Míriam Martins Chaves

The Role of Oxidative Stress in the Aging Process

The aging of organisms is characterized by a gradual functional decline of all organ systems. An appropriate theory must explain four main characteristics of aging: it is progressive, endogenous, irreversible, and deleterious for the individual. The aging of the immune system, or immunosenescence, is manifested by an increased susceptibility to infections with increased morbidity and mortality. Phagocytic capacity, synthesis of reactive oxygen intermediaries, and the intracellular killing efficiency of neutrophils are impaired in the elderly. Among all aging theories, the most updated one describes the free radicals. It implies that progressive aging is associated with higher levels of oxidative biomolecules reacted with free radicals. Although reactive oxygen species (ROS) are predominantly implicated in causing cell damage, they also play a major physiological role in several aspects of intracellular signaling and regulation. ROS include a number of chemically reactive molecules derived from oxygen. Not only oxygen, but also nitrogen can be deleterious species. The overproduction of reactive nitrogen species (RNS) is called nitrosative stress. ROS/RNS are known to play a dual role in biological systems since they can be either harmful or beneficial to living systems.

HMGB1, TLR and RAGE: a functional tripod that leads to diabetic inflammation.

Introduction: Despite advances in treatment of diabetes mellitus, its prevalence continues to rise globally. Medications available are unable to control the vascular complications. Proposals for new therapeutic targets must take into account the hyperglycemia-induced signaling pathways that give rise to the inflammatory profile of the disease. Areas covered: How high-mobility-group box-1 (HMGB1) protein, acting as an activator of Toll-like receptors (TLR) and receptors for advanced glycation end products (RAGE), creates a functional tripod that contributes to increased production of pro-inflammatory mediators, and sustains the chronic inflammatory state associated with diabetes. The interaction of TLR2 and TRL4 with host-derived ligands, which links diabetic complications with the innate immune response, and the activation of RAGE, which induces a cascade of metabolic responses, leading to the production and secretion of pro-inflammatory cytokines. Expert opinion: Considering the involvement of the innate immune system, in association with the role of HMGB1 as an activator of TLR and RAGE, diabetes should be considered and treated as a metabolic and immunological disease, triggered by hyperglycemia. HMGB1 plays a central role in mediating injury and inflammation, and interactions involving HMGB1–TLR–RAGE constitute a tripod that trigger NF-κB activation. Blockade or downregulation of HMGB1, and/or control of the inflammatory tripod, represent a promising therapeutic approach for the treatment of diabetes.

Increase of reactive oxygen (ROS) and nitrogen (RNS) species generated by phagocyting granulocytes related to age.

In this present paper the age-induced effect on reactive oxidizing species generated by oxygen (ROS) and nitrogen (RNS) was studied using human phagocyting granulocytes. The ROS and RNS were quantified, respectively, in a chemiluminescence assay and by the measurement of nitrite production. The age-induced reactive oxidizing species generation was studied in healthy subjects ranging from 20 to 80 years old, divided into six age groups: group I, 20-29 years old; group II, 30-39 years old; group III, 40-49 years old; group IV, 50-59 years old; group V, 60-69 years old; and group VI, 70-80 years old. Our results demonstrate a parallelism between generation of the ROS and RNS induced by the age. A significant increase of ROS production was observed from 40 years old (age groups III, IV, V and VI while for RNS this increase was observed only from 50 years old (groups IV, V and VI). These data suggest an increase of oxidizing species generation (ROS/RNS) related to age. The increased generation of ROS (40-49 years old) was induced before the increasing of RNS (50-59 years old) and it may have consequences on inflammation and host defences.

Therapeutic monoclonal antibodies: scFv patents as a marker of a new class of potential biopharmaceuticals

Anticorpos monoclonais representam a classe de maior crescimento em produtos de biofarmacos e possuem varias aplicacoes em pesquisa medica, diagnostico, terapias e ciencia basica. A producao de anticorpos monoclonais recombinantes revolucionou a geracao de imunoglobulinas e sua utilizacao implica em avanco estrategico, afetando o mercado farmaceutico global de proteinas terapeuticas. No presente trabalho, uma revisao sobre scFv e a relacao do seu numero de patentes foi analisada. Os resultados mostram que varios paises apresentam patentes de scFv com destaque para os Estados Unidos, China e Reino Unido. Os alvos desses anticorpos tambem foram avaliados e as analises revelaram que a maioria e destinado a terapias contra o câncer.

