ALZHEIMER'S DISEASE AND ITS RELATIONSHIP WITH THE MICROBIOTA-GUT-BRAIN AXIS.

BACKGROUND: Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease, characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. Several pathways enable bidirectional communication between the central nervous system (CNS), the intestine and its microbiota, constituting the microbiota-gut-brain axis. OBJECTIVE: Review the pathophysiology of AD, relate it to the microbiota-gut-brain axis and discuss the possibility of using probiotics in the treatment and/or prevention of this disease. METHODS: Search of articles from the PubMed database published in the last 5 years (2017 to 2022) structure the narrative review. RESULTS: The composition of the gut microbiota influences the CNS, resulting in changes in host behavior and may be related to the development of neurodegenerative diseases. Some metabolites produced by the intestinal microbiota, such as trimethylamine N-oxide (TMAO), may be involved in the pathogenesis of AD, while other compounds produced by the microbiota during the fermentation of food in the intestine, such as D-glutamate and fatty acids short chain, are beneficial in cognitive function. The consumption of live microorganisms beneficial to health, known as probiotics, has been tested in laboratory animals and humans to evaluate the effect on AD. CONCLUSION: Although there are few clinical trials evaluating the effect of probiotic consumption in humans with AD, the results to date indicate a beneficial contribution of the use of probiotics in this disease.

A doença de Alzheimer e sua relação com o eixo microbiota-intestino-cérebro / Alzheimer's disease and its relationship with the microbiota-gut-brain axis

ABSTRACT Background: Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disease, characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. Several pathways enable bidirectional communication between the central nervous system (CNS), the intestine and its microbiota, constituting the microbiota-gut-brain axis. Objective: Review the pathophysiology of AD, relate it to the microbiota-gut-brain axis and discuss the possibility of using probiotics in the treatment and/or prevention of this disease. Methods: Search of articles from the PubMed database published in the last 5 years (2017 to 2022) structure the narrative review. Results: The composition of the gut microbiota influences the CNS, resulting in changes in host behavior and may be related to the development of neurodegenerative diseases. Some metabolites produced by the intestinal microbiota, such as trimethylamine N-oxide (TMAO), may be involved in the pathogenesis of AD, while other compounds produced by the microbiota during the fermentation of food in the intestine, such as D-glutamate and fatty acids short chain, are beneficial in cognitive function. The consumption of live microorganisms beneficial to health, known as probiotics, has been tested in laboratory animals and humans to evaluate the effect on AD. Conclusion: Although there are few clinical trials evaluating the effect of probiotic consumption in humans with AD, the results to date indicate a beneficial contribution of the use of probiotics in this disease.

RESUMO Contexto: A doença de Alzheimer (DA) é uma doença neurodegenerativa progressiva e irreversível, caracterizada pelo acúmulo de placas amiloides e emaranhados neurofibrilares no cérebro. Diversas vias possibilitam uma comunicação bidirecional entre o sistema nervoso central (SNC), o intestino e sua microbiota, constituindo o eixo microbiota-intestino-cérebro. Objetivo Revisar a fisiopatogenia da DA, relacioná-la com o eixo microbiota-intestino-cérebro e discutir sobre a possibilidade do uso de probióticos no tratamento e/ou prevenção desta doença. Métodos: Busca de artigos da base de dados PubMed publicados nos últimos 5 anos (2017 a 2022) para estruturar a revisão narrativa. Resultados A composição da microbiota intestinal influencia o SNC, resultando em modificações no comportamento do hospedeiro e pode estar relacionada com o desenvolvimento de doenças neurodegenerativas. Alguns metabólitos produzidos pela microbiota intestinal, como o N-óxido de trimetilamina (TMAO), podem estar envolvidos na patogênese da DA, enquanto, outros compostos produzidos pela microbiota durante a fermentação de alimentos no intestino, como o D-glutamato e os ácidos graxos de cadeia curta, são profícuos na função cognitiva. O consumo de microrganismos vivos benéficos à saúde, os probióticos, tem sido testado em animais de laboratório e humanos para avaliação do efeito na DA. Conclusão Embora haja poucos ensaios clínicos que avaliem o efeito do consumo de probióticos em humanos com DA, os resultados até o momento indicam uma contribuição benéfica do uso de probióticos nesta doença.

Proinflammatory Action of a New Electronegative Low-Density Lipoprotein Epitope.

