Developmental stage modifies diet-induced peripheral insulin resistance in rats.

In the present study, the effects of age and diet on glucose disappearance and tissue-specific glucose uptake (R'g) were examined under basal or hyperinsulinemic, euglycemic conditions in male Sprague-Dawley rats. Rats were equicalorically fed either a high-starch diet (68% of kcal), high-fat diet (HFD; 45% of kcal), or high-sucrose diet (68% of kcal), beginning at either 5 (W; weanling), 10 (Y; young), 18 (M; mature), or 58 wk (O; older) of age for 5 wks (n = 6-9. group(-1) x diet(-1)). Body weight gain was not significantly different among dietary groups within a given age. Significant (P< 0.05) age effects were observed on basal and clamp free fatty acid concentrations. Significant diet effects were observed on basal and clamp triglyceride concentrations. There were significant diet and age effects on basal skeletal muscle R'g. This interaction was primarily due to an age-associated increase in basal R'g microg x g(-1). min(-1)) in HFD (gastrocnemius R'g: 0.9+/-0.2 in W, 1.1+/-0.2 in Y, 1.8+/-0.2 in M, 2.5+/-0.2 in O). Both age and diet significantly decreased insulin-stimulated muscle R'g. However, whereas age-associated reductions in both glucose-6-phosphate concentration and glycogen synthase activity were observed, significant diet effects were observed on glucose-6-phosphate concentrations only. Age significantly reduced basal and clamp adipose tissue R'g when expressed per gram of tissue but significantly increased R'g when expressed per total fat pad mass. These data suggest that diet-induced changes in peripheral glucose metabolism are modulated by age.

Adipose tissue-derived tumor necrosis factor activity correlates with fat cell size but not insulin action in aging rats.

Adipose tissue-derived tumor necrosis factor (AT-TNF) protein and messenger RNA (mRNA) has been shown to correlate with insulin resistance in some studies. However, in a study using different aged Fischer 344 rats, AT-TNF activity correlated more strongly with cell size than with fasting plasma insulin. The present study was undertaken to more carefully examine the relationship among AT-TNF, adipose cell size, and insulin action using more precise measures of insulin action. Basal and hyperinsulinemic, euglycemic clamps were performed in male Sprague Dawley rats at four different ages (8, 13, 21, and 61 weeks old). [3-(3)H]glucose and 2-deoxy-D-[1-(14)C]glucose were used to assess glucose kinetics and tissue-specific glucose uptake. Because TNF activity represents the summation of TNF synthesis, secretion, and the amount of soluble inhibitors present, TNF activity was measured using a bioassay, in addition to measuring TNF protein and mRNA levels. AT-TNF activity increased significantly with age, as did the glucose infusion rate, a measure of whole body insulin resistance. However, AT-TNF activity did not correlate with any parameter of insulin action measured during the hyperinsulinemic, euglycemic clamps. In epididymal fat, AT-TNF activity correlated with: glucose infusion rate: r = -0.50, P = 0.17; rate of appearance: r = -0.19, P = 0.35; rate of disappearance: r = 0.08, P = 0.69. As was noted before, AT-TNF activity correlated well with fat cell size (r = 0.76, P < 0.001 in epididymal fat; r = 0.58, P = 0.007 in SUB fat). These data suggest that although AT-TNF activity and insulin resistance increase with age, the two are not functionally related. These data do not eliminate the potential role of nonadipose TNF in the regulation of insulin action.

Synthesis and accumulation of a receptor regulatory protein associated with lipid droplet accumulation in 3T3-L1 cells.

Synthesis and accumulation of the recently identified prostaglandin F2alpha receptor regulatory protein (FPRP) was found to correlate closely with lipid droplet accumulation by 3T3-L1 preadipose cells. FPRP, a transmembrane glycoprotein, has been shown to regulate the binding of ligand to certain seven-transmembrane receptors. Anti-FPRP immunohistochemistry, Western blotting, and metabolic labeling/immunoprecipitation experiments demonstrated that FPRP was not detectable in undifferentiated 3T3-L1 cells. Interestingly, low levels of FPRP mRNA were detected in the undifferentiated 3T3-L1 cells. After induction of adipose differentiation, FPRP mRNA increased approximately 3 fold whereas FPRP synthesis increased approximately 50 fold. Differentiation induction with either dexamethasone/insulin/isobutylmethylxanthine or the thiazolidinedione derivative ADD 4743 were both effective at inducing FPRP accumulation and accumulation of lipid droplets. By co-immunohistochemical and lipid staining, greater than 99% of the cells accumulating lipid droplets possessed FPRP. FPRP mRNA and protein are also found in rat adipose tissue. Treatment of 3T3-L1 cells with an FPRP anti-sense oligonucleotide during differentiation decreased FPRP accumulation and resulted in a decrease in lipid droplets without altering the level of induction of a late marker of adipocyte differentiation, glycerol-3-phosphate dehydrogenase activity. Transient expression of an FPRP cDNA in undifferentiated 3T3-L1 cells was insufficient to induce lipid droplet accumulation.

