MINIREVIEW

Intestinal Pathophysiology in Autism

JOHN F. WHITE 1
Department of Physiology, Emory University, Atlanta, Georgia 30322

Autism Is a life-long developmental disorder affecting as many in about one-third of autistic children (4), is referred to in
as 1 In 500 children. The causes for this profound disorder are different terms, including "regressive autism" (5).
largely unknown. Recent research has uncovered pathology In
the gastrointestinal tract of autistic children. The pathology,
It is generally agreed that there are multiple causes for
reported to extend from the esophagus to the colon, Is de- autism. Some of the causes of autism are well established,
scribed here along with other studies pointing to a connection including a strong genetic link in the cases, of tuberous
between diet and the severity of symptoms expressed In au- sclerosis, fragile X, and some other disorders (1). The view
tism. The evidence that there Is Impaired Intestinal permeability that there is a strong genetic basis for the disorder stems
In autism Is reviewed, and various theories are discussed by
which a leaky gut could develop. Lastly, some possible ways In from the observation that siblings of autistic offspring have
which Impaired gastrointestinal function might Influence brain a higher incidence of autism than the general population. A
function are discussed. Exp Bioi Mad 228:639-649, 2Q03 strong genetic link has been particularly inferred from stud-
Key words: autism; intestine; casein; celiac disease; endoscopy; ies of autistic children born as twins. Thus, monozygotic
gluten; glycosaminoglycan; immunoglobulin; secretin; vaccine (identical) twins demonstrate a concordance for autism ex-
ceeding 90% (6). The concordance of less than 100% has
been interpreted as evidence that some other factor such as

A
utism is the most prevalent of a subset of disorders an environmental challenge must be congruent with the ge-
organized under the umbrella of pervasive develop- netic susceptibility before the disorder is expressed (7). Re-
mental disorder (PDD). Autism is a serious devel- cently, the apparently large strength of the genetic influence
opmental disorder characterized by profound deficits in lan- in autism was called into question when a database of in-
guage, communication, and socialization, resistance to formation on a large cohort of families having at least two
learning, and displays of stereotypical behavior including siblings with autism was evaluated. A remarkably high pro-
perseveration. The disorder is accompanied by mental re- portion of twin pairs was observed. For example, there was
tardation in three out of four patients (1). Boys are three a 10-fold greater frequency of monozygotic. twins than the
times more likely than girls to receive the diagnosis. One highest population frequencies that could be expected. It
out of three autistic individuals experience epileptic seizures was concluded that twins have an increased risk of autism.
(2). The diagnosis of autism is usually conferred when the Accordingly, Greenberg et at. (8) suggested that earlier es-
child is 2 to 3 years old after extensive evaluation according timates of the role of genes in autism, predicated in part on
to the criteria of the Diagnostic Statistical Manual IV the twin data, may have been overstated. i.e., it was not
(DSM-IV) (3). The children either exhibit a failure to ad- appreciated that the incidence is higher in twins. Further-
vance from birth or, after a period of apparently normal more, it was concluded that the influential genes may reside
growth, suffer a loss of newly acquired skills (language, in the parents genome rather than that of the autistic off-
eye-to-eye contact, and sociability). The latter pattern, seen spring (8). For example, environmental conditions in the
womb such as competition for nutrients may be the greater
influence.
Notwithstanding these interesting new findings, the
J To whom requests for reprints should be addressed at Department of Physiology, cause of the disorder for the great majority of autistic indi-
Emory University, Atlanta, Georgia 30322. E-mail: jfwhite@physio.emory.edu
viduals has not been determined. The term "autistic syn-
1535-3702/03/2286-0639$15.00
drome" is intended to describe a pattern of similar behaviors
Copyright 2003 by the Society for Experimental Biology and Medicine produced by a variety of different insults. The need to un-
derstand the causes of autism and the underlying patho-

