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PARATHYROID SECRETORY PROTEIN: IMMUNOCYTOCHEMICAL LOCALISATION WITHIN CELLS THAT CONTAIN PARATHYROID HORMONE
371
(10200).2 The biological function of P.S.P. is unknown but the pattern of secretion of P.S.P. in response to stimulatory or inhibitory concentrations of calcium is indistinguishable from that of P.T.H.13 P.T.H. and P.S.P. constitute over 90% of the newly synthesised protein released by parathyroid glands during incubation in vitro.’3 These observations suggest that1 P.S.P. and P.T.H. are co-secreted from the parathyroid gland. To test this hypothesis we prepared antisera to both proteins and compared the patterns of cellular immunostaining in successive sections of parathyroid glands treated with one or the other antiserum.
roidism in utero harm the fetus? Follow-up of children exposed in utero to antithyroid drugs has not yet revealed any significant developmental abnormalities either physically or intellectually.89 However, such interference with the fetal pituitarythyroid axis is undesirable and every effort should be made to use the smallest dose of drug at all stages during pregnancy. Careful monitoring will show that therapy can be discontinued in the later months of pregnancy in some patients. Department of Medicine, Gardiner Institute, University of Glasgow, Western Infirmary, Glasgow G11 6NT and Radioimmunoassay Unit, Department of Biochemistry, Royal Infirmary, Glasgow
Antisera
L. C. K. Low W. A. RATCLIFFE W. D. ALEXANDER
were
raised in rabbits
(New
Zealand
white) by repeated
subcutaneous injection4of bovine P.T.H.5 and P.s.P. The P.s.p. was prepared by electrophoretic separation of proteins isolated from a phenol of parathyroid; details of the procedure will be published elsewhere. Antisera to P.T.H. and P.s.p. bound 50% of the corresponding radioiodinated antigens at dilutions of 1 :40 000 and 1:500, respec-
extract
tively. PARATHYROID SECRETORY PROTEIN: IMMUNOCYTOCHEMICAL LOCALISATION WITHIN CELLS THAT CONTAIN PARATHYROID HORMONE
SIR,—In-vitro studies show that bovine parathyroid glands synthesise and secrete a major protein, parathyroid secretory protein (P.s.P.),1 a dimer consisting of two identical subunits of M.w. 70 000, differing in molecular weight from both parathyroid hormone (9500) and the biosynthetic precursors of parathyroid hormone, pre-pro- P.T.H. (13 000) and pro-p.T.H.
Slices of bovine parathyroid gland were fixed for 2 h in 0.2 mol/1 phosphate-buffered 2% p-formaldehyde containing 0.2% picric acid,6 washed in phosphate-buffered saline, dehydrated in ethanol, and embedded in ’Araldite’. After removal of embedding plastic by saturated sodium hydroxide in ethanol,7 serial sections under 1 µm in thickness were cut and stained8 by the antisera. The ultrastructural Habener, J. F., Kemper, B. W., Rich, A., Potts, J. T., Jr. Rec. Prog. Horm. Res. 1977, 33, 249. 3. Habener, J. F., Potts, J. T., Jr. Endocrinology, 1976, 98, 197. 4. Habener, J. F., Potts, J. T., Jr. in Hormones In Blood (edited by H. N.
2.
Antoniades), Cambridge, Mass. 1977. Aurbach, G. D., Dawaon. B. F., Niall, H. -D. Deftos, J., Potts, J. T., Jr. Biochemistry, 1971, 10, 2779. 6. Stefanini, M., De Martino, L. J., Zamboni, L. Nature, 1967, 216, 173. 7. Lane, B. P., Europa, D. L.J. Histochem. Cytochem. 1965, 13, 579. 8. Coons, A. H., Leduc, E. H., Connolly, J. M.J. exp. Med. 1955, 102, 49. 5. Keutmann, H. T.,
8. Burrow, G. N., and others. Am. J. Dis. Child. 1968, 116, 161. 9. McCarroll, A. M., and others. Archs Dis. Childh. 1976, 51, 532. 1. Kemper, B., Habener, J. F., Rich, A., and Potts, J. T., Jr. Science, 1974,
184,167.
