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Gut Check - The bacteria in your intestines are welcome guests

John Travis

New York and London are famous for both their congestion and the diverse origins of their residents. But if you're looking for the ultimate teeming metropolis of immigrants, check out the large intestine. In people, some 500 to 1,000 kinds of bacteria reside in this part of the gastrointestinal (GI) tract, and these gut microbes outnumber all the cells in your body, perhaps by as much as a factor of 10.

"The density of this society is mind-boggling," says Jeffrey I. Gordon of Washington University School of Medicine in St. Louis.

It's a society overlooked by most microbiologists, who generally stick to the myriad bacteria that cause disease. Yet some scientists argue that it's shortsighted to ignore what they call the microflora living in our intestines.

"What these bacteria do definitely makes a very significant contribution to our health�or lack thereof," says Mark Schell of the University of Georgia in Athens, who studies an intestinal microbe called Bifodobacterium longum.

Shell and a few other researchers have recently begun to probe exactly what individual microbes do for or to the intestine.

Consider Bacteriodes thetaiotaomicron. Although not as well known, it's more than 1,000 times as abundant in the guts of people and mice as the extensively studied bacterium Escherichia coli. Some researchers have proposed that in return for a steady food supply, B. thetaiotaomicron breaks down indigestible complex carbohydrates into easily absorbed sugars and produces other substances, such as vitamins, that benefit its host.

There may be much more to this microbe-host relationship, however. About a decade ago, Gordon chose B. thetaiotaomicron as a prototypical germ for studying how microbes influence the GI tract. This bacterium normally becomes a predominant member of the intestinal community about the time an animal is weaned from its mother's milk. Gordon's research team has discovered that the microbe can turn on specific intestinal genes, promote the growth of blood vessels necessary for the gut's function, and trigger production of a chemical that may kill competing bacteria. Investigators are now asking just how much gut bacteria regulate the developing and adult human body.

"Bacteria do an awful lot for us and with us," says Gordon. "Most people's views of bacteria are of menacing, disease-promoting entities. Au contraire, I think that most of our encounters with bacteria are mutually beneficial, friendly, and part of our normal biology. . . . They've insinuated themselves into our biology and coevolved with us."

Sweet-talking germ

Perhaps the best way to understand the significance of intestinal microorganisms is to see what happens when an animal doesn't have them. During the past 50 years, researchers have created germfree mice and rats by delivering the animals by cesarean section into sterile environments and maintaining them there. "It's a very demanding technology," says Gordon. Scientists have generally used such germfree animals to study how particular pathogens cause diseases.

One of the most striking aspects of a germfree rodent is that it must consume about 30 percent more calories to maintain its body weight than a typical rodent does. Germfree animals are also unusually susceptible to infections, presumably because the microflora in a normal gut ward off foreign pathogens.

As a way to study animals hosting a simplified society of gut bacteria, Gordon and his colleagues have introduced B. thetaiotaomicron into germfree mice. Their first significant discovery was that the bacterium could change what sugars the intestine makes.

The surfaces of intestinal cells of typical mice are coated with complex sugars containing the simple sugar fucose and B. thetaiotaomicron consumes the fucose for energy. In germfree mice, however, fucose production ceases around the time of weaning.

If B. thetaiotaomicron colonizes a germfree mouse before weaning, however, normal fucose synthesis continues throughout life, the researchers found. Through a still undiscovered signal, the microbe apparently induces the intestinal cells to make one of its favorite foods. The bacterium even has a fucose sensor that informs it when this food source is scarce, according to Gordon and his colleagues.

The capacity of B. thetaiotaomicron to instruct intestinal cells to make fucose was just a hint of things to come. To get a more comprehensive picture of the bacterium's influence, Gordon's group turned to microchip-size devices, called DNA microarrays, that monitor the activity of thousands of genes at once (SN: 3/8/97, p. 144).

With such instruments, the scientists took a snapshot of the gene activity in the mouse intestine. By comparing tissue from germfree mice and mice hosting B. thetaiotaomicron, the team found that the presence of the bacterium significantly reduces or boosts the activity of about 100 of the approximately 25,000 rodent genes in the microarray survey.

