Breast Milk, a Source of Beneficial Microbes and Associated Benefits for Infant Health

Katríona E. Lyons 1,2, C. Anthony Ryan 3,4, Eugene M. Dempsey 3,4,5, R. Paul Ross 3 and Catherine Stanton 1,3,*

1 Teagasc Food Research Centre, Moorepark, Fermoy, Co. Cork P61 C996, Ireland; katriona.Lyons@teagasc.ie

2 School of Microbiology, University College Cork, Cork T12 YN60, Ireland

3 APC Microbiome Ireland, University College Cork, Cork T12 YT20, Ireland

4 Department of Neonatology, Cork University Maternity Hospital, Cork T12 YE02, Ireland

5 INFANT Research Centre, University College Cork, Cork T12 DFK4, Ireland

* Correspondence: catherine.stanton@teagasc.ie; Tel.: +353-254-2606

Abstract:

Human breast milk is considered the optimum feeding regime for newborn infants due to its ability to provide complete nutrition and many bioactive health factors. Breast feeding is associated with improved infant health and immune development, less incidences of gastrointestinal disease and lower mortality rates than formula fed infants. As well as providing fundamental nutrients to the growing infant, breast milk is a source of commensal bacteria which further enhance infant health by preventing pathogen adhesion and promoting gut colonisation of beneficial microbes. While breast milk was initially considered a sterile fluid and microbes isolated were considered contaminants, it is now widely accepted that breast milk is home to its own unique microbiome. The origins of bacteria in breast milk have been subject to much debate, however, the possibility of an entero-mammary pathway allowing for transfer of microbes from maternal gut to the mammary gland is one potential pathway. Human milk derived strains can be regarded as potential probiotics; therefore, many studies have focused on isolating strains from milk for subsequent use in infant health and nutrition markets. This review aims to discuss mammary gland development in preparation for lactation as well as explore the microbial composition and origins of the human milk microbiota with a focus on probiotic development.

Keywords: mammary gland; breast milk; human milk oligosaccharides (HMOs), human milk microbiome; lactation; probiotic; entero-mammary pathway

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Contributions to human breast milk microbiome and enteromammary transfer of Bifidobacterium breve

Kattayoun Kordy 1,2☯, Thaidra Gaufin3‡, Martin Mwangi3☯, Fan Li1,2,3☯, Chiara Cerini1‡, David J. Lee1☯, Helty Adisetiyo1‡, Cora Woodward3‡, Pia S. Pannaraj1,2‡, Nicole H. Tobin3☯, Grace M. Aldrovandi3☯*

1 Department of Pediatrics, Children’s Hospital of Los Angeles, Los Angeles, CA, United States of America,

2 Department of Pediatrics, University of Southern California, Los Angeles, CA, United States of America,

3 Department of Pediatrics, University of California Los Angeles, Los Angeles, CA, United States of America

☯ These authors contributed equally to this work.

‡ These authors also contributed equally to this work.

* GAldrovandi@mednet.ucla.edu

Abstract

Increasing evidence supports the importance of the breast milk microbiome in seeding the infant gut. However, the origin of bacteria in milk and the process of milk microbe-mediated seeding of infant intestine need further elucidation. Presumed sources of bacteria in milk include locations of mother-infant and mother-environment interactions. We investigate the role of mother-infant interaction on breast milk microbes. Shotgun metagenomics and 16S rRNA gene sequencing identified milk microbes of mother-infant pairs in breastfed infants and in infants that have never latched. Although breast milk has low overall biomass, milk microbes play an important role in seeding the infant gut. Breast milk bacteria were largely comprised of Staphylococcus, Streptococcus, Acinetobacter, and Enterobacter primarily derived from maternal areolar skin and infant oral sites in breastfeeding pairs. This suggests that the process of breastfeeding is a potentially important mechanism for propagation of breast milk microbes through retrograde flux via infant oral and areolar skin contact. In one infant delivered via Caesarian section, a distinct strain of Bifidobacteria breve was identified in maternal rectum, breast milk and the infant’s stool potentially suggesting direct transmis- sion. This may support the existence of microbial translocation of this anaerobic bacteria via the enteromammary pathway in humans, where maternal bacteria translocate across the maternal gut and are transferred to the mammary glands. Modulating sources of human milk microbiome seeding potentially imply opportunities to ultimately influence the develop- ment of the infant microbiome and health.

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PERFECT SOLUTION FOR GESTATIONAL DIABETES

1. RESEARCH TASKS AND OBJECTIVES

People's pursuit of healthy living has increased the demand for nutrition and health. Healthy life depends on three major support: reasonable nutrition intake, proper regular exercise, and healthy psychological status.Nutritional health management is influenced by many factors such as individual, age, gender, physical condition, medical history, living habits and environmental conditions.Different people need different nutritional diet management programs to deal with the same health problem, personalized needs are fundamentally more in line with the concept of nutrition theory, while products of prevention and treatment with specific diseases as the core are becoming new intervention model to be promoted in society.

