Macroencapsulation of Limosilactobacillus reuteri DSPV002C as nutritional supplement for piglets: Storage stability and survival in gastrointestinal conditions.

The aim of this study was to evaluate the protective effect of the encapsulation of Limosilactobacillus reuteri DSPV002C in macrocapsules made from industrial materials during production, storage and under simulated gastrointestinal conditions in vitro and in vivo. The production of macrocapsules involved the evaluation of different wall materials (matrix), namely, gelatin and pregelatinized starch, different inoculums, matrix ratios, and diverse cryoprotectants (whey permeate and maltodextrin). The different macrocapsules were arranged in molds of similar size to pig pelleted food and lyophilized. Then, the viability of the macrocapsules was assessed over time during storage at different temperatures (freezing, refrigeration and room temperature) and atmospheres (vacuum and non-vaccum). The macrocapsules with 10% w/v gelatin+5% w/v pregelatinized starch, permeated (10%, w/v), with a 9:1 inoculum:matrix ratio (GS7.5P9), stored under freezing conditions and vacuum, exhibited the highest viability of L. reuteri DSPV002C (9.3 log CFU/cap until 210 d). Under simulated gastrointestinal conditions, the encapsulated inoculum showed less viability loss (0.58±0.09 log CFU/ml, 26.53%), compared to the free culture (1.56±0.16 log CFU/ml, 2.85%). Finally, by administering GS7.5P9 to pigs, the tolerance of the bacteria to the gastrointestinal environment was verified, with viable counts equal to or greater than 3.72 log CFU/g of fecal matter throughout the trial. In this study, a high-density carrier probiotic macrocapsule of L. reuteri DSPV002C was obtained, which displayed a long shelf life, a suitable shape to be included in pig feed and an adequate survival of viable cells at the site of action.

PEGylation of Chrysin Improves Its Water Solubility while Preserving the In Vitro Biological Activity.

Chrysin is a natural flavonoid that despite having numerous biological properties, its therapeutic value is limited due to its very low solubility in aqueous media. In this work, chrysin was conjugated with methoxypolyethylene glycols (mPEGs) of different molecular weights (350, 500, 750, and 2000 g/mol), affording PEGylated chrysins with high yields and excellent purities. In all cases, an increase in the water solubility of the conjugates was observed, which was highest when 500 g/mol of mPEG was used in the PEGylation reaction. Furthermore, in aqueous solution, PEGylated chrysins formed aggregates of ellipsoid shape. Electrochemical studies showed that the redox properties were conserved after PEGylation. While in vitro antibacterial and antifungal studies probed that the intrinsic activity was conserved, in vitro antitumor activities against HepG2 (liver carcinoma cells) and PC3 (prostate cancer cell) showed that PEGylated chrysins retained the cytotoxic activity and the ability of induction of apoptosis for the evaluated human cancer cells.

Extraction of phenolic compounds from the shells of pecan nuts with cytotoxic activity through apoptosis against the colon cancer cell line HT-29.

The water extraction of phenolic compounds from two varieties ("Mahan" and "Marameck") of pecan nutshells (Carya illinoinensis) without and with sonication, varying the solvent/solid ratio (S), the pH, and the refluxing time (t), was studied. Additionally, the in vitro cytotoxicity and the determination of the cell death mechanism of the extracts against the colon cancer cell line HT-29 were investigated. The content of total phenolic compounds (TPC) of "Marameck" nutshells resulted higher than for the "Mahan" variety, and the pH increase resulted in higher TPC contents for both cultivars. The optimized conditions for TPC extraction without and with sonication resulted: S = 33 ml/g, pH = 12, and t = 9.6 min, and yielded ≈ 70 and 90 mg/g of TPC for "Mahan" and "Marameck" nutshells, respectively. The optimized extracts of pecan nutshells without sonication from both cultivars presented similar cytotoxicity against HT-29 colon cancer cells (IC50  ≈ 50 µg/ml), higher than for sonicated extracts (IC50 ≈ 88 and 138 µg/ml for "Mahan" and "Marameck," respectively). Cell death through apoptosis was the main mechanism of cell death induced by the nutshell extracts. PRACTICAL APPLICATION: The extraction of phenolic compounds (TPC) from the residues of two varieties of pecan nutshells ("Mahan" and "Marameck") was studied. An optimal combination of variables within the pH range that minimizes the solvent-to-solid ratio (S) and the time of refluxing (t), saving at the same time, water and energy, was set up. The phenolic compound extracts obtained from the residues of the pecan nuts exhibit cytotoxic effects against colon cancer cells and could be of interest as an alternative treatment of different types of cancer. Additionally, these extracts may be of importance to the food industry as they can be used as antioxidant agents in food formulation. Also, the high levels of anthocyanidins obtained from the pecan nut extracts after proanthocyanidins' strong acid hydrolysis can be purified and employed as natural red dyes.

