non-immunological gut

Gut, 1992,33,987-993

PROGRESS REPORT

Non-immunological defence mechanisms of the gut

S A Sarker, K Gyr

AbstractNon-immunological defence mechanismsrepresent an important line of intestinaldefence in addition to humoral and cellularimmunity. This review summarises the evi-dence for the role of the non-immunologicaldefence system. Protective factors that havebeen amply documented are gastric juice,intestinal motility, and intestinal flora.Components of pancreatic juice, lysozyme,and epithelial cell turnover may also beinvolved. Special attention is given to gastricacid, infection with Helicobacter pylon, andhypochlorhydria and their association withinfectious diarrhoea. Epidemic hypochlorhy-dria is discussed since this increases sensitivityto intestinal infections in third world countries.

TABLE I Non-immunological defencemechanisms

Gastric acidIntestinal motilityIntestinal microfloraLysozymePancreatic secretionsBile

Medical Department,Kantonsspital Liestal,University of Basel, 4410Liestal, SwitzerlandS A SarkerK GyrCorrespondence to:Professor K Gyr, MedicalDepartment, KantonsspitalLiestal, University of Basel,4410 Liestal, Switzerland.

Accepted for publication8 October 1991

It is generally accepted that humoral and cellularimmunity plays a key role in the defencemechanisms of the gut. In addition, the non-

immunological defence system (Table I) repre-sents an important and often first line of defence.It probably affords sufficient protection, at leastin people living in developed countries with goodhygiene. This could explain observations thatinfectious bacterial disorders of the gastro-intestinal tract are not more frequently observedin patients with humoral and cell mediatedimmune deficiency.' In some instances, patientstotally lacking secretory immunoglobulins main-tain a perfectly functioning gut.2The acidity of the stomach, the motility of the

intestine, the antibacterial effects of pancreaticenzymes,3'- the normal intestinal flora,lysozyme,6 and the intestinal secretions are alleffective antimicrobial factors that contribute tothe non-specific host defence system. Further-more, normal epithelial cell turnover andintestinal motility act together to purge theintestinal tract of harmful micro-organisms.Changes in the non-immunological defence

factors can lead to increased susceptibility of thehost to infections. For example, cholera andsalmonella infections are more common inachlorhydric patients,78 and slowing down theintestinal motility with belladonna alkaloidsprolongs symptomatic shigellosis.9

Gastric acidThe roles of gastric acid in health and disease are

manifold. Firstly, it facilitates digestion ofdietary nutrients and augments dietary iron andcalcium absorption. Secondly, it acts as a major

non-immunological defence factor againstexogenous pathogenic micro-organisms, andalso suppresses colonisation of the proximalbowel by the oropharyngeal and faecal flora."102The following sections will concentrate on thebactericidal activity of gastric acid, clinical con-sequences of hypochlorhydria, the effect ofgastrointestinal infections on gastric acid secre-tion, the possible effect of Helicobactor pylorion acid secretion, the gastric acid barrier inmalnutrition, and the epidemiology of hypo-chlorhydria.

BACTERICIDAL PROPERTIES OF GASTRIC JUICEThe low pH of the gastric juice (<3.0) isresponsible for its bactericidal properties.Exogenous bacteria introduced into the stomachwhen the pH is less than 3.0 are usually des-troyed within 15 minutes.'3 This bactericidalactivity is retained up to pH 4.0.There are a number of studies that show the

bactericidal activity of gastric juice in vivo. Forexample, intrayastric bacterial counts, which arenormally <10 organisms per ml,'4 rise to >105organisms per ml when acidity is reduced."Neutralisation of acid by pretreatment with 2 gsodium bicarbonate reduced the infective dose ofVibrio cholerae 10000 fold'6"' in healthy volun-teers. Infections with cholera were more likely tooccur in dogs if the bacteria were given togetherwith bicarbonate.'8 Similarly, the frequency ofenteric multiplication of the vaccine strain ofShigella flexneri increases threefold with sodiumbicarbonate neutralisation of gastric juice. 19With Campylobacter jejuni, a higher incidence ofillness was observed in a group of healthyvolunteers when the organisms were ingestedwith sodium bicarbonate.20 The use of H2receptor antagonists and proton pump inhibitors(omeprazole) leads to increased intragastricbacterial counts."52' The small bowel colonisa-tion after cimetidine therapy is also considered tobe related to reduced gastric acidity due to thedrug.22 Gastric alkalisation of critically illpatients with Mylanta II was found to be associ-ated with intragastric bacterial and fungalcolonisation.23