High fat diets elevate adipose tissue-derived tumor necrosis factor-alpha activity.

Adipose tissue-derived tumor necrosis factor-alpha (AT-TNF) has been associated with genetic models of insulin resistance and obesity. It is presently unknown if secreted AT-TNF protein is bioactive or whether it can be increased by environmentally induced obesity. In this study, male Wistar rats were fed either a low fat (LF; 12% of energy from corn oil) or a high fat (HF; 45% of energy from corn oil) diet for 5 weeks. From previous data, it is known that after 3 weeks, HF fed animals are obese and insulin resistant compared with the LF group. Hence, animals were killed at 1 week of HF feeding, during the acute response to the diet, and at 5 weeks, when differences in body fat are manifest. Weight gain was significantly increased by diet (P = 0.03) and time (P < 0.0001). AT-TNF bioactivity was measured on secreted protein collected from medium of minced, incubated epididymal (EPI), mesenteric (MES), and retroperitoneal (RETRO) fat pads. AT-TNF bioactivity was significantly increased by diet (P = 0.003) in the RETRO pad and tended to increase (P = 0.07) in EPI. AT-TNF activity was unaffected by diet or time in the MES pad. In the RETRO pad, TNF activity correlated negatively with RETRO fat cell number (r = -0.46, P = 0.002). Secreted AT-TNF protein did not correlate with AT-TNF activity but instead decreased in RETRO with time but not diet. In EPI, secreted AT-TNF protein decreased with the HF diet. Thus, these data suggest that high fat diets and obesity can influence AT-TNF bioactivity and secretion but in an apparent fat pad-specific manner.

Prevention of diet-induced obesity in transgenic mice overexpressing skeletal muscle lipoprotein lipase.

Transgenic (Tg) FVB/N mice were produced that overexpress human lipoprotein lipase (LPL) in skeletal muscle using the muscle creatine kinase promoter and enhancers. It was hypothesized that, by overexpressing LPL in muscle, high fat feeding-induced obesity would be prevented by diverting lipoprotein-derived triglyceride fatty acids away from storage in adipose tissue to oxidation in muscle. Mice were examined both at 6 wk of age before high fat (HF) feeding and at 19 wk of age after 13 wk of HF (46.1% fat) or high carbohydrate (HC) feeding (11.5% fat). At 6 wk in heterozygous Tg mice, LPL was increased 11-fold in white muscle and 2.5-fold in red muscle, but not in cardiac muscle or spleen, brain, lung, kidney, or adipose tissue. Plasma triglycerides (mg/dl) were lower in Tg mice (87 +/- 7 vs. 117 +/- 7, P < 0.0001), and glucose increased (201 +/- 9 vs. 167 +/- 8 mg/dl, P = 0.029). There were no differences in body weight between Tg and nontransgenic (nTg) mice; however, carcass lipid content (% body wt) was significantly decreased in male Tg mice at 6 wk (7.5 +/- 1.0 vs. 9.0 +/- 1.0%, P = 0.035). Body composition was not different in female Tg mice at 6 wk. Overall, when Tg mice were fed either a HC or HF diet for 13 wk, plasma triglycerides (P < 0.001) and free fatty acids (P < 0.001) were decreased, whereas plasma glucose (P = 0.01) and insulin (P = 0.05) were increased compared with nTg mice. HF feeding increased carcass lipid content twofold in both male (10.3 +/- 1.1 vs. 21.4 +/- 2.6%, HC vs. HF, P < 0.001) and female nTg mice (6.7 +/- 0.9 vs. 12.9 +/- 1.8%, P = 0.01). However, the targeted overexpression of LPL in skeletal muscle prevented HF diet-induced lipid accumulation in both Tg male (10.2 +/- 0.7 vs. 13.5 +/- 2.2%, HC vs. HF, P = NS) and female Tg mice (6.8 +/- 0.6 vs. 10.1 +/- 1.4%, P = NS). The potential to increase LPL activity in muscle by gene or drug delivery may prove to be an effective tool in preventing and/or treating obesity in humans.

