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Treatment of prepubertal gonadotrophin-deficient boys with recombinant human follicle-stimulating hormone
THE LANCET
Sinus node dysfunction
Normal sinus node function
110 Chagas
Idiopathic
Chagas
IDC
HC
Change in beats per minute
70
20
–30
–80
–130 patient
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
17 18 19 20 21
22 23 24 25 26
Chronotropic effects of IgG before (black bars) and after (white bars) addition of atropine in patients with abnormal (left) and normal (right) sinus node function IDC=idiopathic dilated cardiomyopathy, HC=healthy controls.
contraction rate (figure). This effect was partially or totally reversed by atropine, suggesting it may be due to stimulation of M2 cholinergic receptors. IgG from patient 7, in the presence of atropine, led to a marked positive effect thought to be due to the presence of anti-adrenoceptor-stimulating antibodies (the effect was partially reversed by metoprolol). In two other patients with sinus-node dysfunction (3 and 5), the cardiomyocyte contraction rate was not significantly affected by IgG, but increased after atropine addition, suggesting that the IgG fraction contained antibodies which reacted simultaneously with the M2 cholinergic receptor and the -adrenergic receptor; their effects cancelling out and becoming apparent only after addition of atropine. IgG from only two patients (12 and 11) with normal sinus-node function had either a negative action on cardiomyocytes or led to notable changes after addition of atropine. By contrast there was a positive effect in 14 patients with normal sinusnode function suggesting the presence of anti- adrenoceptor-stimulating antibodies. Our in-vitro results support the hypothesis that the sinus node may be the target of an autoimmune response, although it is not known whether such effects are important in vivo. IgG from patients with Chagas’ myocarditis caused both sinus bradycardia and second degree atrio-ventricular nodal block in rabbit heart (Langendorf preparation). 5 The association of autoimmune disease of the sinotrial node with chronic cardiac Chagas’ disease suggests that sinusnode dysfunction may be related to a chronic infectious process. The PO ribosomal protein of Trypanosoma cruzi3 is possibly involved in the generation of cholinergic antibodies. 1
2
Maisch B, Lotze U, Scheneide J, Kochsiek K. Antibodies to human sinus node in sick sinus syndrome. Pacing Cardiac Electrophysiol 1986; 9: 1101–09. Magnusson Y, Wallukat G, Waagstein F, Hjalmarson A, Hoebeke J. Autoimmunity in IDC: characterization of antibodies against the Beta-1 adrenoreceptor with positive chronotropic effect. Circulation 1994; 89: 2760–67.
Vol 350 • July 26, 1997
3
4
5
Ferrari I, Levin MJ, Wallukat G, et al. Molecular mimicry between the immunodominant ribosomal protein PO of Trypanosoma cruzi and a functional epitope on human 1-adrenergic receptor. J Exp Med 1995; 182: 59–65. Elies R, Ferrari I, Wallukat G, et al. Structural and functional analysis of the B cell epitope recognised by anti-receptor autoantibodies in patients with Chagas’ disease. J Immunol 1996; 157: 4203–11. Farias de Oliveira S, Pedrosa RC, Nascimento JHM, Campos de Carvalho AC, Masuda MO. Depression of cardiac automaticity and A-V conduction by IgG fraction of chronic chagasic patients with arrhythmia is mediated by muscarinic receptors. Mem Inst Oswaldo Cruz 1995; 90 (suppl 1): 151.
División Cardiología, Hospital Ramos Mejía and Instituto de Ingeniería Genética y Biología Molecular (INGEBI), CONUCET, 1221 Buenos Aires, Argentina, (Pablo A Chiale)
Treatment of prepubertal gonadotrophin-deficient boys with recombinant human folliclestimulating hormone Taneli Raivio, Jorma Toppari, Antti Perheentupa, Alan S McNeilly, Leo Dunkel
Sertoli cells stop dividing in early puberty but, before that, their number is probably dependent on follicle-stimulating hormone (FSH). Administration of FSH to immature nonhuman primates increases the number of Sertoli cells and induces growth of testes. 1 In human beings a gene mutation inactivating the FSH receptor leads to suppression of spermatogenesis and small testis volume. 2 Adults with prepubertal onset of gonadotrophin deficiency often have small testis volume, and a low sperm count, 3 which may result in infertility. We studied the effect of recombinant human FSH (r-hFSH) on growth of testes in prepubertal gonadotrophin-deficient boys.
