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

Immunoregulatory Effects of Porcine Plasma Protein Concentrates on Rat Intestinal Epithelial Cells and Splenocytes

This study was funded by APC Europe and 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]. CHC, CJA and BO 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.Blood contains proteins which have interest as products that may regulate immune function. For this reason some protein-based products are currently used as nutritional supplements for animals, for instance two porcine concentrates, spray dried serum (SDS), and an immunoglobulin concentrate (IC). These products have shown to protect against colonic inflammation in rodents. In the present study we characterize the ability of these products to modulate immune function in isolated cells, namely intestinal epithelial cells (IEC18 cells) and rat spleen cells. Our data indicate that both porcine protein concentrates indeed alter immune cell function, based on the secretion of the modulators known as cytokines. In intestinal epithelial IEC18 cells they promoted the secretion of GRO alpha and MCP-1 cytokines. In spleen cells they mainly inhibited the production of TNF, a key proinflammatory cytokine. In addition, the IC product augmented the release of IL-10, an anti-inflammatory cytokine. Taken together, our data indicate that the immunomodulatory effects observed in vivo are consistent with the direct actions of the protein concentrates on epithelial cells, T lymphocytes, and monocytes. Serum protein concentrates have been shown to exert in vivo anti-inflammatory effects. Specific effects on different cell types and their mechanism of action remain unraveled. We aimed to characterize the immunomodulatory effect of two porcine plasma protein concentrates, spray dried serum (SDS) and an immunoglobulin concentrate (IC), currently used as animal nutritional supplements with established in vivo immunomodulatory properties. Cytokine production by the intestinal epithelial cell line IEC18 and by primary cultures of rat splenocytes was studied. The molecular pathways involved were explored with specific inhibitors and gene knockdown. Our results indicate that both products induced GRO alpha and MCP-1 production in IEC18 cells by a MyD88/NF-kappa B-dependent mechanism. Inhibition of TNF production was observed in rat primary splenocyte cultures. The immunoglobulin concentrate induced IL-10 expression in primary splenocytes and lymphocytes. The effect on TNF was independent of IL-10 production or the stimulation of NF-kB, MAPKs, AKT, or RAGE. In conclusion, SDS and IC directly regulate intestinal and systemic immune response in murine intestinal epithelial cells and in T lymphocytes and monocytes.APC EuropeMinistry of Economy and CompetitivityEuropean Commission SAF2017-88457-R AGL2017-85270R 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 IIIEuropean Commissio

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

COVID-19 outbreaks in a transmission control scenario: challenges posed by social and leisure activities, and for workers in vulnerable conditions, Spain, early summer 2020

Severe acute respiratory syndrome coronavirus 2 community-wide transmission declined in Spain by early May 2020, being replaced by outbreaks and sporadic cases. From mid-June to 2 August, excluding single household outbreaks, 673 outbreaks were notified nationally, 551 active (>6,200 cases) at the time. More than half of these outbreaks and cases coincided with: (i) social (family/friends’ gatherings or leisure venues) and (ii) occupational (mainly involving workers in vulnerable conditions) settings. Control measures were accordingly applied

Estudio de la función inmunológica de la fosfatasa alcalina no específica de tejido

Las fosfatasas alcalinas (APs, ortofosfórico monoéster fosfohidrolasa, EC 3.1.3.1) son una superfamilia de enzimas (glicoproteinas) ampliamente distribuidas, se encuentra desde en bacterias hasta en el hombre. Las fosfatasas alcalinas son enzimas homodiméricas que catalizan la hidrólisis grupos fosfato (R-OP) con la liberación de fosfato inorgánico (Pi) y un alcohol, azúcar, fenol…(R-OH) a pH alcalino. En el sitio catalítico contienen 3 iones metálicos, dos de Zn y uno de Mg, necesarios para la actividad enzimática. Aunque las principales caracteríasticas del mecanismo catalítico se conservan entre especies, cuando se comparan las APs de mamíferos y bacterias, se observa que las APs de mamíferos tienen mayor actividad específica, un mayor pH alcalino óptimo y una menor estabilidad al calor. Además, las APs de mamíferos se encuentran ancladas a la membrana y son inhibidas por L-amino ácidos y péptidos mediante un mecanismo acompetitivo. En esta Tesis Doctoral se propone caracterizar la implicación de la fosfatasa alcalina no específica de tejido en la actividad, diferenciación y proliferación de células del sistema inmunológico y más concretamente de linfocitos T. Se utilizarán para ello distintas herramientas incluyendo ratones heterocigotos para la expresión de TNAP e inhibidores específicos de AP. Además se estudiará la implicación en la respuesta inflamatoria de la TNAP expresada en linfocitos, utilizando un modelo de colitis por transferencia de linfocitos naïve de ratones heterocigotos para la TNAP a ratones Rag-1-/-, deficientes en linfocitos T y B maduros.Tesis Univ. Granada. Programa Oficial de Doctorado en: Biomedicin

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

Germ-free and Antibiotic-treated Mice are Highly Susceptible to Epithelial Injury in DSS Colitis

Background and Aims: Intestinal microbiota is required to maintain immune homeostasis and intestinal barrier function. At the same time, intraluminal bacteria are considered to be involved in inflammatory bowel disease and are required for colitis induction in animal models, with the possible exception of dextran sulphate sodium [DSS] colitis. This study was carried out to ascertain the mechanism underlying the induction of colitis by DSS in the absence of bacteria. Methods: Conventional and germ-free [GF] Naval Medical Research Institute [NMRI] mice were used, plus conventional mice treated with an antibiotic cocktail to deplete the intestinal microbiota [‘pseudo-GF’ or PGF mice]. The differential response to DSS was assessed. Results: Conventional mice developed DSS-induced colitis normally, whereas GF mice showed only minimal inflammation [no colonic thickening, lower myeloperoxidase activity, IL-6, IL-17, TNF- α, and IFN-γ secretion by splenocytes and mesenteric cell cultures, etc.]. However, these mice suffered enhanced haemorrhage, epithelial injury and mortality as a consequence of a weakened intestinal barrier, as shown by lower occludin, claudin 4, TFF3, MUC3, and IL-22. In contrast, PGF mice had a relatively normal, albeit attenuated, inflammatory response, but were less prone to haemorrhage and epithelial injury than GF mice. This was correlated with an increased expression of IL-10 and Foxp3 and preservation barrier-related markers. Conclusions: We conclude that enteric bacteria are essential for the development of normal DSSinduced colitis. The absence of microbiota reduces DSS colonic inflammation dramatically but it also impairs barrier function, whereas subtotal microbiota depletion has intermediate effects at both levels.Ministerio de Economía y Competividad [Spain]European Union (EU) SAF2008-01432 AGL2008-04332 SAF2011-22922 SAF2011-22812 BFU2014-57736-P AGL2014-58883-RFundación Ramón Areces [Spain]Junta de Andalucía CTS164 CTS235 CTS6736Ministerio de Educación [Spain]Swedish Research CouncilInstituto de Salud Carlos IIISpanish GovernmentEuropean Union (EU) BFU2007-30688-E/BF

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