Pregnant women, newborns and children are a social health symbol and an important part of social productivity and overall national strength.Paying attention to nutrition and health in the early stages of life is a major livelihood issue and an important socio-economic issue.Although there are a series of effective maternal and child nutrition improvement interventions in China, the actual nutrition management support in providing health services is often insufficient, and there are also lack of corresponding evaluation indicators, technical service standards, norms and professional and technical personnel.

In October 2018, the first phase of the “Perfect Pregnant Mother Training Camp” exploration project provided a practical basis for this study. In this study, we will undertake a centralized, group-based health management model combine with nutrition, exercise, psychology, and on-line health management, in which the core value lies in providing more professional and personalized nutrition intervention programs and products through exploration of health management models and the interpretation of multidimensional data , and exploring the importance of incorporating adult chronic disease prevention and control into the early life health management system.

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A prospective, proof-of-concept, clinical study on gut microbiota in patients with metabolic syndrome, type 2 diabetes and obesity

1. STUDY TYPE

- A prospective, proof-of-concept, interventional study on gut microbiota composition and function in patients with metabolic syndrome,and/or type 2 diabetes and/or obesity
- treated with standard therapy

2. SPONSOR-INVESTIGATOR

Profit study

3. LOCAL INVESTIGATORS / SUBINVESTIGATORS

- Principal Investigator: Prof. Antonio Gasbarrini
- Sub-Investigators: to be defined

4. PROTOCOL VERSION AND DATE

- Version 1, 08th July 2019

5. TRIAL MANAGEMENT AND COORDINATING FACILITY

- Fondazione Policlinico Universitario Agostino Gemelli IRCCS
- Center for Digestive Diseases 
- Internal Medicine and Gastroenterology Unit
- Largo Agostino Gemelli, 8
- 00168 Rome
- Italy

CLINICAL STUDY PROTOCOL
INVESTIGATOR SIGNATURE PAGE

Study Title: A prospective, proof-of-concept, clinical study on gut microbiota in patients with metabolic syndrome, type 2 diabetes and obesity

The Principal-Investigator has approved the protocol version dated and confirms hereby to conduct the study according to the protocol, current version of the World Medical Association Declaration of Helsinki, ICH-GCP guidelines, ISO 14155 norm and the local legally applicable requirements.

1. BACKGROUNDGut microbiota and metabolic syndrome (MS)

Recently, the role of gut microbiota in metabolic syndrome and associated diseases gained interest among researchers. Metabolic syndrome is defined by a clustering of metabolic disorders that include central adiposity with visceral fat accumulation, dyslipidemia, insulin resistance, dysglycemia and non-optimal blood pressure levels and it is associated with an increased risk of cardiovascular diseases and type 2 diabetes. In fact, gut microbiota could have a role in regulation of body weight and energy homeostasis, influencing extraction of calories from food and their storage in host adipose tissue. Furthermore, gut microbiota in obese and lean individuals differs in the ability to play these functions[1]. At the moment, the first line standard therapy for MS consists in dietary and lifestyle intervention.

2. OBJECTIVES

The primary aim of this study is to evaluate the effects on microbiota of current standard medical treatment for MS, type 2 diabetes and obesity, including microbiome composition characterization based on metagenomic analysis of stools and saliva and genotyping of blood and functional characterization based on metabolomics analysis on urine and blood.

Secondary aims will include clinical efficacy of treatments and correlation with microbiome and metabolome characterization.

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The Role of Two Human Milk Oligosaccharides, 2′-Fucosyllactose and Lacto-N-Neotetraose, in Infant Nutrition

Badriul Hegar,1 Yulianti Wibowo,2 Ray Wagiu Basrowi,2 Reza Gunadi Ranuh,3 Subianto Marto Sudarmo,3 Zakiudin Munasir,1 Alpha Fardah Atthiyah,3 Ariani Dewi Widodo,4 Supriatmo,5 Muzal Kadim,1 Ahmad Suryawan,3 Ninung Rose Diana,6 Christy Manoppo,7 and Yvan Vandenplas8

1Cipto Mangunkusumo Hospital, Faculty of Medicine, University of Indonesia, Jakarta, Indonesia

2Nestle Nutrition Institute, Indonesia

3Soetomo Hospital, Faculty of Medicine University Airlangga, Surabaya, Indonesia

4Harapan Kita Hospital, Jakarta, Indonesia 5Adam Malik Hospital, Sumatera Utara, Indonesia 6Kariadi Hospital, Semarang, Indonesia