263
THE LANCET
A
B
3 2 1 0
5 Testis volume (mL)
200
Patient A Patient B Patient C
Serum inhibin B (pg/mL)
Serum FSH (IU/L)
4
C
150 100 50
13·5
14·5
4 3 229% 2 102% 1 0
0 12·5
96%
12·5
13·5 Age (year)
14·5
12·5
13·5
14·5
Serum FSH (A), inhibin B (B) concentrations, and testis volumes (C) against age in three gonadotrophic-deficient boys measured at 3-month intervals Shaded lines showed the ranges of serum FSH and Inhibin B concentrations in healthy prepubertal boys (Tanner stage G1P1).
We studied three boys (A, 12·8; B, 13·2; and C, 13·2 years old) with gonadotrophin deficiency. Patients A and C had idiopathic, and patient B acquired (surgery for craniopharyngioma at age 10 years) hypopituitarism. Other pituitary-hormone deficiencies were adequately replaced. The boys were given r-hFSH (Gonal-F, Serono) subcutaneously (1·5 IU/kg) three times a week for 12 months. At 3-month intervals, puberty was staged according to Tanner, testis volume measured (0·52⫻length⫻width2), and a blood sample drawn for serum gonadotrophin, sex steroids, and inhibin B concentrations. During the study, without any increase in serum luteinising hormone (LH) or sex-steroid concentrations, serum FSH and inhibin B concentrations increased to within the range observed in healthy prepubertal boys (figure, A and B) and the testis volume increased (p<0·02, figure, C). Patient B had the largest initial testis volume, most likely due to endogenous FSH secretion before his operation. Patient A, with unilateral cryptorchidism, representing the group of hypogonadotrophic males with the poorest prognosis in terms of sperm count3 had the highest relative increase in testis volume (final volume 229% of the initial volume; from 0·8 mL to 2·5 mL). This study shows that in prepubertal boys, r-hFSH treatment stimulates production of inhibin B, which reflects Sertoli-cell function, 4 and induces growth of testes. The volume of the prepubertal testis consists mainly of seminiferous tubules, the length of which correlates with the number of Sertoli cells. 5 We did not do a biopsy of the gonads before or during the r-hFSH treatment; nevertheless, our findings suggest that FSH is capable of inducing combined Sertoli-cell proliferation and elongation of seminiferous tubules, which may potentially increase the sperm-producing capacity later in life. This study was supported by the Foundation for Paediatric Research, Helsinki, Finland. 1
2
3
4
Arslan M, Weinbauer GF, Schlatt S, Shahab M, Niescglag E. FSH and testosterone, alone or in combination, initiate testicular growth and increase the number of spermatogonia and Sertoli cells in a juvenile non-human primate (Macaca mulatta). J Endocrinol 1993; 136: 235–43. Tapanainen JS, Aittomaki K, Min J, Vaskivuo T, Huhtaniemi IT. Men homozygous for an inactivating mutation of the folliclestimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility. Nat Genet 1997; 15: 205–06. Finkel MD, Phillips JL, Snyder PJ. Stimulation of spermatogenesis by gonadotropins in men with hypogonadotropic hypogonadism. New Engl J Med 1985; 313: 651–55. Anawalt BD, Bebb RA, Matsumoto AM, et al. Serum inhibin B
264
5
levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J Clin Endoctinol Metab 1996; 81: 3341–45. Rey RA, Campo SM, Bedecarras P, Nagle CA, Chemes HE. Is infancy a quiescent period of testicular development? Histological, morphometric and functional study of the seminiferous tubules of the Cebus monkey from birth to end of puberty. J Clin Endocrtinol Metab 1993; 76: 1325–31.