QUANTIFICATION OF THE POPULATION AND PHAGOCYTARY ACTIVITY OF HEMOCYTES OF RESISTANT AND SUSCEPTIBLE STRAINS OF Biomphalaria glabrata AND Biomphalaria tenagophila INFECTED WITH Schistosoma mansoni

Among the determinant factors in the resistance and susceptibility of Biomphalaria to Schistosoma mansoni, hemocytes play an important role. Aiming at studying S. mansoni/Biomphalaria interactions related to hemocytes, the first step is certainly connected with the standardization of this cell population in uninfected Biomphalaria. In this way, quantification of this cell population in hemolymph, as well as its phagocitary capacity, have been determined for the first time. Furthermore, using susceptible and resistant strains of B. glabrata and B. tenagophila, the hemocytegram and phagocytary capacity of hemocytes after infection with S. mansoni were determined too. Resistant and susceptible strains of B. glabrata (BA and BH, respectively), as well as resistant and susceptible strains of B. tenagophila (TAIM and CF, respectively) were infected with 10 miracidia of the LE and SJ strains of S. mansoni, respectively. These infected snails and respective uninfected controls were assessed in relation to the number of circulating hemocytes and alteration in the phagocytary capacity, by using Zymozan and MTT. Reading was taken by means of a spectrophotometer at 5 hours and 1, 2, 5, 10, 20 and 30 days after infection. The results showed a decrease in population of the circulating phagocytary cells, 5 hours after infection. One day post-infection, the circulating cells of the susceptible snails showed an increased metabolic activity, but the same event could not be observed in the resistant strains. In the subsequent observation periods, significant differences among the strains studied could not be observed until the end of the experiment.

Modulation of the Reactive Oxygen Species (ROS) generation mediated by cyclic AMP-elevating agents or Interleukin 10 in granulocytes from type 2 diabetic patients (NIDDM): a PKA-independent phenomenon

UNLABELLED SUMMARY-BACKGROUND: The present study investigates the hypothesis that cells from ill patients and from healthy subjects may have different reactivity under metabolic stimulation as a consequence of an disease-induced metabolic adaptation. METHODS Granulocytes either from healthy subjects or from type II-Non Insulin Dependent Diabetes Mellitus (NIDDM) patients were compared in their capacities to generate Reactive Oxygen Species (ROS). The ROS generation was comparatively determined in a chemiluminescence assay, luminol-dependent, after cell incubation in the presence of either cyclic AMP - elevating agents or Interleukin 10. In some experiments the cells were pretreated with H89 compound (a PKA inhibitor) or with diphenylene iodonium (DPI), a NADPH-oxidase inhibitor. RESULTS Our results showed an increased ROS generation in granulocytes from diabetic patients in absence of cyclic AMP-elevating agents or IL-10. In the presence of cyclic AMP-elevating agents was observed an inverse metabolic response in granulocytes from diabetic patients in comparison to cells from healthy subjects. The granulocytes were pre-incubated in the presence of cyclic AMP-elevating agents--amminophylline (AMF) or dibutyryl cyclic AMP (dbcAMP)--or interleukin 10 (IL-10). The AMF, dbcAMP and IL-10 inhibited ROS production by granulocytes from healthy subjects. By contrast, AMF and dbcAMP activated cells from diabetic patients while IL-10 had no effect. The inhibition of ROS induced by AMF, dbcAMP or IL-10 was promptly abolished by the pretreatment of the cells with either PKA H89 inhibitor or NADPH-oxidase inhibitor (DPI) in granulocytes from healthy subjects. In relation to the granulocytes from type 2 diabetics patients, the activation of ROS generation mediated by AMF and dbcAMP was fully abolished by NADPH-oxidase DPI-inhibitor, but not by PKA H89 inhibitor. CONCLUSIONS Our present results reinforce the hypothesis that cells from ill patients (type II diabetic) when compared to cells from healthy subjects have different reactivity under metabolic stimulation. ROS production by human granulocytes was modulated by cyclic AMP elevating agents and IL-10. The inhibition of the ROS production in cells from healthy subjects was PKA-dependent while the activation in granulocytes from patients was PKA-independent. This inverse metabolic response, in cells from patients, suggests the use of an alternative metabolic pathway PKA-independent, possible cAMP/Epac/PKB-dependent. The correlation between activation of ROS production in granulocytes from diabetic patients and pathogenesis of diabetes can be suggested, however, further and extensive studies are needed for demonstrating this suggestion.

