Glycomacropeptide Protects against Inflammation and Oxidative Stress, and Promotes Wound Healing in an Atopic Dermatitis Model of Human Keratinocytes.

Keratinocytes are actively implicated in the physiopathology of atopic dermatitis (AD), a skin allergy condition widely distributed worldwide. Glycomacropeptide (GMP) is a milk-derived bioactive peptide generated during cheese making processes or gastric digestion. It has antiallergic and skin barrier restoring properties when it is orally administered in experimental AD. This study aimed to evaluate the effect of GMP on the inflammatory, oxidative, proliferative, and migratory responses of HaCaT keratinocytes in an in vitro AD model. GMP protected keratinocytes from death and apoptosis in a dose dependent manner. GMP at 6.3 and 25 mg/mL, respectively, reduced nitric oxide by 50% and 83.2% as well as lipid hydroperoxides by 27.5% and 45.18% in activated HaCaT cells. The gene expression of TSLP, IL33, TARC, MDC, and NGF was significantly downregulated comparably to control by GMP treatment in activated keratinocytes, while that of cGRP was enhanced. Finally, in an AD microenvironment, GMP at 25 mg/mL stimulated HaCaT cell proliferation, while concentrations of 0.01 and 0.1 mg/mL promoted the HaCaT cell migration. Therefore, we demonstrate that GMP has anti-inflammatory and antioxidative properties and stimulates wound closure on an AD model of keratinocytes, which could support its reported bioactivity in vivo.

Protective Effect of Glycomacropeptide on the Inflammatory Response of U937 Macrophages.

Macrophages play crucial roles in inflammation and oxidative stress associated with noncommunicable diseases, such as cardiovascular diseases, diabetes, and cancer. Glycomacropeptide (GMP) is a bioactive peptide derived from milk κ-casein that contains abundant sialic acid and has shown anti-inflammatory, antioxidative, anti-obesity, and anti-diabetic properties when is orally administered. The aim of this study was to evaluate the effect of GMP on the regulation of the inflammatory response in human macrophages and the participation of sialic acid in this activity. GMP pretreatment decreased by 35%, 35%, and 49% the production of nitrites, interleukin (IL)-1ß, and tumor necrosis factor (TNF)-α, respectively, in activated human macrophages U937. The same effect was obtained when cells were pretreated with asialo GMP, and no change on the gene expression of the lectins associated with the recognition of sialic acids, SIGLEC5, 7, and 9, was induced by GMP on macrophages, which suggests that sialic acid might not be involved in this immunoregulatory effect. Interestingly, GMP increased 8.9- and 3.5-fold the gene expression of the canonical anti-inflammatory protein SOCS3 and the antioxidant enzyme HMOX1, respectively, in U937 cells. Thus, GMP exerts anti-inflammatory and antioxidative activities on activated macrophages in a sialic acid-independent manner, which might be related to its in vivo reported bioactivity.

Impact of genetic polymorphisms related to innate immune response on respiratory syncytial virus infection in children.

Respiratory syncytial virus (RSV) causes lower respiratory tract infections and bronchiolitis, mainly affecting children under 2 years of age and immunocompromised patients. Currently, there are no available vaccines or efficient pharmacological treatments against RSV. In recent years, tremendous efforts have been directed to understand the pathological mechanisms of the disease and generate a vaccine against RSV. Although RSV is highly infectious, not all the patients who get infected develop bronchiolitis and severe disease. Through various sequencing studies, single nucleotide polymorphisms (SNPs) have been discovered in diverse receptors, cytokines, and transcriptional regulators with crucial role in the activation of the innate immune response, which is implicated in the susceptibility to develop or protect from severe forms of the infection. In this review, we highlighted how variations in the key genes affect the development of innate immune response against RSV. This data would provide crucial information about the mechanisms of viral infection, and in the future, could help in generation of new strategies for vaccine development or generation of the pharmacological treatments.

Effects of a Modern Kefir on Conditions Associated with Moderate Severe Spastic Quadriparesis Cerebral Palsy.

Cerebral palsy (CP) in children constitutes a set of movement and body posture disorders caused by brain injury, which in turn is associated with a series of intestinal, respiratory, and malnutrition conditions. Twenty-four children were selected and included for the present study and subdivided into two groups: (1) children who included modern kefir (containing 12 probiotic species) in their diet; and (2) control group (not including kefir in their diet). The group supplemented with modern kefir received a beverage with multi probiotic species and the control group received commercial yogurt (which included the 2 typical lactic acid bacteria) for 7 weeks. Anthropometric variables, resting energy expenditure, presence, and diagnosis of functional digestive disorders (FDD), frequency of respiratory problems, presence of elevated C-reactive protein, differential count of leukocytes were evaluated. A significant increase in weight and height was found in the kefir group at the final time point. In addition, kefir intake promoted a significant reduction in functional constipation and stool hardness and increased the absolute value of blood lymphocytes. Since the fermented milk beverage modern kefir improves constipation, which is the most important FDD in children with CP and the nutritional and immune status, it could be considered an important strategy to improve health in these children.