INTESTINAL PATHOPHYSIOLOGY IN AUTISM 639

physiology has become more acute since the number of cephalography (EEG) revealed no neurological abnormali-
diagnosed cases has risen markedly in recent years (9-1 I). ties in the children.
Gillberg and Wing (12) reviewed studies originating from In a more recent publication, these same researchers
several different countries in the period from 1966 to 1997 reported their observations on an expanded group of 60
and reported that 18 studies of non-U.S. populations showed children affected with various developmental disorders
a highly significant (P < 0.001) increase in prevalence of (15). The subject group included 50 children diagnosed with
autism of 3.8% per year. In a more recent review, Wing and autism (including the 12 children from the original study) as
Potter (13) offered evidence that, historically, autism has weIl as five with Asperger's syndrome (autism without re-
been underdiagnosed. They attributed most of the rise in the tardation) and two with disintegrative disorder. All but one
incidence of autism to changes in diagnostic criteria and of the 60 children had GI symptoms, including abdominal
increasing awareness of autism spectrum disorders. How- pain, constipation, diarrhea, and bloating. Findings were
ever, they acknowledged the possibility "that there is a real compared with those from a group of 37 developmentally
and continuing rise" in the number of cases. Unfortunately, normal (nonautistic) children with similar GI symptoms (the
the number of weIl-controIled clinical studies of the autistic control group). It was observed that ileal LNH presented in
population that have provided clues to the etiology of the 93% of affected children and 14.3% of control children.
disorder are very limited. Possibly, a breakthrough will re- Colonic LNH was present in 30% of affected children and
sult after publication of several studies reporting new ob- 5.4% of control children. Hyperplasia of the intestinal
servations of significant gastrointestinal pathology in autism lymph nodes was found in 88.5% of biopsies of affected
or in attention deficit hyperactivity disorder (14-17). In this children. Active inflammation of the ileum (ileitis) was ob-
review, the new observations, particularly those on the in- served in 8% and chronic inflammation of the colon (colitis)
testinal tract, are described, as well as earlier studies indi- was seen in 88% of affected children. The authors charac-
cating a link between gastrointestinal function and autism. terized the pathology as "a subtle new variant of inflamma-
Possible scenarios that could produce the impaired intestinal tory bowel disease that lacks the specific diagnostic features
function are discussed, and some possible ways in which of either Crohn's disease or ulcerative colitis," In a com-
altered gut function could influence behavior are consid- ment on the Wakefield paper, Sabra et al. (16) reported
ered. Recent efforts to identify potential susceptibility genes identical pathology (LNH) in the terminal ileum of two
for autism are not discussed in this review. The hope of the patients diagnosed with food aIlergies and attention deficit
author is that this review will serve to spur interest in the hyperactivity disorder.
study of gastrointestinal function as it relates to autism. The In yet another study, Horvath and coworkers (17) used
ultimate goals are to understand the etiology of autism and endoscopy with biopsy to examine the upper GI tract of 36
to provide clinical remedies for this severely handicapping children diagnosed with autism and experiencing abdominal
disorder. pain, chronic diarrhea, bloating, nighttime awakening, or
unexplained irritability, Abnormal findings included reflux
Children with Autism Have Gastrointestinal esophagitis in 25 of the children, chronic gastritis in IS, and
(GI) Pathology chronic duodenitis in 24. Low activity of intestinal carbo-
hydrate digestive enzymes was observed in 21 children,
In a study published in 1998, 12 children diagnosed whereas 27 exhibited increased exocrine secretion of pan-
with autism (all of the regressive pattern) and exhibiting a creatic-biliary fluid after intravenous administration of the
variety of GI ailments, including abdominal pain, diarrhea, GI hormone secretin. Secretin, a peptide hormone released
and bloating, were examined extensively (14). The GI by endocrine cells within the duodenal mucosa, promotes
symptoms had developed coincident with the onset of au- sodium bicarbonate and water secretion by the pancreas. It
tistic behavior, according to the parents. Endoscopy re- is important to note that this study describes altered function
vealed that 10 of the 12 children displayed ileal lymphoid in the upper GI tract of autistic children, whereas the lym-
!!,odular hyperplasia (LNH), Lymph nodules are encapsu- phoid nodular hyperplasia described by Wakefield et al.
lated bodies lying within the submucosa of the intestinal (14, 15) was observed in the lowest portion of the small
wall. Lymph nodules contain lymphocytes and neutrophils. intestine, namely the ileum. The results of these different
The fluid absorbed from the intestinal lumen by the action studies taken together suggest that significant and wide-
of the absorptive epithelial cells is filtered through the spread GI pathophysiology may accompany autism, at least
lymph nodes. Here, antibodies are formed. Of the 12 chil- within a subpopulation of patients. As discussed below, the
dren, eight also displayed abnormalities in the mucosa, the pathology may be central to the etiology of autism. Alter-
region consisting of the absorptive epithelium, underlying natively, it may simply be a secondary consequence of the
connective tissue, and muscularis mucosae. Mucosal abnor- disorder. In either case, it is possible that such widespread
malities included granularity, loss of vascular pattern, and pathology plays a major role in the symptomatology of the
patchy erythema (nonspecific colitis). The findings were disorder in the affected children. It is crucial that these
supported by histological examinations of mucosal biopsies. endoscopic analyses be conducted in other laboratories. In
Cerebral magnetic resonance imaging (MRI) and electroen- the meantime, these limited reports constitute a potentially
640 INTESTINAL PATHOPHYSIOLOGY IN AUTISM