Some of the intestinal genes triggered by the microbe help mammals absorb and metabolize sugars and fats, Gordon and his colleagues reported in 2001. Other activated genes fortify the cellular barrier that prevents bacteria, both dangerous and friendly, from sneaking out of the intestine into other tissues and the bloodstream. And yet other affected genes determine how the intestine detoxifies compounds and how the gut matures.

"We were amazed at the breadth of normal intestinal functions affected by a single microbe," says study coauthor Lora V. Hopper. Gordon adds, "It's difficult to anticipate the full range of host functions that might be manipulated by these microbes."

The genetic activity that the researchers didn't see in the bacteria-colonized mice was interesting, too. Even though the originally germfree mice had never encountered B. thetaiotaomicron before, there was no increase in activity of the genes underlying an immune or inflammatory response. That's a reflection of the microbe's still mysterious skill at convincing a host that it's a friendly visitor and not a danger, says Gordon.

Raising fences

Among the intestinal genes activated by B. thetaiotaomicron is one suspected to stimulate the growth of new blood vessels. The finding spurred Gordon's group to investigate the microbe's control over the system of blood vessels that runs through the GI tract. These blood vessels are crucial to a body's absorption of nutrients.

The researchers discovered that their germfree mice have a poorly formed network of the capillaries that normally supply the inner intestinal surface with its blood supply. This could partly explain the difficulty that germfree animals have absorbing nutrients.

The team also found that it could stimulate germfree mice to grow a normal network of intestinal capillaries by exposing the animals to either a full complement of microflora or just B. thetaiotaomicron. The investigators reported the finding in the Nov. 26, 2002 Proceedings of the National Academy of Sciences.

This is a vivid illustration that the physical development of the gut can depend on the microbes that normally inhabit animals, says Gordon. The researchers also found cells in the mouse gut that seems to work with the microbes to spur vessel growth.

In the small intestine, so-called Paneth cells normally secrete antimicrobial compounds (SN: 8/26/00, p. 135: Available to subscribers at http://www.sciencenews.org/20000826/fob8.asp). This keeps the intestine healthy by protecting other cells that continually replenish the gut lining. Gordon's team created germfree versions of mutant mice that lack Paneth cells and found that B. thetaiotaomicron couldn't trigger the maturation of blood vessels in such rodents. While most investigators have regarded Paneth cells simply as defenders against invading bacteria, it makes sense that these cells mediate interactions between a host and its natural microflora, says Gordon. "What better cell to respond to the presence or absence of a microbe?" he remarks.

The Paneth cell is at the heart of another microbe-intestine interaction uncovered recently by Gordon's group. One of the intestinal genes triggered in germfree mice by B. thetaiotaomicron encodes a protein called angiogenin 4 or Ang4. Cancer researchers are particularly interested in this protein, because they have evidence that it nourishes tumors by creating new blood vessels. Gordon's team suspected that Ang4 plays a role in the intestinal blood vessel maturation that they had documented earlier. Indeed, it turned out that Paneth cells make Ang4 and secrete it when they detect bacteria.

While the suspicion that Ang4 makes intestinal blood vessels has not yet been proven, it looks like the protein has a more certain role. It can kill several bacteria and fungi that cause diseases in mammals, Gordon, Hooper, and their colleagues report in the March Nature Immunology. In contrast, B. thetaiotaomicron and other common residents of the mouse intestines are largely resistant to Ang4.

"One interpretation of the interaction between host defense and the resident flora is that the resident bacteria that are resistant to Paneth-cell secretions stimulate these host-defense mechanisms to prevent competition by nonresident bacteria. The host in turn benefits by decreasing its exposure to potential pathogens," says Tomas Ganz of the University of California, Los Angeles in a commentary accompanying the March report.

Hooper agrees that the normal inhabitants of the gut may use Paneth cells and Ang4 to raise what she calls an "electric fence" to keep out competing microbes. Beyond fending off foreign pathogens, such fences may also keep typical intestinal microbes within the gut. "Anything can become a pathogen if it crosses the fence," she says.

Eating leftovers

Scientists have estimated that the hundreds of bacterial species within the human gut may together possess as many unique genes as a person does, and perhaps far more. "How much of our biology is dependent on metabolic traits encoded in the collective genomes of our microbial partners?" asks Gordon.

