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Seminars in Immunology 19 (2007) 59–69

Review

Use of axenic animals in studying the adaptation of mammalsto their commensal intestinal microbiota

Karen Smith, Kathy D. McCoy ∗, Andrew J. MacphersonDepartment of Medicine, McMaster University, Hamilton, Ontario, Canada

bstract

Vertebrates are essentially born germ-free but normally acquire a complex intestinal microbiota soon after birth. Most of these organisms are non-athogenic to immunocompetent hosts; in fact, many are beneficial, supplying vitamins for host nutrition and filling the available microbiologicaliche to limit access and consequent pathology when pathogens are encountered. Thus, mammalian health depends on mutualism between hostnd flora. This is evident in inflammatory conditions such as inflammatory bowel disease, where aberrant responses to microbiota can resultn host pathology. Studies with axenic (germ-free) or deliberately colonised animals have revealed that commensal organisms are required forhe development of a fully functional immune system and affect many physiological processes within the host. Here, we describe the technical

equirements for raising and maintaining axenic and gnotobiotic animals, and highlight the extreme diversity of changes within and beyond themmune system that occur when a germ-free animal is colonized with commensal bacteria.

2006 Elsevier Ltd. All rights reserved.

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eywords: Axenic; Germ-free; Gnotobiotic; Schaedler flora; Commensal bactommensal mutualism; Inflammatory bowel disease

. Introduction

Vertebrates normally acquire a complex intestinal microbiotaoon after birth and also become colonized with microorgan-sms on other body surfaces. Most of these organisms areon-pathogenic to immunocompetent hosts. The intestine rep-esents an especially attractive niche, rich in carbon and mineralnd solute sources, and is maintained at a stable temperaturen mammals. The consequences of microbial colonization haveeen addressed by studies of the differences between adultnimals that are experimentally maintained under axenic (germ-ree) conditions and the same strain colonized with a ‘normal’nvironmental microbiota. Most of these studies have beenarried out with mammals, which will be the focus of thiseview.

Since germ-free animals have no competition for colonization

y incoming microorganisms, it is relatively easy to deliber-tely colonize germ-free animals with a few defined microbialpecies [1]. Following selective colonization by one or more

∗ Corresponding author at: HSC 3N5C, Department of Medicine, McMasterniversity, 1200 Main St. West, Hamilton, Ontario, L8N 3Z5, Canada.el.: +1 905 525 9140x22588.

E-mail address: kmccoy@mcmaster.ca (K.D. McCoy).

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044-5323/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.oi:10.1016/j.smim.2006.10.002

Deriving and maintaining germ-free animals; Mucosal immune system; Host-

nown bacterial species the animals are defined as having gno-obiotic status. A special example of this in mice is the modifiedchaedler flora of 8 specified bacteria, which is widely used byommercial breeders and animal facilities to provide defined,imited and balanced colonization of specific pathogen-free ani-

als [2–4]. It is important to note that specific pathogen-freeSPF) status of rodent colonies refers to the absence of knownathogens that may produce clinical or subclinical infectionshat bias research results [5]. Testing for pathogens requires aelection of the pathogens to be screened and consideration ofhe frequency and sample size for testing. These issues are alsoelated to the sensitivity and specificities of the screening tests.1

t is important to understand that SPF animals (1) are normallyolonized with commensal bacteria and (2) the diversity of theolonizing commensals is rarely accurately defined. Even if ani-als are delivered to a facility with a modified Schaedler flora,

ne cannot assume that this gnotobiotic status will continue:

ndeed it is highly likely that the flora will become diversifiednless animal handling is carried out under the most scrupu-ously clean conditions. Continued SPF status provides very

1 Please see http://www.lal.org.uk/pdffiles/LAfel2.PDF and http://ehs.cdavis.edu/animal/vet care/vc rhmp.cfm for discussion of issues in relationo SPF screening.

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ittle information about the diversity of intestinal or other envi-onmental commensals.

Our objectives in this review are to provide a background tohe historical and technical aspects of axenic and gnotobioticusbandry and to show the extreme diversity of changes withinnd beyond the immune system that occur when a germ-freenimal is colonized with commensal bacteria.

. History of studies of mutualism with environmentalicrobes in mammals and the development of axenic

xperimental animal colonies

Interest in the effects of commensal microorganisms on theirammalian hosts has a long history. In 1874, Billroth pub-

ished microscopic studies showing that microorganisms couldot be detected in the meconium of newborn babies, but theseuickly appeared in the first stools [6]. Escherich confirmedhese observations with microbiological cultures [7]. These earlynvestigators pioneered many subsequent studies showing thate are all born ‘germ-free’ and acquire our commensal organ-

sms in a colonization sequence soon after birth. The diversityf colonization is influenced by lactation, and later by nutrition.erm-free animals mature physically without the influences of

ny microbial colonization.It has also been known for over a century that high densi-

ies of intestinal bacteria are non-pathogenic as long as they areontained within the lower intestinal lumen. Using both clinicalbservations and animal experiments, Cushing and Livingoodhowed that traumatic perforation of the lower small intestiner the colon leads to peritonitis and septicaemia [8]. Debilitatednd immunocompromised individuals or animals also frequentlyuffer septicaemia with commensal organisms. Our mutualismith commensals therefore requires both intact anatomy and

unctional (innate) immunity: the practical consequence of thiss that lower intestinal surgery or abdominal trauma was highlyazardous before the availability of antibiotics.

The development of axenic animal models also has a longistory. Initial studies of mutualism between eukaryotic androkaryotic organisms were carried out with plants, and Pas-eur considered that microbes would be essential for long-termiability of both plants and animals [9]. This levelled a challengeor experimenters to see whether animals could be raised in aterile environment. This was initially accomplished by asep-ic Caesarean section of guinea pigs or mice and hand-rearingver several weeks, and later over a full life span so that theerm-free colonies could be potentially maintained by asepticnterbreeding [10–12]. A program that was able successfully to

aintain experimental animals germ-free over successive gener-tions was started at Notre Dame University in 1928 and detailsf the early challenges and accomplishments are reviewed byeyniers [13].

Of course, Pasteur was quite right that microbes are requiredor mammalian health in the sense that germ-free animals are

unctionally immature in many systems (Table 1). Whilst thiss not lethal in the sheltered environment of an aseptic flexiblelm isolator in which the animals are given ad libitum (sterile)ood and water, correct functioning of most body systems and

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unology 19 (2007) 59–69

voiding exquisite sensitivity to mucosal pathogens requires thenimal to be colonized with a commensal microbiota. Indeedhe early studies were able to show that the intestinal floraere important in supplying vitamins for host nutrition [14]

nd filling the available microbiological niche to limit accessnd consequent pathology when pathogens are encountered2].

