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Familial autoimmune enteropathy with circulating anti-bullous pemphigoid antibodies and chronic autoimmune hepatitis
The Journal o f Pediatrics Volume 125, Number 6, Part 1
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Fig. 4. Small bowel biopsy specimen. Severe villous atrophy, crypt hyperplasia, and inflammatory cells in the chorion are present. Note the focal epithelial desquamation. (Hematoxylin-eosia stain; X63.) bronchioles. The liver was enlarged and showed extensive hematopoiesis and cholestasis. The pancreas had diffuse interstitial infiltration by lymphocytes. Patient 2. Patient 2 was the younger brother of patient 1. When patient 2 was 3 weeks of age, severe secretory diarrhea (132 ml/kg per day) developed; stool concentrations of sodium were 80 to 125 mmol/L and of chloride were 80 to 135 mmol/L. The infant was treated with TPN, but it had no effect on the number of stools. A biopsy specimen from the intestine demonstrated total villous atrophy with crypt hyperplasia, diffuse inflammation of the lamina propria, and focal epithelial desquamation (Fig. 1). Serum concentrations of proteins, albumin, and immunoglobulins (IgG, IgM, IgA) were low; the IgE value was high. c0-Antitrypsin clearance was increased. The infant was treated with TPN from the first month of life. Mild eczema occurred from 2 to 16 months of age. On the basis of the presence of anti-epithelial cell antibody, various immunosuppressive treatments were tested, including intravenous administration of immune globulins (400 mg/kg per day), prednisone (600 mg/m 2 per day for 3 days and then 2 mg/kg per day), prednisone and cyclosporine, cyclosporine alone, and a combination of prednisone, cyclosporine, and azathioprine. Stool outflow decreased during immunosuppressive therapy, but the histologic findings of jejunal and colonic biopsies before and after treatment remained unchanged. At the age of 16 months, the child was still completely dependent on TPN. Small and large bowel abnormalities had improved but the changes persisted; kidney biopsy specimen showed focal interstitial lymphoid infiltrates and fibrosis without any significant tubular lesion. Three of 10 glomeruli were sclerotic and one had segmental hyalinosis. Immunofluorescence studies revealed mild diffuse me-
sangial and focal subendothelial granular deposits of IgM and the complement subunit C 1q. The liver was enlarged (5 cm below the costal margin) and firm. Serum alanine aminotransferase activity was two to three times the normal value, and anti-smooth muscle antibody titer had fluctuated from 1:160 to 1:320 since the sixth month of life. Liver biopsy results (Fig. 2) showed a moderately aggressive chronic hepatitis with extensive bridging fibrosis, mild piecemeal necrosis, and moderate lymphoid portal inflammation; giant cells and mild intracytoplasmic biliary deposits were present in the lobules. There had been no biochemical or clinical evidence of endocrinopathy since the sixth month of age. Repeated thyroid plasma thyroxine and thyrotropin concentrations and endocrine pancreatic function tests (blood glucose, insulin, glucagon) were always normal. Because of the hepatic disorders, treatment with prednisolone (1.5 mg/kg per day) and azathioprine (1.5 mg/kg per day) was initiated. When the child was 18 months of age, the diarrhea decreased until one or two normal stools were passed each day; however, TPN was maintained because he did not feed well. At the age of 24 months, he was dependent on TPN but also ate a little. He was treated with prednisone (0.5 mg/kg every other day) and azathioprine (1.5 mg/kg every day). The child had normal development with respect to weight, height, and head circumference. Mental development was also normal and the child walked at t8 months of age. Biopsy specimens from the small and large bowel demonstrated short regenerated villi, slight crypt hyperplasia, and mild focal inflammation. The liver remained enlarged and firm, 'alanine aminotransferase values remained elevated (four to six times normal), and the anti-smooth muscle antibody titer was 1:320. A liver biopsy specimen again showed chronic aggressive