L.
Immunofluorescence and electronmicroscopy studies of localisation ofP.T.H. and P.S.P. in bovine parathyroid glands.
(a) Staining with anti-P.s.p. serum; and (b) section successive to that shown in (a) stained with anti-P.T.H. serum. (c) Part of a chief cell in thin section after immunocytochemical staining by peroxidase/antiperoxidase method area
of chief cell in
a
successive thin section but stained with anti-P.T.H.
serum.
with anti-p.s.P.; and
(d)
same
372 localisation of P.T.H. and P.s.P. was accomplished with the unlabelled antibody enzyme method of Sternberger et al. Ribbons of consecutive thin sections mounted on nickel grids were incubated sequentially with anti-p.T.H. or anti-p.s.p. serum, sheep anti-rabbit-globulin serum, and peroxidase-antiperoxidase complex (a gift of Dr L. A. Sternberger, Edgewood Arsenal, Maryland). The peroxidase was then revealed with diaminobenzidine and hydrogen peroxide in tris buffer.1O Immunoreactive deposit was made electron dense by exposing sections to 2% osmium tetroxide in distilled water. The specificity of immunostaining was checked wun antisera that were cross-absorbed (i.e., absorption of anti-P.T.H. serum with P.T.H. or P.S.P. and of anti-P.S.P. sera with P.S.P. or P.T.H.). incubated with anti-P.T.H. or with anti-p.s.p. allowed us to follow the same cells through several sections and to compare the morphological distribution of immunoreactive P.T.H. and P.s.P.
The studiesof consecutivesemithinsections
As the figure shows, immunofluorescence after the application of anti-p.T.H. and anti-p.s.p. sera is limited to an identical population of cells. Furthermore, the two staining patterns in single cells seem superimposable, with finely granular fluorescence restricted to the cytoplasm of the parenchymal (chief) cells. Electronmicroscopy showed that the electron-dense reaction product was confined to the secretory granules of the chief cells. Positive reactions were completely inhibited when the antisera had been absorbed with the homologous antigens, whereas cross-absorptions did not affect the positive reaction. These observations demonstrate that antigenic sites reacting with anti-p.s.p. and anti-p.T.H. sera are present in the same population of chief cells and within the same subcellular compartment. We conclude that the two major proteins secreted by the parathyroid glands are both contained in the same secretory granules of the chief cells. We thank A. M. Lucini, M. Sidler, P. C. Dee, and H. Chang for
The question whether the word should be "opportunist" or "opportunistic" I leave to the etymologists among your readers. Department of Pathology, University College,
p. 207) and Dr Howie1 sugbetween tuberculosis and bronchial that the association gested carcinoma may be more than fortuitous, and I agree. Many factors seem to initiate neoplasms,2 and the chronic immune irritation generated by tuberculosis and sarcoidosis3 is one of these. Mycobacterial infections in Texas were found to be three times more common among cancer patients than among the local population. 9 of the 65 malignant cases had lung cancer, 7 had lymphomas, and 19 had squamous-cell carcinoma of the head and neck.4 A similar increase in the incidence of malignant tumours was reported in Denmark, among 244 cases of respiratory sarcoidosis. 9 of the 48 patients with tumours had lung cancer, and 6 had lymphomas. Partly because there was less sunlight, only 7 of these had skin
SIR,-Both Dr Sakula (July 22,
cancer.5 Atypical
St. Luke’s Hospital, Guildford, Surrey GU1
Endocrine Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts, U.S.A.
JOEL F. HABENER JOHN T. POTTS, JR.
PULMONARY TUBERCULOSIS AND BRONCHIAL CARCINOMA
SIR,—Dr Sakula (July 22, p. 207) takes
me to task for my of the word "opportunist" as applied to tuberculosis complicating bronchial carcinoma. Although I confess to trailing my coat in making such a statement, there is ample support for this use of the word opportunist. In a reference cited in my first letter there is the flat statement that "mycobacteria act as opportunistic pathogens in persons with malignant diseases". Smith2states that "opportunist infections result from microorganisms, both traditional pathogens and those that in the and are past might have been considered nonpathogens commonest in patients whose immune responses are compromised"-e.g., those with lung cancer. Gowing3 and a Ciba symposium4 give similar definitions. But, as Gowing3 and also Smith2state, a satisfactory definition is by no means easy to frame and Dr Sakula will find support for this use of the word opportunist in Stedman’s Medical Dictionary.5
use
...
