Intranasal Liposomal Formulation of Spike Protein Adjuvanted with CpG Protects and Boosts Heterologous Immunity of hACE2 Transgenic Mice to SARS-CoV-2 Infection.

Mucosal vaccination appears to be suitable to protect against SARS-CoV-2 infection. In this study, we tested an intranasal mucosal vaccine candidate for COVID-19 that consisted of a cationic liposome containing a trimeric SARS-CoV-2 spike protein and CpG-ODNs, a Toll-like receptor 9 agonist, as an adjuvant. In vitro and in vivo experiments indicated the absence of toxicity following the intranasal administration of this vaccine formulation. First, we found that subcutaneous or intranasal vaccination protected hACE-2 transgenic mice from infection with the wild-type (Wuhan) SARS-CoV-2 strain, as shown by weight loss and mortality indicators. However, when compared with subcutaneous administration, the intranasal route was more effective in the pulmonary clearance of the virus and induced higher neutralizing antibodies and anti-S IgA titers. In addition, the intranasal vaccination afforded protection against gamma, delta, and omicron virus variants of concern. Furthermore, the intranasal vaccine formulation was superior to intramuscular vaccination with a recombinant, replication-deficient chimpanzee adenovirus vector encoding the SARS-CoV-2 spike glycoprotein (Oxford/AstraZeneca) in terms of virus lung clearance and production of neutralizing antibodies in serum and bronchial alveolar lavage (BAL). Finally, the intranasal liposomal formulation boosted heterologous immunity induced by previous intramuscular vaccination with the Oxford/AstraZeneca vaccine, which was more robust than homologous immunity.

Anhydroecgonine methyl ester (AEME), a cocaine pyrolysis product, impairs glutathione-related enzymes response and increases lipid peroxidation in the hippocampal cell culture.

Crack cocaine smokers inhale, alongside with cocaine, its pyrolysis product, anhydroecgonine methyl ester (AEME). We have previously described AEME neurotoxic effect and its additive effect when co-incubated with cocaine. Our aim was to evaluate, the effect of AEME, cocaine and AEME-cocaine combination on glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) activities after 3 and 6 h of exposure, periods previous to neuronal death. Lipid peroxidation was evaluated through malonaldehyde (MDA) levels at 3, 6, 24 and 48 h of exposure. All treated groups reduced neuronal viability after 24 h of exposure. AEME and cocaine decreased GPx, GR and GST activities after 3 and 6 h, with an increase in MDA levels after 48 h. AEME-cocaine combination decreased the enzymes activities after 3 and 6 h, showing an additive effect in MDA levels after 48 h. These data show that the glutathione-related enzymes imbalance caused by AEME, cocaine or AEME-cocaine combination exposure preceded neuronal death and lipid peroxidation. Moreover, the additive effect on lipid peroxidation observed with AEME-cocaine exposure after 48 h, suggest a higher neurotoxic effect after crack cocaine use when compared to cocaine alone.

Anhydroecgonine methyl ester (AEME), a cocaine pyrolysis product, impairs glutathione-related enzymes response and increases lipid peroxidation in the hippocampal cell culture

Crack cocaine smokers inhale, alongside with cocaine, its pyrolysis product, anhydroecgonine methyl ester (AEME). We have previously described AEME neurotoxic effect and its additive effect when co-incubated with cocaine. Our aim was to evaluate, the effect of AEME, cocaine and AEME-cocaine combination on glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) activities after 3 and 6h of exposure, periods previous to neuronal death. Lipid peroxidation was evaluated through malonaldehyde (MDA) levels at 3, 6, 24 and 48h of exposure. All treated groups reduced neuronal viability after 24h of exposure. AEME and cocaine decreased GPx, GR and GST activities after 3 and 6h, with an increase in MDA levels after 48h. AEME-cocaine combination decreased the enzymes activities after 3 and 6h, showing an additive effect in MDA levels after 48h. These data show that the glutathione-related enzymes imbalance caused by AEME, cocaine or AEME-cocaine combination exposure preceded neuronal death and lipid peroxidation. Moreover, the additive effect on lipid peroxidation observed with AEME-cocaine exposure after 48h, suggest a higher neurotoxic effect after crack cocaine use when compared to cocaine alone.

Anhydroecgonine methyl ester (AEME), a cocaine pyrolysis product, impairs glutathione-related enzymes response and increases lipid peroxidation in the hippocampal cell culture

Crack cocaine smokers inhale, alongside with cocaine, its pyrolysis product, anhydroecgonine methyl ester (AEME). We have previously described AEME neurotoxic effect and its additive effect when co-incubated with cocaine. Our aim was to evaluate, the effect of AEME, cocaine and AEME-cocaine combination on glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione S-transferase (GST) activities after 3 and 6h of exposure, periods previous to neuronal death. Lipid peroxidation was evaluated through malonaldehyde (MDA) levels at 3, 6, 24 and 48h of exposure. All treated groups reduced neuronal viability after 24h of exposure. AEME and cocaine decreased GPx, GR and GST activities after 3 and 6h, with an increase in MDA levels after 48h. AEME-cocaine combination decreased the enzymes activities after 3 and 6h, showing an additive effect in MDA levels after 48h. These data show that the glutathione-related enzymes imbalance caused by AEME, cocaine or AEME-cocaine combination exposure preceded neuronal death and lipid peroxidation. Moreover, the additive effect on lipid peroxidation observed with AEME-cocaine exposure after 48h, suggest a higher neurotoxic effect after crack cocaine use when compared to cocaine alone.

