role of Vpr in HIV-induced immune disease and suggest a novel mechanism of LTNP

Mercury accumulation, structural damages, and antioxidant and immune status changes in the gilthead seabream (Sparus aurata L.) exposed to methylmercury

In aquatic systems, mercury (Hg) is an environmental contaminant that causes acute and chronic damage to multiple organs. In fish, practically all of the organic Hg found is in the form of methylmercury (MeHg), which has been associated with animal and human health problems. This study evaluates the impact of waterborne- exposure to sublethal concentrations of MeHg (10 lgL-1) in gilthead seabream (Sparus aurata). Hg was seen to accumulate in liver and muscle, and histopathological damage to skin and liver was detected. Fish exposed to MeHg showed a decreased biological antioxidant potential and increased levels of the reactive oxygen molecules compared with the values found in control fish (non-exposed). Increased liver antioxidant enzyme activities (superoxide dismutase and catalase) were detected in 2 day- exposed fish with respect to the values of control fish. However, fish exposed to MeHg for 10 days showed liver antioxidant enzyme levels similar to those of the control fish but had increased hepato-somatic index and histopathological alterations in liver and skin. Serum complement levels were higher in fish exposed to MeHg for 30 days than in control fish. Moreover, head–kidney leukocyte activities increased, although only phagocytosis and peroxidase activities showed a significant increase after 10 and 30 days, respectively. The data show that 30 days of exposure to waterborne MeHg provokes more significant changes in fish than a short-term exposure of 2 or 10 days.Versión del editor1,927

Evaluation of Fish Quality and Safety by Proteomics Techniques

19 pages, 1 table, 1 figureStock depletion, new trends in farming practices, the globalization of markets, and the development of novel products and production methods represent new challenges for seafood quality and safety. Fortunately, genomics, proteomics, and high-throughput microarray technologies have fundamentally changed our ability to study the molecular basis of aspects related to food authenticity, safety, and quality as well as changes induced by processing in food matrices. Furthermore, knowledge about the localization, structure, modification, function, and interactions of the proteins expressed by a genome from any tissue used as a source of food can offer precious information in order to improve its quality, safety, and nutritional properties. Although fish protein databases are still scarce, especially when compared with those available for edible plants and terrestrial animals, proteomics studies of model aquatic organisms are helping us to understand problems related to the quality and safety of seafood. The main objective of this chapter is to present a compilation of studies related to fish quality aspects by means of proteomics tools describing new perspectives and challenges for the use of proteomics-based biomarkers in the identification of the causes of quality flaws and their preventionN