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Lgg - summatim




Second, updated edition Maija Saxelin, Ph.D.
Published by
Valio Ltd, R&D
P.O. Box 30,
FIN-00039
Helsinki, Finland
Tel. +358 10 381 121
Fax +358 10 381 3019
http://www.valio.com
Lay-out by
Imageneering
Printed in Finland by
Hämeen Kirjapaino Oy
2002
Valio Ltd 2002


Table of contents


Duodenum <103Jejunum Large intestine 1010—1012 Stool 1010—1012 Figure 1. Bacterial levels in different sections of the gastrointes-
tinal tract (cfu/g).

Bacterial concentrations at different sites of the gastrointestinal tract vary greatly (Fig. 1). The mucous membrane of the mouth and the surfaces of the teeth have high concentrations of bacteria, which pass, with saliva and chewed food, into the oesophagus and thereafter into the stomach, where the food is mixed with gastric juices and fl uidised. The acidity of the gastric juice effectively destroys most of the bacteria that come into contact with it. Food stays in the stomach for around four hours and is gradually released into the small intestine. The proximal part of the small intestine is also acidic due to the acid entering from the stomach. In addition, bile acids secreted into the proximal part of the small intestine destroy bacteria, so the bacteria level is relatively low. As acidity decreases and the bile acids are diluted, the bacteria level in the terminal part of the small intestine rises. The small intestine, several metres long, is densely proliferated with microvilli, which increase the internal surface area of the mucous membrane so much so that, if it were spread out, the small intestine would cover the area of a tennis court. The large surface area enables the effi cient breakdown of food and the subsequent absorption of nutrients through the mucous membrane into growth of external • systemic effects production, low pH) Immune – non-pathogenic boost, formation, lowers Lowers gasproduction Sulphate reducers Anaerobic gram pos. cocci decomposition andabsorption of food Synthesis ofvitamins Bacteria quantity log cfu/g of stool
Figure 2. The most important microbe groups, their quantities, and rough division according
to their potential for harmful and benefi cial effects (1).

the blood stream. Most of the system's immunological tissue is connected with the small intestine and can be found immediately under the epithelial cells of the mucous membrane.
The digestive tract pushes food and chyme forward by powerful peristaltic con- tractions. Moving from the small intestine to the large intestine, peristalsis slows down and sodium and chloride ions are absorbed with water into the blood stream. As a result, the contents of the bowel become more solid. At the same time the bacteria level also rises very sharply. The large intestine has an extensive bacterial metabolism. Bacteria break down the nutrients remaining in the food, such as par- tially digested proteins and fi bre components. Around half of the bulk of stools consists of bacterial mass. Between 400 and 500 species of bacteria have been r ec- L. rhamnosus L. rhamnosus GG • Group of L. rhamnosus • Can be distinguished Lactobacillus species strains in whose total from other strains of the same species by fenotype homology is >70% or genotype methods - common morphology - typical fermentation - similar biochemical profile (API50CH) - homo- or hetero- - genome analysis - probiotic characteristics: - catalase-negative adhesion, colonisation, immunological effects etc.
G+C%= the proportion of guanine and cytosine in DNA Table 1. The common and distinguishing characteristics of bacteria genera, species and
strains using the Lactobacillus rhamnosus GG strain as an example. The lactic acid bacteria
include 15 bacteria genera for which either homofermentative or heterofermentative lactic
acid production is typical. They are gram-positive cocci, rods or coccobacilli which are non-
spore forming and do not form catalase. Lactobacillus is one of the genera forming the lactic
acid bacteria group.

ognised in the large intestine and in stools. Moreover, between 100 and 1,000 times more anaerobic bacteria are present than aerobic. Genome-based research meth- ods have shown that human intestines have numerous, though as yet unidentifi ed, species of bacteria, which do not grow in the culture media currently in use. Fig. 2 presents the most common bacteria genera or groups and their main infl uence on the bacterial metabolism of the bowel.
Lactobacilli are part of the normal intestinal fl ora. They can be found in the stomach and in the proximal part of the small intestine, because lactobacilli are spe- cies that tolerate acidity relatively well. The most common species recognised on the mucous membrane of the bowel are the Lactobacillus acidophilus group (L. acidophilus, L. gasseri, L. jenseni, L. crispatus), L. casei, L. paracasei, L. rhamnosus, L. agilis, L. salivarius, L. plantarum, L. pseudoplantarum, L. buchneri and L. reu- teri. It has not been possible to identify all intestinal Lactobacillus species (2, 3). Lactobacillus rhamnosus GG (ATCC 53103), or more briefl y Lactobacillus GG or LGG, is a probiotic strain that has been isolated from a healthy human intestinal fl ora. Its probiotic effects on human well-being have been widely researched and documented in scientifi c journals. The term probiotic means live microorganisms which, when administered in adequate amounts, confer health benefi ts on the host (4). A probiotic must always be a certain bacterial strain (cf. Table 1) or a combina- tion of known strains whose composition remains stable and whose effects have been demonstrated in studies performed on humans and documented in scientifi c journals. There is a great deal of research data about Lactobacillus GG in the care and preventive treatment of different intestinal symptoms. This summary describes the effects of Lactobacillus GG in healthy intestines, its known clinical uses, and the mechanisms underlying these effects.



1.1 Colonises temporarily LGG Colony
Figure 3. A) Lactobacillus GG colonies among other stool lactobacilli fl ora in an
MRS-agar dish.
B) Lactobacillus GG bacteria (light microscopy, gram staining).
In healthy intestines so-called colonisation resistance prevails. This naturally prevents exogenous microbes, both harmful and harmless to the intestine, from establishing themselves permanently in the digestive tract. Colonisation resistance depends on chemical (e.g. gastric acid, bile acids, enzymes), physical-biological (adhesion, prevention/elimination of harmful bacteria, peristalsis) and immunological factors. Anaerobic bacteria in particular are involved in maintaining colonisation resistance. Resistance easily breaks down, for example, as a consequence of antibiotic or other medical treatment, and this can cause diarrhoea and other intestinal disorders.
Lactobacillus GG tolerates intestinal conditions, such as stomach acidity and bile acids, better than ordinary yoghurt bacteria (5). It adheres both to the intestinal mucus (6-8) and to epithelial cells and tissues in vitro (9) and ex vivo (10, 11). Lactobacillus GG also produces antimicrobial material (12 – 14). Because of its typi- cal colony morphology and other characteristic features, it is possible to analyse Lactobacillus GG from stool and biopsy samples, even though these contain a great many other lactobacilli (Fig. 3). In addition to the colony morphology genetic rec- ognition of the strain is needed to confi rm its identity (15). The capacity of Lacto- bacillus GG to stay alive within the digestive tract has been shown in many studies both in healthy people and in cases of illness (5, 16 – 19).
The attachment of Lactobacillus GG to the mucous membrane of the intestine has been shown by taking biopsy samples from the surface of the large intestine and by identifying Lactobacillus GG in them (20, 21). These studies demonstrated that the Lactobacillus GG strain adheres temporarily to the mucous membrane and stays there for about a week. The colonisation is not permanent because Lactoba- cillus GG triggers an immune response in the mucous membrane, which prevents permanent colonisation (22, 23). Lactobacillus GG, given immediately after birth, was still present in stools in approximately half of premature (24) and full-term (25) babies 2-4 weeks after the dosage ended. Administration of Lactobacillus GG to mothers at the time of delivery yielded a long-lasting colonisation (26). Thus, the permanent colonisation of the intestines of newly born babies may be possible.
1.2 Adapts to healthy intestinal fl ora The composition of human intestinal microfl ora appears fairly constant. Conven- tional culture methods only measure bacterial groups or genera and it is evidently not yet possible to cultivate some of the intestinal bacteria. Changes in the compo- sition of healthy intestinal microfl ora may occur on a species and strain level and cannot be measured by conventional culture methods (27). Lactobacillus GG enhanced the adhesion of bifi dobacteria in vitro (28). Milk products fermented with Lactobacillus GG, or Lactobacillus GG given in powder form, have been shown either to increase signifi cantly the quantity of bifi dobacte- ria and lactobacilli (29, 30) or else showed no changes in the composition of the fl ora (5, 31). However, in a recent study using the FISH method, mentation of Lactobacillus GG capsules increased the level of total anaerobic fl ora, especially bifi dobacteria, bacteroides and clostridia (32), but the level of lactobacilli/ enterococci did not increase. Although Lactobacillus GG becomes part of the bow- el's microbial fl ora, it does not displace all other lactobacilli. The relative proportion varies from individual to individual but it usually accounts for less than a quarter of the total quantity of lactobacilli (33, 34). However, the overall proportion of Lactoba- cillus GG may be greater on the mucous membrane of the intestine (20, 21).
1.3 Improves colonisation resistance Mortality %
Colonized with LGG Days after infection
Figure 4. The mortality of mice with germ-free intestines vs.
Lactobacillus GG colonised intestines following infection with
Salmonella typhimurium (41).

