THE INTESTINAL MICROVILLUS Ultrastructural Variability in Coeliac Disease and Cow's Milk Intolerance

Abstract. In 8 patients with coeliac disease, 2 patients with cow's milk intolerance and 10 patients with other gastrointestinal disturbances, intestinal biopsies were examined in order to compare the extent of alteration of surface vs. crypt microvilli (MV). In those diseases associated with the ingestion of a noxious agent, namely coeliac disease and cow's milk intolerance, a marked and abrupt difference between the severely damaged surface epithelium and its MV, and the preserved crypt epithelium was noted. The results of the examinations give reason to suppose that these morphological variations are caused by the higher gliadin or milk protein concentration over the avillous surface epithelium as opposed to the lower one in the elongated crypts.     

Malabsorption syndrome with cow's milk intolerance. Clinical findings and course in 54 cases.

Fifty-four infants with the malabsorption syndrome and cow's milk intolerence seen during 1962-1971 were investigated. All had diarrhoea and failed to thrive. Most had vomiting and about 20% had atopic eczema and recurrent respiratory infections. Laboratory investigations revealed malabsorption, raised serum IgA, and precipitins to cow's milk. Biopsies showed that the jejunal mucosa was damaged, and in about half the cases was flat. The patient did well on human milk but reacted clinically to cow's milk challenge, either in a few hours or gradually during 3-4 weeks. Some patients showed first a quick, but later a slow, reaction. Clinical symptoms of cow's milk intolerance disappeared at the age of about one year. At that time 81% had normal faecal fat, but only 29% had a normal proximal jejunal mucosa. Many of the patients developed intolerances to other food proteins, such as soya and wheat, if these were given during the sensitive period. Forty-two patients have been followed up for 2 years on a normal gluten-containing diet. Of these, 37 have a normal or nearly normal jejunal mucosa and 5 (12%) have subtotal villous atrophy indicative of coeliac disease. It is concluded that the malabsorption syndrome with cow's milk intolerance is a clear-cut clinical entity. However, the symptomatology, results of laboratory tests, and jejunal biopsy findings closely resemble those of other entities where damage to the intestinal mucosa causes a malabsorption snydrome. Follow-up studies showed that the disease is transient, but about 10% of the patients have coeliac disease, regarded in such cases as the primary disorder.     

The importance of cow's milk protein intolerance in chronic diarrhoea of children

A survey is given of the cases diagnosed as cow's milk protein intolerance in the last 5 years. The diagnosis was established on the basis of the regression of clinical symptoms after elimination from the diet of cow's milk and their recurrence after milk challenge. In more than half of the cases intestinal biopsy was carried out; three patients were rebiopsied after milk challenge. Intestinal biopsy is indicated solely in cases when the exclusion of coeliac disease is necessary for the correct diagnosis.     

Cow's milk protein intolerance: clinical and pathogenic aspects (author's transl)

Cow's milk protein intolerance is a transient food intolerance of early infancy. Ingestion of cow's milk protein causes an enteropathy of variable degree. Clinical manifestations are primarily gastrointestinal, although dermal and respiratory symptoms add to the clinical syndrome. Three types are found: an acute anaphylactic reaction, a chronic mild form, and a chronic severe form which is of utmost practical importance in severe protracted diarrhoea of infancy. A graduated diagnostic procedure is proposed, taking into account clinical and morphological reactions to cow's milk proteins. Pathogenesis is immunologically mediated. A concept of transient food protein intolerance is developed which has to be separated from the permanent intolerance of gluten in coeliac disease. Prognosis of cow's milk protein intolerance is excellent after elimination of the offending agent. Breast feeding seems to be effective in prevention of the disease.     

