Eva Salinas

Mast cells (MCs) are immune cells that produce numerous immunological mediators involved in inflammatory and allergic responses. Increased numbers of MCs are observed in chronic inflammatory reactions in organs such as the colon. There, MCs seem to participate in deleterious immune responses and tissue damage, but the detailed mechanisms of their activation are not known, mostly because procedures to obtain MC primary cultures from the colonic mucosa are expensive and time-consuming and present low yield. Here we describe a protocol to obtain MCs from the colonic mucosa (cmMCs) of C57BL/6 mice with high yield, viability and purity. Mucosal colon cells were dispersed by enzymatic digestion, and cmMCs were isolated by Percoll continuous-gradient centrifugation. This method allowed for the purification of 1,446,667 ± 112,442 cell/g of mucosal tissue, with 87.22% viability and 95.16% purity. The mucosal-like phenotype was predominant in isolated cmMCs, characterized by weak toluidine blue staining but strong expression of MC protease-1 (Mcpt1). Activation assays showed that freshly isolated cmMCs increased intracellular calcium and showed degranulation in response to ATP or IgE-antigen-dependent FcεRI cross-linking. This highly reproducible technique is cost-effective and requires no specialized equipment. This protocol could be applied in research related to inflammatory bowel disease, colon cancer or food allergies.

Glycomacropeptide (GMP) is a milk-derived bioactive peptide with demonstrated prebiotic properties. It is composed of a 64-amino acid framework and different carbohydrate molecules, which may serve as carbon sources for beneficial bacteria. Lacticaseibacillus rhamnosus GG (LGG) is a widely used probiotic strain that promotes intestinal barrier function, prevents pathogen colonisation and exerts anti-inflammatory and immunomodulatory activities. In this study, we explored the effect of GMP supplementation to LGG cultures on growth, adhesion and biofilm-forming properties of the bacterium. Prebiotic GMP promoted LGG growth and acidifying activities, mainly under anaerobiosis, without modifying the binding-mucus ability. GMP supplementation also increased LGG-biofilm biomass 3.8- and 3.4-fold under aerobic and anaerobic conditions. Besides, GMP treatment increased LGG-biofilm volume 4.7-fold, thickness by 39% and roughness by 110%. Thus, incorporating GMP into food or nutraceutical formulations might represent a viable strategy to enhance the probiotic efficacy of LGG strain, potentially improving its performance in functional products.

Introduction: Atopic dermatitis (AD) is a chronic inflammatory skin disorder resulting from the interplay of genetic and environmental factors, with a dysregulated type-2 immune response. The association between AD onset and intestinal dysbiosis supports research into nutritional interventions such as fermentable fibers intake. Agave-derived fructans (AFs) display prebiotic activity, modulating gut microbial communities that may positively influence immune functions. In this study, we evaluated the anti-inflammatory and immunomodulatory effects of oral AFs in a rat AD model. Methods: AD-like lesions were induced in the ear of Wistar rats by frequent application of 2,4-dinitrochlorobenzene (DNCB). AFs (0.1, 1, 5 g/kg) from Agave tequilana Weber var. azul were orally administered for 13 days. Inflammation, pruritus, gene expression of transcriptional factors of immune response, and staphylococcal colonization were evaluated in lesional skin. Cytokine expression, relative abundance of the main bacterial phyla and genera, and levels of short-chain fatty acids were analyzed in the intestinal milieu. Results: Treatment with AFs at 0.1 g/kg significantly reduced ear thickness at 1- and 6-hours post-DNCB application. Similarly, ear edema at 1 hour was attenuated, and inhibition of the NF-κB inflammatory pathway was detected. After AFs treatment at 0.1 g/kg, serum IgE levels were normalized to those of control animals. All AFs significantly decreased dermal mast cell and eosinophil counts, as well as epidermal thickening, with greater efficacy observed at lower doses. The scratching behavior remained unchanged across groups. AFs reduced Staphylococcus aureus abundance in lesional skin and restored Staphylococcus epidermidis levels to baseline. In lesional tissue, AFs downregulated Gata3 , Rorc , Il4 , and Il17a mRNA expression, while promoting a regulatory immune profile in mesenteric lymph nodes, characterized by increased Foxp3 , Il10 , and Tgfb1 expression. Administration of AFs at 0.1 and 1 g/kg promoted fecal abundance of Bifidobacterium and cecal acetic acid concentrations, whereas doses of 1 and 5 g/kg upregulated Firmicutes, Lactobacillus , and propionic acid levels. All doses reduced Proteobacteria abundance. Conclusion: AFs exhibit anti-inflammatory, immunoregulatory, and microbiota-modulatory properties in both the gut and skin compartments, in a non-linear dose-response manner. These findings suggest that the intake of AFs may contribute to the therapeutic management of AD.

Kombucha is a fermented beverage with high contents of antioxidants and probiotics. Enteric damage due to uncontrolled consumption of non‐steroidal anti‐inflammatory drugs (NSAIDs) represents a significant cause of morbidity and mortality. This study aimed to evaluate the preventive effects of kombucha against indomethacin‐induced enteric damage. Wistar rats were divided into control (Ctrl), supplemented with kombucha (Komb), indomethacin‐induced enteric damage (Indo), and indomethacin‐induced enteric damage and supplemented with kombucha (Indo‐Komb) groups. We analyzed ulcer index, histological changes, gene expression of intestinal barrier function genes, and microbiota phyla in cecal content. Kombucha supplementation reduced ulcer index and intestinal inflammation, which improved survival ( p = 0.0291), maintained body weight, ( p < 0.001) and avoided the drop in levels of hemoglobin, hematocrit, and erythrocytes ( p < 0.001). Histopathological analyses of intestines revealed impaired integrity of villi with inflammatory infiltrates, which were significantly prevented with kombucha supplementation ( p = 0.0024 and p = 0.0017, respectively). Rats that received kombucha upregulated the expression of tight junction genes Muc2 , Cldn1 , and Ocln ( p = 0.027, p = 0.024, p < 0.001, respectively). The upregulation of the oxidative and inflammatory markers Nos2 , Mpo , and Tnf was prevented by kombucha intake ( p < 0.001). Additionally, kombucha ameliorated the dysbiosis in indomethacin‐induced enteric damaged rats by reduction of Proteobacteria ( p < 0.001) while increasing Firmicutes abundance. This study demonstrated that kombucha consumption is a viable strategy to delay NSAID‐induced enteric damage through enhancement of intestinal barrier integrity and prevention of dysbiosis.

Gut microbiota exert functions of high importance in the intestine. Furthermore, there is increasing evidence for its role in immune regulation and maintenance of homeostasis in many physiological processes taking place in distant tissues. In particular, in this review, we explore the impact of metabolites produced by the gut microbiota on the development of atopic dermatitis (AD). Probiotics and prebiotics balance the microbiota and promote the generation of bacterial metabolites, such as short-chain fatty acids and tryptophan derivates, which promote the regulation of the exacerbated AD immune response through regulatory T cells and IL-10 and TGF-β cytokines. Metabolites also have a direct action on keratinocytes once they reach the bloodstream. Besides, probiotics decrease the levels of metabolites associated with AD onset, such as phenols. Understanding all these crosstalk processes between the gut and the skin reveals a number of possibilities, mainly through the manipulation of the gut microbiome, which may represent therapeutic strategies that can contribute to the standard treatments of AD patients to improve their quality of life.

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.

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)-1β, 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.

Ortega-Rivera OA, et al. J Breast Cancer. 2023 Feb;26:e9. https://doi.org/10.4048/jbc.2023.26.e9

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.