Identification of stemness-related glycosylation changes in head and neck squamous cell carcinoma.

BACKGROUND: Altered glycosylation is a hallmark of cancer associated with therapy resistance and tumor behavior. In this study, we investigated the glycosylation profile of stemness-related proteins OCT4, CIP2A, MET, and LIMA1 in HNSCC tumors. METHODS: Tumor, adjacent normal tissue, and blood samples of 25 patients were collected together with clinical details. After tissue processing, lectin-based glycovariant screens were performed. RESULTS: Strong correlation between glycosylation profiles of all four stemness-related proteins was observed in tumor tissue, whereas glycosylation in tumor tissue, adjacent normal tissue, and serum was differential. CONCLUSIONS: A mannose- and galactose-rich glycosylation niche associated with stemness-related proteins was identified.

Narrowing down the number of potential plant-based probiotic candidates by successive in vitro, technological and in vivo assays.

The interest on plant-based fermented food is in raise in Western countries. The aim of this study was to select interleukin (IL)-10 inducing strains for the development of potential probiotic plant-based fermented foods. Departing from a collection of 52 lactic acid bacteria (LAB) strains derived from plant material, in vitro co-culture with murine macrophages allowed us to narrow down the number of candidates to 21 strains able to induce IL-10 secretion. 14 of these strains were able to promote the production of tumour necrosis factor-α too. The capacity to induce IL-6 was used to further reduce the number of strains to 4, from which Lactiplantibacillus plantarum subsp. plantarum LpAv was selected to ferment oat and carrots. L. plantarum LpAv was able to ferment oat and carrots until reaching counts of ca. 108 and 109 cfu/ml. Fermented oat and carrots were orally administered to mice for 10 consecutive days and challenged with a single infective dose of Salmonella enterica serovar. Typhimurium. Counts of L. plantarum LpAv in fermented carrots were 9.23±0.05 cfu/ml and 9.27±0.01 cfu/ml, at day 1 and 10 of the feeding period. Fermented carrots were able to confer enhanced protection (80% of survival) against infection, when compared to control mice (less than 25% of survival). However, L. plantarum LpAv administered as pure culture was not able to confer protection against Salmonella infection. L. plantarum LpAv was selected among 52 plant-derived LAB and it was able to ferment oat and carrots, being only fermented carrots able to confer enhanced protection against Salmonella infection. A succession of in vitro to in vivo tests is proposed as a tool to narrow down the number of candidates when searching for potential novel probiotics from a collection of autochthonous strains.

Occurrence of bacteria with technological and probiotic potential in Argentinian human breast-milk.

Breast milk can be a source of potential probiotic bacteria, but the technological capacity of isolates obtained from this source is not always guaranteed. We aimed at isolating lactobacilli from breast milk samples collected in Argentina, focusing on isolates with functional and technological potential as probiotics. Fourteen Lactobacillus and one Bifidobacterium isolates were obtained from 164 samples donated by 104 mothers. The isolates preliminarily identified by MALDI-TOF, and then the identity was confirmed by partial 16S rRNA gene sequencing. Hydrophobicity was determined (hexadecane and xylene partition). The strains were also co-cultured with murine RAW 264.7 macrophages for screening the capacity to induce the anti-inflammatory cytokine interleukin (IL)-10. Hydrophobicity ranged from 7.4 and 95.9%. The strains Lactobacillus gasseri (70a and 70c) and Lactobacillus plantarum (73a and 73b) were the strains with a higher capacity to induce IL-10 production by macrophages. The technological application was evaluated by freezing dried in 10% lactose or 10% polydextrose. The survival was assessed after accelerated (37 °C, 4 weeks) or long-term (5 and 25 °C, 12 months) storage. Except for Lactobacillus gallinarum 94d, strains lost less than 1 Log10 order cfu/g after long-term (12 months) storage at 5 °C in lactose and polydextrose as protectants. A low correlation between survival to accelerated and long-term storage tests was observed. L. gasseri (70a and 70c) and L. plantarum (73a and 73b) deserve further studies as potential probiotics due to their capacity to induce IL-10 from murine macrophages and their hydrophobicity. In special, L. plantarum 73a was able to confer enhanced protection against Salmonella infection by promoting the immunity of the small intestine.