Familial autoimmune enteropathy with circulating anti-bullous pemphigoid antibodies and chronic autoimmune hepatitis A l a i n L a c h a u x , MD, R. Bouvier, MD, E. C o z z a n i , MD, I. L o r a s - D u c l a u x , MD, J. Kanitakis, MD, M. C h e v a l l i e r , MD, a n d D. Kaiserlian, PhD From the Service d'H6patogastroent6rologie et Nutrition P6diatriques, Pavilion S, the Laboratoire d'Anatomie Pathologique, BOtiment 40, and INSERMU346, Unit~ Peau Humaine et ImmunitY, Pavilion R, H6pital Edourdo Herriot, and the Laboratoire d'lmmunopathotogie, and INSERM U 404, Unit6 Immunologie et Vaccination, Pasteur Institut, Lyon, France In a f a m i l y of four children (two boys and two girls), the two brothers had severe, p r o t r a c t e d w a t e r y d i a r r h e a b e g i n n i n g at 2 a n d 3 weeks of life, r e s p e c t i v e l y . D u o d e n a l m u c o s a in both patients showed t o t a l villous a t r o p h y a n d severe inf l a m m a t o r y infiltration of the entire b o w e l . The first p a t i e n t also had l y m p h o i d c e l l infiltration of the p a n c r e a s a n d d i e d at 6 weeks of a g e . The s e c o n d b o y is a l i v e at 2 years of a g e and is i m m u n o c o m p e t e n t , but still r e c e i v e s t o t a l parenteral nutrition. Indirect i m m u n o f l u o r e s c e n c e studies r e v e a l e d c i r c u l a t i n g antib o d i e s to enterocytes, smooth muscle, thyroid, and islet cells. Bullous p e m p h i g o l d a n t i b o d i e s (230 and 180 kd), s p e c i f i c for h e m i d e s m o s o m a l proteins a n d usually a s s o c i a t e d with a s u b e p i d e r m a l blistering skin disease, were d e t e c t e d by d i r e c t and i n d i r e c t i m m u n o f l u o r e s c e n c e studies a n d by Western i m m u n o b l o t . A d i a g n o s i s of a u t o i m m u n e hepatitis was m a d e , b a s e d on e v i d e n c e of c h r o n i c a c t i v e hepatitis and c i r c u l a t i n g anti-smooth muscle a n t i b o d y . Immunosuppressive treatments i n d u c e d partial c l i n i c a l remission of the d i a r r h e a but no resolution of the small b o w e l injury. At 16 months of age, remission of the diarrhea occurred, but persistent a u t o i m m u n e hepatitis led us to m a i n t a i n t r e a t m e n t with p r e d n i s o n e a n d azathioprine, a n d later with c y c l o s p o r i n e . In this child, as in other patients with a u t o i m m u n e disease, the link b e t w e e n a u t o a n t i b o d i e s a n d o r g a n d a m a g e remains uncertain but i m m u n o s u p p r e s s i v e t r e a t m e n t is indic a t e d . (J PEDIATR1994;125:858-62) A u t o i m m u n e enteropathy is a well-defined entity, t, 2 but the role of the circulating antibodies to gut epithelial cells in the pathogenesis of the disease remains unknown. W e report a new case t h a t meets the criteria for A I E and a u t o i m m u n e hepatitis; the findings include circulating antibodies to bullous pemphigoid antigen.
Submitted for publication April 27, 1994; accepted July 12, 1994. Reprint requests: Alain Lachaux, MD, Service d'H+patogastroent6rologie et Nutrition P6diatriques, Pavilion S, H6pital Edouard Herriot, 69437 Lyon, Cedex 03 France. Copyright | 1994 by Mosby-Year Book, Inc. 0022-3476/94/$3.00 + 0 9/20/59060
858
CASE REPORTS Patient 1. Patient 1 was the first child of unrelated parents and the elder brother of patient 2. He had severe, protracted diarrhea beginning at 2 weeks of age and died at 6 weeks of age despite total parenteral nutrition. Autopsy findings revealed total villous atroAECA AIE TBS TPN
Anti-epithelial cell antibody Autoimmune enteropathy Tris-buffered saline solution Total parenteral nutrition
phy over the entire small bowel and marked lymphoid cell infiltration in the lamina propria of the entire digestive tract. Scattered lymphoid infiltrates were also present in the kidney and around the
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Fig. 2. Liver biopsy specimen shows aggressive hepatitis: a portal tract with bridging fibrosis, lymphoid infiltration, and a few giant cells. (Trichrome stain; X160.)
