Pemafibrate abrogates SLD in a rat experimental dietary model, inducing a shift in fecal bile acids and microbiota composition.

BACKGROUND AND AIMS: Drugs resolving steatotic liver disease (SLD) could prevent the evolution of metabolic dysfunction associated SLD (MASLD) to more aggressive forms but must show not only efficacy, but also a high safety profile. Repurposing of drugs in clinical use, such as pemafibrate and mirabegron, could facilitate the finding of an effective and safe drug-treatment for SLD. APPROACH AND RESULTS: The SLD High Fat High Fructose (HFHFr) rat model develops steatosis without the influence of other metabolic disturbances, such as obesity, inflammation, or type 2 diabetes. Further, liver fatty acids are provided, as in human pathology, both from dietary origin and de novo lipid synthesis. We used the HFHFr model to evaluate the efficacy of pemafibrate and mirabegron, alone or in combination, in the resolution of SLD, analyzing zoometric, biochemical, histological, transcriptomic, fecal metabolomic and microbiome data. We provide evidence showing that pemafibrate, but not mirabegron, completely reverted liver steatosis, due to a direct effect on liver PPARα-driven fatty acid catabolism, without changes in total energy consumption, subcutaneous, perigonadal and brown fat, blood lipids and body weight. Moreover, pemafibrate treatment showed a neutral effect on whole-body glucose metabolism, but deeply modified fecal bile acid composition and microbiota. CONCLUSIONS: Pemafibrate administration reverts liver steatosis in the HFHFr dietary rat SLD model without altering parameters related to metabolic or organ toxicity. Our results strongly support further clinical research to reposition pemafibrate for the treatment of SLD/MASLD.

Nutritional, molecular, and functional properties of a novel enzymatically hydrolyzed porcine plasma product.

In the present study, an enzymatically hydrolyzed porcine plasma (EHPP) was nutritionally and molecularly characterized. EHPP molecular characterization showed, in contrast to spray-dried plasma (SDP), many peptides with relative molecular masses (Mr) below 8,000, constituting 73% of the protein relative abundance. IIAPPER, a well-known bioactive peptide with anti-inflammatory and antioxidant properties, was identified. In vivo functionality of EHPP was tested in C. elegans and two different mouse models of intestinal inflammation. In C. elegans subjected to lipopolysaccharide exposure, EHPP displayed a substantial anti-inflammatory effect, enhancing survival and motility by 40% and 21.5%, respectively. Similarly, in mice challenged with Staphylococcus aureus enterotoxin B or Escherichia coli O42, EHPP and SDP supplementation (8%) increased body weight and average daily gain while reducing the percentage of regulatory Th lymphocytes. Furthermore, both products mitigated the increase of pro-inflammatory cytokines expression associated with these challenged mouse models. In contrast, some significant differences were observed in markers such as Il-6 and Tnf-α, suggesting that the products may present different action mechanisms. In conclusion, EHPP demonstrated similar beneficial health effects to SDP, potentially attributable to the immunomodulatory and antioxidant activity of its characteristic low Mr bioactive peptides.

G9a Inhibition Promotes Neuroprotection through GMFB Regulation in Alzheimer's Disease.

Epigenetic alterations are a fundamental pathological hallmark of Alzheimer's disease (AD). Herein, we show the upregulation of G9a and H3K9me2 in the brains of AD patients. Interestingly, treatment with a G9a inhibitor (G9ai) in SAMP8 mice reversed the high levels of H3K9me2 and rescued cognitive decline. A transcriptional profile analysis after G9ai treatment revealed increased gene expression of glia maturation factor ß (GMFB) in SAMP8 mice. Besides, a H3K9me2 ChIP-seq analysis after G9a inhibition treatment showed the enrichment of gene promoters associated with neural functions. We observed the induction of neuronal plasticity and a reduction of neuroinflammation after G9ai treatment, and more strikingly, these neuroprotective effects were reverted by the pharmacological inhibition of GMFB in mice and cell cultures; this was also validated by the RNAi approach generating the knockdown of GMFB/Y507A.10 in Caenorhabditis elegans. Importantly, we present evidence that GMFB activity is controlled by G9a-mediated lysine methylation as well as we identified that G9a directly bound GMFB and catalyzed the methylation at lysine (K) 20 and K25 in vitro. Furthermore, we found that the neurodegenerative role of G9a as a GMFB suppressor would mainly rely on methylation of the K25 position of GMFB, and thus G9a pharmacological inhibition removes this methylation promoting neuroprotective effects. Then, our findings confirm an undescribed mechanism by which G9a inhibition acts at two levels, increasing GMFB and regulating its function to promote neuroprotective effects in age-related cognitive decline.