Children’s Hospital, University of Helsinki, Fin 00290 Helsinki, Finland, (T Raivio); Departments of Physiology and Pediatrics, University of Turku, Turku, Finland; and Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh, UK
Detection of gene deletion in single metastatic tumour cells in lymphnode tissue by fluorescent in-situ hybridisation Svetlana Pack, Alexander O Vortmeyer, Eugenia Pak, Lance A Liotta, Zhengping Zhuang
Pathological identification of malignant tumour cells in lymphnodes is often difficult although changes may be obvious in the presence of gland-forming metastatic adenocarcinoma or pigmented metastatic malignant melanoma.1 Very few tumour cells or the presence of one or two atypical cells may cause malignant disease to be suspected, but not provide sufficient evidence for correct diagnosis. We report the detection of genetic alterations in morphologically obscure neoplastic cells by single cell genetic analysis with fluorescent in-situ hybridisation (FISH). A 57-year-old man presented at the Clinical Centre, National Institutes of Health, with a 8 ⫻7⫻4 cm renal-cell carcinoma (RCC) in the left kidney. During nephrectomy, a 1⫻0·5⫻0·5 cm lymphnode was resected and submitted for intraoperative consultation. Both a touch preparation and frozen section were made. Examination of both preparations did not show evidence of metastatic tumour. The remainder of the frozen tissue was fixed in formalin and embedded in paraffin. We analysed cells from one of the touch preparations with FISH with the P1 clone containing the von Hippel Lindau (VHL) tumour suppressor gene for detection of VHL-gene deletion (figure, top). This analysis showed two VHL-gene alleles present in most cells with a few cells containing only one allele (figure, middle). Examination of tumour tissue from the same patient also revealed VHL-
Vol 350 • July 26, 1997
Sinus node dysfunction
Normal sinus node function
110 Chagas
Idiopathic
Chagas
IDC
HC
Change in beats per minute
70
20
–30
–80
–130 patient
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16
17 18 19 20 21
22 23 24 25 26
Chronotropic effects of IgG before (black bars) and after (white bars) addition of atropine in patients with abnormal (left) and normal (right) sinus node function IDC=idiopathic dilated cardiomyopathy, HC=healthy controls.
contraction rate (figure). This effect was partially or totally reversed by atropine, suggesting it may be due to stimulation of M2 cholinergic receptors. IgG from patient 7, in the presence of atropine, led to a marked positive effect thought to be due to the presence of anti-adrenoceptor-stimulating antibodies (the effect was partially reversed by metoprolol). In two other patients with sinus-node dysfunction (3 and 5), the cardiomyocyte contraction rate was not significantly affected by IgG, but increased after atropine addition, suggesting that the IgG fraction contained antibodies which reacted simultaneously with the M2 cholinergic receptor and the -adrenergic receptor; their effects cancelling out and becoming apparent only after addition of atropine. IgG from only two patients (12 and 11) with normal sinus-node function had either a negative action on cardiomyocytes or led to notable changes after addition of atropine. By contrast there was a positive effect in 14 patients with normal sinusnode function suggesting the presence of anti- adrenoceptor-stimulating antibodies. Our in-vitro results support the hypothesis that the sinus node may be the target of an autoimmune response, although it is not known whether such effects are important in vivo. IgG from patients with Chagas’ myocarditis caused both sinus bradycardia and second degree atrio-ventricular nodal block in rabbit heart (Langendorf preparation). 5 The association of autoimmune disease of the sinotrial node with chronic cardiac Chagas’ disease suggests that sinusnode dysfunction may be related to a chronic infectious process. The PO ribosomal protein of Trypanosoma cruzi3 is possibly involved in the generation of cholinergic antibodies. 1
2
Maisch B, Lotze U, Scheneide J, Kochsiek K. Antibodies to human sinus node in sick sinus syndrome. Pacing Cardiac Electrophysiol 1986; 9: 1101–09. Magnusson Y, Wallukat G, Waagstein F, Hjalmarson A, Hoebeke J. Autoimmunity in IDC: characterization of antibodies against the Beta-1 adrenoreceptor with positive chronotropic effect. Circulation 1994; 89: 2760–67.
