Microvillus inclusion disease: progressive mucosal pathology. A scanning and transmission electron microscopic study, and thoughts about possible pathogenesis.

An infant of African-American descent presented in the immediate newborn period with secretory diarrhea, the cause of which turned out to be microvillus inclusion disease (MID). Small intestinal mucosal biopsies at 6 weeks of age were diagnostic for MID by electron microscopy and repeat biopsies from the small intestine at 15 months demonstrated the seeming relentless progression of this disorder, when a normal structure and organization of small intestinal mucosa was no longer recognizable. Since the child could not tolerate any form of enteral nutrition, a small intestinal transplant was contemplated, but could not be done. The patient did not survive the consequences of an overwhelming sepsis, which resulted in multi-organ failure.

The hereditary hyperbilirubinaemias.

The presence of jaundice in the neonate, infant or young child presents a broad differential diagnosis. The 'disease' may be benign, as in breast-milk jaundice, or potentially fatal, as in hereditary fructose intolerance. The cause of the jaundice may be a primary hepatic disorder, such as extrahepatic biliary atresia, or secondary to a non-hepatic cause, such as haemolysis or sepsis. There may be significant hepatic injury and dysfunction, as in fulminant viral hepatitis, or simply elevation of plasma bilirubin, as in Gilbert's syndrome. In this chapter we will discuss the familial hyperbilirubinaemia syndromes. This diverse group of disorders is characterized by hepatic dysfunction in the absence of hepatocellular injury. The first section of the chapter will discuss the unconjugated hyperbilirubinaemias: Crigler-Najjar syndrome I, Crigler-Najjar syndrome II and Gilbert's syndrome. The discovery of the gene for bilirubin uridine diphosphate glucuronosyltransferase has increased our understanding of the genetic heterogeneity and clinical presentation of the Crigler-Najjar syndromes. The remainder of the chapter will discuss the conjugated hyper-bilirubinemias: Rotor syndrome and Dubin-Johnson syndrome. These rare diseases share many clinical features; however, they can be readily distinguished by biochemical markers in the urine and bile.

Other hereditary diseases and the liver.

In this chapter, an abbreviated account is presented on the subject of hereditary diseases and the liver. However, it is incomplete because Alagille syndrome, storage disorders, alpha-1-antitrypsin deficiency and Wilson disease are not included as they appear in other chapters of this volume. Biliary atresia is omitted because all available evidence does not support any significant genetic association. Molecular biological techniques have enabled linkage of several liver cholestatic disorders to chromosomal loci, and further characterization of the canalicular bile salt transporter (cBST) will advance our understanding of pathogenetic mechanisms involved in benign and progressive cholestatic syndromes. Disorders that have been treated as separate entities may have common 'roots', exemplified by the concept of the ductal plate malformation in fibropolycystic disease. Whereas the majority of disorders referred to in this chapter present early in life, there are several that are associated with liver failure in the neonatal period, which makes early recognition particularly important. Liver transplantation offers a cure for many hereditary disorders affecting the liver but it is not applicable to all.

The scanning electron microscope: how valuable in the evaluation of small bowel mucosal pathology in chronic childhood diarrhea?

Data are presented on scanning electron microscopy (SEM) on small intestinal biopsies of children with chronic diarrhea. In particular, there were 230 patients aged 3 months to 13 years with the following diagnoses: chronic nonspecific diarrhea, cow's milk protein intolerance, soy protein intolerance, giardiasis, cystic fibrosis, gluten-sensitive enteropathy, isolated lactase deficiency, isolated sucrase-isomaltase lactase deficiency, microvillus inclusion disease, rotavirus enteritis, protracted diarrhea of infancy, chylomicron retention disease, visceral myopathy and villous asthenia. Examination of biopsied intestinal mucosa by SEM has yielded important new information and insights on structural pathology and ultrastructural topography. Many of the observed changes helped to better understand the pathophysiology of some of the diarrheal disorders. SEM was also able to detect new features such as mycoplasma-like microorganisms and the absence of the glycocalyx. To adequately assess small bowel mucosal pathology at the ultrastructural level, scanning electron microscopy is an indispensable tool.