7Kandou Hospital, Manado, Indonesia

8KidZ Health Castle, UZ Brussel, Vrije Universiteit Brussel, Brussels, Belgium

Abstract

Human breast milk contains numerous biomolecules. Human milk oligosaccharides (HMOs) are the third most abundant component of breast milk, after lactose and lipids. Amongst the synthetized HMOs, 2′-fucosyllactose (2′-FL) and lacto-N-neotetraose (LNnT) are widely studied and are considered safe for infant nutrition. Several studies have reported the health benefits

of HMOs, which include modulation of the intestinal microbiota, anti-adhesive effect against pathogens, modulation of the intestinal epithelial cell response, and development of the immune system. The amount and diversity of HMOs are determined by the genetic background of the mothers (HMO secretors or non-secretors). The non-secretor mothers secrete lower HMOs than secretor mothers. The breastfed infants of secretor mothers gain more health benefit than those of non-secretor mothers. In conclusion, supplementation of infant formula with 2′-FL and LNnT is a promising innovation for infant nutrition.

Keywords: Human milk; Oligosaccharide; 2′-fucosyllactose; Lacto-N-neotetraose; Infant; Breast feeding

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PERFECT PREGNANCY CAMP REPORT

the perfect pregnant mother training camp project introductionProject time PHASE ONE

October 3-7, 2018

PHASE TWO

October 11-15, 2018

Five groups of families were invited to partici-pate in two sessions of the five-day campa, which included accommodation, three meals a day (plus meals), customized courses and keeping biological samples.

1.TEAM INTRODUCTION

the project team is composed of multi-disciplinary medical staff of Peking union medical college hospital, Seven Tian Zhen team(七田真团队) of being yoga and fetal education teacher during pregnancy , and Nie Qiaole team （聂巧乐团队）of music childbirth course. Professor Ma Liangkun is the project leader.

1) Peking union medical college hospital team: responsible for project planning, operation, specimen collection and processing, data collection, data analysis, professional course design, teaching and other contents.

2) Seven Tian Zhen team: responsible for teaching yoga and fetal education during pregnancy.

3) Nie Qiaole team: responsible for the teaching of music childbirth course.

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The Bacterial Ecosystem of Mother’s Milk and Infant’s Mouth and Gut

Elena Biagi1*†, Sara Quercia 1†, Arianna Aceti2, Isadora Beghetti2, Simone Rampelli1, Silvia Turroni1, Giacomo Faldella2, Marco Candela1, Patrizia Brigidi1 and Luigi Corvaglia2

1 Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy, 2 Neonatal Intensive Care Unit, Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy

The progressive building of the infants’ gut microbiota is pivotal for educating their immune system. Human breast milk is among the first sources of microbes for the assembly of the infant’s microbiota, but research struggles to give a demonstration for the origin of bacteria in milk. Aiming at contributing to the knowledge on assembly of the mother’s milk and infant’s microbiome, here we characterized the oral, gut and milk ecosystems in a homogeneous cohort of 36 healthy mother–infants pairs, by 16S rRNA next-generation sequencing. A limited number of operational taxonomic units (OTUs) was shared among the three ecosystems, including not only OTUs assigned to the well- known immune-modulating Bifidobacterium genus, but also specific Streptococcus and Staphylococcus OTUs, which were dominant in the infant’s mouth ecosystem. The high conservation of these OTUs among the three ecosystems seems to call for a worth exploring ecological role through targeted and/or culture-dependent techniques. Notwithstanding the limitations of a 16S rRNA gene-based molecular characterization, we might hypothesize that the baby’s mouth, being the transition point for the milk to reach the intestine, could play a role in both the gut microbiota assembly, via deglutition, and mother’s milk duct colonization, during suction.

Keywords: infant gut microbiota, infant oral microbiota, milk microbiota, microbiota assembly, breastfeeding, term infants

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Effect of Lactobacillus paracasei subsp. paracasei, L. casei 431 on immune response to influenza vaccination and upper respiratory tract infections in healthy adult volunteers: a randomized, double-blind, placebo-controlled, parallel-group study1–4

Lillian Jespersen, Inge Tarnow, Dorte Eskesen, Cathrine Melsaether Morberg, Birgit Michelsen, Susanne Bu¨gel, Lars Ove Dragsted, Ger T Rijkers, and Philip C Calder

ABSTRACT

Background: Probiotics can modulate the immune system in healthy individuals and may help reduce symptoms related to respiratory infections.