Jejunal mucosa lymphoid cell subsets and the expression of major histocompatibility complex antigens in children

Using monoclonal antibodies with the immunoperoxidase technique the distribution pattern of class I and class II antigens of the major histocompatibility complex (MHC), and of the lymphocyte subsets have been studied in intestinal biopsies from children without mucosal lesions, from children with coeliac disease (CD) and from infants with cow's milk protein intolerance (CMPI). The staining of the intestinal mucosa for class I antigens is unaltered irrespective of the histological picture or the clinical diagnosis. Class II antigens are only partially or not expressed at all by epithelial cells in untreated cocliac disease and in some cases of cow's milk protein intolerance. The number and the composition of the lamina propria lymphocytes in both CD and CMPI are different from the normal situation. An increase of all lamina propria lymphocyte subsets is observed in untreated CD. A decrease of OKT4⁺ lymphocytes is observed in the lamina propria of CMPI patients. These changes may be involved in the pathogenesis of these diseases.Abbreviations MHC major histocompatibility complex - CD coeliac disease - CMPI cow's milk protein intolerance - IEL intra-epithelial lymphocytes     

Kinetics and clinical use of the combined D-xylose and NBT-PABA test

The oral D-xylose- and NBT-PABA-test are frequently applied as screening methods for malassimilation syndromes. After oral load time-related serum concentrations are used as reference values. In this study both substances were given simultaneously as isotonic solutions after a fasting period of 6 hours. A standard meal was offered 20 minutes later. Blood samples were taken prior to and 60, 90 and 120 minutes after administration of the test-substances. 43 pediatric patients were studied. 11 patients with cystic fibrosis, 10 patients with underlying intestinal disease such as coeliac disease and cows milk intolerance and 22 patients with other than gastrointestinal diseases were compared. In children with coeliac disease and cows milk intolerance 1-hour PABA concentrations were in the range of the control group whereas 1-hour D-xylose concentrations were lower than 15 mg/dl. In patients with cystic fibrosis the maximum concentrations of D-xylose were within the limits of normal values while 1-hour PABA concentrations were lower than 0.15 mg/dl in cases of relevant exocrine pancreatic insufficiency. The results of this study show that the combined D-xylose and NBT-PABA-test has proved its practical value in cases of suspected malassimilation and its discriminatory property between patients with relevant pancreatic insufficiency and patients with diminished intestinal absorptive capacity due to coeliac disease or cow's milk intolerance.     

The diagnostic significance of antibodies to various cow's milk proteins (fluorescent immunosorbent test)

Antibodies of various immunoglobulin classes to different cow's milk proteins were studied with the fluorescent immunosorbent test in 601 newborns, infants, children and adults (A). The antibody levels, expressed as the geometric mean (gm) of four antibody titres to casein, -lactoglobulin, -lactalbumin and bovine serum albumin, showed a clear dependence on age. They were compared with the antibody levels in children with cow's milk protein intolerance (C), other gastrointestinal disorders (B) and coeliac disease (D). The 20 children with cow's milk protein intolerance clearly differed (significance level 2×10^-11) from those of the two control groups (A, B) insofar as the criterion adopted was not the titre against a single protein but the gm of the four antibody titres, and insofar as allowance was made for the age of the patients.All patients with cow's milk protein intolerance also showed elevated gm titres of IgE, IgA and IgM antibodies. However, since a number of children in the control groups also showed higher values, particularly with regard to IgE antibodies, the determination of the IgE, IgA and IgM antibodies adds little to the diagnosis and at best provides a further discriminatory aid.Although antibody titres fall immediately after placing the child on a milk-free diet, it is a matter of months before they become negative (titre <120). After challenge titres rise again. In a longitudinal study of 25 children with acute gastroenteritis (E) it was shown that the antibody titres remained unchanged during and after the attack. This contradicts the often expressed opinion that the cow's milk antibodies frequently observed in healthy infants are induced as a consequence of gastroenteritis. In contrast to the other groups, all 26 children with proven coeliac disease (D) had antibodies to gliadin, irrespective of whether their gm cow's milk antibody titre was high or low.Computer Center of the University of Basle     