Loss of the glycocalyx of enterocytes in small intestine: a feature detected by scanning electron microscopy in children with gastrointestinal intolerance to dietary protein.

Observations are reported on biopsied small bowel mucosa by scanning and transmission electron microscopy in 10 infants and children with chronic diarrhea and intolerance to dietary protein(s). The most striking and consistent finding was widespread loss of glycocalyx from the surface of enterocytes, exposing the tips of the microvilli. Concomitantly, disaccharidase activities were markedly depressed in nine of the 10 patients, and such enzyme activities showed a tendency to return toward normal after removal of the offending dietary antigen(s) in concert with efforts to rebuild the glycocalyx. By light microscopy, no discernible structural damage was seen in eight of the 10 patients; two showed blunting of villi, but there was also loss of glycocalyx. The pathogenesis of the loss of the glycocalyx is unknown. Its finding is rather unique, because it has not been observed in any other condition characterized by chronic diarrhea in children. It is possible that the loss of the glycocalyx is a sequel to cell damage by a hitherto not defined interaction between host and dietary protein. Immunologic processes may be involved.

Intestinal alkaline phosphatase activity in relation to age in humans.

Early studies of a few subjects suggested that intestinal alkaline phosphatase (IAP) activities in children 5 years of age or less were higher than in older individuals. To further investigate this finding, the IAP and disaccharidase activities of 298 subjects (133 were 5 years of age or less) with normal intestinal histology were assayed. Ninety-five of the children had serum alkaline phosphatase determined. The youngest individual with a low lactase activity was 5 years of age, which supported the earlier findings. When the whole population was tested, there was no correlation between the intestinal and serum alkaline phosphatase values. The mean IAP activity of subjects 1 year old and less was greater than in older individuals, but there was greater statistical dispersion and the data were not normally distributed. When studying the natural logarithm of the data, a wide dispersion of values about the mean in the 0 to 3 year-old age group was observed. This qualitative behavior is characteristic of functions involving the base of the natural logarithm and of processes that "age" in a simple way.

Intestinal disaccharidase and alkaline phosphatase activity in giardiasis.

We report results on determinations of small intestinal brush-border enzyme activities in 22 children (aged 11 months to 14 years) with giardiasis. In particular, activities of disaccharidases (lactase, sucrase, maltase) and of alkaline phosphatase were investigated. Forty-one percent of the patients, irrespective of age, had a demonstrable depression of disaccharidase activities, usually in a combination involving two or more enzymes. A depression of intestinal alkaline phosphatase activity was present in 33% of patients, and only in those who demonstrated disaccharidase deficiencies. Mild villus atrophy was present in two mucosal specimens, whereas all others showed normal villus morphology by light microscopy. The results obtained in this study suggest that giardiasis in otherwise healthy children does not cause marked structural damage to the small bowel mucosa, as seen by the light microscope. However, some form of damage to the brush border does occur frequently, as evidenced by a depression of brush-border enzymes. This damage most likely contributes to the diarrhea and also to the carbohydrate intolerance in these patients.

Scanning electron microscopy of soy protein-induced damage of small bowel mucosa in infants.

Observations are reported by scanning electron microscopy (SEM) of soy protein-induced villous atrophy and mucosal recovery in two infants aged 5 weeks and 4 1/2 months. Whereas, by light microscopy, the mucosal lesions appeared similar, i.e., a flat mucosa, their appearance by SEM was different: the damage appeared more severe in the younger infant, although there was a shorter period of exposure to soy protein. The mucosal architecture was restudied 6 weeks after the initial biopsy. The degree of mucosal reconstruction was more advanced in the older infant--the one who showed less severe damage by SEM on the first biopsy. Although these investigations by SEM of damaged small bowel mucosa in soy protein intolerance did not contribute definite information to clarify the pathogenesis of villous atrophy in this condition, the injury was consistent with a lectin-induced toxicity, similar to the one postulated for celiac disease. SEM seems eminently suited to study of the effect of interactions between environment and host at mucosal surfaces; and finer gradations of damage to small bowel mucosa can be determined better by SEM, while this is not possible by light microscopy. Of interest was the rather extensive colonization of the mucosal surface by microorganisms in one of the two patients. However, the contribution of microbial colonization to mucosal damage could not be assessed.