In addition to the prevention of the adhesion and the colonisation of pathogens, colonisation resistance also means that intestinal bacteria are not translocated to the blood circulation and other sterile body sites. Animal experiments have shown that the addition of Lactobacillus GG to animal feed improves colonisation resist- ance and protects the intestine from harmful bacteria. Salmonella levels were con- siderably lower in the intestines of mice that received Lactobacillus GG than in the placebo group. Furthermore, the life spans of Salmonella-infected ex-germ-free mice were considerably extended by Lactobacillus GG (Fig. 4). Lactobacillus GG also protected the mice from Candida albicans infection, reduced the growth of yeast and prolonged the life of the mice. The protective infl uence was based on both immunological and non-immunological factors (35, 36). Lactobacillus GG was also shown to prevent the attachment of Clostridium diffi cile onto the wall of ham- sters' intestines and, in combination with xylitol, to protect hamsters from death caused by C. diffi cile (37). Lethally irradiated mice died of bacteraemia of intestinal origin, but no cases of lactobacilli or Lactobacillus GG bacteraemia were observed. Rather, oral Lactoba- cillus GG intake was reported to prolong the survival of the mice (38). The infl u- ence of different probiotics on the extent of liver injury, bacterial translocation and intestinal fl ora in an acute liver injury model with rats was studied (39). The liver injury was induced by intraperitoneal injection of the rats with D-galactosamine. The bacteria were administered rectally eight days before the liver injury. Lacto- bacillus GG, which was one of the studied strains, reduced signifi cantly the bacte- rial translocation to portal and arterial blood, and the liver and mesenteric lymph nodes. The liver injury, measured as alanine aminotransferase, was less serious in the Lactobacillus GG group compared to the control group (39). In another experi- mental study, liver injury was caused by chronic alcohol consumption in rats. The blood of the rats had a lower level of endotoxin and a less injured liver when they received Lactobacillus GG in their diet (40).
In a study with mice, the translocation rate of Salmonella to several organs was signifi cantly reduced by the administration of Lactobacillus GG (41). In a study with neonatal rabbits, Lactobacillus GG was shown to signifi cantly reduce small- bowel colonisation by Escherichia coli. It also reduced the frequency of intestinal bacterial translocation in the mesenteric lymph nodes and in the spleen (42). In vitro studies support the theory of improvement of colonisation resistance by Lactobacillus GG. Although adhesion of Salmonella typhimurium on intesti- nal mucus was enhanced by Lactobacillus GG (43), the invasion to the cells was reduced (41). Furthermore, Lactobacillus GG reduced the adhesion of enterpatho- genic Escherichia coli on intestinal mucus (43) and on intestinal cells (44). The translocation of E. coli through Caco-2 enterocyte monolayer was also reduced by pre-incubation of the monolayer with the probiotic (45). These results show that Lactobacillus GG is not an invasive organism. It strengthens the barrier mechanisms in the intestine either directly or via the modifi cation of intestinal microecology.


1.4 Reduces harmful metabolism in the colon • inhibits the invasion of harmful microbes • prevents the growth of harmful bacteria Boosting immune response
• reinforces the mucosal barrier
• promotes recovery in infections
• promotes the immunological
Effect on large intestine enzyme activity
• lowers activity of carcinogen- activating enzymes • decreases secretion of toxic compounds• increases quantity of short chain fatty acids Binding mycotoxins and
bacterial toxins

Figure 5. The effects of Lactobacillus GG on intestinal microecology.
Intestinal bacteria break down the components of food into a more easily digest- ible form, affect the local immune response of the mucous membrane and promote colonisation resistance against pathogens. The intestinal microfl ora have also been shown to participate in the metabolism of harmful compounds in the human diet and in breaking down drugs as well as toxins (46). The Western diet, with its high fat and low fi bre content, supposedly increases the risk of colon cancer. Colonic microfl ora have been shown to be linked to this risk. The hydrolytic enzymes of the bacteria change pre-carcinogenic compounds in food into carcinogenic com- pounds. Epidemiological studies have demonstrated that a high consumption of fer- mented milk products may reduce the risk of colon cancer. Those in the risk group had less lactic acid bacteria in their intestines than those in the low-risk group (47). The mechanism may be the protective effect of calcium or conjugated linolic acid, the low activity of hydrolytic enzymes in lactic acid bacteria (48), and the effect of lowering the pH of bowel contents. A diet containing Lactobacillus GG-fermented milk has been shown to lower the activity of hydrolytic enzymes (β-glucuronidase, glycocholic acid hydrolase, nitroreductase) and tryptic activity in the colon con- tents, and the urinary secretion of toxic compounds. Some of these studies have also found a lowering of the pH of stools and a decrease in the amount of ammonia (29, 30, 49-52). All these factors together (Fig. 5) suggest that Lactobacillus GG, and particularly the fermented milk products that contain it, change the bowel contents so as to lower the risk of tumour formation.
Further support for the idea has been obtained from experimental studies. In one study (53) intestinal tumours were chemically induced in rats that were being fed on a high-fat diet. When the rats' diet contained Lactobacillus GG, signifi cantly fewer tumours formed in their large intestines, and the number of tumours per tumour-bearing rat was signifi cantly lower than those in the placebo group (Fig. 6). This work showed that the initiation of tumour formation can be reduced or delayed by Lactobacillus GG, but that lactobacilli have no effect on the advance of tumours that have already begun (53). In another experimental study, bladder cancer cells were transferred to mice and the effect of oral administration of Lactobacillus GG on the development of tumour formations was studied (54). The administration of Lactobacillus GG, or saline as a placebo, was started immediately after implantation of the tumour cells or one week later. Early administration of Lactobacillus GG reduced the size of the tumours signifi cantly or totally inhibited their formation. The levels of spleen T-lymphocytes (CD3, CD4, CD8a) and natural killer cells were signifi cantly higher in the Lactobacillus GG group compared to the placebo group. The levels of lym- focytes and granulocytes were also higher in the tumours of the animals in the Lactobacillus GG group. The conclusion was that Lactobacillus GG may inhibit the growth of tumours via an immune response (54). Afl atoxins (AF's) are a group of structurally similar toxins produced by the common moulds Aspergillus fl avus and A. nomius. The toxins are potentially car- cinogenic and harmful in food and feed. They can be produced in conditions con- ductive to the growth of the mould. The risk of the growth of fungus is higher in conditions with high relative humidity and temperature, and without competing Small bowel
Small bowel
Figure 6. The effect of Lactobacillicus GG on the formation of chemically induced
tumours in rats (53).