Postbiotics: facts and open questions. A position paper on the need for a consensus definition.

The past definitions of probiotics and prebiotics have been reviewed and updated recently. According to these concepts, probiotics comprise live microorganisms that confer a health benefit on the host when administered in adequate amounts, whereas a prebiotic is a substrate that is selectively utilised by host microorganisms, conferring a health benefit. The words probiotics and prebiotics can be found on labels of many foods and supplements. Consumers have a growing awareness of these terms' meanings, and many countries are increasingly using them for regulation purposes. At the same time, there is increasing evidence on the health effects of non-viable microorganisms and the metabolites that they can produce by fermentation or by their action on food components. Different terms have been used in the literature to refer to these bioactive compounds, which do not fall under the known categories of probiotics, prebiotics or synbiotics. The tentative term postbiotics has been the most used one so far. However, no definition of the term has gained international consensus to date. This work aims to provide information on the facts and the open questions about the so-called postbiotics.

Rapid review shows that probiotics and fermented infant formulas do not cause d-lactic acidosis in healthy children.

AIM: Extensive ongoing research on probiotics and infant formulas raises a number of safety questions. One concern is the potential influence of d-lactic acid-containing preparations on the health of infants and children. The aim of this review was to summarise the available knowledge on the ingestion of d-lactic acid-producing bacteria, acidified infant formulas and fermented infant formulas as a potential cause of paediatric d-lactic acidosis. METHODS: A Medline database search was performed in July 2017, with no restrictions on the language, article type or publication date. The 1715 search results were screened for clinical trials, review articles, case series and case reports of relevance to the topic. RESULTS: We identified five randomised controlled trials from 2005 to 2017 covering 544 healthy infants and some case reports and experimental studies. No clinically relevant adverse effects of d-lactic acid-producing probiotics and fermented infant formulas were described in healthy children. However, a harmless, subclinical accumulation of d-lactate was theoretically possible. The only known cases of paediatric d-lactic acidosis occurred in patients with short bowel syndrome or, historically, in infants fed with acidified formulas. CONCLUSION: Our main finding was that probiotics and fermented formulas did not cause d-lactic acidosis in healthy children.

Human milk oligosaccharide categories define the microbiota composition in human colostrum.

Human milk oligosaccharides (HMOs) are structurally diverse unconjugated glycans with a composition unique to each lactating mother. While HMOs have been shown to have an impact on the development of infant gut microbiota, it is not well known if HMOs also already affect milk microbial composition. To address this question, we analysed eleven colostrum samples for HMO content by high-pressure liquid chromatography and microbiota composition by quantitative PCR. Higher total HMO concentration was associated with higher counts of Bifidobacterium spp. (ρ=0.63, P=0.036). A distinctive effect was seen when comparing different HMO groups: positive correlations were observed between sialylated HMOs and Bifidobacterium breve (ρ=0.84, P=0.001), and non-fucosylated/non-sialylated HMOs and Bifidobacterium longum group (ρ=0.65, P=0.030). In addition to associations between HMOs and bifidobacteria, positive correlations were observed between fucosylated HMOs and Akkermansia muciniphila (ρ=0.70, P=0.017), and between fucosylated/sialylated HMOs and Staphylococcus aureus (ρ=0.75, P=0.007). Our results suggest that the characterised HMOs have an effect on specific microbial groups in human milk. Both oligosaccharides and microbes provide a concise inoculum for the compositional development of the infant gut microbiota.

Late preterm birth has direct and indirect effects on infant gut microbiota development during the first six months of life.

AIM: Preterm infants display aberrant gut microbial colonisation. We investigated whether the differences in gut microbiota between late preterm and full-term infants results from prematurity or external exposures. METHODS: This study comprised 43 late preterm infants (340/7 -366/7 ) and 75 full-term infants based on faecal samples collected following birth and at two to four weeks and six months of age. We assessed clinically relevant bacteria using quantitative polymerase chain reaction. Logistic regression analyses were performed to determine whether the observed differences in gut microbiota were attributable to prematurity or perinatal exposure. RESULTS: The prevalence of bifidobacteria differed in the intestinal microbiota of the full-term and late preterm neonates. Differences in the presence of specific species were detected at the age of six months, although the microbiota alterations were most prominent following delivery. As well as prematurity, the mode of birth, intrapartum and neonatal antibiotic exposure, and the duration of breastfeeding had an additional impact on gut microbiota development. CONCLUSION: The gut microbiota composition was significantly different between late preterm and full-term infants at least six months after birth. Antibiotic exposure was common in late preterm infants and modulated gut colonisation, but preterm birth also affected gut microbiota development independently.

