Clinica Chimica Acta xxx (2015) xxx–xxx

CCA-13820; No of Pages 9

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Clinica Chimica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /c l inch im

Gut microbiota biomodulators, when the stork comes by the scalpel

Vito LeonardoMiniello ⁎, Angela Colasanto, Fernanda Cristofori, Lucia Diaferio, Laura Ficele,Maria Serena Lieggi,Valentina Santoiemma, Ruggiero FrancavillaDepartment of Paediatrics, Aldo Moro University of Bari-Giovanni XXIII Hospital, Via Amendola 270, 70126 Bari, Italy

Abbreviations:B,Bifidobacterium; CS, Caesarean section;sal associated molecular patterns; DCs, dendritic cells; EEuropean Society for Paediatric Allergology and Clinical ImSociety for Paediatric Gastroenterology Hepatology aAgriculture Organization; FoxP3, forkhead box protein 3; FOgalacto-oligosaccharides; GALT, gut-associated lymphoibiomodulators; HMOs, human milk oligosaccharides; sIgA,ible Treg; IFN, interferon; IL, interleukin; IECs, intestinal epiride; NEC, necrotizing enterocolitis; nTreg, natural Treg; NFκB; NOD, nucleotide-binding oligomerization domain; NLRsmal dendritic cells; HMO, oligosaccharides; PAMPs, pathogePRRs, pattern recognition receptors; DCreg, regulatory dendtight junctions; TLRs, toll-like receptors; TGF, transforminggrfactor; Th1, type 1 helper T cells; Th2, type 2 helper T cells;W⁎ Corresponding author at: Azienda Ospedaliero Unive

Ospedale Giovanni XXIII, Via Amendola 270, 70126 Bari, IE-mail address: vito.miniello@libero.it (V.L. Miniello).

http://dx.doi.org/10.1016/j.cca.2015.01.0220009-8981/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: Miniello VL, et al, Gdx.doi.org/10.1016/j.cca.2015.01.022

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 September 2014Received in revised form 22 January 2015Accepted 25 January 2015Available online xxxx

Keywords:Gut microbiotaCaesarean deliveryDysbiosisProbioticsPrebioticsGut microbiota biomodulators

Themicrobial communities that reside in the human gut (microbiota) and their impact on human health and dis-ease are nowadays one of themost exciting new areas of research. Awell-balancedmicrobial intestinal coloniza-tion in early postnatal life is necessary for the development of appropriate innate and adaptive immuneresponses and to establish immune homeostasis later in life. Although the composition and functional character-istics of a ‘healthy’ gut microbiota remain to be elucidated, perturbations in the microbial colonization of aninfant's gastrointestinal tract have been associated with an increased risk of short- and long-term immunologi-cally mediated diseases. Emerging evidence suggests that gut microbiota biomodulators, such as probiotics, pre-biotics, synbiotics, and postbiotics may support disease prevention in infants who tend to have a delayed and/oraberrant initial colonization with reduced microbiota diversity (delivery by caesarean section, premature deliv-ery, and excessive use of perinatal antibiotics). Under these dysbiosis conditions probiotics could act as ‘surro-gate’ colonizers to prevent immune-mediated diseases. This review focuses on the influence of delivery modeon the colonization of the infant gastro-intestinal tract. In particular, it examines themanipulation of the gut mi-crobiota composition through the use of gutmicrobiota biomodulators, in themanagement of aberrant initial gutcolonization and subsequent consequences for the health of the offspring.

© 2015 Elsevier B.V. All rights reserved.

“Pray for us now and at the hour of our birth.”[Thomas Stearns Eliot]

1. Introduction

Caesarean section (CS) is the most common surgical procedure per-formed onwomenworldwide. According to theWorldHealthOrganiza-tion (WHO), CS rate (percentage of births managed by CS) exceeding

C, Clostridium; CAMPs, commen-PS, exopolysaccharide; ESPACI,munology; ESPGHAN, Europeannd Nutrition; FAO, Food andS, fructo-oligosaccharides; GOS,d tissue; GMB, gut microbiotaimmunoglobulin A; iTreg, induc-thelial cells; LPS, lipopolysaccha--κB, nuclear transcription factor-, NOD-like receptors; nDCs, nor-n associated molecular patterns;ritic cells; Treg, regulatory T; TJs,owth factor; TNF, tumornecrosisHO,World Health Organization.rsitaria Policlinico Consorziale-taly. Tel.: +0039 080 5594075.

ut microbiota biomodulators

15% lacks medical justification and it could be linked with adverse ma-ternal and child health consequences [1]. CS rate is elevated especiallyin tertiary centermostly because of risk pregnancy and pretermdeliver-ies. Contrary to widespread belief, Caesarean delivery is not safer thanvaginal delivery for premature and small for gestational age infants[2]. A recent research showed that small for gestational age babies deliv-ered early by CS had higher rates of respiratory distress syndrome thansimilar preterm babies who were born vaginally [3].

Although since 1985 WHO has recommended that the rate is notto exceed 10–15%, there is no empirical evidence for an optimumrange of percentages, despite of a growing body of research showinga negative effect of high rates. It should be noted that the pro-posed upper limit of 15% is not a target to be achieved but rather athreshold not to be exceeded. Nevertheless, the rates inmost developedcountries and in many urban areas of lesser-developed countries areabove that threshold. Rates of CS have increased beyond the recom-mended level especially in high-income areas such as North America,Italy, France, Germany, and the United Kingdom of Great Britain. Anestimated one-third of all births in the United States occur by CS,many of which are elective. Across Europe, there are differences be-tween countries: Italy has the highest Caesarean rate of Europe (38%in 2008) while in the Nordic countries CS rate is 14% [4]. Furthermore,the cost of the global “excess” CS was estimated to amount approxi-mately 2.32 billions $, while the cost of the global “needed” CS was ap-proximately 432 million $ [5].

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Vaginal delivery and exclusive breast-feeding during the firstmonths of life have short- and long-term beneficial effects, such asprotection against infectious diseases, reduced infant morbidity andmortality, and low incidence of immunological disorders. The humanimmune system undergoes major development during infancy and ishighly related to the microbes that colonize the intestinal tract in earlypostnatal life [6]. Gut microbiota has protective, metabolic, trophic,and immunological functions. Neonates born by vaginal delivery are ex-posed to the mother's vaginal and intestinal flora as they pass throughthe birth canal and typically harbor communities of bacteria that resem-bled those of the mother's microbiota. On the other hand, infants deliv-ered via CS are colonized by bacteria that are most similar to the skincommunities of the mothers [7]. Such perturbed microbiota composi-tion (dysbiosis) can alter immune regulatory networks that normallyrestrain intestinal inflammation, and may contribute to a number of in-testinal and extraintestinal immune-mediated diseases. Neonataldysbiosis has been proposed as one of the environmental factors thatmay play a role in the increasing incidence of both allergic and autoim-mune diseases [8,9].