A more recent aspect of the history of germ-free experi-entation needs to be mentioned. Human inflammatory bowel

isease (Crohn’s disease and ulcerative colitis) affects about 1n 2000 individuals and results in chronic relapsing and remit-ing intestinal inflammation, typically from the second to thirdecade throughout life. We now know from reverse genetictudies in human populations [15–17] that one of the suscep-ibility genes for Crohn’s disease encodes a eukaryotic receptorNOD2/CARD15) for the muramyl dipeptide component ofeptidoglycan, which is part of the capsular structure of prokary-tes [18,19]. In other words, altered host responsiveness to theicrobiota can underlie the condition. It is obviously not pos-

ible to experimentally sterilize the intestine of human patients,lthough strategies that surgically divert the fecal stream leado improvement in downstream inflammation. The generalityf the effect is established by another important line of exper-mentation for inflammatory bowel disease in the developmentf a large variety of animal models. In many different targetedesions of innate or adaptive immune function and immuneegulation, as well as deficiencies of intestinal epithelial per-eability, spontaneous chronic intestinal inflammation results

n the experimental rodent strains maintained under conditionshere they are colonized with intestinal microbes. Three land-ark papers were published in the same issue of Cell in 1993

etailing chronic intestinal inflammatory consequences of tar-eted deficiency in IL-10, IL-2 and the alpha chain of the T celleceptor [20–22], although subsequently a much larger varietyf these defects in different layers of host immunity have beenhown to behave similarly: reviewed in [23]. Where tested, inost of these models the chronic inflammation is abrogated if the

nimals are maintained in germ-free conditions [23], support-ng the concept that mutualism with the intestinal microbiota isnely balanced, and disrupting the protection mechanisms leads

o chronic intestinal immunopathology driven by prokaryoticnflammatory molecules.

. Methods of deriving and maintaining germ-freenimals and carrying out axenic experiments

Germ-free colonies of rodents are generally bred and main-ained in flexible film isolators. These are ventilated withEPA-filtered sterile air under positive pressure and are fit-

ed with a side port containing a double door system to allowntiseptic connexion of a transport drum to import sterile food,ater and bedding (Fig. 1). There are some exceptions to this

xperimental setup in which segments of intestine are rendered

erm-free by microsurgery [24,25], or germ-free piglets that areand-reared in aseptic conditions after Caesarian section, butontinuous maintenance of rodents in a flexible film isolator ishe dominant system and will be described in detail.

K. Smith et al. / Seminars in Immunology 19 (2007) 59–69 61

Table 1

Observation Reference Evidence of reversibility bydeliberate colonization

Nutritional differences Require exogenous Vitamin K in diet [38,39]Require vitamin B in diet [40]Decreased body fat percentage [41]Voluntary intake of food normal or increased [42–44]

Vascular Reduced total blood volume [45,46]Decreased cardiac output [45]Decreased regional blood flow to skin, liver,lungs, digestive tract

[42,47]

Increased cholesterol in blood stream [48]Increased red blood cell count andhematocrit values

[45,49]

Hepatic Liver is smaller [49]Increased ferritin [50]Increased cholesterol [51]

Respiratory Thinner alveolar and capsule wall andreduced reticuloendothelial elements

[52]

Fluid balance Increased water intake [42]Metabolic rate Decreased basal metabolic rate [53,54]

Intestinal physiology Osmolarity of the small intestine is reduced [42]Oxygen tension and electripotential in thesmall intestine is elevated

[42]

Absorptive function Increased absorption of vitamins andminerals

[55–57]

Endocrine function Decreased Iodine uptake by thyroid [53]Decreased motor activity andhyporesponsiveness to epinephrine,norepinephrine and vasopressin

[58]

Exocrine function Increased trypsin and chymotrypsin andinvertase in faeces

[59–61]

Increased mucoproteins andmucopolysaccharides

[59]

Intestinal motility Increased muscular tissue (muscle cells areelongated and hypertrophied) in cecum

[42]

Cecal muscle strips less sensitive to amines [62]Decreased spontaneous contraction of cecalmuscle strips

[62]

Altered myenteric neurons [63]Structural differences in the primary plexusof Auerbach

[63]

Increased transit time of contents [64,65]

Intestinal function Rate of absorption of ingested materialsaltered

[46,55]

Increased Enteroglucagon cells [66] [66,67]Altered enzyme expression [68–77] [70–74,78]No cyclical or branched chain fatty acids inintestinal contents

[79]

Excrete largely unsaturated fatty acids [79]Decreased fatty acids in intestinal contents [79]No urobilin in urine [80] [80]Increased bilirubin in faeces [80]Faeces have large amounts of mucin [81]

Electrolysis Cecal contents are more alkaline [82] [82]Urinary calcium and citrate are high,phosphate is low

[57,83] [83]

Slightly reduced sodium and low chlorideion concentration in intestinal contents

[84–86] [85,86]

Metabolism Response to anaesthesia altered [87]More urea and little ammonia in intestinalcontents

[44,79] [88]

62 K. Smith et al. / Seminars in Immunology 19 (2007) 59–69

Table 1 (Continued )

Observation Reference Evidence of reversibility bydeliberate colonization

Increased excretion of free amino acids andurea

[44]

Increased nitrogen in faeces and cecalcontents

[41,44,79]

Increased oxidation-reduction potential ofcecal contents

[82] [82]

Excrete low amount of acetic acids [89]�-aspartlyglycine present in faeces [61] [90]Cecal contents have more hexosomines [91]

Intestinal morphology Total mass of intestine is decreased [41,49,92,93]Total surface area of small intestine isdecreased

[92,94]

Villi of the small intestine are slender anduniform

[95]

Ileal villi are shorter [96]Duodenal villi are longer [97]Crypts of small intestine are shorter [95,96]Lamina propria of small intestine is thinnerand less cellular

[95,96]

Cellular renewal rate is decreased in laminapropria of the small intestine

[96]

Cecum is larger [98–101] [100]Cecal wall is thinner [42]Cecal mucosa is thinner and raised in shortirregular villi

[76]

Tend to develop intestinal volvuli atileo-caecal-colic junction

[42]

Intestinal epithelium characteristics Proportion of epithelial cells normal orslightly elevated in small intestine

[102]

Epithelial cells covering the villi are moreuniform and have longer microvilli

[96]

Epithelium of small intestine shows slowerrate of cell turnover

[96,97,103–105] [104]

Decreased cellular renewal rate in Peyer’spatch

[96]

Increased number of goblet cells in cecum [106]Decreased RELMb in colonic epithelium [107] [107]Different lectin composition in mucus incolon

[108]

Mucosal immunity Plasma cells are rare in small intestine [95]Decreased IgA [109–112] [109,112]More diverse repertoire of CD8 IEL of smallintestine in rats (no difference in mice [113])

[114] [114]

Reduced number and cytotoxicity of IEL ofsmall intestine

[95,113,115–117] [95,113,115–117]

Decreased expression of activation markerson intestinal macrophages

[118]

Decreased MHC II on epithelial cells ofsmall intestine

[119,120] [119,120]

Lower levels of nitric oxide in small intestine [121]Increased 5-HT in small intestine [122]Decreased Histamine in small intestine andintestinal contents

[122]

Secondary lymphoid tissue Reduced lymphocytic tissue and lymphaticsystem

[49,100,123–127] [100,127]

Peyer’s patch are reduced in number and size [49]MLN are smaller, less cellular and do nothave germinal centres

[95] [95]

Systemic immunity Fewer germinal centres and plasma cells [102,127–130] [102,127]Low Immunoglobulin levels [110,111,123,124,126,131–140] [133,134,139,140]

IgM may be normal (or decreased [110]) [111]Decreased IgM and IgG response toDNP-BSA

[141]

K. Smith et al. / Seminars in Immunology 19 (2007) 59–69 63

Table 1 (Continued )

Observation Reference Evidence of reversibility bydeliberate colonization

Delayed and reduced primary antibody titresagainst heat-killed E. coli

[142]