CLINICAL CONSEQUENCES OF HYPOCHLORHYDRIAOne possible serious consequence of achlor-hydria or hypochlorhydria was suggested by theeminent British gastroenterologist Sir ArthurHurst in 1934, when he mentioned the possi-bility that 'bacillary and amoebic dysentery

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occur much more commonly in people withachlorhydria and hypochlorhydria.' He furtherspeculated that the same might be true fortyphoid fever and cholera.24 This observation hasbeen confirmed by a series of studies showinghypochlorhydria as a predisposing factor for thegrowth of the organisms responsible for typhoidand non-typhoid salmonellosis,7 bacillarydysentery,9 giardiasis,25 and cholera826 and ofother infectious agents responsible for gastro-enteritis." Another report suggests thatClostridium difficile infection (pseudomem-branous colitis) may also be associated withhypochlorhydria.27

It has also been found that reduction in acidsecretion in atrophic gastritis allows bacterialcolonisation of the stomach, which is mostextreme in achlorhydria patients with perniciousanaemia.'1 28 The bacterial flora in the stomach ofpatients with pernicious anaemia consist ofenterobacteriaceae, enterococci, streptococci,staphylococci, lactobacilli, and less frequentlyobligatory anaerobes, such as clostridia andbacteroides.29 An association of hypochlorhydriaafter a course of omeprazole therapy and recur-rent enteritis has recently been reported.30Gastric resection and/or vagotomy frequentlyleads to the colonisation of the stomach andsmall intestine by faecal types of bacteria.3' 32During epidemics ofcholera in Israel33 and Italy,34the attack rates were significantly higher insubjects with hypochlorhydria due to surgicalresection of the stomach and chronic ingestion ofantacids.

It is widely, though not unanimously,assumed that there is an increased risk of gastriccancer and premalignant changes in perniciousanaemia 536 and after partial gastrectomy.37 38The increased risk of cancer is probably relatedto overgrowth of nitrate reducing bacteriaproducing nitrites and N-nitrosamine com-pounds,3' 39 which have already been shown to becarcinogenic. A number of recent studies alsodemonstrated that reduction in gastric acid out-put caused by different types of drugs, includingH2 receptor antagonists, usually results insignificant bacterial overgrowth and intragastricbiochemical changes, including the productionof nitrite and nitrosamine compounds. '

EFFECT OF GASTROINTESTINAL INFECTIONS ONGASTRIC ACID SECRETIONIt is known that bacterial, viral, and parasiticinfections suppress gastric acid production inman41-- and in animals.45 ' In fact, bacterialinfections, including salmonellosis, tuberculo-sis, and bronchopneumonia, are associated withsuppression of histamine stimulated acid secre-tion.4'42 Normochlorhydria was restored uponeradication of infection in most of the cases.Recently, H pylorn has been associated withhypochlorhydria and gastritis.4748 This organismwill be discussed in the next section in greaterdetail.

There is little information on the effect ofparasitic infections on gastric acid output. InBrazil, a decreased basal and stimulated acidoutput has been observed in patients withChagas disease.49 Decreased basal and stimulated

acid output was also noted in patients withschistosoma50 or giardia infections.5' Impairedgastric acid secretion was seen in people infectedwith ancylostoma4349 or Diphyllobothriumlatum.52The mechanism of the suppression of acid

secretion in infection is not well understood. Ithas frequently been thought to be related todirect morphological effects of the infection onthe gastric mucosa.4' ` 5' On the other hand,suppression of acid secretion was found to bemore marked when there was fever," whichindicates that fever rather than infection per semay be responsible for suppression. A transienthypochlorhydria has been shown in people keptin a heated cabinet with a rise of body tempera-ture from 370 to 39°C. The hypochlorhydriaobserved in infection might be modulatedthrough the stress induced release of endogenousprostaglandins (PGE2), which has been found tosuppress acid secretion.6