Effect of gamma radiation on the activity of hemocytes and on the course of Schistosoma mansoni infection in resistant Biomphalaria tenagophila snails

High doses of gamma radiation (10 Krad) in Biomphalaria tenagophila snails (Taim strain), which have been found to be resistant to Schistosoma mansoni, were not sufficient to impair their resistance to the parasite. The number of hemocytes, as well as their phagocytic activity, were not affected by irradiation, thus showing resemblance with mammal macrophages, which are resistant to gamma irradiation also.

Ascorbic acid, alpha-tocopherol, and beta-carotene reduce oxidative stress and proinflammatory cytokines in mononuclear cells of Alzheimer's disease patients.

Abstract Objectives The in vitro effect of a vitamin complex in generating and reducing oxidative species in peripheral blood mononuclear cells (PBMNC) and plasma of patients with Alzheimers disease (AD) and healthy subjects (HS) was evaluated. Methods Two concentrations of a vitamin complex ([A] and [20A]) with ascorbic acid, alpha-tocopherol, and beta-carotene were incubated with either mononuclear cells or plasma. The generation of oxidizing species was measured in a luminol-dependent chemiluminescence assay and the reducing response by the MTT dye reduction assay. The levels of cytokines (interleukin [IL]-1β, IL-6, and IL-4) were measured by sandwich enzyme-linked immunosorbent assay. Results Our results demonstrated that the increase in the vitamin complex concentration reduced the reactive oxygen species (ROS) production and enhanced cellular reduction capacity in cells of AD patients in concentration [20A]. Plasma reduction capacity rose significantly for both groups (AD and HS). Concentration [A] did not alter the IL-1β production, increased IL-4 production in both groups and lowered IL-6 production in AD cells. Concentration [20A] increased pro-inflammatory cytokines (IL-1β and IL-6) and decreased IL-4 production by PBMNC of HS leading to a pro-inflammatory status. Discussion The antioxidant vitamin complex was effective in reducing oxidative stress in PBMNC of AD patients by lowering ROS production, improving cellular antioxidant capacities and modifying cytokine induced inflammation.

From hyperglycemia to AGE-RAGE interaction on the cell surface: a dangerous metabolic route for diabetic patients.

Background: Diabetes mellitus constitutes a serious public health problem. Recent advances in therapies are unable to control its vascular complications. New medications have been suggested but without comprehensive knowledge of the signaling pathways induced by hyperglycemia. Objective: To review the mechanisms of the inducer and effector phases of the metabolic cascade in diabetes, with emphasis on the steps that have been targeted to date The route from hyperglycemia to advanced glycation end products (AGE)–receptor of advanced glycation end products (RAGE) complex formation is suggested as a target for new therapies. Methods: A review of literature, including historical papers and recent manuscripts. Results: All the components of the suggested route are initially activated or indirectly formed due to hyperglycemia via a two-phase cascade that represents a dangerous metabolic route, especially for diabetics. Recent experiments with new drugs and clinical trials targeting this pathway are examined. Conclusions: In order to control vascular injury in diabetes, several steps in the cascade need to be inhibited simultaneously together with aggressive glycemic control.

Correlation between NADPH Oxidase and Protein Kinase C in the ROS Production by Human Granulocytes Related to Age

Background: Aging may be defined as gradual and progressive changes in an organism that increase the probability of death. Accumulating evidence now indicates that the sum of deleterious free radical reactions going on continuously throughout cells and tissues constitutes the aging process or is a major contributor to it. Objective: The aim of this paper was to study the correlation between NADPH oxidase and protein kinase C (PKC) in the reactive oxygen species (ROS) production related to age. Methods: The age-induced ROS generation was studied in healthy subjects ranging in age from 20 to 80 years, divided into six age groups: (1) 20–29, (2) 30–39, (3) 40–49, (4) 50–59, (5) 60–69, and (6) 70–80 years. The ROS were quantified using a chemiluminescence assay (luminol dependent) and the results expressed as RLU/s at maximum peak and total chemiluminescence (integral under the curve RLU/s). Results: Our results demonstrate a significant increase of the ROS production from 40 years of age (age groups 3–6). In the age groups 1 and 2, we did not observe a significant difference (p > 0.05). These data suggest an increase of the ROS production from 40 to 49 years of age which may be induced by the PKC activity. The selective PKC inhibitor (calphostin C) abrogated the stimulatory effect of phorbol-12,13-dibutyrate on the ROS production. However, the NADPH oxidase inhibitor diphenylene iodonium did not inhibit the total ROS production by granulocytes in relation to age. Conclusions: These data suggest a correlation between age-related PKC activity, NADPH oxidase phosphorylation, and ROS production. The above correlations between unspecific and inflammatory responses related to age are discussed.