In other words, the potential disadvantages of CS include alteredbacterial profile of the neonate/infant intestinal microbiota, which, inturn, leads to immune dysfunction and increased tendency forimmune-mediated diseases. Rising CS rates in Western countriesmake such a potential relation an important public health concern.

2. “Homo bacteriens” [10]

Human beings are born into, and develop in, a microbial world.Some scientists believe that early in the history of the planet, differenttypes of microbes joined together to form a new type of organism.These microbes were engulfed by larger bacteria, forming a microbialsymbiosis and the host cell protected the smaller microbe inside,while benefiting from the skills of its new partner. In 1981 LynnMargulis proposed that the main organelles of the eukaryotic cellwere primitive prokaryotic cells that had been engulfed by a different,bigger prokaryotic cell. The “endosymbiotic theory of eukaryote evolu-tion” claims that mitochondria were originally separate organismsthat entered into a symbiotic relationship with eukaryotic cells throughendosymbiosis.

Humans coexist with an enormous quantity of microbial organismscollectively termed microbiota. The microbiota represents an ensembleof microorganisms that resides at various sites on and inside the humanbody, including skin, nares, oral cavity, urogenital tract, and gut. The gutmicrobiota (formerly called gut microflora) represents the best studiedmicrobial ecosystem coexisting with human subjects. The gut ecosys-tem plays a key role in the maturation of the immune system and inother physiological processes including mucosal barrier function.Under normal conditions, this immunologically and metabolically ac-tive ‘foreign’ body exists in a state of symbiotic tolerance with its hostand remains relatively stable over time.

Because of more than 100 trillion microbes, ten-fold the numberof human cells, including at least 1000 separate bacterial specieswhich constantly interact with themselves and their host, the intestinehas evolved to our main immune organ. It is estimated that themicrobiota-encoded microbiome (the collective gene set of all coloniz-ing microbes) contains 8 million genes, 150 times more unique genesthan are encoded by the human genome. Consequently, our immunesystem is charged with the critical task of distinguishing between ben-eficial and pathogenic microbes [11]. Given this important role, whileenabling the uptake of large amounts of nutritional products, it is notsurprising that the intestine harbors over 70% immune-competent cells.

3. The multi-omics approach

The development of the neonatal fecal microbiota has been stud-ied by culture-dependent techniques from the 1950s onwards. A

Please cite this article as: Miniello VL, et al, Gut microbiota biomodulatorsdx.doi.org/10.1016/j.cca.2015.01.022

considerable portion of the microbial cosmos inside our body iscomposed of bacteria that cannot be cultivated by current micro-biological methods. However, recent technological advances as well asa growing interest in the human gut ecosystem have led to a surgeof progress in this area. Over the last few years a number of crucial tech-nological innovations have been introduced to shed more light onthe composition and functionality of human gut microbiota, offeringa complementary support to the classical microbiology. Thanks totheir ability to identify a large number of species that cannot becultivated, they allowed a more complete and rapid assessment ofthe gastrointestinal ecosystem and the approach to the study ofhuman microbiota has become multidimensional. Although therehave been over 50 large bacterial families (phyla) described to date,the human gut microbiota is dominated by only two of them: theBacteroidetes and the Firmicutes, whereas Proteobacteria, Actinobacteria,Fusobacteria, Cyanobacteria and Verrucomicrobia are present in minorproportions.

Integrative multi-omics approach, defined as the study of relatedsets of biological molecules in a comprehensive fashion, yields a globalpicture of the microbial community structure and the metabolic statusof the gut ecosystem, which is paramount to establishing correla-tions with host physiology. The multi-omics approach includes dataobtained fromgenomics (i.e., the large-scale genotyping of single nucle-otide), transcriptomics (i.e., the measurement of all gene expressionvalues), proteomics (i.e., the identification of all proteins present in acell) and metabolomics (i.e., the identification and quantification of allmetabolites) of bacteria, host cells and intestinal contents. By omics-technologies we can gain important new insight how early-life eventslike mode of delivery, type of feeding or genetic backgroundmay inter-fere with the colonization pattern.

The metabolomic approach, in which a large number of small mole-cule metabolites can be defined in a biological sample, offers a promis-ing avenue to ‘fingerprint’ microbiota functional status and a powerfulstrategy to elucidate the molecular mechanisms involved in host-microbial interactions in the complex gut ecosystem [12]. Since fecalsamples contain endogenous human metabolites, gut microbiota me-tabolites, and other compounds, the use of omics-technologies opensthe possibility of applying fecal metabolomics to study the gut microbi-ota functions related to human health. Changes in themetabolomic pro-file of feces reflect, among others, quantitative and qualitative changesof intestinal microorganisms. A growing number of studies have dem-onstrated that fecal metabolome profiling, obtained by high-resolutionnuclear magnetic resonance spectroscopy-based metabolomics, canreveal the significant metabolites differentiating comparative groups.Comparative analysis of individual human gut microbiomes hasrevealed the existence of a distinct infant and adult-type microbiota.The fecal microbiota is commonly used as a reflection of the intestinalbacterial composition. Profiling the fecal metabolome can produceinformation that may be used to point to the existence of a distinctneonatal colonization pattern, markedly influenced by several post-natal factors such as the mode of delivery (vaginal versus caesareandelivery) and infant feeding (breast milk versus infant formula). Theperformance of metagenomic studies have provided the basis forthe use of dietary interventions aimed at counteracting microbiotaaberrancies. Fecal metabolomics, in fact, may explore the effects ofnew therapeutic strategies to optimize the microbial ecosystem by gutmicrobiota biomodulators such as probiotics, prebiotics, synbiotics orpostbiotics.

The potential clinical value of such biomarkers for the early assess-ment of perturbations in the infant microbial colonization remains,however, a complex task. Understanding the influence of micro-bial communities during colonization is complicated by multiple fac-tors: the complexity to assess a ‘healthy’ gut microbiota, knowledge ofits composition under eubiosis or dysbiosis conditions, and the diffi-culty in gaining a global view of microbe–microbe and microbe–hostinteractions.

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4. Here comes the stork, and there goes the breastfeeding!