The small bowel mucosa in disease states characterized by chronic diarrhea: observations by scanning electron microscopy.

This paper summarizes observations by scanning electron microscopy (SEM) of surface ultrastructure of small bowel mucosa in patients (mostly children) with disorders characterized by chronic diarrhea. Included are chronic nonspecific diarrhea; conditions associated with villous damage, such as gluten-, milk protein- and soy protein-intolerance; giardiasis; cystic fibrosis and Crohn's disease. SEM has proven most useful to characterize pathologic processes on the surface of the small bowel mucosa, and thus has helped gain more insight to explain clinical symptoms. This was particularly true for chronic nonspecific diarrhea. It is predicted that SEM will become a valuable tool to aid in the diagnosis of diseases of the intestinal tract, which is considered one of the principal domains for such surface ultrastructure research.

Chronic nonspecific diarrhea in children: investigation of the surface morphology of small bowel mucosa utilizing the scanning electron microscope.

Investigations by scanning electron microscopy into changes of surface morphology of small bowel mucosa in children with chronic nonspecific diarrhea are reported. The study population comprised 56 patients, ranging in age from 5 months to 7 years; 65% were between 10 and 28 months old, and 64% of the patients were boys. The major findings were: microorganisms on the mucosal surface; excessive extrusion of cell cytoplasm and of enterocytes (cell shedding); presence of excessive mucus on the mucosal surface; damage to the brush border; and partial villous atrophy. The latter lesion was found in only four patients. All these changes are considered pathologic and, for the most part, are presumed to be due to the presence of antigens, in particular, microorganisms. A depression of disaccharidase activities was encountered in 64% of the patients, but prevalence was without regard to age. Most common was a combined depression of lactase, sucrase, and maltase, as well as an isolated depression of lactase. The possibility has to be considered that enteroadherent microorganisms which are usually not considered pathogenic, and microorganisms such as Mycoplasma, may emerge as intestinal pathogens in susceptible children. It is feasible that genetic traits of the host and environmental factors facilitate adherence and colonization of the small bowel mucosa which, in turn, produces chronic diarrhea. Further studies are needed to confirm the preliminary information contained in this report.

Malabsorption in giardiasis: presence of a luminal barrier (mucoid pseudomembrane). A scanning and transmission electron microscopic study.

Investigations of the small bowel mucosa by scanning and transmission electron microscopy are reported in two coetaneous children with giardiasis. Serum immunoglobulins and mucosal architecture by light microscopy were normal in both, as well as activities of lactase, sucrase, and maltase. One of the children had suggestive evidence of malabsorption. Scanning and transmission electron microscopic examination of biopsied mucosa of the child with malabsorption suggested the following: increased secretory activity in crypts and increased cell damage, producing excess mucus containing extruded cytoplasm. It is hypothesized that the physicochemical properties of the mucoid material permitted ready interaction with the mucosal surface, the biochemical qualities of which could have been altered by the presence of the parasites. Close adherence of this mucoid layer to the surface gave it the aspect of a pseudomembrane which, by covering wide areas of the mucosal surface, probably functioned as an effective luminal barrier for nutrients. This pseudomembrane was not seen by light microscopy. Excessive secretion of mucus, its physicochemical and immunologic properties, and mucus-mucosal surface interaction could have been engendered by genetic and environmental factors, which need further study. Although some mucus was present in the mucosa of the other child, it was confined to the base of the villi, and did not cover the mucosal surface. The presence of a luminal barrier (mucoid pseudomembrane) may be added to the list of mechanisms implicated in causing malabsorption in giardiasis.

Influence of dietary cholesterol, saturated and unsaturated lipid on 3-hydroxy-3-methylglutaryl CoA reductase activity in rabbit intestine and liver.