microfl ora. In laboratory tests Lactobacillus GG has been shown to bind AFB (55, 56) and AFM (57). The binding of AF's seems to be mainly extra-cellular and stable, removing about 80% of the AFB from a liquid growth medium (58, 59). AFM is the main form found in the milk of lactating animals, indicating the contamination of feed by AFB . Pierides et al (57) demonstrated that lactobacilli and lactococci can potentially be used for the binding and removal of afl atoxin M from milk. In an in vivo study AFB was injected with Lactobacillus GG bacteria into chicken duodena, and the concentration of AFB in the luminal fl uid and tissues of sacrifi ced animals was analysed after one minute. Half the toxin concentration was removed from the luminal fl uid. The complex was stable under the luminal condi- tions for a one-hour test period and it reduced the uptake by the intestinal tissue by 74% (60). Based on these studies, is would seem possible to remove AF's from the intestine by Lactobacillus GG on a signifi cant level and to reduce the toxic load of the intestine via excreted bacteria. Most alcohol is metabolised in the liver but recently the role of oro-gastroin- testinal bacteria has also been realised. Many intestinal facultative anaerobic and aerobic bacteria can oxidise ethanol to acetaldehyde, which itself is harmful to the mucous membrane. Nosova et al. (61) studied the capacity of intestinal bifi dobac- teria, lactobacilli and Lactobacillus GG to oxidise ethanol to acetaldehyde. In gen- er al, lactobacilli had weak oxidation potential, the most active being Lactobacillus GG in anaerobic conditions. It also had the highest ability to degrade acetaldehyde to acetate. The degradation of acetaldehyde by bacteria was generally inhibited by ethanol, but Lactobacillus GG was not very sensitive to that chemical. The circum- stances on the colon mucous membrane to oxidise acetaldehyde are favourable but the level of probiotics should be fairly high, and the relevance of the results remains to be shown in human trials. 1.5 Does it alleviate constipation? Lactic acid bacteria are generally considered to alleviate constipation. However, there is little clinical proof of their effi cacy in severe constipation, and many stud- ies have been conducted without good research procedures or statistical analysis. Lactobacillus GG-fermented milk products have been seen to slightly increase the water content of stools but they have had no effect on the frequency in defecation of healthy volunteers (29, 51), nor did they have any effect on transit time in those suffering from constipation (62). However, Lactobacillus GG enhanced the laxative effects of rye fi bre and had a tendency to reduce intestinal symptoms caused by the fi bre (62). Suggestions of an increase in bowel activity were obtained in one Lactobacillus GG study with and without lactulose (63).
In Japanese studies, the daily consumption of an Lactobacillus GG-fermented milk product was shown to signifi cantly increase the level and ratio of faecal bifi do- bacteria and lactobacilli and to reduce the level of lecitinase negative clostridia. The consumption of the product also increased signifi cantly defecation frequency and relieved discomfort after the bowel movement. There was a tendency to increase the faecal moisture and decrease pH and ammonia content (30, 64). These results indicate that not all studies are necessarily applicable to other cultures with a totally different diet composition from that of the West. The innate and adaptive immune systems are the two compartments traditionally described as important for the immune response. Macrophages, neutrophils, natu- ral killer (NK) cells and a serum complement represent the main components of the innate system, in charge of the fi rst line of defence against many microorgan- isms. However, there are many agents that this system is unable to recognize. The adaptive system (B and T cells) provides an additional means of defence, while cells of the innate system modulate the beginning and subsequent direction of adaptive immune responses. Several soluble compounds (cytokines, interleukins, interferons) are involved in the modulation of the immune system. In vitro Lactobacillus GG induced the expression and production of the proin- fl ammatory Th-1-type cytokines TNF-α, IL-1β, IL-6 and IL-18 in peripheral blood mononuclear cells, but not the Th-2 type cytokine IL-4 and relatively little IL-10 (65, 66). Lactobacillus GG also activated the transcription factor NF-κB, which is the central activator of innate immune response, and the Toll-like receptors TLR1 and TLR2, which mediate bacterial recognition and cellular signalling (67, 68). The results suggest that Lactobacillus GG is able to activate innate immune responses.
In an animal study, orally administered Lactobacillus GG bacteria had dose- and duration-dependent immunomodulatory effects on the proliferative activity of B and T murine spleen lymphocytes ex vivo. A dose relevant to human nutrition enhanced T-cell proliferation at the optimal concanavalin A concentration and B-cell proliferation at the optimal and supraoptimal concentrations of lipopolysaccharide (69). In a human intervention, Lactobacillus GG enhanced signifi cantly the forma- tion of the phagocytic receptors CR1, CR3, FcγRIII and FcαR in neutrophile blood cells in healthy humans but suppressed the response of milk-hypersensitive human volunteers during a milk challenge. The conclusion was that probiotic bacteria appear to modulate the non-specifi c immune response differently in healthy sub- jects and hypersensitive subjects by immunostimulation in healthy and by down- r egulation in hypersensitive ones (70). Human administration of Lactobacillus GG combined with an oral rotavirus vac- cine enhanced the formation of rotavirus specifi c IgM-secreting cells and rotavi- rus specifi c IgA in sera (71). There was also a trend towards a greater increase in antigen-specifi c IgA response when Lactobacillus GG was given with an oral Sal- monella typhi Ty21 vaccine (72). In another study with Salmonella typhi vaccine, Lactobacillus GG enhanced signifi cantly the IgG and IgA response to the vaccine (73). In children with rotavirus infection, Lactobacillus GG increased the formation of immunoglobulin secreting cells in all immunoglobulin classes and in rotavirus specifi c antibody-secreting IgA cells (74-76).
These studies show that Lactobacillus GG both activates the innate immune response and enhances adaptive immunity, especially during infections. 3.1 Respiratory infections Otitis media
Figure 7. The effect of Lactobacillicus GG on the prevalence of respiratory infections and
frequency of antibiotic treatment in children. The children drank either LGG milk or ordinary
milk during daily meals for a period of seven months (77).

Day care centres expose children to infections, especially of the upper respiratory tract. Overall, more than 90% of child absenteeism from day care is caused by infec- tious diseases. In addition to discomfort to the children and inconvenience to their families, illnesses are costly to society. The greatest costs result from the parents' absence from work because of a child's illness. A long-term study was made to see if consumption of Lactobacillus GG had an effect on infections in children (77). A total of 571 children from 18 day care cen- tres in Helsinki, Finland, participated in the study. During the seven-month research period, half the children were given pasteurised milk that contained Lactobacillus GG (5-10x105 cfu/ml) to drink with all meals, and the other half were given ordi- nary milk. The average milk consumption was 260 ml/day. The children's health w as carefully monitored: symptoms in the respiratory and digestive tract, as well as absences from the day care centres, were recorded daily by the parents. Doctors' diagnoses and antibiotic treatments were also reported. Children in the Lactobacil- lus GG group had fewer days of absence from day care because of illness (4.9 vs. 5.8 days, p=0.03), an 11% difference. There was also a relative reduction of 17% in the number of children who suffered from respiratory tract infections with com- plications, especially ear infections, in the Lactobacillus GG group (Fig. 7). The number of children who received antibiotic treatment for respiratory infections was 19% lower in the Lactobacillus GG group than in the placebo group. The con- clusion was that Lactobacillus GG may reduce children's respiratory infections and their severity. Risk of
dental caries (%)

Figure 8. The effect of Lactobacillus GG on
the risk of dental caries. The children drank
either LGG milk or ordinary milk during daily
meals for a period of seven months (79).

Teeth are in continuous interaction with the surrounding world, mainly saliva and whatever you put in your mouth. Milk provides calcium and phosphates in the mouth, which causes remineralisation of places demineralised by caries. Milk and dairy products are important elements in children's nutr ition and dental health, since teeth at this point are particularly vulnerable to attack from caries, having just begun the mineralisation process. Lactobacilli are common bacteria in the oral cavity, but they are generally regarded as potentially cariogenic, growing together with streptococcus mutans. However, in in vitro studies, Lactobacillus GG showed slow or no fermentation of sucrose and lactose (34), and suppressed the growth of the mutans- group streptococci, which are the indicator bacteria of dental caries (78). The long-term effect of Lactobacillus GG on the risk of caries was studied in 18 day care centres in Helsinki, Finland (79). In a randomised, placebo-controlled study children were given pasteurised milk that contained Lactobacillus GG (5-10x105 cfu/ml) or standard milk as a placebo, fi ve days a week for seven months with their day care meals. The children's oral health was recorded at baseline and at the end, and mutans-group streptococci were cultivated from saliva-dental plaque samples. The risk was classifi ed as high if the child had a score of decayed/missed/fi lled teeth (dmft) or initial caries of >0 and a mutans streptococci count >105 cfu/ml, as moderate if either of these was detected, and as no risk if dmft was 0 and the mutans streptococci count <105 cfu/ml. The results showed less dental caries in the Lactobacillus GG group at the end of the study and lower mutans streptococci counts. The risk of dental caries was 44% lower in the Lactobacillus GG compared to the placebo (OR=0.56, p=0.01; Fig. 8). The conclusion was that the milk contain- ing the probiotic Lactobacillus GG bacteria may have benefi cial effects on chil- dren's dental health beyond the effect of standard milk. LGG and diarrhoea 4.
4.1 Preventive treatment 4.1.1 Acute diarrhoea in children Figure 9. The effect of Lactobacillus GG
on the occurrence of acute diarrhoea. Chil-
dren hospitalised for non-diarrhoea rea-
sons were given either Lactobacillus GG or
placebo throughout their stay (81).