Decreased delayed type hypersensitivity inresponse to sheep red blood cells

[143]

Decreased response to T cell mitogens [141,144] [144]Normal IgM response to sheep red bloodcells

[141]

Normal IgM response to phosphorylcholine [145]Normal IgM and IgG response toDNP-lys-Ficoll

[141]

Normal response to Ova [146,147]Normal response to DNP-KLH [148]Normal response to Alum-bovine gammaglobulin

[135]

Good graft vs. host reaction [149]Enhanced antibody response to ferritin [150]Increased antibody response to DNP-Ficoll [151]

Infection susceptibility/response More susceptible to Shigella flexneri [127]More susceptible Bacillus anthracis [152]Less able to contain Leishmania [153]More susceptibile to Listeria [154]Lower antibody response to Escherichia coli [138,142]Normal response to Plasmodium berghi [155,156]Increased susceptibility to Coxsackie B [157] [157]Slightly more susceptible to Influenza A [158]Normal response to some viruses [159,160]Normal response to Trypanosoma Lewisi [161]Respond to Candida albicans [162]Normal antibody response to Serratiamarcescens

[150]

Viral infection results in comparableslightly higher interferon production

Fig. 1. Flexible-film isolator for axenic husbandry. The interior of the isolatoris ventilated under positive pressure with HEPA-filtered air (A), and animals aremanipulated using the gloves (B) set into the side of the plastic film. The sideport with external (C) and internal (inset) doors to allow sterile connexions withthe isolator is also shown.

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Transport drums are used for preparation of sterile materialso supply axenic isolators. They are designed to fit into an animalnit autoclave and consist of a steel cylinder, with an area thats perforated, open at one end (Fig. 2A). The perforated sections sealed with a fiber filter (to permit the penetration of steam inhe autoclave) and the open end is sealed with an impermeablelm or an autoclavable plastic cap once the contents are insideFig. 2B). Special arrangements of perforated steel trays allowood pellets to be autoclaved in the drum, and a trap arrangements used to catch excess condensation when autoclaving waterFig. 2). The drums need a trolley system to be rolled in andut of the autoclave and within the animal unit, owing to theireight.Once an autoclaved drum has been prepared it is maneuvered

nto place and the external door of the double door airlock onhe isolator is removed. To achieve a sterile connexion, the areaetween the drum and the isolator must be sterilized. This ischieved by a cylindrical plastic sleeve, which is strapped oraped between the airlock and the drum (Fig. 3A). Before thenner door of the airlock is opened, or the membrane sealing

he drum broken, the inside of the transfer sleeve is sterilizedy spraying with a 2% peracetic acid mist under high pressureFig. 3B). This is a very effective sterilant (although an extremelyorrosive and irritant chemical) widely used in the food industry:

64 K. Smith et al. / Seminars in Immunology 19 (2007) 59–69

Fig. 2. Transport drums for autoclaving materials to be used in germ-free husbandry. (A) View of a drum filled with cages, the inside of the drum showing underlyingp matera d prioa e’ str

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erforations in the steel structure required for sterilizing steam to reach internalround the outside and contents in place, sealed with transparent film at one enutoclave food pellets is also shown. Note the steam sensitive control ‘SteriGag

0 min after misting the inside of the sleeve is sterile. Follow-ng sterilization the door and seal can be opened and materialsmported into the isolator.

It is potentially easy to contaminate germ-free animals andeticulous protocols and experimental controls are required toake sure this does not happen. These are done on a number

f different levels: (a) the autoclave printout with details of thectual cycle is taped onto the drum and checked before connex-on; (b) the drum is always opened first and the steam sensitiveontrol strip is checked before opening the inner door of thesolator; (c) samples of the imported materials are taken forerobic and anaerobic bacterial culture at each connexion; (d)amples of animal feces and bedding are taken from the isolatoror bacterial culture at each connexion; (e) sentinel animals areemoved on a monthly basis for direct bacteriology, microscopynd 16S PCR testing of intestinal contents to test for culturablend unculturable organisms.

So far this outline of axenic animal husbandry has covered

aintenance of an established colony. At the outset the isolatorust be cleaned and sterilized (this time by the use of a 3%

eracetic acid mist). The initial colony of germ-free animals isbtained using a special (lighter!) version of the transport drum

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ig. 3. (A) Transport drum connected to the port of the isolator using a plastic sleeve (he drum remains in place until sterilization is complete). The inset shows the glovesnside of the connecting sleeve with 2% peracetic acid using a pressure spray. Followleeve is left for 60 min for sterilization to be complete. The membrane of the drum ihe contents are checked: provided the results are satisfactory the internal door of the

ials during the autoclave cycle. (B) View of a drum with the fiber filter wrappedr to autoclaving. The internal arrangement of (removable shelves) required to

ip that has been placed inside each drum.

rom another facility or commercial supplier, and imported insimilar fashion to the materials through a sterilized sleeve, asescribed above.

The power of modern experimentation with mice dependsn the wide availability of different strains with known geneticesions that allows the experimenter to determine the effect ofdefined gene product on the whole animal in vivo. To do this

n the setting of axenic animals requires germ-free rederivationf strains that are usually only available as colonized animals.here are two ways of doing this. Many units euthanise a preg-ant female at term of the strain to be derived, and quicklyass the body through antiseptic into an isolator: the pups arehen urgently delivered, resuscitated and placed with a germ-ree foster mother that has newly delivered a litter of her own.he drawbacks of this approach are that considerable skill is

equired to decide the timing of the Caesarian section so that theups will breath following surgery and resuscitation. Secondly,cceptance by the foster mother requires a considerable element

f luck.

An alternative method of germ-free rederivation, which werefer, is to carry out an embryo transfer at the two-celltage, using pseudopregnant germ-free females as recipients

with the external door removed, but the internal door and the membrane sealingattached to the opposite slide of the sleeve. (B) The procedure of misting the

ing this, the access port used to spray the inside of the sleeve is sealed and thes then pierced and the internal control strips for effective steam sterilization ofisolator is then opened and the contents of the drum are imported.

K. Smith et al. / Seminars in Immunology 19 (2007) 59–69 65

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ig. 4. Experimental protocol for superovulation of the embryo donor females (Stemales.

Fig. 4). The donor females of the appropriate strain are super-vulated with sequential injections of pregnant mare’s serumonadotrophin (PMSG) and, 48 h later, chorionic gonadotrophinnd then mated with stud males of the same strain. Thirty-sixours after mating the oviducts are dissected and the embryosushed out by cannulation of the infundibulum under a dis-ecting microscope. Harvested two cell stage embryos (whichust have been fertilized) are collected with a micropipette andashed five times in antibiotic-containing medium: these are

hen transferred into the oviducts of a germ-free recipient femalehat was mated with a vasectomised germ-free male 60 h previ-usly. Whilst this procedure is technologically more demandinghan Caesarian section, it avoids difficulties of timing of deliv-ry (which occurs spontaneously at term) and the mother willsually take care of the new born pups. Further, risks of trans-ission of transplacental pathogens are avoided, and sufficient

ffspring are derived in some cases for the experiments to be car-ied out without the requirement for subsequent interbreeding tostablish a germ-free colony of that strain.

Regardless of method, rederived strains are kept in isolationway from the main breeding germ-free colony until their axenictatus has been repeatedly confirmed by the methods describedreviously.