Adipose tissue-derived tumor necrosis factor-alpha activity is elevated in older rats.

High levels of adipose tissue-derived tumor necrosis factor-alpha (AT-TNF) mRNA and protein have previously been associated with genetic models of obesity and insulin resistance. Because there are endogenous TNF inhibitors it is unknown if AT-TNF activity is also increased. We hypothesized that AT-TNF activity would increase in older animals because of an accumulation of fat mass. We chose to study 2 different-aged male Fischer 344 rats, 3-month-old (young) and 14-month-old (mature) because fat mass should be quite different but insulin action on glucose metabolism similar. Indeed, mature rats had over 1.5-fold more fat mass, but whole body insulin resistance, as estimated by fasting plasma insulin, was similar to young rats. Mature rats had twice as much AT-TNF activity as the young in both the epididymal (EPI) and retroperitoneal (Retro) fat pads (p < .0005). AT-TNF correlated with fasting plasma insulin in Retro only (r = .48, p = .04). AT-TNF activity strongly correlated with cell size in both EPI and Retro (r = .79 and .81, respectively, p < .0001). Because cytokines can be regulated at several levels, AT-TNF activity, protein, and mRNA were measured. AT-TNF protein levels were higher in young rats, suggesting that these animals may secrete an inhibitor that reduces AT-TNF activity. There were no significant differences in AT-TNF mRNA between groups. Since TNF has been shown to affect several key genes in tissue culture, mRNA for lipoprotein lipase, hormone-sensitive lipase, and Glut4 were measured. No differences were found between groups. In summary, AT-TNF activity increased in mature animals in relation to adipose cell size.

Tumor necrosis factor-alpha eliminates binding of NF-Y and an octamer-binding protein to the lipoprotein lipase promoter in 3T3-L1 adipocytes.

TNF alpha has been shown to reduce lipoprotein lipase (LPL) activity in adipose tissue. Regulation of LPL by TNF alpha occurs at the level of LPL gene transcription and posttranscriptionally. To elucidate further the transcriptional mechanism of TNF alpha inhibition of LPL gene transcription, transfection analysis was used to locate the site(s) of the LPL promoter that imparts the TNF alpha response. Transient transfections using LPL promoter deletions fused to luciferase in differentiated 3T3-L1 cells with and without TNF alpha treatment indicated that a DNA region downstream of -180 bp confers the TNF alpha effect. Electrophoretic mobility shift assays using two 32P-labeled LPL probes spanning the region between -180 and +44 bp revealed the loss of several LPL DNA-protein interactions after TNF alpha treatment, including the binding of NF-Y to the CCAAT box and a protein to the octamer consensus sequence. Protein binding to the OCT-1 consensus sequence is unaffected until after 4 h of TNF alpha treatment. In addition, the amount of mRNA for OCT-1 is not altered with TNF alpha treatment. These results indicate that TNF alpha regulates at least two DNA-binding proteins on the proximal promoter, thereby inhibiting LPL gene transcription.

The role of the inferior colliculus in a genetic model of audiogenic seizures.

Previous studies have shown the functional importance of the inferior colliculus (IC) for the propagation and initiation of audiogenic seizures in several models of epilepsy in rats. A review of the cell types and cytoarchitecture of the IC, including its three major subdivisions, is presented. Significant increases in GABA levels and the number of GABAergic neurons are found in the central nucleus of the IC (ICCN) of genetically epilepsy-prone rats (GEPR-9s) as compared to Sprague-Dawley rats that do not display audiogenic seizures. Two independent anatomical methods were used to determine the number of GABAergic neurons, immunocytochemistry and in situ hybridization. In both types of preparation, the labeled cells in the ICCN appeared to be of different sizes but the number of small cells with diameters less than 15 microns showed the greatest increase. Nissl-stained sections showed that the total number of neurons in the ICCN was increased in GEPR-9s and indicated that the increase in GABAergic neurons was not due to a change in the phenotype of collicular neurons from non-GABAergic to GABAergic. The number of small neurons in Nissl-stained sections of the ICCN was shown to correlate with seizure severity in the offspring of crosses made between Sprague-Dawley rats and GEPR-9s. Furthermore, the GEPR-3s that display moderate seizures showed a significant increase in the number of small neurons in the ICCN, and the magnitude of this increase was predicted from this correlation. Finally, the use of knife cuts through the midbrain indicated that the ICCN sends an important projection to the external nucleus and that this projection plays a vital role in the propagation of seizure activity from the site of seizure initiation in the ICCN. It remains to be resolved how the increase in small GABAergic neurons in the ICCN is responsible for the known pharmacological defects observed at GABAergic synapses.

