Objective: The mechanisms of atherosclerosis development in the apolipoprotein E-deficient (apoE−/−) mice have been clearly described. In this genetically modified strain, oxidative stress contributes with atherosclerotic lesion progression. However, there are controversial criteria on the vulnerability of female apoE−/− mouse to oxidative stress and atherosclerosis during the fertile period. Thus, the aim of the present work was to examine the implication of vascular oxidative stress during early atherosclerosis development in young apoE−/− female mice. Methods: AQ1 Please provide a brief methodology Results: Hematoxylin/eosin staining demonstrated the presence of early atherosclerotic lesions in the apoE−/− mice, which progressed with age. Dihydroethidium staining of aortic sections revealed a significant increase of superoxide anion generation in apoE−/− compared with the wild type mice. The increment of this reactive oxygen species was associated, at least, with the overexpression of Nox2 mRNA levels. In addition, the 8-week-old apoE−/− mice had a significant increment of lipid and protein damage, as well as a disruption of superoxide dismutase and catalase activity, together with a deprivation of reduced glutathione and an impairment of nitric oxide availability. Conclusion: The present study shows that early atherosclerotic lesion formation in young apoE−/− female mice is associated with an increment of Nox2-mediated reactive oxygen species generation with the subsequent redox disruption, suggesting a key role of oxidative stress during atherogenesis in fertile apoE−/− female mice.

La retencion de las lipoproteinas de baja densidad (LDL) por parte de los proteoglicanos arteriales constituye un evento molecular clave en el inicio y desarrollo de la aterosclerosis. Este proceso promueve la oxidacion de las LDL y con ello el desarrollo de una respuesta inflamatoria cronica que contribuye a la progresion de la enfermedad. En el Centro de Inmunologia Molecular se obtuvo un anticuerpo monoclonal quimerico, llamado chP3R99, que reconoce proteoglicanos sulfatados de la pared arterial y es capaz de inhibir la retencion y oxidacion de las LDL. Ademas, la inmunizacion el anticuerpo chP3R99 previene la formacion de lesiones ateroscleroticas mediante la induccion de anticuerpos autologos anti-proteoglicanos. Sin embargo, no se conocen en su totalidad los mecanismos moleculares de accion de este anticuerpo. Por lo que el presente trabajo tuvo como objetivo evaluar el efecto de la inmunizacion con el anticuerpo chP3R99 sobre el infiltrado de macrofagos, poblacion celular que desencadena la respuesta inflamatoria en el tejido vascular. Se llevo a cabo un esquema de inmunizacion preventiva, donde se administro una dosis de 100 µg s.c. del anticuerpo en conejos tratados posteriormente con 2 mL/kg i.v. de Lipofundin, un inductor de lesiones ateroscleroticas. Los resultados mostraron que el tratamiento con el anticuerpo inhibio el infiltrado de macrofagos y redujo significativamente (p<0,05) la expresion genica de la enzima oxido nitrico sintasa y la actividad de la enzima mieloperoxidasa. Estos resultados demuestran la potencialidad de la inmunizacion con el anticuerpo chP3R99 de atenuar la respuesta inflamatoria, factor clave en el desarrollo de la aterosclerosis. Palabras clave: Aterosclerosis, macrofagos, inflamacion, anticuerpo monoclonal. Abstract The retention of low-density lipoproteins (LDL) by arterial proteoglycans represent a key molecular event in atherosclerosis development. This process promotes the oxidation of LDL and the development of a chronic inflammatory response, leading to atherosclerosis progression. At the Center of Molecular Immunology, it was obtained a chimeric monoclonal antibody, named chP3R99, which recognizes sulfated proteoglycans of the arterial wall, and able to inhibit the retention and oxidation of LDL. In addition, the immunization with this antibody prevents atherosclerotic lesions development by mean a generation of anti-proteoglycans autologous antibodies. Nonetheless, the molecular mechanisms of action of this antibody remain in part unknown. Thus, the present work was aimed to evaluate the effect of chP3R99 immunization on the macrophage infiltration, because of the key role of this cellular population during vascular inflammation. We carried out a preventive immunization where we administered a dose of 100 µg, s.c. in rabbits later treated with 2 mL/kg i.v. of Lipofundin, an inductor of atherosclerotic lesions. The results showed that the treatment with the chP3R99 antibody inhibited the macrophage infiltration, at the same time that reduced the gene expression of inducible nitric oxide synthase and myeloperoxidase activity. These results demonstrate the potential of chP3R99 immunization to reduce the inflammatory response, a key factor in atherosclerosis development. Keywords: Atherosclerosis, macrophages, inflammation, monoclonal antibody

