Unraveling the Mechanisms of Ch-SeNP Cytotoxicity against Cancer Cells: Insights from Targeted and Untargeted Metabolomics.

Although chitosan-stabilized selenium nanoparticles (Ch-SeNPs) have emerged as a promising chemical form of selenium for anticancer purposes, gathering more profound knowledge related to molecular dysfunctions contributes significantly to the promotion of their evolution as a chemotherapeutic drug. In this sense, metabolites are the end products in the flow of gene expression and, thus, the most sensitive to changes in the physiological state of a biological system. Therefore, metabolomics provides a functional readout of the biochemical activity and cell state. In the present study, we evaluated alterations in the metabolomes of HepG2 cells after the exposure to Ch-SeNPs to elucidate the biomolecular mechanisms involved in their therapeutic effect. A targeted metabolomic approach was conducted to evaluate the levels of four of the main energy-related metabolites (adenosine triphosphate (ATP); adenosine diphosphate (ADP); nicotinamide adenine dinucleotide (NAD+); and 1,4-dihydronicotinamide adenine dinucleotide (NADH)), revealing alterations as a result of exposure to Ch-SeNPs related to a shortage in the energy supply system in the cell. In addition, an untargeted metabolomic experiment was performed, which allowed for the study of alterations in the global metabolic profile as a consequence of Ch-SeNP exposure. The results indicate that the TCA cycle and glycolytic pathways were impaired, while alternative pathways such as glutaminolysis and cysteine metabolism were upregulated. Additionally, increased fructose levels suggested the induction of hypoxia-like conditions. These findings highlight the potential of Ch-SeNPs to disrupt cancer cell metabolism and provide insights into the mechanisms underlying their antitumor effects.

Short-chain fatty acids during pregnancy in multiple sclerosis: A prospective cohort study.

BACKGROUND AND PURPOSE: Short-chain fatty acids (SCFAs) can have pro- or anti-inflammatory properties, but their relationship with multiple sclerosis (MS) relapses during pregnancy remains unknown. This study aimed to explore SCFA profiles in MS patients during pregnancy and to assess their association with the appearance of relapses during pregnancy and postpartum. METHODS: We prospectively included 53 pregnant MS patients and 21 healthy control women. Patients were evaluated during pregnancy and puerperium. SCFAs were measured by liquid chromatography-mass spectrometry. RESULTS: Sixteen patients (32%) had relapses during pregnancy or puerperium, and 37 (68%) did not. All MS patients showed significant increases in acetate levels during pregnancy and the postpartum period compared to non-MS women. However, propionate and butyrate values were associated with disease activity. Their values were higher in nonrelapsing patients and remained similar to the control group in relapsing patients. The variable that best identified active patients was the propionate/acetate ratio. Ratios of <0.36 during the first trimester were associated with higher inflammatory activity (odds ratio = 165, 95% confidence interval = 10.2-239.4, p < 0.01). Most nonrelapsing patients showed values of >0.36, which were similar to those in healthy pregnant women. CONCLUSIONS: Low propionate/acetate ratio values during the first trimester of gestation identified MS patients at risk of relapses during pregnancy and the postpartum period.

Transcriptome Analysis Identifies Novel Mechanisms Associated with the Antitumor Effect of Chitosan-Stabilized Selenium Nanoparticles.

Selenium nanoparticles (SeNPs) have been receiving special attention in recent years due to their antioxidant capacity and antitumor properties. However, the mechanisms associated with these properties remain to be elucidated. For this reason, a global transcriptome analysis has been designed in this work and it was carried out using human hepatocarcinoma cells and chitosan-stabilized SeNPs (Ch-SeNPs) to identify new targets and pathways related to the antitumor mechanisms associated with Ch-SeNPs. The results obtained confirm the alteration of the cell cycle and the effect of Ch-SeNPs on different tumor suppressors and other molecules involved in key mechanisms related to cancer progression. Furthermore, we demonstrated the antioxidant properties of these nanoparticles and their capacity to induce senescence, which was further confirmed through the measurement of ß-galactosidase activity.