Axenic experiments can be as simple as directly comparingissues or cell populations of germ-free mice removed from areeding isolator with the same strain colonized with an intesti-al flora. Alternatively, the animals can be manipulated undererm-free conditions or colonized with deliberate bacterial con-amination or by the addition of a female containing a definedora to the same cage. Such experiments are normally carriedut in smaller ‘surgical’ isolators, which are loaded from thereeding isolator and moved into an experimental room. This

etup minimizes the risk to the breeding stock from the greaterisk of bacterial contaminations during manipulations. Surgicalsolators can also be moved into rooms with biosafety levels 2r 3 for work with biologically hazardous agents.

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) and transfer of two cell stage embryos into recipient germ-free pseudopregnant

. The spectrum of differences between germ-free andolonized animals

Numerically, most environmental organisms are in the lowerntestine, and the effects on the intestinal mucosal immune sys-em between germ-free and colonized status are profound (andell known to most immunologists). For example, the contentf intestinal IgA-secreting plasma cells is reduced in germ-freenimals, and the Peyer’s patches are reduced in size and the num-er of lymphoid follicles that they contain. The T cell contentf the mucosal immune system is also reduced in germ-free ani-als: particularly the CD4+ cells of the lamina propria, but also

ome subsets of the intraepithelial compartment, particularly the� T cell receptor CD8��-bearing subset [26–28]. Experimentsomparing the gene expression profiles of the intestinal epithe-ial layer between colonized and germ-free animals have shownhat programming of expression of intestinal epithelial cells ishaped by the presence of intestinal bacteria and that upregu-ated genes contribute to secretion of antibacterial molecules athe intestinal surface and central regulation of host metabolism29–32]. There are a large number of additional studies that showiverse changes in intestinal morphology, absorptive function,lectrolyte handling, bile metabolism, motility, and enteroen-ocrine and exocrine function in the germ-free state, which areeferenced in Table 1. In most cases these observations have ante-ated expression profiling and gene-targeting technology so thenderlying host signaling mechanisms are not known.

The short-range interactions on the intestine or mucosalmmune system itself are by no means the only consequencesf bacterial colonization. Central systemic lymphoid structuresave a hypoplastic structure, with reduced B and T cell contentnd poorly formed high endothelial venules in germ-free ani-

als [33,34]. Since live commensal bacteria are generally absent

rom systemic secondary lymphoid structures, and SPF mice aremmunologically ignorant of these organisms [35,36] the effectsre likely to be due to microbial molecules that are absorbed into

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he body: this has been directly shown for a bacterial polysaccha-ide (PSA) of Bacteroides fragilis which can normalize the CD4

cell content of the spleen when given as a purified compound37]. The practical consequence is the function of the immuneystem in response to immunization, tolerisation or systemicnfection by viral pathogens is altered in the germ-free state (seeable 1).

As with the short-range effects of colonization in the intes-ine, a much larger range of consequences of the germ-free stateave also been described in many different non-immune sys-ems. These include effects on body metabolism, electrolyte anduid handling, the vasculature, the liver, the endocrine systemnd behaviour (Table 1). In most cases the detailed mechanismsnderlying the physical and functional alterations are not yetully understood.

This astonishing range of immune and non-immune hosthanges demonstrate that mammalian bodies are powerfullyhaped by the presence of commensal microorganisms. Normalost function of many systems is dependent on colonization,ut intestinal inflammatory immunopathology can also resultrom defective mutualism. The critical tools of strain combina-ion experiments and axenic/gnotobiotic technology will allows to understand the mechanisms that underlie host-commensalutualism in health and to improve the therapies of the defects

hat occur in inflammatory bowel disease.

eferences

[1] Schaedler RW, Dubos R, Costello R. Association of germfree mice withbacteria isolated from normal mice. J Exp Med 1965;122:77–83.

[2] Dubos R, Schaedler RW. The effect of the intestinal flora on the growthrate of mice, and on their susceptibility to experimental infections. J ExpMed 1960;111:407–11.

[3] Orcutt RP, Gianni FJ, Judge RJ. Development of an ‘Altered Schaedlerflora’ for NCI gnotobiotic rodents. Microecol Ther 1987;17:59.

[4] Dewhirst FE, Chien CC, Paster BJ, Ericson RL, Orcutt RP, Schauer DB,et al. Phylogeny of the defined murine microbiota: altered Schaedler flora.Appl Environ Microbiol 1999;65:3287–92.

[5] Baker DG. Natural pathogens of laboratory mice, rats, and rabbits andtheir effects on research. Clin Microbiol Rev 1998;11:231–66.

[6] Billroth T. Untersuchungen uber die Vegetationsformen von Coccobac-teria septica. Berlin; 1874.

[7] Escherich T. Die Darmbakterien des Neugeborenen und Sauglings.Fortschritte der Medizin 1885;3:515–47.

[8] Cushing H, Livingood LE. Experimental and surgical notes upon thebacteriology of the upper portion of the alimentary canal, with observa-tions on the establishment there of an amicrobic state as a preliminary tooperative procedures on the stomach and small intestine. John’s HopkinsHospital Reports 1900;9:543–9.

[9] Pasteur L. Observations relatives a la note de M. Duclaux. Compt Rend1885;100:69.

[10] Cohendy M. Experiences sur la vie sans microbes. Compt Rend1912;154:533–6.

[11] Cohendy M. Experiences sur la vie sans microbes, elevage aseptique decobages. Compt Rend 1914;158:1283–4.

[12] Glimstedt G. Bakterienfrei Meerschweinchen. Aufzucht, Lebensfahigkeitund Wachstum, nebst Untersuchung uber das lymphatische Gewebe. Acta

Pathol Microbiol Scand Suppl 1936;30:1–295.

[13] Reyniers JA. The pure culture concept and gnotobiotics. Ann N Y AcadSci 1959;78:3–16.

[14] Gustafsson BE. Vitamin K deficiency in germfree rats. Ann N Y AcadSci 1959;78:166–74.

unology 19 (2007) 59–69

[15] Ogura Y, Bonen DK, Inohara N, Nicolae DL, Chen FF, Ramos R, et al.A frameshift mutation in NOD2 associated with susceptibility to Crohn’sdisease. Nature 2001;411:603–6.

[16] Hugot JP, Chamaillard M, Zouali H, Lesage S, Cezard JP, Belaiche J, etal. Association of NOD2 leucine-rich repeat variants with susceptibilityto Crohn’s disease. Nature 2001;411:599–603.

[17] Hampe J, Cuthbert A, Croucher PJP, Mirza MM, Mascheretti S, FisherS, et al. Association between insertion mutation in NOD2 gene andCrohn’s disease in German and British populations. Lancet 2001;357:1925–8.

[18] Inohara N, Nunez G. NODs, intracellular proteins involved in inflamma-tion and apoptosis. Nat Rev Immunol 2003;3:371–82.

[19] Girardin SE, Boneca IG, Viala J, Chamaillard M, Labigne A, ThomasG, et al. Nod2 is a general sensor of peptidoglycan through muramyldipeptide (MDP) detection. J Biol Chem 2003;278:8869–72.

[20] Kuhn R, Lohler J, Rennick D, Rajewsky K, Muller W. Interleukin-10-deficient mice develop chronic enterocolitis. Cell 1993;75:263–74.

