194
3. Libert F,
Ludgate M, Dinsart C, Vassart G. Thyroperoxidase, but not the thyrotropin
receptor, contains sequential epitopes recognised by autoantibodies m recombinant peptides expressed in the pUEX vector.J Clin Endocrinol Metab 1991; 73: 857-60. 4. Harfst E, Johnstone AP, Gout I, Taylor AH, Waterfield MD, Nussey SS. The use of the amplifiable high-expression vector pEE14 to study the interactions of autoanubodies with recombinant human thyrotrophm receptor. Mol Cell Endocrinol (in press). 5. Xie Y-B, Wang H, Segaloff DL. Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity. J Biol Chem 1990; 265: 21411-14. 6. Dirmikis S, Munro DS. Studies on the binding activity for the long-acting thyroid stimulator. J Endocrinol 1973; 58: 577-90. 7. Adlkofer F, Kotulla P, Schleusener H. Binding of thyrotrophin to low molecular weight fragments of human thyroid membranes. Acta Endocrinol (Copenh) 1980, 94: 58-63.
Influenza vaccination in asthma Inhibition of adenylate cyclase activity stimulated by autoantisera or (B) TSH.
(A)
Inhibition of adenylate cyclase activity in a CHO clone expressing full-length hTSHR protein, by homogenates of non-transfected CHO cells (CHO), two individual clones which express full length hTSHr (FL and FL+; about 30 000 and 80 000 TSH binding sites per cell, respectively), and a clone expressing even higher levels of the hTSHRR extracellular region (EX). Value for 100% inhibition is taken as cyclic AMP concentrations in assay buffer alone, and 0% is value for TSH or antisera in absence of cell homogenates n = 11-18, means and SEM (vertical bars) shown
high-expression pEE14 system (Celltech), as we have already described for full-length hTSHr (FL-hTSHr).4 The resultant protein could be seen, by immunoprecipitation and immunoblotting, to be a glycoprotein of about 55 000 molecular weight. The binding functions of this product were then investigated by its ability to absorb cAMP stimulating activity from TSH and autoantibodies. Cells containing recombinant protein were freeze-thawed and sonicated in hypotonic buffer containing protease inhibitors (106 cells/ml) and incubated with either bovine TSH (’Thyrotropin’, Armour, at a fmal concentration of 50 RU/ml) or untreated Graves’ serum samples (final dilution 1 in 10) for 30 min at 37°C; the concentrations of agonist were chosen to give about half-maximum stimulation of cAMP production. After centrifugation, the supernatant (250 µl) was tested for its cAMPstimulating activity as describedexcept that intracellular cAMP was measured after washing of the cells and extraction with 70% ethanol. The results (figure) clearly show that the external hTSHr domain was potent in reducing the activity of autoantibodies, but had very little effect on the activity of TSH. By contrast, cells containing full-length hTSHr, especially after amplification of expression, were equipotent in absorbing both TSH and autoantibodies (figure). These results are surprising in view of the predictions from the cDNA sequence; the similar lutropin (LH)/chorionic gonadotropin receptor external region binds labelled chorionic gonadotropin with high affinityHowever, our data are compatible with results obtained from early studies6,7 on the absorption of autoantibodies by human thyroid membranes; mechanical disruption of these membranes caused the solubilisation of a part of the hTSHr that retained the capacity to bind antibodies but which had a much reduced affinity for TSH. This had a sedimentation coefficient of 4S (about 50 000 molecular weight-the so-called 4S-LAA) suggesting that it is a large part of the hTSHr external domain fractured during preparation at or near the membrane insertion. We conclude that the hTSHr extracellular region contains epitopes for stimulating autoantibodies but is insufficient to constitute the high-affinity TSH binding site. Division of Biochemical Medicine, St George’s Hospital Medical School, London SW17 ORE, UK
ELIZABETH HARFST A. P. JOHNSTONE S. S. NUSSEY
SIR,-A large number of people are vaccinated with influenza vaccine every winter, since the UK Chief Medical Officer recommends an annual influenza vaccination for people at special risk, especially elderly patients, those with chronic pulmonary disease, chronic heart disease, chronic renal disease, and diabetes mellitus, and those receiving immunosuppressant therapy. Influenza vaccines are of two main types, live attenuated and inactivated. The inactivated vaccine is available in two forms, split virus vaccine and surface antigen vaccine. These vaccines have many side-effects. Because asthma exacerbations can be triggered by upper-respiratory-tract infections, a large number of asthmatics are vaccinated against influenza each year, but a viral aetiology has not been confirmed in more than 20% of such exacerbations in adults. Influenza vaccination is known to increase bronchial reactivity and cause exacerbations of asthma. 2,3 Some of our patients had exacerbations of asthma after such vaccination and three were admitted, one of whom needed assisted ventilation in the intensive care unit. These cases have been reported to the Committee on Safety of Medicines. We do not recommend an annual influenza vaccine in stable asthmatics, because of the potential side-effects. Perhaps those patients whose asthma is unstable, and who had frequent exacerbations in the previous year after upper-respiratory-tract infections, should be vaccinated with sub-unit vaccine. These vaccines are prepared by separating surface antigens from the core virus by selective solubilisation,4have fewer side-effects, and are well tolerated by a group of moderately severe asthmatics.5 WAJAHAT U. HASSAN
1. Brewis RAL, Gibson GJ, Geddes DM. Respir Med 1990; 17: 648. 2 Dejongste JC, Degenhart HC, Neijens HJ, et al. Bronchial responsiveness and leucocyte reactivity after influenza vaccine m asthmatic patients. Eur J Respir 1984; 65: 196. 3. Oukette JJ, Reed CE. Increased response of asthmatic subjects to methacholine after influenza vaccine. J Allergy 1965; 36: 558-63. 4. Backmayer H. Selective solubilisation of haemagglutinin and neuroaminidase from influenza viruses. Intervirology 1975; 5: 260-68. 5. Albazzaz MK, Harvey JE, Grilli EA, Caul EO, Roome AP. Subunit influenza vaccination in adults with asthma: effects on clinical state, airway reactivity and antibody response. Br Med J 1987; 294: 1196.