Herpesvirus Antibodies, Vitamin D and Short-Chain Fatty Acids: Their Correlation with Cell Subsets in Multiple Sclerosis Patients and Healthy Controls.

Although the etiology of multiple sclerosis (MS) is still unknown, it is commonly accepted that environmental factors could contribute to the disease. The objective of this study was to analyze the humoral response to Epstein-Barr virus, human herpesvirus 6A/B and cytomegalovirus, and the levels of 25-hydroxyvitamin D (25(OH)D) and the three main short-chain fatty acids (SCFA), propionate (PA), butyrate (BA) and acetate (AA), in MS patients and healthy controls (HC) to understand how they could contribute to the pathogenesis of the disease. With this purpose, we analyzed the correlations among them and with different clinical variables and a wide panel of cell subsets. We found statistically significant differences for most of the environmental factors analyzed when we compared MS patients and HC, supporting their possible involvement in the disease. The strongest correlations with the clinical variables and the cell subsets analyzed were found for 25(OH)D and SCFAs levels. A correlation was also found between 25(OH)D and PA/AA ratio, and the interaction between these factors negatively correlated with interleukin 17 (IL-17)-producing CD4+ and CD8+ T cells in untreated MS patients. Therapies that simultaneously increase vitamin D levels and modify the proportion of SCFA could be evaluated in the future.

Acetate correlates with disability and immune response in multiple sclerosis.

BACKGROUND: Gut microbiota has been related to multiple sclerosis (MS) etiopathogenesis. Short-chain fatty acids (SCFA) are compounds derived from microbial metabolism that have a role in gut-brain axis. OBJECTIVES: To analyse SCFA levels in plasma of MS patients and healthy donors (HD), and the possible link between these levels and both clinical data and immune cell populations. METHODS: Ninety-five MS patients and 54 HD were recruited. Patients were selected according to their score in the Expanded Disability Status Scale (EDSS) (49 EDSS ≤ 1.5, 46 EDSS ≥ 5.0). SCFA were studied in plasma samples by liquid chromatography-mass spectrometry. Peripheral blood mononuclear cells were studied by flow cytometry. Gender, age, treatments, EDSS and Multiple Sclerosis Severity Score (MSSS) were evaluated at the recruitment. RESULTS: Plasma acetate levels were higher in patients than in HD (p = 0.003). Patients with EDSS ≥ 5.0 had higher acetate levels than those with EDSS≤ 1.5 (p = 0.029), and HD (p = 2.97e-4). Acetate levels correlated with EDSS (r = 0.387; p = 1.08e-4) and MSSS (r = 0.265; p = 0.011). In untreated MS patients, acetate levels correlated inversely with CD4+ naïve T cells (r =  - 0.550, p = 0.001) and directly with CD8+ IL-17+ cells (r = 0.557; p = 0.001). CONCLUSIONS: Plasma acetate levels are higher in MS patients than in HD. In MS there exists a correlation between plasma acetate levels, EDSS and increased IL-17+ T cells. Future studies will elucidate the role of SCFA in the disease.

How oral probiotics affect the severity of an experimental model of progressive multiple sclerosis? Bringing commensal bacteria into the neurodegenerative process.