Vol 350 • July 26, 1997
3
4
5
Ferrari I, Levin MJ, Wallukat G, et al. Molecular mimicry between the immunodominant ribosomal protein PO of Trypanosoma cruzi and a functional epitope on human 1-adrenergic receptor. J Exp Med 1995; 182: 59–65. Elies R, Ferrari I, Wallukat G, et al. Structural and functional analysis of the B cell epitope recognised by anti-receptor autoantibodies in patients with Chagas’ disease. J Immunol 1996; 157: 4203–11. Farias de Oliveira S, Pedrosa RC, Nascimento JHM, Campos de Carvalho AC, Masuda MO. Depression of cardiac automaticity and A-V conduction by IgG fraction of chronic chagasic patients with arrhythmia is mediated by muscarinic receptors. Mem Inst Oswaldo Cruz 1995; 90 (suppl 1): 151.
División Cardiología, Hospital Ramos Mejía and Instituto de Ingeniería Genética y Biología Molecular (INGEBI), CONUCET, 1221 Buenos Aires, Argentina, (Pablo A Chiale)
Treatment of prepubertal gonadotrophin-deficient boys with recombinant human folliclestimulating hormone Taneli Raivio, Jorma Toppari, Antti Perheentupa, Alan S McNeilly, Leo Dunkel
Sertoli cells stop dividing in early puberty but, before that, their number is probably dependent on follicle-stimulating hormone (FSH). Administration of FSH to immature nonhuman primates increases the number of Sertoli cells and induces growth of testes. 1 In human beings a gene mutation inactivating the FSH receptor leads to suppression of spermatogenesis and small testis volume. 2 Adults with prepubertal onset of gonadotrophin deficiency often have small testis volume, and a low sperm count, 3 which may result in infertility. We studied the effect of recombinant human FSH (r-hFSH) on growth of testes in prepubertal gonadotrophin-deficient boys.
263
THE LANCET
A
B
3 2 1 0
5 Testis volume (mL)
200
Patient A Patient B Patient C
Serum inhibin B (pg/mL)
Serum FSH (IU/L)
4
C
150 100 50
13·5
14·5
4 3 229% 2 102% 1 0
0 12·5
96%
12·5
13·5 Age (year)
14·5
12·5
13·5
14·5
Serum FSH (A), inhibin B (B) concentrations, and testis volumes (C) against age in three gonadotrophic-deficient boys measured at 3-month intervals Shaded lines showed the ranges of serum FSH and Inhibin B concentrations in healthy prepubertal boys (Tanner stage G1P1).