Tegernsee giant p
SIR,-Dr Nerlich (Oct 5, p 886) and Dr Schwindinger (Dec 7, 1454) and their colleagues speculate on the possible coexistence of
juvenile gigantism and polyostotic fibrous dysplasia in the Tegemsee giant, but are unaware that this association has already been reported.1 The first of the two patients described was a boy born in Nottingham in 1930, whom I saw in Manchester Royal Infirmary in 1954, and who was still alive, though severely disabled by bone disease, in the late 1960s. Department
1 Ohmon M, Endo T, Onaya T. Development of chicken antibodies toward the human thyrotropin receptor peptides and their bioactivities. Biochem Biophys Res Commun
1991; 174: 399-403. 2. Kosugi S, Ban T, Akamizu T, Kohn LD. Site-directed mutagenesis of a portion of the extracellular domain of the rat thyrotropin receptor important m autoimmune thyroid disease and nonhomologous with gonadotropm receptors. Relationship of functional and immunogenic domains J Biol Chem 1991; 266: 19413-18.
ALLAN F. HENDERSON NIALL P. KEANEY
Royal Infirmary, Sunderland SR2 7JE, UK
of Medicine,
Rayne Institute, University College and Middlesex School of Medicine, London WC1 E 6JJ, UK 1. Falconer
OLIVER WRONG
MA, Cope CL. Fibrous dysplasia of bone with endocrine disorders pigmentation. Q J Med 1942; 11: 121-54.
cutaneous
and
3. Libert F,
Ludgate M, Dinsart C, Vassart G. Thyroperoxidase, but not the thyrotropin
receptor, contains sequential epitopes recognised by autoantibodies m recombinant peptides expressed in the pUEX vector.J Clin Endocrinol Metab 1991; 73: 857-60. 4. Harfst E, Johnstone AP, Gout I, Taylor AH, Waterfield MD, Nussey SS. The use of the amplifiable high-expression vector pEE14 to study the interactions of autoanubodies with recombinant human thyrotrophm receptor. Mol Cell Endocrinol (in press). 5. Xie Y-B, Wang H, Segaloff DL. Extracellular domain of lutropin/choriogonadotropin receptor expressed in transfected cells binds choriogonadotropin with high affinity. J Biol Chem 1990; 265: 21411-14. 6. Dirmikis S, Munro DS. Studies on the binding activity for the long-acting thyroid stimulator. J Endocrinol 1973; 58: 577-90. 7. Adlkofer F, Kotulla P, Schleusener H. Binding of thyrotrophin to low molecular weight fragments of human thyroid membranes. Acta Endocrinol (Copenh) 1980, 94: 58-63.
Influenza vaccination in asthma Inhibition of adenylate cyclase activity stimulated by autoantisera or (B) TSH.