hepatitis. Treatment with cyclosporine was started. To date, after 6 months of cyclosporine therapy, the patient is doing well, alanine aminotransferase levels are normal, and the diarrhea has not recurred. METHODS Direct immunofluorescence tests. We performed direct immunofluorescence tests on apparently normal skin of patient 2. Biopsy tissue was snap-frozen in liquid nitrogen, and 4 #m thick cryostat sections were prepared on a cryomicrotome and placed onto glass slides. The following steps were then performed: (1) incubation with bovine serum albumin 1% in phosphate-buffered saline solution (15 minutes); (2) incubation with a mixture of fluorescein isothiocyanateconjugated anti-IgG, anti-IgA, anti-IgM, and anti-C3. After a final wash in phosphate-buffered saline solution, the sections were examined with a Zeiss immunofluorescence microscope equipped with epi-illumination. Indirect immunofluorescence studies. We studied normal human gut and skin for detection of A E C A and anti-basement membrane zone antibodies. Circulating antibodies were detected by standard indirect immunofluorescence, performed on 4 #m cryostat sections of normal tissue including human skin split by NaC1, 1.0 mol/L. 3 The sections were first incubated for 30 minutes at room temperature with serial dilution of patient's serum followed by a
30-minute incubation with fluorescein isothiocyanate-conjugated goat anti-human total Ig antibodies (Dakopatts, Copenhagen, Denmark). Sodium dodccyl sulfate-polyacrylamidc gel electrophoresis and immunoblot analysis. Epidermal proteins were obtained from normal human keratinocyte cultures according to a previously described technique3: proteins were extracted from the culture fasks after incubation for 30 m~nutes at 40 ~ C, with a lysis buffer (10 mmol Tris HC1 [pH 7.8] per liter, with 2% sodium dodecyl sulfate and 5% B-mercaptoethanol, and 100 tool ethylenediamine tetraacetic acid per liter, 200 mol phenylmethylsulphonyl fluoride per liter, 1 mmol leupeptin per liter, and 1 mmol pepstatin per liter). Epidermal proteins were separated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (acrylamide concentration, 6%) under reducing conditions as previously described.4 The proteins were electrophoretically transferred to nitrocellulose filters. The efficiency of transblotting was checked by ponceau red staining of transferred epidermal and standard proteins. Nitrocellulose strips were then sequentially incubated with the following solutions: (1) 5% dried milk in 10 mmol Tris HC1 per liter and 140 mmol SaC1 [pH 7.4] per liter Tris-buffered saline solution/milk) for 1 hour at 37 ~ C or overnight at 40 ~ C; (2) 1:100
The Journal of Pediatrics Volume 125, Number 6, Part 1
dilution of each serum sample in TBS/milk for 1 hour at 37 ~ C; (3) 1:500 dilution of a biotinylated goat antihuman immunoglobulin (Amersham, Buckinghamshire, United Kingdom) in TBS/milk for 30 minutes at room temperature; and (4) 1:3000 dilution of streptavidin-alkaline phosphatase substrate solution (diethanolamine buffer, nitroblue tetrazolium, 5-bromo-4-chloro-3-indoyl phosphate, Amersham). Intervening washes were performed in TBS with 0.1% polyoxyethylenesorbitan monolaurate (Tween 20). RESULTS Immunologic studies. Serum immunoglobulin levels (IgG, IgA, and IgM) were norma ! or initially low, whereas IgE levels were always elevated until 24 months of age. Antibodies to vaccines were present. Radioallergosorbent test results were negative for allergies to milk, soybean, wheat, rice, fish, chicken, and peanuts. No T- or B-cell immune function deficiency was detected. The CD3 +, CD4 +, CD8 +, and B-lymphocyte counts were normal, as were the T-lymphocyte stimulation test results (phytohemagglutinin, pokeweed mitogen, and concanavalin A), complement factor levels, and neutrophil count. Results of serum immune complex testing were negative. Immunohistochemical staining of biopsy specimens from the patient's Colon and small bowel revealed the presence of CD3 +, CD4 +, CD8 +, and CD25 + cells in the epithelium and the lamina propria. No chromosomal abnormalities were found in blood lymphocytes. Anti-epithelial cell antibody. The presence of circulating AECA in patient 2 was demonstrated by the intense labeling of biopsy specimens from both small and large intestinal epithelium of normal human gut. The labeling persisted with dilution up to 1:100 of the patient's serum and was observed both in cytoplasm and at the membranes of enterocytes; goblet cells were not stained. The circulating autoantibodies to intestinal epithelial cells were IgGs and remained unchanged throughout the course of the disease. Bullous pempbigold antibody. Direct immunofluorescence of cryostat skin sections revealed the presence of linear IgG deposits at the dermoepidermal junction. Indirect immunofluorescence using the patient's serum showed the presence of circulating IgG autoantibodies binding to the epidermal side of a skin specimen split by NaC1, 1.0 mol/L. Immunoblot analysis of the reactivity of circulating autoantibodies with epidermal protein extracts demonstrated that the autoantibodies recognized the bullous pemphigoid antigens Agl and Ag2, of molecular weights 230 and 180 kd, respectively. Other tissue antibodies. High titers of thyroid microsomal and islet cell antibodies were transiently present. Smooth