A growing number of studies support that the bidirectional interactions between the gut microbiota, the immune system and the CNS are relevant for the pathophysiology of MS. Several studies have reported alterations in the gut microbiome of MS patients. In addition, a variety of studies in animal models of MS have suggested that specific members of the gut commensal microbiota can exacerbate or ameliorate neuroinflammation. Probiotics represent oral nontoxic immunomodulatory agents that would exert benefits when using in combination with current MS therapy. Here we investigate the effect of Vivomixx on the gut microbiome and central and peripheral immune responses in a murine model of primary progressive MS. Vivomixx administration was associated with increased abundance of many taxa such as Bacteroidetes, Actinobacteria, Tenericutes and TM7. This was accompanied by a clear improvement of the motor disability of Theiler's virus infected mice; in the CNS Vivomixx reduced microgliosis, astrogliosis and leukocyte infiltration. Notably, the presence of Breg cells (CD19+CD5+CD1dhigh) in the CNS was enhanced by Vivomixx, and while spinal cord gene expression of IL-1ß and IL-6 was diminished, the probiotic promoted IL-10 gene expression. One of the most significant findings was the increased plasma levels of butyrate and acetate levels in TMEV-mice that received Vivomixx. Peripheral immunological changes were subtle but interestingly, the probiotic restricted IL-17 production by Th17-polarized CD4+ T-cells purified from the mesenteric lymph nodes of Theiler's virus infected mice. Our data reinforce the beneficial effects of oral probiotics that would be coadjuvant treatments to current MS therapies.

Antimycobacterial Effect of Selenium Nanoparticles on Mycobacterium tuberculosis.

Tuberculosis (TB) remains the leading cause of death from a single infection agent worldwide. In recent years, the occurrence of TB cases caused by drug-resistant strains has spread, and is expected to continue to grow. Therefore, the development of new alternative treatments to the use of antibiotics is highly important. In that sense, nanotechnology can play a very relevant role, due to the unique characteristics of nanoparticles. In fact, different types of nanoparticles have already been evaluated both as potential bactericides and as efficient drug delivery vehicles. In this work, the use of selenium nanoparticles (SeNPs) has been evaluated to inhibit the growth of two types of mycobacteria: Mycobacterium smegmatis (Msm) and Mycobacterium tuberculosis (Mtb). The results showed that SeNPs are able to inhibit the growth of both types of mycobacteria by damaging their cell envelope integrity. These results open a new opportunity for the use of this type of nanoparticles as antimycobacterial agents by themselves, or for the development of novel nanosystems that combine the action of these nanoparticles with other drugs.

Strategies for Membrane Protein Analysis by Mass Spectrometry.

Membrane proteins are of utmost importance in different cellular processes including: cell signaling, substrate transport, homeostasis control, immune surveillance, etc. In addition, they represent between 60% and 70% of the therapeutic targets currently used. Therefore, the identification and characterization of these proteins is crucial in many fields of research. Although proteomics has undergone an extraordinary advance in recent years thanks to the development of mass spectrometry, the methods used for the identification and quantification of soluble proteins generally fail to be used for membrane proteins, mainly due to their hydrophobic character.In this chapter, we revised the different alternatives, modifications and improvements that have been developed over the years with the aim of adapting the methods used in proteomics to the particular study of membrane proteins, thus allowing to increase the number of membrane proteins identified, as well as their coverage.

Effects of chitosan-stabilized selenium nanoparticles on cell proliferation, apoptosis and cell cycle pattern in HepG2 cells: comparison with other selenospecies.

Selenium is an essential element that plays an important role in many biological functions. Many studies have reported the potential beneficial effects of Se intake for cancer therapy and prevention, which are not only dose-dependent but also closely related to the properties of specific selenospecies. Selenium nanoparticles are considered a novel selenium compound with excellent antioxidant properties; however, little is known about the properties of selenium nanoparticles in comparison to other well-studied selenospecies. Here, we combined different independent bioanalytical approaches to carry out a comparison between the effects of selenium nanoparticles and other selenocompounds (inorganic and organic selenospecies) using an in-vitro model. The bioanalytical characterization of different parameters such as cell proliferation, apoptosis and cell cycle pattern on HepG2 cells has shown the unique properties of this relatively novel compound that support and complete prior evidences for future applications as chemotherapeutic agent.

Characterization of protective extracellular membrane-derived vesicles produced by Streptococcus pneumoniae.