9.
Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J., Meyer, H. G. J. Histochem. Cytochem. 1970, 18, 315. Graham, R. C., Karnovsky, M. J. ibid. 1966, 14, 291.
10. 1. Ortbols, D. W., Marr, J. J. Am. Rev. resp. Dis. 1978, 117, 39. 2. Smith, H. Br. med. J. 1973, ii, 107. 3. Gowing, N. in Hodgkin’s Disease (edited by D. Smithers). London, 1973. 4. Wolstenholme, G. E. W., Porter, R. Ciba Foundation Symposium on Systemic Myeoses; p. 15. London, 1968. 5. Stedman’s Medical Dictionary; p. 990. New York, 1976.
3NT
GERALD A. MACGREGOR
MARKER FOR PINEAL TUMOURS?
and may be difficult to idenmarker would be of considerable importance in the diagnosis and monitoring of these lesions. The finding of high melatonin concentrations in the serum of a patient with histologically proven pineocytoma 10 1’ prompted examination of serum from similar patients on the West Midlands Cancer Registry. Sera were assayed for melatonin, chorionic gonadotrophin (H.c.G.) and its subunits, carcinoembryonic antigen, and a-fetoprotein. The five additional patients (three male, two female) were aged 9-72 years (mean 25). Blood-samples were taken at noon from these patients who, 6 months to 12 years previously, had had 4000-5000 rad directed at the pineal, the diagnosis having been confirmed radiologically. Melatonin levels were 183, 131, 142, 182, and 140 pg/ml; normal mid-day values are 20+3 pg/ml (mean ±2 s.D.). In one patient the a-gonadotrophin subunit was not significantly raised at 6.4 µg/l (normal <6.3), and in two patients &bgr;-H.C.G. concentrations were raised at 4.9 and 3.3 µg/l (normal <2.5). Carcinoembryonic antigen and oc-fetoprotein levels were normal in all patients. These results substantiate an earlier report’o 11 that gonadotrophin subunits may be increased in patients with pineal tumours, but the concentrations in our patients were not sufficiently abnormal to provide a useful index of tumour activity. On the other hand, measurement of melatonin in serum may be useful, but the effects of other diseases and of radiotherapy require further elucidation before its value can be- fully
SIR,-Pineal
MARIELLA RAVAZZOLA LELIO ORCI
mycobacteriaor a’protoplast form of Myco-
bacterium tuberculosis7 may be a cause of sarcoidosis.
technical assistance. These studies were supported in part by grants from the U.S. Public Health Service and the Swiss National Science Foundation (grant 3.120.77). J. F. H. is an investigator of the Howard Hughes Medical Institute. Institute of Histology and Embryology, University of Geneva Medical School, 1211 Geneva 4, Switzerland
T. M. HEALY
Dublin 2, Ireland
tify, 1,9
tumours are rare
so a tumour
1. 2. 3. 4.
Howie, A. J. Lancet, 1978, i, 881. MacGregor, G. A. ibid. 1973, i, 1185. MacGregor, G. A. Br. med. J. 1972, i, 375. Field, R., Bodey, G. P., Groschel, D. Archs intern. Med. 1976, 136, 67. 5. Brincker, H., Wilbek, E. Br. J. Cancer, 1974, 29, 247. 6. Strickland, G. T., Moser, K.Am.J. Med. 1967, 43, 131. 7. Mitchell, D. N., Rees, R. J. W., Goswami, K. K. A. Lancet, 1976, ii, 761. 8. Wurtman, R. H., Moskowitz, M. A. New Engl. J. Med. 1977, 296, 1329. 9. Wurtman, R. J. Moskowitz, M. A. ibid. p. 1383. 10. Barber, S. G., Smith, J. A., Hughes, R. C. Br. med. J. 1978, ii, 328.