H PYLORI AND GASTRIC ACID SECRETIONThere is increasing interest in the role ofH pylonin the pathogenesis of peptic ulcer andgastritis.57-59 H pylori is found in 90-100% ofpatients with duodenal ulcers, 70% with gastriculcers,'"6' and 6-31% of children complaining ofvomiting and upper abdominal pain.62 The roleofH pylon in these disorders is indicated by theobservation that its eradication in ulcer diseasereduces the chance of subsequent relapse.6364Hunt et al recently reported the effect ofH pylonon acid production, using 14C-aminopyrinuptake by isolated guinea pig parietal cells.H pylori caused a reduction in basal acid secre-tion ofabout 80%, and histamine stimulated acidsecretion was reduced to 50% within 15 minutesof inoculation with H pylori.6' There are alsoreports describing H pylori associated gastrichypochlorhydria in man.47461 Although a strik-ing association between the presence ofH pyloriand hypochlorhydria exists, the question ofcause and effect is still obscure. In 1983,Marshall, serving as his own volunteer, renderedhimself temporarily hypochlorhydric withcimetidine and then swallowed cells ofH pylon'cultured from a patient with gastritis. Althoughgastritis and H pylori were detected in his followup tissue specimens,6768 a definite association ofthe organism with hypochlorhydria could not beestablished, as gastric acid secretion before andafter challenge was not monitored in that experi-ment. The possible association ofH pylon' withthe high prevalence of hypochlorhydria in poorcountries is further alluded to in 'Epidemiology.'Because of poor environmental conditions indeveloping countries, people are very oftenexposed to enteric pathogens such as V cholerae,Shigella, and Escherichia coli. Low gastric acidproduction has been found to be associated witha high risk of cholera and E coli diarrhoea inthose communities. 69 It is not unlikely thatH pylori associated hypochlorhydria leads toincreased susceptibility to enteric pathogens. Alongitudinal prospective study is thus warrantedto investigate the association ofH pylori with theincidence of hypochlorhydria and of infectiousdiarrhoea.

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Non-immunological defence mechanisms ofthe gut

GASTRIC ACID BARRIER IN MALNUTRITIONThe gastric acid barrier was found to be impairedin malnourished children and in protein depletedexperimental animals."272 The effect of malnutri-tion on gastric acidity and gastric bacterialcolonisation was studied in 35 severely mal-nourished Bangladeshi children by Gilmanet al.73 Gastric acid output, both basal and afterbetazole stimulation, was found to be signifi-cantly lower in malnourished children than inthe better nourished counterparts. Hypochlor-hydria was accompanied with Gram negativebacterial colonisation in 81% of the mal-nourished children, whereas none of 20 betternourished children had Gram negative colonisa-tion of their gastric juice. After three weeks ofnutritional rehabilitation, gastric acid secretionstill remained depressed in the malnourishedchildren. The factors responsible for the de-creased acid output in those children remainunclear. Although anaemia has been postulatedto be associated with reduced gastric acid out-put,74 the study in Bangladeshi children did notshow any correlation between acid output andhaematocrit. However, the low gastric acid out-put may be related to chronic gastritis. In otherstudies, evidence for this was found in biopsyspecimens from the gastric fundus of mal-nourished children.7072 No data are available onparietal cell numbers and their changes uponrehabilitation in malnourished children. Theprolonged depression in acid concentrationobserved in the study with Bangladeshi childrensuggests that nutritional support for a periodlonger than three weeks is required to ensure fullrecovery of acid secretion.

EPIDEMIOLOGY OF HYPOCHLORHYDRIAThe prevalence of hypochlorhydria varieswidely, but is high in third world countries. Areduced acid output has been shown in mostmalnourished children in Indonesia.70 Nigeria,75South Africa,72 and Bangladesh.73 Relative hypo-chlorhydria has been demonstrated to occur inthe first week of life and in people over the age of60.76 This might explain the increased incidenceof small bowel colonisation in infants77 and theincreased risk for salmonella infection in theelderly.7"79

Nalin et al8 reported hypochlorhydria in 41%of Bangladeshi patients convalescing fromcholera. A high gastric pH (>4 0) has also beenobserved in people of countries in Africa, Southand Central America, and possibly southern andeastern Europe.'3 In contrast, an acidic andsterile stomach is normal in the United Statesand north western Europe. These observationsmay suggest nutritional factors, environmentalcontamination, and a possible association withan infectious agent as the cause of hypochlor-hydria in third world countries. Several reportshave described spontaneous outbreaks of hypo-chlorhydria, sometimes resulting in completeanacidity, in previously healthy people withnormal gastric acid secretion808' or patients withZollinger-Ellison syndrome.8283 Two reportspreviously described epidemic outbreaks ofhypochlorhydria associated with gastritis, whererecent evidence suggests an association with

