- Email: [email protected]
TREATMENT OF MEMBRANOUS NEPHROPATHY WITH PREDNISOLONE AND CHLORAMBUCIL
1015 the intracellular accumulation of unesterified cholesterol is best shown by histochemical (filipin) staining.4 Combining the two approaches further increases efficiency and reliability. The few "variant" families5 (about 10%) with less pronounced abnormalities apart, we conclude that prenatal diagnosis can now be offered to a large majority of couples at risk for type C Niemann-Pick disease.
typical patients,S while
Neurochemistry Laboratory, Fondation Gillet-Mérieux, and INSERM U189, Centre Hospitalier Lyon-Sud, Faculté de Médecine Lyon-Sud, 69310 Pierrep Bénite, France
MARIE T. VANIER ROBERT M. ROUSSON
Cell Culture
Laboratory, Hôpital Debrousse, Lyon
GINETTE MANDON
Cytogenetics Laboratory, Hôpital St Vincent de Paul, Paris
AGNES CHOISET
Department of Histopathology, Hospital for Sick Children, London
BRIAN D. LAKE
NINCDS, National Institutes of Health, Bethesda, Maryland, USA
Filipin staining for unesterified cholesterol in chorionic villus cells from a fetus (case 6) affected with type C Niemann-Pick disease.
1. Pentchev
2.
Washington, Paris, and Lyon, and stored in culture medium at room temperature during transport to Lyon (up to 48 h). At-risk samples were analysed between the second and the fourth passage, together with at least two control samples cultured concomitantly. The assay also included skin fibroblast cultured from the index case and from a control. Lipoprotein-stimulated cholesteryl ester formation was studied with unfrozen human serum as lipoprotein sourceThe wide range of values observed in the 15 controls (380-3900 pmol cholesteryl 3H-oleate formed per mg cellular protein in 6 h) was attributed to a large inter-individual variation in normal chorionic villus cells rather than to slight modifications in culture conditions or the use of different batches of biological reagents. Four at-risk samples (fetuses 1, 3, 6, and 7) showed very low levels of esterification (20-120 pmol/mg) while the other three were normal. In parallel experiments, cells grown in ’Labtek’ chambers (Miles) and preincubated for 3 days in lipoprotein-deficient serum were challenged with medium supplemented with 10% human serum for 24 h. After fixation, the cells were stained with filipin to demonstrate intracellular unesterified cholesterol by fluorescence microscopy.4 Chorion villus cells from fetuses 1,3,6, and 7 showed massive perinuclear storage of unesterified cholesterol (figure), while a very low fluorescence was observed in control samples and in the other at-risk samples. An additional deficiency in esterification of non-lipoprotein cholesterol3 and a low rate of sphingomyelin degradation in situ3 were demonstrated in samples 1, 3, 6, and 7 (data not shown). The latter fetuses were predicted to be affected and these pregnancies were terminated at the request of the parents. Biochemical confirmation of the diagnosis in the abortuses was obtained by demonstration of an increased concentration of unesterified cholesterol, sphingomyelin, bis(monoacylglycero)phosphate, and glucosylceramide in livers in each case as well as a study of fetal skin fibroblasts in case 7 (serum-induced cholesterol esterification 60 pmol/mg protein; controls 3520 [SD 1530]; massive intracellular accumulation of free cholesterol on filipin staining). Morphological, histochemical, and ultrastructural studies in cases 1, 3, and 6 confirmed storage cells in bone marrow and liver, with neuronal storage in the brain. The two children bom so far have been confirmed as unaffected; one predicted normal pregnancy continues. The demonsration of abnormalities in intracellular processing of exogenous cholesterol in cultured cells has proved to be an important breakthrough in type C Niemann-Pick disease, with special reference to diagnosis. Esterification studies using a non-lipoprotein and a lipoprotein cholesterol source showed the superiority of the lipoprotein source for discrimination of less
PETER G. PENTCHEV
PG, Comly ME, Kruth HS, et al. Group C Niemann-Pick disease: faulty regulation of low density lipoprotein uptake and cholesterol storage in cultured fibroblasts. FASEB J 1987; 1: 40-45. Liscum L, Faust JR. LDL-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick disease type C fibroblasts. J Biol Chem 1987;
262: 17002-08. 3. Vanier MT, Wenger DA, Comly ME, Rousson R, Brady RO, Pentchev PG. Niemann-Pick disease type C clinical variability and diagnosis based on defective cholesterol esterification. Clin Genet 1988; 33: 331-48. 4. Kruth HS, Comly ME, Butler JD, et al. Type C Niemann-Pick disease. Abnormal metabolism of low density lipoprotein in homozygous and heterozygous
fibroblasts. J Biol Chem 1986; 261: 16769-74. 5. Vanier MT, Pentchev PG, Rousson R. Pathophysiological approach of Niemann-Pick
disease type C: definition of a biochemical heterogeneity and re-evaluation of the lipid storage process. In: Salvayre R, Douste-Blazy L, Gatt S, eds. Lipid storage disorders: biological and medical aspects. (Life Sci Ser vol CL). New York: Plenum Press, 1988: 175-85.