An inbred epilepsy-prone substrain of BALB/c mice shows absence of the corpus callosum, an abnormal projection to the basal forebrain, and bilateral projections to the thalamus.

BALB/c mice lack a corpus callosum in about 11% of the population. Two inbred substrains of BALB/c mice, epilepsy-prone (EP) and epilepsy-resistant (ER), have been examined to determine whether these substrains differ in regard to corpus callosum morphology. Further, this study addressed the issue of whether misrouted cortical axons form an aberrant pathway instead of the corpus callosum. Initial studies that examined fresh brain tissue of adult animals revealed normal corpora callosa in all ER mice but deficient or absent corpora callosa in all EP mice. Subsequently, Dil crystals were placed in the motor cortices of aldehyde-fixed brains of 2-week-old animals to investigate cortical projections in both inbred substrains of mice. Fluorescent microscopy revealed that all of the ER animals had normal corpora callosa, whereas all EP animals exhibited either reduced corpora callosa (partially callosal) or an absence (acallosal) of this structure. Both acallosal and partially callosal EP mice displayed an extensive, aberrant projection to the basal forebrain as well as bilateral projections to midline and intralaminar thalamic nuclei. The fibers projecting to the basal forebrain arose from the cortex, coursed toward the midline before turning ventrally along the midline, and appeared to terminate in the medial septal nucleus and the nucleus of the diagonal band. ER animals lacked this aberrant cortical projection to the basal forebrain. Electron microscopic results obtained from EP mice indicated that labeled axons in this aberrant pathway formed axosomatic, axodendritic, and axospinous synapses with the neurons in the medial septal/diagonal band complex. The function of the aberrant projection to the basal forebrain remains unknown but it may provide an abnormal excitatory input to a region that provides cholinergic and GABAergic input to the cerebral cortex and hippocampus. The additional projections to midline and contralateral intralaminar thalamic nuclei in EP mice may function to intensify the synchronization of bilateral discharges.

Thromboxane production in copper-deficient and marginal platelets: influence of superoxide dismutase and lipid hydroperoxides.

Platelet thromboxane (TX) production was examined in response to dietary copper. Groups of eight rats were fed copper-deficient, -marginal, and -adequate diets providing 0.5, 1.7, and 7.5 micrograms Cu/g, respectively, with controlled dietary Se and vitamin E. Platelets were purified and washed by centrifugation. Separate platelet samples from each rat were challenged with 10 micrograms/ml of collagen and 1 unit/ml (27.3 nM) of thrombin in Tyrode's buffer, 2.0 mM Ca2+. Platelet copper-dependent superoxide dismutase (CuSOD) activity showed a significant depression with reduced diet copper, but platelet glutathione peroxidase activity was unaffected. Challenged platelet TX production showed a significant 1.5- to 2.5-fold increase in response to both dietary copper deficiency and marginality, with highly significant negative correlations between challenged platelet TX production and platelet CuSOD activity and between TX production and copper status (liver copper). Endogenous (unchallenged) platelet lipid hydroperoxide concentrations, measured as free fatty acid hydroperoxides by a glutathione-disulfide-specific glutathione reductase recycling assay, showed a nonsignificant 47-67% increase in copper deficiency. Pooled data showed a significant 71% increase in platelet lipid hydroperoxides in copper deficiency. Platelet TX production showed a significant correlation with endogenous lipid hydroperoxides. The results suggest that dietary copper insufficiency increases platelet TX synthesis through changes in CuSOD in a dose-responsive (diet copper and platelet CuSOD activity) manner, and that platelet TX synthesis is influenced by lipid hydroperoxides (peroxide tone).