important new avenue of research in the effort to understand ated with reactivity of antigliadin antibodies with their cer-
autism, its causes, and symptomatology. ebellar Purkinje cells (22). The incidence of ataxia in celiac
In contrast with the findings above, a very recent study patients is 6% to 10%. Ataxia is not a diagnostic indicator
in the United Kingdom, which examined the early medical of autism. CNS effects of gluten (or gluten metabolites) in
records of 66 children who were later diagnosed with autism neurological disorders may be quite common. Thus, a recent
and 30 children later diagnosed with "possible autism," re- text chapter on autism listed several reports that associated
ported that they did not indicate the presence of 01 inflam- celiac disease with "an extraordinary range of psychiatric
mation, celiac disease, food intolerance, or recurrent 01 and neuropathologic conditions" (23).
symptoms at a higher rate than were reported in the early In two separate studies involving a large number of
medical records of 449 (control) children who did not sub- autistic patients, it was noted that an improvement of social,
sequently develop autism (18). The authors concluded that cognitive, and communication skills occurred when they
there was not a substantial association between GI illness in were placed on a diet free of gluten and cow's milk or a diet
children and the development of autism. However, the au- free of cow's milk alone. In the first study, several objective
thors acknowledged that some children may have had sub- measures of behavior were reported to improve when au-
clinical 01 symptoms that were overlooked and, further- tistic children were placed on an elimination diet free of
more, that severe 01 disease may be associated with autism cow's milk (24). Significant symptom improvement was
in certain individuals. exhibited by 36 autistic patients in five of seven objective
behavior scales 8 weeks after placement on a cow's milk
Earlier Studies Point to a Linkage Between Gut elimination diet. In another study, 15 subjects with autistic
Function and Autism syndromes were placed on a diet for a period of 4 years that
There have long been indications that autistic children was reportedly free of gluten and casein (25). Casein is the
had impaired 01 tract function. Anecdotal reports by parents major protein of milk. Previously, all of the children exhib-
extending back more than 30 years described evidence that ited pathological urine patterns and increased levels of pep-
their autistic children suffered disturbed GI tract function tides in their urine. Differences in urinary peptides levels in
and intolerance to certain foods (19). In a recent survey of autistic patients had been reported by others in earlier stud-
500 parents of autistic children, almost one-half reported ies (26, 27). The same research group subsequently reported
that their children had loose stools or frequent diarrhea (20). that some of the urinary peptides derived from gluten and
Food intolerance was noted particularly for wheat and casein (28). After 1 year on the diet, a statistically signifi-
cow's milk. In one early study, an autistic child was coin- cant improvement in social, cognitive, and communication
cidentally afflicted with celiac disease, a disorder charac- skills was observed. Also, urine patterns and urine peptide
terized by marked atrophy of the intestinal villi caused by a levels normalized. Further significant improvements in be-
response of the intestinal immune system to gliadin, a pep- havior were observed after 4 years on the diet. Unfortu-
tide in gluten (19). The villi, which are projections of the nately, neither study controlled for the possibility that the
intestinal mucosa into the lumen of the intestine, become improvements were dependent upon parallel instructional
shortened and denuded of surface epithelium by dietary glu- and other behavioral interventions. Also, objective mea-
ten in affected individuals. Gluten is a protein found in sures of the extent to which the diet was free of gluten and
wheat and barley (21); celiac disease is treated by eliminat- casein were not reported. Nevertheless, these structured
ing gluten from the diet. When the autistic child was reex- studies as well as anecdotal reports by parents of perceived
posed to gluten in the diet after a period on a gluten-free behavioral improvements after dietary restrictions have
diet, his autistic symptoms were observed to worsen. The stimulated research to determine whether the intestinal mu-
observation inspired a study in which transcephalic direct cosal barrier was incompetent in autistic individuals, allow-
current (TDC) potentials were measured in several autistic ing selected foods or their metabolic products, impermeant
children with histories of episodes of colic, diarrhea, dehy- in the normal human gut, to gain access to the interstitial
dration, and food intolerance but lacking the diagnosis of fluid and either initiate immune reactions or produce pa-
celiac disease (19). TOC potentials are slowly changing thology.
voltages recorded from the surface of the head and origi- The small intestinal mucosa normally acts as a barrier
nating from the cortex of the brain. When the children were to prohibit many substances within the intestinal lumen
exposed to an oral dose (I g) of gliadin, frontal voltage was from entering the blood (29). The luminal membrane of the
significantly inhibited compared with that produced by I g simple columnar epithelial cells, which line the mucosal
of sugar. Normal children and normal siblings of autistic surface (primarily enterocytes and goblet cells), as well as
children did not show this response. This report suggests the tight junctional complexes linking the epithelial cells to
that gliadin or its metabolites gains access to and has a their neighbors are the principal barriers to the free move-
direct effect upon the central nervous system (CNS) in these ment into the blood of dietary foods and the products of
children. A CNS effect of gluten is also suggested by a their hydrolysis released during intraluminal digestion.
recent report that in (nonautistic) celiac patients, dietary These architectural features embody the "intestinal luminal
gluten produces ataxia (lack of coordination) and is associ- barrier." A functional method of assessing the physical in-