Responses of Mast Cells to Pathogens: Beneficial and Detrimental Roles.

Mast cells (MCs) are strategically located in tissues close to the external environment, being one of the first immune cells to interact with invading pathogens. They are long living effector cells equipped with different receptors that allow microbial recognition. Once activated, MCs release numerous biologically active mediators in the site of pathogen contact, which induce vascular endothelium modification, inflammation development and extracellular matrix remodeling. Efficient and direct antimicrobial mechanisms of MCs involve phagocytosis with oxidative and non-oxidative microbial destruction, extracellular trap formation, and the release of antimicrobial substances. MCs also contribute to host defense through the attraction and activation of phagocytic and inflammatory cells, shaping the innate and adaptive immune responses. However, as part of their response to pathogens and under an impaired, sustained, or systemic activation, MCs may contribute to tissue damage. This review will focus on the current knowledge about direct and indirect contribution of MCs to pathogen clearance. Antimicrobial mechanisms of MCs are addressed with special attention to signaling pathways involved and molecular weapons implicated. The role of MCs in a dysregulated host response that can increase morbidity and mortality is also reviewed and discussed, highlighting the complexity of MCs biology in the context of host-pathogen interactions.

Protective Effect of Glycomacropeptide on Food Allergy with Gastrointestinal Manifestations in a Rat Model through Down-Regulation of Type 2 Immune Response.

Glycomacropeptide (GMP) is a bioactive peptide derived from milk κ-casein with immune-modulatory and anti-inflammatory properties. Food allergy (FA) is an adverse immune reaction with a broad spectrum of manifestations. Allergen intake induces persistent intestinal inflammation and tissue damage. In this study, the anti-allergic activity of GMP was evaluated using a rat ovalbumin (OVA)-induced FA model with gastrointestinal manifestation. Rats were orally GMP treated from 3 days prior and during FA development. The severity of food anaphylaxis and diarrheal episodes, antibody production and histamine level were measured. Histopathological changes, inflammation and predominant cytokine profile at intestine were analyzed. Oral GMP intake decreased clinical signs and diarrhea severity induced by allergen, with a significant reduction in intestinal edema and expression level of IL-1ß and TNF-α. Prophylaxis with GMP also diminished serum anti-OVA IgE and IgG1, and histamine levels. GMP treatment markedly decreased eosinophil infiltration, mast cell and goblet cell hyperplasia, total IgE expression in intestine, and prevented histological changes in villi, crypts and internal muscularis layer. The treatment effectively suppressed IL-5, IL-13 and GATA3 expression and skewed the intestinal cytokine profile toward type 1 and regulatory. These results suggest that GMP may protect against FA through down-regulating the type 2 inflammatory response.

Glycomacropeptide Ameliorates Indomethacin-Induced Enteropathy in Rats by Modifying Intestinal Inflammation and Oxidative Stress.

Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy is considered a serious and increasing clinical problem without available treatment. Glycomacropeptide (GMP) is a 64-amino acid peptide derived from milk κ-casein with numerous biological activities. The aim of this study was to investigate the protective effect of GMP on NSAID enteropathy in rats. Enteropathy was induced by seven days oral indomethacin administration. Rats were orally GMP treated from seven days previous and during the establishment of the enteropathy model. Changes in metabolism, hematological and biochemical blood alterations, intestinal inflammation and oxidative damage were analyzed. Integrity barrier markers, macroscopic intestinal damage and survival rate were also evaluated. GMP treatment prevented anorexia and weight loss in animals. Furthermore, prophylaxis with GMP ameliorated the decline in hemoglobin, hematocrit, albumin and total protein levels. The treatment had no therapeutic efficacy on the decrease of occludin and mucin (MUC)-2 expression in intestinal tissue. However, GMP markedly decreased neutrophil infiltration, and CXCL1, interleukin-1ß and inducible nitric oxide synthase expression. Nitric oxide production and lipid hydroperoxide level in the small intestine were also diminished. These beneficial effects were mirrored by preventing ulcer development and increasing animal survival. These results suggest that GMP may protect against NSAID enteropathy through anti-inflammatory and antioxidant properties.

Glycomacropeptide Bioactivity and Health: A Review Highlighting Action Mechanisms and Signaling Pathways.