Recent studies have provided a greater understanding of how thecomposition of an individual's microbiota changes throughout develop-ment, especially during the first year of life. Bacterial colonization of theinfant gut occurs in several distinct phases, startingwith birth, followedby the introduction of oral feeding and weaning [13]. By the end of thefirst year of life, microbial ecosystems in each infant usually convergetoward a profile characteristic of the adult gastrointestinal tract [14].Nevertheless, bacterial colonizers might have substantial and lasting ef-fects on the immune response, irrespective of the composition of themature microbiota.

The neonatal colonization pattern is markedly influenced by severalpost-natal environmental factors such as the mode (vaginal vs caesare-an) and the place (home-born vs hospital-born) of delivery, the mater-nal (parental) colonization, the number of siblings, infant feeding andthe use of antibiotics, which may contribute to variations in normalphysiology or to disease predisposition [15]. The first and most impor-tant phase of normal colonization occurs when the newborn infantpasses through the birth canal and ingests a healthy bolus of maternalvaginal and colonic microorganisms. Although most literature sug-gested that the gastrointestinal tract of a normal fetus is sterile (the“sterile womb” paradigm) recent studies suggest that maternal micro-bial transfer to the offspring begins during pregnancy, providing a pio-neer microbiota [16].

The immune system undergoes major development during infancyand is highly related to the microbes that colonize the intestinal tract.Awell-balancedmicrobial intestinal colonization (eubiosis) is necessaryto establish immune homeostasis later in life [8]. With full colonization,a symbiotic relationship develops between colonizing bacteria, theunderlying epithelial and the gut-associated lymphoid tissue (GALT),comprised of Peyer's patches, isolated lymphoid follicles andmesentericlymph nodes. It is therefore safe to assume that appropriate develop-ment of the immune system requires colonization of the gut with nor-mal microbiota. The patterns of microbial colonization associated withhealth are difficult to define. Although we are far from understandingwhat constitutes a “healthy gut microbiota” based onmicrobial compo-sition [17] normal colonization is most likely to occurwhen the infant isborn full term by a vaginal delivery and is exclusively breastfed duringthe first six months of life.

Breast feeding is critical to early colonization of the newborn gutand to the development of mucosal immune-protective function. Inbreastfed vaginally delivered infants the gutmicrobiota is not only seed-ed and imprinted bymaternal bacterial species, but its compositionmayalso be shaped by the maternal seric immunoglobulin (sIg) A pool thatis influenced, in turn, by the maternal microbiota. In addition, humancolostrum and milk, which had been traditionally considered sterile,provide a continuous supply of symbiotic commensal to the infant gut[18]. Human colostrum and milk contain approximately 200 differentoligosaccharides (HMO), differing in their structure, size, and sequence,which are believed to provide substrate for the production of short-chain fatty acids, leading to the proliferation of health-promoting bacte-rial genera such as Bifidobacteria and Lactobacillus (prebiotic effect) [19].Therefore type of feeding (breastfeeding vs formula) is a key factor indetermining the bifidobacterial communities after birth and duringearly infancy in exclusively breastfed infants delivered by natural deliv-ery [20]. One of the proposed mechanisms by which Bifidobacteria me-diate some health benefits is the production of exopolysaccharide(EPS), a polysaccharide coating on the outside of the cell envelope,which is critical for in vivo cell survival [21]. Recent data assign a pivotalrole for EPS in modulating various aspects of bifidobacterial–host inter-action (immune-modulator), including the ability of indigenous bacte-ria to remain immunologically silent (immune evasion facilitator) and,in turn, provide pathogen protection. Differences inmetabolic capabilitybetween different commensal strains in the gut habitatmay involve dis-tinct responses to the host colonic epithelium. A recent study

Please cite this article as: Miniello VL, et al, Gut microbiota biomodulatorsdx.doi.org/10.1016/j.cca.2015.01.022

demonstrated that co-cultures of Bifidobacterium and Bacteroides couldbehave differently against EPS as a function of the specific characteris-tics of the strains fromeach species [22] Bifidobacterium (B) longum sub-species infantis is a species predominant in the gastrointestinal tracts ofhealthy breast-fed infants. Recent studies have suggested that HMOstimulates gene transcriptions in this health-promoting (probiotic-like) commensal, which induces intestinal production of anti-inflammatory cytokines such as interleukin (IL)-10, while reducing pro-duction of pro-inflammatory cytokine such as tumor necrosis factor(TNF)-α [23]. Therefore, HMOs are known to be more than just “foodfor bugs”.

Diet has a dominant role over other possible variables in shaping thegutmicrobiota. The intestine of breast-fed infants was traditionally con-sidered to be dominated by Bifidobacteria but recent studies using high-throughput 16S rDNA techniques demonstrated that Proteobacteriadominate the infant intestinal microbiota [24]. However, breast-fedinfants harbor a fecal microbiota by more than two times increased innumbers of Bifidobacteria when compared to formula-fed infants.Among Bifidobacteria, B. breve, B. bifidum, B. longum, and B. adolescentiswere isolated in both formula- and breast-fed infants, whereas B.infantis is typical of breast-feds and B. fragilis of formula-fed infants[15]. Moreover, the bottle-fed babies have been demonstrated to carrya less stable and uniform population when compared to the breast-fedones [25] and have less beneficial microbes like Bifidobacteria andLactobacilli spp. and higher levels of pathobionts (any disease-causingmicroorganism) like Enterobacteriaceae, compared to breast-fed infants[26]. The higher counts and incidences of Clostridia in formula-fed in-fants are accompanied by a predominance of C. perfringens, C.paraputrificum, C. clostridiiforme, and C. tertiumwhile themost commonspecies is usually C. difficile in breast-fed infants.

5. Translating the hygiene hypothesis to the microbiota hypothesis

Over the last decades there has been inWestern industrialized coun-tries a tremendous increase in the prevalence of both allergies (asthma,atopic dermatitis, food allergy and allergic rhinitis), in which the im-mune response is dominated by type 2 helper T (Th2) cells, and autoim-mune diseases (type 1 diabetes, Crohn's disease, and celiac disease) inwhich the immune response is dominated by type 1 helper T (Th1) cells.

Microbes are one of the environmental factors that play an impor-tant role in shaping normal and pathologic immune responses. The‘hygiene hypothesis’ was first formulated in 1989 by Strachan who re-ported an inverse relationship between family size and developmentof atopic disorders. The British epidemiologist proposed that a lower in-cidence of infection in early childhood, transmitted by unhygienic con-tact with older siblings, could be a cause of the rise in allergic diseases[27].