TREATMENT OF MEMBRANOUS NEPHROPATHY WITH PREDNISOLONE AND CHLORAMBUCIL
SIR,-We used combined treatment with prednisolone and chlorambucil in a 52-year-old man with idiopathic membranous nephropathy, with the protocol used by Mathieson et al.1 Treatment was six alternating monthly cycles of prednisolone and chlorambucil, as originally described by Ponticelli et al.2 We had encouraging results in reducing proteinuria during the first two cycles, but at the end of the third cycle, before the start of chlorambucil, the patient had a temperature above 39°C. We excluded possible infective causes for the fever, which was persistent and did not respond to antipyretic compounds. We assumed that the raised temperature was due to partial adrenal insufficiency caused by the abrupt discontinuation of prednisolone and that it would resolve once steroids were re-started. This side-effect, which was not reported by Ponticelli or Mathieson or their colleagues, is serious and could affect the course of treatment, which otherwise seems to be effective. Though abrupt discontinuation of steroids has been described in relation to duration of therapy and total dose,3 such discontinuation can also produce life-threatening events because of moderate or severe adrenal insufficiency. Clinic of Nephrology, Second Hospital IKA, Thessaloniki 55132, Greece
C. MAVROMATIDIS S. SPAIA F. CHRISTIDOU G. VAYONAS
PW, Turner AN, Maidment CGH, Evans DJ, Rees AJ. Prednisolone and chlorambucil treatment in idiopathic membranous nephropathy with deteriorating renal function. Lancet 1988; ii: 869-72. 2. Ponticelli C, Zucchelli P, Imbasciati E, et al. Controlled trial of methylprednisolone and chlorambucil in idiopathic membranous nephropathy. N Engl J Med 1984; 310: 946-50. 3. Christy NP. HPA failure and glucocorticoid therapy. Hosp Pract 1984; 19: 77. 1 Mathieson
typical patients,S while
Neurochemistry Laboratory, Fondation Gillet-Mérieux, and INSERM U189, Centre Hospitalier Lyon-Sud, Faculté de Médecine Lyon-Sud, 69310 Pierrep Bénite, France
MARIE T. VANIER ROBERT M. ROUSSON
Cell Culture
Laboratory, Hôpital Debrousse, Lyon
GINETTE MANDON
Cytogenetics Laboratory, Hôpital St Vincent de Paul, Paris
AGNES CHOISET
Department of Histopathology, Hospital for Sick Children, London
BRIAN D. LAKE
NINCDS, National Institutes of Health, Bethesda, Maryland, USA
Filipin staining for unesterified cholesterol in chorionic villus cells from a fetus (case 6) affected with type C Niemann-Pick disease.
1. Pentchev
2.