[21] Sadlack B, Merz H, Schorle H, Schimpl A, Feller AC, Horak I. Ulcera-tive colitis-like disease in mice with a disrupted interleukin-2 gene. Cell1993;75:253–61.

[22] Mombaerts P, Mizoguchi E, Grusby MJ, Glimcher LH, Bhan AK, Tone-gawa S. Spontaneous development of inflammatory bowel disease in Tcell receptor mutant mice. Cell 1993;75:274–82.

[23] Mueller C, Macpherson AJ. Layers of mutualism with commensal bac-teria protect us from intestinal inflammation. Gut 2006;55:276–84.

[24] Parrott DM, Ferguson A. Selective migration of lymphocytes within themouse small intestine. Immunology 1974;26:571–88.

[25] Rhee KJ, Sethupathi P, Driks A, Lanning DK, Knight KL. Role of com-mensal bacteria in development of gut-associated lymphoid tissues andpreimmune antibody repertoire. J Immunol 2004;172:1118–24.

[26] Helgeland L, Vaage JT, Rolstad B, Halstensen TS, Midtvedt T, BrandtzaegP. Regional phenotypic specialization of intraepithelial lymphocytes in therat intestine does not depend on microbial colonization. Scand J Immunol1997;46:349–57.

[27] Helgeland L, Vaage JT, Rolstad B, Midtvedt T, Brandtzaeg P. Micro-bial colonization influences composition and T-cell receptor V betarepertoire of intraepithelial lymphocytes in rat intestine. Immunology1996;89:494–501.

[28] Guy-Grand D, Griscelli C, Vassalli P. The mouse gut T lymphocyte, anovel type of T cell. Nature, origin, and traffic in mice in normal andgraft-versus-host conditions. J Exp Med 1978;148:1661–77.

[29] Hooper LV, Gordon JI. Commensal host-bacterial relationships in the gut.Science 2001;292:1115–8.

[30] Hooper LV, Stappenbeck TS, Hong CV, Gordon JI. Angiogenins, a newclass of microbicidal proteins involved in innate immunity. Nat Immunol2003;4:269–73.

[31] Stappenbeck TS, Hooper LV, Gordon JI. Developmental regulation ofintestinal angiogenesis by indigenous microbes via Paneth cells. ProcNatl Acad Sci USA 2002;99:15451–5.

[32] Backhed F, Ding H, Wang T, Hooper LV, Koh GY, Nagy A, et al. Thegut microbiota as an environmental factor that regulates fat storage. ProcNatl Acad Sci USA 2004;101:15718–23.

[33] Bauer H, Horowitz RE, Levenson SM, Popper H. The response of lym-phatic tissue to the microbial flora. Studies on germfree mice. Am J Path1963;42:471–9.

[34] Manolios N, Geczy CL, Schrieber L. High endothelial venule morphol-ogy and function are inducible in germ-free mice: a possible role forinterferon-gamma. Cell Immunol 1988;117:136–51.

[35] Macpherson AJ, Gatto D, Sainsbury E, Harriman GR, HengartnerH, Zinkernagel RM. A primitive T cell-independent mechanism ofintestinal mucosal IgA responses to commensal bacteria. Science2000;288:2222–6.

[36] Konrad A, Cong Y, Duck W, Borlaza R, Elson CO. Tight mucosal com-

partmentation of the murine immune response to antigens of the entericmicrobiota. Gastroenterology 2006;130:2050–9.

[37] Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL. An immunomodula-tory molecule of symbiotic bacteria directs maturation of the host immunesystem. Cell 2005;122:107–18.

n Imm

K. Smith et al. / Seminars i

[38] Wostmann BS. The germfree animal in nutritional studies. Annu Rev Nutr1981;1:257–79.

[39] Wostmann BS, Knight PL. Antagonism between vitamins A and K in thegermfree rat. J Nutr 1965;87:155–60.

[40] Sumi Y, Miyakawa M, Kanzaki M, Kotake Y. Vitamin B-6 deficiency ingermfree rats. J Nutr 1977;107:1707–14.

[41] Levenson SM, Tennant B. Some metabolic and nutritional studies withgermfree animals. Fed Proc 1963;22:109–19.

[42] Gordon HA. Is the germ-free animal normal? In: Coates ME, editor. Thegerm free animal in research. London and New York: Academic Press;1968. p. 127–50.

[43] Luckey TD, Reyniers JA, Gyorgy P, Forbes M. Germfree animals andliver necrosis. Ann N Y Acad Sci 1954;57:932–5.

[44] Combe E, Sacquet E. Influence of the axenic state on various nitrogencompounds contained in the caecum of albino rats receiving vari-able amounts of proteins. C R Acad Sci Hebd Seances Acad Sci D1966;262:685–8.

[45] Gordon HA, Wostmann BS, Bruckner-Kardoss E. Effects of microbialflora on cardiac output and other elements of blood circulation. Proc SocExp Biol Med 1963;114:301–4.

[46] Laroche MJ, Cottart A, Sacquet E, Charlier H. Absorption of some chem-ical substances by the gastrointestinal tract of germ-free and conventionalrats. Ann Pharm Fr 1964;22:333–8.

[47] Wostmann BS, Knight PL, Keeley LL, Kan DF. Metabolism and functionof thiamine and naphthoquinones in germfree and conventional rats. FedProc 1963;22:120–4.

[48] Danielsson H, Gustafsson B. On serum-cholesterol levels and neutralfecal sterols in germ-free rats; bile acids and steroids 59. Arch BiochemBiophys 1959;83:482–5.

[49] Gordon HA. Morphological and physiological characterization ofgermfree life. Ann N Y Acad Sci 1959;78:208–20.

[50] Reddy BS, Wostmann BS, Pleasants JR. Iron, copper, and manganese ingermfree and conventional rats. J Nutr 1965;86:159–68.

[51] Wostmann BS, Wiech NL, Kung E. Catabolism and elimination of choles-terol in germfree rats. J Lipid Res 1966;7:77–82.

[52] Gordon HA. Germ-free animals in research: an extension of the pureculture concept. Triangle 1965;7:108–21.

[53] Desplaces A, Zagury D, Sacquet E. Study of the thyroid function of thegerm-free rat. C R Hebd Seances Acad Sci 1963;257:756–8.

[54] Wostmann BS, Bruckner-Kardoss E, Knight Jr PL. Cecal enlargement,cardiac output, and O2 consumption in germfree rats. Proc Soc Exp BiolMed 1968;128:137–41.

[55] Heneghan JB. Influence of microbial flora on xylose absorption in ratsand mice. Am J Physiol 1963;205:417–20.

[56] Ford DJ, Coates ME. Absorption of glucose and vitamins of the B complexby germ-free and conventional chicks. Proc Nutr Soc 1971;30:10A–1A.

[57] Reddy BS, Pleasants JR, Wostmann BS. Effect of intestinal microfloraon calcium, phosphorus and magnesium metabolism in rats. J Nutr1969;99:353–62.

[58] Baez S, Gordon HA. Tone and reactivity of vascular smooth muscle ingermfree rat mesentery. J Exp Med 1971;134:846–56.

[59] Loesche WJ. Protein and carbohydrate composition of cecal contents ofgnotobiotic rats and mice. Proc Soc Exp Biol Med 1968;128:195–9.

[60] Borgstrom B, Dahlqvist A, Gustafsson BE, Lundh G, Malmquist J.Trypsin, invertase and amylase content of feces of germfree rats. ProcSoc Exp Biol Med 1959;102:154–5.

