Neuroprotective effects of whey and buttermilk-based formulas on a DSS-induced colitis murine model.

Inflammatory bowel disease is a gut-brain axis disorder that comprises chronic inflammatory conditions affecting the gastrointestinal tract, where alterations in the mood of patients are common. Gut-brain axis is a bidirectional communication that link gut and brain. The close association between inflammatory bowel disease and neuroinflammation has far-reaching implications, as is increasingly recognized as a contributing factor to neuropsychiatric and neurodegenerative diseases. The increasing prevalence and high economic cost, together with the loss of life quality of people suffering from these diseases, point to the need to find alternatives to alleviate them. Exploring new therapeutic avenues prompts us to consider the potential benefits of milk fractions, taking advantage of the use of dairy by-products, such as whey and buttermilk. This study examines the impact of cow's whey- and buttermilk-based formulas supplemented with bovine lactoferrin and milk fat globule membrane on the expression of cytokines, as well as on the components of immune and serotonergic system of the brain in a murine model of DSS-induced colitis. Our results show the potential of these dairy by-products, especially whey, as functional foods in ameliorating neuroinflammation and safeguarding the central nervous system function amid the neurological complications induced or concomitant with intestinal inflammatory processes.

Lactoferrin modulates oxidative stress and inflammatory cytokines in a murine model of dysbiosis induced by clindamycin.

Antibiotics, specifically clindamycin, cause intestinal dysbiosis, reducing the microbiota with anti-inflammatory properties. Furthermore, clindamycin can induce alterations in the immune responses and oxidative stress. Lactoferrin, among other activities, participates in the maintenance of intestinal homeostasis and reduces dysbiosis induced by antibiotic treatment. The aim of this study was to analyze the effect of native and iron-saturated bovine LF in a murine model of dysbiosis induced by clindamycin. Six groups of male C57BL/6 mice were treated with saline (control), clindamycin (Clin), native lactoferrin (nLF), iron-saturated lactoferrin (sLF), nLF/Clin or sLF/Clin. Oxidation caused in the intestinal cells of the ileum of animals subjected to different treatments was analyzed, focusing on lipid peroxidation and protein carbonyl content. The expression of inflammatory mediators was determined by qRT-PCR. Treatment with clindamycin did not modify lipid peroxidation, but significantly increased protein carbonyl levels up to almost 5-fold respect to the control, an effect that was reversed by orally administering sLF to mice. Furthermore, clindamycin increased the expression of interleukin-6 and TNF-α by 1- and 2-fold change, respectively. This effect was reversed by treatment with nLF and sLF, decreasing the expression to basal levels. In conclusion, this study indicates that lactoferrin can prevent some of the effects of clindamycin on intestinal cells and their associated immune system.

Regulation of monocyte apoptosis and DNA extrusion in monocyte extracellular traps by PSGL-1: Relevance in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by severe organ damage and lacking curative treatment. While various immune cell types, especially dysfunctional B and T cells and neutrophils, have been related with disease pathogenesis, limited research has focused on the role of monocytes in SLE. Increased DNA extracellular traps, apoptosis and necrosis have been related to lupus pathogenesis. Our goal is to analyze the contribution of P-selectin glycoprotein ligand 1 (PSGL-1) in SLE monocytes to disease pathogenesis by investigating the control exerted by PSGL-1 on monocyte apoptosis and DNA extrusion in extracellular traps (METs). Monocytes from active disease patients (aSLE) exhibited reduced levels of PSGL-1. Importantly, lower PSGL-1 levels in SLE monocytes associated with several clinical characteristics, including anti-dsDNA autoantibodies, lupus anticoagulant, clinical lung involvement, and anemia. Monocytes from SLE patients showed higher susceptibility to apoptosis than healthy donors (HD) monocytes and PSGL-1/P-selectin interaction decreased secondary necrosis in HD but not in aSLE monocytes. Regarding METs, aSLE monocytes exhibited higher susceptibility to generate METs than HD monocytes. The interaction of HD monocytes with P-selectin induced Syk activation and reduced the levels of DNA extruded in METs. However, in aSLE monocytes, PSGL-1/P-selectin interaction did not activate Syk or reduce the amount of extruded DNA. Our data suggest a dysfunctional PSGL-1/P-selectin axis in aSLE monocytes, unable to reduce secondary necrosis or the amount of DNA released into the extracellular medium in METs, potentially contributing to lupus pathogenesis.