11. Kubo, O., and others J. Neurosurg. 1977, 47, 101.
(10200).2 The biological function of P.S.P. is unknown but the pattern of secretion of P.S.P. in response to stimulatory or inhibitory concentrations of calcium is indistinguishable from that of P.T.H.13 P.T.H. and P.S.P. constitute over 90% of the newly synthesised protein released by parathyroid glands during incubation in vitro.’3 These observations suggest that1 P.S.P. and P.T.H. are co-secreted from the parathyroid gland. To test this hypothesis we prepared antisera to both proteins and compared the patterns of cellular immunostaining in successive sections of parathyroid glands treated with one or the other antiserum.
roidism in utero harm the fetus? Follow-up of children exposed in utero to antithyroid drugs has not yet revealed any significant developmental abnormalities either physically or intellectually.89 However, such interference with the fetal pituitarythyroid axis is undesirable and every effort should be made to use the smallest dose of drug at all stages during pregnancy. Careful monitoring will show that therapy can be discontinued in the later months of pregnancy in some patients. Department of Medicine, Gardiner Institute, University of Glasgow, Western Infirmary, Glasgow G11 6NT and Radioimmunoassay Unit, Department of Biochemistry, Royal Infirmary, Glasgow
Antisera
L. C. K. Low W. A. RATCLIFFE W. D. ALEXANDER
were
raised in rabbits
(New
Zealand
white) by repeated
subcutaneous injection4of bovine P.T.H.5 and P.s.P. The P.s.p. was prepared by electrophoretic separation of proteins isolated from a phenol of parathyroid; details of the procedure will be published elsewhere. Antisera to P.T.H. and P.s.p. bound 50% of the corresponding radioiodinated antigens at dilutions of 1 :40 000 and 1:500, respec-
extract
tively. PARATHYROID SECRETORY PROTEIN: IMMUNOCYTOCHEMICAL LOCALISATION WITHIN CELLS THAT CONTAIN PARATHYROID HORMONE
SIR,—In-vitro studies show that bovine parathyroid glands synthesise and secrete a major protein, parathyroid secretory protein (P.s.P.),1 a dimer consisting of two identical subunits of M.w. 70 000, differing in molecular weight from both parathyroid hormone (9500) and the biosynthetic precursors of parathyroid hormone, pre-pro- P.T.H. (13 000) and pro-p.T.H.
Slices of bovine parathyroid gland were fixed for 2 h in 0.2 mol/1 phosphate-buffered 2% p-formaldehyde containing 0.2% picric acid,6 washed in phosphate-buffered saline, dehydrated in ethanol, and embedded in ’Araldite’. After removal of embedding plastic by saturated sodium hydroxide in ethanol,7 serial sections under 1 µm in thickness were cut and stained8 by the antisera. The ultrastructural Habener, J. F., Kemper, B. W., Rich, A., Potts, J. T., Jr. Rec. Prog. Horm. Res. 1977, 33, 249. 3. Habener, J. F., Potts, J. T., Jr. Endocrinology, 1976, 98, 197. 4. Habener, J. F., Potts, J. T., Jr. in Hormones In Blood (edited by H. N.
2.
Antoniades), Cambridge, Mass. 1977. Aurbach, G. D., Dawaon. B. F., Niall, H. -D. Deftos, J., Potts, J. T., Jr. Biochemistry, 1971, 10, 2779. 6. Stefanini, M., De Martino, L. J., Zamboni, L. Nature, 1967, 216, 173. 7. Lane, B. P., Europa, D. L.J. Histochem. Cytochem. 1965, 13, 579. 8. Coons, A. H., Leduc, E. H., Connolly, J. M.J. exp. Med. 1955, 102, 49. 5. Keutmann, H. T.,
8. Burrow, G. N., and others. Am. J. Dis. Child. 1968, 116, 161. 9. McCarroll, A. M., and others. Archs Dis. Childh. 1976, 51, 532. 1. Kemper, B., Habener, J. F., Rich, A., and Potts, J. T., Jr. Science, 1974,
184,167.
L.
Immunofluorescence and electronmicroscopy studies of localisation ofP.T.H. and P.S.P. in bovine parathyroid glands.