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muscle antibodies (titer 1:160 to 1:320) were present during the course of the disease. No mitochrondria, liver, kidney microsomal, kidney brush border, or nuclear antibodies were detected. DISCUSSION The pathogenic significance of circulating AECA in patients with autoimmune enteropathy is unknown. In previous studies of children with AIE, the antibody concentration did not necessarily correlate with the severity of the disease. 17 In our patient, no good correlation between antibody titers and the course of the disease was noted. Early in the disease, during different treatments, and at the age of 24 months when the diarrhea was in remission, the titer of circulating A E C A was approximately the same. Thus the possibility remains that the antibody may have been the effect rather than the cause of the bowel injury. This is compatible with autoimmune disease T-cell activation as the primary event and not necessarily as the cause of the organ damage through B-cell activation and antibody production. The association of AIE with diseases such as nephropathy, 1-6 type I diabetes, v and hemolytic anemia 5 is now well recognized; however, the presence of bullous pemphigoid antibodies (with or without skin disease) has not been described in infants with AIE. Bullous pemphigoid is an autoimmune blistering skin disease characterized histologically by subepidermai bullae and immunologically by in vitro deposition of autoantibodies and complement components along the epidermal basement membrane zone; autoantibodies are also present in the serum. The targets of these autoantibodies are components of hemidesmosomes, namely the bullous pemphigoid antigens A g l and Ag2, of molecular weights 230 kd and 180 kd, respectively.3 Western immunoblot studies have demonstrated antibodies against the 230 and 180 kd bullous pemphigoid antigens, different from those previously described in AIE. The pathogenic significance of circulating bullous pemphigoid antibodies is uncertain. 8 Thus, even though bullous lesions did not develop on our patient's skin, he had high levels of IgE and episodes of eczema. Duodenal and colonic biopsy specimens showed epithelial desquamation. An artefact seems unlikely because of the repeated observations on many biopsy specimens. There was no evidence that bullous pemphigoid antibodies had a role in the pathogenesis of tbe gut disease. The possibility of an antigen common to gut mucosa and skin may be considered, and bullous pemphigold antibodies may be synthesized in contact with the gut or skin epithelial cell basal hemidesmosome. 8, 9 Thus it seems worthwhile to screen for these antibodies in the evaluation of patients with AIE. Other studies have revealed smooth muscle antibodies9, 10
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without liver involvement in patients with AIE. In another patient, serum aminotransferase levels were moderately elevated and an atypical liver and kidney microsomal antibody was present, but the liver biopsy specimen revealed a nonaggressive hepatitis with giant cell transformation. 6 Thus our patient had increased alanine aminotransferase levels with smooth muscle antibodies and a histologic appearance of chronic active hepatitis compatible with the diagnosis of autoirnmune hepatitis. Patient 1 died at 6 weeks of age and probably had A I E ; the gut lesions observed at autopsy looked similar to those revealed by endoscopic biopsies in patient 2. In addition, lymphocyte infiltration can involve different organs, and the pancreatic lesions should be interpreted as another target organ. Perhaps this localization of the disease might explain the frequency of type I diabetes observed in patients with AIE. In this noninbred family, two boys wer e affected and the two sisters were healthy. In previous reports, familial occurrence was frequent and A I E affected male subjects almost exclusively. T h e predominance of mate subjects in much of the published data 111 and in this family is in accordance with a sex-linked pattern of inheritance. We adopted a conservative approach to immunosuppressive treatment. The triple-agent immunosuppressive treatment (including prednisolone, cyclosporine, and azathioprine) seemed to be the most effective for reducing stool outflow, but after the diarrhea remitted we chose azathioprine and prednisone because of the autoimmune hepatitis. Secondarily, we introduced cyclosporine alone to control the disease, with apparent benefit. 2-5 In our patient, the link between autoantibodies and widespread organ damage remains uncertain. Further studies of other patients with similar disorders will increase our understanding of this disease, and may elucidate
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whether the autoantibodies present in the patient's blood are causative or occur as a consequence of tissue damage. REFERENCES 1. Colletti RB, Guillot AP, Rosen S, et al. Autoimmune enterop-
2.
3.
4. 5.
6.
7.
8. 9. 10.
11.
athy and nephropathy with circulating anti-epithelial cell antibodies. J PEDIATR 1991;118:858-64. Sanderson IR, Phillips AD, Spencer J, Walker-Smith JA. Response to autoimmune enteropathy to cyclosporin A therapy. Gut 1991;32:1421-5. Machado P, Michalaki H, Roche P, Gaucherand M, Thivolet J, Nicolas JF. Serological diagnosis of bullous pemphigoid (BP): comparison of the sensitivity of indirect immunofluorescence on salt-split skin to immunoblotting. Br J Dermatol 1992;126:236-42. Leammli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680-5. Seidman EG, Lacaille F, Russo P, Galeano N, Murphy G, Roy CC. Successful treatment of autoimmune enteroPathy with cyclosporine. J PEmATR 1990;117:929-32. Mitton SG, Mirakion R, Larcher VF, Dillon M J, WalkerSmith JA~ Enteropathy and renal involvement in an infant with evidence of widespread autoimmune disturbance. J Pediatr Gastroenterol Nutr 1989;8:397-400. Catassi C, Mirakian R, Natalini G, et al. Unresponsive enteropathy associated with circulating enterocyte autoantibodies in a boy with common variable hypogammaglobulinemia and type I diabetes. J Pediatr Gastroenterol Nutr 1988;7:608-13. Besnard V, Nicolas JF. Patbogenesis of bullous pemphigoid: new insights. Eur J Dermatol 1993;3:285-8. Unsworth D J, Walker-Smith JA. Autoimmunity in diarrhoea1 disease. J Pediatr Gastroenterol Nutr 1985;4:375-80. Savage MO, Mirakian R, Wozniak ER, et al. Specific autoantibodies to gut epithelium in two infants with severe protracted diarrhoea. J Pediatr Gastroenterol Nutr 1985;4: 187-95. Powell BR, Buist NRM, Stenzel P. An X-linked syndrome of diarrhea, polyendocrinopathy, and fatal infection in infancy. J PEDIATR 1982;100:731-37.