The electronegative low-density lipoprotein, LDL (-), is an endogenously modified LDL subfraction with cytotoxic and proinflammatory actions on endothelial cells, monocytes, and macrophages contributing to the progression of atherosclerosis. In this study, epitopes of LDL (-) were mapped using a phage display library of peptides and monoclonal antibodies reactive to this modified lipoprotein. Two different peptide libraries (X6 and CX8C for 6- and 8-amino acid-long peptides, respectively) were used in the mapping. Among all tested peptides, two circular peptides, P1A3 and P2C7, were selected based on their high affinities for the monoclonal antibodies. Small-angle X-ray scattering analysis confirmed their structures as circular rings. P1A3 or P2C7 were quickly internalized by bone marrow-derived murine macrophages as shown by confocal microscopy. P2C7 increased the expression of TNFα, IL-1 ß and iNOS as well as the secretion of TNFα, CCL2, and nitric oxide by murine macrophages, similar to the responses induced by LDL (-), although less intense. In contrast, P1A3 did not show pro-inflammatory effects. We identified a mimetic epitope associated with LDL (-), the P2C7 circular peptide, that activates macrophages. Our data suggest that this conformational epitope represents an important danger-associated molecular pattern of LDL (-) that triggers proinflammatory responses.

GFP-SCFV: expression and possible applications as a tool for experimental investigations of atherosclerosis.

Experimental studies on atherosclerosis are crucial for investigating its pathophysiology, defining new therapeutic targets, and developing new drugs and diagnostic tools. Thus, many imaging markers have been developed and introduced in experimental studies. The main advantage of these new tools is that they allow the noninvasive diagnosis of atherosclerotic vascular disease. Here, we describe the cloning, expression, purification, and stabilization of a chimeric protein specifically designed to probe cells and tissues for the presence of LDL(-), a relevant marker of atherosclerosis. The DNA sequence that encodes the anti-LDL(-) scFv, previously obtained from a hybridoma secreting an anti-LDL(-) monoclonal antibody, was inserted into the bacterial vector pET-28a(+) in tandem with a DNA sequence encoding GFP. The recombinant protein was expressed in high yields in E. coli as inclusion bodies. The applicability of GFP-scFv was assessed by ELISA, which determined its affinity for LDL(-) and confocal microscopy, that showed macrophage uptake of the protein along with LDL(-). In conclusion, our data suggest that the anti-LDL(-) GFP-scFv chimeric protein could be useful in studies on atherogenesis as well as for developing diagnostic tools for atherosclerosis.

Reduced plasma zinc levels, lipid peroxidation, and inflammation biomarkers levels in hemodialysis patients: implications to cardiovascular mortality.

Despite the fact that low plasma zinc (Zn) levels play important roles in the oxidative stress, the relationships between lipid peroxidation and inflammation biomarkers with low plasma Zn levels have not been investigated in chronic kidney disease (CKD) patients. The aim of this study was to evaluate the Zn plasma levels, electronegative LDL [LDL(-)] levels, and inflammation markers as predictors of cardiovascular (CV) mortality in hemodialysis (HD) patients. Forty-five HD patients (28 men, 54.2 ± 12.7 years, 62.2 ± 51.4 months on dialysis and BMI 24.3 ± 4.1 kg/m(2)) were studied and compared to 20 healthy individuals (9 men, 51.6 ± 15.6 years, BMI 25.2 ± 3.9 kg/m(2)) and followed for 24 months to investigate the risks for CV mortality. LDL(-) levels were measured by ELISA, plasma Zn levels by atomic absorption spectrophotometry, C-reactive protein (CRP) level by immunoturbidimetric method, and tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and plasminogen activator inhibitor-1 (PAI-1) levels by a multiplex assay kit. HD patients presented low plasma Zn levels (54.9 ± 16.1 µg/dL) and high-LDL(-) (0.18 ± 0.12 U/L) and TNF-α (5.5 ± 2.2 pg/mL) levels when compared to healthy subjects (78.8 ± 9.4µ g/dL, 0.10 ± 0.08U/L, 2.4 ± 1.1 pg/mL, respectively, p < 0.05). Zn plasma levels were negatively correlated to TNF-α (r = -0.49; p = 0.0001) and LDL(-) (r = -0.33; p = 0.008). During the 2 years, 24.4% of the patients died, all due to CV disease. Analysis by the Cox model showed that high CRP, TNF-α, IL-6 levels, and long duration of HD were significant predictors of mortality. In conclusion, reduced Zn levels were associated with lipid peroxidation and inflammation, and we confirm here in a Brazilian cohort of HD patients that inflammation markers are strong predictors of CV death.

Lipid peroxidation is associated with the severity of periodontal disease and local inflammatory markers in patients with type 2 diabetes.