(a) Staining with anti-P.s.p. serum; and (b) section successive to that shown in (a) stained with anti-P.T.H. serum. (c) Part of a chief cell in thin section after immunocytochemical staining by peroxidase/antiperoxidase method area
of chief cell in
a
successive thin section but stained with anti-P.T.H.
serum.
with anti-p.s.P.; and
(d)
same
372 localisation of P.T.H. and P.s.P. was accomplished with the unlabelled antibody enzyme method of Sternberger et al. Ribbons of consecutive thin sections mounted on nickel grids were incubated sequentially with anti-p.T.H. or anti-p.s.p. serum, sheep anti-rabbit-globulin serum, and peroxidase-antiperoxidase complex (a gift of Dr L. A. Sternberger, Edgewood Arsenal, Maryland). The peroxidase was then revealed with diaminobenzidine and hydrogen peroxide in tris buffer.1O Immunoreactive deposit was made electron dense by exposing sections to 2% osmium tetroxide in distilled water. The specificity of immunostaining was checked wun antisera that were cross-absorbed (i.e., absorption of anti-P.T.H. serum with P.T.H. or P.S.P. and of anti-P.S.P. sera with P.S.P. or P.T.H.). incubated with anti-P.T.H. or with anti-p.s.p. allowed us to follow the same cells through several sections and to compare the morphological distribution of immunoreactive P.T.H. and P.s.P.
The studiesof consecutivesemithinsections
As the figure shows, immunofluorescence after the application of anti-p.T.H. and anti-p.s.p. sera is limited to an identical population of cells. Furthermore, the two staining patterns in single cells seem superimposable, with finely granular fluorescence restricted to the cytoplasm of the parenchymal (chief) cells. Electronmicroscopy showed that the electron-dense reaction product was confined to the secretory granules of the chief cells. Positive reactions were completely inhibited when the antisera had been absorbed with the homologous antigens, whereas cross-absorptions did not affect the positive reaction. These observations demonstrate that antigenic sites reacting with anti-p.s.p. and anti-p.T.H. sera are present in the same population of chief cells and within the same subcellular compartment. We conclude that the two major proteins secreted by the parathyroid glands are both contained in the same secretory granules of the chief cells. We thank A. M. Lucini, M. Sidler, P. C. Dee, and H. Chang for
The question whether the word should be "opportunist" or "opportunistic" I leave to the etymologists among your readers. Department of Pathology, University College,
p. 207) and Dr Howie1 sugbetween tuberculosis and bronchial that the association gested carcinoma may be more than fortuitous, and I agree. Many factors seem to initiate neoplasms,2 and the chronic immune irritation generated by tuberculosis and sarcoidosis3 is one of these. Mycobacterial infections in Texas were found to be three times more common among cancer patients than among the local population. 9 of the 65 malignant cases had lung cancer, 7 had lymphomas, and 19 had squamous-cell carcinoma of the head and neck.4 A similar increase in the incidence of malignant tumours was reported in Denmark, among 244 cases of respiratory sarcoidosis. 9 of the 48 patients with tumours had lung cancer, and 6 had lymphomas. Partly because there was less sunlight, only 7 of these had skin
SIR,-Both Dr Sakula (July 22,
cancer.5 Atypical
St. Luke’s Hospital, Guildford, Surrey GU1
Endocrine Unit, Massachusetts General Hospital and Department of Medicine, Harvard Medical School, Boston, Massachusetts, U.S.A.
JOEL F. HABENER JOHN T. POTTS, JR.
PULMONARY TUBERCULOSIS AND BRONCHIAL CARCINOMA
SIR,—Dr Sakula (July 22, p. 207) takes
me to task for my of the word "opportunist" as applied to tuberculosis complicating bronchial carcinoma. Although I confess to trailing my coat in making such a statement, there is ample support for this use of the word opportunist. In a reference cited in my first letter there is the flat statement that "mycobacteria act as opportunistic pathogens in persons with malignant diseases". Smith2states that "opportunist infections result from microorganisms, both traditional pathogens and those that in the and are past might have been considered nonpathogens commonest in patients whose immune responses are compromised"-e.g., those with lung cancer. Gowing3 and a Ciba symposium4 give similar definitions. But, as Gowing3 and also Smith2state, a satisfactory definition is by no means easy to frame and Dr Sakula will find support for this use of the word opportunist in Stedman’s Medical Dictionary.5
use
...
