Metabolism of sulfated mucopolysaccharide in cultured fibroblasts from cystic fibrosis patients

Volume 77 Number 4

Table I. Flank masses in the infant Enlarged spleen Renal cystic lesions Perinephric abscess Hydronephrosis Nephroblastoma and other renal neoplasms Adrenal cyst Adrenocortical neoplasm Neuroblastoma Pheochromocytoma Retroperitoneal sarcoma Hepatic neoplasm and hamartoma Abnormal hepatic lobe Mesenteric cyst Choledochal cyst Pancreatic cyst Enteric duplication

Cysts have been classified as parasitic, epithelial, endothelial, and pseudocysts. The review by V a n de Water and Fonkalsrud 1 tabulates 6 cases of pseudocyst, one hamartoma, and one lymphogenous cyst, occurring in infants. The pathogenesis of pseudocyst is unclear, but resolution of h e m a t o m a or abscess may be operative in the ultimate development of this entity. T h e 20-day-old girl reported by Van de Water and Fonkalsrud ~ had a clinical course and lesion similar to our patient. Escherichia

Metabolism of sulfated mucopolysaccbaride in cultured fibroblasts from cystic fibrosis patients Ulrich Wiesmann, M.D.,* and Elizabeth F. Neufeld, Ph.D. ~'* BETHESDA, MD.

From the Pediatric Metabolism Branch and the Section on Intermediary Metabolism, National Institute of Arthritis and Metabolic Diseases, National Institutes o[ Health. ~Fellow of the Cystic Fibrosis Foundation. r address: Building 10, Room 9B-17, National Institutr o] Health, Bethesda, Md. 20014.

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coli was cultured from the lesion which was probably a liquefied pyogenic abscess, rather than a pseudocyst. Perinephric abscesses are generally extensions of intrarenal inflammatory lesions and distinguished from adrenal abscess by separation from the adrenal gland by fascial planes. T h e adrenal abscess described in this case must be added to the list of abnormal masses in the lateral portion of the abdomen in infants.

SUMMARY A case of adrenal abscess is presented as an example of a rare lesion which mimics neuroblastoma. Although careful evaluation of history, physical findings, and laboratory data may suggest an inflammatory process, diagnosis depends upon exploratory surgery and through pathologic study. The authors thank Dr. Parker Allen for interpretation and description of the x-rays and Mrs. H. Lesehnik for preparing the manuscript. REFERENCE 1. Van de Water, J. M., and Fonkalsrud, E. W. : Adrenal cysts in infancy, Surgery 60: 1267, 1966.

A L T I-I O 1~ a H cystic fibrosis is a genetic disorder which affects primarily the exocrine glands, abnormalities have recently been observed in fibroblasts cultured from the skin of patients with cystic fibrosis and of some of their relatives. These cells stain metachromatically with toluidine blue), 2 contain more mucopolysaccharide, both sulfated and unsulfated, than do fibroblasts from normal individuals, 3 and in the presence of radioactive sulfate and acetate, accumulate excess radioactive mucopolysaccharide. 2 The observations are sufficiently similar to data obtained on fibroblasts from patients affected with mucopolysaccharidoses, such as the Hurler and Hunter syndromes, 4 to suggest that some disturbance of mucopolysaccharide metabolism might be a primary defect in cystic fibrosis. We have examined the turnover of sul-

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f a t e d m u c o p o l y s a c c h a r i d e in fibroblasts from 13 patients w i t h cystic fibrosis. T h e d a t a show t h a t the sulfated m u c o p o l y s a c c h a r i d e m e t a b o l i s m of their fibroblasts is readily differentiated f r o m t h a t of H u r l e r a n d H u n t e r cells, b u t differs only slightly, if at all, from the normal. T h e results would seem to exclude a n alteration in sulfated mucopolysacc h a r i d e m e t a b o l i s m as a p r i m a r y manifestation of the cystic fibrosis gene. METHODS

F i b r o b l a s t lines were started f r o m skin biopsies, or, in the case of a n e w b o r n control subject, f r o m foreskin o b t a i n e d at circumcision. T h e cultures were grown at 37 ~ C. in 100 ram. F a l c o n Petri dishes in an a t m o s p h e r e of 95 p e r cent air a n d 5 p e r cent CO2. T h e y were fed twice weekly with Eagle's M i n i m u m Essential m e d i u m in Earle's salts ( w i t h MgC12 i n s t e a d of M g S O ~ a n d N a H C O 3 r e d u c e d to 1.6 Gm. p e r liter) reinforced with t 0 p e r cent fetal calf serum ( G r a n d I s l a n d Biologieals Co., C h a g r i n Falls, O h i o ) a n d ' nonessential amino acids, and c o n t a i n i n g antibiotics as follows: penicillin, 100 U . p e r milliliter, streptomycin, 100 U. p e r milliliter, a n d nystatin, 60 U. p e r milliliter. Experiments were u n d e r t a k e n 4 days after t r a n s p l a n t a t i o n , when cells were con-