A fi fteen-month study surveyed the incidence of diarrhoea among under-nourished Peruvian children living in poor conditions (80). One half of a group of 204 chil- dren received a Lactobacillus GG dose six times a week at home and the other half, a placebo. Altogether, 954 diarrhoea episodes were recorded and the infectious agent was determined in 58% of the cases. Pathogenic bacteria were isolated in about one half of the cases, parasites in one half, and viruses in one third. Mixed infections were therefore very common. The Lactobacillus GG group was found to have signifi cantly fewer diarrhoea episodes caused by the adenovirus; ference was found in the incidence of other pathogens. Looking at the data as a whole, the incidence of diarrhoea in the Lactobacillus GG group was 5.2 episodes per child per year, compared with 6.0 episodes in the placebo group (p=0.028). Diarrhoea prevention was most effective in children aged 18-29 months (4.9 epi- sodes LGG vs. 6.2 episodes placebo, p=0.004) and was primarily of benefi t to chil- dren who were not breastfed. Lactobacillus GG had no effect on the duration of diarrhoea in this study (80). Another, short-term clinical study, to evaluate the reduction of the risk of diar- rhoea by Lactobacillus GG, was made in a Polish hospital (81). Children hospital- ised for reasons other than diarrhoea were given Lactobacillus GG or a placebo twice daily during their hospital stay. The risk of diarrhoea was reduced signifi - cantly in the Lactobacillus GG group compared to the placebo group (6.7% vs. 33.3%, RR 0.2, p=0.002). Surprisingly, there was an equal prevalence of rotavirus infection in both groups, but the administration of Lactobacillus GG signifi cantly reduced the risk of rotavirus gastroenteritis (1/45 vs. 6/36, RR 0.13, p=0.02; Fig. 9). This result poses an interesting question as to the potential of Lactobacillus GG to protect against rotavirus after a non-diarrhoeal infection.
4.1.2 Antibiotic-associated side effects Possibly the most common indication for the clinical use of probiotics is their abil- ity to prevent the side effects of antibiotics, such as diarrhoea and abdominal pain. Antibiotics change the composition of the bowel microfl ora, allowing the possibil- ity for opportunistic pathogens such as Clostridium diffi cile to proliferate. Antibiot- ics also interfere with the metabolism of the microfl ora, for instance, by impeding the formation of short-chain fatty acids in the colon. Probiotics are therefore well suited for maintaining or restoring the balance of the bacterial fl ora. The effect of Lactobacillus GG taken in a capsule form has been proved to reduce the side effects of antibiotics in children. In a randomised, double-blind, placebo-controlled study, common acute infections in 188 children were treated by commonly used antibiotics, and under the care of family physicians (82). Half the patients received 1 – 2 Lactobacillus GG capsules (1x1010 cfu) once a day, the Symptoms (%)
Figure 10. The effect of Lactobacillus GG on
intestinal symptoms caused by antibiotics (82).

other half received identical placebo capsules without the bacteria (one capsule for children <12 kg, two capsules for those >12 kg). Any gastrointestinal complaints were monitored via telephone interviews. Signifi cantly less diarrhoea and daily def- ecations were reported in the Lactobacillus GG group than in the control group. Furthermore, the stools were more solid and the study group had less abdominal pain than the placebo group (Fig. 10). Lactobacillus GG did not cause any side effects in this or in other studies. Another study was conducted in Finland with children prescribed oral antibiot- ics for the treatment of acute respiratory infections (83). The children were ran- domised to receive either one placebo (n=58) or one Lactobacillus GG (n=61) capsule twice a day (2x1010 cfu). The parents kept a daily symptom diary at home and recorded stool frequency and consistency. In cases of diarrhoea, stool samples were analysed for adenovirus, rotavirus, calicivirus and astrovirus as well as for Salmonella, Shigella, Yersinia, Campylobacter, Clostridia diffi cile, Staphylococcus aureus and yeasts. Within two weeks of antimicrobial treatment the incidence of diar- rhoea was 5% in the Lactobacillus GG group and 16% in the placebo group (p=0.05). In diarrhoeal episodes two cases of C. diffi cile were found (one in each group) and three cases of Norwalk-like calicivirus were positive (one in the Lactobacillus GG group, two in the placebo group). No other pathogens were recovered (83). In a small study with adult volunteers Lactobacillus GG reduced signifi cantl y diarrhoea caused by erythromycin and somewhat reduced abdominal pain (84). In the study, volunteers took a Lactobacillus GG-fermented milk product or a placebo yoghurt (post-pasteurised yoghurt without the living bacteria) in the morning and evening, half an hour after they had taken an antibiotic. Armuzzi et al. studied the effect of Lactobacillus GG on gastrointestinal discom- fort caused by the antibiotic treatment of Helicobacter pylori (85, 86). In a pilot study (86) 120 asymptomatic volunteers carrying H. pylori were randomised to the eradication therapy with pantoprazole, clarithromycin and tinidazole for one week or the same regimen supplemented with Lactobacillus GG (6x109 cfu/sachet) for two weeks. Lactobacillus GG was taken 2 h after breakfast and dinner, mixed with water. Bloating, diarrhoea and taste disturbances were the most frequent side effects during the eradication week and were signifi cantly reduced in the Lactobacillus GG group. The same pattern was observed throughout the follow-up period. The overall assessment of treatment tolerability showed a signifi cant trend in favour of the Lactobacillus GG-supplemented group (p=0.03). In another, double-blinded, placebo-controlled study, 60 healthy asymptomatic H. pylori positive volunteers were randomised to one week therapy with rebeprazole, clarithromycin, tinidazole and Lactobacillus GG (6x109 cfu/sachet) for two weeks, or to the same regimen with a placebo preparation (85). Again, diarrhoea, nausea and taste disturbances were signifi cantly reduced in the Lactobacillus GG group com- pared to the placebo (RR=0.1, 0.3 and 0.5 respectively). An overall assessment of treatment tolerability showed a signifi cant difference in favour of the Lactobacillus GG group (p=0.04). There was no difference between the groups in the success of the eradication of H. pylori (in both studies it was about 80%), but supplementation with Lactobacillus GG helped to improve the tolerability of the antibiotics. A randomised, double-blinded, placebo-controlled study was performed with 267 initially hospitalised adult patients treated with intravenous or oral antibiotics for a presumed or proven infection (cellulites, pneumonia, urinary tract infection and pyelonephritis) (87). The main groups of antibiotics were β-lactams (cephalosporins 60%, penicillin 27%) and fl uoroquinolones (39%). Lactobacillus GG (1x1010 cfu) or placebo capsules were given twice a day. The Lactobacillus GG intervention had no effect either on the incidence or on the duration of mild or severe diarrhoea. Broad-spectrum antibiotics, especially for immunocompromised patients, can Ref. 90, MD plate
E-test, AB Biodisc
for gram posit., Radiometer
Amoxycillin / Clavulanate Trimethoprim / Sulphamethoxazole Table 2. The antibiotic sensitivity of Lactobacillus GG in MIC (minimum inhibitory concentra-
tion) values.