Genistein loaded in self-assembled bovine serum albumin nanovehicles and their effects on mouse mammary adenocarcinoma cells.

Antitumor activity of plant-derived flavonoids has been researched during recent decades. Among them, genistein (Gen) stands out for showing cytotoxic activity against breast cancer cells. However, its low water solubility, limited bioavailability, and fast metabolism hinder its administration in chemopreventive therapies. To overcome these obstacles, bovine serum albumin nanovehicles (BSAnp) were obtained by a heat-induced self-assembly process at 70 °C and two aqueous medium pH (9.0 and 11.0) and assayed for the Gen loading. Thus, in this work, Gen loading in BSAnp was studied by spectroscopic techniques and compared with the one obtained for its stereoisomer, chrysin (Chrys). Results revealed that Gen binds to BSAnp via fluorescence quenching mechanism forming inclusion complexes. Compared to Chrys, Gen binding to BSAnp involved more molecules, whereas the association constant was similar for both flavonoids. In general, flavonoid loading in protein systems was strongly affected by the combined effects of BSA conformational state (native vs. aggregated), nanovehicle size, and flavonoid chemical structure. To evaluate the antitumor properties freeze-dried powders were obtained, and they were assayed in vitro after reconstitution by XTT technique and Annexin V-FITC flow cytometry against mouse mammary adenocarcinoma F3II cells. Gen-loaded BSAnp produced a significant decrease in cell viability compared with unloaded BSAnp systems, being the highest cytotoxic effects found for the lowest sized Gen-loaded BSAnp. The leading cytotoxicity mechanism for Gen-loaded systems was apoptosis. Summarizing, it can be concluded that BSAnp constitute versatile nanovehicles for potential flavonoid incorporation in pharmaceutical and nutraceutical matrices.

Relative expression of genes associated with adhesion to bovine mammary epithelial cells by Streptococcus uberis.

Streptococcus uberis is one of the most prevalent environmental pathogens of bovine mastitis. Biofilm growth ability by S. uberis looks to depend first upon the adherence of cells to a surface. The S. uberis ability to adhere to mammary gland epithelia might provide an advantage to colonize the lactating mammary gland. The objectives of this study were (a) to select S.uberis strains according to their ability to form biofilm, (b) to determine adherence to and internalization into MAC-T cells and (c) to investigate the expression profile adherence genes in these S. uberis strains. For the assays, the MAC-T bovine mammary epithelial cell line was used. Relative expression of genes acdA, lmb, scpA, sua, fbp and lbp was quantified by RT-qPCR. We observed that the RC38 strain from clinical bovine mastitis showed in the six genes higher values than control in both conditions. While the strain with greater ability to adhere, from clinical mastitis and biofilm producer (RC29) evidenced higher values in group 1 (G1) (bacteria after the initial contact with MAC-T cells) and decrease in group 2 (G2) (both adhered and internalized bacteria) than control. Strains with a moderate or strong capacity for biofilm production showed significantly lower relative expression values in the G2. In all adherence associated genes, strain RC19 showed relative expression values incremented in G1, while in G2 decreased expression. In conclusion, we did not find a single profile of relative expression because the relative expression levels of each gene differed depending on the strain and the co-culture stage of S. uberis cells from which RNA was obtained.

Assessment of the potential utility of different regions of Streptococcus uberis adhesion molecule (SUAM) for mastitis subunit vaccine development.