Objective: The objective of the study was to investigate the effect of the probiotic strain Lactobacillus paracasei subsp. paracasei, L. casei 431 (Chr. Hansen A/S) (hereafter, L. casei 431) on immune response to influenza vaccination and respiratory symptoms in healthy adults. Design: A randomized double-blind, placebo-controlled trial was conducted in 1104 healthy subjects aged 18–60 y at 2 centers in Germany and Denmark. Subjects were randomly assigned to receive an acidified milk drink containing $109 colony-forming units of

L. casei 431 (n = 553) or placebo (n = 551) for 42 d. After 21 d, subjects received the seasonal influenza vaccination. The primary out- come was seroprotection rate (anti-influenza antibody titers by hem- agglutination inhibition) 21 d after vaccination. Other outcomes were seroconversion rate and mean titers, influenza A–specific antibodies and incidence, and duration and severity of upper respiratory symp- toms. Antibiotic use and use of health care resources were recorded. Results: There was no effect of L. casei 431 on immune responses to influenza vaccination. Generalized linear mixed modeling showed a shorter duration of upper respiratory symptoms in the probiotic group than in the placebo group (mean 6 SD: 6.4 6 6.1 vs. 7.3 6 9.7 d, P = 0.0059) in the last 3 wk of the intervention period. No statistically significant differences were found for incidence or severity. Conclusions: Daily consumption of L. casei 431 resulted in no ob- servable effect on the components of the immune response to influenza vaccination but reduced the duration of upper respiratory symptoms. The trial was registered at www.isrctn.com as ISRCTN08280229.

Am J Clin Nutr 2015;101:1188–96.

Keywords: immune function, probiotics, upper respiratory tract infection, Lactobacillus paracasei subsp. paracasei, vaccination re- sponse, L. casei 431

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Human milk and infant intestinal mucosal glycans guide succession of the neonatal intestinal microbiota

David S. Newburg1 and Lorenzo Morelli2

Infants begin acquiring intestinal microbiota at parturition. Initial colonization by pioneer bacteria is followed by active succession toward a dynamic ecosystem. Keystone microbes engage in reciprocal transkingdom communication with the host, which is essential for human homeostasis and health; therefore, these bacteria should be considered mutualists rather than commensals. This review discusses the maternal role in providing infants with functional and stable microbiota. The initial fecal inoculum of microbiota results from the prox- imity of the birth canal and anus; the biological significance of this anatomic proximity could underlie observed differences in microbiota between vaginal and cesarean birth. Secondary sources of inocula include mouths and skin of kin, animals and objects, and the human milk microbiome, but guiding microbial succession may be a primary role of human milk. The unique glycans of human milk cannot be digested by the infant, but are utilized by mutualist bacteria. These prebiotic glycans support expansion of mutualist microbiota, which manifests as differences in microbiota among breastfed and artificially fed infants. Human milk glycans vary by maternal genotype. Milks of genetically distinct mothers and variations in infant mucosal glycan expression support discrete microbiota. Early coloniza- tion may permanently influence microbiota composition and function, with ramifications for health.

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Modulation of vaccine response by concomitant probiotic administration

Catherine Maidens, Caroline Childs, Agnieszka Przemska, Iman Bin Dayel & Parveen Yaqoob

Department of Food and Nutritional Sciences, The University of Reading, Reading, UK

Evidence suggests that probiotic bacteria modulate both innate and adaptive immunity in the host, and in some situations can result in reduced severity of common illnesses, such as acute rotavirus infection and respiratory infections. Responses to vaccination are increasingly being used to provide high quality information on the immunomodulatory effects of dietary components in humans. The present review focuses on the effect of probiotic administration upon vaccination response. The majority of studies investigating the impact of probiotics on responses to vaccination have been conducted in healthy adults, and at best they show modest effects of probiotics on serum or salivary IgA titres. Studies in infants and in elderly subjects are very limited, and it is too early to draw any firm conclusions regarding the potential for probiotics to act as adjuvants in vaccination. Although some studies are comparable in terms of duration of the intervention, age and characteristics of the subjects, most differ in terms of the probiotic selected. Further well designed, randomized, placebo-controlled studies are needed to understand fully the immunomodulatory properties of probiotics, whether the effects exerted are strain-dependent and age-dependent and their clinical relevance in enhancing immune protection following vaccination.

Keywords: antibody, immune response, immunomodulation, probiotic, vaccine

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Efficacy of daily intake of Lactobacillus casei Shirota on respiratory symptoms and influenza vaccination immune response: a randomized, double-blind, placebo-controlled trial in healthy elderly nursing home residents1–3

Karolien Van Puyenbroeck, Niel Hens, Samuel Coenen, Barbara Michiels, Caroline Beunckens, Geert Molenberghs, Paul Van Royen, and Veronique Verhoeven

ABSTRACT

Background: Age is associated with immune dysregulation, which results in an increased infection rate and reduced effectiveness of vaccination.

