Premature Birth Infants Present Elevated Inflammatory Markers in the Meconium

Introduction: Prematurity, a well-established risk factor for various intestinal diseases in newborns, results in increased morbidity and mortality. However, the intestinal inflammatory status of preterm (PT) infants has been poorly characterized. Here we have broadly described the intestinal and systemic inflammatory status of PT children. Materials and Methods: Meconium and plasma from 39 PT and 32 full term (T) newborns were studied. Fecal calprotectin, polymorphonuclear leukocyte elastase (PMN-E), TNF, IL-17A, IL-8, IP-10, MCP-1, MIP-1, IL-1β, IL-1α, and E-selectin and the enzymatic activities of myeloperoxidase (MPO) and alkaline phosphatase (AP) in meconium were measured. Plasma levels of AP, hepatocyte growth factor, nerve growth factor (NGF), proinflammatory cytokines, leptin, adiponectin, PAI-1, and resistin were also determined. Correlations with gestational age (GA) and birth weight (BW) were studied. Results: Neutrophil derived PMN-E, MPO and calprotectin were increased in the meconium of PT compared to T newborns, while AP was decreased. No significant differences were found in other inflammatory parameters. Considering data from all children, GA and BW showed inverse correlation with neutrophil markers, while AP directly correlated with BW. Plasma levels of IL-1β and NGF were enhanced in PT infants, and were also negatively correlated with BW. PT children additionally showed neutropenia and decreased adiponectin, leptin, haematocrit, and haemoglobin. These parameters (neutrophils, adiponectin, and so forth) were positively correlated with GA and BW, while IL-8, MCP-1, PAI-1, and plasma AP were negatively correlated. PT children showing postnatal morbidity exhibited increased meconium MPO and MIP-1α. Conclusion: PT neonates present a significant elevation of intestinal inflammatory parameters, characterized by the presence of neutrophil markers, associated with mild systemic inflammation.Ministry of Economy and CompetitivityEuropean Commission SAF2017-88457-R AGL2017-85270-R BFU2014-57736-P AGL2014-58883-RJunta de Andalucia CTS235 CTS164University of Granada (Contrato Puente Program-Plan Propio)Ministry of Education [Spain]Instituto de Salud Carlos III European Commissio

Leptin-resistant Zucker rats with trinitrobenzene sulfonic acid colitis present a reduced inflammatory response but enhanced epithelial damage

The study was funded the following grants of the Ministerio de Economía y Competitividad and the Fondo Europeo de Desarrollo Regional FEDER (SAF2011-22922, SAF2011-22812, BFU2014- 57736-P, and AGL2014-58883-R) and Junta de Andalucía (CTS164, CTS235, and CTS6736). B. Rivero-Guti errez, R. Gámez-Belmonte, and M. Arredondo-Amador were supported by fellowships from the Ministerio de Educación.The role of leptin in the development of intestinal inflammation remains controversial, since proinflammatory and anti-inflammatory effects have been described. This study describes the effect of the absence of leptin signaling in intestinal inflammation. Experimental colitis was induced by intrarectal administration of trinitrobenzene sulfonic acid (TNBS) to lean and obese Zucker rats (n = 10). Effects on inflammation and mucosal barrier were studied. Bacterial translocation and LPS concentration were evaluated together with colonic permeability to 4-kDa FITC-dextran. Obese Zucker rats showed a lower intestinal myeloperoxidase and alkaline phosphatase activity, reduced alkaline phosphatase sensitivity to levamisole, and diminished colonic expression of Nos2, Tnf, and Il6, indicating attenuated intestinal inflammation, associated with attenuated STAT3, AKT, and ERK signaling in the colonic tissue. S100a8 and Cxcl1 mRNA levels were maintained, suggesting that in the absence of leptin signaling neutrophil activation rather than infiltration is hampered. Despite the lower inflammatory response, leptin resistance enhanced intestinal permeability, reflecting an increased epithelial damage. This was shown by augmented LPS presence in the portal vein of colitic obese Zucker rats, associated with induction of tissue nonspecific alkaline phosphatase, LPS-binding protein, and CD14 hepatic expression (involved in LPS handling). This was linked to decreased ZO-1 immunoreactivity in tight junctions and lower occludin expression. Our results indicate that obese Zucker rats present an attenuated inflammatory response to TNBS, but increased intestinal epithelial damage allowing the passage of bacterial antigens.Ministerio de Economía y Competitividad and the Fondo Europeo de Desarrollo Regional FEDER (SAF2011-22922, SAF2011-22812, BFU2014- 57736-P, and AGL2014-58883-R)Junta de Andalucía (CTS164, CTS235, and CTS6736)Ministerio de Educació