Investigators have begun to address that question. For example, Schell recently worked with scientists at the Nestl� Research Center in Lausanne, Switzerland, to unravel some of the genetic secrets of B. longum. This microbe typically colonizes the intestines of a newborn mammal, thrives during the breast-feeding period, and then subsides after weaning, when B. thetaiotaomicron and other bacteria take hold. Nestl� incorporates B. longum into some of its products, such as infant formulas and yogurts, to promote gastrointestinal health.

In the Oct. 29, 2002 Proceedings of the National Academy of Sciences, Schell and his colleagues unveiled the entire DNA sequence of B. longum and identified a large roster of genes for enzymes that break apart sugars and other edible substances. Some of these enzymes may degrade complex sugars found in breast milk, speculates Schell. Others, such as ones that apparently break down plant gums, may help the bacterium survive later in its host's life when B. longum is in the minority in the intestines.

The bacterium appears to break down substances that B. thetaiotaomicron and other Bacteriodes can't handle. "It seems to be more specialized for the leftovers of metabolism," says Schell. In a strategy similar to Gordon's, investigators at Nestl� are now using germfree mice to evaluate B. longum's impact on intestinal genes.

Gordon's team is drawing its own insights from the group's recent deciphering of B. thetaiotaomicron's genome. Among that microbe's nearly 4,800 genes, several hundred encode proteins that bind carbohydrates, enzymes that degrade bonds between sugars, or enzymes that create new sugars, the investigators reported in the March 28 Science. And the activity of many of these genes appears to be regulated by genes encoding molecules related to known environmental sensors, suggesting that the microbe can monitor the contents of the intestines and quickly deploy the molecular machinery needed for it to digest nutrients.

"This organism has a sweet tooth. It knows how to process carbohydrates," says Gordon.

Over the next 5 to 10 years, predicts Schell, researchers will decode the genomes of many more intestinal microbes. Investigators may also begin to address such issues as whether a person's diet changes his or her intestinal microflora. "I think the gut population of a vegetarian is clearly going to be different" from that of a meat eater, says Schell.

Gordon offers an even more provocative question: Do intestinal microbes influence a person's weight? "Over time, could relatively minor differences in the ability to extract nutrients in some individuals predispose them to obesity?" he asks.

The complicated nation of bacteria within our intestines is a "window into our biology and how we've evolved as a species," concludes Gordon.

http://www.sciencenews.org/articles/20030531/bob9.asp

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Heather is the Administrator of the IBS Message Boards. She is the author of Eating for IBS and The First Year: IBS, and the CEO of Heather's Tummy Care. Join her IBS Newsletter. Meet Heather on Facebook!

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FoodInfo Online Features

15 October 2001

Prebiotics for improved gut health

Glenn R Gibson

Food Microbial Sciences Unit, School of Food Biosciences,
The University of Reading, UK

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The bacterial microbiota in the human large intestine is thought to compromise 95% of the total cells in the body, representing 1012 cells/g dry weight contents. Through the activities of the resident microflora, the colon plays a major role in host nutrition and welfare (Gibson and Roberfroid 1999). Dietary modulation of the human gut flora can be of great benefit to health. In recent years, the functional food concept has moved away from mineral and vitamin supplementation towards the situation where improved gut (microbial) functionality is the main current driving force. The colon is the most intensely populated region of the gastrointestinal tract (Figure 1) and is therefore the main target for such dietary intervention.

The gastrointestinal tract is a sterile environment at birth and bacterial colonisation begins during the delivery process (organisms are transferred to the newborn gut from the maternal faecal or vaginal flora and/or the environment). Initial bacteria to colonise the colon are facultatively anaerobic organisms, such as Escherichia coli and streptococci. These first colonisers metabolise any traces of oxygen in the gut, thereby reducing the environment into one of strong anaerobic conditions. The bacteria that then further colonise the gut depend largely upon the feeding profile of the infant. The breast fed infant has bifidobacteria as the numerically predominant genus, whereas formula feeds give rise to a more complex, adult-like, gut flora with clostridia, bacteroides, bifidobacteria and streptococci as prevalent genera (Salminen et al. 1998). A major reason for these differences is that breast milk contains a 'bifidus' factor, which stimulates growth of bifidobacteria; this is a glycoprotein containing glucose, galactose, fructose, and N-acetyl glucosamine. Breast fed infants generally have less gastrointestinal problems than their formula-fed counterparts and this may well be attributed to the powerful anti-pathogen effects exerted by bifidobacteria. The final phase of microflora acquisition occurs at weaning, when a complex microflora develops. Currently, there is much interest in modulating the constituents of infant formulae, such that bifidobacteria are much more effectively stimulated.