Akkermansia muciniphila: a novel functional microbe with probiotic properties.

Akkermansia muciniphila is an intestinal anaerobe which has been proposed as a new functional microbe with probiotic properties. However, the species is not included in the European Union qualified presumption of safety (QPS) list and has not yet been assessed. Moreover, products containing A. muciniphila are not on the market and are thus controlled by the Novel Foods Regulation, which requires extensive safety assessment. This review addresses the safety aspects of the use of A. muciniphila based on published information on its functions in humans and predictions based on its activity in model animals. Further, comprehensive studies related to A. muciniphila and its safety properties have gradually appeared and are summarised here. Many of the criteria required for novel food safety assessment in Europe can thus be fulfilled. However, studies focusing on the toxicological properties of A. muciniphila, including long-term and reproduction studies, have not so far been reported and are discussed in the light of the observation that most, if not all, healthy subjects are known to carry this intestinal anaerobe. As this also applies to other beneficial bacteria found in the human intestinal tract, the A. muciniphila case can be seen as a model for the comprehensive safety evaluations required by the European authorities.

The case in favour of probiotics before, during and after pregnancy: insights from the first 1,500 days.

Successful human reproduction requires microbial homeostasis in the female reproductive tract, and colonisation of the newborn with beneficial microbes. In order to prevent several complications associated with dysbiosis, the administration of probiotics is more often being considered. The objective of the enclosed review was to examine the rationale for probiotic utility before and during pregnancy and in the early phase of infant life. The conclusions emerged from a panel of researchers who met during the International Scientific Association for Probiotics and Prebiotics (ISAPP) workshop held in Washington, DC, USA in 2015. The group concluded based upon the current literature, that a case can be made for the use of a specific sets of probiotic organisms during the first 1,500 days of life, with the goal of a healthy pregnancy to term, and a healthy start to life with lowered risk of infections and inflammatory events. The key to successfully translating these recommendations to practice is that products be made available and affordable to women in developed and developing countries.

Role of probiotics in reducing the risk of gestational diabetes.

Overweight and obesity currently constitute a major threat to human well-being. Almost half of the female population are currently overweight. Pregnant overweight women are at risk of gestational diabetes affecting the health of the mother and the child, in both the short and long term. Notwithstanding the extensive scientific interest centred on the problem, research efforts have thus far been unable to devise preventive strategies. Recent scientific advances point to a gut microbiota dysbiosis, with ensuing low-grade inflammation as a contributing element, in obesity and its comorbidities. Such findings would suggest a role for specific probiotics in the search for preventive and therapeutic adjunct applications in gestational diabetes. The aim of the present paper was to critically review recent demonstrations of the role of intestinal microbes in immune and metabolic regulation, which could be exploited in nutritional management of pregnant women by probiotic bacteria. By modulating specific target functions, probiotic dietary intervention may exert clinical effects beyond the nutritional impact of food. As this approach in pregnancy is new, an overview of the role of gut microbiota in shaping host metabolism, together with the definition of probiotics are presented, and finally, specific targets and potential mechanisms for probiotics in pregnancy are discussed. Pregnancy appears to be the most critical stage for interventions aiming to reduce the risk of non-communicable disease in future generations, beyond the immediate dangers attributable to the health of the mother, labour and the neonate. Specific probiotic interventions during pregnancy provide an opportunity, therefore, to promote the health not only of the mother but also of the child.

The impact of early gut microbiota modulation on the risk of child disease: alert to accuracy in probiotic studies.