[61] Norin KE, Gustafsson BE, Midtvedt T. Strain differences in faecal tryp-tic activity of germ-free and conventional rats. Lab Anim 1986;20:67–9.

[62] Strandberg K, Sedvall G, Midtvedt T, Gustafsson B. Effect of some bio-logically active amines on the cecum wall of germfree rats. Proc Soc ExpBiol Med 1966;121:699–702.

[63] Dupont JR, Jervis HR, Sprinz H. Auerbach’s plexus of the rat cecum in

relation to the germfree state. J Comp Neurol 1965;125:11–8.

[64] Abrams GD, Bishop JE. Effect of the normal microbial flora on gastroin-testinal motility. Proc Soc Exp Biol Med 1967;126:301–4.

[65] Sacquet E, Garnier H, Raibaud P. Study of rate of the gastrointestinaltransit of spores of a strictly thermophilic strain of Bacillus subtilis in the

unology 19 (2007) 59–69 67

holoxenic rat, the axenic rat and the cecectomized axenic rat. C R SeancesSoc Biol Fil 1970;164:532–7.

[66] Arantes RM, Nogueira AM. Increased intracellular content ofenteroglucagon in proximal colon is related to intestinal adaptation togerm-free status. Cell Tissue Res 2001;303:447–50.

[67] Gregor M, Stallmach A, Menge H, Riecken EO. The role of gut-glucagon-like immunoreactants in the control of gastrointestinal epithelial cellrenewal. Digestion 1990;46(Suppl. 2):59–65.

[68] Yolton DP, Stanley C, Savage DC. Influence of the indigenous gastroin-testinal microbial flora on duodenal alkaline phosphatase activity in mice.Infect Immun 1971;3:768–73.

[69] de Both NJ, Plaisier H. The influence of changing cell kinetics onfunctional differentiation in the small intestine of the rat. A study ofenzymes involved in carbohydrate metabolism. J Histochem Cytochem1974;22:352–60.

[70] Dai D, Nanthkumar NN, Newburg DS, Walker WA. Role of oligosac-charides and glycoconjugates in intestinal host defense. J PediatrGastroenterol Nutr 2000;30(Suppl. 2):S23–33.

[71] Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. Molec-ular analysis of commensal host-microbial relationships in the intestine.Science 2001;291:881–4.

[72] Nanthakumar NN, Dai D, Meng D, Chaudry N, Newburg DS, Walker WA.Regulation of intestinal ontogeny: effect of glucocorticoids and lumi-nal microbes on galactosyltransferase and trehalase induction in mice.Glycobiology 2005;15:221–32.

[73] Nanthakumar NN, Dai D, Newburg DS, Walker WA. The role of indige-nous microflora in the development of murine intestinal fucosyl- andsialyltransferases. FASEB J 2003;17:44–6.

[74] Lopez-Boado YS, Wilson CL, Hooper LV, Gordon JI, Hultgren SJ, ParksWC. Bacterial exposure induces and activates matrilysin in mucosalepithelial cells. J Cell Biol 2000;148:1305–15.

[75] Kozakova H, Stepankova R, Rehakova Z, Kolinska J. Differences in ente-rocyte brush border enzyme activities in ageing rats reared in germ-freeand conventional conditions. Physiol Res 1998;47:253–8.

[76] Jervis HR, Biggers DC. Mucosal enzymes in the cecum of conventionaland germfree mice. Anat Rec 1964;148:591–7.

[77] Dahlqvist A, Bull B, and BE, Gustafsson. Rat intestinal 6-bromo-2-naphthyl glycosidase and disaccharidase activities. I. Enzymic propertiesand distribution in the digestive tract of conventional and germ-free ani-mals. Arch Biochem Biophys 1965;109:150–8.

[78] Bry L, Falk PG, Midtvedt T, Gordon JI. A model of host-microbialinteractions in an open mammalian ecosystem. Science 1996;273:1380–3.

[79] Evrard E, Hoet PP, Eyssen H, Charlier H, Sacquet E. Faecal lipids ingerm-free and conventional rats. Br J Exp Pathol 1964;45:409–14.

[80] Gustafsson BE, Lanke LS. Bilirubin and urobilins in germfree, ex-germfree, and conventional rats. J Exp Med 1960;112:975–81.

[81] Carlstedt-Duke B, Hoverstad T, Lingaas E, Norin KE, Saxerholt H, Stein-bakk M, et al. Influence of antibiotics on intestinal mucin in healthysubjects. Eur J Clin Microbiol 1986;5:634–8.

[82] Wostmann BS, Bruckner-Kardoss E. Oxidation-reduction potentials incecal contents of germfree and conventional rats. Proc Soc Exp Biol Med1966;121:1111–4.

[83] Gustafsson BE, Norman A. Urinary calculi in germfree rats. J Exp Med1962;116:273–84.

[84] Asano T. Modification of cecal size in germfree rats by long-term feedingof anion exchange resin. Am J Physiol 1969;217:911–8.

[85] Asano T. Inorganic ions in cecal content of gnotobiotic rats. Proc Soc ExpBiol Med 1967;124:424–30.

[86] Asano T. Anion concentration in cecal content of germ-free and conven-tional mice. Proc Soc Exp Biol Med 1969;131:1201–5.

[87] Quevauviller A, Laroche MJ, Cottart A, Sacquet E, Charlier E. Anestheticactivity and comparative metabolism of hexobarbital in the germ-free and

conventional mouse. Ann Pharm Fr 1964;22:339–44.

[88] Ducluzeau R, Raibaud P, Dickinson AB, Sacquet E, Mocquot G. Hydrol-ysis of urea in vitro and in vivo, in the cecum of gnotobiotic rats, bydifferent bacterial strains isolated from the digestive tract of conventionalrats. C R Acad Sci Hebd Seances Acad Sci D 1966;262:944–7.

6 n Imm

8 K. Smith et al. / Seminars i

[89] Hoverstad T, Midtvedt T. Short-chain fatty acids in germfree mice andrats. J Nutr 1986;116:1772–6.

[90] Welling GW, Groen G, Tuinte JH, Koopman JP, Kennis HM. Biochemicaleffects on germ-free mice of association with several strains of anaerobicbacteria. J Gen Microbiol 1980;117:57–63.

[91] Lindstedt G, Lindstedt S, Gustafsson BE. Mucus in intestinal contents ofgermfree rats. J Exp Med 1965;121:201–13.

[92] Meslin JC, Sacquet E, Guenet JL. Action of bacterial flora on the mor-phology and surface mucus of the small intestine of the rat. Ann BiolAnim Biochim Biophys 1973;13:203–14.

[93] Meslin JC, Sacquet E. Effects of microflora on the dimensions of entero-cyte microvilli in the rat. Reprod Nutr Dev 1984;24:307–14.

[94] Gordon HA, Bruckner-Kardoss E. Effect of normal microbial flora onintestinal surface area. Am J Physiol 1961;201:175–8.

[95] Glaister JR. Factors affecting the lymphoid cells in the small intesti-nal epithelium of the mouse. Int Arch Allergy Appl Immunol1973;45:719–30.

[96] Abrams GD, Bauer H, Sprinz H. Influence of the normal flora on mucosalmorphology and cellular renewal in the ileum. A comparison of germ-freeand conventional mice. Lab Invest 1963;12:355–64.