Green Alga Ulva spp. Hydrolysates and Their Peptide Fractions Regulate Cytokine Production in Splenic Macrophages and Lymphocytes Involving the TLR4-NFkB/MAPK Pathways

Hydrolysates of food protein sources have immunomodulatory effects, which are of interest for use as functional foods. In this study, we have characterized the immune regulatory effect on rat splenocytes, macrophages and T lymphocytes of Ulva spp. hydrolysates and their peptide fractions with or without in vitro gastrointestinal digestion and/or ultrafiltration. IL-10 was induced in almost all conditions and cell types obtained from wild type animals. The induction was in general increased by ultrafiltration and in vitro gastrointestinal digestion. TNF was also induced in basal conditions. In turn, TNF and IFN- production was attenuated by the hydrolysate products in lipopolysaccharide or concanavalin A immune stimulated cells. Inhibitors for the activation of NF B, MAPK p38 and JNK inhibited IL-10 induction in rat splenocytes. The response was dramatically attenuated in TLR4-/- cells, and only modestly in TLR2-/- cells. Food peptides from Ulva spp. genus exert anti-inflammatory effects in immune cells mediated by TLR4 and NF B. Similarity with the immunomodulatory profile of protein hydrolysates from other sources suggests a common mechanism.This work was supported by funds from the Ministry of Economy and Competitivity, partly with Fondo Europeo de Desarrollo Regional FEDER funds [SAF2017-88457-R, AGL2017-85270-R, BFU2014-57736-P, AGL2014-58883-R] and by Junta de Andalucía [CTS235, CTS164]. C.H.-C. and R.G.-B. were supported by the University of Granada (Contrato Puente Program—Plan Propio) and the Ministry of Education [Spain], respectively. CIBERehd is funded by Instituto de Salud Carlos III

Deficiency in Tissue Non-Specific Alkaline Phosphatase Leads to Steatohepatitis in Mice Fed a High Fat Diet Similar to That Produced by a Methionine and Choline Deficient Diet

Funding: This research was funded by the Ministry of Economy and Competitivity of Spain, partly with Fondo Europeo de Desarrollo Regional FEDER funds [BFU2014-57736-P, AGL2014-58883-R, SAF2017-88457-R, AGL2017-85270-R] and by Junta de Andalucía [CTS235, CTS164]. MTG, RGB and CHC were supported by fellowships from the Ministry of Education. CIBERehd is funded by Instituto de Salud Carlos III. Institutional Review Board Statement: The study was conducted according to the guidelines of the Guide for the Care and Use of Laboratory Animals, and approved by the Animal Welfare Committee of the University of Granada (registry number: CEEA 01/03/2017–029). Informed Consent Statement: Not applicable for studies not involving humans. Acknowledgments: We gratefully acknowledge the assistance of Mercedes González and the rest of the group.The liver expresses tissue-nonspecific alkaline phosphatase (TNAP), which may participate in the defense against bacterial components, in cell regulation as part of the purinome or in bile secretion, among other roles. We aimed to study the role of TNAP in the development of hepatosteatosis. TNAP+/− haplodeficient and wild type (WT) mice were fed a control diet (containing 10% fat w/w) or the same diet deficient in methionine and choline (MCD diet). The MCD diet induced substantial weight loss together with hepatic steatosis and increased alanine aminotransferase (ALT) plasma levels, but no differences in IL-6, TNF, insulin or resistin. There were no substantial differences between TNAP+/− and WT mice fed the MCD diet. In turn, TNAP+/− mice receiving the control diet presented hepatic steatosis with alterations in metabolic parameters very similar to those induced by the MCD diet. Nevertheless, no weight loss, increased ALT plasma levels or hypoglycemia were observed. These mice also presented increased levels of liver TNF and systemic resistin and glucagon compared to WT mice. The phenotype of TNAP+/− mice fed a standard diet was normal. In conclusion, TNAP haplodeficiency induces steatosis comparable to that produced by a MCD diet when fed a control diet.Ministry of Economy and Competitivity of SpainEuropean Commission BFU2014-57736-P AGL2014-58883-R SAF2017-88457-R AGL2017-85270-RJunta de Andalucia CTS235 CTS164Ministry of EducationInstituto de Salud Carlos III European Commissio

Función de la fosfatasa alcalina no específica de tejido en la pancreatitis aguda experimental y en la activación de macrófagos