H pylori. Ramsey et al84 reported an outbreak ofhypochlorhydria in 17/35 volunteers participat-ing in a study of food stimulated gastric acidsecretion. Gledhill et aP5 observed four cases ofhypochlorhydria in five previously healthyvolunteers participating in a study involvingintragastric pH measurements. The clustering ofcases in both the studies suggests a possibleinfective cause. Both of the studies were con-ducted before 1983, when MarshallI6 andWarren87 had not yet described the curvedbacillusHpylori (previously called C pyloridis). Aretrospective examination of the gastric mucosalbiopsies obtained from the volunteers participat-ing in the study conducted by Gledhill et al,however, did detect H pylori. A rising antibodytitre against H pylori was also demonstrated inthe convalescent gastritis sera obtained from thesubjects participating in the investigation byRamsey and his group.16 Both observationssuggest the association of H pylorn with hypo-chlorhydria.

Intestinal factors in the defence mechanismsof the gut

EPITHELIAL CELL TURNOVERThe mammalian intestine is covered by auniversal epithelium that can either absorb orsecrete. An increase in the rate of epithelialsurface renewal may occur as a result of bacterial,viral, or parasitic infections. The luminal loss ofepithelial cells, connective tissue stroma, andlymphocytes with exuded fluid tends to dilute,dissolve, or wash away the various infectiveagents adhering to the mucosa and between thecells. This loss of intestinal surface cells actstogether with interdigestive motor activity(MMC) to cleanse the intestine and thus preventstasis and bacterial colonisation.

INTESTINAL MOTILITYOne important host mechanism limiting theproliferation of organisms in the small intestineis gut motility. In humans, the well definedphase III activity of the interdigestive motorcomplex (MMC) occurs every 84-112 minutesand migrates down the upper intestinal tractwith a speed of about 6-8 cm/minute. Thecaudate moving band of intense contractionduring phase III has been called the 'intestinalhousekeeper.8889 The contraction movesdigestive contents rapidly down the smallintestine. At the port of entry, gastric aciddestroys most of the organisms from the outsideenvironment. In the upper intestine this'housekeeper' mechanism gets rid of micro-organisms that managed to escape the gastricacid barrier, and thus prevents stagnation andbacterial overgrowth. The other important roleof this movement is to maintain an appropriatedistribution of the endogenous enteric micro-flora and to prevent migration oforganisms fromthe colon.88`99'

Altered intestinal motility might have adverseeffects on the normal ecology of the intestine. Inconditions associated with increased motility, areduction of the normal gut flora could occur. A

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considerable reduction of the anaerobic bacterialflora has indeed been observed in some cholerapatients.92 This reduction, however, is likely toreflect not only a specific effect on gut motilitybut much more the high flow rate of the gutcontents in this disease condition. On the otherhand, decreased motility might lead to stasis andcreate an ideal culture medium for the develop-ment of bacterial overgrowth in the smallintestine.' The presence of such overgrowth indiabetic patients93 could in fact be related todelayed intestinal motility.94On the other hand, motility disturbances have

been reported in the small bowel overgrowthsyndrome'"9596 which may be the result ratherthan the cause ofincreased bacterial growth. Themechanism is still not clear. However, humoralfactors may play a role.No thorough studies have yet been performed

on the effect of enteric infection on intestinalmotility in man. Studies in animal models haveprovided some information, for example thatbacterial enterotoxins may produce abnormalpatterns of smooth muscle motor activity. Inrabbits, cholera toxin has been found to producean abnormal electrical pattern called the migrat-ing action potential complex (MAPC), whichconsists of bursts of intense activity.97 Experi-ments on invasive bacteria in the same animalmodel showed a different pattern of electricalactivity characterised by repetitive bursts ofaction potentials.98 The authors suggested thatMAPC is characteristic of toxigenic bacteria, andrepetitive bursts of invasive bacteria. The find-ing suggests that the disturbed motility observedin infectious diarrhoea is an important factor in

its pathogenesis.