Early-life exposure to older siblings or pets is protective against thedevelopment of allergic disease. Exposure to siblings and pets appearto differentially impact gutmicrobiota composition and diversity duringearly life, by promoting a diverse and healthy gut microbiota.

According to the hygiene hypothesis infectious agents at the earlystages of life act as pivotal challenges for the immune system and maymodulate Th2-like allergic responses by promoting immune deviation(toward a Th1 response) and/or enhancing immune regulation, de-pending on the nature of the microbial agents, the time and durationfrom exposure to infection, and the genetic background of the host [28].

Although the relationship between the Th1-promoting effects of in-fections and the consequent inhibition of allergy provides an explana-tion for the hygiene hypothesis, the mechanism has been challengedbecause some Th1-type autoimmune diseases have also increasedover the past decades. Since the first exposure to microorganismsseems to be crucial to establish immune homeostasis, delay in exposureto bacterial colonizers and, in turn, the lack of a regulatory mechanismmay be involved and may even be more relevant than infections(the ‘microbiota hypothesis’). This ‘new’ hygiene hypothesis starts

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with the assumption that altered gutmicrobial communities, closely as-sociatedwith the loss of ancient and coevolvedmicrobes (“old friends”)lead to the disruption of the gut immune homeostasis [29].

In 1986, Tim Mosmann and Bob Coffman identified two subsets ofactivated T helper lymphocytes which differed from each other intheir pattern of cytokine production and their functions: those thatmade interferon (IFN)-γ as their signature cytokine (Th1 cells) andthose that produced IL-4 (Th2 cells). Today four (and possibly more)distinct T helper-cell subsets have been shown to exist, Th1, Th2,Th17, and regulatory T (Treg) cells. The key role of Treg cells, whichhave immunosuppressive functions and distinct cytokine profiles isnow well established [30]. There are a number of different Tregcells that can be divided into natural Treg (nTreg) cells and inducibleTreg (iTreg) cells. nTreg cells come from the thymus, whereas iTregcells arise in the periphery. iTreg cells include IL-10-producing Th1cells and transforming growth factor (TGF)-β-producing Th3. Anti-inflammatory cytokines including IL-10 and TGF-β are crucial in theattenuation or containment of inflammatory process and confer sup-pressive effects on the immune response. Gut commensals can induceTreg cells allowing the host to tolerate the massive burden of antigenspresented to the gut and ensuring that innocuous antigens do not trig-ger excessive inflammatory immune response (tolerance).

Early microbial colonization has been proposed as a major driver forthe normal age-related maturation of both Th1 and Treg cells pathwaysthat appear important in suppressing early propensity for Th2 allergicresponses. The absence of a proper immunosuppressive mechanismcan result in an imbalance between Th1 and Th2 cells and, in turn,Th1- or Th2-mediated inflammatory diseases.

Upon delivery, the neonate is exposed to a wide variety of microbes,many of which are provided by themother during and after the passagethrough the birth canal, a heavily colonized ecosystem. Perturbations inthemicrobial colonization such as a delayed colonizationwithbeneficialbacteria and reduced diversity of the intestinal microbiota, might inter-fere with the development of immunologic tolerance.

Gut dysbiosis and low microbial diversity in infancy were observedto precede onset of allergic disease. A reduced diversity and lowercounts of Bifidobacteria and Lactobacilli seem to characterize infantswho will develop allergy [31]. Whether dysbiosis (the basis for the‘new’ hygiene hypothesis) is a cause or effect of these immunedisordersremains to be determined.

Recent studies suggest that healthy infant immune developmentmay depend on the establishment of a diverse gut microbiota ratherthan the presence or absence of specific microbial strains. The biodiver-sity hypothesis, an extension of both the hygiene and microbiota hy-potheses, provides a link between changes in altered microbiota anddisease development.

6. A favorable “trialogue”

A comprehensive review of the functions of the intestinalmicrobiotais beyond the scope of this review, yet here we wish to focus on the im-munologic functions because of their importance in development of theintestinal barrier and the possible pathogenesis of several immuno-mediated diseases.

The human intestine allows the absorption of nutrients while alsofunctioning as a barrier, by preventing antigens and pathogens fromentering themucosal tissues and potentially causing disease. Themuco-sal barrier, which consists of only a single layer of epithelial cells andmucus, is one of themost important components of the innate immunesystem. The intestinal epithelium constitutes the largest exposedsurface area of the human body. Intestinal epithelial cells (IECs) partic-ipating in mucosal barrier function are conventional enterocytes, gobletcells, entero-endocrine cells and Paneth cells. The luminal secretion ofmucins and non-specific antimicrobial peptides by goblet cells andPaneth cells, respectively, establishes a physical and biochemical barrierto microbial contact with the epithelial surface and underlying immune

Please cite this article as: Miniello VL, et al, Gut microbiota biomodulatorsdx.doi.org/10.1016/j.cca.2015.01.022

cells [32,33]. The mucosal barriers (membrane and mucus) providethe first line of defense and directly communicate with microbiota inthe gut [34]. A crucial component for the maintenance of the intestinalbarrier is the intercellular junctional complex (tight junctions, adherensjunctions and desmosomes). Tight junctions (TJs) seal the space be-tween epithelial cells, thus preventing paracellular diffusion of microor-ganisms and other antigens across the epithelium. TJs are not staticbarriers but highly dynamic structures that are constantly beingremodeled due to interactions with external signals, such as cytokines,food and bacterial components, originating in the lumen, epitheliumand lamina propria [35].

Altered intestinal barrier function and increased intestinal epithelialpermeability can be consequences of disrupted early colonization. Theintegrity of the epithelial barrier depends on homeostatic regulatorymechanisms, includingmucosal induction of Treg cells, where commen-salmicrobiota–host interactions play decisive roles. An immature intes-tinal epithelial barrier may predispose infants to allergic diseases.Understanding the factors that regulate gut barrier maturation mayyield insight into strategies to prevent these diseases. The gut matura-tion process is associated with a decrease in intestinal permeabilityand a consequent decrease in the transport of macromolecules acrossthe intestinal barrier. This phenomenon, called gut closure, takes placewithin the first postnatal days. Intestinal permeability decreases fasterin breastfed babies than in formula-fed infants [26]. Tight junctions ofIECs take some weeks to mature and close the gut to whole proteinsand pathogens. Any delay or insult to the gut that changes this processpredisposes the infant to infection, inflammatory states, and allergicsensitization.