The resident gut microbiota ferments substances, mainly provided by the diet, that cannot be digested by the host in the small gut. These include, resistant starch, non-starch polysaccharides (dietary fibre), oligosaccharides, proteins, amino acids, etc. In a typical adult, around 80 g of food ingested each day reaches the large intestine and is therefore susceptible to fermentation by the gut flora. The two main types of fermentation that are carried out in the gut are saccharolytic and proteolytic. The main end products of carbohydrate metabolism are, the short chain fatty acids, acetate, propionate, and butyrate. These may be further metabolised systemically or locally to provide energy generation for the host. The end products of proteolytic fermentation include, phenolic compounds, amines, and ammonia, all of which are toxic. The proximal colon (right side) is essentially a site of saccharolytic fermentation, whereas the more distal (left side) sees more proteolytic fermentation. This is probably a major reason why many gastrointestinal disorders (including colon cancer, ulcerative colitis) predominate distally.

Dietary modulation of the human gut flora has been carried out for many years. In humans, there are positive aspects to the gut fermentation, which may improve certain aspects of host health. The microflora contains certain bacteria that can be perceived as health promoting, as well as pathogenic. For instance, bifidobacteria and lactobacilli may help to improve resistance to gut infections by inhibiting the growth of harmful microorganisms (that may onset both acute and chronic gut disorder), reduce blood lipid levels, improve the immune response, and be involved in protection against gut cancers (Gibson and Roberfroid 1999; Sanders 1998). The definitive health outcomes, and their mechanisms of effect, are being gradually uncovered and there is currently much interest in increasing numbers and activities of these bacteria in the large gut, preferably at the expense of more harmful species. The manner in which this can be achieved is through dietary supplementation.

The use of probiotics has been widely supported. In this case, foodstuffs such as fermented milk products containing viable cultures perceived as beneficial (e.g. lactobacilli, bifidobacteria) are used to proliferate populations in the colon. Probiotics are defined as live microbial feed supplements, which beneficially affect the host animal by improving its intestinal microbial balance (Fuller 1989). To be effective, probiotics must be capable of being prepared in a viable manner and on large scale (e.g. for industrial purposes), whilst during use and under storage the probiotic should remain viable and stable, be able to survive in the intestinal ecosystem, and the host animal should gain beneficially from harbouring the probiotic. Clearly, the organisms used should be generally regarded as safe.

Whilst records indicate that probiotics have been ingested by humans for thousands of years, the work of Metchnikoff in the Pasteur Institute at the start of the 20th Century was probably the first realistic look at their use. He observed that Bulgarian peasants who consumed fermented dairy products exhibited longevity (Metchnikoff, 1907). In his thesis 'The Prolongation of Life' he attributed this to their elevated intake of so called soured milks � what we now recognise as probiotics.

Many probiotic products now exist in Europe. The most popular food vehicles are fermented milks, other drinks, or as lyophilised forms. Examples include: Danone's Actimel (containing Lactobacillus casei Immunitas) or Bio (containing bifidobacteria); Yakult (L. casei Shirota); Nestle's LC1 (L. johnsonii); and Seven Seas' Multibionta (containg Bifidobacterium bifidum, B. longum, and L. acidophilus).