INTESTINAL PATHOPHYSIOLOGY IN AUTISM 641

 tegrity of the luminal barrier is to measure the ability of (lg's). Of the five classes of antibodies (lgA, IgM, IgG, IgD,
small sugar molecules, orally administered, to gain entry and IgE), IgA antibodies are secreted by the digestive tract.
into the blood and, eventually, to be excreted into the urine Whether intact dietary protein molecules or their derivative
(30). The sugar permeability test involves the simultaneous peptides penetrate the mucosal barrier through the M cells
oral administration of two sugars, usually mannitol and or through and between the enterocytes, their interaction
lactulose, followed by the measurement of the ratio of man- with the immune system is indicated by the fact that, in two
nitol:lactulose recovered in the urine. Mannitol is a mono- reports, autistic patients reportedly exhibited significantly
saccharide that is relatively poorly absorbed by the human higher levels of IgA for dietary casein (24, 36), gluten (36),
intestine because it has no affinity for the glucose-galactose lactalbumin (24), and I)-lactoglobulin (24). IgG was el-
carrier protein molecules that reside in the apical (luminal) evated for casein (24) and gluten (36); IgM was increased
brush border membrane of small intestinal enterocytes. for casein (24). Removal of the dietary challenge reduced
Mannitol molecules pass through the luminal membrane by the immune reaction. This response of the immune system
way of aqueous pores in the brush border membrane (31). provides additional evidence that the autistic intestine is
Lactulose, a larger dissacharide molecule, also lacks affinity abnormally permeable to gluten and casein.
for the carrier and is too large to pass through the pores.
Lactulose molecules, which do reach the blood, de-so by The Leaky Gut Hypothesis
passing between the epithelial cells (i.e., the paracellular The idea that the integrity of the intestinal mucosal
pathway) and through zones of cell extrusion at the tip of lining, referred to as the intestinal mucosal barrier, is com-
the villus. The paracellular pathway is also the presumed promised in autism is embodied in the "leaky gut hypoth-
route of passage that peptides such as gliadin take through esis." According to this hypothesis, the intestinal mucosa is
a damaged intestinal mucosa. Using the sugar permeability abnormally permeable in autism. Digestion products of
test, abnormal intestinal permeability has been found in pa- natural foods such as cow's milk and bread are able to enter
tients with recognized intestinal disorders (32, 33). More the blood through the leaky mucosa and induce antigenic
recently, D'Eufemia et al. (34) applied the test to patients responses, as well as interfere directly with the central ner-
with autism. They reported that intestinal permeability was vous system. It has been established that digestion of dietary
significantly increased (i.e., greater permeability to lactu- gluten and casein in the lumen of the small intestine by the
lose relative to mannitol) in nine of 21 high-functioning action of pancreatic and intestinal peptidases releases short
autistic patients (4-16 years of age) when compared with a chain peptides, which are structurally similar to endorphins.
group of 40 healthy, age-matched children (34). The change These products are called exorphins to reflect their dietary
in the urinary mannitol:lactulose ratio in 43% of the autistic origin (37). Gliadomorphins are a family of exorphins re-
children was accounted for solely by an increase in the leased from the partial digestion of the wheat protein glia-
lactulose permeability; there was no difference in mannitol din. Similarly, casomorphins are a family of exorphins re-
recovery between the groups. The authors proposed that the leased upon partial digestion of the milk protein casein (38).
elevated lactulose permeability reflects damage to the tight Casomorphins and gliadomorphins are potent psychosis-
junctions linking the intestinal epithelial cells in the affected inducing factors (39). They are also very stable epitopes.
autistic patients. These results, in conjunction with those Noting that schizophrenic patients had difficulty with di-
implicating dietary gluten and casein causally in autistic etary gluten, Dohan (40) hypothesized that there is a defect
symptoms, are integrated into the view that products result- in the intestinal barrier of these patients that allows passage
ing from the incomplete hydrolysis of dietary gluten and of neuroactive peptides of food origin into the blood and
casein penetrate the mucosal barrier through abnormally then into the cerebrospinal fluid to interfere directly with the
leaky tight junctions. function of the CNS. This hypothesis may be applicable to
An additional route by which dietary proteins such as autistic patients as well because one member of the family
gluten and casein may enter the submucosa is via the mi- of casomorphins, [3-casomorphins-7, is reported to be el-
crofold (M) cell pathway (35). The immune system of the evated in the urine of autistic patients (28). Evidence that
-GI tract is organized in the lymph nodules, which, in the the exorphins are able to enter the mammalian CNS was
ileum, are aggregated in groups called Peyer's patches. As reported by Hemming (41) who detected orally adminis-
noted above, the lymph nodules lie below the epithelial cell tered gluten fragments in rat brains. Furthermore, brain opi-
layer, displacing the muscularis mucosae and forming folds ate receptors reportedly bind gluten exorphins (37). When
in the mucosa. The epithelial cell layer contains, in addition infused into the blood stream of rats, [3-casomorphin-7 ac-
to enterocytes and goblet cells, M cells. M cells are special- tivates an immediate early gene (c-Fos) in several regions of
ized to transport antigens and microorganisms transcellu- the rat brain, indicating that the exorphin not only gains
larly from the lumen into the subepithelial space. In the access to the brain but activates brain cells as well (42).
subepithelial space, the antigens and microorganisms inter- Further supporting the notion that natural foods passing
act with the cells of the immune system (35). The B cells of through a leaky intestinal mucosa play a role in producing
the lymph nodules respond to the presence of antigens in the the behavioral symptomatology of autism, the studies cri-
diet by secreting antibodies, also called immunoglobulins tiqued above reported that improvements in behavior, in-