9.
Sternberger, L. A., Hardy, P. H., Jr., Cuculis, J. J., Meyer, H. G. J. Histochem. Cytochem. 1970, 18, 315. Graham, R. C., Karnovsky, M. J. ibid. 1966, 14, 291.
10. 1. Ortbols, D. W., Marr, J. J. Am. Rev. resp. Dis. 1978, 117, 39. 2. Smith, H. Br. med. J. 1973, ii, 107. 3. Gowing, N. in Hodgkin’s Disease (edited by D. Smithers). London, 1973. 4. Wolstenholme, G. E. W., Porter, R. Ciba Foundation Symposium on Systemic Myeoses; p. 15. London, 1968. 5. Stedman’s Medical Dictionary; p. 990. New York, 1976.
3NT
GERALD A. MACGREGOR
MARKER FOR PINEAL TUMOURS?
and may be difficult to idenmarker would be of considerable importance in the diagnosis and monitoring of these lesions. The finding of high melatonin concentrations in the serum of a patient with histologically proven pineocytoma 10 1’ prompted examination of serum from similar patients on the West Midlands Cancer Registry. Sera were assayed for melatonin, chorionic gonadotrophin (H.c.G.) and its subunits, carcinoembryonic antigen, and a-fetoprotein. The five additional patients (three male, two female) were aged 9-72 years (mean 25). Blood-samples were taken at noon from these patients who, 6 months to 12 years previously, had had 4000-5000 rad directed at the pineal, the diagnosis having been confirmed radiologically. Melatonin levels were 183, 131, 142, 182, and 140 pg/ml; normal mid-day values are 20+3 pg/ml (mean ±2 s.D.). In one patient the a-gonadotrophin subunit was not significantly raised at 6.4 µg/l (normal <6.3), and in two patients &bgr;-H.C.G. concentrations were raised at 4.9 and 3.3 µg/l (normal <2.5). Carcinoembryonic antigen and oc-fetoprotein levels were normal in all patients. These results substantiate an earlier report’o 11 that gonadotrophin subunits may be increased in patients with pineal tumours, but the concentrations in our patients were not sufficiently abnormal to provide a useful index of tumour activity. On the other hand, measurement of melatonin in serum may be useful, but the effects of other diseases and of radiotherapy require further elucidation before its value can be- fully
SIR,-Pineal
MARIELLA RAVAZZOLA LELIO ORCI
mycobacteriaor a’protoplast form of Myco-
bacterium tuberculosis7 may be a cause of sarcoidosis.
technical assistance. These studies were supported in part by grants from the U.S. Public Health Service and the Swiss National Science Foundation (grant 3.120.77). J. F. H. is an investigator of the Howard Hughes Medical Institute. Institute of Histology and Embryology, University of Geneva Medical School, 1211 Geneva 4, Switzerland
T. M. HEALY
Dublin 2, Ireland
tify, 1,9
tumours are rare
so a tumour
1. 2. 3. 4.
Howie, A. J. Lancet, 1978, i, 881. MacGregor, G. A. ibid. 1973, i, 1185. MacGregor, G. A. Br. med. J. 1972, i, 375. Field, R., Bodey, G. P., Groschel, D. Archs intern. Med. 1976, 136, 67. 5. Brincker, H., Wilbek, E. Br. J. Cancer, 1974, 29, 247. 6. Strickland, G. T., Moser, K.Am.J. Med. 1967, 43, 131. 7. Mitchell, D. N., Rees, R. J. W., Goswami, K. K. A. Lancet, 1976, ii, 761. 8. Wurtman, R. H., Moskowitz, M. A. New Engl. J. Med. 1977, 296, 1329. 9. Wurtman, R. J. Moskowitz, M. A. ibid. p. 1383. 10. Barber, S. G., Smith, J. A., Hughes, R. C. Br. med. J. 1978, ii, 328.
11. Kubo, O., and others J. Neurosurg. 1977, 47, 101.