An alternative approach has been investigated where the commensal bifidobacteria and/or lactobacilli are selectively promoted by the intake of certain non-viable substrates, known as prebiotics. Gibson and Roberfroid (1995) first described a prebiotic as a "non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health." As diet is the main factor controlling the intestinal microflora, it is possible to modulate the microflora composition through foods. A prebiotic substrate is selectively utilised by beneficial components of the indigenous gut flora but does not promote growth of potential pathogens, such as toxin producing clostridia, proteolytic bacteroides, and toxigenic Escherichia coli. In this manner, a "healthier" microflora composition is obtained, whereby the bifidobacteria and/or lactobacilli become predominant in the intestine and exert possible health-promoting effects (similar to the situation that prevails in the breast fed infant gut). For a dietary substrate to be classed as a prebiotic, three criteria are required:

1) the substrate must not be hydrolysed or absorbed in the stomach or small intestine;

2) it must be selective for beneficial commensal bacteria in the colon, such as the bifidobacteria;

3) the substrate should induce beneficial luminal/systemic effects within the host.

The premise behind prebiotics is therefore to stimulate certain indigenous bacteria in the gut, rather than introducing exogenous species, as is the case with probiotics. Ingesting a diet containing non-digestible carbohydrates that are selectively fermented by indigenous beneficial bacteria, is the prebiotic principal. Any dietary component that reaches the colon intact is a potential prebiotic, however much of the interest in the development of prebiotics is aimed at non-digestible oligosaccharides, such as fructooligosaccharides (FOS), trans-galactooligosaccharides (TOS), isomaltooligosaccharides (IMO), xylooligosaccharides (XOS), soyoligosaccharides (SOS), glucooligosaccharides (GOS), and lactosucrose. In Europe, FOS, GOS and lactulose have been shown to be prebiotics, through numerous volunteer trials, as evidence by their ability to change the gut flora composition after a short feeding period (Gibson et al. 2000). The Japanese market is more widespread.

As prebiotics exploit the use non-viable dietary components to improve gut health, the range of foods into which they can be added is much wider than that for probiotics, where culture viability needs to be maintained. This has the advantage that heat stability, or exposure to oxygen is not an issue. As such, virtually any carbohydrate containing food is susceptible to supplementation. Examples are shown in Table 1.

On the contrary, it may be possible to intake prebiotics more naturally through the diet. Many fruit and vegetables contain prebiotic oligosaccharides, such as FOS. Examples are onion, garlic, banana, asparagus, leek, and Jerusalem artichoke. However, the likely situation is that levels are too low to have any significant effect. Our (unpublished) data indicate that at least 4g/d, but more preferably 8g/d of FOS, would be needed to significantly elevate bifidobacteria in the human gut. Hence, there exists much value in the approach of dietary fortification. However, it is important to ensure that the prebiotic effect is maintained in the food product. Figure 2 shows data from a recent volunteer trial carried out at the University of Reading (Tuohy et al. 2001). Here, shortbread containing 7g/d FOS was fed to human subjects and the effects upon faecal bacteria determined as compared to a placebo (FOS not added). The nature of the trial was a crossover approach in that volunteers took active and placebo shortbread, but neither they nor the investigators were aware of which was ingested. Moreover, the bacteriology was carried out using a (culture independent) probing approach that relied upon differences in 16SrRNA profiles for the confirmation of identity. The data clearly show that the use of FOS exerted a profound effect upon bifidobacteria.

It is the case that many new products are being, and have been, developed that exploit the prebiotic approach. As mentioned, their use is more widespread than for probiotics. Hence, it is likely that the eventual market value will outstrip that of the live microbial approach (currently estimated at over 1 billion euro p.a. in Europe). There is therefore a huge potential for the use of functional foods, and in particular prebiotics in the food industry. However, it is important that new product developments have satisfactory scientific evidence to back their claims. One difficulty in the past has been the limitations imposed by cultural microbiology when applied to a complex ecosystem, such as the human gut.

Gut microbiology is conventionally carried out by plating faecal microorganisms onto selective agars designed to recover numerically predominant groups. However, the agars used are only semi-selective, do not recover non-culturable bacteria (which may represent over 50% of the overall diversity), and allow operator subjectivity in terms of microbial characterisation, which is usually based on limited phenotypic procedures. As such, alternative mechanisms, based around molecular principles, to more effectively characterise the microflora involved in fermentation studies, need to be applied. The use of such methods gives a much clearer picture of the gut microbiota and the effects of prebiotics. Traditional methods of bacterial enumeration are therefore being replaced with these molecular techniques (Tannock 1999; Vaughan et al. 2000). One such method is fluorescent in situ hybridisation (FISH) using specific rRNA probes, as employed in the study mentioned above (see Figure 3). The application of post-genomic principles in gut microbial studies will help fully explore human gut microflora diversity, develop reliable model systems, test a new generation of purpose designed prebiotic molecules with enhanced functionality, and determine the effectiveness of dietary intervention in the clinical situation.