642 INTESTINAL PATHOPHYSIOLOGY IN AUTISM

eluding reduced incidence of seizures in autism, were seen the epithelium overlying the follicles; neutrophils also in-
after the introduction of a diet free of gluten (34) or a diet filtrated the crypt epithelium that lies between the villi and
free of milk and gluten (25, 36). Schizophrenic patients, provides a constant supply of new epithelial cells for the
who likewise often exhibit elevated levels of IgA's for glia- villi. Also, the intestinal epithelium was significantly more
din, casein, and [3-lactoglobulin (43) and appear to have a ulcerated and eroded in autistic versus normal subjects.
higher than usual incidence of celiac disease (44), have also Lastly, the basement membrane thickness was increased,
been reported to benefit behavioraIly from a similar diet whereas the density of sulfated glycosaminoglycans in the
(45-47). Unfortunately, a gluten-free and casein-free diet basement membrane and epithelium was greatly reduced.
(the "gfcf' diet), although reportedly ameliorating behav- Taken together, these findings indicate pathological inflam-
iors for many of the affected schizophrenic individuals, does mation of the intestine in these autistic subjects. Although
not totaIly eliminate those behaviors. Hence, the diet does the findings would seem to provide evidence that the mu-
not represent a cure. Moreover, the failure to completely cosal barrier is compromised in autism, no direct evidence
eliminate the behaviors raises the possibility that elevated of an alteration of the mucosal intestinal barrier, as could
exorphins cause permanent damage to the infant brain. have been gained using the sugar permeability test, was
sought in these studies. It remains to be seen whether a
What is the Nature of the Change in Intestinal dietary antigen plays a direct role in altering intestinal per-
Permeability in Autism? meability (see below) or simply gains access to the blood
Pathological inflammation of the intestinal mucosa has through a mucosa that has been made more permeable as a
long been recognized as a primary symptom in celiac dis- consequence of some other sequence of events.
ease and inflammatory bowel disease. The recent work de-
tailed above indicates that there is also pathological inflam- Possible Pathways by Which Intestinal Function
mation of the ileum in autism (14-17), although there is no May Become Impaired
evidence to support the view that gluten or casein cause the Below, several hypotheses are described that have been
inflammation. Intestinal inflammation can be regarded as advanced to account for the intestinal pathophysiology ob-
the consequence of the disruption of the complex interaction served in autism. Evidence for each of these hypotheses is
between all of the ceIls of the mucosa (immune and non- based upon a very limited number of basic and clinical
immune), as weIl as the extraceIlular matrix, the normal research studies of varying rigor and conclusivity. None of
interactions being mediated by ceIl surface and paracrine the hypotheses has gained sufficient support to broadly in-
molecules (48). The intestinal inflammation seen in Crohn's fluence clinical treatment of autistic patients.
disease and ulcerative colitis is associated with disruption of Antigens in the Diet. Inasmuch as gliadin, the pep-
the glycosaminoglycans that comprise vascular tissue, con- tide derived from gluten, is known to extensively damage
nective tissue, and the basal lamina of epithelia (49). Gly- the intestinal mucosa in celiac disease and there is some
cosaminoglycans are polysaccharides, which include hepa- limited evidence that autistic symptoms of many individuals
rin sulfate, dermatin sulfate, and chondroitin sulfate, as weIl are exacerbated by a diet that includes gluten, it is possible
as sialic acid residues. As highly anionic structural compo- that gliadin produces the increase in intestinal permeability
nents of the connective tissue matrix and waIl of blood seen in autism. Children with cow's milk aIlergy also ex-
vessels, they render the tissues electrostaticaIly negative and hibit inflammation of the intestinal lamina propria and par-
thereby restrict blood proteins from exiting into the inter- tial villous atrophy after ingesting milk; inflammation is
stitial space and intestinal lumen. Deficiencies in the activ- reversed when milk is excluded from the diet (55, 56).
ity of the enzyme phenylsulfotransferase, which normaIly Hence, milk proteins may also cause changes in gut perme-
maintains sulfation, have been reported in autistic subjects ability, accounting for the intolerance to this food in autism.
(50,51). The disruption of glycosaminoglycans and associ- The work of D'Eufemia et al. (34) described above offered
ated loss of tissue sulfation observed to occur in inflamma- some support for the view that gluten enhances intestinal
tory bowel disease (52) may contribute to the generalized permeability by increasing the permeability of the paracel-
increase in intestinal permeability that occurs with leakage lular pathway, i.e., the route between the enterocytes. There
of protein and fluid across the waIl of the intestine in that was no evidence that the enterocytes themselves were ren-
disease, although such an association is far from estab- dered more permeable, as occurs in celiac disease, wherein
lished. In Crohn's disease, blood proteins are lost into the a near total loss of the intestinal cell lining is manifest. In
intestinal lumen and are excreted into the feces; the extent keeping with this distinction, the pathology reported to oc-
of protein loss correlates positively with the length of the cur in the ileum of autistic subjects (14-17) is much more
bowel that is inflamed (53). In the study by Wakefield et al. subtle than that observed in celiac disease. These consider-
(14) examination of histological sections of intestinal tissue ations do not disaIlow a role for gluten and casein in intes-
from autistic patients revealed a near doubling of the num- tinal hyperpermeability. For example, these proteins or their
ber of lymphoid follicles, as weIl as follicle enlargement and digestive products (gliadomorphins and casomorphins) may
merging of adjacent follicles. More recently, Furlano et al. access the submucosa via the M cell pathway and produce
(54) reported that neutrophils and lymphocytes infiltrated antigenic responses that damage the mucosa. Two cytokines