To characterize further the behavior of the rate-limiting enzyme of cholesterol biosynthesis in animal species, we studied the kinetic properties and the influence of dietary lipid on intestinal and hepatic HMG-CoA reductase activity in the rabbit. In intestinal crypt and villous cells isolated by a dual buffer technique, the KM value was 4.2 and 4.6 microM, respectively for DL-HMC-CoA. The specific activity of HMG-CoA reductase in the jejunum was 0.86 nmol/mg per hr, and evenly distributed between crypt and villous cells. By contrast, reductase activity was considerably lower in the ileum: in villous cells it was 0.40 nmol/mg per hr, and in crypt cells only 0.26 nmol/mg per hr. Liver microsomes had a KM value of 3.0 microM, while the reductase activity averaged 2 nmol/mg per hr. An unexpected finding was the uneven distribution of HMG-CoA reductase in the various lobes of the liver in the single animal. The addition of 1% cholesterol to the diet for 48 hours was followed by an average decline of 73% (P less than 0.005) of HMG-CoA reductase activity in villous and crypt cells of the jejunum. In the ileum, the decrease was less marked (38%, P less than 0.01). Whereas the addition of 5% corn oil to a 1% cholesterol diet did not have an additional suppressant effect on intestinal reductase, the addition of 5% coconut oil to 1% cholesterol caused further decrease of HMG-CoA reductase in jejunum and ileum (P less than 0.05). The 1% cholesterol diet resulted in a 25% decrease of hepatic reductase after 24 hours, whereas after 6 days, the enzyme activity was reduced by 90% of normal. Both 5% corn oil or 5% coconut oil, in addition to 1% cholesterol, further suppressed hepatic reductase activity. The weight of the experimental evidence presented in these studies suggests that cholesterol has a major regulatory effect on both intestinal and hepatic reductase in the rabbit.

Congenital pancreatic lipase deficiency.

A 5 1/2-year-old boy is reported with congenital lipase deficiency and the presence of colipase. He presented with greasy-oily stools since infancy, but growth and development have been normal. No other cause for exocrine pancreatic insufficiency could be found. Intraluminal (jejunal) fat digestion was defective, but some hydrolytic products of dietary long-chain triglyceride were present. The di- and monoglycerides were probably generated by pregastric lipases, although this was not measured directly. Amylase activity was depressed to some extent, a finding which could not be explained. Our studies do not clarify the issue of whether or not the absence of pancreatic lipase is explained as an inherited defect of lipase synthesis, or if it was acquired in utero or in the early postnatal period.

[Diagnostic value of laboratory and scintigraphic investigations in young infants with cholestatic jaundice (author's transl)]. / Möglichkeiten der klinisch-chemischen und nuklearmedizinischen Diagnostik des Cholestase-Syndroms beim jungen Säugling.

The diagnostic value of laboratory and scintigraphic examination techniques in young infants with cholestatic jaundice will be discussed. The correct diagnosis of neonatal hepatitis or extrahepatic biliary atresia cannot be derived from such investigations as determination of bilirubin, enzyme activities, immunologic or serologic parameters or scintigraphy of the liver. Only quantitative changes of serum LP-X before and after administration of cholestyramin and the modified rose-bengal test may help to establish a correct diagnosis in cholestatic jaundice during the first 6 weeks of life.

Quantitative changes of serum lipoprotein-X after cholestyramine administration in infants with cholestatic biliary tract and liver disease.

Lipoprotein-X (LP-X) was determined before and after the administration of cholestyramine in fifty-five infants with persistent cholestatic jaundice to differentiate between intra- and extrahepatic disease. In twenty-seven infants with biliary atresia, serum LP-X prior to cholestyramine ranged from 0.87 to 11.42 g/l (mean: 3.43 g/l; the average concentration was significantly lower (P less than 0.001) in males. After cholestyramine, LP-X rose in twenty-three, remained the same in two, and decreased slightly in two infants. Serum LP-X was present in twenty of the twenty-eight infants with intrahepatic cholestasis prior to cholestyramine in concentrations from 0.84 to 14.19 g/l (mean: 3.13 g/l). After cholestyramine, LP-X decreased in all by an average of 78% (P less than 0.005). The other eight infants did not have LP-X before or after cholestyramine. This study shows that LP-X in the serum of infants with cholestatic jaundice indicates severe cholestasis, but is not itself diagnostic of biliary atresia. The differentiation of biliary atresia from other diseases is readily achieved, as the administration of cholestyramine for 2-3 weeks causes a marked decrease of serum LP-X in patients with patent extrahepatic bile ducts. The absence of serum LP-X excludes biliary atresia.