PSGL-1, ADAM8, and selectins as potential biomarkers in the diagnostic process of systemic lupus erythematosus and systemic sclerosis: an observational study.

Background: Early diagnosis and treatment of Systemic lupus erythematosus (SLE) and Systemic sclerosis (SSc) present significant challenges for clinicians. Although various studies have observed changes in serum levels of selectins between healthy donors and patients with autoimmune diseases, including SLE and SSc, their potential as biomarkers has not been thoroughly explored. We aimed to investigate serum profiles of PSGL-1 (sPSGL-1), ADAM8 (sADAM8) and P-, E- and L-selectins (sP-, sE- and sL-selectins) in defined SLE and SSc patient cohorts to identify disease-associated molecular patterns. Methods: We collected blood samples from 64 SLE patients, 58 SSc patients, and 81 healthy donors (HD). Levels of sPSGL-1, sADAM8 and selectins were analyzed by ELISA and leukocyte membrane expression of L-selectin and ADAM8 by flow cytometry. Results: Compared to HD, SLE and SSc patients exhibited elevated sE-selectin and reduced sL-selectin levels. Additionally, SLE patients exhibited elevated sPSGL-1 and sADAM8 levels. Compared to SSc, SLE patients had decreased sL-selectin and increased sADAM8 levels. Furthermore, L-selectin membrane expression was lower in SLE and SSc leukocytes than in HD leukocytes, and ADAM8 membrane expression was lower in SLE neutrophils compared to SSc neutrophils. These alterations associated with some clinical characteristics of each disease. Using logistic regression analysis, the sL-selectin/sADAM8 ratio in SLE, and a combination of sL-selectin/sE-selectin and sE-selectin/sPSGL-1 ratios in SSc were identified and cross-validated as potential serum markers to discriminate these patients from HD. Compared to available diagnostic biomarkers for each disease, both sL-selectin/sADAM8 ratio for SLE and combined ratios for SSc provided higher sensitivity (98% SLE and and 67% SSc correctly classified patients). Importantly, the sADAM8/% ADAM8(+) neutrophils ratio discriminated between SSc and SLE patients with the same sensitivity and specificity than current disease-specific biomarkers. Conclusion: SLE and SSc present specific profiles of sPSGL-1, sE-, sL-selectins, sADAM8 and neutrophil membrane expression which are potentially relevant to their pathogenesis and might aid in their early diagnosis.

An artificial intelligence-driven predictive model for pediatric allogeneic hematopoietic stem cell transplantation using clinical variables.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) is a procedure with high morbidity and mortality. Identifying patients for maximum benefit and risk assessment is crucial in the decision-making process. This has led to the development of predictive risk models for HSCT in adults, which have limitations when applied to pediatric population. Our goal was to develop an automatic learning algorithm to predict survival in children with malignant disorders undergoing HSCT. METHODS: We studied allogenic HSCTs performed on children with malignant disorders at a third-level hospital between 1991 and 2021. Survival was analyzed using the Kaplan-Meier method, log-rank test for the univariate analysis, and Cox regression for the multivariate analysis. A prognostic index was constructed based on these findings. Lastly, we constructed a predictive model using a random forest algorithm to forecast 1-year survival after HSCT. RESULTS: We analyzed 229 HSCTs in 201 patients with a median follow-up of 1.64 years. Variables that impacted on the multivariate analysis were older age (hazard ratio [HR] 1.40, 95% confidence interval [CI] 1.12-1.76, p = .003), oldest period of HSCT (HR 0.46, 95% CI 0.29-0.73, p < .001), and mismatched donor (HR 2.65, 95% CI 1.51-4.65, p = .001). Our prognostic index was associated with 3-year overall survival (OS; p < .001). A random forest was developed using as variables: diagnosis, age, year of HSCT, time from diagnosis to HSCT, disease stage, donor type, and conditioning. This achieved 72% accuracy in predicting 1-year OS. CONCLUSIONS: Our index and random forest was effective in predicting 1-year survival. However, further validation in diverse populations is necessary to establish their generalizability.

Antirotaviral activity of dairy byproducts enriched in fractions from hyperimmune bovine colostrum: the effect of thermal and high hydrostatic pressure treatments.