Objective: We assessed whether an intervention with Lactobacillus casei Shirota (LcS) in elderly nursing home residents reduced their susceptibility to respiratory symptoms and improved their immune response to influenza vaccination.

Design: Between October 2007 and April 2008, a randomized, dou- ble-blind, placebo-controlled trial was conducted in 737 healthy people aged 65 y in 53 nursing homes in Antwerp, Belgium. Volunteers were randomly assigned to receive a probiotic (n = 375; 2 bottles of fermented milk that contained 6.5 • 109 live LcS/bottle) or a placebo (n = 362; similar drink with no bacteria) for 176 d. After 21 d, all subjects received an influenza vaccination. Primary outcome parameters were the number of days with respi- ratory symptoms, the probability of respiratory symptoms, and anti- influenza antibody titer by hemagglutination inhibition after vaccination.

Results: Univariate and multivariate modeling showed no effect of the probiotic on clinical outcome parameters. Generalized linear mixed modeling showed no effect of the probiotic itself on the probability of respiratory symptoms [OR of probiotic: 0.8715; 95% CI: 0.6168, 1.2887). No significant difference regarding the influenza-vaccination immune response was shown.

Conclusion: The results of this study show that daily consumption of a fermented milk drink that contains LcS has no statistically or clinically significant effect on the protection against respiratory symptoms. This trial was registered at clinicaltrials.gov as NCT00849277. Am J Clin Nutr 2012;95:1165–71.

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Lactobacillus plantarum CECT7315 and CECT7316 stimulate immunoglobulin production after influenza vaccination in elderly

M. Bosch1, M. Méndez2, M. Pérez3, A. Farran4,5, M. C. Fuentes1 and J. Cuñé1

1AB-Biotics, S. A. Cerdañola del Vallés. Spain. 2Serveis Residencials d’estades temporals i respir. Llars Mundet. Barcelona. Spain. 3Centre Assistencial Dr. Emili Mira i López. Recinte Torribera. Santa Coloma de Gramenet. Spain. 4Centre d’Ensenyament de Nutrició Humana i Dietètica (CESNID). Santa Coloma de Gramenet. Spain. 5Departament de Nutrició i Bromatologia. Universitat de Barcelona. Barcelona. Spain.

Abstract

Objective: The effectiveness of influenza vaccination in preventing illness is lower in the elderly; this is why the ability of Lactobacillus plantarum CECT 7315/7316 to stimulate the response to influenza vaccination in elderly was evaluated.

Research methods and procedures: A randomized, double-blind, placebo-controlled human trial including 60 institutionalized volunteers aged 65-85 years was performed. All the volunteers were vaccinated with a trivalent influenza vaccine (A/Wisconsin/67/2005 NYMC X-161B (H3N2), A/Solomon Islands/3/2006 (H1N1) and B/Malaysia/2506/2004) for the Spanish vaccine campaign 2006/2007. The consumption of the probiotic began between three and four months after the vaccination. Volunteers were randomly assigned to one of three following groups: group A (receiving 5*109 cfu/day of L. plantarum CECT 7315/7316 in 20 g powdered skim milk), group B (receiving 5*108 cfu/day of L. plantarum CECT 7315/7316 in 20 g powdered skim milk) and group C or placebo (20 g powered skim milk). The participants consumed the probiotic during 3 months.

Results: The consumption of L. plantarum CECT

7315/7316 during 3 months after influenza vaccination increased the levels of influenza-specific IgA and IgG antibodies. Moreover, a trend towards an increase in influenza-specific IgM antibodies was also observed.

Conclusion: L. plantarum CECT7315/7316 has an immunostimulating effect and could be used to improve the response to influenza vaccination in elderly.

Key words: Lactobacillus plantarum CECT 7315/ 7316. Immunoestimulation. Influenza. Vaccine. Elderly.

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Vaccine Adjuvant Properties of Probiotic Bacteria

Author: Paul V Licciardi

Specialty: Immunology, Infectious Diseases, Clinical Immunology, Pediatrics

Institution: Pneumococcal Laboratory, Murdoch Childrens Research Institute

Address: Melbourne, Australia

Institution: Department of Paediatrics, University of Melbourne Address: Melbourne, Australia

Author: Mimi L.K. Tang

Specialty: Immunology, Microbiology, Infectious Diseases, Pediatrics, Clinical Immunology Institution: Department of Allergy and Immunology, Royal Children's Hospital, Melbourne

Address: Melbourne, Australia

Institution: Pneumococcal Laboratory, Murdoch Children's Research Institute

Address: Melbourne, Australia

Institution: Department of Paediatrics, University of Melbourne Address: Melbourne, Australia

Abstract:

Vaccine-preventable diseases are still responsible for the deaths of more than 1 million children under the age of 5 years annually, mostly in developing countries. A substantial number of these deaths are due to pneumococcal bacteria and infections with rotavirus. Important issues faced by the WHO, governments, vaccine manufacturers, and international organizations such as UNICEF and the Global Alliance for Vaccines and Immunization (GAVI) are the cost-effective introduction of these life-saving vaccines in resource-poor countries where there is a considerable disease burden, and achieving high rates of completion of vaccination schedules remains elusive. Problems with vaccine coverage and vaccine delivery in these regions are significant, as in some cases large proportions of the target population do not receive adequate vaccination. Consequently, there is a need to develop more effective vaccination strategies that can provide adequate protection with reduced schedules. To date, emphasis has been placed on identifying novel vaccine antigens and adjuvants that induce stronger protective immune responses, as well as developing mucosally-administered vaccines. These approaches would have enormous benefits in allowing safe administration of vaccines in remote areas and may overcome the necessity for multiple doses. In this regard, the use of probiotic bacteria as novel mucosal adjuvants to enhance existing vaccine specific immune responses offers an exciting new approach. In this review, we discuss the evidence for the role of probiotics in enhancing vaccine responses and provide justification for further investigation into their clinical effects and mechanisms of action.

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Oligosaccharides in 4 Different Milk Groups, Bifidobacteria, and Ruminococcus obeum

mGiovanni V. Coppa, mOrazio Gabrielli, mLucia Zampini, mTiziana Galeazzi, mAnna Ficcadenti,

mLucia Padella, mLucia Santoro, ySara Soldi, zAntonio Carlucci, §Enrico Bertino, and jjLorenzo Morelli

Abstract

Objectives: The aim was of this study is to identify a link between the total amount of breastmilk oligosaccharides and faecal microbiota composition of newborns at the end of the first month of life, with special attention paid to bifidobacteria, and establish the role, if any, of the different oligosaccharides in determining the gut microbiota composition.

Subjects and Methods: Milk oligosaccharide groups were identified by high-performance anionexchange chromatography analysis. DPCRNA from newborns’ faecal samples at 30 days of life was isolated and processed by polymerase chain reaction analyses that allow the identificationof 6 species of bifidobacteria (adolescentis, bifidum, breve, catenulatum, longum, infantis) and Ruminococcus spp; denaturing gradient gel electrophoresis analysis was also performed.

Results: No substantial differences in bifidobacteria species composition within milk groups 1, 2, and 3 were observed; however, infants fed with group 4 milk show a microbiota characterised by a greater frequency of Bifidobacteria adolescentis and the absence of Bifidobacteria catenulatum. For the first time, a high percentage of the Ruminococcus genus in infants fed with all milk groups was found.

Conclusions: Our data show that milk groups 1, 2, and 3, containing an amount of oligosaccharides ranging within 10 to 15 g/L, share a substantially identical composition of the intestinalmicrobiota in breast-fed infants, despite quali-quantitative difference in oligosaccharides content. Newborns taking milk with only 5 g/L of oligosaccharides (group 4) harbour a different intestinal microbiota.

Key Words: Bifidobacterium, breast-fed infants, human milk oligosaccharides, intestinal microbiota, Ruminococcus

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Lactobacillus crispatus M247-Derived H2O2 Acts as a Signal Transducing Molecule Activating Peroxisome Proliferator Activated Receptor-ç in the Intestinal Mucosa

SANDRA VOLTAN,* DIEGO MARTINES,‡ MARINA ELLI,§ PAOLA BRUN,* STEFANO LONGO,§ ANDREA PORZIONATO,ǁ VERONICA MACCHI,ǁ RENATA D’INCÀ,‡ MELANIA SCARPA,* GIORGIO PALÙ,* GIACOMO C. STURNIOLO,‡ LORENZO MORELLI,§ and IGNAZIO CASTAGLIUOLO*

*Department of Histology, Microbiology and Medical Biotechnologies, ‡Department of Surgical and Gastroenterological Sciences, University of Padua;

§AAT—Advanced Analytical Technologies, Piacenza; ǁDepartment of Human Anatomy and Physiology, University of Padua, Italy

Background & Aims:

Accumulating evidence indi- cates that the peroxisome proliferator activated recep- tor (PPAR)-ç is a major player in maintaining intes- tinal mucosa homeostasis, but whether PPAR-ç is directly involved in probiotic-mediated effects and the molecular events involved in its activation are not known. Methods: We investigated the role of PPAR-ç in the immunomodulatory effects of Lactobacillus crispatus M247 on intestinal epithelial cells (IEC) and the role of probiotic-derived H2O2 on PPAR-ç activ- ity. Results: L crispatus M247 supplementation in mice significantly increased PPAR-ç levels and tran- scriptional activity in the colonic mucosa. L crispatus M247 induced PPAR-ç nuclear translocation and en- hanced transcriptional activity in epithelial (CMT-93) cells, as demonstrated by the increased luciferase ac- tivity of a PPAR-ç–responsive element, PPAR-ç– responsive gene up-regulation, and reduced activity of an nuclear factor-nB–responsive element. Pharma- cologic PPAR-ç inhibition or silencing by small in- terfering RNA cancelled the L crispatus M247–medi- ated effects in CMT-93 cells. Because Lactobacillus strains producing little H2O2 failed to activate PPAR-ç, we investigated the role of L crispatus M247– derived H2O2 in PPAR-ç activation. L crispatus M247 induced a transient rise in intracellular H2O2 and PPAR-ç transcriptional activity was cancelled by antioxidant or H2O2 scavenger. Toll-like receptor (TLR)-2 was not required for PPAR-ç up-regulation mediated by L crispatus M247 in mice, although the protective effects of L crispatus M247 on dextran sodium sulfate-induced colitis were less pronounced in TLR-2—/— mice. Conclusions: L crispatus M247 uses H2O2 as a signal transducing molecule to induce PPAR-ç activation in IEC, directly modulating epithe- lial cell responsiveness to inflammatory stimuli.

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Proteomic investigation of the aggregation phenomenon in Lactobacillus crispatus

Rosa A. Siciliano a,⁎, Giuseppina Cacace a, Maria F. Mazzeo a, Lorenzo Morelli c, Marina Elli c,

Mauro Rossi b, Antonio Malorni a

a Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell'Alimentazione del CNR, Avellino, Italy

b Laboratorio di Immunobiologia, Istituto di Scienze dell'Alimentazione del CNR, Avellino, Italy

c AAT-Advanced Analytical Technologies S.r.l., spin off company of Università Cattolica del Sacro Cuore, Piacenza, Italy

Abstract

Aggregation process affects the ability of Lactobacillus crispatus, a probiotic, to survive into the gastro-intestinal environment and to adhere to the intestinal mucosa. To elucidate mechanisms underlying this process, a comparative proteomic study was carried out on a wild type strain M247 and its spontaneous isogenic mutant Mu5, which had lost the aggregative phenotype. Results highlighted an overall lower amount of enzymes involved in carbohydrate transport and metabolism in strain M247 compared to strain Mu5, suggesting a reduction in the general growth rate, probably caused by nutrient limitation in cell aggregates, coherently with the phenotypic traits of the strains. Moreover, the up-regulation of a putative elongation factor Tu in the wild type M247 strain could suggest a role of this particular protein in the adhesion mechanism of L. crispatus.

Keywords: Probiotics; Adhesion; Aggregation; Elongation factor EF-Tu; Lactobacillus; Proteome

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Aggregating Phenotype in Lactobacillus crispatus Determines Intestinal Colonization and TLR2 and TLR4 Modulation in Murine Colonic MucosaA

Sandra Voltan,1 Ignazio Castagliuolo,1* Marina Elli,4 Stefano Longo,4 Paola Brun,1 Renata D’Inc`a,2 Andrea Porzionato,3 Veronica Macchi,3 Giorgio Palu`,1 Giacomo C. Sturniolo,2

Lorenzo Morelli,4 and Diego Martines2

Department of Histology, Microbiology, and Medical Biotechnologies,1 Department of Gastroenterological Sciences,2 and Department of Human Anatomy and Physiology,3 University of Padua, Padua, Italy, and AAT-Advanced Analytical Technologies S.r.l., Piacenza, Italy4