Dietary supplementation with spray-dried animal plasma improves vaccine protection in aged mice.

Background: Senescence is characterized by an aggravated inflammatory state that reduces vaccine responsiveness. Dietary supplementation with spray-dried porcine plasma (SDP) exerts anti-inflammatory effects in different mucosal areas. We aimed to determine if the anti-inflammatory properties of SDP improve the efficiency of immunization in senescent animals. Methods: Experiments were performed in 2-month-old and 6-month-old male SAMP8 mice fed control or SDP (8%) feeds for 4 months. The mice received nasal doses of 2.5 µg of Staphylococcus aureus enterotoxin B (SEB) or vehicle every 15 days (i.e., 3 times). Fifteen days after the last dose, a lethal shock was induced by intraperitoneal administration of SEB and LPS. Results: Immunization increased anti-SEB IgA in intestinal and bronchoalveolar fluid (p < 0.05). After the lethal shock, all immunized aged mice that were supplemented with SDP survived, in contrast to only 66% of those fed the control feed (p < 0.05). Moreover, after the lethal challenge, aged mice showed higher expression levels of pro-inflammatory cytokines (Il-6, Tnf-α, Ifn-γ, and Il-1ß) in jejunal and (Tnf-α, and Il-1ß) in lung tissues (p < 0.05), which were reduced by SDP supplementation (p < 0.05). Furthermore, in senescent mice, SDP supplementation augmented Il-4 and Il-10 expression in both tissues (p < 0.05). Conclusion: SDP reduces the mucosal inflammation associated with aging, improving vaccine protection in senescent mice.

Dietary intake of table olives exerts antihypertensive effects in association with changes in gut microbiota in spontaneously hypertensive rats.

Arbequina table olive (AO) consumption lowers blood pressure (BP) in spontaneously hypertensive rats (SHR). This study evaluates whether dietary supplementation with AO induced changes in the gut microbiota that are consistent with the purported antihypertensive effects. Wistar-Kyoto rats (WKY-c) and SHR-c received water, while SHR-o were supplemented by gavage with AO (3.85 g kg-1) for 7 weeks. Faecal microbiota was analysed by 16S rRNA gene sequencing. SHR-c showed increased Firmicutes and decreased Bacteroidetes compared to WKY-c. AO supplementation in SHR-o decreased BP by approximately 19 mmHg, and reduced plasmatic concentrations of malondialdehyde and angiotensin II. Moreover, reshaped faecal microbiota associated with antihypertensive activity by lowering Peptoniphilus and increasing Akkermansia, Sutterella, Allobaculum, Ruminococcus, and Oscillospira. Also promoted the growth of probiotic strains of Lactobacillus and Bifidobacterium and modified the relationship of Lactobacillus with other microorganisms, from competitive to symbiotic. In SHR, AO promotes a microbiota profile compatible with the antihypertensive effects of this food.

The Neuroprotective Effects of Spray-Dried Porcine Plasma Supplementation Involve the Microbiota-Gut-Brain Axis.

Dietary supplementation with spray-dried porcine plasma (SDP) reduces the Alzheimer's disease (AD) hallmarks in SAMP8 mice. Since gut microbiota can play a critical role in the AD progression, we have studied if the neuroprotective effects of SDP involve the microbiota−gut−brain axis. Experiments were performed on two-month-old SAMP8 mice fed a standard diet and on six-month-old SAMP8 mice fed a control diet or an 8% SDP supplemented diet for four months. Senescence impaired short- and long-term memory, reduced cortical brain-derived neurotrophic factor (BDNF) abundance, increased interleukin (Il)-1ß, Il-6, and Toll-like receptor 2 (Tlr2) expression, and reduced transforming growth factor ß (Tgf-ß) expression and IL-10 concentration (all p < 0.05) and these effects were mitigated by SDP (all p < 0.05). Aging also increased pro-inflammatory cytokines in serum and colon (all p < 0.05). SDP attenuated both colonic and systemic inflammation in aged mice (all p < 0.05). SDP induced the proliferation of health-promoting bacteria, such as Lactobacillus and Pediococcus, while reducing the abundance of inflammation-associated bacteria, such as Johnsonella and Erysipelothrix (both q < 0.1). In conclusion, SDP has mucosal and systemic anti-inflammatory effects as well as neuroprotective properties in senescent mice; these effects are well correlated with SDP promotion of the abundance of probiotic species, which indicates that the gut−brain axis could be involved in the peripheral effects of SDP supplementation.