Nowadays, rotaviruses remain a major health burden, especially in developing countries, and strategies complementary to vaccination are needed. In this view, dairy fractions have attracted great scientific interest, due to their high content of bioactive compounds. The objective of this study was to evaluate the antiviral activity of whey and buttermilk enriched in proteins from hyperimmune bovine colostrum (HBC) against rotavirus. The enriched fractions were spray-dried and subsequently tested for their neutralizing activity against the bovine rotavirus WC3 strain in vitro, using differentiated Caco-2/TC7 cells. The highest antirotaviral activity was observed when whey and buttermilk were enriched in purified immunoglobulin G (IgG), showing complete rotavirus neutralization at concentrations of 3 and 6 mg mL-1 for whey and buttermilk, respectively. Additionally, the use of a crude immunoglobulin fraction also gave satisfactory results. The inhibitory activities of all samples significantly decreased after the application of heat, except for the IgG-enriched buttermilk which showed a slight increase of activity following the application of short-time treatments (75 or 85 °C for 20 s). This sample also showed a significant increase of activity (13%) after the application of low-intensity high hydrostatic pressure treatment (400 MPa for 5 min). The maximum loss of bioactivity was observed at 600 MPa for 10 min (31 and 20% for whey- and buttermilk-based formulas, respectively). This study provides relevant information on the potential of whey, buttermilk, and HBC to be part of functional products as complementary strategies to combat rotavirus infections.

Whey and Buttermilk-Based Formulas Modulate Gut Microbiota in Mice with Antibiotic-Induced Dysbiosis.

SCOPE: Diet is one of the main factors that modifies intestinal microbiota composition. The search for foods that can reverse situations of intestinal dysbiosis such as that induced by antibiotics is of great interest. Buttermilk and whey are the main by-products produced by the dairy industry containing bioactive compounds. The aim of this study is to investigate the ability of whey and buttermilk-based formulas supplemented with lactoferrin and milk fat globule membrane (MFGM) to modulate the effects of clindamycin on mouse intestinal microbiota. METHODS AND RESULTS: Male C57BL/6 mice are treated with saline (control), clindamycin (Clin), a formula containing whey (F1) or buttermilk (F2), Clin+F1 or Clin+F2, and their fecal microbiota profiles are analyzed by sequencing of 16S rRNA gene using the MinION device. Clin induces alterations in both the composition and metabolic functions of the mice intestinal microbiota. The treatment with F1 or F2 reverses the effects of clindamycin, restoring the levels of Rikenellaceae and Lactobacillaceae families and certain pathways related to short-chain fatty acids production and tetrahydrofolate biosynthesis. CONCLUSION: Whey and buttermilk supplemented with lactoferrin and MFGM may be a bioactive formula for functional foods to prevent or restore microbiota alterations induced by antibiotic administration.

Dairy By-Products and Lactoferrin Exert Antioxidant and Antigenotoxic Activity on Intestinal and Hepatic Cells.

The dairy industry generates a large volume of by-products containing bioactive compounds that may have added value. The aim of this study was to evaluate the antioxidant and antigenotoxic effects of milk-derived products, such as whey, buttermilk, and lactoferrin, in two human cell lines: Caco-2 as an intestinal barrier model and HepG2 as a hepatic cell line. First, the protective effect of dairy samples against the oxidative stress caused by menadione was analyzed. All these dairy fractions significantly reversed the oxidative stress, with the non-washed buttermilk fraction presenting the greatest antioxidant effect for Caco-2 cells and lactoferrin as the best antioxidant for HepG2 cells. At concentrations that did not impact cell viability, we found that the dairy sample with the highest antigenotoxic power against menadione, in both cell lines, was lactoferrin at the lowest concentration. Additionally, dairy by-products maintained their activity in a coculture of Caco-2 and HepG2, mimicking the intestinal-liver axis. This result suggests that the compounds responsible for the antioxidant activity could cross the Caco-2 barrier and reach HepG2 cells on the basal side, exerting their function on them. In conclusion, our results show that dairy by-products have antioxidant and antigenotoxic activities, which would allow revaluing their use in food specialties.

Low P-Selectin Glycoprotein Ligand-1 Expression in Neutrophils Associates with Disease Activity and Deregulated NET Formation in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the generation of anti-DNA autoantibodies due to exposure of immune cells to excessive amounts of extracellular DNA. Lack of P-selectin in mice induces the development of a lupus-like syndrome and patients with cutaneous lupus have reduced P-selectin expression in skin vessels. Using flow cytometry we analyzed in healthy donors and patients the expression of P-selectin Glycoprotein Ligand-1 (PSGL-1) in circulating neutrophils and the implication of PSGL-1/P-selectin interaction in neutrophil extracellular traps (NETs) generation. We found a statistical significance that neutrophils from active SLE patients have a reduced expression of PSGL-1 and low levels of PSGL-1 in neutrophils from SLE patients associated with the presence of anti-dsDNA antibodies, clinical lung involvement, Raynaud's phenomenon, and positive lupus anticoagulant. PSGL-1 is present along the DNA in the NET. In healthy donors, neutrophil interaction with immobilized P-selectin triggers Syk activation, increases the NETs percentage and reduces the amount of DNA extruded in the NETs. In active SLE patients, neutrophil interaction with P-selectin does not activate Syk or reduce the amount of DNA extruded in the NETs, that might contribute to increase the extracellular level of DNA and hence, to disease pathogenesis.