[97] Lesher S, Walburg Jr HE, Sacher Jr GA. Generation cycle in the duodenalcrypt cells of germ-free and conventional mice. Nature 1964;202:884–6.

[98] Wostmann B, Bruckner-Kardoss E. Development of cecal distention ingerm-free baby rats. Am J Physiol 1959;197:1345–6.

[99] Pleasants JR. Characteristics of the germ-free animal. In: Coates ME, edi-tor. The germ free animal in research. London and New York: AcademicPress; 1968. p. 113–25.

[100] Hudson JA, Luckey TD. Bacteria induced morphologic changes. ProcSoc Exp Biol Med 1964;116:628–31.

[101] Nuttall GHF, Thierfelder H. Thierisches Leben ohne Baktieren in Ver-dauungskanal. Hoppe-Seyl Z Physiol Chem 1895;21:109–21.

[102] Gordon HA, Bruckner-Kardoss E. Effect of the normal microbial floraon various tissue elements of the small intestine. Acta Anat (Basel)1961;44:210–25.

[103] Kenworthy R, Allen WD. Influence of diet and bacteria on smallintestinal morphology, with special reference to early weaning andEscherichia coli. Studies with germfree and gnotobiotic pigs. J CompPathol 1966;76:291–6.

[104] Khoury KA, Floch MH, Hersh T. Small intestinal mucosal cell prolifera-tion and bacterial flora in the conventionalization of the germfree mouse.J Exp Med 1969;130:659–70.

[105] Guenet JL, Sacquet E, Gueneau G, Meslin JC. Action of total microfloraof the rat on mitotic activity of Lieberkuhn’s crypts. C R Acad Sci HebdSeances Acad Sci D 1970;270:3087–90.

[106] Gustafsson BE, Maunsbach AB. Ultrastructure of the enlargec cecum ingermfree rats. Z Zellforsch Mikrosk Anat 1971;120:555–78.

[107] He W, Wang ML, Jiang HQ, Steppan CM, Shin ME, Thurnheer MC, et al.Bacterial colonization leads to the colonic secretion of RELMbeta/FIZZ2,a novel goblet cell-specific protein. Gastroenterology 2003;125:1388–97.

[108] Szentkuti L, Enss ML. Comparative lectin-histochemistry on the pre-epithelial mucus layer in the distal colon of conventional and germ-freerats. Comp Biochem Physiol A Mol Integr Physiol 1998;119:379–86.

[109] Moreau MC, Ducluzeau R, Guy-Grand D, Muller MC. Increase in thepopulation of duodenal immunoglobulin A plasmocytes in axenic miceassociated with different living or dead bacterial strains of intestinalorigin. Infect Immun 1978;21:532–9.

[110] Fahey JL, Sell S. The Immunoglobulins of Mice. V. The metabolic(catabolic) properties of five immunoglobulin classes. J Exp Med1965;122:41–58.

[111] Arnason BG, Salomon JC, Grabar P. Anti-liver antibodies and serumimmunoglobulins in the standard and axenic mice; a comparative studyafter acute hepatic necrosis induced by carbon tetrachlorode (Cc14). C RHebd Seances Acad Sci 1964;259:4882–5.

[112] Crabbe PA, Nash DR, Bazin H, Eyssen H, Heremans JF. Immuno-histochemical observations on lymphoid tissues from conventional andgerm-free mice. Lab Invest 1970;22:448–57.

[113] Umesaki Y, Setoyama H, Matsumoto S, Imaoka A, Itoh K. Differentialroles of segmented filamentous bacteria and clostridia in develop-

unology 19 (2007) 59–69

ment of the intestinal immune system. Infect Immun 1999;67:3504–11.

[114] Helgeland L, Dissen E, Dai KZ, Midtvedt T, Brandtzaeg P, Vaage JT.Microbial colonization induces oligoclonal expansions of intraepithelialCD8 T cells in the gut. Eur J Immunol 2004;34:3389–400.

[115] Kawaguchi M, Nanno M, Umesaki Y, Matsumoto S, Okada Y, Cai Z, et al.Cytolytic activity of intestinal intraepithelial lymphocytes in germ-freemice is strain dependent and determined by T cells expressing gammadelta T-cell antigen receptors. Proc Natl Acad Sci USA 1993;90:8591–4.

[116] Imaoka A, Matsumoto S, Setoyama H, Okada Y, Umesaki Y. Prolifera-tive recruitment of intestinal intraepithelial lymphocytes after microbialcolonization of germ-free mice. Eur J Immunol 1996;26:945–8.

[117] Umesaki Y, Setoyama H, Matsumoto S, Okada Y. Expansion of alpha betaT-cell receptor-bearing intestinal intraepithelial lymphocytes after micro-bial colonization in germ-free mice and its independence from thymus.Immunology 1993;79:32–7.

[118] Mikkelsen HB, Garbarsch C, Tranum-Jensen J, Thuneberg L.Macrophages in the small intestinal muscularis externa of embryos, new-born and adult germ-free mice. J Mol Histol 2004;35:377–87.

[119] Umesaki Y, Okada Y, Matsumoto S, Imaoka A, Setoyama H. Segmentedfilamentous bacteria are indigenous intestinal bacteria that activateintraepithelial lymphocytes and induce MHC class II molecules and fuco-syl asialo GM1 glycolipids on the small intestinal epithelial cells in theex-germ-free mouse. Microbiol Immunol 1995;39:555–62.

[120] Matsumoto S, Setoyama H, Umesaki Y. Differential induction of majorhistocompatibility complex molecules on mouse intestine by bacterialcolonization. Gastroenterology 1992;103:1777–82.

[121] Sobko T, Reinders C, Norin E, Midtvedt T, Gustafsson LE, Lundberg JO.Gastrointestinal nitric oxide generation in germ-free and conventionalrats. Am J Physiol Gastrointest Liver Physiol 2004;287:G993–7.

[122] Beaver MH, Wostmann BS. Histamine and 5-hydroxytryptamine in theintestinal tract of germ-free animals, animals harbouring one micro-bial species and conventional animals. Br J Pharmacol Chemother1962;19:385–93.

[123] Bos NA, Meeuwsen CG, Hooijkaas H, Benner R, Wostmann BS, Pleas-ants JR. Early development of Ig-secreting cells in young of germ-freeBALB/c mice fed a chemically defined ultrafiltered diet. Cell Immunol1987;105:235–45.

[124] Bos NA, Meeuwsen CG, Wostmann BS, Pleasants JR, Benner R. Theinfluence of exogenous antigenic stimulation on the specificity repertoireof background immunoglobulin-secreting cells of different isotypes. CellImmunol 1988;112:371–80.

[125] Bos NA, Meeuwsen CG, Van Wijngaarden P, Benner R. B cell repertoirein adult antigen-free and conventional neonatal BALB/c mice. II. Analysisof antigen-binding capacities in relation to VH gene usage. Eur J Immunol1989;19:1817–22.

[126] Hooijkaas H, Benner R, Pleasants JR, Wostmann BS. Isotypes andspecificities of immunoglobulins produced by germ-free mice fedchemically defined ultrafiltered “antigen-free” diet. Eur J Immunol1984;14:1127–30.

[127] Sprinz H, Kundel DW, Dammin GJ, Horowitz RE, Schneider H, FormalSB. The response of the germfree guinea pig to oral bacterial challengewith Escherichia coli and Shigella flexneri. Am J Pathol 1961;39:681–95.