Influence of cow's milk proteins and gluten on human duodenal mucosa in organ culture

Forty-five duodenal biopsies from 33 children and 3 adult patients were maintained in organ culture for 24 h and exposed to various cow's milk proteins and gluten. In 10 of 11 celiac patients with a flat duodenal mucosa, and in 2 of 4 patients with partial villous atrophy, a significant reduction in the mean enterocyte height was found after in vitro gluten exposure, compared to culture in basic culture medium. Three patients had coexisting celiac disease and cow's milk protein intolerance. alpha-Lactalbumin and beta-lactoglobulin exhibited toxic effects on flat biopsies from two of these patients, and casein was toxic in one. In 10 patients with cow's milk protein intolerance, a significant reduction in enterocyte height was noted in one case with gluten, and in three patients with casein and lactoglobulin, whereas lactalbumin did not affect the tissues. In seven control patients having a normal duodenal mucosa, no in vitro influences were noted, whereas in four patients with partial villous atrophy, a toxic reaction to gluten was seen in one and a reduced enterocyte height was seen after lactoglobulin exposure in another. In vitro toxicity induced by gluten corresponded well with the diagnosis of celiac disease, whereas toxic reactions to cow's milk proteins during organ culture were inconsistent in cow's milk intolerance, except for cases in which a marked enteropathy was documented.     

CHANGING PATTERN OF COW'S MILK INTOLERANCE: An Analysis of the Occurrence and Clinical Course in the 60s and mid-70s

ABSTRACT. Verkasalo, M., Kuitunen, P., Savilahti, E. and Tiilikainen, A. (Children's Hospital, University of Helsinki, Finland). Changing pattern of cow's milk intolerance. Acta Paediatr Scand, 70: 289, 1981.–The rapid changeover to commercial adapted infant formulae which took place in Finland between 1973 and 1975 was studied as a factor in the occurrence of severe intestinal cow's milk intolerance (CMI). Of infants treated for CMI in 1962-73, ninety-three percent (25/27) were on homemade or unadapted formulae. The admission rate for CMI in these years was 0.22/1 000 liveborn infants breast fed less than six months. During 1974-77 the corresponding figure was 0.56, with 85 % of the patients (18/26) on adapted cow's milk formulae. The patients treated before 1974 had a longer symptomatic period before admission, greater growth retardation and more severe intestinal damage than those seen during and after 1974. This is believed to reflect mainly the increasing awareness of CMI on the part of both laymen and the medical profession. In the history of 2/3 of the patients at least one of the following conditions was noted: non-breast feeding, infectious gastroenteritis, praematurity, 21-trisomy, prior intra-abdominal surgery, Hirschsprung's disease, and atopic disease in family members. The long follow-up averaging over four years revealed four patients with coeliac disease. In one of these the proximal jejunal mucosa was normal after two years on gluten-containing diet, but he showed a mucosal relapse as late as between 2 to 4 years on normal diet.     

Confirming persistence of gluten intolerance in children diagnosed as having coeliac disease in infancy.

In young infants the clinical and investigative features of coeliac disease (CD) may be mimicked by other conditions such as cow's milk intolerance or secondary disaccharidase deficiency. It is therefore especially important to confirm a diagnosis of CD by later gluten challenge in such infants. Sixteen children in whom the diagnosis of CD had been made before the age of 12 months had an oral gluten challenge, after being treated with a gluten-free diet for periods of one month to 5 years. In 15 we showed intestinal xylose malabsorption by the one-hour blood xylose level within 1-28 days of starting ingestion of gluten. One child, with a persistently normal one-hour blood xylose test after gluten challenge for 3 months, had normal absorption and normal jejunal histology after 18 months on a gluten-containing diet; she is considered not to have CD. The one-hour blood xylose test before and after gluten challenge can help to confirm the diagnosis in coeliac patients diagnosed in infancy.     