cause serious D-lactic acidosis due to the intestinal lactobacilli producing D-lactic acid. Lactobacillus GG produces L-lactic acid and has been successfully used to treat one such case (88). The susceptibility of LGG to antibiotics
Although Lactobacillus GG is susceptible to the most common antibiotics (89, 90) (Table 2), it has been shown to survive in the intestines during antibiotic treatment in most test subjects. Lactobacillus GG was isolated in stools in 75, 76 and 57% of the test subjects being treated with erythromycin, ampicillin and penicillin respec- tively (5, 33, 84). The survival of Lactobacillus GG can be explained by the antibi- otic and bacterial preparations being taken at different times, and possibly by the lower antibiotic level in the bowel than in the blood stream. Some species of lacto- bacilli are naturally resistant to vancomycin, including all strains of the species L. rhamnosus, L. casei, L. plantarum and L. reuteri. It is also pertinent to ask whether the genes responsible for vancomycin resistance can be transferred to other bacte- ria. Vancomycin-resistance genes in Lactobacillus GG were shown to differ from the van genes in enterococci, and were not transferred to enterococci (90, 91). Antibiotic-resistance genes can sometimes be transferred via plasmids. Lactobacil- lus GG does not carry plasmids and is safe in that sense, too (91). 4.1.3 Traveller's diarrhoea Intestinal troubles are a common complaint among those travelling from cold or cool climates to warm and tropical countries. Lactic acid bacteria are often used to prevent intestinal troubles while travelling, even though few studies have been conducted on their effi cacy. The fi rst Lactobacillus GG study was conducted on Finnish tourists (n=756) who visited two resorts in Turkey (92). An average of 43.8% of the travellers had diarrhoea. Lactobacillus GG taken twice a day signifi cantly reduced the incidence of diarrhoea in those staying one week in one of the resorts but not in the other. No explanation for the difference in effectiveness between the resorts was found, but it is possible that the dose (1x109 cfu twide daily) of Lactobacillus GG used in the study was too low.
The second study was carried out with American tourists (n=245) whose desti- nations were primarily in Asia, East Africa, South America, India and Central America (93). One Lactobacillus GG capsule per day provided statistically signifi cant pro- tection. In the Lactobacillus GG group the average incidence of diarrhoea was 3.9%, whereas in the placebo group it was 7.4% (p=0.05), i.e. a protection factor of 47%. Travellers who had previously suffered from tourist diarrhoea benefi ted the most. The best protection against traveller's diarrhoea is still good personal hygiene such as hand washing, drinking bottled water and drinks without ice cubes, and the consumption of adequately cooked, hot food. However, Lactobacillus GG provides extra protection.
4.2 Treatment studies 4.2.1 Rotavirus diarrhoea Duration of
diarrhoea,days

Figure 11. The effect of Lactobacillus GG on the duration of acute
diarrhoea. Ambulatory children were given Lactobacillus GG mixed in
milk or mother milk substitute for a maximum of fi ve days (101).

Lactobacillus GG accelerates recovery in acute diarrhoea. Studies have been pri- marily conducted on children with rotavirus, which is the most common cause of diarrhoea in western countries. Lactobacillus GG accelerated by about one day the recovery of children hospitalised with acute diarrhoea (Table 3). The acceleration of recovery was generally noted on the second day: children treated with Lactoba- cillus GG defecated less often and their stools were more solid than those in the placebo group (94). Children treated at home, with Lactobacillus GG administra- tion starting on the second day after the onset of diarrhoea, suffered symptoms for approximately half as long as the placebo group (Fig. 11). In addition, these children spread the virus for a shorter time than those in the placebo group, since after six days signifi cantly fewer of them excreted rotavir us in their stools than in Duration of diarrhoea
days or hours (SD)
LGG group
39 Fermented milk 2.8 (1.2) Lactophilus(1 2.6 (1.4) Yoghurt starter 0.04 2.6 (1.3) Placebo Inactivated powder after 2 days treatment after 2 days treatment 123 Within ORS: single or 17.7 (12.2–25.6) h 30.4 (23.6–39.3) h multiple dose, after (after ORS or placebo) Rotavirus posit.
1) Other L.rhamnosus strain2) Ambulatory Table 3. Lactobacillus GG in the treatment of acute diarrhoea. Hospitalised patients were
given LGG twice a day after oral rehydration (ORS), if not otherwise stated.

the placebo group. The effect of Lactobacillus GG in the treatment of rotavirus diar- rhoea has been confi rmed through a multi-centre study carried out by the ‘diarrhoea working group' of the European Society of Paediatric Gastroenterology, Hepatology and Nutrition (95). In a recent systematic review of published studies, Lactobacillus GG was shown to be, so far, the only probiotic strain with a consistent effect on the duration of diarrhoea and on the risk of diarrhoea lasting >3 days (96).
The best recovery is obtained when Lactobacillus GG is administered as early as possible after the symptoms of diarrhoea have appeared. If rehydration is needed, then Lactobacillus GG treatment is best started at the same time as oral rehydra- tion (95, 97). The effect of the treatment was the same whether Lactobacillus GG was administered in powder form (capsule/opened) or in the form of a fermented dairy product (cf. Table 3). It is also worth mentioning that heat-inactivated Lacto- bacillus GG accelerated the recovery in acute diarrhoea as effectively as the living bacteria (75) but the immune effect differed.
4.2.2 Other types of acute diarrhoea Studies carried out in Thailand and Pakistan using Lactobacillus GG in the treat- ment of acute diarrhoea showed that recovery from watery diarrhoea was acceler- ated, but not from diarrhoea with bloody stools (98, 99). Nor did Lactobacillus GG succeed in removing Klebsiella oxytoca from the intestines of premature babies (100). On the other hand, an Italian study (101) and the European multicenter study (95) showed a signifi cant effect of Lactobacillus GG both in rotavirus infections and in cases where the cause of the diarrhoea was unknown. Similarly, in a study performed in Petroskoi (Russia), the difference was signifi cantly in favour of the Lactobacillus GG group, even though only 27% of the patients had rotavirus diar- rhoea. About a fi fth had diarrhoea caused by known bacteria and in about half the cases the aetiology was unknown (102). Therefore, it seems that Lactobacillus GG is effective not only in rotavirus diarrhoea but also in some infections where the aetiology is unknown. If the mucous membrane is profoundly infl amed or even destroyed, the effectiveness of Lactobacillus GG remains unclear.
4.2.3 Are all lactobacilli effective? Lactobacillus GG was compared with another L. rhamnosus strain (Lactophilus®, Laboratoires Lyocentre, France), traditionally used in the prevention and treatment of children's diarrhoea in Finland, and with a common yoghurt starter culture powder (76). Only Lactobacillus GG was found to accelerate recovery from diarrhoea. This suggests that different bacterial strains within the same species have signifi cant ferences in their effect. The clinical effi cacy of every single strain must therefore be proved in carefully conducted studies, preferably made by several study groups.
4.3 Mechanisms behind the effects 4.3.1 Infections - enhancing immune response and balancing intestinal microfl ora Figure 12. The effect of Lactobacillus GG on immune
response.
A) Total number of immunoglobulin secreting
cells (ICS) in the acute phase of rotavirus diarrhoea and
B) total number of rotavirus specifi c IgA secreting cells
(sASC) three weeks after infection. Points represent indi-
vidual patients, horizontal lines the means (75).