The Journal o[ Pediatrics October 1970

fluent but not overgrown (ca. 1 mg. cell p r o t e i n p e r P e t r i dish). L a b e l i n g with a~SO, a n d m e a s u r e m e n t of radioactivity incorp o r a t e d into mucopolysaccharide were perf o r m e d as previously d e s c r i b e d ) W i t h the exception of Patients L. D. a n d D. R., the patients with cystic fibrosis were p a r t of a g r o u p a d m i t t e d to t h e N a t i o n a l I n s t i t u t e of Arthritis a n d M e t a b o l i c Diseases for p a r t i c i p a t i o n in other research projects; they are therefore older t h a n average, a n d most of t h e m have only m i l d or m o d e r a t e clinical manifestations. RESULTS

C u l t u r e d skin fibroblasts i n c o r p o r a t e ino r g a n i c sulfate into m u c o p o l y s a c c h a r i d e , p a r t of which is r e t a i n e d within the cell for e v e n t u a l degradation, whereas most is secreted into the external m e d i u m 4, "~; incorp o r a t i o n of a5SO4 into b o t h i n t r a c e l l u l a r a n d e x t r a c e l l u l a r m u c o p o l y s a c c h a r i d e was therefore measured. T o correct for some variation b e t w e e n experiments in the specific activity of the sulfate, results a r e expressed as a ratio of i n c o r p o r a t i o n into fibroblasts f r o m patients to i n c o r p o r a t i o n into fibroblasts f r o m the skin of a n o r m a l v o l u n t e e r (20-yearold w o m a n ) arbitrarily selected as t h e reference line. A 3 day exposure to 35SO4 was

T a b l e I. A c c u m u l a t i o n a n d secretion of r a d i o a c t i v e m u c o p o l y s a c c h a r i d e b y fibroblasts d e r i v e d f r o m patients w i t h cystic fibrosis Intracellular accumulation

Secretion

No. obserSeverity of Ratio ~ Patient Ratio* Range rations Sex disease D.R. 8 M Moderate 1.9 1.5-2.2 5 1.2 M.N. 24 F Moderate 1.6 1.4-2.0 4 1.3 H.R. 25 M Moderate 1.6 1 1.5 G.P. 14 M Moderate 1.5 1 1.5 L.D. 6 M Mild 1.4 1 P.J. 13 M Mild 1.4 1 1.3 G.N. 23 M Mild 1.3 1.2-1.5 3 1.4 A.J. 13 M Mild 1.2 1 1.1 M.R. 15 M Severe 1.1 1 0.9 M.P. 14 M Severe 1.1 0.9-1.2 2 1.2 W.P. 11 F 9 Moderate 1.1 0.9-1.2 2 C.R. 24 M Mild 0.9 I 0.9 B.S. 15 M Mild 0.8 0.5-1.1 3 0.8 *Ratio of radioactive mucopolysaccharide(in counts per minute per milligram of cell protein) accumulated or secreted in 3 days by fibroblasts from a given individual to those from a normal control subject. The secreted mucopolysaccharideincludes mucopolysaccharidesoluble in the medium as well as that which adheres to the cells and is released by trypsinizatlon. Age (yr.)

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selected on the basis of preliminary experiments which showed no change in this ratio after 2 days. As can be seen in Table I, 6 lines of fibroblasts from patients with cystic fibrosis accumulated within + 20 per cent of normal; 6 lines, 1.3 to 1.6 times the normal level, and one line twice the normal level. Fibroblasts from 3 other normal control Subjects (males, ages r days, 19 and 21 years, respectively) did not differ from the reference line by more than + 10 per cent. Table I also shows that the amount of radioactive mucopolysaccharide secreted by cystic fibrosis cells does not differ much from

200

I

the normal amount; the mean secretion for 11 cystic fibrosis lines was 1.2 times the amount secreted by the normal reference line. Again, fibroblasts from the 3 other normal control subjects did not differ from the reference line by more than 20 per cent. These results contrast sharply with the greatly increased intracellular accumulation of 3~SO4-1abeled mucopolysaccharide in cells from individuals affected with mucopolysaccharide-storage disorders. In fibroblasts from 7 patients with the Hunter syndrome and from 6 with the Hurler syndrome, which have been studied extensively in this laboratory, intracellular accumula-

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Fig. 1. Incorporation of a~S04 into intracellular mucopolysaccharide by fibroblasts from a normal subject, from a patient with cystic fibrosis (D. R.), and from a patient with the Hurler syndrome.