Modulation of AggR levels reveals features of virulence regulation in enteroaggregative E. coli.

Enteroaggregative Escherichia coli (EAEC) strains are one of the diarrheagenic pathotypes. EAEC strains harbor a virulence plasmid (pAA2) that encodes, among other virulence determinants, the aggR gene. The expression of the AggR protein leads to the expression of several virulence determinants in both plasmids and chromosomes. In this work, we describe a novel mechanism that influences AggR expression. Because of the absence of a Rho-independent terminator in the 3'UTR, aggR transcripts extend far beyond the aggR ORF. These transcripts are prone to PNPase-mediated degradation. Structural alterations in the 3'UTR result in increased aggR transcript stability, leading to increased AggR levels. We therefore investigated the effect of increased AggR levels on EAEC virulence. Upon finding the previously described AggR-dependent virulence factors, we detected novel AggR-regulated genes that may play relevant roles in EAEC virulence. Mutants exhibiting high AggR levels because of structural alterations in the aggR 3'UTR show increased mobility and increased pAA2 conjugation frequency. Furthermore, among the genes exhibiting increased fold change values, we could identify those of metabolic pathways that promote increased degradation of arginine, fatty acids and gamma-aminobutyric acid (GABA), respectively. In this paper, we discuss how the AggR-dependent increase in specific metabolic pathways activity may contribute to EAEC virulence.

Reply to Nifli, A.-P. Comment on "Rosell-Cardona et al. Dietary Spray-Dried Porcine Plasma Reduces Neuropathological Alzheimer's Disease Hallmarks in SAMP8 Mice. Nutrients 2021, 13, 2369".

Thank you for your comments on our recent work of the effects of supplementation with spray-dried porcine plasma (SDP) on neuropathological markers of Alzheimer's disease (AD) [...].

Dietary Spray-Dried Porcine Plasma Reduces Neuropathological Alzheimer's Disease Hallmarks in SAMP8 Mice.

Alzheimer's disease (AD) is characterized by the aberrant processing of amyloid precursor protein (APP) and the accumulation of hyperphosphorylated tau, both of which are accompanied by neuroinflammation. Dietary supplementation with spray-dried porcine plasma (SDP) has anti-inflammatory effects in inflammation models. We investigated whether dietary supplementation with SDP prevents the neuropathological features of AD. The experiments were performed in 2- and 6-month-old SAMP8 mice fed a control diet, or a diet supplemented with 8% SDP, for 4 months. AD brain molecular markers were determined by Western blot and real-time PCR. Senescent mice showed reduced levels of p-GSK3ß (Ser9) and an increase in p-CDK5, p-tau (Ser396), sAPPß, and the concentration of Aß40, (all p < 0.05). SDP prevented these effects of aging and reduced Bace1 levels (all p < 0.05). Senescence increased the expression of Mme1 and Ide1 and pro-inflammatory cytokines (Il-17 and Il-18; all p < 0.05); these changes were prevented by SDP supplementation. Moreover, SDP increased Tgf-ß expression (p < 0.05). Furthermore, in aged mice, the gene expression levels of the microglial activation markers Trem2, Ym1, and Arg1 were increased, and SDP prevented these increases (all p < 0.05). Thus, dietary SDP might delay AD onset by reducing its hallmarks in senescent mice.

Dietary Supplementation with Spray-Dried Porcine Plasma Attenuates Colon Inflammation in a Genetic Mouse Model of Inflammatory Bowel Disease.