Cow's milk protein intolerance in childhood. Review of 30 cases]

Retrospective clinical study of 30 cases of cow's milk protein intolerance with a long term follow-up (3-141/2 years) in 22 cases. The onset of the disease is usually in the first 6 months, following a short exposure to cow's milk proteins. A later onset is also possible, particularly following an intestinal infection which seems to favor the sensibilisation. Usually the digestive disturbances predominate and are often associated with respiratory, cutaneous and other general problems (especially unexplained prolonged fever). In about half the cases, laboratory studies reveal signs of exsudative enteropathy or generalized malabsorption with lesions of varying severity of the jejunal mucosa. Clinical observation and investigation allows us to divide the patients into three groups according to different manifestations: 1. acute anaphylactic, 2. chronic and benign (colitis), 3. chronic and severe, with accompanying malnutrition and intestinal malabsorption. This last category leads occasionally to diagnostic difficulties in distinguishing it from coeliac disease. Whatever manifestations encountered, the disease is usually transitory, and disappears between the age of 2 and 21/2 years, but can also persist for a much longer time. The long-term follow-up study showed complete catch-up growth, absence of further gastro-intestinal problems, and a low incidence of other allergic diseases (18%).     

Cellular immunity in children with coeliac disease

The experimental evidence implicating defective cell-mediated immunity in coeliac disease, a condition where symptomatology is believed to be due to immunological reaction to wheat gluten, is often inconsistent and sometimes controversial.Studies of certain parameters of cellular immunity in four groups of pediatric patients were performed: coeliac patients on normal diet; coeliac patients consuming gluten-free diet; children with cow's milk allergy and a control group consisting of children with gastrointestinal complaints not due to gluten or cow's milk sensitivity.In all these assays no significant differences were found between treated or untreated coeliac children, infants with milk allergy or the gastro-intestinal control groups. On the basis of this study we could find no evidence of impairment of cell-mediated immunity in coeliac children. This conclusion is compatible with the hypothesis that intestinal damage may be due to a subpopulation of lymphocytes sensitive to gluten in persons with normal immune systems. In adults where abnormalities of cell-mediated immunity have sometimes been noted, the reason could be a loss of lymphocytes from the damaged mucosa of the gastrointestinal tract following prolonged antigenic stimulation. This indicates the need for strict adherence to a gluten-free-diet.Supported in part by a grant to A.A. by Stiftung Volkswagenwerk, FRG.     

Cow's milk protein-sensitive enteropathy. Clinical and histological results of the cow's milk provocation test

Nineteen infants suspected of having cow's milk protein-sensitive enteropathy were studied. They all showed failure to thrive, diarrhoea and/or vomiting when fed a diet of cow's milk, and improved when their diet was changed to casein hydrolysate. Jejunal biopsy was done before and 18--23 hours after a milk challenge. Of the 19 infants, 12 presented histological evidence of cow's milk protein intolerance. Eight suffered from vomiting and diarrhoea within 9 days of the milk challenge, but in 4 cases the histological abnormalities were not accompanied by clinical symptoms. In one case a chicken meat intolerance was documented. The histological appearance of the intestinal mucosa after chicken challenge was identical to that observed after milk challenge. In our opinion, repeated intestinal biopsies before and after an acute challenge is the best method to establish the diagnosis not only of cow's milk protein intolerance but also of intolerance to other alimentary proteins.     

Biochemical quantification of crypt hyperplastic villous atrophy by aldolase activity assay

Aldolase activity with the two substrates fructose-1-phosphate and fructose-1,6-diphosphate was measured in the homogenate of small intestinal biopsy specimens from children with different malabsorptive diseases (celiac disease, cow's milk protein intolerance, infectious diarrhea, giardiasis, and Crohn's disease) and controls. It is demonstrated that the ratio of fructose-1,6-diphosphate/fructose-1-phosphate activity, which reflects the relative amounts of the crypt enzyme aldolase A (EC 4.1.2.13) and the villous enzyme aldolase B (EC 4.1.2.7), correlates very well with both the ratio of crypt to villous height (correlation factor r = 0.92) and the mitotic index (r = 0.80).     