PANCREATIC JUICE AND BILE

Pancreatic exocrine insufficiency has beendocumented in protein calorie malnutrition inanimals' "'1 and in man.` Patients with pan-creatic insufficiency were reported to have more

severe and prolonged episodes of acute diar-rhoeal illness. 102 103

The basis of the possible anti-infective roleof the exocrine pancreas has been elucidatedin a series of studies in animals by Gyr andFelsenfeld.315 In protein depleted animals withexocrine pancreatic insufficiency, a challengewith V cholerae produced a longer lasting diar-rhoea and more prolonged excretion of theorganism than in pair-fed controls. 0" 105 Oraladministration of pancreatic extract to theprotein-depleted animals was found to modifythe course of the diarrhoea by reducing theseverity and duration.4 These studies thussuggest an involvement of exocrine pancreaticsecretion in the local defence against choleraunder conditions of protein deficiency.

In vitro experiments by the same groupshowed a bactericidal activity of pancreaticlipase, which damaged the cell wall and thecytoplasm of V cholerae.3 Earlier, Dutta and Ozashowed that extracts of the intestinal fluid ofadult rabbits degraded cholera toxins.'06 Anti-bacterial activity of the canine pancreatic fluidagainst E Coli, Shigella spp, Salmonella spp, andKlebsiella pneumoniae has been observed by

Rubinstein et all07 (Table II). Human duodeno-pancreatic secretions were also found to contain afactor that enhances the bactericidal activity ofclioquinol and certain other drugs, includingchloroquinodole, chloramphenicol, and trime-thoprim.'08 The observation has recently beenconfirmed by Bassi et al, who also demonstratedbacteriostatic properties of pure pancreatic juice(PPJ).109 In his study the minimum inhibitoryconcentration (MIC) of the antibiotic mezlocillinagainst E coli was found to be four times lesswhen PPJ was added, suggesting the ability ofPPJ to enhance the activity of this antibiotic. Thefactors or substances present in PPJ associatedwith the antibacterial properties are not yet fullyelucidated. Mett's investigations'08 suggest a heatstable substance with a molecular weight of lessthan 8000 Da.The association of exocrine pancreatic

insufficiency'0' and bacterial overgrowth"0 in theproximal bowel has been observed in childrenwith protein energy malnutrition. The effect oforal pancreatic enzymes on the intestinal florahas also been studied by Gyr et al in proteindeficient monkeys.5 The study demonstratedthat pancreatic extracts hastened the restorationof the normal intestinal flora in protein depletedmonkeys to their pre-dietary state. The mecha-nism of the restoration of intestinal ecology bythe pancreatic extract is still unknown.The biological effects of bile salts on infec-

tion have not been thoroughly investigated.Although in vitro studies demonstrated thatdeconjugated bile acids exert a potential inhibi-tory effect on micro-organisms,"' "2 in vivostudies in support of this observation are lacking.IgA in bile is considered to be involved ineliminating harmful antigens from the circula-tion by the disposal of IgA antigen complexesinto the gut lumen."'"" But further work isneeded to elucidate the role of bile salts in localdefence against enteric pathogens.

LYSOZYMELysozyme is an enzyme which has been identi-fied in the digestive system in the ductal cells of

TABLE II In vitro activity ofcanine pancreaticfluid againstvarious human faecal and urinary isolates*"'

Mean no ofbacteria, loglodml atdifferent incubation times:**

Oh 2h 3h 6h 24h

Escherichia coli (24 strains) PF 4-1 3.5 0 0 0C 4.0 4-8 7-2 8-6 9.2

Shigella sp PF 4-2 3-4 2-6 1.1C 4-1 4-3 3-4 2-2

Salmonella typhimurium PF 4-1 4-1 3-9 0(6strains) C 4-1 4-7 7.5 9-0

Klebsiella pneumoniae PF 4-1 4-1 3-9 0(12 strains) C 4-1 6-0 8-2 9-1

Pseudomonas aeruginosa PF 4-1 4-2 4-2 4-3(16strains) C_ 4.0 5 7 8.1 8-9

Staphylococcusfaecalis PF 3-1 5.7 6-3 7-1(9strains) C 4-1 5.2 7-4 8-2

Staphylococcus, coagulase+ PF 4-1 3-7 3-6 3-2(12 strains) C 4-2 5 3 6.5 9 4

Staphylococcus, coagulase- PF 3.5 3-2 3-7 4-3(9 strains) C 3-4 3-6 4-8 8-2

Bacterioidesfragilis PF 4-2 4-7 7-3 9.7(2strains) C 4-1 4.5 7.3 9.3

SD in all instances c6.4* 102 and therefore not included in table.PF= pancreatic fluid; C=control.*Adapted from Rubinstein et al 1985.**Difference ofmore than 2 log unit is considered significant.