Food-derived bioactive peptides are reported as beneficial and safe for human health. Glycomacropeptide (GMP) is a milk-protein-derived peptide that, in addition to its nutritional value, retains many biological properties and has therapeutic effects in several inflammatory disorders. GMP was shown under in vitro and in vivo conditions to exert a number of activities that regulate the physiology of important body systems, namely the gastrointestinal, endocrine, and immune systems. This review represents a comprehensive compilation summarizing the current knowledge and updated information on the major biological properties associated with GMP. GMP bioactivity is addressed with special attention on mechanisms of action, signaling pathways involved, and structural characteristics implicated. In addition, the results of various studies dealing with the effects of GMP on models of inflammatory diseases are reviewed and discussed.

Overexpression of inflammatory-related and nitric oxide synthase genes in olfactory bulbs from frontal lobe epilepsy patients.

Neuroinflammation has been shown to constitute a crucial mechanism in the pathophysiology of epileptic brain and several genes of inflammatory mediators have been detected in surgically resected hippocampus tissue but not in non-related seizure brain regions. Interestingly, it has been reported an olfactory dysfunction in frontal lobe epilepsy (FLE). Our aim was to quantify the gene expression of inflammatory-related and nitric oxide synthase genes in olfactory bulbs (OB) tissue from FLE patients. RNA was isolated from OB resection of FLE patients and autopsy subjects without any neurological disease (n = 7, each). After cDNA synthesis, we performed qPCR for interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), nuclear factor κB p65 (RELA), Toll-like receptor 4 (TLR 4), its agonist high mobility group box 1 (HMGB 1) as well nitric oxide synthase isozymes (NOS 1, 2 and 3). We found a significant increase in gene expression of pro-inflammatory cytokines (IL-1ß, IL-6 and TNFα), TLR4 receptor and in its agonist HMGB1 and the downstream transcription factor NFκB p65. Moreover, we observed an increase of both NOS1 and NOS3 and a slightly increase of NOS2; however, it was not significant. Our study describes the overexpression of inflammatory-related genes and NOS isozymes in OB from FLE patients. Even though, the number of patients was limited, our findings could point out that neuroinflammation and nitrosative stress-related genes in the OB could be produced in general manner in all brain regions and thus contribute in part, to the olfactory dysfunction observed in FLE patients.

Status epilepticus triggers early mitochondrial fusion in the rat hippocampus in a lithium-pilocarpine model.

Many reports investigating the hippocampus have demonstrated an increase in neuronal damage, cellular loss, oxidative stress and mitochondrial DNA damage during status epilepticus (SE); however, information regarding alterations in mitochondrial fission and fusion events in SE is lacking. The aim of the present study was to examine the possible imbalance between mitochondrial fission and fusion in the hippocampus of male rats after acute seizure mediated by SE. In this study, we used ninety animals were randomly divided into control and SE groups and subjected to the lithium-pilocarpine model of epilepsy. Hippocampi were obtained at 3, 24 and 72h after SE, and the cytoplasmic and mitochondrial fractions of the cells were used to analyze changes in the Drp1 and Fis1 fission proteins and the Mfn1 and Opa1 fusion proteins by western blot analysis. Moreover, changes in the expression of fission and fusion mRNA transcripts were evaluated by real-time PCR. Mitochondrial morphology was also analyzed using standard transmission electron microscopy. Our data showed that the fission-related mRNA Drp1 was down-regulated rapidly after SE, while Fis1 did not show any significant changes in expression. Moreover, the mitochondrial fusion-associated proteins Mfn1 and Opa1 exhibited an increase in expression at 72h after SE. Electron microphotography revealed several morphological changes, such as swollen mitochondria and damage of the inner mitochondrial membrane, at 24h; at 72h elongation of some mitochondrial was also observed. Our results suggest that after the initiation of SE, the main regulator of the fission mRNA Drp1 is down-regulated, which in turn regulates mitochondrial fission and leads to an increase in the Mfn1 and Opa1 proteins to induce mitochondrial fusion, suggesting an imbalance of the fission and fusion processes.

Anticonvulsant Effect of Time-Restricted Feeding in a Pilocarpine-Induced Seizure Model: Metabolic and Epigenetic Implications.