INTESTINAL PATHOPHYSIOLOGY IN AUTISM 643

 released by mononuclear cells of the immune system upon and the prevalence of autism. Fully five separate studies
exposure to antigens have been reported to increase intes- have failed to find any significant correlation between these
tinal permeability in experimental preparations. Thus, inter- variables (9, II, 68-70). For example, after reviewing the
feron-v increased epithelial permeability in cultured cell case history of 473 autistic children in London (UK) over a
lines of intestinal origin grown into confluent monolayers 20-year period starting from 1979, Taylor et al. (11) found
(57-59). Similarly, tumor necrosis factor a (TNF-a) re- no significant change in the proportion of children with
leased from mononuclear cells of infants with cow's milk developmental regression or bowel problems before and af-
allergy elevated tissue conductance and the transepithelial ter introduction (in 1988) of the MMR vaccine. Similarly,
flux of the macromolecular marker horseradish peroxidase Dales et at. (9) found no correlation between the upward
(HRP), as well as the flux of mannitol and sodium ions in a trend of early autism caseload in the period of 1980 to 1994
cultured cell line (60). The effect produced by TNF-a was and childhood MMR immunization rates in California
potentiated by interferon-v, The changes in intestinal per- school children given the vaccine by the 17th or 24th month
meability induced by the cytokines are suggestive of an of age. Hence, this approach affords little support for a role
increased transepithelial transit through the paracellular of the MMR vaccine in producing autism. However, it is
pathway. In a separate study, the effect of interferon-v was noteworthy that Edwardes and Baltzan (71) reported that the
seen to operate through elevation of cytosolic levels of nitric data of Dales et at. (9) reveal a positive correlation between
oxide in the target tissues (57). The manner in which food MMR vaccination rates and autism incidence when the data
antigens may alter intestinal function and produce intestinal for the children vaccinated at 17 months are examined in
pathology in allergic individuals was recently discussed isolation. Nevertheless, Edwardes and Baltzan felt that im-
(61). Lacking is any evidence that gliadin and milk proteins, munization should be maintained. Without question, the
or their products, produce these cytokines in the autistic gut. MMR vaccine has been very efficacious in limiting child-
Vaccines. The intestinal pathology noted in the study hood measles, mumps, and rubella infection. Vaccination
of Wakefield et al. (14), namely, the hyperplastic lymph against these diseases is central to the establishment and
nodes in the ileum and colon, suggests that the immune maintenance of the public health. For this reason, it is para-
system of the gut has been seriously challenged in the au- mount that concerns regarding the safety of the MMR vac-
tistic patients. Although Wakefield et al. did not draw a cine be fully alleviated as may only occur after gastroen-
conclusion as to the cause of the pathology, they noted terologic measurements, including endoscopy, are per-
parental reports that the children had received the trivalent formed before and after vaccination.
measles-mumps-rubella (MMR) vaccine before undergoing Pharmaceutical formulations of selected infant vac-
behavioral regression. Likewise, Fudenburg (50) and Gupta cines have contained small amounts of an antimicrobial pre-
(52) noted in separate reports a close temporal association servative, thimerosal ([ {o-carboxyphenyl) thio] ethylmer-
between the administration of the MMR vaccine and the cury sodium salt), a compound consisting largely of the
onset of autistic symptoms in patients under their care. The organomercurial ethylmercury. The role of mercurials in
genomic RNA of the vaccine strain of measles virus was producing damage to the CNS has been documented (72). In
detected in peripheral mononuclear cells of three out of nine addition, the organomercurial methyl mercury and inor-
autistic patients examined (62). Very recently, 75 of 91 ganic HgCl z have been shown to increase the ionic conduc-
patients with developmental disorders and diagnosed with tance and the mannitol permeability of isolated segments of
ileal LNH and enterocolitis were reported positive for rat colon (73). There is also evidence that HgCl z can influ-
measles virus in their intestinal tissue compared with five of ence the immune system of the gut. Thus, a single large oral
70 control patients (63). The measles virus was localized dose of HgCl z given to rats that were immunized against
within cells of the immune system of hyperplastic lymph ovalbumin caused a significantly enhanced immune re-
follicles. Previously, Lewin et al. (64) and Miyamoto et al. sponse to ovalbumin (elevated serum IgE and IgG) and
(65) reported detection of measles virus in tissue from pa- damaged the DNA in the intestinal epithelial and lymph
tients with Crohn's disease. In contrast, Iizuka et al. (66) node cells (74). HgCl z may act in part to increase the in-
found no evidence of measles virus in a population of testinal permeability to ovalbumin. Additional studies are
Crohn's patients. The reason for these very different results needed to determine whether methylated mercurials in
is unclear and underlines the need for further investigations. lower doses produce similar effects as HgClz. These obser-
Parenthetically, after measles immunization of children, the vations notwithstanding, there is no objective evidence di-
primary cytokine produced is interferon-v (67). The effect rectly implicating thimerosal in adverse responses to vac-
of interferon-v to increase permeability of monolayers of cines. The phaseout underway in thimerosal usage as a pre-
cultured intestinal cells was discussed above. The studies servative in pharmaceutical vaccine formulations will
suggesting that the MMR vaccine may be an etiological reduce the exposure of children to this toxin.
factor in autism have enormous implications for public Impairment in Gut Development. Nelson et al.
health. Hence, they have prompted others to exam retro- (75) recently reported that several neuropeptides (that in-
spectively several epidemiological databases of different hibit or excite neurons) and neurotrophins (that alter neuron
populations to exam the relationship between vaccinations metabolism) were very significantly elevated in the blood of