Several studies have shown that the administration of Lactobacillus GG enhances immune response during rotavirus diarrhoea. Lactobacillus GG signifi cantly increased both non-specifi c immune response (in immunoglobulin classes IgG, IgA, IgM) in the acute stage of diarrhoea and in the quantity of rotavirus-specifi c anti- body-secreting cells in the follow-up stage. Three weeks after the infection, 90% of those who received Lactobacillus GG had a rotavirus-specifi c IgA response, com- pared with only 46% in the placebo group (Fig.12). It seems, therefore, that bacte- rial treatment gives additional protection against re-infection. This and later studies (74-76) show that the infl uence of Lactobacillus GG is specifi cally mediated through an enhanced immune response. Lactobacillus GG has also been found to induce an enhanced response with an oral rotavirus vaccine (71). Not all lacto- bacilli, however, increase the immune response, which in part explains the dif- ferences in their effects (76). The effect of Lactobacillus GG on innate defence systems might also contribute to the accelerated recovery from diarrhoea, e.g. enhanced production of induced nitric oxide (103), mucin production (44) and increased rate of enterocyte proliferation (104).
Another possible contributing factor in shortening the duration of diarrhoea is the balancing of intestinal microfl ora. Acute osmotic diarrhoea may be followed by bacterial imbalance and the overgrowth of specifi cally urease-producing bacteria. These may release ammonia, which is toxic to the intestinal mucous membrane. However, urease activity was not elevated in those subjects treated with Lactoba- cillus GG (105). Lactobacillus GG adheres to intestinal mucus (106) and is able to survive in the bowel even during acute diarrhoea, making it suitable for balancing intestinal microfl ora (19, 105). 4.3.2 Antibiotics and balancing intestinal fl ora Data has been obtained on the effi cacy of antimicrobial medication and Lactobacillus GG in the treatment of shigellosis and on their infl uence on the composition of bowel microfl ora (107). After ten days of treatment, 79% of the children had recovered in the group that received Lactobacillus GG and 67% in the group that only received medici- nal treatment (p<0.05). Due to the paucity of the material (n=31) no far-reaching con- clusions can be drawn about the clinical signifi cance of the treatment. At the start of treatment, the subjects' intestinal microfl ora was completely unbalanced, i.e. the quantity of aerobic bacteria was greater than that of anaerobic bacteria (Fig. 13). Furthermore, there were hardly any lactobacilli at all and the rela- tive proportion of subordinate bacteria had risen considerably. After fi ve days of treatment, the quantity of lactobacilli had increased and the microfl ora had par- tially normalised in both groups that received Lactobacillus GG. After ten da Log cfu/g
Treatment day 1
Log cfu/g
Treatment day 5
Log cfu/g
Treatment day 10
Anaerobic bacteria Treatment groups
LGG = Lactobacillus GG twice day
LGG+TS = combined treatmentTS = trimethoprim-sulfa, 36 mg/kg/day Figure 13. The effect of Lactobacillus GG on the quantity of faecal aerobic and
anaerobic bacteria as well as total lactobacilli during the antimicrobial treatment of
shigellosis (108).

treatment, the level of anaerobes was normal in the Lactobacillus GG group, was slightly normalised in those who received the combined lactobacilli and medicinal treatment, and was still low in those who had only received the medicinal treat- ment. Moreover, lactobacilli were still absent from the intestines of those who had only received the medicinal treatment (Fig.13). An intestinal microfl ora imbalance, and particularly a defi ciency of anaerobic bacteria, increases the translocation of intestinal bacteria from the lumen to the tissues and increases the risk of infections and bacteraemia (37). Lactobacillus GG is resistant to trimethoprim-sulfamethoxa- zole, so it is well able to balance the intestinal fl ora during the treatment.
Changes in the intestinal microbe population can also be measured as changes in its metabolic activity. Bacterial metabolism produces short-chain fatty acids from carbohydrates and proteins, particularly acetate, propionate and butyrate. Most of these are absorbed by the mucous membrane as an energy source for colonocytes. Short-chain fatty acids lower the pH of the bowel contents, and butyrate in particu- lar is considered to have a protective infl uence on the mucous membrane (108). In premature babies who received antibiotic treatment, Lactobacillus GG did not cause any signifi cant changes in the production of short-chain fatty acids (109). However, with medicinal treatment against Salmonella and Shigella, Lactobacillus GG normalised the production of short-chain fatty acids, which points to a normali- sation of intestinal microfl ora (110).
4.4 Indications in Clostridium diffi cile treatment C. diffi cile is an opportunistic pathogen which can also be found in normal human microfl ora. It does not usually cause any symptoms. However, when the microfl ora balance is disturbed - for example, as a consequence of antibiotic treatment - the C. diffi cile population can increase considerably and the toxin it produces can cause varying degrees of chronic diarrhoea and even pseudomembraneous colitis. C. dif- fi cile diarrhoea recurs in about 10-20% of subjects treated with antibiotics (van- comycin or metronidazole), and more effective treatments are scarce. The use of Lactobacillus GG in the treatment of recurrent C. diffi cile diarrhoea has been reported in around 40 subjects (111-113). A positive treatment response was achieved with a single treatment in 84% of the cases, and with repeat treatment in 94%. In the preliminary results from a placebo-controlled pilot study, a signifi cant effect was obtained in those who had C. diffi cile colitis for the fi rst time, but not in cases which had recurred often (114, 115). Further studies are underway.
The histopathology of C. diffi cile colitis and its effect on the intestinal microfl ora has been studied in an animal experiment (37). C. diffi cile combined with an anti- biotic (ampicillin) is inevitably fatal in hamsters. As it was known that Lactobacillus GG maintains normal intestinal microfl ora and that xylitol prevents the adhesion of C. diffi cile, the effectiveness of the combination treatment was tested in hamsters. It was found that this could prevent both the development of enter ocolitis in animals (in 4 animals out of 5) and their death. Animals not undergoing the combination treatment died within 2.5 days. In the hamsters with enterocolitis, the anaerobic microfl ora of the epithelium of the bowel was almost completely destroyed and coliform, facultative bacteria had become the dominant microfl ora in the contents of the bowel. In those hamsters that survived without enterocolitis, the dominant microfl ora were anaerobic bacteria, and C. diffi cile was found in only low concen- trations in the bowel lumen of two animals (37).
permeability of the 5.
Transport of
intact protein

Without Peyer's patches With Peyer's patches P<0.002 P<0.001 Figure 14. The effect of Lactobacillus GG on mucosal
permeability. Fourteen-day old rats were gavaged daily
with cow milk +/- Lactobacillus GG, and the jejunal
permeability was analysed when the rats were twenty-
one days old (118).

When intestinal infl ammation and microfl ora imbalance occur, the permeability of the mucous membrane increases, and large antigen molecules (116) and intestinal bacteria (37) can migrate across the mucous membrane into the system. Similarly, it has been shown that sensitivity to food antigens increases after acute diarrhoea, because antigens are abnormally transported across the intestinal mucous mem- brane (117). Furthermore, experimental studies with rat pups show that both for- eign antigens in the diet or rotavirus infection increase the permeability of the immature mucous membrane, with no antigen-specifi c local response. When test animals received Lactobacillus GG in their diet, the maturation of the mucous membrane occurred normally: the transport of antigens was strongly reduced and occurred in a controlled route via Peyer's patches (Fig. 14). The result w enhancement of a local, antigen-specifi c IgA response (116, 118). It has also been shown in humans that Lactobacillus GG enhances a local, antigen-specifi c IgA response to food antigens (31). Such an enhanced response is important as regards the tolerance of food antigens. Chronic non-steroidal anti-infl ammatory drugs destroy gastrointestinal mucosa, leading to ulceration. The protective effect of fermented milk drinks on indomet- acin-induced alterations of mucosal permeability has been studied (119). The fer- mented milk drinks contained active or heat-inactivated strains of Lactobacillus GG, L. helveticus and L. acidophilus (>107 cfu/g each). Four gastrointestinal perme- ability tests were carried out in randomized order on 16 healthy adults: 1) basal, 2) after indometacin, 3) after indometacin when the fermented milk drink with living bacteria was consumed for fi ve days, 4) after indometacin when the fermented milk drink with heat-inactivated bacteria was consumed for fi ve days. Gastric perme- ability was measured by sucrose urinary excretion, and intestinal permeability by lactulose/mannitol excretion. Indometacin signifi cantly increased both gastric and intestinal permeability. The fermented milk with living bacteria signifi cantly reduced abnormal gastric permeability, but not the intestinal permeability induced by indometacin. The drink with the heat-inactivated bacteria had no effect.
6.1 Speeds recovery in allergy Score of atopic
symptoms

Formula + LGG
Figure 15. The effect of Lactobacillus GG on the
atopic dermatitis of milk allergic children, during a
milk elimination diet (121).