In terms of new developments, it is important that the definitive health bonuses associated with prebiotic intake be determined. This is especially relevant given the broad applicability of their use. It is likely that prevention of acute gastroenteritis through fortification of certain gut microbiota components is an important aspect. Moreover, improved protection from more chronic gut disorders that have been associated with bacteria (inflammatory bowel disease, colon cancer, irritable bowel syndrome) may also be possible. It may also be the case that certain target populations, such as infants, the elderly, hospitalised persons, are more susceptible to the approach. Finally, the use of synbiotics, where both probiotics and prebiotics are combined, may offer the dual benefits of both approaches, whilst the use of a selective substrate may help long term persistence of the live microorganisms.

References

Franks, A.H., Harmsen, H.J., Raangs, G.C., Jansen, G.J., Schut, F. and Welling, G.W. (1998) Variations of bacterial populations in human faeces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Applied and Environmental Microbiology 64, 3336-3345.

Fuller, R. (1989) Probiotics in man and animals. Journal of Applied Bacteriology 66, 365-378.

Gibson, G.R., Berry Ottaway, P. and Rastall, R.A. (2000) Prebiotics: New Developments in Functional Foods. Chandos Publishing Limited, Oxford.

Gibson, G.R. and Roberfroid, M.B. (1995) Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics. Journal of Nutrition 125, 1401-1412.

Gibson, G.R. and Roberfroid, M.B. (1999) Colonic Microbiota, Nutrition and Health. Kluwer Academic Publishers, Dodrecht.

Metchnikoff, E. (1907) The Prolongation of Life. William Heinemann, London.

Salminen, S., Bouley, C., Boutron-Ruault, M-C., Cummings, J.H., Franck, A., Gibson, G.R., Isolauri, E., Moreau, M.C., Roberfroid, M.B. and Rowland, I.R. (1998) Functional food science and gastrointestinal physiology and function. British Journal of Nutrition 80, S147-S171.

Sanders, M.A. (1998) Overview of functional foods: emphasis on probiotic bacteria. International Dairy Journal 8, 341-347.

Tannock, G.W. (1999) A fresh look at the intestinal microflora. In Probiotics A Critical Review, pp.5-14 [GW Tannock, editor] Wymondham: Horizon Scientific Press.

Tuohy, K.M., Kolida, S., Lustenberger, A. and Gibson, G.R. (2001) The prebiotic effects of biscuits containing partially hydrolyzed guar gum and fructooligosaccharides � A human volunteer study. British Journal of Nutrition � in press.

Vaughan, E.E., Schut, F., Heilig, H.G.H.J., Zoetendal, E.G., deVos, W.M. and Akkermans, A.D.L. (2000) A molecular view of the intestinal ecosystem. Current Issues in Intestinal Microbiology 1, 1-12.

About the author

Glenn Gibson is Professor of Food Microbiology and Head of Food Microbial Sciences Unit at The University of Reading. Prior to this he was with the Institute of Food Research, Reading and MRC Dunn Laboratories, Cambridge. The Research Unit runs numerous projects on gut microbiology and food safety. Specific interests include, the bacteriology of acute and chronic gut disorders (e.g. gastroenteritis, ulcerative colitis, bowel cancer) and the use of molecular approaches to facilitate characteisation/enumeration of microorganisms. The group are extensively researching prebiotics and probiotics as dietary microflora management tools - in gut model systems and through human studies.

� IFIS Publishing 2004 - All Rights Reserved

http://www.foodsciencecentral.com/library.html#ifis/3731

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Heather is the Administrator of the IBS Message Boards. She is the author of Eating for IBS and The First Year: IBS, and the CEO of Heather's Tummy Care. Join her IBS Newsletter. Meet Heather on Facebook!

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