Washington, Paris, and Lyon, and stored in culture medium at room temperature during transport to Lyon (up to 48 h). At-risk samples were analysed between the second and the fourth passage, together with at least two control samples cultured concomitantly. The assay also included skin fibroblast cultured from the index case and from a control. Lipoprotein-stimulated cholesteryl ester formation was studied with unfrozen human serum as lipoprotein sourceThe wide range of values observed in the 15 controls (380-3900 pmol cholesteryl 3H-oleate formed per mg cellular protein in 6 h) was attributed to a large inter-individual variation in normal chorionic villus cells rather than to slight modifications in culture conditions or the use of different batches of biological reagents. Four at-risk samples (fetuses 1, 3, 6, and 7) showed very low levels of esterification (20-120 pmol/mg) while the other three were normal. In parallel experiments, cells grown in ’Labtek’ chambers (Miles) and preincubated for 3 days in lipoprotein-deficient serum were challenged with medium supplemented with 10% human serum for 24 h. After fixation, the cells were stained with filipin to demonstrate intracellular unesterified cholesterol by fluorescence microscopy.4 Chorion villus cells from fetuses 1,3,6, and 7 showed massive perinuclear storage of unesterified cholesterol (figure), while a very low fluorescence was observed in control samples and in the other at-risk samples. An additional deficiency in esterification of non-lipoprotein cholesterol3 and a low rate of sphingomyelin degradation in situ3 were demonstrated in samples 1, 3, 6, and 7 (data not shown). The latter fetuses were predicted to be affected and these pregnancies were terminated at the request of the parents. Biochemical confirmation of the diagnosis in the abortuses was obtained by demonstration of an increased concentration of unesterified cholesterol, sphingomyelin, bis(monoacylglycero)phosphate, and glucosylceramide in livers in each case as well as a study of fetal skin fibroblasts in case 7 (serum-induced cholesterol esterification 60 pmol/mg protein; controls 3520 [SD 1530]; massive intracellular accumulation of free cholesterol on filipin staining). Morphological, histochemical, and ultrastructural studies in cases 1, 3, and 6 confirmed storage cells in bone marrow and liver, with neuronal storage in the brain. The two children bom so far have been confirmed as unaffected; one predicted normal pregnancy continues. The demonsration of abnormalities in intracellular processing of exogenous cholesterol in cultured cells has proved to be an important breakthrough in type C Niemann-Pick disease, with special reference to diagnosis. Esterification studies using a non-lipoprotein and a lipoprotein cholesterol source showed the superiority of the lipoprotein source for discrimination of less
PETER G. PENTCHEV
PG, Comly ME, Kruth HS, et al. Group C Niemann-Pick disease: faulty regulation of low density lipoprotein uptake and cholesterol storage in cultured fibroblasts. FASEB J 1987; 1: 40-45. Liscum L, Faust JR. LDL-mediated suppression of cholesterol synthesis and LDL uptake is defective in Niemann-Pick disease type C fibroblasts. J Biol Chem 1987;
262: 17002-08. 3. Vanier MT, Wenger DA, Comly ME, Rousson R, Brady RO, Pentchev PG. Niemann-Pick disease type C clinical variability and diagnosis based on defective cholesterol esterification. Clin Genet 1988; 33: 331-48. 4. Kruth HS, Comly ME, Butler JD, et al. Type C Niemann-Pick disease. Abnormal metabolism of low density lipoprotein in homozygous and heterozygous
fibroblasts. J Biol Chem 1986; 261: 16769-74. 5. Vanier MT, Pentchev PG, Rousson R. Pathophysiological approach of Niemann-Pick
disease type C: definition of a biochemical heterogeneity and re-evaluation of the lipid storage process. In: Salvayre R, Douste-Blazy L, Gatt S, eds. Lipid storage disorders: biological and medical aspects. (Life Sci Ser vol CL). New York: Plenum Press, 1988: 175-85.
TREATMENT OF MEMBRANOUS NEPHROPATHY WITH PREDNISOLONE AND CHLORAMBUCIL
SIR,-We used combined treatment with prednisolone and chlorambucil in a 52-year-old man with idiopathic membranous nephropathy, with the protocol used by Mathieson et al.1 Treatment was six alternating monthly cycles of prednisolone and chlorambucil, as originally described by Ponticelli et al.2 We had encouraging results in reducing proteinuria during the first two cycles, but at the end of the third cycle, before the start of chlorambucil, the patient had a temperature above 39°C. We excluded possible infective causes for the fever, which was persistent and did not respond to antipyretic compounds. We assumed that the raised temperature was due to partial adrenal insufficiency caused by the abrupt discontinuation of prednisolone and that it would resolve once steroids were re-started. This side-effect, which was not reported by Ponticelli or Mathieson or their colleagues, is serious and could affect the course of treatment, which otherwise seems to be effective. Though abrupt discontinuation of steroids has been described in relation to duration of therapy and total dose,3 such discontinuation can also produce life-threatening events because of moderate or severe adrenal insufficiency. Clinic of Nephrology, Second Hospital IKA, Thessaloniki 55132, Greece
C. MAVROMATIDIS S. SPAIA F. CHRISTIDOU G. VAYONAS
PW, Turner AN, Maidment CGH, Evans DJ, Rees AJ. Prednisolone and chlorambucil treatment in idiopathic membranous nephropathy with deteriorating renal function. Lancet 1988; ii: 869-72. 2. Ponticelli C, Zucchelli P, Imbasciati E, et al. Controlled trial of methylprednisolone and chlorambucil in idiopathic membranous nephropathy. N Engl J Med 1984; 310: 946-50. 3. Christy NP. HPA failure and glucocorticoid therapy. Hosp Pract 1984; 19: 77. 1 Mathieson