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The Journal of Pediatrics October 1970

I

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Low accumulating Hurler 70

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Fig. 2. Loss of radioactivity from intracellular mucopolysaccharide during chase with unlabeled medium. Fibroblast lines as in Fig. 1.

tion after 3 days in 8~SO4 varies from 3.5 to 9 times the normal level and continues to increase with time. Fibroblasts from one Hurler line, however, consistently accumulate only 2 to 3 times as much as the normal in 3 days and are therefore not readily differentiated in their accumulation pattern from the fibroblasts of the cystic fibrosis patient (D. R.) which accumulate twice as much (Fig. 1). The difference becomes obvious if one performs a pulse-chase experiment--i.e., if one prelabels the cells, then replaces the radioactive medium with nonradioactive (Fig. 2). In cells from normal individuals or patients with cystic fibrosis, most of the intracellular mucopolysaccharide

has a half-life of 8 hours, whereas a minor component has a half-life of 4 days. In the Hurler cells, there characteristically is but little disappearance of radioactive mucopolysaccharide in the first 8 hours of chase; the entire pool has a half-life of 4 days. Since the disappearance of the labeled mucopolysaccharide occurs by degradation, 5 one must conclude that the kinetics of mucopolysaccharide degradation in fibroblasts from patients with cystic fibrosis and from normal individuals are identical. DISCUSSION T h e intracellular sulfated mucopolysaccharide is not the precursor of mucopolysac-

Volume 77 Number 4

charide secreted into the medium but is material in the process of degradation. 5 Presumably, there exists within the cell a finite pool of mucopolysaccharide destined for secretion, but kinetic studies suggest that it is small, turns over rapidly, and thus accounts for little of the total intracellular mucopolysaccharide. T h e level of radioactivity in the sulfated mucopolysaccharide is an indirect measure of the concentration of these molecules. When all the unlabeled mucopolysaccharide has been replaced by radioactive mucopolysaccharide, a steady state is reached in the labeling pattern. Such a plateau is approached in 2 to 3 days in normal and most cystic fibrosis cell lines, whereas 'it is not reached at all in Hurler and Hunter lines for as long as experiments have been carried out. 5 Thus, if cystic fibrosis cells have 10 times as much sulfated mucopolysaccharide as normal cells, one would expect 10 times as much radioactivity in the mucopolysaccharide after a 3 day exposure to isotope. Since such accumulation did not occur, we conclude that under our culture conditions the pools of sulfated mucopolysaccharide in fibroblasts from normal individuals and from patients with cystic fibrosis are very similar. This appears to be the case even for the fibroblasts of Patient L. D., shown by Matalon 6 to accumulate excessive amounts of mucopolysaccharide under his experimental conditions. Intracellular accumulation of mucopolysaccharide is a function of biosynthesis, secretion, and degradation. I t must depend not only on the availability of metabolites and energy but also on complex processes such as the turnover of lysosomes, the organelles in which mucopolysaccharide degradation occurs. In the Hurler and H u n t e r syndromes, the accumulation of sulfated mucopolysaccharide is due to the lack of genotype-specific macromolecules required f o r adequate degradation of mucopolysaccharide.~, 7 T h e occasional increase in accumulation in fibroblasts from patients with cystic fibrosis, as in Patient D. R., must be due to some other cause, since degradation

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689

in these cells follows a normal course. One possibility might be an increased number of lysosomes, each lysosome functioning adequately. Some preliminary experiments have shown that growing normal fibroblasts in 0.08M sucrose, a procedure believed to stimulate lysosome formation, s increases the accumulation of sulfate-labeled mucopolysaccharide manyfold with only a slight change in the pattern of degradation. In addition to metachromasia and increased accumulation of mucopolysaccharid% several metabolic anomalies have been observed in fibroblasts from cystic fibrosis patients: increased secretion of mucopolysaccharide, 9 increased storage of glycogen, 1~ decreased collagen production, 1~ and a longer generation time. 12 Fibroblasts from a few patients with cystic fibrosis, however, show no increased mucopolysaccharide content or secretion.2, 9 These diverse observations, as well as our unexpectedly negative findings with respect to sulfa}ed mucopolysaccharide metabolism, can be readily understood if it is assumed that there is some as yet undefined defect caused by the cystic fibrosis gene, which has consequences in a number of metabolic pathways, including those of mucopolysaccharide turnover. Because these consequences may be far removed from the primary defect, their manifestation should be dependent on the total metabolic environment, as affected both by the remainder of the cell's genotype and by the conditions of tissue culture. We are indebted to Dr. R. 1V[atalon for the fibroblast culture of Patient D. L., to Dr. J. Houck for the biopsy of Patient D. R., to Dr. W. B. Uhlendorf for starting all other cystic fibrosis lines, and to Dr. P. di Sant' Agnese for help and encouragement. REFERENCES

1. Danes, B. S., and Bearn, A. G.: A genetic cell marker in cystic fibrosis of the pancreas, Lancet 1: 1061, 1968. 9. Danes, B. S., and Bearn, A. G.: Cystic fibrosis of the pancreas: A study in cell culture, J. Exp. Med. 129: 775, 1969. 3. Matalon, R., and Dorfman, A.: Acid mu~opolysaccharides in cultured fibroblasts of cystic

690

4. 5.