Streptococcus uberis is one of the most prevalent pathogens causing clinical and subclinical mastitis worldwide. Among bacterial factors involved in intramammary infections caused by this organism, S. uberis adhesion molecule (SUAM) is one of the main virulence factors identified. This molecule is involved in S. uberis internalization to mammary epithelial cells through lactoferrin (Lf) binding. The objective of this study was to evaluate SUAM properties as a potential subunit vaccine component for prevention of S. uberis mastitis. B epitope prediction analysis of SUAM sequence was used to identify potentially immunogenic regions. Since these regions were detected all along the gene, this criterion did not allow selecting a specific region as a potential immunogen. Hence, four fractions of SUAM (-1fr, 2fr, 3fr and 4fr), comprising most of the protein, were cloned and expressed. Every fraction elicited a humoral immune response in mice as predicted by bioinformatics analysis. SUAM-1fr generated antibodies with the highest recognition ability towards SUAM native protein. Moreover, antibodies against SUAM-1fr produced the highest proportion of internalization inhibition of S. uberis to mammary epithelial cells. In conclusion, SUAM immunogenic and functionally relevant regions were identified and allowed to propose SUAM-1fr as a potential candidate for a subunit vaccine for S. uberis mastitis prevention.

Candida albicans up-regulates the Fas-L expression in liver Natural Killer and Natural Killer T cells.

After Candida albicans arrival to the liver, the local production of proinflammatory cytokines and the expanded intrahepatic lymphocytes (IHL) can be either beneficial or detrimental to the host. Herein we explored the balance between protective inflammatory reaction and liver damage, focusing our study on the contribution of TNF-α and Fas-Fas-L pathways in the hepatocellular apoptosis associated to C. albicans infection. A robust tissue reaction and a progressive increase of IL-1ß, IL-6 and TNF-α were observed in infected animals. Blocking the biological activity of TNF-α did not modify the number of apoptotic cells observed in C. albicans infected animals. Fas-L molecule was up regulated on purified hepatic mononuclear cells and its expression progressed with the infection. In the IHL compartment, the absolute number of Fas-L+ NK and NKT cells increased on days 1 and 3 of the infection. C. albicans was also able to up regulate Fas-L expression in normal liver NK and NKT cells after in vitro contact. The innate receptor TLR2 was involved in this phenomenon. In the interplay between host factors and evasion strategies exploited by pathogens, the mechanism supported here could represent an additional way that allows this fungus to circumvent protective immune responses in the liver.

Abrogation of spontaneous liver tolerance during immune response to Candida albicans: contribution of NKT and hepatic mononuclear cells.

Hepatic mononuclear cells (HMC) are a heterogeneous population with innate immune properties involved in the response to several pathogens. Herein, during the primary infection with Candida albicans, we observed dynamic changes in CD3+, NK+ and NKT+ intrahepatic lymphoid subsets and a significant increase in the absolute number of antigen-presenting cells (APC). The liver tolerogenic microenvironment sustained by higher levels of IL-10, transforming growth factor-ß and IL-4 was severely modified upon the robust IFN-γ production after the fungal colonization. NKT cells purified from infected animals released significant amounts of IFN-γ and the production of this cytokine was exacerbated after a second contact with the fungus. Interestingly, C. albicans per se was unable to activate tolerogenic NKT cells from naive animals. In vitro experiments performed with HMC cells depleted of the CD11b/c+ population revealed that in the absence of APC, NKT cells are unable to produce IFN-γ in response to C. albicans. Our findings constitute the first evidence that this innate lymphocyte population is involved in the pathogenesis of C. albicans infection.

Immune neuroendocrine interactions during a fungal infection in immunocompetent or immunosuppressed hosts.

The yeast Candida albicans belongs to the microflora of healthy individuals, although it can infect a variety of tissues ensuing changes in the host's immune status. To evaluate the effect of neuroendocrine input on the early immune response during the fungal infection, we use a 3-day paradigm of chronic varied stress in Wistar rats infected with C. albicans. We find that stress mediators contribute to the spread of the fungus and downregulate critical functions of phagocytic cells at the infection site. Phenotypic and functional alterations of effector cells account for the decreased resistance to candidiasis and condition the development of the adaptive response. Stressed hosts exhibit a higher fungal burden in kidneys and livers associated with hyphal forms. The hepatic inflammatory reaction is compromised with severe steatosis, increment of functional enzymes, marked lipid peroxidation and hepatocyte apoptosis. Moreover, infection-related sickness symptoms are significantly increased by exposure to stress with anorexia, weight loss, lack of leptin and depletion of glycogen depots. Food deprivation exacerbates the liver injury. Stress mediators perturb the complex immune and metabolic program that operates early during fungal spread and promotes severe tissue damage.