M1 and M3 muscarinic receptors may play a role in the neurotoxicity of anhydroecgonine methyl ester, a cocaine pyrolysis product.

The smoke of crack cocaine contains cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). AEME possesses greater neurotoxic potential than cocaine and an additive effect when they are combined. Since atropine prevented AEME-induced neurotoxicity, it has been suggested that its toxic effects may involve the muscarinic cholinergic receptors (mAChRs). Our aim is to understand the interaction between AEME and mAChRs and how it can lead to neuronal death. Using a rat primary hippocampal cell culture, AEME was shown to cause a concentration-dependent increase on both total [(3)H]inositol phosphate and intracellular calcium, and to induce DNA fragmentation after 24 hours of exposure, in line with the activation of caspase-3 previously shown. Additionally, we assessed AEME activity at rat mAChR subtypes 1-5 heterologously expressed in Chinese Hamster Ovary cells. l-[N-methyl-(3)H]scopolamine competition binding showed a preference of AEME for the M2 subtype; calcium mobilization tests revealed partial agonist effects at M1 and M3 and antagonist activity at the remaining subtypes. The selective M1 and M3 antagonists and the phospholipase C inhibitor, were able to prevent AEME-induced neurotoxicity, suggesting that the toxicity is due to the partial agonist effect at M1 and M3 mAChRs, leading to DNA fragmentation and neuronal death by apoptosis.

Neurotoxicity of anhydroecgonine methyl ester, a crack cocaine pyrolysis product.

Smoking crack cocaine involves the inhalation of cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). Although there is evidence that cocaine is neurotoxic, the neurotoxicity of AEME has never been evaluated. AEME seems to have cholinergic agonist properties in the cardiovascular system; however, there are no reports on its effects in the central nervous system. The aim of this study was to investigate the neurotoxicity of AEME and its possible cholinergic effects in rat primary hippocampal cell cultures that were exposed to different concentrations of AEME, cocaine, and a cocaine-AEME combination. We also evaluated the involvement of muscarinic cholinergic receptors in the neuronal death induced by these treatments using concomitant incubation of the cells with atropine. Neuronal injury was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The results of the viability assays showed that AEME is a neurotoxic agent that has greater neurotoxic potential than cocaine after 24 and 48 h of exposure. We also showed that incubation for 48 h with a combination of both compounds in equipotent concentrations had an additive neurotoxic effect. Although both substances decreased cell viability in the MTT assay, only cocaine increased LDH release. Caspase-3 activity was increased after 3 and 6 h of incubation with 1mM cocaine and after 6 h of 0.1 and 1.0mM AEME exposure. Atropine prevented the AEME-induced neurotoxicity, which suggests that muscarinic cholinergic receptors are involved in AEME's effects. In addition, binding experiments confirmed that AEME has an affinity for muscarinic cholinergic receptors. Nevertheless, atropine was not able to prevent the neurotoxicity produced by cocaine and the cocaine-AEME combination, suggesting that these treatments activated other neuronal death pathways. Our results suggest a higher risk for neurotoxicity after smoking crack cocaine than after cocaine use alone.

Peripheral oxidative stress biomarkers in mild cognitive impairment and Alzheimer's disease.

Oxidative stress has been associated with normal aging and Alzheimer's disease (AD). However, little is known about oxidative stress in mild cognitive impairment (MCI) patients who present a high risk for developing AD. The aim of this study was to investigate plasma production of the lipid peroxidation marker, malonaldehyde (MDA) and to determine, in erythrocytes, the enzymatic antioxidant activity of catalase, glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) in 33 individuals with MCI, 29 with mild probable AD and 26 healthy aged subjects. GR/GPx activity ratio was calculated to better assess antioxidant defenses. The relationship between oxidative stress and cognitive performance was also evaluated by the Mini Mental State Examination (MMSE). AD patients showed higher MDA levels than both MCI and healthy elderly subjects. MCI subjects also exhibited higher MDA levels compared to controls. Catalase and GPx activity were similar in MCI and healthy individuals but higher in AD. GR activity was lower in MCI and AD patients than in healthy aged subjects. Additionally, GR/GPx ratio was higher in healthy aged subjects, intermediate in MCI and lower in AD patients. No differences in GST activity were detected among the groups. MMSE was negatively associated with MDA levels (r = -0.31, p = 0.028) and positively correlated with GR/GPx ratio in AD patients (r = 0.68, p < 0.001). MDA levels were also negatively correlated to GR/GPx ratio (r = -0.31, p = 0.029) in the AD group. These results suggest that high lipid peroxidation and decreased antioxidant defenses may be present early in cognitive disorders.