(A)
Inhibition of adenylate cyclase activity in a CHO clone expressing full-length hTSHR protein, by homogenates of non-transfected CHO cells (CHO), two individual clones which express full length hTSHr (FL and FL+; about 30 000 and 80 000 TSH binding sites per cell, respectively), and a clone expressing even higher levels of the hTSHRR extracellular region (EX). Value for 100% inhibition is taken as cyclic AMP concentrations in assay buffer alone, and 0% is value for TSH or antisera in absence of cell homogenates n = 11-18, means and SEM (vertical bars) shown
high-expression pEE14 system (Celltech), as we have already described for full-length hTSHr (FL-hTSHr).4 The resultant protein could be seen, by immunoprecipitation and immunoblotting, to be a glycoprotein of about 55 000 molecular weight. The binding functions of this product were then investigated by its ability to absorb cAMP stimulating activity from TSH and autoantibodies. Cells containing recombinant protein were freeze-thawed and sonicated in hypotonic buffer containing protease inhibitors (106 cells/ml) and incubated with either bovine TSH (’Thyrotropin’, Armour, at a fmal concentration of 50 RU/ml) or untreated Graves’ serum samples (final dilution 1 in 10) for 30 min at 37°C; the concentrations of agonist were chosen to give about half-maximum stimulation of cAMP production. After centrifugation, the supernatant (250 µl) was tested for its cAMPstimulating activity as describedexcept that intracellular cAMP was measured after washing of the cells and extraction with 70% ethanol. The results (figure) clearly show that the external hTSHr domain was potent in reducing the activity of autoantibodies, but had very little effect on the activity of TSH. By contrast, cells containing full-length hTSHr, especially after amplification of expression, were equipotent in absorbing both TSH and autoantibodies (figure). These results are surprising in view of the predictions from the cDNA sequence; the similar lutropin (LH)/chorionic gonadotropin receptor external region binds labelled chorionic gonadotropin with high affinityHowever, our data are compatible with results obtained from early studies6,7 on the absorption of autoantibodies by human thyroid membranes; mechanical disruption of these membranes caused the solubilisation of a part of the hTSHr that retained the capacity to bind antibodies but which had a much reduced affinity for TSH. This had a sedimentation coefficient of 4S (about 50 000 molecular weight-the so-called 4S-LAA) suggesting that it is a large part of the hTSHr external domain fractured during preparation at or near the membrane insertion. We conclude that the hTSHr extracellular region contains epitopes for stimulating autoantibodies but is insufficient to constitute the high-affinity TSH binding site. Division of Biochemical Medicine, St George’s Hospital Medical School, London SW17 ORE, UK
ELIZABETH HARFST A. P. JOHNSTONE S. S. NUSSEY
SIR,-A large number of people are vaccinated with influenza vaccine every winter, since the UK Chief Medical Officer recommends an annual influenza vaccination for people at special risk, especially elderly patients, those with chronic pulmonary disease, chronic heart disease, chronic renal disease, and diabetes mellitus, and those receiving immunosuppressant therapy. Influenza vaccines are of two main types, live attenuated and inactivated. The inactivated vaccine is available in two forms, split virus vaccine and surface antigen vaccine. These vaccines have many side-effects. Because asthma exacerbations can be triggered by upper-respiratory-tract infections, a large number of asthmatics are vaccinated against influenza each year, but a viral aetiology has not been confirmed in more than 20% of such exacerbations in adults. Influenza vaccination is known to increase bronchial reactivity and cause exacerbations of asthma. 2,3 Some of our patients had exacerbations of asthma after such vaccination and three were admitted, one of whom needed assisted ventilation in the intensive care unit. These cases have been reported to the Committee on Safety of Medicines. We do not recommend an annual influenza vaccine in stable asthmatics, because of the potential side-effects. Perhaps those patients whose asthma is unstable, and who had frequent exacerbations in the previous year after upper-respiratory-tract infections, should be vaccinated with sub-unit vaccine. These vaccines are prepared by separating surface antigens from the core virus by selective solubilisation,4have fewer side-effects, and are well tolerated by a group of moderately severe asthmatics.5 WAJAHAT U. HASSAN
1. Brewis RAL, Gibson GJ, Geddes DM. Respir Med 1990; 17: 648. 2 Dejongste JC, Degenhart HC, Neijens HJ, et al. Bronchial responsiveness and leucocyte reactivity after influenza vaccine m asthmatic patients. Eur J Respir 1984; 65: 196. 3. Oukette JJ, Reed CE. Increased response of asthmatic subjects to methacholine after influenza vaccine. J Allergy 1965; 36: 558-63. 4. Backmayer H. Selective solubilisation of haemagglutinin and neuroaminidase from influenza viruses. Intervirology 1975; 5: 260-68. 5. Albazzaz MK, Harvey JE, Grilli EA, Caul EO, Roome AP. Subunit influenza vaccination in adults with asthma: effects on clinical state, airway reactivity and antibody response. Br Med J 1987; 294: 1196.
Tegernsee giant p
SIR,-Dr Nerlich (Oct 5, p 886) and Dr Schwindinger (Dec 7, 1454) and their colleagues speculate on the possible coexistence of
juvenile gigantism and polyostotic fibrous dysplasia in the Tegemsee giant, but are unaware that this association has already been reported.1 The first of the two patients described was a boy born in Nottingham in 1930, whom I saw in Manchester Royal Infirmary in 1954, and who was still alive, though severely disabled by bone disease, in the late 1960s. Department
1 Ohmon M, Endo T, Onaya T. Development of chicken antibodies toward the human thyrotropin receptor peptides and their bioactivities. Biochem Biophys Res Commun
1991; 174: 399-403. 2. Kosugi S, Ban T, Akamizu T, Kohn LD. Site-directed mutagenesis of a portion of the extracellular domain of the rat thyrotropin receptor important m autoimmune thyroid disease and nonhomologous with gonadotropm receptors. Relationship of functional and immunogenic domains J Biol Chem 1991; 266: 19413-18.
ALLAN F. HENDERSON NIALL P. KEANEY
Royal Infirmary, Sunderland SR2 7JE, UK
of Medicine,
Rayne Institute, University College and Middlesex School of Medicine, London WC1 E 6JJ, UK 1. Falconer
OLIVER WRONG
MA, Cope CL. Fibrous dysplasia of bone with endocrine disorders pigmentation. Q J Med 1942; 11: 121-54.
cutaneous
and