Dietary supplementation with spray-dried porcine plasma (SDP) can modulate the immune response of gut-associated lymphoid tissue. SDP supplementation reduces acute mucosal inflammation, as well as chronic inflammation associated with aging. The aim of this study was to analyze if SDP supplementation could ameliorate colitis in a genetic mouse model of inflammatory bowel disease (IBD). Wild-type mice and Mdr1a knockout (KO) mice were administered a control diet or an SDP-supplemented diet from day 21 (weaning) until day 56. The histopathological index, epithelial barrier, and intestinal immune system were analyzed in the colonic mucosa. KO mice had higher epithelial permeability, increased Muc1 and Muc4 expression, and lower abundance of E-cadherin and Muc2 (all p < 0.001). SDP prevented these effects (all p < 0.05) and decreased the colonic inflammation observed in KO mice, reducing neutrophil and monocyte infiltration and activation and the percentage of activated T helper lymphocytes in the colonic mucosa (all p < 0.05). SDP also diminished proinflammatory cytokine expression and increased the anti-inflammatory IL-10 concentration in the colonic mucosa (all p < 0.05). In conclusion, dietary supplementation with SDP enhances colon barrier function and reduces mucosal inflammation in a mouse model of IBD.

Dietary Spray-Dried Porcine Plasma Prevents Cognitive Decline in Senescent Mice and Reduces Neuroinflammation and Oxidative Stress.

BACKGROUND: Aging is characterized by chronic, low-grade inflammation that correlates with cognitive decline. Dietary supplementation with spray-dried porcine plasma (SDP) reduces immune activation in rodent models of inflammation and aging. OBJECTIVE: We investigated whether the anti-inflammatory properties of SDP could ameliorate age-related cognitive deterioration and preserve brain homeostasis in an aging mouse model of senescence. METHODS: Male senescence-accelerated prone 8 (SAMP8) mice were used. In Experiment 1, cognitive performance (n  = 10-14 mice/group) was analyzed by the novel object recognition test in 2-mo-old mice (2M group) and in mice fed a control diet or a diet supplemented with 8% SDP for 2 (4M-CTL and 4M-SDP groups) and 4 mo (6M-CTL and 6M-SDP groups). In Experiment 2, the permeability of the blood-brain barrier and junctional proteins in brain tissue was assessed, as well as synaptic density, oxidative stress markers, and inflammatory genes and proteins in mice from the 2M, 6M-CTL, and 6M-SDP groups ( n = 5-11). Statistical analyses included one-factor ANOVA followed by Fisher's posthoc test. RESULTS: 6M-SDP mice had better cognitive performance than 6M-CTL mice in both short-term (P = 0.024) and long-term (P = 0.017) memory tests. In brain tissue, 6M-SDP mice showed reduced brain capillary permeability (P = 0.034) and increased ZO1 and E-cadherin expression (both P <0.04) compared with 6M-CTL mice. SDP also prevented the NFκB activation observed in 6M-CTL mice (P = 0.002) and reduced Il6 expression and hydrogen peroxide concentration (both P <0.03) observed in 6M-CTL mice. SDP also increased the concentration of IL10 (P = 0.027), an anti-inflammatory cytokine correlated with memory preservation. CONCLUSIONS: In senescent SAMP8 mice, dietary supplementation with SDP attenuated cognitive decline and prevented changes in brain markers of neuroinflammation and oxidative stress.

Dietary Animal Plasma Proteins Improve the Intestinal Immune Response in Senescent Mice.

Increased life expectancy has promoted research on healthy aging. Aging is accompanied by increased non-specific immune activation (inflammaging) which favors the appearance of several disorders. Here, we study whether dietary supplementation with spray-dried animal plasma (SDP), which has been shown to reduce the activation of gut-associated lymphoid tissue (GALT) in rodents challenged by S. aureus enterotoxin B (SEB), and can also prevent the effects of aging on immune system homeostasis. We first characterized GALT in a mouse model of accelerated senescence (SAMP8) at different ages (compared to mice resistant to accelerated senescence; SAMR1). Second, we analyzed the SDP effects on GALT response to an SEB challenge in SAMP8 mice. In GALT characterization, aging increased the cell number and the percentage of activated Th lymphocytes in mesenteric lymph nodes and Peyer's patches (all, p < 0.05), as well as the expression of IL-6 and TNF-α in intestinal mucosa (both, p < 0.05). With respect to GALT response to the SEB challenge, young mice showed increased expression of intestinal IL-6 and TNF-α, as well as lymphocyte recruitment and activation (all, p < 0.05). However, the immune response of senescent mice to the SEB challenge was weak, since SEB did not change cell recruitment or the percentage of activated Th lymphocytes. Mice supplemented with SDP showed improved capacity to respond to the SEB challenge, similar to the response of the young mice. These results indicate that senescent mice have an impaired mucosal immune response characterized by unspecific GALT activation and a weak specific immune response. SDP supplementation reduces non-specific basal immune activation, allowing for the generation of specific responses.