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Fig. 4. Small bowel biopsy specimen. Severe villous atrophy, crypt hyperplasia, and inflammatory cells in the chorion are present. Note the focal epithelial desquamation. (Hematoxylin-eosia stain; X63.) bronchioles. The liver was enlarged and showed extensive hematopoiesis and cholestasis. The pancreas had diffuse interstitial infiltration by lymphocytes. Patient 2. Patient 2 was the younger brother of patient 1. When patient 2 was 3 weeks of age, severe secretory diarrhea (132 ml/kg per day) developed; stool concentrations of sodium were 80 to 125 mmol/L and of chloride were 80 to 135 mmol/L. The infant was treated with TPN, but it had no effect on the number of stools. A biopsy specimen from the intestine demonstrated total villous atrophy with crypt hyperplasia, diffuse inflammation of the lamina propria, and focal epithelial desquamation (Fig. 1). Serum concentrations of proteins, albumin, and immunoglobulins (IgG, IgM, IgA) were low; the IgE value was high. c0-Antitrypsin clearance was increased. The infant was treated with TPN from the first month of life. Mild eczema occurred from 2 to 16 months of age. On the basis of the presence of anti-epithelial cell antibody, various immunosuppressive treatments were tested, including intravenous administration of immune globulins (400 mg/kg per day), prednisone (600 mg/m 2 per day for 3 days and then 2 mg/kg per day), prednisone and cyclosporine, cyclosporine alone, and a combination of prednisone, cyclosporine, and azathioprine. Stool outflow decreased during immunosuppressive therapy, but the histologic findings of jejunal and colonic biopsies before and after treatment remained unchanged. At the age of 16 months, the child was still completely dependent on TPN. Small and large bowel abnormalities had improved but the changes persisted; kidney biopsy specimen showed focal interstitial lymphoid infiltrates and fibrosis without any significant tubular lesion. Three of 10 glomeruli were sclerotic and one had segmental hyalinosis. Immunofluorescence studies revealed mild diffuse me-
sangial and focal subendothelial granular deposits of IgM and the complement subunit C 1q. The liver was enlarged (5 cm below the costal margin) and firm. Serum alanine aminotransferase activity was two to three times the normal value, and anti-smooth muscle antibody titer had fluctuated from 1:160 to 1:320 since the sixth month of life. Liver biopsy results (Fig. 2) showed a moderately aggressive chronic hepatitis with extensive bridging fibrosis, mild piecemeal necrosis, and moderate lymphoid portal inflammation; giant cells and mild intracytoplasmic biliary deposits were present in the lobules. There had been no biochemical or clinical evidence of endocrinopathy since the sixth month of age. Repeated thyroid plasma thyroxine and thyrotropin concentrations and endocrine pancreatic function tests (blood glucose, insulin, glucagon) were always normal. Because of the hepatic disorders, treatment with prednisolone (1.5 mg/kg per day) and azathioprine (1.5 mg/kg per day) was initiated. When the child was 18 months of age, the diarrhea decreased until one or two normal stools were passed each day; however, TPN was maintained because he did not feed well. At the age of 24 months, he was dependent on TPN but also ate a little. He was treated with prednisone (0.5 mg/kg every other day) and azathioprine (1.5 mg/kg every day). The child had normal development with respect to weight, height, and head circumference. Mental development was also normal and the child walked at t8 months of age. Biopsy specimens from the small and large bowel demonstrated short regenerated villi, slight crypt hyperplasia, and mild focal inflammation. The liver remained enlarged and firm, 'alanine aminotransferase values remained elevated (four to six times normal), and the anti-smooth muscle antibody titer was 1:320. A liver biopsy specimen again showed chronic aggressive
Familial autoimmune enteropathy with circulating anti-bullous pemphigoid antibodies and chronic autoimmune hepatitis A l a i n L a c h a u x , MD, R. Bouvier, MD, E. C o z z a n i , MD, I. L o r a s - D u c l a u x , MD, J. Kanitakis, MD, M. C h e v a l l i e r , MD, a n d D. Kaiserlian, PhD From the Service d'H6patogastroent6rologie et Nutrition P6diatriques, Pavilion S, the Laboratoire d'Anatomie Pathologique, BOtiment 40, and INSERMU346, Unit~ Peau Humaine et ImmunitY, Pavilion R, H6pital Edourdo Herriot, and the Laboratoire d'lmmunopathotogie, and INSERM U 404, Unit6 Immunologie et Vaccination, Pasteur Institut, Lyon, France In a f a m i l y of four children (two boys and two girls), the two brothers had severe, p r o t r a c t e d w a t e r y d i a r r h e a b e g i n n i n g at 2 a n d 3 weeks of life, r e s p e c t i v e l y . D u o d e n a l m u c o s a in both patients showed t o t a l villous a t r o p h y a n d severe inf l a m m a t o r y infiltration of the entire b o w e l . The first p a t i e n t also had l y m p h o i d c e l l infiltration of the p