Las fosfatasas alcalinas son una superfamilia de enzimas ampliamente distribuidas desde bacterias hasta humanos, capaces de hidrolizar grupos fosfato con liberación de fosfato inorgánico a pH alcalino. En humanos, las fosfatasas alcalinas se dividen en dos grupos: enzimas específicas de tejido, las cuales incluyen la isoenzimas intestinal (IAP), placentaria y de células germinales; y las isoformas de la fosfatasa alcalina no específica de tejido (TNAP). De hecho, el gen ALPL da lugar a tres variantes diferentes de la enzima, las cuales difieren solamente en la fracción glucosídica, concretamente, las isoformas de hígado, hueso y riñón, las cuales se encuentran mayoritariamente en estos órganos [1]. La TNAP es ampliamente conocida por su papel en el desarrollo de huesos y dientes. Las mutaciones en el gen de TNAP conducen a la hipofosfatasia [2, 3]. En el hígado, actúa inhibiendo la secreción biliar y se ve incrementada en caso de colestasis [4]. Además, la TNAP ha demostrado ser crucial en el crecimiento de axones, proliferación neuronal y diferenciación [5, 6]. Por otra parte, algunas evidencias señalan un posible papel neurotóxico de la TNAP, debido a la desfosforilación de la proteína tau fosforilada, un elemento clave en la enfermedad de Alzheimer y otros trastornos neurodegenerativos [7]. El intestino del ratón adulto expresa mayoritariamente IAP aunque TNAP puede ser detectada en el intestino delgado y en mayor medida en el colon. IAP es una de las isoformas mejor caracterizadas. Está involucrada en la homeostasis intestinal, controla la absorción de lípidos [8] y la secreción de bicarbonato [9, 10], limita la translocación bacteriana a través de la mucosa y atenúa la toxicidad y la inflamación mediada por el lipopolisacárido bacteriano (LPS) [4, 11, 12]. Aparte de estas acciones, IAP también previene la disbiosis y las infecciones bacterianas [13]. Además, tanto en su forma endógena como su suplementación oral protege frente el síndrome metabólico [14]. Nuestro grupo de investigación ha descrito un incremento en la expresión de la TNAP en el colon en modelos de enfermedad inflamatoria intestinal, debido tanto al flujo de leucocitos hacia el tejido colónico inflamado como a la expresión aumentada en las células epiteliales [15-17]. En las células epiteliales del colon, a los cambios observados en el patrón de glucosilación, desde tipo hígado en condiciones basales hacia la isoforma tipo hueso o hígado tras la inflamación, se les ha atribuido un papel protector. [17]. La TNAP también ha demostrado afectar al metabolismo de la glucosa y la sensibilidad a la insulina al actuar sobre la producción de adipoquinas [18]. La pancreatitis aguda es uno de las enfermedades gastrointestinales más comunes que requiere de hospitalización [19]. Esta patología suele desarrollarse como una enfermedad moderada y autolimitada (80%), pero a veces puede progresar de manera rápida e incluso fulminante, con un incremento del riesgo de mortalidad substancial [20]. La pancreatitis aguda puede ser causada por una gran variedad de estímulos lesivos, como los cálculos biliares, el consumo excesivo de alcohol y las mutaciones en el gen que codifica el tripsinógeno [21]. Esta enfermedad implica una activación prematura de las enzimas digestivas y la autofagia del páncreas con el consiguiente daño y el reclutamiento de células del sistema inmune en respuesta [22, 23]. Las células inmunes implicadas en la respuesta inflamatoria en la pancreatitis aguda son las células acinares pancreáticas, las células endoteliales, los linfocitos, los monocitos/macrófagos y los neutrófilos [24]. Los últimos representan la primera línea de defensa y son reclutados hacia el espacio intersticial pancreático, seguidos de los monocitos [22, 25]. Además, se han descrito tanto en modelos animales de pancreatitis aguda como en pacientes de esta enfermedad la existencia de cambios en la permeabilidad intestinal, lo cual se correlaciona con la severidad de la patología [26]. Por su parte, la sepsis/endotoxemia es una de las causas de muerte más comunes en las unidades de cuidado intensivo. La sepsis se define como una disfunción orgánica capaz de poner en riesgo la vida y que está provocada por una respuesta inmune alterada a una infección [27]. Asimismo, se asocia con una enorme producción de citoquinas proinflamatorias y antiinflamatorias, siendo los macrófagos uno de los principales productores de estos mediadores [28]. La TNAP se expresa en células del sistema inmune. En los linfocitos B, la TNAP se ha relacionado con el proceso de diferenciación hacia células secretoras de anticuerpos [29, 30]. En los neutrófilos, esta enzima se ha localizado en las vesículas secretoras [31]. Recientemente, nuestro grupo de investigación ha arrojado algo de luz sobre el papel de la TNAP en las células T, por medio de un mecanismo que parece estar de nuevo relacionado con las modificaciones en la glucosilación [32]. Sin embargo, es poca la información que se conoce relativa a la función de la TNAP en los neutrófilos y los macrófagos.Tesis Univ. Granada

Experimental acute pancreatitis is enhanced in mice with tissue nonspecific alkaline phoshatase haplodeficiency due to modulation of neutrophils and acinar cells.