The Anti-Inflammatory Effect of Spray-Dried Plasma Is Mediated by a Reduction in Mucosal Lymphocyte Activation and Infiltration in a Mouse Model of Intestinal Inflammation.

Spray-dried preparations from porcine and bovine plasma can alleviate mucosal inflammation in experimental models and improve symptoms in patients with enteropathy. In rodents, dietary supplementation with porcine spray-dried plasma (SDP) attenuates intestinal inflammation and improves the epithelial barrier function during intestinal inflammation induced by Staphylococcus aureus enterotoxin B (SEB). The aim of this study was to discern the molecular mechanisms involved in the anti-inflammatory effects of SDP. Male C57BL/6 mice were fed with 8% SDP or control diet (based on milk proteins) for two weeks, from weaning until day 33. On day 32, the mice were given a SEB dose (i.p., 25 µg/mouse) or vehicle. SEB administration increased cell recruitment to mesenteric lymph nodes and the percentage of activated Th lymphocytes and SDP prevented these effects). SDP supplementation increased the expression of interleukin 10 (IL-10) or transforming growth factor- ß (TGF-ß) compared to the SEB group. The SEB challenge increased six-fold the expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) and intercellular adhesion molecule 1 (ICAM-1); and these effects were attenuated by SDP supplementation. SEB also augmented NF-κB phosphorylation, an effect that was prevented by dietary SDP. Our results indicate that the anti-inflammatory effects of SDP involve the regulation of transcription factors and adhesion molecules that reduce intestinal cell infiltration and the degree of the inflammatory response.

Oral Serum-Derived Bovine Immunoglobulin/Protein Isolate Has Immunomodulatory Effects on the Colon of Mice that Spontaneously Develop Colitis.

Dietary immunoglobulin concentrates prepared from animal plasma can modulate the immune response of gut-associated lymphoid tissue (GALT). Previous studies have revealed that supplementation with serum-derived bovine immunoglobulin/protein isolate (SBI) ameliorates colonic barrier alterations in the mdr1a-/- genetic mouse model of IBD. Here, we examine the effects of SBI on mucosal inflammation in mdr1a-/- mice that spontaneously develop colitis. Wild type (WT) mice and mice lacking the mdr1a gene (KO) were fed diets supplemented with either SBI (2% w/w) or milk proteins (Control diet), from day 21 (weaning) until day 56. Leucocytes in mesenteric lymph nodes (MLN) and in lamina propria were determined, as was mucosal cytokine production. Neutrophil recruitment and activation in MLN and lamina propria of KO mice were increased, but were significantly reduced in both by SBI supplementation (p < 0.05). The increased neutrophil recruitment and activation observed in KO mice correlated with increased colon oxidative stress (p < 0.05) and SBI supplementation reduced this variable (p < 0.05). The Tact/Treg lymphocyte ratios in MLN and lamina propria were also increased in KO animals, but SBI prevented these changes (both p < 0.05). In the colon of KO mice, there was an increased production of mucosal pro-inflammatory cytokines such as IL-2 (2-fold), IL-6 (26-fold) and IL-17 (19-fold), and of chemokines MIP-1ß (4.5-fold) and MCP-1 (7.2-fold). These effects were significantly prevented by SBI (p < 0.05). SBI also significantly increased TGF-ß secretion in the colon mucosa, suggesting a role of this anti-inflammatory cytokine in the modulation of GALT and the reduction of the severity of the inflammatory response during the onset of colitis.

Dietary intervention with serum-derived bovine immunoglobulins protects barrier function in a mouse model of colitis.