Host–microbiota interactions and cross-talk between differentmembers of the gut microbiota are far from completely understoodalthough they represent a crucial factor in the development andmaintenance of immune system. In a normal state of eubiosis, intes-tinal epithelial TJs provide an effective barrier against paracellularpenetration of noxious substances and antigens present in the gas-trointestinal lumen, including bacteria, bacterial toxins and bacterialby-products. Bifidobacteria appear to play an important role in main-taining the gut barrier. An increase in Bifidobacteria in ob/ob micewas associated with a significant improvement of gut permeabilityand with an increase in tight junction mRNA expression. Anti-inflammatory cytokines such as IL-10 and TGF-β may attenuate orprotect against intestinal inflammation by preserving the TJ barrierfunction.

The intestinal epithelium is fundamental tomaintain barrier integri-ty and to participate in food degradation and absorption, but it can alsodecipher signals coming from microbial communities in the gut lumenand ‘educate’ the subjacent immune system to set the tone of mucosalimmunity accordingly [34]. In response to the gut microbiota, the hostwill secrete a variety of cytokines and host defense effector moleculesthat can in turn shape the indigenous microbiota community andprime host responses to environmental stimuli.

Several data confirm that gutmicrobiota is engaged in a dynamic in-teraction with the intestinal innate and adaptive immune system, af-fecting different aspects of its development and function. Over thepast two decades, immunologists have begun to appreciate the com-plexity of the gut innate immune system, its importance as the firstwave of defensive action against perceived harmful microbes and itsfunctions in triggering antigen-specific responses by engaging the adap-tive immune system. In order to maintain homeostasis, the innate im-mune cells will continuously need to identify self versus non-self.Discrimination between specific microbesmay be a feature of the adap-tive immune system,which can recognize discretemolecular sequencesand mount both pro- and anti-inflammatory responses depending onthe nature of the antigen. Through sophisticated pathways IECs cansense and respond to microbial stimuli to reinforce their barrier func-tion and to participate in the coordination of appropriate immune re-sponses, ranging from tolerance to anti-pathogen immunity. Central to

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this process are innate immune receptor molecules referred to as ‘pat-tern recognition receptors’ (PRRs). Among these evolutionarilyconserved molecules the best known are the toll-like receptors (TLRs),that reside on the cell surface, and cytosolic nucleotide-binding oligo-merization domain (NOD)-like receptors (NLRs) [36]. TLRs, present onIECs, dendritic cells (DCs), macrophages, neutrophils, and other cellsbelonging to innate immune system, act as primary sensors that detecta wide variety of microbial components and promote immune re-sponses through recognition of their respective bacterial commensal-or pathogen-associated molecular patterns (CAMPs and PAMPs respec-tively) such as lipopolysaccharide (LPS), bacterial DNA, peptidoglycan,muramyl dipeptide, lipoteichoic acids, N-formylmethionine and flagel-lin. Binding of the PRRs to their respective ligands initiates a widespectrum of responses from phagocytosis to production of a variety ofcytokines, which in turn shape and enhance the adaptive immuneresponses.

Both CAMPs and PAMPs are sensed by TLRs. This requires discrimi-native responses to commensals in comparison with pathogens to en-sure tolerance and protective immunity, respectively. Members of thetoll-like receptor and NOD-like receptor families provide distinct path-ways for the recognition of microbial ligands or endogenous signals as-sociated with pathogenesis. The strategic distribution of TLRs and NLRson and within IECs has a considerable effect on whether bacterial mo-lecular patterns will be recognized.

While classically thought to promote immunity, it now appears thatPRRs signaling can also restrain immune responses as a number of fac-tors allow the intestinal epithelium to tolerate commensal organisms.Signaling through surface at the apical pole of IECs induces the inhibi-tion the nuclear transcription factor-κB (NF-κB), whereas TLR signalingfrom the basolateral pole triggers sequential activation of NF-κB that in-duces the release of paracellular secretion of proinflammatory cytokinesand anti-microbial peptides.

Conversely, when pathogenic bacteria invade IECs they introducePAMPs into the cytosol where they are recognized by NLRs. Restrictedaccess to cytosolic NLRs, therefore, aims to constrain interactions withcommensal bacteria. Transmembrane TLRs 1, 2, 4, 5, and 6 and cytosolicNOD1 and NOD2 detect and translate signals of PAMPs into rapid hostdefenses.

A characteristic feature ofmucosal tolerance is the induction and ex-pansion of Treg cells, which limit excessive proinflammatory responses.These include the masking or modification of CAMPs that are usuallyrecognized by TLRs and the inhibition of the NFκB inflammatorypathway. Adaptive immunity efficiently tailors immune responses todiverse types of microbes, whether to promote mutualism or hostdefense. Recent studies suggest that certain symbiotic bacteria inhibitthe NF-κB pathway. Commensal species such as B. infantis have beenshown to differentially induce Treg cells and result in the production ofthe anti-inflammatory cytokine IL-10, whereas other resident symbi-onts may promote the development of Th cells, including Th17 cells,and result in a low-grade inflammatory response (physiologic inflam-mation). Round et al. showed one of the mechanisms by which the im-mune system differentiates between the microbiota and infectiveagents to avoid triggering intestinal immunity. According to the authors,a symbiosis factor (polysaccharide A) of the human gut commensal B.fragilis activates Treg cells to promote immunologic tolerance [37].Therefore, TLRs represent dynamic signaling systems that mediate di-verse immunologic outcomes, distinguishing between beneficial andpathogenic microbes to engender host–bacterial mutualism and/or toensure host defenses.

The crosstalk between colonizing bacteria, intestinal epithelial cellsand immune cells (‘trialogue’) has shown to have a crucial role in stim-ulating appropriate programming of mucosal immunity to drivetolerogenic responses [34,35,38,39]. As a pivotal link in innate and adap-tive immune responses, DCs play an extremely important role for therecognition of antigenic structures of alimentary or microbial origin.DCs in the lamina propria can extend their appendices between

Please cite this article as: Miniello VL, et al, Gut microbiota biomodulatorsdx.doi.org/10.1016/j.cca.2015.01.022

epithelial cells, and, via TLR-2 and TLR-4 on their surface, they can sam-ple commensal-bacterial molecular patterns.