Tissue nonspecific alkaline phosphatase (TNAP) has a well established role in bone homeostasis and in hepatic/biliary conditions. In addition, TNAP is expressed in the inflamed intestine and is relevant to T and B lymphocyte function. TNAP KO mice are only viable for a few days, but TNAP+/- haplodeficient mice are viable. Acute pancreatitis was induced by repeated caerulein injection in WT and TNAP+/- mice. TNAP+/- mice presented an increased expression of Cxcl2, Ccl2, Selplg (P-selectin ligand), Il6 and Il1b in the pancreas. Freshly isolated acinar cells showed a dramatic upregulation of Cxcl1, Cxcl2, Ccl2, Il6, Selpg or Bax in both pancreatitis groups. TNAP+/- cells displayed a 2-fold higher expression of Cxcl2, and a smaller increase in Il6. These findings could be partly replicated by in vitro treatment of primary acinar cells with caerulein. Furthermore, the proinflammatory effect on acinar cells could be partially reproduced in wild type cells treated with the TNAP inhibitor levamisole. TNAP mRNA levels were also markedly upregulated by pancreatitis in acinar cells. Neutrophil infiltration (MRP8+ cells) and activation (IL-6 and TNF production in LPS treated primary neutrophils) were increased in TNAP+/- vs WT mice. Neutrophil depletion greatly attenuated inflammation, indicating that this cell type is mainly responsible for the higher inflammatory status of TNAP+/- mice. In conclusion, our results show that altered TNAP expression results in heightened pancreatic inflammation, which may be explained by an augmented response of neutrophils and by a higher sensitivity of acinar cells to caerulein injury

Molecular action mechanism of anti-inflammatory hydrolysates obtained from brewers' spent grain

Background: Brewers’ spent grain (BSG) is a relevant, protein-rich by-product of the brewing process. Protein hydrolysates from different sources exert immune-regulatory actions activating toll-like receptors (TLRs), nuclear factor kappa B (NFκB), and mitogen-activated protein kinases (MAPKs). Effects of gastrointestinal digestion have been poorly studied. Here, we studied the immune-regulatory effect of BSG hydrolysates, and their in-vitro-digested products, on rat splenocytes, macrophages, and T lymphocytes. Results: In primary cultures of rat spleen cells, BSG hydrolysates induced interleukin 10 and tumor necrosis factor production in basal conditions. Under stimulation with lipopolysaccharide or concanavalin A, hydrolysates further induced interleukin 10 production. Tumor necrosis factor and interferon-γ were inhibited in lipopolysaccharide‑ and concanavalin-A-stimulated cells respectively. In vitro gastrointestinal digestion attenuated the observed effects. Splenic macrophages and T lymphocytes behaved in a similar fashion. In spleen cells from TLR2−/− and TLR4−/− mice, immune-regulatory effects were greatly reduced or abrogated. The study of signal transduction pathways indicated a major involvement of NFκB, and the contribution of MAPKs p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinases 1 and 2. Conclusion: BSG hydrolysates, like those obtained from other food sources, regulate the immune response, involving TLR2 and TLR4 and the activation of NFκB and MAPKs, an effect partly maintained after in vitro gastrointestinal digestion. Our data support the hypothesis of a shared, rather unspecific, mechanism of action of protein hydrolysates.Fil: Cian, Raúl Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Tecnología de los Alimentos; ArgentinaFil: Hernández Chirlaque, Cristina. Universidad de Granada; EspañaFil: Gámez Belmonte, Reyes. Universidad de Granada; EspañaFil: Drago, Silvina Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Tecnología de los Alimentos; ArgentinaFil: Sánchez de Medina, Fermín. Universidad de Granada; EspañaFil: Martínez Augustin, Olga. Universidad de Granada; Españ

Green Alga Ulva spp. Hydrolysates and Their Peptide Fractions Regulate Cytokine Production in Splenic Macrophages and Lymphocytes Involving the TLR4-NFκB/MAPK Pathways.