Gastrointestinal occult hemorrhage and gastroduodenitis in cow's milk protein intolerance

We are reporting on four infants with cow's milk protein intolerance who presented with hypochromic anemia and occult gastrointestinal hemorrhage. Esophagogastroduodenoscopy revealed erosive gastritis or gastroduodenitis in all cases. Management with a cow's milk-free diet led to a favorable clinical and hematological outcome; the endoscopic inflammatory image disappeared after the cow's milk-free diet trial. Challenge with cow's milk led to gastrointestinal symptoms and to impaired D-xylose absorption. The data presented suggest that some patients with cow's milk intolerance may have gastroduodenitis resulting in occult gastrointestinal hemorrhage and hypochromic anemia.     

Incidence and symptoms of cow's milk protein intolerance following acute gastroenteritis in young infants. Effect of a hypoallergenic diet

In a prospective randomized study we investigated in 28 mainly bottle-fed infants younger than 60 days whether in acute gastroenteritis a hypoallergenic formula could prevent the development of cow's milk protein intolerance. Group 1 (14 infants) was fed with a formula adapted to human milk, Group 2 (14 infants) got a semi-elementary formula (Alfaré). After 3 months group II was exposed to cow's milk protein with a standardized challenge and the incidence of CMPI in both groups was calculated. All cases with the acute form of CMPI occurred in group II (5/12) whereas in group I only one infant suffered from the protracted mild form of the disease. Inspite of the relatively small number of probands we conclude from our results that in infants who are not totally breast-fed in the post-enteritic period feeding with a formula adapted to human milk is preferable to hypoallergenic semi-elementary preparations. An allergen free period of 3 months seems to induce symptoms of cow's milk intolerance, probably as a booster-effect to early sensibilisation.     

Leucocyte Migration Inhibition in Cow's Milk Protein Intolerance

ABSTRACT. The leucocyte migration inhibition (LMI) was determined in an assay after in vitro challenge with beta-lactoglobulin. The assay was considered positive when migration inhibition index was greater than 20 % (mean +3 SD of healthy infants). Ninety-eight infants with protracted diarrhoea and failure to thrive, 16 healthy, 12 malnourished, and 16 infants suffering from acute gastroenteritis were studied. Of the 98 patients with protracted diarrhoea, 12 fulfilled Goldman's criteria for cow's milk protein intolerance, 63 had lactose malabsorption, and in 15 no associated causative factor was identified. The mean index of migration inhibition in the cow's milk allergic group (58.83 ± 11.98) was higher than in healthy controls (8.25 ± 3.91), the difference being statistically significant ( p < 0.05). The test was positive in all patients with caw's milk protein intolerance. The assay was also positive in four other patients suffering from protracted diarrhoea, two of whom had lactose malabsorption. All the infants with acute gastroenteritis and malnutrition had values within the normal range. The migration inhibition index in five patients with cow's milk intolerance had declined to 24.74 ± 4.87 in assays performed 1-6 weeks after return of clinical tolerance to cow's milk ( p < 0.05) but the test was still within the postive range in three of the five infants. These results suggest that this cell mediated immune assay is a sensitive test for the diagnosis of cow's milk protein intolerance in infants. The specificity needs to be reassessed in the light of more objective criteria for the diagnosis of cow's milk protein intolerance.     