6. 7.

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fibrosis of the pancreas, Biochem. Biophys. Res. Commun. 33: 954, 1968. Neufeld, E. F., and Pratantoni, J. C.: Inborn errors of mucopolysaccharide metabolism, Science. In press. Fratantoni, J. C., Hall, C. W., and Neufeld, E. F.: The defect in Hurler's and Hunter's syndromes: Faulty degradation of mucopolysaccharide, Proe. Nat. Acad. Sci. U. S. A. 60: 699, 1968. Matalon, R.: PersonaI communication. Fr~,tantoni, J. C., Hall, C. W., and Neufeld, E. F.: The defect in Hurler and Hunter syndromes. II. Deficiency of specific factors involved in mucopolysaccharide degradation, Proc. Nat. Acad. Sci. U. S. A. 64" 360, 1969. Dingle, J. T., Fell, H. B., and Glauert, A. M.: Endocytosis of sugars in embryonic skeletal

Extra posterior cervical skin: A possible sign of chromosomal aberration

L a w r e n c e R. S h a p i r o , M . D . , Lillian Y. F. Hsu, M . D . , a n d K u r t H i r s c h h o r n , M . D . "x" NEW

YORK

AND

THIELLS~

N.

Y.

Excess S K I N at the back of the neck, r a t h e r t h a n webbing, has been noted in m a n y cases of trisomy 181 a n d has been referred to as one of the 10 c a r d i n a l signs of D o w n ' s syndrome3 W e have recently seen 3 infants with extra From the Mount Sinai School of Medicine and Letehworth Village. Supported in part by a grant [rom the Birth DeJects Institute, The New York State Department o[ Health, United States Public Health Service Grant HD-02552, and the Association [or the Aid to Crippled Children. Reprint address: L. R. Shapiro, Cytogenetics Laboratory, Letchworth Village, ThleIls, N. Y. 10984. *Career scientist 1-513 o] the Health Research Council o] the City of New York.

The Journal o[ Pediatrics October 1970

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10.

11. 12.

tissues in organ culture. IV. Lysosomal and other biochemical effects, J. Cell Sci. 4: 139, 1969. Danes, B. S., and Bearn, A. G.: Cystic fibrosis: Distribution of mucopolysaccharides in fibroblast cultures, Biochem. Biophys. Res. Commun. 36: 919, 1969. Pallavicini, J. C., Wiesmann, U., Uhlendorf, W. B., and di Sant' Agnese, P. A.: Glycogen content of tissue culture fibroblasts from patients with cystic fibrosis and other heritable disorders, J. P~DIAT. 77" 280, 1970. Houck, J. c.: Personal communication. Raft, E. C., and Houck, J. C.: Migration and proliferation of diploid human fibroblasts following wounding of confluent monolayers, J. Cell Physiol. 74: 235, 1969.

posterior cervical skin, but not webbing, each of w h o m h a d a chromosomal a b n o r m a l i t y other t h a n trisomy 18, trisomy 21, or translocation mongolism. CASE REPORTS Case 1. The first infant was 8 weeks of age when he was referred for evaluation of a peculiar facies a n d redundant skin in the posterior cervical region (Fig. 1). Chromosome analysis revealed 49 chromosomes with an XXXXY sex chromosome constitution. This was confirmed by buccal smear and autoradiographic analysis,a Case 2. The second infant was 6 months of age when she was seen for evaluation of multiple congenital anomalies; she was noted to have marked redundancy of the skin of the posterior cervical region (Fig. 2). In addition, she had microcephaly, micrognathia, preauricular skin tags, a cleft soft palate, a significant heart murmur, low-placed and widely spaced nipples, cubitus valgus, long fingerlike thumbs, bilateral dislocated hips, hypotonia, and lymphedema of the pretibial regions and dorsum of the feet. Chromosome and autoradiographic analysis revealed 47 chromosomes with an additional G group chromosome (47,XX,G+) which has been interpreted and Presented by us as an example Of trisomy 22. 4 Case 3. The third infant was seen at 3 days of age because she had a peculiar facies, narrow palpebral fissures, widely spaced and low-set