Hydrolysates of food protein sources have immunomodulatory effects, which are of interest for use as functional foods. In this study, we have characterized the immune regulatory effect on rat splenocytes, macrophages and T lymphocytes of Ulva spp. hydrolysates and their peptide fractions with or without in vitro gastrointestinal digestion and/or ultrafiltration. IL-10 was induced in almost all conditions and cell types obtained from wild type animals. The induction was in general increased by ultrafiltration and in vitro gastrointestinal digestion. TNF was also induced in basal conditions. In turn, TNF and IFN-γ production was attenuated by the hydrolysate products in lipopolysaccharide or concanavalin A immune stimulated cells. Inhibitors for the activation of NFκB, MAPK p38 and JNK inhibited IL-10 induction in rat splenocytes. The response was dramatically attenuated in TLR4-/- cells, and only modestly in TLR2-/- cells. Food peptides from Ulva spp. genus exert anti-inflammatory effects in immune cells mediated by TLR4 and NFκB. Similarity with the immunomodulatory profile of protein hydrolysates from other sources suggests a common mechanism

Molecular action mechanism of anti‐inflammatory hydrolysates obtained from brewers' spent grain

Background: Brewers’ spent grain (BSG) is a relevant, protein-rich by-product of the brewing process. Protein hydrolysates from different sources exert immune-regulatory actions activating toll-like receptors (TLRs), nuclear factor kappa B (NFκB), and mitogen-activated protein kinases (MAPKs). Effects of gastrointestinal digestion have been poorly studied. Here, we studied the immune-regulatory effect of BSG hydrolysates, and their in-vitro-digested products, on rat splenocytes, macrophages, and T lymphocytes. Results: In primary cultures of rat spleen cells, BSG hydrolysates induced interleukin 10 and tumor necrosis factor production in basal conditions. Under stimulation with lipopolysaccharide or concanavalin A, hydrolysates further induced interleukin 10 production. Tumor necrosis factor and interferon-γ were inhibited in lipopolysaccharide‑ and concanavalin-A-stimulated cells respectively. In vitro gastrointestinal digestion attenuated the observed effects. Splenic macrophages and T lymphocytes behaved in a similar fashion. In spleen cells from TLR2−/− and TLR4−/− mice, immune-regulatory effects were greatly reduced or abrogated. The study of signal transduction pathways indicated a major involvement of NFκB, and the contribution of MAPKs p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinases 1 and 2. Conclusion: BSG hydrolysates, like those obtained from other food sources, regulate the immune response, involving TLR2 and TLR4 and the activation of NFκB and MAPKs, an effect partly maintained after in vitro gastrointestinal digestion. Our data support the hypothesis of a shared, rather unspecific, mechanism of action of protein hydrolysates.Fil: Cian, Raúl Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Tecnología de los Alimentos; ArgentinaFil: Hernández Chirlaque, Cristina. Universidad de Granada; EspañaFil: Gámez Belmonte, Reyes. Universidad de Granada; EspañaFil: Drago, Silvina Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Ingeniería Química. Instituto de Tecnología de los Alimentos; ArgentinaFil: Sánchez de Medina, Fermín. Universidad de Granada; EspañaFil: Martínez Augustin, Olga. Universidad de Granada; Españ

Green Alga Ulva spp. Hydrolysates and Their Peptide Fractions Regulate Cytokine Production in Splenic Macrophages and Lymphocytes Involving the TLR4-NFκB/MAPK Pathways

Hydrolysates of food protein sources have immunomodulatory effects, which are of interest for use as functional foods. In this study, we have characterized the immune regulatory effect on rat splenocytes, macrophages and T lymphocytes of Ulva spp. hydrolysates and their peptide fractions with or without in vitro gastrointestinal digestion and/or ultrafiltration. IL-10 was induced in almost all conditions and cell types obtained from wild type animals. The induction was in general increased by ultrafiltration and in vitro gastrointestinal digestion. TNF was also induced in basal conditions. In turn, TNF and IFN-γ production was attenuated by the hydrolysate products in lipopolysaccharide or concanavalin A immune stimulated cells. Inhibitors for the activation of NFκB, MAPK p38 and JNK inhibited IL-10 induction in rat splenocytes. The response was dramatically attenuated in TLR4−/− cells, and only modestly in TLR2−/− cells. Food peptides from Ulva spp. genus exert anti-inflammatory effects in immune cells mediated by TLR4 and NFκB. Similarity with the immunomodulatory profile of protein hydrolysates from other sources suggests a common mechanism