Effect of in vitro gastrointestinal digestion on the antibacterial activity of bioactive dairy formulas supplemented with lactoferrin against Cronobacter sakazakii.

Milk is a source of proteins with high nutritional value and relevant biological activities. Bioactive milk proteins, like lactoferrin, are important for newborn development and can also be used as ingredients in functional products to improve health. Lactoferrin is essential in infant's diet, since protects against infections and promotes immune system maturation. Bovine lactoferrin is used to supplement formula milk in order to strengthen baby's defences against some pathogenic bacteria. Thus, lactoferrin supplemented formula can be a barrier against emergent pathogens, such as Cronobacter sakazakii, which has caused great concern in the last few years. Milk proteins generate bioactive peptides in the digestion process, and it is known that industrial processing can modify their susceptibility to digestion. Treatments such as heating have been shown to denature whey proteins and make them more easily digestible. Therefore, the aim of this study was to analyze the effect of technological treatments and gastrointestinal digestion on the antibacterial activity against C. sakazakii of proteins present in dairy formulas supplemented with lactoferrin. Commercial bovine lactoferrin has been shown to have antibacterial activity against C. sakazakii, both in the native state and after static in vitro gastrointestinal digestion. In addition, the digests obtained from dairy formulas subjected to technological treatments, either homogenization or pasteurization, have higher antibacterial activity than non-treated formulas. The release of low molecular weight peptides during the in vitro gastric digestion is probably the cause that would explain the enhanced antibacterial activity of the digested dairy formulas.

Lactoferrin modulates gut microbiota and Toll-like receptors (TLRs) in mice with dysbiosis induced by antibiotics.

Background: Antibiotic administration can result in gut microbiota and immune system alterations that impact health. Bovine lactoferrin is a milk protein with anticancer, anti-inflammatory, antimicrobial and immune modulatory activities. The aim was to study the ability of native and iron-saturated lactoferrin to reverse the effects of clindamycin on gut microbiota and intestinal Toll-like receptor (TLR) expression in a murine model. Methods: Male C57BL/6 mice were treated with vehicle, clindamycin (Clin), native bovine lactoferrin (nLf), nLf + clindamycin (nLf_Clin), iron-saturated bovine lactoferrin (sLf) and sLf + clindamycin (sLf_Clin). Fecal samples of each group were collected, and bacterial DNA was extracted. Sequencing of 16s rRNA V4 hypervariable gene regions was conducted to assess the microbial composition. mRNA expression levels of TLRs (1-9) were determined in mouse colon by qPCR. Pearson's correlation test was carried out between bacteria showing differences in abundance among samples and TLR2, TLR8 and TLR9. Results: Beta-diversity analysis showed that the microbial community of the vehicle was different from the communities of Clin, nLf_Clin and sLf_Clin. At the family level, Bacteroidaceae, Prevotellaceae and Rikenellaceae decreased in the Clin group, and treatment with nLf or sLf reverted these effects. Clin reduced the expression of TLR2, TLR8 and TLR9 and sLf reverted the decrease in the expression of these receptors. Finally, TLR8 was positively correlated with Rikenellaceae abundance. Conclusion: In a situation of intestinal dysbiosis induced by clindamycin, lactoferrin restores the normal levels of some anti-inflammatory bacteria and TLRs and, therefore, could be a good ingredient to be added to functional foods.

Development of Encapsulation Strategies and Composite Edible Films to Maintain Lactoferrin Bioactivity: A Review.