The composition of the gut microbiota, and thus also the modification of the gut microbiota by specific probiotics or prebiotics early in life, may have an impact on the risk of disease in the child. Above the impact on gut microecology, probiotic effects have been attributed to restoration to normal of increased intestinal permeability, improvement of the intestine's immunological barrier functions, alleviation of the intestinal inflammatory response, and reduced generation of proinflammatory cytokines characteristic of local and systemic allergic inflammation. Recent demonstrations from experimental and clinical studies suggest that the gut microbiota is also involved in the control of body weight and energy metabolism, affecting the two main causes of obesity: energy acquisition and storage, and contributing to insulin resistance and the inflammatory state characterising obesity. Current research focuses both on characterising specific probiotic strains and on how the food matrix and the dietary content interacts with the most efficient probiotic strains. It is important to characterise each probiotic to species and strain level and to select strains with documented properties, the probiotic potential being strain-specific. As any proof of causality requires clinical intervention studies in humans in different populations, rigorous and detailed documentation will enhance reproducibility and circumvent confusion.

Severity of atopic disease inversely correlates with intestinal microbiota diversity and butyrate-producing bacteria.

The reports on atopic diseases and microbiota in early childhood remain contradictory, and both decreased and increased microbiota diversity have been associated with atopic eczema. In this study, the intestinal microbiota signatures associated with the severity of eczema in 6-month-old infants were characterized. Further, the changes in intestinal microbiota composition related to the improvement of this disease 3 months later were assessed. The severity of eczema correlated inversely with microbiota diversity (r = -0.54, P = 0.002) and with the abundance of butyrate-producing bacteria (r = -0.52, P = 0.005). During the 3-month follow-up, microbiota diversity increased (P < 0.001) and scoring atopic dermatitis values decreased (P < 0.001) in all infants. This decrease coincided with the increase in bacteria related to butyrate-producing Coprococcus eutactus (r = -0.59, P = 0.02). In conclusion, the high diversity of microbiota and high abundance of butyrate-producing bacteria were associated with milder eczema, thus suggesting they have a role in alleviating symptoms of atopic eczema.

Pathogen exclusion properties of canine probiotics are influenced by the growth media and physical treatments simulating industrial processes.

AIMS: Manufacturing process used in preparation of probiotic products may alter beneficial properties of probiotics. The effect of different growth media and inactivation methods on the protective properties of canine-originated probiotic bacteria against adhesion of canine enteropathogens was investigated. METHODS AND RESULTS: Three established dog probiotics, Lactobacillus fermentum VET9A, Lactobacillus plantarum VET14A and Lactobacillus rhamnosus VET16A, and their mixture were assessed using the dog mucus pathogen exclusion model. The pathogens used were Enterococcus canis, Salmonella enterica serovar Typhimurium and Clostridium perfringens. The effect of growth media, one reflecting laboratory and the other manufacture conditions, and viability (viable and heat inactivated, 80°C per 30 min) on the pathogen exclusion properties of probiotics were characterized. Greater pathogen exclusion percentages were noted for probiotics growing in conditions reflecting manufacture when compared to laboratory (P < 0·05). Inactivation of probiotics by heat (80°C per 30 min) increased pathogen exclusion compared with their viable forms (P < 0·05). CONCLUSIONS: Manufacturing process conditions such as growth media, incubation temperature and pretreatment methods may significantly affect the protective properties of the tested strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Growing conditions and pretreatment methods should be carefully considered when designing new probiotics to reduce the risk of common infections in dogs. The studied probiotics are promising potential feed additives for dogs.

Assessment of stress tolerance acquisition in the heat-tolerant derivative strains of Bifidobacterium animalis subsp. lactis BB-12 and Lactobacillus rhamnosus GG.

AIMS: The purpose of this study was to investigate the heat-shock response at molecular level in Lactobacillus rhamnosus GG, Bifidobacterium animalis subsp. lactis BB-12 and their heat-tolerant derivatives and to characterize the changes that make the derivatives more robust in terms of heat stress. METHODS AND RESULTS: The study strains were exposed for 2 h to a heat-shock treatment, Bif. animalis subsp. lactis BB-12 and its derivative at 50°C and the Lact. rhamnosus GG and its derivative at 60°C. Protein synthesis before and after heat shock was examined using proteomics and RT-qPCR. The analysis revealed that the regulation of seven proteins in both strain pairs was modified as a response to heat or between the original and the derivative strain. The comparison of wild-type strains and the heat-tolerant derivatives suggests that the acquisition of heat tolerance in the Bif. animalis subsp. lactis BB-12 derivative is due to a slightly increased constitutive level of chaperones, while in Lact. rhamnosus GG derivative, the main reason seems to be a higher ability to induce the production of chaperones. CONCLUSIONS: This study revealed possible markers of heat tolerance in B. lactis and Lact. rhamnosus strains. SIGNIFICANCE AND IMPACT OF STUDY: This study increases our knowledge on how Lactobacillus and Bifidobacterium strains may acquire heat tolerance. These findings may be useful for improving the heat tolerance of existing probiotic strains as well as screening new heat-tolerant strains.