Intestinal DCs induce anti-inflammatory and tolerogenic responsesto harmless antigens such as those derived from the residentmicrobiotaor harmless food. Normal dendritic cells (nDCs) induced by infection orexposure to bacterial components may become DC1-like cells (DC1)and/or regulatory DCs (DCreg). DC1 can activate Th1 cells, which inhibitallergic Th2 responses (immune deviation), whereas tolerogenic DCregcan induce allergen-specific and allergen-non-specific Treg cells throughthe production of immunoregulatory cytokines such as IL-10 and TGF-β(immune regulation) [40]. Treg cells function to dampen the immune re-sponse by expressing forkhead box protein 3 (FoxP3), TGF-β and IL-10.Recently Hansen et al. demonstrated that adult CS born mice had lowerproportions of FoxP3+ regulatory T cells, DCreg, and less IL10 gene ex-pression in mesenteric lymph nodes and spleens [41].

7. Here comes the stork, by the scalpel

Over the last 20 years numerous reports have been published on theepidemic of immune-mediated disease statesworldwide, but specifical-ly in developed countries and among children. Although genetic suscep-tibility plays an irrefutable role in immune disorders, their prevalencehas beenmuch faster than any possible shift in genetic constitution. Re-cent evidences indicate that environmental factors in the gut, and espe-cially qualitative and quantitative disturbance in early colonization(dysbiosis), lead to the disruption of the gut immune homeostasis.

The gut microbiota promotes human health, but can also drive dis-ease. Perturbations in the microbial colonization of an infant's gastroin-testinal tract (delivery by CS, premature delivery, and excessive use ofperinatal antibiotics) have been associatedwith dysbiosis and increasedexpression of autoimmune and allergic diseases [15].

A recent meta-analysis investigated the potential effect of CS onasthma. Reviewing a total of 20 articles, Thavagnanam et al. reported astatistically significant 20% increase in the subsequent risk of developingasthma in children born by CS [42]. It is possible that this associationmay be different in the atopic and non-atopic asthma phenotypessince research evidence suggests that risk factors associated with asth-ma might be different for each asthma phenotype. However, accordingto some studies emergency CS and instrumental vaginal delivery (for-ceps and vacuum extraction) may contribute to the development ofasthma rather than the mode of delivery, possibly because of theprolonged maternal stress associated with these deliveries [43].

The rising incidence of childhood-onset Type 1 diabetes in devel-oped countries is likely to be a result of changes in perinatal environ-mental factors. The hypothesis that infants delivered by CS do nothave the same exposure to maternal bacteria as infants delivered vagi-nally is one of the possible causal pathways. Cardwell et al. investigatedthe evidence of an increased risk of childhood-onset type 1 diabetes inchildren born by CS by systematically reviewing the published literatureand performing a meta-analysis with adjustment for recognized con-founders. This analysis demonstrated a 20% increase in the risk ofchildhood-onset type 1 diabetes after CS delivery that cannot be ex-plained by known confounders (maternal age, birth weight, gestationalage, birth order, maternal diabetes or breastfeeding) [44].

Recently, a positive association between elective (not emergency)CS and later celiac disease was shown, indicating that the commensalmicrobiota of the newborn may play a role in the development of celiacdisease [45].

Colonization differences linked to differing modes of delivery seemto be taking the hygiene hypothesis to an entirely new level. Microbiotaassociatedwith disease is defined by lower species diversity, fewer ben-eficial microbes and/or the presence of pathobionts. During natural de-livery the vaginal and the fecal maternal microbiota (mainly Bacteroidesand Bifidobacteria) are transferred from mother to child through thebirth channel. Conversely, CS-delivered infants, deprived of contactwith the maternal vaginal microbiota, experience a deficiency and a

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delay in colonization of Bacteroides, Bifidobacterium and Lactobacillusand a higher presence of facultative anaerobes such as Clostridium spe-cies, compared with vaginally born infants [7,46].

The total microbiota diversity is lower in CS infants, probably largelyas a consequence of the lack of one of the major gut phylum, theBacteroidetes, during the first two years of life. Bacteroidetes are impor-tant for priming the immune system to respond appropriately to trig-gers. Recently Jakobsson et al. showed that infants born via CS hadlower total bacterial diversity during the first two years of life, a lowerabundance and diversity of the phylum Bacteroidetes, lower circulatinglevels of Th1-associated chemokines (CXCL10 and CXCL11) during in-fancy, and reduced Th1 responses, compared with children deliveredvaginally [47]. Reduced diversity of the gut microbial communities dur-ing infancy was shown to be associated with increased risk of allergicdisorders (allergic sensitization, allergic rhinitis, and peripheral bloodeosinophilia), in the first six years of life [48].

Emerging evidences support the role of C. difficile in the associationbetween mode and place of delivery and atopic outcomes [49,50]. Theassociations between the presence of C. difficile in the feces at 1 monthpostpartum and the development of wheeze, eczema, and sensitizationto food allergens in the first 2 years of life were confirmed to persistthroughout the first 7 years of life, in a more recent study [46]. More-over, colonization with Clostridia, at the age of 5 and 13 weeks, was as-sociated with an increased risk of developing atopic dermatitis in thesubsequent 6 months of life [50].

Imbalanced gut flora can result in the production of LPS, the majorcomponent of the outer membrane of Gram-negative bacteria (C. diffi-cile, enteropathogenic Escherichia coli, Pseudomonas aeruginosa, Klebsiel-la pneumoniae, and others). The release of LPS induces an increase inintestinal TJ permeability, mucosal immune dysregulation and en-hanced production of proinflammatory cytokines (IL-1β, IL-6 and TNF-α). These cytokines cause direct disruption of the intestinal TJ barrierleading to paracellular permeation of luminal antigens into circulation,which may contribute to systemic inflammation, resulting in a furtherincrease in TJ permeability [51].

Although several studies have monitored the bacterial communitiesin preterm infants, the early microbial colonization of the immature gutis not yet completely understood. The gut of a preterm infant, cared forin the relatively aseptic neonatal intensive care environment and usual-ly receiving antibiotics shortly after birth, shows delayed colonizationwith a low diversity of bacterial community species- and strain-levelcomposition. The fact that most preterm infants are sparsely colonized,as opposed to the more complex/diverse pattern of colonization foundin healthy term neonates, could predispose to continued overgrowthof potentially pathogenic species, especially under antibiotic pressure.Reduction of normal commensal microbiota diversity due to over-growth by infectious agents may impair beneficial stimulation ofgastro-intestinalmucosal development and the innate and adaptive im-mune responses. The gut microbiota of preterm infants delivered by CShave been reported to differ from those of infants delivered vaginally,both in the timing and composition of colonization.Moreover, maternalcomplications appear to significantly influence the composition of thegut microbiota of premature infants. Neonates with prenatal exposureto a non-sterile intrauterine environment (prolonged premature rup-ture of membranes and maternal chorioamnionitis) were found tohave a relatively higher abundance of potentially pathogenic bacteriain the stool compared to neonates without those exposures [52,53].