Allergies have increased and are still increasing in western countries. In Finland approximately 2.5% of small children suffer from allergy caused by cow's-milk pro- tein. In recent years there has been intensive research into how this trend could be altered through bacterial treatment. Studies on the treatment of atopic and food allergies have suggested that by restoring the permeability of the intestinal mucous membrane, by modulating the local immune response and by using bacteria that suitably alter the food antigens, an immune response that has gone awry can be guided back in the right direction (120).
A randomised, placebo-controlled study on children with an atopic eczema with allergy to milk showed that the intensity and extension of the rash and subjective symptoms decreased signifi cantly faster when their milk elimination diet contained Lactobacillus GG (Fig. 15). The intestinal infl ammation was measured using the cytokine content of their stools. Tumour necrosis factor-α was found to fall signifi - cantly more rapidly in the Lactobacillus GG group compared to the placebo, indi- cating a faster recovery from infl ammation. Lactobacillus GG also helped those children who were only fed on mother's milk and where the bacteria were admin- istered to the mothers (121).
In another clinical study, Lactobacillus GG was given to infants who manifested atopic eczema during exclusive breastfeeding, and who had no exposure to any infant food or substitute formula (122). They were weaned to a probiotic (Lactoba- cillus GG or bifi dobacteria) -supplemented extensively hydrolysed whey protein formula or to the same formula without probiotics. The skin condition, the growth and concentrations of circulating cytokines and chemokines as well as soluble cell surface adhesion molecules in serum and methyl-histamine and eosinophilic pro- tein X in the urine were determined. According to results after two months, the atopic eczema was signifi cantly improved in the probiotic groups compared to the placebo. The median score of atopic dermatitis during breastfeeding was 16 (7-25) and decreased in the Lactobacillus GG group to 1 (0.1-8.7), vs. 13.4 (4.5-18.2) in the placebo group (p=0.01). The concentrations of serum soluble CD4 decreased in the same period in the probiotic groups but not in the placebo group, and the serum tumour growth factor-β tended to increase in the Lactobacillus GG group. Before intervention, the urine eosinophilic protein X correlated signifi cantly with the clinical score of atopic symptoms. Its concentration decreased signifi cantly in the Lactobacillus GG group during supplementation, which supports the clinical results. In conclusion, the data confi rmed that Lactobacillus GG supplementation during the weaning period counteracted infl ammatory responses and helped to tolerate new dietary antigens.
6.2 Prevents the risk of allergy in infancy An interesting question is whether the development of allergic diseases can be prevented in early infancy by modulating the intestinal microfl ora with probiotic bacteria. To evaluate this, a group of families at high risk of allergy was selected (123). The only inclusion criterion was a family history of atopic disease: one or more family members with atopic eczema, allergic rhinitis or asthma. In all, 159 eczema (%)
Figure 16. The effect of Lactobacillus
GG on the incidence of atopic eczema in

infants. Pregnent mothers took Lactoba-cillus GG or identical placebo capsules daily for 2 – 4 weeks before the delivery. After the birth it was given to the new-born baby or alternatively to the breast- feeding mother (123). mothers were randomised to receive two Lactobacillus GG (1010 cfu) or placebo capsules daily for 2-4 weeks before the expected date of the birth. After the birth, either the breastfeeding mother or the infant consumed the bacteria for six months. The children were clinically examined until they were two years old and the incidence of atopic diseases calculated. Parents reported any symptoms observed in their children which might be related to atopic disease. Sensitisation to common dietary and respiratory antigens was measured by the skin prick test and total and antigen-specifi c IgE assays. Altogether, 132 families with atopic diseases completed the study. Atopic eczema was found in 46 out of 132 children (35%) at the age of two years, asthma in six and allergic rhinitis in one child. Almost every other baby in the placebo group developed an atopic disease, but only one in four in the Lactobacillus GG group (Fig. 16). The mean duration of breastfeeding was as long in both the atopic (7 mo) and the non-atopic (6.7 mo) group. Surprisingly, there was no difference in the effect, no matter whether Lactobacillus GG was given directly to the infant or to the breast-feeding mothers. Concentration of total IgE as well as fre- quencies of increased antigen-specifi c IgE concentrations and of positive skin-prick tests were similar between the Lactobacillus GG group and the placebo group. It is possible that the risk of allergy in infants can be reduced by maintaining a good bacterial balance in pregnant mothers. The addition of probiotics to the diet of the nursing mothers enhanced the protective effect of breast milk. In a randomised, placebo-controlled study (124) with 62 mother-child pairs, lus GG increased the level of anti-infl ammatory TGF-β2 in breast milk signifi cantly, compared to the placebo group. The risk of developing atopic eczema during the fi rst two years of life of the infants was signifi cantly reduced in the probiotic group compared to the placebo group (15% vs. 47%; relative risk 0.32, p=0.0098). 6.3 Mechanisms behind the effects The mechanisms by which probiotics have an effect in the prevention and allevia- tion of allergy are not yet fully understood but many factors have been found (125). Microbial fl ora has an effect on the development of immune response and the bal- ance of T-helper cell types (Th1/Th2). The balance in turn determines the develop- ment of oral tolerance. Th-2 type immune cells produce interleukin (IL)-4, which is essential for B-cell differentiation into IgE-producing cells, and IL-5, which is impor- tant for the activity of eosinophil lymphocytes. Intestinal permeability also is dis- turbed, allowing the absorption of antigenic macromolecules (126).
Food antigens, like caseins, enhanced the mitogen-induced proliferation of lym- phocytes of atopic children, but caseins degraded by Lactobacillus GG had a mod- erating effect (127). Caseins degraded by Lactobacillus GG also down-regulated the IL-4 production of lymphocytes compared to the control (128, 129). T-cell acti- vation was suppressed in vitro by Lactobacillus GG-degraded caseins, production of IL-2 mRNA was suppressed and the production of IL-2 protein reduced. At the same time, the levels of IL-4 and IFN-γ were reduced. The mechanism was based on the inhibition of the translocation of protein kinase C (one of the markers of cell activation) in the peripheral blood mononuclear cells of healthy children (130). Oral administration of Lactobacillus GG reduced the soluble CD4+, a marker of T-cell activation (122) and the secretion of IL-10, which is associated with the Th1/Th2 balance in a concentration-dependent manner (130). Not only the degraded caseins but also the cell-free homogenates of probiotic bacteria are shown to affect cell proliferation (131), indicating that the degradation compo- nent of bacteria may possibly play a role in the modifi cation of immune response. Since it degrades milk proteins, Lactobacillus GG may also form bioactive peptides, which may in turn have an infl uence on the digestive tract (132). Normal immune response
Figure 17. The effect of Lactobacillus GG on immune response of healthy persons, during
gastrointestinal infection and on milk-hypersensitised persons.