644 INTESTINAL PATHOPHYSIOLOGY IN AUTISM
69 children who developed autism compared with the blood and, when secretin was infused intravenously, they ob-
of 54 children who developed normally. Measurements served hypersecretion of pancreatic fluid. These observa-
were performed on archived neonatal blood samples drawn tions may be explained by a lower than normal release of
routinely at birth from infants born in California. Among a secretin in the autistic patients upon stimulation of the se-
group of eight neuroactive compounds measured by immu- cretin cells. Consequently, the reduced blood levels of se-
noaffinity chromatography vasoactive intestinal peptide cretin could allow gastric HCl secretion to increase abnor-
(VIP), calcitonin gene-related peptide (CGRP), brain- mally due to relief from secretin inhibition of gastric acid
derived neurotrophic factor (BDNF), and neurotrophin 4/5 secretion and could also reduce pancreatic alkali secretion.
(NT 4/5) were elevated. The blood levels of neuropeptides These separate effects would, in turn, increase acidity of the
and neurotrophins did not correlate with the degree of men- intestinal luminal contents. The greater luminal acidity
tal impairment or with the presentation of seizures or with could then elevate intestinal permeability excessively by
the subtype of autism (regressive versus nonregressive). altering the integrity of the tight junctional complexes. In
Ninety percent of the autistic children had a concentration this respect, it has been observed that exposure of the rat
of VIP that exceeded the control range; similar percentages intestinal mucosa to acidic saline for 30 min resulted in
for the other compounds were: NT 4/5 87%, CGRP 81%, severe injury to the tips of the villi and elevated lumen-to-
and BDNF 65%. In 94% of the autistic infants, both a neu- blood passage of the serum protein albumin (78). If hypo-
ropeptide (VIP or CGRP) and a neurotrophin (BDNF or NT secretion of secretin does occur in autism, then it might be
4/5) were elevated. The findings were not exclusive for clinically beneficial to elevate serum secretin in these pa-
autism---children diagnosed with mental retardation without tients. Interestingly, Horvath et al. (79) reported that within
autism exhibited similar blood patterns of neuroactive com- 5 weeks of receiving intravenous secretin infusions suffi-
pounds. Hence, the altered levels of neuropeptides and neu- cient to elevate the pancreatic secretory rate, three patients
rotrophins may be a necessary, but not a sufficient, condi- exhibited marked dimunition of GI symptoms and improve-
tion for impairment of gut development in autism. ment in behavior, including expansion of expressive lan-
VIP, a member of the VIP-glucagon-secretin family of guage. This surprising result caused considerable interest.
neuropeptides, is a neurotransmitter and neuromodulator However, when several laboratories attempted to replicate
important in cerebral growth, neurogenesis, and astrocyto- their results in trials with large numbers of patients, a ben-
genesis. In addition, VIP is present in the peripheral nervous eficial effect of secretin was not seen whether patients were
system, endocrine pancreas, and in the enteric nervous sys- given secretin in single doses (80-83) or in multiple doses
tem (ENS) of the intestine, among other sites. The ENS is (84). Hence, the great weight of evidence argues against a
an independently acting nerve network within the walls of role for hyposecretion of the hormone secretin in autism.
the gastrointestinal tract that regulates secretion and motil-
ity. Although the source of the elevated blood neuropeptides How Might Impaired Intestinal Function
and neurotrophins is unknown, the enhanced secretion of Influence Autistic Behavior?
VIP (an inhibitory agonist in the ENS) by enteric neurons Autistic individuals frequently exhibit mental retarda-
may impair gut development, motility, and secretion and tion as well as unusual behaviors such as perseveration.
thereby play a role in producing the GI symptoms noted in These are indications of impaired CNS function (I, 85). The
autism. In this respect, it may be noteworthy that 20% of studies described above provide evidence that, in autism,
autistic children examined in a recent study had decreased much of the GI tract, extending from the esophagus to the
serum levels of IgA, the Ig formed in the gut (76). Perhaps, colon, possesses pathology (14-17). The possibility cannot
in these patients, the immune system of the GI tract is be discounted that any GI pathology that exists is unrelated
inadequately developed, or less responsive to stimuli, leav- to the function of the CNS. However, the reports that, in
ing them more susceptible to antigens. some autistic children, a diet free of gluten and casein pro-
Hyperacidity of the Intestinal Luminal Contents duces a dimunition of autistic behaviors is intriguing and
Due to Hyposecretion of Secretin. As noted above, raises the possibility that altered GI tract function may at
there is some limited evidence, as yet unverified, that se- least increase the severity of the behavioral symptoms (24,
cretion of the GI hormone secretin is impaired in autism 25, 86). Indeed, impairment of brain development in very
(17). Well-documented functions of secretin include stimu- young children through gut-associated pathways may be
lation of pancreatic sodium bicarbonate and water secretion irreversible. Hence, it is valuable to consider two potential
and inhibition of gastric acid secretion. These two effects scenarios by which impaired intestinal function may playa
aid in maintaining the pH of the intestinal luminal fluid near role in altering CNS function. In this respect, it is important
neutrality. The catalytic activity of pancreatic digestive en- to recall that, as noted above, the etiology of autism is
zymes is optimal at pH values of 7 to 8 and are lower under multifactorial.
acidic conditions (77). Horvath et ai. (17) observed esoph- Metabolites of Gluten and Milk Interfere with
ageal reflux of gastric acid in autistic patients. This could be Brain Function. According to this theory, it is proposed
caused by gastric hypersecretion of hydrochloric acid. They that gliadomorphins and casomorphins arising from the par-
also noted impaired intestinal hydrolytic enzyme activity, tial luminal digestion of dietary gliadin and casein, respec-