Changes in lens membrane major intrinsic polypeptide during cataractogenesis in aged Hannover Wistar rats.

Cataracts previously have been shown to occur spontaneously in aged Hannover Wistar rats. The morphology and time course of opacification of these cataracts are very similar to those appearing in the human lens, where it has been previously shown that the major intrinsic polypeptide (MIP26K) of the fiber cell membrane undergoes covalent modification during cataractogenesis. To ascertain possible biochemical similarities between the two cataract systems, antisera were made against synthetic peptides corresponding to the sequence of MIP26K to probe Western blots of lens proteins from transparent versus opaque lenses from normal aged rats. The results of this analysis showed that these antisera can detect the presence of covalent changes occurring in the MIP26K molecule during the development of cortical opacities in the normal aged rat.

Detection of growth-stimulating activity in the proximal small intestine during weaning in the suckling rat.

Adaptive changes in enzyme expression and cell proliferation occur in the small intestine of the suckling rat at the beginning of the 3rd postnatal week. This physiological adaptation can be modulated by factors including diet or glucocorticoids. We have previously described an intestinal growth-stimulating fraction derived from the remnant small bowel after resection and found that enteral nutrition is critical for its detection. In view of the similarity between the changes in cell proliferation that occur between 15 and 22 days postnatally and those immediately after resection, we sought to determine whether the small intestinal mucosa of the neonatal rat also contains a similar growth-stimulating fraction. Our results show that extracts of the proximal intestine prepared from 15-day-old rats do contain the growth-stimulating fraction. The activity was not detectable in maternal milk nor in the intestinal extract from 8-day-old rats. When the suckling rats were deprived of solid food, the activity was not detectable in the 15-day-old group. Gel filtration of the acidic extract on a G-25 Sephadex column revealed that the active component is made up of two molecular weight species (approximately 4,500 and 1,500 daltons) similar to that described in the proximal intestine of the postresectional model. These findings suggest that dietary factors may play a role in modulating the proliferative changes that occur at the time of weaning by way of the growth-stimulating fraction.

A growth-stimulating activity derived from the proximal small intestine is associated with an adaptive response.

Luminal nutrition is important for the maintenance of small intestinal structure and function. The equilibrium between crypt cell production and villous cell loss in the mucosal epithelium of the small intestine is altered under certain conditions such as after a small bowel resection. Immediately after resection, there is a marked increase in crypt cell proliferation giving rise to an adaptive hyperplasia in the remnant intestine and for this response luminal nutrition is a critical factor. We have previously demonstrated the presence of a growth-stimulating (GS) activity in a heat-stable acidic extract of the rat proximal intestine 24, 48, and 96 h after resection, which is coincidental with an increase in crypt cell proliferation as measured by thymidine kinase activity. Eight days after resection when the GS activity is no longer detectable, the thymidine kinase activity returns to control values. The molecular weights of the peptides associated with this GS activity are 4500 and 1500, as determined by Sephadex gel filtration. Of note is that the oral intake of food is necessary for the appearance of the GS activity postoperatively. The presence of the GS activity has also been demonstrated upon refeeding after a fast, as well as at weaning in the rat, two physiological situations known to be associated with increased proliferation in the small intestine. This GS activity in the proximal intestine first detected in the resection model may represent a general mechanism by which food controls the cell renewal pattern of the small intestine.

Determination of picomole quantities of hydroperoxides by a coupled glutathione peroxidase and glutathione disulfide specific glutathione reductase assay.

A procedure to quantitate picomole amounts of hydroperoxides based on GSSG formation is described. Hydroperoxides are incubated with GSH and glutathione peroxidase, and the GSSG formed is measured by a GSSG-specific glutathione reductase recycling assay. Prior to analysis the remaining GSH is removed with N-ethylmaleimide. N-Ethylmaleimide inhibition of the recycling assay is prevented by alkaline hydrolysis of the N-ethylmaleimide, without GSSG hydrolysis, at pH 11. The method is rapid, 30 min, with a limit of detection of 142 pmol calculated by linear regression analysis. Stoichiometric amounts of GSSG are produced in response to hydroperoxides. An application of the method is shown with air oxidation of arachidonic acid solutions over 4 h at room temperature. The method is sufficiently sensitive to quantitate the low amounts of hydroperoxides present in fresh arachidonic acid samples.