Yoghurt and probiotic bacteria in dietary guidelines of the member states of the European Union.

Beneficial microbes enter the food supply primarily through fermented foods (largely milks) and addition of probiotics. Fermented milks are a significant component of the diet of some geographical regions, including Europe. The fermentation process serves to preserve safety, nutritional quality and palatability of milk. In addition, the microbes associated with fermented milks and probiotics are associated with human health benefits. However, in the area of health claims only one claim for beneficial microbes has been approved in the European Union, that is for yoghurt to improve lactose tolerance. We searched for health messages that include probiotics or fermented milks in nutrition guidelines and recommendations in thirteen countries of the EU plus Switzerland. Such messages are allowed when they are made by non-commercial government bodies. Our analysis revealed that five EU member states have national nutrition guidelines or recommendations that include either probiotics or fermented milks with live bacteria. This supports that some EU member states recognise health benefits associated with consumption of live microbes, even if commercial marketing claims are not authorised. Harmonisation between recommendations and approved health-claims would benefit consumers and public health.

A method to collect to high volumes of milk from mice

La obtención de leche a partir de ratones de laboratorio (Mus musculus) podría ser interesante para una gran variedad de estudios preclínicos. Son muy escasas las referencias en la literatura científica sobre protocolos que describan como obtener estas muestras, siendo la principal limitación de tales protocolos el pequeño volumen de muestra que es habitual obtener. El objetivo de nuestro estudio fue desarrollar un método funcional para obtener fácilmente cantidades considerables de leche de ratón. Hembras adultas de la variedad no consanguíneas NMRI y de la consanguínea BALB/c con su camada fueron utilizadas en este estudio. La leche fue recogida entre los días 7-12 tras el parto. Las crías fueron separadas de sus madres entre 6-12 horas antes del ordeño para permitir la acumulación de leche en las glándulas. Las hembras fueron anestesiadas usando o una mezcla de midazolam y ketamina, o empleando isoflurano como agente inhalatorio. Para inducir la eyección de la leche, se administro una dosis de 2-8 UI de oxitocina intraperitonealmente. La leche fue recogida usando un sacaleches eléctrico para humanos que fue modificado para adaptarse al pezón de los ratones y para recoger pequeños volúmenes. Con este procedimiento, la cantidad de leche obtenida de los ratones osciló entre los 0,2 y los 1,5 ml. Concluimos por tanto, que este método proporciona una excelente manera de adquirir cantidades considerables de leche de ratón (AU)

Collecting milk samples from mice (Mus musculus) may be interesting for a variety of preclinical research. References in the literature for protocols describing how to milk a dam are scarce, and a major limitation of such protocols is the small sample volume that is generally collected. The aim of our study was to develop a practical protocol to collect substantial amounts of milk from mice. Adult female outbred NMRI and inbred BALB/c mice with nursing litters were used in this study. The milking was carried out on days 7–12 after parturition. The pups were separated from their mothers for 6–12 h before milking to allow accumulation of milk in the glands. Dams were anesthetized using either an injectable mixture of midazolam and ketamine, or by use of the inhalational agent isoflurane. To induce milk flow, the mice were given 2-8 IU of oxytocin intraperitoneally. The milk was collected using an electric human breast pump that was modified to accommodate mouse nipples and to handle small liquid volumes. With this protocol, the total amount of milk collected from each dam per each milking ranged between 0.2 and 1.5 mL. We concluded that this milking method provides an excellent means for acquiring substantial amounts of mouse milk (AU)

Reshaping the gut microbiota at an early age: functional impact on obesity risk?