The colonic MICROBIOTA is a MAJOR MODULATOR of the MUCOSAL IMMUNE SYSTEM; therefore, its MANIPULATION through SUPPLEMENTATION with probiotics MAY significantly affect the host’s IMMUNE responses. Since different probiotics SEEM to exert various effects in vivo, we tested the relevance of the autoaggregation phenotype on the intestinal persistence of lactobacilli and their ability to MODULATE the host’s innate IMMUNE responses. After 14 days of diet SUPPLEMENTATION, the aggregating strain Lactobacillus crispatus M247 but not aggregation-deficient isogenic MUTANT MU5 was recovered FROM the feces and colonic MUCOSA of MICE. This observation was CONFIRMED by strain-specific PCR AMPLIFICATION and by Lactobacillus-specific denaturing gradient gel electrophoresis analysis. Indeed, L. crispa- tus M247 increased Toll-like receptor 2 (TLR2) MRNA levels, while it reduced TLR4 MRNA and protein levels in the colonic MUCOSA, whereas MU5 was ineffective. In colonic epithelial cells (CMT-93 cells) L. crispatus M247 but not MU5 induced TIME-DEPENDENT extracellular signal-regulated kinase-1 (ERK1) tyrosine phosphorylation and TLR MODULATION, which were abolished in the presence of PD98059 (an ERK1 inhibitor). To assess the functional relevance of probiotic-induced TLR MODULATION, we DETERMINED the consequences of L. crispatus preexposure on TLR4 (lipopolysaccharide [LPS]) and TLR2 [PAM3Cys-Ser-(Lys)4] LIGAND-MEDIATED effects in intestinal epithelial cells. Preexposure to L. crispatus M247 blunted LPS-induced interleukin-6 (IL-6) release and inhibition of CMT-93 MIGRATION over a wound edge, whereas it enhanced TLR2-MEDIATED IL-10 up-regulation. In SUMMARY, the aggre- gation phenotype is required for L. crispatus persistence in the colon and for MODULATION of TLR2/TLR4 expression through an ERK-dependent pathway. We speculate that the aggregation phenotype in L. crispatus M247 is required to TEMPER epithelial cell responsiveness to bacterial endotoxins, which thus affects the evolution of intestinal INFLAMMATORY processes.

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The aggregation-promoting factor of Lactobacillus crispatus M247 and its genetic locus

H. Marcotte1,2, S. Ferrari3, C. Cesena3, L. Hammarstro¨ m2, L. Morelli3, G. Pozzi1 and M.R. Oggioni1

1Dipartimento di Biologia Moleculare, Laboratorio di Microbiologia Moleculare e Biotecnologia, Sezione di Microbiologia, Policlinico Le Scotte, Siena, Italy, 2Division of Clinical Immunology, Karolinska Institutet at Huddinge Hospital, Stockholm, Sweden, and 3Istituto di Microbiologia, Facolta` di Agraria, UCSC, Piacenza, Italy

ABSTRACT

H. MARCOTTE, S. FERRARI, C. CESENA, L. HAMMARSTRO¨M, L. MORELLI, G. POZZI AND M.R. OGGIONI. 2004.

Aims: Characterization of the aggregation-promoting factor (APF) of the human intestinal isolate Lactobacillus crispatus M247 and its homologous nonaggregating mutant Mu5.

Methods and Results: Western blot analysis revealed that the supernatant of both M247 and Mu5 contains a 28- kDa protein which cross reacts with the antiserum produced against the APF of Lact. gasseri 4B2. The apf genes of M247 and Mu5 strains were identical and were shown to be 672 nucleotides in length and encoding a protein of 223 amino acids with a predicted molecular weight of 24Æ0 kDa.

Conclusion: Our results shows that the lost of aggregation in Mu5 is not related to a defect in secretion of the APF protein or a mutation in the apf gene.

Significance and Impact of the Study: These results suggest that the mutation in Mu5 may be contained in another molecule involved in aggregation such as a possible receptor for APF.

Keywords: aggregation, aggregation-promoting factor, apf gene, colonization, lactobacilli, nonaggregating mutant.

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Lactobacillus crispatus and its Nonaggregating Mutant in Human Colonization Trials

C. Cesena, * L. Morelli, * M. Alander,T T. Siljander E. Tuomola,1: S. Salminen,1: T. Mattila-Sandholm, T. Vilpponen-Salmela, and A. von Wright-r

*lstituto di Microbiologia, Facolta di Agraria,

U.C.S.C., Via Emilia Parmense 84, |-29100, Piacenza, Italy

TVTT Biotechnology and Food Research, PO. Box 1501, FIN-02044 VTT, Finland

iDepartment of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland

Harju|a Hospital, PO. Box 38, FIN-70101, Kuopio, Finland

ABSTRACT

A wild-type Lactobacillus crispatus, showing a cell aggregation phenotype and its spontaneous nonaggre- gating mutant were compared for their in vitro adhe- sion properties to human ileal mucus and to a cultured human colonic cell line (Caco2) and for their in vivo

colonization and adhesion potential with colonoscopy patients as volunteers in feeding trials. The wild-type strain adhered better to mucus or to Cac02 cells than did the mutant. Altogether, three human trials with the wild type and two with the mutant strain were performed. In two of the trials, the wild type could be recovered from either fecal samples or biopsies taken from the colon, while the mutant strain could not be demonstrated in either of the trials where it was used. The L. crispatus colonies recovered from the trials were often mixed, and several enterococci and lactobacillus strains coaggregating with L. crispatus wild type could be isolated. The results indicate that the surface-medi- ated properties, such as aggregation, of lactobacilli can have a role in adhesion and colonization.

(Key words: probiotics, aggregation, colonization, Lac- tobacillus)

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