a n c r e a s a n d d i e d at 6 weeks of a g e . The s e c o n d b o y is a l i v e at 2 years of a g e and is i m m u n o c o m p e t e n t , but still r e c e i v e s t o t a l parenteral nutrition. Indirect i m m u n o f l u o r e s c e n c e studies r e v e a l e d c i r c u l a t i n g antib o d i e s to enterocytes, smooth muscle, thyroid, and islet cells. Bullous p e m p h i g o l d a n t i b o d i e s (230 and 180 kd), s p e c i f i c for h e m i d e s m o s o m a l proteins a n d usually a s s o c i a t e d with a s u b e p i d e r m a l blistering skin disease, were d e t e c t e d by d i r e c t and i n d i r e c t i m m u n o f l u o r e s c e n c e studies a n d by Western i m m u n o b l o t . A d i a g n o s i s of a u t o i m m u n e hepatitis was m a d e , b a s e d on e v i d e n c e of c h r o n i c a c t i v e hepatitis and c i r c u l a t i n g anti-smooth muscle a n t i b o d y . Immunosuppressive treatments i n d u c e d partial c l i n i c a l remission of the d i a r r h e a but no resolution of the small b o w e l injury. At 16 months of age, remission of the diarrhea occurred, but persistent a u t o i m m u n e hepatitis led us to m a i n t a i n t r e a t m e n t with p r e d n i s o n e a n d azathioprine, a n d later with c y c l o s p o r i n e . In this child, as in other patients with a u t o i m m u n e disease, the link b e t w e e n a u t o a n t i b o d i e s a n d o r g a n d a m a g e remains uncertain but i m m u n o s u p p r e s s i v e t r e a t m e n t is indic a t e d . (J PEDIATR1994;125:858-62) A u t o i m m u n e enteropathy is a well-defined entity, t, 2 but the role of the circulating antibodies to gut epithelial cells in the pathogenesis of the disease remains unknown. W e report a new case t h a t meets the criteria for A I E and a u t o i m m u n e hepatitis; the findings include circulating antibodies to bullous pemphigoid antigen.
Submitted for publication April 27, 1994; accepted July 12, 1994. Reprint requests: Alain Lachaux, MD, Service d'H+patogastroent6rologie et Nutrition P6diatriques, Pavilion S, H6pital Edouard Herriot, 69437 Lyon, Cedex 03 France. Copyright | 1994 by Mosby-Year Book, Inc. 0022-3476/94/$3.00 + 0 9/20/59060
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CASE REPORTS Patient 1. Patient 1 was the first child of unrelated parents and the elder brother of patient 2. He had severe, protracted diarrhea beginning at 2 weeks of age and died at 6 weeks of age despite total parenteral nutrition. Autopsy findings revealed total villous atroAECA AIE TBS TPN
Anti-epithelial cell antibody Autoimmune enteropathy Tris-buffered saline solution Total parenteral nutrition
phy over the entire small bowel and marked lymphoid cell infiltration in the lamina propria of the entire digestive tract. Scattered lymphoid infiltrates were also present in the kidney and around the
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Fig. 2. Liver biopsy specimen shows aggressive hepatitis: a portal tract with bridging fibrosis, lymphoid infiltration, and a few giant cells. (Trichrome stain; X160.)
hepatitis. Treatment with cyclosporine was started. To date, after 6 months of cyclosporine therapy, the patient is doing well, alanine aminotransferase levels are normal, and the diarrhea has not recurred. METHODS Direct immunofluorescence tests. We performed direct immunofluorescence tests on apparently normal skin of patient 2. Biopsy tissue was snap-frozen in liquid nitrogen, and 4 #m thick cryostat sections were prepared on a cryomicrotome and placed onto glass slides. The following steps were then performed: (1) incubation with bovine serum albumin 1% in phosphate-buffered saline solution (15 minutes); (2) incubation with a mixture of fluorescein isothiocyanateconjugated anti-IgG, anti-IgA, anti-IgM, and anti-C3. After a final wash in phosphate-buffered saline solution, the sections were examined with a Zeiss immunofluorescence microscope equipped with epi-illumination. Indirect immunofluorescence studies. We studied normal human gut and skin for detection of A E C A and anti-basement membrane zone antibodies. Circulating antibodies were detected by standard indirect immunofluorescence, performed on 4 #m cryostat sections of normal tissue including human skin split by NaC1, 1.0 mol/L. 3 The sections were first incubated for 30 minutes at room temperature with serial dilution of patient's serum followed by a