Overweight and obesity can currently be considered a major threat to human health and well-being. Recent scientific advances point to an aberrant compositional development of the gut microbiota and low-grade inflammation as contributing factors, in conjunction with excessive energy intake. A high-fat/energy diet alters the gut microbiota composition, which reciprocally engenders excessive energy harvesting and storage. Further, microbial imbalance increases gut permeability, leading to metabolic endotoxemia, inflammation and insulin resistance. Local intestinal immunologic homeostasis is achieved by tolerogenic immune responses to microbial antigens. In the context of amelioration of insulin sensitivity and decreased adiposity, the potential of gut microbiota modulation with specific probiotics and prebiotics lies in the normalization of aberrant microbiota, improved gut barrier function and creation of an anti-inflammatory milieu. This would suggest a role for probiotic/prebiotic interventions in the search for preventive and therapeutic applications in weight management.

The effect of growth media and physical treatments on the adhesion properties of canine probiotics.

AIMS: The manufacturing processes have been reported to influence the properties of probiotics with potential impact on health properties. The aim was to investigate the effect of different growth media and inactivation methods on the properties of canine-originated probiotic bacteria alone and in combination mixture. METHODS AND RESULTS: Three established dog probiotics, Lactobacillus fermentum VET9A, Lactobacillus plantarum VET14A and Lactobacillus rhamnosus VET16A, and their combination mixture were evaluated for their adhesion to dog mucus. The effect of different growth media, one reflecting laboratory and the other manufacturing conditions, and inactivation methods (95°C, 80°C and UV irradiation) on the mucus adhesion of the probiotic strains was characterized. Evaluation of dog probiotics was supported by cell visualization using transmission electron microscopy (TEM). Higher adhesion percentage was reported for probiotic strains growing in laboratory rather than in manufacturing conditions (P < 0.05). Inactivation by heat (95°C, 80°C) decreased the adhesion properties when strains were cultivated in soy-based growth media compared with those grown in MRS broth (P < 0.05). TEM observations uncovered differences in cell-surface components in nonviable forms of probiotic strains as compared with their viable forms. CONCLUSIONS: Manufacturing process conditions such as growth media and pretreatment methods may significantly affect the adhesive ability of the tested strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Growth conditions, growth media, pretreatment methods and different probiotic combinations should be carefully considered for quality control of existing probiotics and for identification of new probiotics for dogs. These may also have an impact on health benefits for the host.

Interaction of Campylobacter spp. and human probiotics in chicken intestinal mucus.

Campylobacter is the most common cause of bacterial food-borne diarrhoeal disease throughout the world. The principal risk of human contamination is handling and consumption of contaminated poultry meat. To colonize poultry, Campylobacter adheres to and persists in the mucus layer that covers the intestinal epithelium. Inhibiting adhesion to the mucus could prevent colonization of the intestine. The aim of this study was to investigate in vitro the protective effect of defined commercial human probiotic strains on the adhesion of Campylobacter spp. to chicken intestinal mucus, in a search for alternatives to antibiotics to control this food-borne pathogen. The probiotic strains Lactobacillus rhamnosus GG and Propionibacterium freudenreichii ssp. shermanii JS and a starter culture strain Lactococcus lactis ssp. lactis adhered well to chicken intestinal mucus and were able to reduce the binding of Campylobacter spp. when the mucus was colonized with the probiotic strain before contacting the pathogen. Human-intended probiotics could be useful as prophylactics in poultry feeding for controlling Campylobacter spp. colonization.

Workshop Report: concepts and methods in the economics of nutrition--gateways to better economic evaluation of nutrition interventions.

Improving health through better nutrition of the population may contribute to enhanced efficiency and sustainability of healthcare systems. A recent expert meeting investigated in detail a number of methodological aspects related to the discipline of nutrition economics. The role of nutrition in health maintenance and in the prevention of non-communicable diseases is now generally recognised. However, the main scope of those seeking to contain healthcare expenditures tends to focus on the management of existing chronic diseases. Identifying additional relevant dimensions to measure and the context of use will become increasingly important in selecting and developing outcome measurements for nutrition interventions. The translation of nutrition-related research data into public health guidance raises the challenging issue of carrying out more pragmatic trials in many areas where these would generate the most useful evidence for health policy decision-making. Nutrition exemplifies all the types of interventions and policy which need evaluating across the health field. There is a need to start actively engaging key stakeholders in order to collect data and to widen health technology assessment approaches for achieving a policy shift from evidence-based medicine to evidence-based decision-making in the field of nutrition.