Dietary supplementation with immunoglobulins from animal plasma has anti-inflammatory effects on intestinal and lung models of acute inflammation. Here, we aimed to establish whether dietary intervention with serum-derived bovine immunoglobulin (SBI) can prevent alterations in intestinal barrier function in a mouse model with a genetic predisposition to inflammatory bowel disease (IBD). Wild-type (WT) mice and mice lacking the mdr1a gene (KO) were fed diets supplemented with either SBI (2% wt/wt) or milk proteins (control diet), from day 21 (weaning) until day 56. The epithelial permeability of distal colon crypts was measured by confocal microscopy using a fluorescent marker. The expression of junctional epithelial E-cadherin and ß-catenin proteins were determined by Western blot and zonula occludens-1 (ZO-1) by immunofluorescence. Mucins (MUC1, MUC2, MUC4), TFF3, cytokines (TNF-α, IFN-γ), and inducible nitric oxide synthase RNA expression were quantified by real-time PCR. SBI blocked the increase in colon crypt permeability and partially prevented the reduction in E-cadherin and ZO-1 expression that characterize the KO mouse model (both P < 0.05). SBI inclusion also reduced the mucosal expression of the inflammatory markers TNF-α, IFN-γ, and inducible nitric oxide synthase (all P < 0.005). The number of goblet cells in the colon of KO mice was low and correlated well with MUC2 and TFF3 expression (P < 0.001), whereas dietary supplementation with SBI attenuated these effects (all P < 0.05). In short, dietary SBI ameliorated colonic barrier alterations and reduced the expression of mucosal inflammatory markers in a genetic model of IBD.

Type I interferon response is delayed in human astrovirus infections.

Type I interferon (IFN) activation and its subsequent effects are important in the response to viral infections. Here we show that human astroviruses (HAstVs), which are important agents of acute gastroenteritis in children, induce a mild and delayed IFN response upon infecting CaCo-2 cells. Although IFN-ß mRNA is detected within infected cells and supernatant from infected cells show antiviral activity against the replication of other well-known IFN-sensitive viruses, these responses occur at late stages of infection once genome replication has taken place. On the other hand, HAstV replication can be partially reduced by the addition of exogenous IFN, and inhibition of IFN activation by BX795 enhances viral replication, indicating that HAstVs are IFN-sensitive viruses. Finally, different levels of IFN response were observed in cells infected with different HAstV mutants with changes in the hypervariable region of nsP1a/4, suggesting that nsP1a/4 genotype may potentially have clinical implications due to its correlation with the viral replication phenotype and the antiviral responses induced within infected cells.

Vasopressin regulation of epithelial colonic proliferation and permeability is mediated by pericryptal platelet-derived growth factor A.

Arginine vasopressin (AVP) has trophic effects on the rat distal colon, increasing the growth of pericryptal myofibroblasts and reducing the colonic crypt wall permeability. This study aimed to reproduce in vitro the effects of AVP observed in vivo using cultures of human CCD-18Co myofibroblasts and T84 colonic epithelial cells. Proliferation of myofibroblasts was quantified by bromodeoxyuridine incorporation; the expression of platelet-derived growth factor A (PDGFA), platelet-derived growth factor B, epidermal growth factor, transforming growth factor-ß and vascular endothelial growth factor was measured by PCR and the expression of epithelial junction proteins by Western blot. Arginine vasopressin stimulated myofibroblast proliferation and the expression of PDGFA without affecting the expression of platelet-derived growth factor B, epidermal growth factor, transforming growth factor-ß or vascular endothelial growth factor. These effects were prevented when AVP receptor inhibitors were present in the medium. Pre-incubation of CCD-18Co cells with anti-PDGF antibody or with an inhibitor of the PDGF receptor abolished the effects of AVP. When colonocytes were incubated with medium obtained from myofibroblasts incubated with AVP, both cell proliferation and the expression of epithelial junction proteins increased; however, direct incubation of colonocytes with AVP did not modify these variables. These results demonstrate that AVP stimulates myofibroblast proliferation and induces PDGFA secretion, implying that PDGFA mediates local myofibroblast proliferation by an autocrine feedback loop and regulates epithelial proliferation and permeability by a paracrine mechanism.

Aldosterone induces myofibroblast EGF secretion to regulate epithelial colonic permeability.