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Non-immunological defence mechanisms ofthe gut 991

salivary glands, in the absorbing epithelium ofthe intestinal tract, and in pancreatic fluid."5The enzyme has been reported to increase inactivity in a variety of infections. It is well knownto lyse bacteria,"6 and might therefore play a rolein mucosal defence against invading organisms.However, further work is required to investigateits mechanism of action and its influenceon specific organisms responsible for gastro-intestinal infection.

INTESTINAL FLORA

LuminalfloraThe intestinal flora is an enormously complexecosystem and consists of both aerobic andanaerobic micro-organisms. Bacteriologicalstudies indicate that the small intestinal flora isrelatively sparse, containing less than 105 cfu perml, while in the colon an individual harboursmore than 400 species. The majority of them areanaerobic - that is, they do not grow in thepresence of oxygen. Motility, immunologicalfactors, and intestinal secretions are all con-sidered to play a role in maintaining the normalgut microflora. An augmentation ofthe intestinalmicroflora was observed in vervet monkeys afterpancreatic. duct ligation."7 On the other hand, asdiscussed above, pancreatic extracts helped torestore the normal flora in protein-deficientmonkeys.4The role of the normal intestinal flora as an

extremely important host defence mechanism isnow beginning to be appreciated. In normalsituations, an individual's intestinal flora ishighly effective in resisting colonisation bypotentially pathogenic invaders. The indigenousflora produces a variety of antimicrobial sub-stances, including colicins"8 and short chainfatty acids,"9 which are potentially bactericidaland bacteriostatic and are therefore consideredto inhibit the growth of invading organisms.

There is considerable evidence for anincreased susceptibility to infection in patientswith reduced bacterial flora.'20'2' Diarrhoeaassociated with the use of antibiotics is common,and is likely to be due to alterations of the normalindigenous flora. In experimental mice, theinfective dose of Salmonella typhimurium wasfound to be reduced 10 000 fold when themicroflora was eradicated by administration ofstreptomycin. 22 The protective role of theintestinal flora in humans is also suggested bythe increased frequency of salmonella infectionsamong Swedish tourists who used prophylacticantibiotics compared with those who took noprophylaxis. 2'The endogenous gut flora also play an impor-

tant role in maintaining the histological structureofthe gut. In fact, it has been demonstrated in thegerm-free animal that the 'normal' histology ofthe gut mucosa is determined by the presence ofthe bacterial flora.'23 Without the resident florathe intestinal wall has been found to be thinnerand to contain only few lymphocytes.

FLORA IN THE EPITHELIUMBesides the organisms distributed along the

intestinal lumen, a unique population of bacteriaexists in the epithelium, consisting of organismsquite different from those colonising the lumen.Electron microscopic studies show that theyare firmly adherent to the mucus layer overlyingthe microvilli of the crypts of the distal smallbowel.'24 125 This flora includes mainlyLactobacillus, Bacteroides and Clostridium spp,but present knowledge is incomplete. The role ofthe epithelial flora in defense is also not yet clear,but it seems possible that it interferes with thecolonisation and multiplication of pathogenicbacteria by competing for nutrients.

ConclusionThis review has summarised evidence for theimportance of the non-immunological defencesystem of the gut and discussed the variousmechanisms that may be involved. The import-ant defence factors whose efficacy has beenamply documented are gastric acidity, intestinalmotility, and the intestinal flora. Components ofpancreatic juice, lysozyme, and epithelial cellturnover are also likely to be involved, butdefinite proof is still lacking. Special attentionhas been paid to gastric acid as a defence factor,the occurrence of hypochlorhydria, and its pos-sible association with H pylon. Epidemic hypo-chlorhydria may be one of the factors thatincreases sensitivity to infectious diarrhoea inthird world countries. Further investigations arenecessary to elucidate the efficacy and relativeimportance of the non-immunological defencesystem.SAS is Fellow from the International Center for DiarrhoealDisease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh.KG is sponsored by the Swiss Development Corporation, Bern,Switzerland.

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