The role of glutathione peroxidase-1 (GPx1) in limiting the oxidative risk for atherogenesis is increasingly recognized. Therapeutic strategies, designed to augment cellular endogenous defense systems have been identified as a promising approach to control oxidative stress-associated diseases, including atherosclerosis. Ozone therapy regulates oxidative metabolism and prevents oxidative stress-associated-chronic diseases. Thus, the present study was aimed to test the hypothesis that ozone-oxidative conditioning up-regulates GPx1 synthesis in apolipoprotein E deficient mice (apoE−/−), protecting the vasculature against atherosclerosis. Male apoE−/− mice were treated with 1mL of ozone/oxygen containing 40μg/mL of ozone by rectal insufflation. As controls, mice were untreated or insufflated with oxygen only. Results showed a significant increase (P<0.05) of aortic GPx1 gene expression in ozone-treated mice, whereas only minor atherosclerotic lesions were observed. Furthermore, GPx1 activity and GSH levels were significant increased (P<0.05) in ozone receiving group compared with controls. In addition, lipid peroxidation was attenuated by ozone treatment, whereas serum lipids were similar among experimental groups. These results altogether suggest that ozone therapy attenuated atherogenesis by a mechanism that involved, at least, the improvement of aortic GPx1 expression/activity. Further studies are needed in order to assess the possible link of cellular redox-sensitive pathways with the antiatherogenic effect of ozone therapy.

Apoptosis seems to play an important role in cancer immunotherapy outcome. We have studied the kinetic pattern of apoptosis induction in H125 human lung carcinoma xenografts after treatment with the monoclonal antibody (MAb) anti-epidermal growth factor receptor (EGFr) IOR EGF/r3. Tumor-bearing nude mice were injected intravenously with a single 8 mg/kg dose of IOR EGF/r3 and tumor specimens were taken up to 30 days post treatment. Apoptosis was measured by morphometric analysis of the histological sections at each tumor specimen over time points. The results showed a significant apoptotic response in tumors within six days after injection of this MAb reaching a peak at 20 days post treatment. The kinetics were very broad, with apoptotic cells present over the entire time-frame. However, the time course of the apoptotic index showed a significant difference to the mitotic index. Finally, the MAb-induced apoptosis was related to tumor growth delay indicating a probable arrest of cell cycle and a corresponding inhibition of tumor progression, which was corroborated by the Ki67 and proliferating cell nuclear antigen (PCNA) biomarkers.

Metabolism of arachidonic acid by cyclooxygenase is one of the primary sources of reactive oxygen species in the ischemic brain. Neuronal overexpression of cyclooxygenase-2 has recently been shown to contribute to neurodegeneration following ischemic injury. In the present study, we examined the possibility that the neuroprotective effects of the cyclooxygenase-2 inhibitor nimesulide would depend upon reduction of oxidative stress following cerebral ischemia. Gerbils were subjected to 5 min of transient global cerebral ischemia followed by 48 h of reperfusion and markers of oxidative stress were measured in hippocampus of gerbils receiving vehicle or nimesulide treatment at three different clinically relevant doses (3, 6 or 12 mg/kg). Compared with vehicle, nimesulide significantly (P<0.05) reduced hippocampal glutathione depletion and lipid peroxidation, as assessed by the levels of malondialdehyde (MDA), 4-hydroxy-alkenals (4-HDA) and lipid hydroperoxides levels, even when the treatment was delayed until 6 h after ischemia. Biochemical evidences of nimesulide neuroprotection were supported by histofluorescence findings using the novel marker of neuronal degeneration Fluoro-Jade B. Few Fluoro-Jade B positive cells were seen in CA1 region of hippocampus in ischemic animals treated with nimesulide compared with vehicle. These results suggest that nimesulide may protect neurons by attenuating oxidative stress and reperfusion injury following the ischemic insult with a wide therapeutic window of protection.

We investigated the relative contribution of COX-1 and/or COX-2 to oxidative damage, prostaglandin E2 (PGE2) production and hippocampal CA1 neuronal loss in a model of 5 min transient global cerebral ischemia in gerbils. Our results revealed a biphasic and significant increase in PGE2 levels after 2 and 24-48 h of reperfusion. The late increase in PGE2 levels (24 h) was more potently reduced by the highly selective COX-2 inhibitor rofecoxib (20 mg/kg) relative to the COX-1 inhibitor valeryl salicylate (20 mg/kg). The delayed rise in COX catalytic activity preceded the onset of histopathological changes in the CA1 subfield of the hippocampus. Post-ischemia treatment with rofecoxib (starting 6 h after restoration of blood flow) significantly reduced measures of oxidative damage (glutathione depletion and lipid peroxidation) seen at 48 h after the initial ischemic episode, indicating that the late increase in COX-2 activity is involved in the delayed occurrence of oxidative damage in the hippocampus after global ischemia. Interestingly, either selective inhibition of COX-2 with rofecoxib or inhibition of COX-1 with valeryl salicylate significantly increased the number of healthy neurons in the hippocampal CA1 sector even when the treatment began 6 h after ischemia. These results provide the first evidence that both COX isoforms are involved in the progression of neuronal damage following global cerebral ischemia, and have important implications for the potential therapeutic use of COX inhibitors in cerebral ischemia.