In vivo studies show that raised aldosterone (Aldo) during low-Na adaptation regulates the growth of pericryptal myofibroblasts and reduces the permeability of the colonic epithelium. The aim of this study was to reproduce in vitro the in vivo condition of increased Aldo using human CCD-18Co myofibroblasts and T84 colonic epithelial cells to measure myofibroblast and epithelial proliferation and the expression of intercellular junction proteins. Proliferation was quantified by measuring 5-bromo-2'-deoxyuridine incorporation. The myofibroblast expression of EGF, VEGFa, and transforming growth factor-ß1 (TGF-ß1) was measured by real-time PCR and the expression of junctional complex proteins by Western blot. Aldo stimulated the proliferation of myofibroblasts by 70% (P < 0.05) and increased EGF mRNA expression by 30% (P < 0.05) without affecting VEGFa and TGF-ß1. EGF concentration in the incubation medium increased by 30% (P < 0.05) 24 h after Aldo addition, and these effects were prevented by the addition of spironolactone. Myofibroblast proliferation in response to Aldo was mediated by EGF receptor (EGFR) and involved both MAPKK and phosphatidylinositol 3-kinase pathways. When T84 cells were incubated with medium from myofibroblasts stimulated with Aldo (conditioned medium), the expression of ß-catenin and claudin IV was increased by 30% (P < 0.05) and proliferation by 40% (P < 0.05). T84 proliferation decreased when α-EGF, or the EGFR antagonist AG1478, was present. Results in vivo indicate that rats fed a low-salt diet showed an increased expression of EGF and EGFR in the colonic mucosa. These results support the view that changes in colonic permeability during low-Na adaptation are mediated by the EGF secreted by myofibroblasts in response to raised Aldo.

Dietary plasma proteins modulate the adaptive immune response in mice with acute lung inflammation.

We examined the effects of oral plasma protein supplements on the pulmonary adaptive immune response in mice challenged with intranasal LPS. C57BL/6 mice were fed a control diet or a diet supplemented with plasma proteins [spray-dried plasma (SDP) 80 g/kg] or with an Ig concentrate [(IC) 20 g/kg] from postnatal d 19 (weaning) until d 34. Mice were challenged with PBS or LPS from Escherichia coli at d 33 and killed 24 h later for leukocyte analyses or at d 34 and killed 6 h later for cytokine determination. LPS induced the activation of T helper (Th) lymphocytes in lung and blood and this response was reduced by SDP and IC (P < 0.05). In both tissues, LPS increased the Th1 and Th2 subpopulations and this effect was inhibited by the two plasma protein supplements (P < 0.05). The LPS challenge increased the expression of all the cytokines studied (P < 0.01). SDP and IC reduced the expression of IFNγ, IL-5, IL-12p40, IL-12p70, IL-13, and IL-17 in both tissues, whereas they increased the percentage of regulatory Th lymphocytes in lung, even in PBS-treated mice (P < 0.05). LPS reduced the concentration of mature TGFß1 (P < 0.05) in the lung but did not modify the expression of IL-10. Mice exposed to LPS and supplemented with SDP or IC showed an increased expression of the anti-inflammatory cytokine IL-10 (P < 0.05). Moreover, the two supplements increased the concentration of IL-10 in intestinal mucosa (P < 0.05). Our results show that plasma supplementation reduces the immune response that characterizes the acute lung inflammation syndrome.

Dietary plasma proteins attenuate the innate immunity response in a mouse model of acute lung injury.

We examined whether oral plasma protein supplements affect the innate immune response in a model of acute lung inflammation. Mice were fed diets supplemented with 8 % spray-dried plasma (SDP) or 2 % plasma Ig concentrate (IC) from day 19 (weaning) until day 34. The mice were challenged with intranasal lipopolysaccharide (LPS) at day 33 (and killed 24 h later for cytokine and leucocyte analyses) or at day 34 (and killed 6 h later for cytokine determinations). In bronchoalveolar lavage fluid (BALF), LPS increased the number of leucocytes by twenty-sevenfold, an effect that was partly prevented by both SDP and IC, and by twentyfold the percentage of activated monocytes, which was partly prevented by SDP. In the lung tissue, LPS increased the infiltrated leucocytes, and this effect was prevented in part by SDP. In unchallenged mice, both SDP and IC diets reduced the percentage of resident neutrophils and monocytes (P < 0·05). In the blood, both SDP and IC completely prevented LPS-dependent monocyte activation (CD14⁺; P < 0·05). LPS dramatically increased the concentration of cytokines (TNF-α, IL-1α, IL-6, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor) and chemokines (CXCL1, CCL2, CCL3 and CCL4) in BALF. The acute response of cytokine production was reduced by 20-80 % by both SDP and IC. For chemokines, plasma supplements had no effect on LPS-induced CXCL1 expression but significantly reduced CCL2, CCL3 and CCL4 production (P < 0·05). The results support the view that dietary plasma proteins can be used to attenuate endotoxin-associated lung inflammation.