El hígado como órgano inmunológico / The liver and immune organ

La prevalencia de enfermedades hepáticas a nivel mundial registra cifras alarmantes. Solo la infección por malaria afecta a 500 millones de personas por año a nivel mundial. Enfermedades de etiología viral como hepatitis B y C, contribuyen al aumento de la casuística y por su caracter de patologías de tipo crónico evolucionan a formas severas como la fibrosis o los procesos neoplásicos. La relevancia del hígado como órgano central en la maquinaria metabólica del organismo y como clave partícipe de la respuesta inflamatoria sistémica, indican la necesidad de preservar sus capacidades funcionales.

Candida albicans-secreted lipase induces injury and steatosis in immune and parenchymal cells.

Virulence depends on opposing reactions between host and pathogen and is intrinsically linked to the host immune status. Virulence factors rely upon microbial attributes that mediate cell damage. While the activity of several Candida albicans hydrolytic enzymes is well characterized, the biological role of lipases is uncertain. In this report, we identified, isolated, and characterized a C. albicans 70 kDa lipase that exhibited maximal activity at physiological pH and temperature. We evaluated the ability of C. albicans lipase to interact with two types of mammalian host cells: macrophages, as crucial immune effector cells involved in fungal control, and hepatocytes, as examples of parenchymal cells compromised during fungal dissemination. Herein, we demonstrate for the first time that an extracellular lipase released by C. albicans directly induced cytotoxicity and promoted the deposition of lipid droplets in the cytoplasm of macrophages and hepatocytes.

Neurocandidiasis, definiendo las interacciones patógeno/huésped en este particular sitio del organismo / Neurocandidiasis, defending host/pathogen intereactions in this particular site of the body

La colonización del sistema Nervioso Central por Candida Alnicans u otras especies de este género es un hecho no poco frecuente y de elevado riesgo para el huésped. La morbi-mortalidad asociada a esta presentación de la micosis y la ausencia de terapias exitosas comprometen aún más los alcances de esta patología. Otros factores que contribuyen a otorgar mayor complejidad a este escenario son las particularidades inherentes a este patógeno oportunista y las características del nicho biológico colonizado. En el presente artículo revisamos aspectos importantes del agente etiológico, sus características más destacadas y las estrategias de agresión/invasión involucradas durante su interacción con el huésped. Las peculiaridades de este sitio considerado de “Inmunoprivilegio”, los mediadores y células que contribuyen a otorgarle tal estatus y su implicancia en la evolución y severidad del proceso también son considerados. El creciente desafío de su diagnóstico, la promoción de alternativas terapéuticas y el desarrollo de nuevas estrategias constituyen un verdadero desafío que convoca a investigadores de distancias disciplinas a comprometer esfuerzos.

Neurocandidiasis, definiendo las interacciones patógeno/huésped en este particular sitio del organismo / Neurocandidiasis, defending host/pathogen intereactions in this particular site of the body

La colonización del sistema Nervioso Central por Candida Alnicans u otras especies de este género es un hecho no poco frecuente y de elevado riesgo para el huésped. La morbi-mortalidad asociada a esta presentación de la micosis y la ausencia de terapias exitosas comprometen aún más los alcances de esta patología. Otros factores que contribuyen a otorgar mayor complejidad a este escenario son las particularidades inherentes a este patógeno oportunista y las características del nicho biológico colonizado. En el presente artículo revisamos aspectos importantes del agente etiológico, sus características más destacadas y las estrategias de agresión/invasión involucradas durante su interacción con el huésped. Las peculiaridades de este sitio considerado de ¶Inmunoprivilegio÷, los mediadores y células que contribuyen a otorgarle tal estatus y su implicancia en la evolución y severidad del proceso también son considerados. El creciente desafío de su diagnóstico, la promoción de alternativas terapéuticas y el desarrollo de nuevas estrategias constituyen un verdadero desafío que convoca a investigadores de distancias disciplinas a comprometer esfuerzos.(AU)