CONTEXT: Periodontitis is the most common lytic disease of bone and is recognized as a common complication of diabetes. Lipid peroxidation (LPO) is increased in diabetes and may be related to modulation of the inflammatory response. LPO levels in patients with diabetes and periodontal disease have not been evaluated. OBJECTIVE: The aim of this study was to evaluate the levels of LPO and its correlation with periodontal status and inflammatory cytokines in type 2 diabetic and nondiabetic patients. DESIGN AND SETTING: This is a cross-sectional study involving Brazilian patients recruited at the State University of São Paulo. PATIENTS: The sample comprised 120 patients divided into four groups based upon diabetic and dyslipidemic status: poorly controlled diabetics with dyslipidemia, well-controlled diabetics with dyslipidemia, normoglycemic individuals with dyslipidemia, and healthy individuals. MAIN OUTCOME MEASURES: Blood analyses were carried out for fasting plasma glucose, glycated hemoglobin, and lipid profile. Periodontal examinations were performed, and gingival crevicular fluid was collected. LPO levels were evaluated by measuring oxidized low-density lipoprotein (ELISA) and malondialdehyde (HPLC). Cytokines were evaluated by the multiplex bead technique. RESULTS: LPO evaluated by malondialdehyde in plasma and gingival crevicular fluid was significantly increased in diabetes groups. Significant correlations between LPO markers and periodontal parameters indicate a direct relationship between these levels and the severity of inflammation and secretion of inflammatory cytokines, particularly in diabetic patients. CONCLUSION: These findings suggest an important association for LPO with the severity of the local inflammatory response to bacteria and the susceptibility to periodontal disease in diabetic patients.

Development of immunoassays for anti-electronegative LDL autoantibodies and immune complexes.

BACKGROUND: Electronegative low-density lipoprotein (LDL-) promotes atherosclerosis through inflammatory and immunologic mechanisms that lead to the production of anti-LDL(-) autoantibodies and to the subsequent formation of immune complexes (IC) and macrophage foam cells. We described the development and validation of an ELISA for the quantification of free anti-LDL(-) autoantibodies and an ELISA for the quantification of IC consisting of LDL(-)-bound IgG in human plasma. METHODS: LDL(-) purified from human plasma, and anti-LDL(-) monoclonal antibody Fab fragments were adsorbed onto ELISA plates to capture anti-LDL(-) autoantibodies and IC-LDL(-), respectively. The performance characteristics of both ELISAs, including the limits of detection and quantification, accuracy and inter- and intra-assay precision were evaluated. Linearity, interference and stability tests were also performed. RESULTS: The calibration range of the anti-LDL(-) assay was 0.004-0.125 mU/l and plasma demonstrated a dilutional linearity when diluted 1:100, 1:200, 1:400 and 1:800. The calibration range of the IC-LDL(-) assay was 0.06-4 U/l, and plasma demonstrated a dilutional linearity when diluted 1:12.5, 1:25, 1:50 and 1:100. Both ELISAs showed intra- and inter-assay precision and recovery within the required limits for immunoassays. CONCLUSION: These ELISAs can be used in clinical studies and for the biochemical investigation of atherosclerosis. In addition, they will enable the comprehensive evaluation of the importance of bound or free autoantibodies against LDL(-) in this disease.

Increased electronegative LDL and decreased antibodies against electronegative LDL levels correlate with inflammatory markers and adhesion molecules in hemodialysed patients.

BACKGROUND: Chronic Kidney Disease (CKD) patients present high levels of electronegative LDL (LDL-) that can modulate the expression of molecules involved in inflammation and it is closely linked to atherosclerosis. We investigated the association between LDL(-) and inflammatory markers in patients undergoing hemodialysis (HD). METHODS: Forty-seven HD patients from a private clinic in Rio de Janeiro, Brazil were studied and compared with 20 age matched healthy individuals. Serum LDL(-) and anti-LDL(-) autoantibody levels were measured by ELISA; TNF-α, IL-6, VCAM-1 and ICAM-1 were determined by a multiplex assay kit. RESULTS: HD patients presented higher IL-6 and TNF-α concentrations (4.1 ± 1.6 and 5.5 ± 2.1 pg/ml, respectively) than healthy subjects (2.6 ± 0.2 and 2.4 ± 1.1 pg/ml, respectively) (p=0.0001). In addition, they presented higher VCAM-1 and ICAM-1 levels and, LDL(-) concentrations were also increased (0.18 ± 0.12 U/l) when compared to healthy individuals (0.10 ± 0.08 U/l) (p<0.02). In contrast, the anti-LDL(-) autoantibody levels were lower in HD patients (0.02 ± 0.01 mg/l) than in healthy subjects (0.05 ± 0.03 mg/l) (p<0.001). There was a positive correlation between LDL(-) and IL-6 (r=0.25, p=0.004) and ICAM-1 (r=0.36; p=0.003). There was also a negative correlation between anti-LDL(-) autoantibodies and TNF-α (r=-0.37; p=0.003) and VCAM-1 (r=-0.50; p=0.0001). CONCLUSIONS: The association between LDL(-) and inflammation and the lower levels of anti-LDL(-) autoantibodies are important risk factors related to atherosclerosis in CKD.