Several studies suggest that cyclooxygenase-2 contributes to the delayed progression of ischemic brain damage. In this study we examined whether the highly selective cyclooxygenase-2 inhibitor DFU reduces neuronal damage when administered several hours after 5 min of transient forebrain ischemia in gerbils. The extent of ischemic injury was assessed behaviorally by measuring the increases in locomotor activity and by histopathological evaluation of the extent of CA1 hippocampal pyramidal cell injury 7 days after ischemia. DFU treatment (10 mg/kg, p.o.) significantly reduced hippocampal neuronal damage even if the treatment is delayed until 12 h after ischemia. These results suggest that selective cyclooxygenase-2 inhibitors may be a valuable therapeutic strategy for ischemic brain injury.

Cyclooxygenase-2 is involved in the inflammatory component of the ischemic cascade, playing an important role in the delayed progression of the brain damage. The present study evaluated the pharmacological effects of the selective cyclooxygenase-2 inhibitor nimesulide on delayed neuronal death of hippocampal CA1 neurons following transient global cerebral ischemia in gerbils. Administration of therapeutically relevant doses of nimesulide (3, 6 and 12 mg/kg; i.p.) 30 min before ischemia and at 6, 12, 24, 48 and 72 h after ischemia significantly (P<0.01) reduced hippocampal neuronal damage. Treatment with a single dose of nimesulide given 30 min before ischemia also resulted in a significant increase in the number of healthy neurons in the hippocampal CA1 sector 7 days after ischemia. Of interest is the finding that nimesulide rescued CA1 pyramidal neurons from ischemic death even when treatment was delayed until 24 h after ischemia (34+/-9% protection). Neuroprotective effect of nimesulide is still evident 30 days after the ischemic episode, providing the first experimental evidence that cyclooxygenase-2 inhibitors confer a long-lasting neuroprotection. Oral administration of nimesulide was also able to significantly reduce brain damage, suggesting that protective effects are independent of the route of administration. The present study confirms the ability of cyclooxygenase-2 inhibitors to reduce brain damage induced by cerebral ischemia and indicates that nimesulide can provide protection when administered for up to 24 h post-ischemia.

La retencion de las lipoproteinas de baja densidad (LDL) por parte de los proteoglicanos arteriales constituye un evento molecular clave en el inicio y desarrollo de la aterosclerosis. Este proceso promueve la oxidacion de las LDL y con ello el desarrollo de una respuesta inflamatoria cronica que contribuye a la progresion de la enfermedad. En el Centro de Inmunologia Molecular se obtuvo un anticuerpo monoclonal quimerico, llamado chP3R99, que reconoce proteoglicanos sulfatados de la pared arterial y es capaz de inhibir la retencion y oxidacion de las LDL. Ademas, la inmunizacion el anticuerpo chP3R99 previene la formacion de lesiones ateroscleroticas mediante la induccion de anticuerpos autologos anti-proteoglicanos. Sin embargo, no se conocen en su totalidad los mecanismos moleculares de accion de este anticuerpo. Por lo que el presente trabajo tuvo como objetivo evaluar el efecto de la inmunizacion con el anticuerpo chP3R99 sobre el infiltrado de macrofagos, poblacion celular que desencadena la respuesta inflamatoria en el tejido vascular. Se llevo a cabo un esquema de inmunizacion preventiva, donde se administro una dosis de 100 µg s.c. del anticuerpo en conejos tratados posteriormente con 2 mL/kg i.v. de Lipofundin, un inductor de lesiones ateroscleroticas. Los resultados mostraron que el tratamiento con el anticuerpo inhibio el infiltrado de macrofagos y redujo significativamente (p<0,05) la expresion genica de la enzima oxido nitrico sintasa y la actividad de la enzima mieloperoxidasa. Estos resultados demuestran la potencialidad de la inmunizacion con el anticuerpo chP3R99 de atenuar la respuesta inflamatoria, factor clave en el desarrollo de la aterosclerosis.  Palabras clave: Aterosclerosis, macrofagos, inflamacion, anticuerpo monoclonal. Abstract The retention of low-density lipoproteins (LDL) by arterial proteoglycans represent a key molecular event in atherosclerosis development. This process promotes the oxidation of LDL and the development of a chronic inflammatory response, leading to atherosclerosis progression. At the Center of Molecular Immunology, it was obtained a chimeric monoclonal antibody, named chP3R99, which recognizes sulfated proteoglycans of the arterial wall, and able to inhibit the retention and oxidation of LDL. In addition, the immunization with this antibody prevents atherosclerotic lesions development by mean a generation of anti-proteoglycans autologous antibodies. Nonetheless, the molecular mechanisms of action of this antibody remain in part unknown. Thus, the present work was aimed to evaluate the effect of chP3R99 immunization on the macrophage infiltration, because of the key role of this cellular population during vascular inflammation. We carried out a preventive immunization where we administered a dose of 100 µg, s.c. in rabbits later treated with 2 mL/kg i.v. of Lipofundin, an inductor of atherosclerotic lesions. The results showed that the treatment with the chP3R99 antibody inhibited the macrophage infiltration, at the same time that reduced the gene expression of inducible nitric oxide synthase and myeloperoxidase activity. These results demonstrate the potential of chP3R99 immunization to reduce the inflammatory response, a key factor in atherosclerosis development. Keywords: Atherosclerosis, macrophages, inflammation, monoclonal antibody