30-minute incubation with fluorescein isothiocyanate-conjugated goat anti-human total Ig antibodies (Dakopatts, Copenhagen, Denmark). Sodium dodccyl sulfate-polyacrylamidc gel electrophoresis and immunoblot analysis. Epidermal proteins were obtained from normal human keratinocyte cultures according to a previously described technique3: proteins were extracted from the culture fasks after incubation for 30 m~nutes at 40 ~ C, with a lysis buffer (10 mmol Tris HC1 [pH 7.8] per liter, with 2% sodium dodecyl sulfate and 5% B-mercaptoethanol, and 100 tool ethylenediamine tetraacetic acid per liter, 200 mol phenylmethylsulphonyl fluoride per liter, 1 mmol leupeptin per liter, and 1 mmol pepstatin per liter). Epidermal proteins were separated by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (acrylamide concentration, 6%) under reducing conditions as previously described.4 The proteins were electrophoretically transferred to nitrocellulose filters. The efficiency of transblotting was checked by ponceau red staining of transferred epidermal and standard proteins. Nitrocellulose strips were then sequentially incubated with the following solutions: (1) 5% dried milk in 10 mmol Tris HC1 per liter and 140 mmol SaC1 [pH 7.4] per liter Tris-buffered saline solution/milk) for 1 hour at 37 ~ C or overnight at 40 ~ C; (2) 1:100
The Journal of Pediatrics Volume 125, Number 6, Part 1
dilution of each serum sample in TBS/milk for 1 hour at 37 ~ C; (3) 1:500 dilution of a biotinylated goat antihuman immunoglobulin (Amersham, Buckinghamshire, United Kingdom) in TBS/milk for 30 minutes at room temperature; and (4) 1:3000 dilution of streptavidin-alkaline phosphatase substrate solution (diethanolamine buffer, nitroblue tetrazolium, 5-bromo-4-chloro-3-indoyl phosphate, Amersham). Intervening washes were performed in TBS with 0.1% polyoxyethylenesorbitan monolaurate (Tween 20). RESULTS Immunologic studies. Serum immunoglobulin levels (IgG, IgA, and IgM) were norma ! or initially low, whereas IgE levels were always elevated until 24 months of age. Antibodies to vaccines were present. Radioallergosorbent test results were negative for allergies to milk, soybean, wheat, rice, fish, chicken, and peanuts. No T- or B-cell immune function deficiency was detected. The CD3 +, CD4 +, CD8 +, and B-lymphocyte counts were normal, as were the T-lymphocyte stimulation test results (phytohemagglutinin, pokeweed mitogen, and concanavalin A), complement factor levels, and neutrophil count. Results of serum immune complex testing were negative. Immunohistochemical staining of biopsy specimens from the patient's Colon and small bowel revealed the presence of CD3 +, CD4 +, CD8 +, and CD25 + cells in the epithelium and the lamina propria. No chromosomal abnormalities were found in blood lymphocytes. Anti-epithelial cell antibody. The presence of circulating AECA in patient 2 was demonstrated by the intense labeling of biopsy specimens from both small and large intestinal epithelium of normal human gut. The labeling persisted with dilution up to 1:100 of the patient's serum and was observed both in cytoplasm and at the membranes of enterocytes; goblet cells were not stained. The circulating autoantibodies to intestinal epithelial cells were IgGs and remained unchanged throughout the course of the disease. Bullous pempbigold antibody. Direct immunofluorescence of cryostat skin sections revealed the presence of linear IgG deposits at the dermoepidermal junction. Indirect immunofluorescence using the patient's serum showed the presence of circulating IgG autoantibodies binding to the epidermal side of a skin specimen split by NaC1, 1.0 mol/L. Immunoblot analysis of the reactivity of circulating autoantibodies with epidermal protein extracts demonstrated that the autoantibodies recognized the bullous pemphigoid antigens Agl and Ag2, of molecular weights 230 and 180 kd, respectively. Other tissue antibodies. High titers of thyroid microsomal and islet cell antibodies were transiently present. Smooth
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muscle antibodies (titer 1:160 to 1:320) were present during the course of the disease. No mitochrondria, liver, kidney microsomal, kidney brush border, or nuclear antibodies were detected. DISCUSSION The pathogenic significance of circulating AECA in patients with autoimmune enteropathy is unknown. In previous studies of children with AIE, the antibody concentration did not necessarily correlate with the severity of the disease. 17 In our patient, no good correlation between antibody titers and the course of the disease was noted. Early in the disease, during different treatments, and at the age of 24 months when the diarrhea was in remission, the titer of circulating A E C A was approximately the same. Thus the possibility remains that the antibody may have been the effect rather than the cause of the bowel injury. This is compatible with autoimmune disease T-cell activation as the primary event and not necessarily as the cause of the organ damage through B-cell activation and antibody production. The association of AIE with diseases such as nephropathy, 1-6 type I diabetes, v and hemolytic anemia 5 is now well recognized; however, the presence of bullous pemphigoid antibodies (with or without skin disease) has not been described in infants with AIE. Bullous pemphigoid is an autoimmune blistering skin disease characterized histologically by subepidermai bullae and immunologically by in vitro deposition of autoantibodies and complement components along the epidermal basement membrane zone; autoantibodies are also present in the serum. The targets of these autoantibodies are components of hemidesmosomes, namely the bullous pemphigoid antigens A g l and Ag2, of molecular weights 230 kd and 180 kd, respectively.3 Western immunoblot studies have demonstrated antibodies against the 230 and 180 kd bullous pemphigoid antigens, different from those previously described in AIE. The pathogenic significance of circulating bullous pemphigoid antibodies is uncertain. 