8. Gut microbiota biomodulators

There is accumulating evidence showing that prenatal and postnatalperiods represent an important window of opportunity to modulateimmune responses. Increased interest in the effects of the intestinal mi-crobiota on human health has resulted in attempts to optimize themicrobial ecosystem by gut microbiota biomodulators such as pro-biotics, prebiotics, synbiotics or postbiotics [54,55]. Probiotics are

Please cite this article as: Miniello VL, et al, Gut microbiota biomodulatorsdx.doi.org/10.1016/j.cca.2015.01.022

defined by the Food and Agriculture Organization (FAO) of the UnitedNations and the WHO (FAO/WHO) as “live microorganisms that,when administered in adequate amounts, confer a health benefit onthe host”. A prebiotic is “a selectively fermented ingredient that allowsspecific changes, both in the composition and/or activity in the gas-trointestinal microbiota that confers benefits upon host well-beingand health”. Human milk oligosaccharides (HMOs) make up a largepart of human milk composition, similar to the level of proteins. Al-though their biological function is not yet fully understood, there is ev-idence that HMOs are important for the prebiotic effect (essentiallybifidogenic) as well as the anti-infective and allergy-preventive proper-ties of human milk. Apart from HMOs, only bifidogenic, non-digestibleoligosaccharides, particularly inulin and its hydrolysis product such asfructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) fulfillall the criteria for prebiotic definition. Due to their complexity, oligosac-charides with structures identical to HMOs are not available as dietaryingredients. As an alternative and in an attempt to mimic the multiplebenefits of HMOs, other, non-HMOs are currently added to infant for-mula, including FOS and GOS. An updated systematic review andmeta-analysis of randomized controlled trials of prebiotics in pre-terminfants reported no adverse effects, indicating that these non-digestible food ingredients that act to promote the growth of selectedbacterial species are safe in babies born by premature delivery [56].More evidence is rising for (in)direct effects of a mixture of neutralshort-chain GOS and long-chain FOS on the immune system. In a recentCochrane review Osborn and Sinn found some evidence that infant for-mula containing prebiotic supplements can help prevent atopic derma-titis in children up to two years, but was unclear whether the use ofprebiotics should be restricted to infants at high risk of allergy or mayhave an effect in low risk populations [57].

In addition, since the quality of existing evidence was generally lowor very low, the authors concluded that more high quality research isneeded before we can recommend routine use of prebiotics for preven-tion of allergy.

Synbiotic is a product that contains both probiotics and prebiotics.Postbiotics are metabolic by-products, dead microorganisms, or othermicrobial-based nonviable products generated by a probiotic microor-ganism that influences the host's biological functions. Since theEuropean Society of Paediatric Gastroenterology Hepatology andNutrition (ESPGHAN) Committee on Nutrition stated in 2010 that “thepresently available data do not permit recommending the routine useof probiotics as food supplements in preterm infants”, heat-killedprobiotics or bacterial lysatesmay be a promising alternative to live bac-teria as a therapy with significantly less risk for adverse outcomes. Ofadditional concern is that in many countries, including the UnitedStates, regulatory agencies such as the U.S. Food and Drug Administra-tion (FDA) have limited ability to intervene with the use of probioticsin premature infants without a specific product marketed for suchpurpose.

Most probiotics taxonomically belong to two genera, Bifidobacteriaand Lactobacilli. However, Lactococcus, Streptococcus, and Enterococcusspecies, as well as some non-pathogenic strains of Escherichia coli(E. coli Nissle 1917), and yeast strains (Saccharomyces boulardii) mayalso act as probiotics.

Mechanisms of probiotic action described to date include improvingthe gut'smicrobial composition andpreserving its stability; competitionwith pathogens for nutrients, growth and adhesion; strengthening ofthe gut mucosal barrier (increased mucous production, repair andmaintenance of tight junctions with reduced permeability); anti-inflammatory effect; modulation of the immune system inside and out-side the gut to convey an advantage to the host [58].

Research studies involving animals and human beings providegood evidence that certain strains of Bifidobacteria and Lactobacilli canbeneficially modulate immune function through a number of differentpathways including effects on mucosal barrier, enterocytes (reducedcell signaling via NF-kB, increased production of TGF-β), local DCs

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Table 1Old and new knowledge about probiotics.

What do we know?

• The human gut microbiota plays a pivotal role in many aspects of humanphysiology including metabolism, nutrient absorption and immune function.

• A well-balanced bacterial colonization of the infant gut has a profound impacton programming short- and long term immune homeostasis.

• In the initial stages of life several factors influence the establishment andcomposition of the gut microbiota: gestational age, delivery mode and place,infant feeding, and the use of antibiotics, among others.

• Establishment of the intestinal microbiota has been shown to differ betweeninfants delivered by Caesarean section and those delivered vaginally.

• Inadequate or abnormal early intestinal colonization (dysbiosis), whetherinduced by Caesarean section, premature delivery, or excessive use of perinatalantibiotics, lead to the breakdown in the gut homeostatic symbiosis and, in turn,to development of immunologically mediated diseases.

• Gut microbiota biomodulators, such as probiotics, prebiotics and synbiotics, caninfluence the intestinal microbiota and modulate the immune response.

• The probiotic immunomodulatory properties seem to be bacteria-specific andenvironment-dependent.

What is still unknown?

• Whether probiotics and prebiotics, alone or together (synbiotics), may supportdisease prevention in infants who tend to have a delayed and/or aberrant initialcolonization with reduced microbial diversity.

• Which are optimal conditions (strain, dose and duration) for clinical microbialapplication.

• Which are potential short- and long-term health effects of orally administeredprobiotic bacteria in preterm babies.

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(promotion of tolerogenic DCs), regulatory T cells (increased TGF-β-producing Th3 cells), T and B cells (increased Th1 differentiation andTh1 skewed responses, inhibition of Th2 allergic responses, increasedlocal IgA production) [59,60].

Indeed, several steps in the NFκB pathway present opportunities forprobiotics to prevent the activation of this nuclear factor and influencedownstream cytokine secretion.