Extracellular vesicles are produced by many pathogenic microorganisms and have varied functions that include secretion and release of microbial factors, which contribute to virulence. Very little is known about vesicle production by Gram-positive bacteria, as well as their biogenesis and release mechanisms. In this work, we demonstrate the active production of vesicles by Streptococcus pneumoniae from the plasma membrane, rather than being a product from cell lysis. We biochemically characterized them by proteomics and fatty acid analysis, showing that these vesicles and the plasma membrane resemble in essential aspects, but have some differences: vesicles are more enriched in lipoproteins and short-chain fatty acids. We also demonstrate that these vesicles act as carriers of surface proteins and virulence factors. They are also highly immunoreactive against human sera and induce immune responses that protect against infection. Overall, this work provides insights into the biology of this important Gram-positive human pathogen and the role of extracellular vesicles in clinical applications. BIOLOGICAL SIGNIFICANCE: Pneumococcus is one of the leading causes of bacterial pneumonia worldwide in children and the elderly, being responsible for high morbidity and mortality rates in developing countries. The augment of pneumococcal disease in developed countries has raised major public health concern, since the difficulties to treat these infections due to increasing antibiotic resistance. Vaccination is still the best way to combat pneumococcal infections. One of the mechanisms that bacterial pathogens use to combat the defense responses of invaded hosts is the production and release of extracellular vesicles derived from the outer surface. Little is known about this phenomenon in Gram-positives. We show that pneumococcus produces membrane-derived vesicles particularly enriched in lipoproteins. We also show the utility of pneumococcal vesicles as a new type of vaccine, as they induce protection in immunized mice against infection with a virulent strain. This work will contribute to understand the role of these structures in important biological processes such as host-pathogen interactions and prevention of human disease.

Airways infection with virulent Mycobacterium tuberculosis delays the influx of dendritic cells and the expression of costimulatory molecules in mediastinal lymph nodes.

Despite tuberculosis resurgence and extensive dendritic cell (DC) research, there are no in vivo studies evaluating DC within regional lymphoid tissue during airways infection with virulent Mycobacterium tuberculosis (Mtb) H37Rv. Using DC-specific antibodies, immunocytochemistry, flow cytometry and Ziehl-Neelsen (ZN) for bacilli staining, we searched for Mtb and DC changes within mediastinal lymph nodes, after intratracheal (ITT) inoculation of virulent Mtb. ZN and immunocytochemistry in frozen and paraffin sections of mediastinal lymph nodes identified Mtb until day 14 after ITT inoculation, associated with CD11c(+) and Dec205(+) DC. Analysing CD11c, MHC-CII, and Dec205 combinations by flow cytometry in MLN suspensions revealed that CD11c(+)/MHC-CII(+) and CD11c(+)/Dec205(+) DC did not increase until day 14, peaked on day 21, and sharply declined by day 28. No changes were seen in control, saline-inoculated animals. The costimulatory molecules evaluated in CD11c(+) DCs followed a similar trend; the CD80 increase was negligible, slightly surpassed by CD40. CD86 increased earlier and the three markers peaked at day 21, declining by day 28. While antigen-specific proliferation was not evident for MLN CD4(+) T cells at 2 weeks postinfection, delayed-type hypersensitivity responses upon ITT inoculation revealed that, as early as day 3 and 7, both the priming and peripheral systemic immune responses were clearly established, persisting until days 14-21. While airways infection with virulent Mtb triggers an early, systemic peripheral response maintained for three weeks, this seems dissociated from regional events within mediastinal lymph nodes, such as antigen-specific T-cell reactivity and a delay in the influx and local activation of DC.

In situ analysis of lung antigen-presenting cells during murine pulmonary infection with virulent Mycobacterium tuberculosis.