High incidence of upper gastrointestinal tract involvement in children with Crohn disease.

This retrospective study of Crohn disease in 230 children and adolescents with a mean age of 12.5 years at the time of diagnosis and an average follow-up of 6.6 years showed that 30% had lesions of the esophagus, stomach, and duodenum. Three patients had Crohn disease isolated to the upper gastrointestinal tract. The 169 patients with both small and large bowel disease were at greater risk (33%, P less than .05) of having upper gastrointestinal lesions than the 37 with isolated small bowel disease and the 21 with disease limited to the colon and/or rectum. An aggregate of symptoms and signs more likely present in those with upper gastrointestinal involvement included: dysphagia, pain when eating, nausea and/or vomiting, and aphthous lesions of the mouth. Furthermore, weight loss was more severe and hypoalbuminemia more frequent. Because upper gastrointestinal series x-ray studies failed to detect upper gastrointestinal lesions in 13 patients of 69 of those with upper gastrointestinal disease, endoscopy should be considered in all children and adolescents in whom a diagnosis of Crohn disease is entertained. Endoscopy and biopsy of the upper gastrointestinal tract should be done in any patient with symptoms suggestive of proximal involvement. Finally, in view of the fact that endoscopy established the diagnosis of Crohn disease in five patients previously thought to have chronic ulcerative colitis, the procedure should routinely be performed in all patients with chronic ulcerative colitis or indeterminate colitis before surgery is performed.

Chronic intermittent elemental diet improves growth failure in children with Crohn's disease.

Growth failure often complicates Crohn's disease in pediatric patients and is principally due to inadequate caloric intake. To assess whether intermittent courses of an elemental diet could reestablish growth, 8 children (aged 9.8-14.2 yr) with Crohn's disease and growth failure entered into a prospective trial. Each patient was studied during an observation year on standard therapy, then for an experimental year during which they received enteral elemental diet 1 out of 4 mo. An age- and disease-matched control group of 4 patients was treated by conventional medical therapy during both years. Elemental diet therapy was administered nocturnally, at home, by continuous nasogastric infusion and increased the daily caloric intake by 25% (p less than 0.01). Anthropometric measurements demonstrated significant height and weight gains in the elemental diet group vs. controls (p less than 0.01). Crohn's disease activity index and prednisone intake decreased significantly in patients receiving elemental diet therapy when compared with themselves and with controls on conventional medical therapy (p less than 0.05). In contrast, the rate of pubertal development was similar in both groups irrespective of the treatment modality. This study demonstrates that chronic intermittent elemental diet effectively reverses growth arrest, while decreasing prednisone requirements and Crohn's disease activity index in pediatric Crohn's disease patients prior to puberty.

Role of food in appearance of growth-stimulating activity in rat proximal intestine after small-bowel resection.

The oral intake of food is important for the observed compensatory hyperplasia in the remnant small intestine after resection but the molecular events governing this response are not known. Peptides, of molecular weight 4500 and 1000 daltons, present in the the proximal intestine for 96 hr after resection and mitogenic for the intestine have been implicated in the adaptive hyperplasia. In this study, the role of food in the appearance of these peptides was assessed. The results show that after resection, rats nourished intravenously demonstrated neither a significant adaptation nor any of the detectable mucosal mitogens, whereas the rats nourished intragastrically demonstrated both hyperplasia and the mitogenic peptide(s). The association of the hyperplasia with the appearance of the mitogenic peptides in the small intestine suggests that they are important in the mechanism by which food promotes the adaptive hyperplasia.

Primary sclerosing cholangitis in children: study of five cases and review of the literature.

Primary sclerosing cholangitis in five children is described and 78 cases in the pediatric age group are reviewed. In 24% of the cases, primary sclerosing cholangitis is not associated with an underlying disease and may appear to be prolonged cholestasis of infancy. When an associated condition is present, chronic inflammatory bowel disease, in particular ulcerative colitis, is most common (47%). Histiocytosis X and a variety of immune disorders account for 15% and 10% of cases, respectively. Primary sclerosing cholangitis should be considered in the differential diagnosis of chronic liver disease in the pediatric age group, even in young infants. Results of this survey demonstrate that neither clinical features nor liver function tests are reliable diagnostic predictors, that histologic changes are often nonspecific, and that cholangiography is essential to establish the correct diagnosis.