We studied three boys (A, 12·8; B, 13·2; and C, 13·2 years old) with gonadotrophin deficiency. Patients A and C had idiopathic, and patient B acquired (surgery for craniopharyngioma at age 10 years) hypopituitarism. Other pituitary-hormone deficiencies were adequately replaced. The boys were given r-hFSH (Gonal-F, Serono) subcutaneously (1·5 IU/kg) three times a week for 12 months. At 3-month intervals, puberty was staged according to Tanner, testis volume measured (0·52⫻length⫻width2), and a blood sample drawn for serum gonadotrophin, sex steroids, and inhibin B concentrations. During the study, without any increase in serum luteinising hormone (LH) or sex-steroid concentrations, serum FSH and inhibin B concentrations increased to within the range observed in healthy prepubertal boys (figure, A and B) and the testis volume increased (p<0·02, figure, C). Patient B had the largest initial testis volume, most likely due to endogenous FSH secretion before his operation. Patient A, with unilateral cryptorchidism, representing the group of hypogonadotrophic males with the poorest prognosis in terms of sperm count3 had the highest relative increase in testis volume (final volume 229% of the initial volume; from 0·8 mL to 2·5 mL). This study shows that in prepubertal boys, r-hFSH treatment stimulates production of inhibin B, which reflects Sertoli-cell function, 4 and induces growth of testes. The volume of the prepubertal testis consists mainly of seminiferous tubules, the length of which correlates with the number of Sertoli cells. 5 We did not do a biopsy of the gonads before or during the r-hFSH treatment; nevertheless, our findings suggest that FSH is capable of inducing combined Sertoli-cell proliferation and elongation of seminiferous tubules, which may potentially increase the sperm-producing capacity later in life. This study was supported by the Foundation for Paediatric Research, Helsinki, Finland. 1
2
3
4
Arslan M, Weinbauer GF, Schlatt S, Shahab M, Niescglag E. FSH and testosterone, alone or in combination, initiate testicular growth and increase the number of spermatogonia and Sertoli cells in a juvenile non-human primate (Macaca mulatta). J Endocrinol 1993; 136: 235–43. Tapanainen JS, Aittomaki K, Min J, Vaskivuo T, Huhtaniemi IT. Men homozygous for an inactivating mutation of the folliclestimulating hormone (FSH) receptor gene present variable suppression of spermatogenesis and fertility. Nat Genet 1997; 15: 205–06. Finkel MD, Phillips JL, Snyder PJ. Stimulation of spermatogenesis by gonadotropins in men with hypogonadotropic hypogonadism. New Engl J Med 1985; 313: 651–55. Anawalt BD, Bebb RA, Matsumoto AM, et al. Serum inhibin B
264
5
levels reflect Sertoli cell function in normal men and men with testicular dysfunction. J Clin Endoctinol Metab 1996; 81: 3341–45. Rey RA, Campo SM, Bedecarras P, Nagle CA, Chemes HE. Is infancy a quiescent period of testicular development? Histological, morphometric and functional study of the seminiferous tubules of the Cebus monkey from birth to end of puberty. J Clin Endocrtinol Metab 1993; 76: 1325–31.
Children’s Hospital, University of Helsinki, Fin 00290 Helsinki, Finland, (T Raivio); Departments of Physiology and Pediatrics, University of Turku, Turku, Finland; and Medical Research Council Reproductive Biology Unit, Centre for Reproductive Biology, Edinburgh, UK
Detection of gene deletion in single metastatic tumour cells in lymphnode tissue by fluorescent in-situ hybridisation Svetlana Pack, Alexander O Vortmeyer, Eugenia Pak, Lance A Liotta, Zhengping Zhuang
Pathological identification of malignant tumour cells in lymphnodes is often difficult although changes may be obvious in the presence of gland-forming metastatic adenocarcinoma or pigmented metastatic malignant melanoma.1 Very few tumour cells or the presence of one or two atypical cells may cause malignant disease to be suspected, but not provide sufficient evidence for correct diagnosis. We report the detection of genetic alterations in morphologically obscure neoplastic cells by single cell genetic analysis with fluorescent in-situ hybridisation (FISH). A 57-year-old man presented at the Clinical Centre, National Institutes of Health, with a 8 ⫻7⫻4 cm renal-cell carcinoma (RCC) in the left kidney. During nephrectomy, a 1⫻0·5⫻0·5 cm lymphnode was resected and submitted for intraoperative consultation. Both a touch preparation and frozen section were made. Examination of both preparations did not show evidence of metastatic tumour. The remainder of the frozen tissue was fixed in formalin and embedded in paraffin. We analysed cells from one of the touch preparations with FISH with the P1 clone containing the von Hippel Lindau (VHL) tumour suppressor gene for detection of VHL-gene deletion (figure, top). This analysis showed two VHL-gene alleles present in most cells with a few cells containing only one allele (figure, middle). Examination of tumour tissue from the same patient also revealed VHL-
Vol 350 • July 26, 1997