[128] Pollard M. The use of germfree animals in virus research. Prog Med Virol1965;7:362–76.

[129] Bauer H, Horowitz RE, Levenson SM, Popper H. The response of thelymphatic tissue to the microbial flora. Studies on germfree mice. Am JPathol 1963;42:471–83.

[130] Olson GB, Wostmann BS. Lymphocytopoiesis, plasmacytopoiesis andcellular proliferation in nonantigenically stimulated germfree mice. JImmunol 1966;97:267–74.

[131] Gustafsson BE, Laurell CB. G Gamma globulins in germ-free rats. J ExpMed 1958;108:251–8.

[132] Wostmann BS. Recent studies on the serum proteins of germfree animals.Ann N Y Acad Sci 1961;94:272–83.

[133] Thorbecke GJ. Some histological and functional aspects of lymphoidtissue in germfree animals. I. Morphological studies. Ann N Y Acad Sci1959;78:237–46.

n Imm

and euthymic mice following alimentary tract colonization and infection

K. Smith et al. / Seminars i

[134] Gustafsson BE, Laurell CB. Gamma globulin production in germfree ratsafter bacterial contamination. J Exp Med 1959;110:675–84.

[135] Sell S. Immunoglobulins of the Germfree Guinea Pig. J Immunol1964;93:122–31.

[136] Freitas AA, Viale AC, Sundblad A, Heusser C, Coutinho A. Normalserum immunoglobulins participate in the selection of peripheral B-cellrepertoires. Proc Natl Acad Sci USA 1991;88:5640–4.

[137] Hooijkaas H, van der Linde-Preesman AA, Bitter WM, Benner R, Pleas-ants JR, Wostmann BS. Frequency analysis of functional immunoglobulinC- and V-gene expression by mitogen-reactive B cells in germfreemice fed chemically defined ultra-filtered “antigen-free” diet. J Immunol1985;134:2223–7.

[138] Ikari NS. Bactericidal antibody to Escherichia coli in germ-free mice.Nature 1964;202:879–81.

[139] Wagner M, Wostmann BS. Serum protein fractions and antibody studiesin gnotobiotic animals reared germfree or monocontaminated. Ann N YAcad Sci 1961;94:210–7.

[140] Wostmann BS, Gordon HA. Electrophoretic studies on the serum proteinpattern of the germfree rat and its changes upon exposure to a conventionalbacterial flora. J Immunol 1960;84:27–31.

[141] Ohwaki M, Yasutake N, Yasui H, Ogura R. A comparative study onthe humoral immune responses in germ-free and conventional mice.Immunology 1977;32:43–8.

[142] Horowitz RE, Bauer H, Paronetto F, Abrams GD, Watkins KC, PopperH. The response of the lymphatic tissue to bacterial antigen. Studies ingermfree mice. Am J Pathol 1964;44:747–61.

[143] MacDonald TT, Carter PB. Requirement for a bacterial flora before micegenerate cells capable of mediating the delayed hypersensitivity reactionto sheep red blood cells. J Immunol 1979;122:2624–9.

[144] Wells C, Balish E. The mitogenic activity of lipopolysaccharide for spleencells from germfree, conventional, and gnotobiotic rats. Can J Microbiol1979;25:1087–93.

[145] Etlinger HM, Heusser CH. T15 dominance in BALB/c mice is not con-trolled by environmental factors. J Immunol 1986;136:1988–91.

[146] Lerner EM. Federation proceedings. Federation of the American societiesfor experimental biology. 1964. p. 286.

[147] Walton KL, Galanko JA, Sartor RB, Fisher NC. T cell-mediated oral tol-erance is intact in germ-free mice. Clin Exp Immunol 2006;143:503–12.

[148] Bos NA, Ploplis VA. Humoral immune response to 2,4-dinitrophenyl–keyhole limpet hemocyanin in antigen-free, germ-free and conventionalBALB/c mice. Eur J Immunol 1994;24:59–65.

[149] Salomon JC. Induction of homologous disease in axenic (germ-free)newborn mice. CR Acad Sci Hebd Seances Acad Sci D 1965;260:4862–4.

[150] Bauer H, Paronetto F, Burns WA, Einheber A. The enhancing effect ofthe microbial flora on macrophage function and the immune response. Astudy in germfree mice. J Exp Med 1966;123:1013–24.

unology 19 (2007) 59–69 69

[151] Bakker R, Lasonder E, Bos NA. Measurement of affinity in serum samplesof antigen-free, germ-free and conventional mice after hyperimmuniza-tion with 2,4-dinitrophenyl keyhole limpet hemocyanin, using surfaceplasmon resonance. Eur J Immunol 1995;25:1680–6.

[152] Taylor MJ, Rooney JR, Blundell GP. Experimental anthrax in the rat. II.The relative lack of natural resistance in germ-free (Lobund) hosts. Am JPathol 1961;38:625–38.

[153] Oliveira MR, Tafuri WL, Afonso LC, Oliveira MA, Nicoli JR, Vieira EC,et al. Germ-free mice produce high levels of interferon-gamma in responseto infection with Leishmania major but fail to heal lesions. Parasitology2005;131:477–88.

[154] Inagaki H, Suzuki T, Nomoto K, Yoshikai Y. Increased susceptibility toprimary infection with Listeria monocytogenes in germfree mice may bedue to lack of accumulation of L-selectin+ CD44+ T cells in sites ofinflammation. Infect Immun 1996;64:3280–7.

[155] Martin LK, Einheber A, Porro RF, Sadun EH, Bauer H. Plasmodiumberghei infections in gnotobiotic mice and rats: parasitologic, immuno-logic and histopathologic observations. Mil Med 1966;131(Suppl.):870–89.

[156] Finerty JF, Evans CB, Hyde CL. Plasmodium berghei and Eperythro-zoon coccoides: antibody and immunoglobulin synthesis in germfreeand conventional mice simultaneously infected. Exp Parasitol 1973;34:76–84.

[157] Schaffer J, Beamer PR, Trexler PC, Breidenbach G, Walcher DN.Response of germ-free animals to experimental virus monocontamina-tion. I. Observation on Coxsackie B virus. Proc Soc Exp Biol Med1963;112:561–4.

[158] Dolowy WC, Muldoon RL. Studies of germfree animals. I. Responseof mice to infection with influenza a virus. Proc Soc Exp Biol Med1964;116:365–71.

[159] Bhatt PN, Jacoby RO, Jonas AM. Respiratory infection in mice withsialodacryoadenitis virus, a coronavirus of rats. Infect Immun 1977;18:823–7.

[160] Tennant RW, Parker JC, Ward TG. Virus studies with germfree mice. Ii.Comparative responses of germfree mice to virus infection. J Natl CancerInst 1965;34:381–7.

[161] Giannini MS. The specific anti-parasite immune responses of germ-free and conventional rats infected with Trypanosoma lewisi. J Parasitol1987;73:144–8.

[162] Balish E, Filutowicz H. Serum antibody response of gnotobiotic athymic

with Candida albicans. Can J Microbiol 1991;37:204–10.[163] De Somer P, Billiau A. Interferon production by the spleen of rats after

intravenous injection of Sindbis virus or heat-killed Escherichia coli. ArchGesamte Virusforsch 1966;19:143–54.