INTESTINAL PATHOPHYSIOLOGY IN AUTISM 645

tively, are absorbed through a leaky gut, enter into the CNS, normally absorbed in the ileum of the small intestine. Pa-
and interfere with normal brain function because their func- thology in the ileal mucosa of autistic patients, as observed
tional properties mimic the opioid hormone [3-endorphin. to occur in the studies noted above, could interfere with the
This hypothesis developed from the pioneering work of transport process for vitamin BJ2 in the ileal absorptive
Dohan (40), who proposed that schizophrenia was caused cells. If absorption is severely inhibited, the resulting lower
by a dietary overload of peptides from gluten and milk. blood vitamin BJ2 could interfere with the formation of
Others have disputed the role of gluten in causing schizo- myelin, the lipoprotein material surrounding the axon of
phrenia (87). Panskepp (88), in developing his opiate excess myelinated nerve fibers (98). Myelin is necessary for nor-
hypothesis of autism, noted that young animals exposed to mal conduction of the action potential in myelinated nerve
low doses of opiate drugs displayed behavioral symptoms fibers. Wakefield et al. (14) observed that, in conjunction
similar to those seen in autistic children. The influence of with the intestinal pathology observed in his autistic popu-
opioids on human brain function has been described (27). lation, vitamin B 12 absorption was significantly reduced in
Gliadomorphins and casomorphins are not hydrolyzed by all eight autistic individuals in which urinary methylmalonic
proteolytic enzymes, hence, are very stable families of com- acid excretion was measured. Methylmalonic acid is nor-
pounds that can produce long-lasting effects on the CNS mally converted to succinyl coenzyme A by a vitamin B I 2
(89). Casomorphins are detectable in human cerebrospinal dependent mutase. Dietary vitamin BJ2 deficiency results in
fluid (CSF) (90). One member of this family, [3-casomor- impaired mutase activity and spillover of methylmalonic
phin-7, caused behavioral changes when injected into rats acid into the urine. Wakefield et al. (14) proposed that nerve
(91). myelogenesis, which is dependent upon vitamin B 12' may
If exorphins do interfere with CNS activity, it is rea- be impaired in autistic children as a result. Direct evidence
sonable to expect that autistic individuals might have of vitamin B I2 deficiency and impaired myelogenesis in
greater levels of endogenous endorphins (which include en- autism is lacking at this time.
kephalins and l3-endorphin) in their cerebrospinal fluid.
Gillberg (92) reported that six of 20 autistic youths pos- Future Directions for Research
sessed significantly elevated levels of enkephalins in a spe- This review has focused on recent reports of GI pathol-
cific fraction of the CSF obtained by spinal puncture (92). ogy in infantile autism. Collectively, the reports represent a
These six patients also exhibited decreased sensitivity to potentially important new advance in our understanding of
pain. The level of endorphins in lumbar CSF is considered autism and related developmental disorders. However, there
to reflect the level of l3-endorphin in the brain (93). In a is very consideable controversy surrounding the published
subsequent study of 31 autistic children, Gillberg et al. (94) findings, as well as many of the related issues dealt with in
found that CSF [3-endorphins were statistically lower than this review. For this reason, there is a great deal that needs
in a group of nonage-matched (adult) individuals. Contrast- to be done. It is essential that others examine the GI tract of
ing with these reports, Nagamitsu et al. (93) found that symptomatic autistic children to substantiate the findings of
levels of [3-endorphin in the CSF of 19 autistic individuals Wakefield and of Horvath and their respective coworkers.
did not differ significantly from the levels of [3-endorphin in Research into the GI pathology must be extended to detail
age-matched controls. Hence, there is conflicting evidence the alterations in morphology, metabolism, and transepithe-
whether endogenous [3-endorphins, at least, are elevated in lial transport capacity at the cellular level and to evaluate
autism. This does not preclude the possibility that dietarily the reputed pathology as a tool in the clinical diagnosis of
derived exorphins play a prominent role in producing the autism. The extent to which the barrier functions of the
behavioral symptoms of autism. Nevertheless, a great deal small intestine and colon are affected needs intensive ex-
of work is needed to establish that exorphins are absorbed amination. The causative agent(s), whether exogenous or
into the blood and produce changes in CNS function. endogenous, must be identified. It is important to exten-
It is perhaps relevant to this topic that untreated celiac sively evaluate the efficacy of gluten- and casein-free diets
patients, who, like autistic patients, exhibit intestinal patho- in ameliorating autistic symptoms and whether such diets
physiology, commonly also exhibit psychiatric symptoms, ameliorate the gut pathology similar to the manner in which
including anxiety and depression (95-97). Imposition of a a gluten-free diet reverses the extensive mucosal pathology
gluten-free diet produces a rapid elimination of these symp- in celiac disease. All potential links between the GI pathol-
toms in celiac patients (95). Likewise, Dohan (44) has re- ogy and the autistic symptoms must be given careful and
ported anecdotal evidence of a high incidence of celiac dis- rigorous examination. Even if it develops that the GI pa-
ease in schizophrenic patients. Hence, autism may be only thology is unrelated to the etiology of autism or its unique
the most recent of several pathological conditions discov- behavioral symptomatology, the discoveries appear to
ered to possess a strong brain-gut connection (54). verify what parents and physicians have long suspected,
Vitamin 8 12 Deficiency Impairs Nervous Sys- namely, that many autistic children have abnormal GI func-
tem Development. According to this theory, impaired tion. Routine endoscopic examination of newly diagnosed
intestinal absorption of vitamin B 12 produces a deficiency autistic patients, coupled with an effective treatment to nor-
resulting in impaired nerve function. Dietary vitamin B 12 is malize GI tract morphology and function, may prove effec-

646 INTESTINAL PATHOPHYSIOLOGY IN AUTISM

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