8 Thus, even though bullous lesions did not develop on our patient's skin, he had high levels of IgE and episodes of eczema. Duodenal and colonic biopsy specimens showed epithelial desquamation. An artefact seems unlikely because of the repeated observations on many biopsy specimens. There was no evidence that bullous pemphigoid antibodies had a role in the pathogenesis of tbe gut disease. The possibility of an antigen common to gut mucosa and skin may be considered, and bullous pemphigold antibodies may be synthesized in contact with the gut or skin epithelial cell basal hemidesmosome. 8, 9 Thus it seems worthwhile to screen for these antibodies in the evaluation of patients with AIE. Other studies have revealed smooth muscle antibodies9, 10
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without liver involvement in patients with AIE. In another patient, serum aminotransferase levels were moderately elevated and an atypical liver and kidney microsomal antibody was present, but the liver biopsy specimen revealed a nonaggressive hepatitis with giant cell transformation. 6 Thus our patient had increased alanine aminotransferase levels with smooth muscle antibodies and a histologic appearance of chronic active hepatitis compatible with the diagnosis of autoirnmune hepatitis. Patient 1 died at 6 weeks of age and probably had A I E ; the gut lesions observed at autopsy looked similar to those revealed by endoscopic biopsies in patient 2. In addition, lymphocyte infiltration can involve different organs, and the pancreatic lesions should be interpreted as another target organ. Perhaps this localization of the disease might explain the frequency of type I diabetes observed in patients with AIE. In this noninbred family, two boys wer e affected and the two sisters were healthy. In previous reports, familial occurrence was frequent and A I E affected male subjects almost exclusively. T h e predominance of mate subjects in much of the published data 111 and in this family is in accordance with a sex-linked pattern of inheritance. We adopted a conservative approach to immunosuppressive treatment. The triple-agent immunosuppressive treatment (including prednisolone, cyclosporine, and azathioprine) seemed to be the most effective for reducing stool outflow, but after the diarrhea remitted we chose azathioprine and prednisone because of the autoimmune hepatitis. Secondarily, we introduced cyclosporine alone to control the disease, with apparent benefit. 2-5 In our patient, the link between autoantibodies and widespread organ damage remains uncertain. Further studies of other patients with similar disorders will increase our understanding of this disease, and may elucidate
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whether the autoantibodies present in the patient's blood are causative or occur as a consequence of tissue damage. REFERENCES 1. Colletti RB, Guillot AP, Rosen S, et al. Autoimmune enterop-
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athy and nephropathy with circulating anti-epithelial cell antibodies. J PEDIATR 1991;118:858-64. Sanderson IR, Phillips AD, Spencer J, Walker-Smith JA. Response to autoimmune enteropathy to cyclosporin A therapy. Gut 1991;32:1421-5. Machado P, Michalaki H, Roche P, Gaucherand M, Thivolet J, Nicolas JF. Serological diagnosis of bullous pemphigoid (BP): comparison of the sensitivity of indirect immunofluorescence on salt-split skin to immunoblotting. Br J Dermatol 1992;126:236-42. Leammli UK. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 1970;227:680-5. Seidman EG, Lacaille F, Russo P, Galeano N, Murphy G, Roy CC. Successful treatment of autoimmune enteroPathy with cyclosporine. J PEmATR 1990;117:929-32. Mitton SG, Mirakion R, Larcher VF, Dillon M J, WalkerSmith JA~ Enteropathy and renal involvement in an infant with evidence of widespread autoimmune disturbance. J Pediatr Gastroenterol Nutr 1989;8:397-400. Catassi C, Mirakian R, Natalini G, et al. Unresponsive enteropathy associated with circulating enterocyte autoantibodies in a boy with common variable hypogammaglobulinemia and type I diabetes. J Pediatr Gastroenterol Nutr 1988;7:608-13. Besnard V, Nicolas JF. Patbogenesis of bullous pemphigoid: new insights. Eur J Dermatol 1993;3:285-8. Unsworth D J, Walker-Smith JA. Autoimmunity in diarrhoea1 disease. J Pediatr Gastroenterol Nutr 1985;4:375-80. Savage MO, Mirakian R, Wozniak ER, et al. Specific autoantibodies to gut epithelium in two infants with severe protracted diarrhoea. J Pediatr Gastroenterol Nutr 1985;4: 187-95. Powell BR, Buist NRM, Stenzel P. An X-linked syndrome of diarrhea, polyendocrinopathy, and fatal infection in infancy. J PEDIATR 1982;100:731-37.