Milk allergy is widely believed to be exclusive to young children. However, the latest studies have shown that a clear immune response can be observed in lactose- tolerant adults who show or feel symptoms during exposure to milk (133). This manifested itself as the boosting of a non-specifi c immune response (increasing of phagocyte receptors and boosting of phagocytoses). Lactobacillus GG adminis- tered in conjunction with milk exposure reduced the infl ammation response sig- nifi cantly. In the healthy control group, milk did not cause a phagocyte response; milk with Lactobacillus GG, however, increased the non-specifi c immune response instead of lowering it (70). This refl ects the balancing effect of Lactobacillus GG with regard to immune responses. On one hand, it increases immunological defences and boosts immune responses in healthy subjects and in those with infec- tions (see chapter 2); and on the other, it reduces the hyperactive immune response in allergies (Fig. 17).
The bacteria are transferred from a mother to her child at birth. There are indi- cations that the intestinal fl ora of atopic infants differs from the fl ora of healthy infants. At three weeks of age infants who later developed an atopic disease had a lower level of intestinal bifi dobacteria than non-atopic ones (134). Lactobacillus GG has been shown to enhance the growth of bifi dobacteria in newborn babies (25) and in milk-hypersensitive adults (32).
LGG and promising 7.1 Rheumatoid arthritis In children with chronic arthritis, Lactobacillus GG has been proved to enhance the IgA class local immune response, increase the specifi c IgA response to food antigens, and normalise high urease enzyme activity in stools. High urease activity indicates an imbalance in the intestinal microfl ora. All changes were transient and related to the short-term (10 days) use of Lactobacillus GG (135, 136). These results suggest that Lactobacillus GG has the ability to strengthen the intestinal immune barrier of the mucous membrane in chronic arthritis. In a double-blind, placebo- controlled, randomised study Lactobacillus GG or placebo capsules were taken by 21 patients with rheumatoid arthritis (137). Clinical examinations were made and blood samples taken fi ve times during the one-year study. The activity of the arthri- tis was evaluated by laboratory tests, functioning ability, the number of swollen and tender joints, a physician's assessment and subjective evaluation by the patient. At the end of the study the number of swollen and tender joints tended to be reduced in the Lactobacillus GG group compared to the placebo group. The activity of the arthritis tended to decrease more in the Lactobacillus GG group compared to pla- cebo, and the patients in the Lactobacillus GG group also needed less medication for rheumatoid arthritis. Due to the limited number of patients, the results were not statistically signifi cant but the tendency towards a benefi cial effect was clear (137). 7.2 Infl ammatory bowel diseases There are several chronic intestinal diseases without known aetiology, such as Crohn's disease, ulcerative colitis and pouchitis. They are collectively called infl amma- tory bowel diseases (IBD). In addition to the genetic background and autoimmune nature of the disease, the role of intestinal microfl ora in the development of these dis- eases is also speculated (138). IBD is thought to be caused by an aggressive immune response to luminal bacteria and is characterised by a Th-1 type cytokine pattern. Crohn's disease can appear in any section of the digestive tract but is most often found in the bowel. The clinical description includes increased permeability of the intestinal mucous membrane and disturbed processing and transport of food antigens. Because Lactobacillus GG is known to restore the permeability of the mucous membrane, its effect was studied in patients with Crohn's disease. The study confi rmed that Lactobacillus GG increased local, antigen-specifi c immune response in the mucous membrane and in this way corrected the permeability disturbance of the membrane (135, 136). In a small, open-label pilot study Lacto- bacillus GG was given in enterocoated tablets to four children with mildly to moderately active Crohn's disease for six months. The results showed a signifi cant improvement in clinical activity and improved intestinal permeability (139). There is still a dearth of randomised, double-blind, placebo-controlled trials. Human in vivo administration of Lactobacillus GG led to a decrease in the ini- tially strong proliferative response of peripheral blood CD4+ T-lymphocytes towards foreign intestinal fl ora and their bacterial components (Bacteroides fragilis and E. coli). The secretion of IL-10 (Th-2 type cytokine) by peripheral blood CD4+ T-lym- phocytes increased and the level of IFN-γ and TNF-α (Th-1 type cytokines) was reduced (140, 141). These results indicate that adjunct administration of Lactobacil- lus GG might have a benefi cial effect in the treatment of IBD. Preliminary results from an open-label pilot study in the treatment of refractory "pouchitis" with capsules fi lled with Lactobacillus GG and fructooligosaccharide report a benefi cial effect as an adjunct therapy to antibiotics (142). Placebo-control- led studies are in progress.
To study experimentally the potential effect of Lactobacillus GG on colon infl ammation, this was given to rats with acetic acid-induced colitis, without signifi - cant health improvement. The need of host-specifi c lactobacilli strains to protect the colon is still an open question, since another, rat-specifi c lactobacilli strain had benefi cial effects (143). Theoretically, Lactobacillus GG might suppress infl amma- tion via the induction of nitrogen oxide (NO) production in enterocytes (103). NO is an important part of the defence system in the enter ocytes of the mucosa. Compounds that induce the epithelial cells to produce NO are known to help the epithelial cell defence systems and to suppress infl ammation. However, they have short-term effects, and if NO is induced by intestinal fl ora, the effect might be more long-term and might support the normal cell functions and defences. NO also enhances mucin formation, which characteristic has also been demonstrated to take place with Lactobacillus GG (44).
7.3 Irritable bowel syndrome Irritable bowel syndrome (IBS) is a widespread functional disorder of the diges- tive tract. Among the symptoms are bloating, abdominal pain, constipation, faecal urgency and diarrhoea. Its aetiology is unknown and therapeutic options are lim- ited. There are only a few trials, which have studied the potential benefi ts of pro- biotics in improving the symptoms caused by IBS. A pilot study was made with enterocoated Lactobacillus GG tablets (1010 cfu). The study was a randomised dou- ble-blinded placebo-controlled and crossover setting with 24 volunteers. The inter- vention was a two-week run-in with the placebo, followed by 8-wk interventions with Lactobacillus GG or placebo, a two-week wash-out, and an 8-wk cross-over, changing the products. IBS medication (used by 83%) was discontinued at the beginning of the trial. Symptoms were recorded in daily diaries and by periodic questionnaires. The effi cacy of the placebo (during the run-in period) varied from 0% (nausea) to 29% (constipation and bloating). Lactobacillus GG intake did not have any signifi cant effects on the symptoms. The study group consisted of patients with bloating as the main symptom. It was noted, however, that there tended to be a reduction in the number of unformed bowel motions with Lactobacillus GG treatment for patients with diarrhoea (144). In preliminary open-label studies the capsules fi lled with Lactobacillus GG and fructooligosaccharides relieved the gas-production in patients with IBS (145) and lactose malabsorption (146). Placebo-controlled studies are in progress.
7.4 Cystic fi brosis One interesting area of application for bacterial therapy is in the treatment of cystic fi brosis. In a preliminary report, an Italian research group (147) has shown that taking Lactobacillus GG bacteria daily for six months signifi cantly reduced the number of pulmonary infections and abdominal pains, and particularly improved weight gain in children suffering from Pseudomonas infection. Further study con- fi rmed the benefi ts for Pseudomonas-infected patients. The incidence and duration of their infections were signifi cantly reduced, pulmonary function improved and weight gain increased compared to the placebo group (148). Final reports of the results are still missing.
LGG quantity
log cfu

Buttermilk,
Capsule,
drink, 2 dl
Figure 18. Lactobacillus GG doses obtained from Gefi lus® products and Lactoba-
cillus GG levels in stools, when the products are taken daily.

We are traditionally accustomed to thinking that food is food and medicine is medi- cine with no overlap between the two. At the end of the 1980s and particularly during the 1990s interest in this ‘grey area' increased greatly. Nowadays such products are termed functional, i.e. foods that have an effect on health beyond their nutritional value. Their development has aroused wide interest and there are already hundreds of foods on the market that, in addition to nutrition, also have health-maintaining or even therapeutic effects. The effi cacy of the active ingredient used in a functional food or of a product that contains it has to be demonstrated in humans. There has to be a suffi cient quantity of the active ingredient in the food.
The quantity of Lactobacillus GG varies according to the type of product and the manufacturer. Finnish Lactobacillus GG products (Gefi lus®) have been shown to con- tain suffi cient Lactobacillus GG to colonise the bowel (16-18, 33, 77)(Fig. 18). It has been observed that milk and apparently other protective compounds in food improve the survival of Lactobacillus GG through the stomach, i.e. there is a buffering effect. Consequently the quantity of Lactobacillus GG in powder form or in capsules has to be greater ( 1010 cfu/day) than in milk-based products (108 – 109 cfu/day). The lowest dose, with which the clinical effi cacy of Lactobacillus GG in powder form has been documented, was 3x109 cfu twice a day, in the prevention and treat- ment of acute diarrhoea (81, 101). On the other hand, clinical effi cacy was achieved with dairy products, which had a corresponding quantity of Lactobacillus GG (see Table 3). In healthy children even a lower level ( 108 cfu/day) of Lactobacillus GG in milk reduced the risk of respiratory infection (77) and dental caries (79). It is a matter of individual preference whether one chooses to consume probiotic bacte- ria in everyday food or in a more pharmaceutical form. 1. Gibson GR. Dietary modulation of the human gut microfl ora using prebiotics. Br J Nutr
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cfu = colony forming units
log = logarithm
n.s. = not signifi cant
n = quantity
SD = standard deviation
P = statistical signifi cance
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