Scarce information exists about the role of lung antigen-presenting cells (APCs) in vivo during pulmonary tuberculosis. As APCs activate cellular immunity, following intratracheal inoculation with virulent Mycobacterium tuberculosis, we assessed in situ lung APC recruitment, distribution, granuloma involvement, morphology and mycobacterial burden by using MHC-CII, CD14, scavenger receptor class A (SRA), the murine dendritic cell (DC)-restricted marker CD11c and Ziehl-Neelsen staining. CD11c(+) DC and CD14(+) cell recruitment into lungs appeared by day 14, continuing until day 60. MHC-CII(+) cells increased since day 7, persisting until day 60. Thus, virulent mycobacteria delays (14-21 days) lung APC recruitment compared to model antigens and nonvirulent bacilli (24-48 h). Regarding granuloma constitution, highly bacillary CD14(+) and SRA(+) cells were centrally located. MHC-CII(+) cells were more peripheral, with less mycobacteria. CD11c(+) cells were heterogeneously distributed within granulomas, with scarce bacilli. When labelling lung suspensions for MHC-CII and classifying cells as macrophages or DC, then staining for Ziehl-Neelsen, a remarkable segregation was found regarding bacillary burden. Most macrophage-like cells contained numerous bacilli, while DC had no or scarce mycobacteria. This implies differential APC contributions in situ during pulmonary tuberculosis regarding mycobacterial uptake, granuloma involvement and perhaps bacillary growth.

Leiomiosarcoma asociado con el virus de Epstein-Barr en un adulto con trasplante renal / Leiomyosarcoma associated with epstein-barr virus in an adult with renal transplant

Recientemente se informó la asociación del virus de Epstein-Barr (VEB) y los tumores de músculo liso, principalmente en niños inmunosuprimidos; pero el papel que juega en la patogenia de estos tumores, no se ha esclarecido. Informamos un caso en que establecimos la presencia de VEB y leiomiosarcoma en un hombre de 28 años con insuficiencia renal crónica terminal que en 1994, recibió un trasplante renal. En 1996 ingresó con lesiones nodulares en ambas bases pulmonares, hígado, bazo, car anterior del muslo izquierdo y ganglios retroperitoneales; un año después falleció. En las biopsias de muslo e hígado se observó leiomiosarcoma. Las reacciones de inmunoperoxidasa fueron positivas para vimentina y para actina de músculo liso. La hibridación in situ fue positiva para antígenos nucleares del VEB (EBNA-2) en células neoplásicas. Este caso corresponde al primer sarcoma observado en pacientes trasplantados en nuestra institución como un caso poco frecuente de leiomiosarcoma asociado a VEB en adulto

A cholangiolocellular carcinoma of the liver: ultrastructural and immunohistochemical characterization / Un carcinoma colangiolocelular hepático, caracterización ultraestructural e inmunohistoquímica

Se estudió mediante microscopía de luz, electrónica e inunohistoquímica un caso de colangiolocarcinoma en un hombre de 68 años de edad. El tumor se encontraba constituido por células neoplásicas formando estructuras parecidas a ductos. El estudio inmunohistoquímico mostró positividad para células ductales (citoqueratina 7) y hepatocitos (fibrinógeno) en las células neoplásicas. El estudio ultraestructural mostró células con características de células epiteliales de conducto y hepatocitos. De estos hallazgos se concluye que los colangiolocarcinomas puede derivar de una célula transicional común, probablemente similar a las células ovales observadas en los modelos de carcinogénesis experimental en hígado de ratas

Rapeseed diet and hepatocyte hypertrophy: an experimental morphometric study

En un estudio previo se sugirió que la dieta suplementada con semilla de nabo evita el desarrollo de cirrosis experimental en la rata. En el presente trabajo se demostró por morfometría que la semilla de nabo produce hipertrofia de los hepatocitos. Este crecimiento, a juzgar por los hallazgos con microscopía electrónica, dependió fundamentalmente de la superficie citoplásmica, y en menor grado, del núcleo del hepatocito. La combinación de semilla de nabo con tetracloruro de carbono (CC14) produjo igualmente hipertrofia; sin embargo, aquélla no modificó el cuadro histológico de cirrosis inducida por CC14. El hecho de que la hipertrofia provocada por la semilla de nabo sea básicamente a expensas de organelos membranosos sustenta la idea de que algún ingrediente de ésta modifica la actividad de síntesis o degradación proteínica del hepatocito.