A new generation of antiepileptic drugs has emerged; however, one-third of epilepsy patients do not properly respond to pharmacological treatments. The purpose of the present study was to investigate whether time-restricted feeding (TRF) has an anticonvulsant effect and whether this restrictive diet promotes changes in energy metabolism and epigenetic modifications in a pilocarpine-induced seizure model. To resolve our hypothesis, one group of rats had free access to food and water ad libitum (AL) and a second group underwent a TRF schedule. We used the lithium-pilocarpine model to induce status epilepticus (SE), and behavioral seizure monitoring was analyzed. Additionally, an electroencephalography (EEG) recording was performed to verify the effect of TRF on cortical electrical activity after a pilocarpine injection. For biochemical analysis, animals were sacrificed 24 h after SE and hippocampal homogenates were used to evaluate the proteins related to metabolism and chromatin structure. Our results showed that TRF had an anticonvulsant effect as measured by the prolonged latency of forelimb clonus seizure, a decrease in the seizure severity score and fewer animals reaching SE. Additionally, the power of the late phase EEG recordings in the AL group was significantly higher than the TRF group. Moreover, we found that TRF is capable of inducing alterations in signaling pathways that regulate energy metabolism, including an increase in the phosphorylation of AMP dependent kinase (AMPK) and a decrease in the phosphorylation of Akt kinase. Furthermore, we found that TRF was able to significantly increase the beta hydroxybutyrate (ß-HB) concentration, an endogenous inhibitor of histone deacetylases (HDACs). Finally, we found a significant decrease in HDAC activity as well as an increase in acetylation on histone 3 (H3) in hippocampal homogenates from the TRF group. These findings suggest that alterations in energy metabolism and the increase in ß-HB mediated by TRF may inhibit HDAC activity, thus increasing histone acetylation and producing changes in the chromatin structure, which likely facilitates the transcription of a subset of genes that confer anticonvulsant activity.

Lipoprotein N-acyl transferase (Lnt1) is dispensable for protein O-mannosylation by Streptomyces coelicolor.

A protein glycosylation system related to that for protein mannosylation in yeast is present in many actinomycetes. This system involves polyprenyl phosphate mannose synthase (Ppm), protein mannosyl transferase (Pmt), and lipoprotein N-acyl transferase (Lnt). In this study, we obtained a series of mutants in the ppm (sco1423), lnt1 (sco1014), and pmt (sco3154) genes of Streptomyces coelicolor, which encode Ppm, Lnt1, and Pmt, to analyze their requirement for glycosylation of the heterologously expressed Apa glycoprotein of Mycobacterium tuberculosis. The results show that both Ppm and Pmt were required for Apa glycosylation, but that Lnt1 was dispensable for both Apa and the bacteriophage φC31 receptor glycosylation. A bacterial two-hybrid assay revealed that contrary to M. tuberculosis, Lnt1 of S. coelicolor does not interact with Ppm. The D2 catalytic domain of M. tuberculosisPpm was sufficient for complementation of an S. coelicolor double mutant lacking Lnt1 and Ppm, both for Apa glycosylation and for glycosylation of φC31 receptor. On the other hand, M. tuberculosisPmt was not active in S. coelicolor, even when correctly localized to the cytoplasmic membrane, showing fundamental differences in the requirements for Pmt activity in these two species.

The O-mannosylation and production of recombinant APA (45/47 KDa) protein from Mycobacterium tuberculosis in Streptomyces lividans is affected by culture conditions in shake flasks.

BACKGROUND: The Ala-Pro-rich O-glycoprotein known as the 45/47 kDa or APA antigen from Mycobacterium tuberculosis is an immunodominant adhesin restricted to mycobacterium genus and has been proposed as an alternative candidate to generate a new vaccine against tuberculosis or for diagnosis kits. In this work, the recombinant O-glycoprotein APA was produced by the non-pathogenic filamentous bacteria Streptomyces lividans, evaluating three different culture conditions. This strain is known for its ability to produce heterologous proteins in a shorter time compared to M. tuberculosis. RESULTS: Three different shake flask geometries were used to provide different shear and oxygenation conditions; and the impact of those conditions on the morphology of S. lividans and the production of rAPA was characterized and evaluated. Small unbranched free filaments and mycelial clumps were found in baffled and coiled shake flasks, but one order of magnitude larger pellets were found in conventional shake flasks. The production of rAPA is around 3 times higher in small mycelia than in larger pellets, most probably due to difficulties in mass transfer inside pellets. Moreover, there are four putative sites of O-mannosylation in native APA, one of which is located at the carboxy-terminal region. The carbohydrate composition of this site was determined for rAPA by mass spectrometry analysis, and was found to contain different glycoforms depending on culture conditions. Up to two mannoses residues were found in cultures carried out in conventional shake flasks, and up to five mannoses residues were determined in coiled and baffled shake flasks. CONCLUSIONS: The shear and/or oxygenation parameters determine the bacterial morphology, the productivity, and the O-mannosylation of rAPA in S. lividans. As demonstrated here, culture conditions have to be carefully controlled in order to obtain recombinant O-glycosylated proteins with similar "quality" in bacteria, particularly, if the protein activity depends on the glycosylation pattern. Furthermore, it will be an interesting exercise to determine the effect of shear and oxygen in shake flasks, to obtain evidences that may be useful in scaling-up these processes to bioreactors. Another approach will be using lab-scale bioreactors under well-controlled conditions, and study the impact of those on rAPA productivity and quality.