Lactoferrin (LF) is a whey protein with various and valuable biological activities. For this reason, LF has been used as a supplement in formula milk and functional products. However, it must be considered that the properties of LF can be affected by technological treatments and gastrointestinal conditions. In this article, we have revised the literature published on the research done during the last decades on the development of various technologies, such as encapsulation or composite materials, to protect LF and avoid its degradation. Multiple compounds can be used to conduct this protective function, such as proteins, including those from milk, or polysaccharides, like alginate or chitosan. Furthermore, LF can be used as a component in complexes, nanoparticles, hydrogels and emulsions, to encapsulate, protect and deliver other bioactive compounds, such as essential oils or probiotics. Additionally, LF can be part of systems to deliver drugs or to apply certain therapies to target cells expressing LF receptors. These systems also allow improving the detection of gliomas and have also been used for treating some pathologies, such as different types of tumours. Finally, the application of LF in edible and active films can be effective against some contaminants and limit the increase of the natural microbiota present in meat, for example, becoming one of the most interesting research topics in food technology.

Comparative effect of bovine buttermilk, whey, and lactoferrin on the innate immunity receptors and oxidative status of intestinal epithelial cells.

Milk contains bioactive molecules with important functions as defensive proteins; among them are the whey protein lactoferrin and proteins of the milk fat globule membrane (MFGM) present in buttermilk. The aim of this study has been to investigate the effects of lactoferrin, whey, and buttermilk as modulators of intestinal innate immunity and oxidative stress on intestinal epithelial cells, to evaluate its potential use for the development of functional foods. The mRNA expression levels of innate immune system Toll-like receptors (TLR2, TLR4, and TLR9), lipid peroxidation (malondialdehyde + 4-hydroxyalkenals) and protein expression levels of carbonyl were analyzed in enterocyte-like Caco-2/TC7 cells treated for 24 h with different concentrations of lactoferrin, whey, or buttermilk. None of the substances analyzed caused oxidative damage; however, whey significantly decreased the levels of lipid peroxidation. Furthermore, both lactoferrin and whey reduced the oxidative stress induced by lipopolysaccharide. With respect to TLR receptors, lactoferrin, whey, and buttermilk specifically altered the expression of TLR2, TLR4, and TLR9 receptors, with a strong decrease in the expression levels of TLR4. These results suggest that lactoferrin, whey, and buttermilk are potentially interesting ingredients for functional foods because they seem to modulate oxidative stress and the inflammatory response induced by the activation of TLRs.

Effect of hydrolysis and microwave treatment on the antibacterial activity of native bovine milk lactoferrin against Cronobacter sakazakii.

Bovine lactoferrin (bLF) is an iron-binding glycoprotein used in functional and therapeutic products due to its biological properties, the most important being its antimicrobial activity. In this study, hydrolysates of bovine lactoferrin (bLFH) obtained with pepsin, chymosin and microbial rennet were assayed against Cronobacter sakazakii (104 CFU/mL) in different media: phosphate buffered saline (PBS), bovine skim milk and whey, and reconstituted powdered infant formula (PIFM). The results obtained have shown that hydrolysis of bLF enhances its antibacterial activity against C. sakazakii. The three types of bLFH dissolved in PBS reduced C. sakazakii growth from a concentration of 0.1 mg/mL and inhibited it completely above 0.5 mg/mL, after 4 and 8 h of incubation at 37 °C. The three bLFH (1 and 2 mg/mL) did not show any antibacterial activity in skim milk, whey and reconstituted PIFM after 8 h of incubation at 37 °C. However, C. sakazakii growth was completely inhibited in whey when pepsin and chymosin bLFH (2 mg/mL) were combined with undigested bLF (2 mg/mL), after 8 h of incubation at 37 °C. On the other hand, the combination of any of the three hydrolysates with bLF showed very low activity in skim milk and practically no activity in reconstituted PIFM. Furthermore, the effect of temperature after reconstitution (4, 23 and 37 °C), on the antibacterial activity of bLF (2.5 and 5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also investigated. bLF at 5 mg/mL significantly reduced (p < .05) the proliferation of C. sakazakii in reconstituted PIFM at 37 °C until 2 h. C. sakazakii did not grow at 4 °C for 6 days in reconstituted PIFM with or without bLF. The effect of microwave heating (450, 550 and 650 W for 5, 10 and 15 s) on the antibacterial activity and stability of bLF (2.5 mg/mL) in reconstituted PIFM contaminated with C. sakazakii (10-102 CFU/mL) was also studied. The antibacterial activity of bLF was maintained after treatments at 450 and 550 W for 5 s, which kept 94 and 89% of bLF immunoreactivity, respectively. Moreover, microwave treatments of reconstituted PIFM with or without bLF, at 650 W for 5 s, and at 450, 550 and 650 W for 10 and 15 s, completely inactivated C. sakazakii.