Leucocyte migration inhibition in cow's milk protein intolerance

The leucocyte migration inhibition (LMI) was determined in an assay after in vitro challenge with beta-lactoglobulin. The assay was considered positive when migration inhibition index was greater than 20% (mean +3 SD of healthy infants). Ninety-eight infants with protracted diarrhoea and failure to thrive, 16 healthy, 12 malnourished, and 16 infants suffering from acute gastroenteritis were studied. Of the 98 patients with protracted diarrhoea, 12 fulfilled Goldman's criteria for cow's milk protein intolerance, 63 had lactose malabsorption, and in 15 no associated causative factor was identified. The mean index of migration inhibition in the cow's milk allergic group (58.83 +/- 11.98) was higher than in healthy controls (8.25 +/- 3.91), the difference being statistically significant (p less than 0.05). The test was positive in all patients with cow's milk protein intolerance. The assay was also positive in four other patients suffering from protracted diarrhoea, two of whom had lactose malabsorption. All the infants with acute gastroenteritis and malnutrition had values within the normal range. The migration inhibition index in five patients with cow's milk intolerance had declined to 24.74 +/- 4.87 in assays performed 1-6 weeks after return of clinical tolerance to cow's milk (p less than 0.05) but the test was still within the positive range in three of the five infants. These results suggest that this cell mediated immune assay is a sensitive test for the diagnosis of cow's milk protein intolerance in infants. The specificity needs to be reassessed in the light of more objective criteria for the diagnosis of cow's milk protein intolerance.     

Antibodies to gliadin as a screening test for coeliac disease. A prospective study.

The diagnostic value of gliadin antibody determination using the fluorescent immunosorbent test was examined in a prospective study of 57 children with gastrointestinal disease. Antibodies to gliadin were found in all 20 patients with active coeliac disease, whereas 7 of these children (37%) had a normal xylose absorption test despite a flat small gut mucosa. Only 4 (14%) of 28 children with other gastrointestinal conditions had antibodies to gliadin, invariably in low titre. After at least 2 years on a gluten-free diet none of 9 children with coeliac disease in remission had demonstrable gliadin antibodies. The gliadin antibodies disappear slowly, within 6 to 24 months, after withdrawal of gliadin from the diet. 0.8% (5/606) of a healthy control group of children, adolescents and adults (not biopsied) had gliadin antibodies in low titre. Increased mean cow's milk antibody titres were demonstrable in 8 (40%) of 20 patients with active coeliac disease as well as in 9 (32%) of 28 patients with other gastrointestinal lesions. Our studies show that determination of circulating gliadin antibodies is a worthwhile screening test in suspected cases of coeliac disease. In patients so selected there is a definite indication for small intestinal biopsy to confirm the diagnosis.     

Prospective, controlled, multi-center study on the effect of an amino-acid-based formula in infants with cow's milk allergy/intolerance and atopic dermatitis

Cow's milk allergy/intolerance is treated by complete avoidance of cow's milk proteins. Because cow's milk is an important food for infants, its avoidance may lead to an increased risk of growth impairment. Whilst there is evidence for the beneficial effects of extensively hydrolyzed cow's milk formulae (eHF) in infants with cow's milk allergy/intolerance, little is known about the effects of amino-acid-based formulae (AA) in such infants. We therefore performed a prospective, controlled, multi-center trial to study the efficacy of AA in comparison with eHF, on the growth and clinical symptoms of 73 infants (median age 5.7 months) with cow's milk allergy/intolerance and atopic dermatitis. Cow's milk allergy/intolerance was proven in all infants by double-blind, placebo-controlled food-challenge. We observed a significant improvement in the SCORAD index in both groups, from a mean of 24.6, at entry, to a mean of 10.7 (p < 0.0001) after 6 months. In the AA group there was a significant increase in the length standard deviation score (p < 0.04), whilst there was no difference in the eHF group. The weight-for-length values were stable in both groups. The energy intake during the study was similar in both groups. Both an AA and eHF resulted in a significant clinical improvement in infants with an early onset of symptoms of cow's milk allergy/intolerance. Feeding an AA resulted in improved growth compared with feeding eHF, despite similar dietary intakes, and may therefore be considered as a beneficial alternative in infants with severe cow's milk allergy intolerance.