As the composition of the human microbiota at each body site isdistinct, different probiotics are likely needed for diseases at differentbody sites. In a double-blind randomized placebo-controlled study weshowed that oral administration of a probiotic strain in children withatopic dermatitis can modulate in vivo the exhaled breath condensatecytokine pattern [61]. Decreased capacity to generate INF-γ is a hall-mark of the atopic state. Our findings suggested that Lactobacillus reutericould stimulate INF-γ production and lower pro-inflammatory IL-4levels in allergic paediatric patients. This is the first study, to our knowl-edge, showing that a probiotic strain could modulate the cytokinepattern at sites in the body that are distant from that of the initial inter-action of the probiotic.

Clinical evidence suggests that probiotics can act as surrogate colo-nizers [13] and help prevent the expression of allergic diseases. Basedon the established fact of altered gut colonization in cases of CS, it hasbeen recently suggested that CS-delivered babies might benefit fromthe dietary supplementation with probiotics and/or prebiotics.

To evaluate the effect of probiotic and prebiotic supplementation inpreventing allergies, Finnish researchers conducted a clinical trial ofmore than 1200motherswhose infants would be at high risk to developallergies. According to American Academy of Paediatrics, ESPGHAN andEuropean Society for Paediatric Allergology and Clinical Immunology(ESPACI) an infant is defined as “high risk” for developing allergic dis-ease if there is at least one first degree relative (parent or sibling) witha documented allergic condition (atopic dermatitis, asthma, allergicrhinitis, or food allergy). During the last month of their pregnancies,the mothers took daily doses of a probiotic mixture (Lactobacillusrhamnosus GG, Lactobacillus rhamnosus LC705, Bifidobacterium breveBb99, Propionibacterium freudenreichii) or a placebo, and their infantswere given the same probiotic mixture plus a prebiotic (GOS) or a pla-cebo for thefirst 6months of their lives [62]. The childrenwere followedfor 5 years and evaluated for incidence of allergic diseases. Although noallergy-preventive effect was extended to age 5 years by perinatal sup-plementation with the probiotics and synbiotic in high-risk babies, it isnoteworthy that less IgE-associated allergic disease occurred only inCaesarean-delivered children (24.3% vs 40.5%; p = .035). Therefore,the authors concluded that “protection was conferred only to CSbabies”.

The crucial challenge is to clearly determine which probiotic organ-isms are beneficial and exert a preventive or therapeutic effect. Forthose that can duly be termed ‘probiotics’, the range of applicationshas to be defined more precisely than has been done so far. Differentspecies of the genera Bifidobacterium and Lactobacillus can evoke differ-ent responses in the host, but not all strains of the same species can beconsidered beneficial.

Acute inflammatory necrosis of the intestinal tract, necrotizing en-terocolitis (NEC) is the most common acquired gastrointestinal diseaseand the secondmost common cause of death among preterm infants inneonatal intensive care units. Probiotic supplementation has potentialas an effective way to prevent or minimize NEC [63]. However NeenaModi suggested that “all infants recruited to trials or receivingnew ther-apies receive long-term follow-up designed to test specific hypothesesrelating to safety, later outcomes and biological effector mechanisms”[64].

There has recently been an increase in the number of studiesreporting the use of probiotics in both experimental animal modelsand clinical applications [65]. Since all probiotics are not the same inprotectingpremature infants fromNEC, Underwood et al. tested two ge-netically different strains of Bifidobacteria, normal inhabitants of the

Please cite this article as: Miniello VL, et al, Gut microbiota biomodulatorsdx.doi.org/10.1016/j.cca.2015.01.022

gastrointestinal tract: B. infantis and B. animalis lactis [66]. The studywas conducted in premature infants born between 24 to 33 weeks ges-tation andweighing less than 1500 g. In both the formula-fed and breastmilk-fed newborns, greater increases in fecal Bifidobacteria occurred inthe B. infantis groups than in the B. lactis groups. In addition, only breastmilk-fed babies who received B. infantis experienced improved benefi-cial bacterial diversity over dose/time and a decline of harmful bacteriaknown as γ-Proteobacteriawhich typically increase at the onset of NEC.It is therefore inaccurate to generalize findings observed in a single pro-biotic species to all probiotics since their efficacy is widely variable andmultifaceted.

Improper use of the term “probiotic” and failure to recognize the im-portance of the dose specificity and strain specificity of effects is a con-cern [67]. Several evidences stress the importance of consideringspecific species and strain interactions andnot simply higher taxonomicdivisions in the relationship among intestinalmicrobial populations andtheir different responses to probiotics. The ability of probiotics to influ-ence the immune system differs greatly depending on the strain inquestion. One probiotic strain is not like another when it comes to im-mune function. Different strains activate different subtypes of T-helpercells while others specifically induce another subtype. Concerns havealso been raised about the quality of probiotic products. Some productshave been found to contain smaller numbers of live microorganismsthan expected. In addition, some products have been found to containbacterial strains other than those listed as ingredients.

9. Conclusions

In this reviewwehighlight advances in our understanding of the im-mune mechanisms by which the dynamic interplay of the gut microbi-ota and its host favors a mutual relationship. Increasing evidencesuggests that a qualitative and quantitative disturbance in colonization(dysbiosis) is associated with dysfunction of immune responses(Table 1). Inadequate or abnormal early intestinal colonization,whetherinduced by Caesarean section, premature delivery, or excessive use ofperinatal antibiotics, lead to the breakdown in this homeostatic symbi-osis and, in turn, to development of various chronic inflammatory disor-ders (Fig. 1). Noteworthy, in recent decades, there has been an increasein the proportion of CS-delivered infants beyond the recommended by

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Fig. 1. Delayed and/or aberrant initial colonization lead to the disruption of the gut immune homeostasis (dysbiosis) and, in turn, to the development of various chronic inflammatorydisorders.

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WHO level of 15%. Emerging evidences support the administration ofgut microbiota biomodulators (probiotics, prebiotics and synbiotics)to optimize the composition of themicrobiota and to strengthen the in-tegrity of the mucosal barrier, thereby restoring immunomodulatoryfunction in cases of aberrant homeostasis. The concept of probioticshas attracted increasing attention in recent years since several clinicalstudies, suggesting that probiotics may convert a dysbiosis to a symbio-sis by balancing potential pathogens with health-promoting bacteria,have been published.

By building on our current knowledge of strain specific immuno-modulatory properties it may becomepossible to design clinically effec-tive, bacteria based strategies to maintain and promote health. Futureresearch is especially needed on the identification of probiotic appropri-ate strains for a immunomodulatory function in cases of aberrant ho-meostasis, and especially on potential short- and long-term healtheffects, in preterm babies.

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