738
assay, but only 2 of these gave the
same
value in ELI SA. Of the 51 24 (47%) had ELISA
ELISA-positive, non-neutralising samples,
values of more than 0-01 but less than 01 IU/ml; 27 (53%) had ELISA values greater than 0’ 1 IU/ml. None of the samples with an ELISA value of less than 0-01 IU/ml had a higher value in the tissue culture assay. The implication of these findings is that, even assuming that an ELISA value of 0. 1 IU/ml or more is necessary to confer protection, 23% of subjects assessed by this method would be wrongly assumed not to need immunisation. Thus, ELISA estimation of antitoxin to diphtheria seems to detect a high proportion of specific IgG that cannot neutralise the toxin. In our studies with purified fragments of diphtheria toxin we have shown that fragment A is very immunogenic in several species (unpublished) including man,s and with the intact toxin this fragment contains the immunodominant epitopes.6 Since antibodies to fragment A play no part in protection, it is perhaps not surprising that there is so little correlation between ELISA and toxin neutralisation assays. In contrast to fragment A, antibodies to fragment B can neutralise the toxin. The monoclonal antibodies against the intact toxin that are neutralising are directed against fragment B or the intact toxin, but not fragment A.7 Thus, an ELISA with purified fragment B as antigen might provide a better correlation with neutralising activity. It should be emphasised, however, that most neutralising antibodies will probably be directed against the intact toxin, and clearly the best way to detect these would be to use a functional toxin neutralisation assay. Such assays correlate very well with toxin neutralisation in vivo for
subject. Lymphocytes were isolated by standard methods$ with 3Hwas assessed9 binding &bgr;-adrenoceptor dihydroalprenolol as a ligand at the saturating concentration of the
10
nmol/l.
Lymphocytes from black subjects with asthma had significantly lower binding (fmol/106 cells) than those from whites with asthma (p = 0-03, unpaired t test):
These data suggest that differences in &bgr;-receptors may be a factor in the differences in asthma prevalence and mortality between black and white subjects. Although the results need to be expanded to include more complete binding and functional studies, they pose the intriguing question of possible ethnically determined biochemical differences underlying susceptibility to, and outcome of, asthma, as well as differences in response to standard treatments.
Support from grants ES-04696 (NIEHS), ES-02366 Charles A. Dana Foundation is acknowledged. Department of Environmental Health, SC-34, University of Washington, Seattle, Washington 98195, USA
diphtheria antitoxins.8
1. Weiss
Division of Bacteriology, National Institute for Biological Standards and Control, South Mimms, Hertfordshire EN6 3QG, UK
2
2 3.
4.
5.
6.
7
8.
JANE Q. KOENIG LUCIO G. COSTA GRACE KAYLOR
KB, Wagener DK. Changing patterns of asthma mortality: identifying target populations at risk. JAMA 1990; 264: 1683-87. Sly M, O’Donnell R Regional distribution of deaths from asthma Ann Allergy 1989; 62: 347-54.
D. SESARDIC M. J. CORBEL
3. McWorter
4. 1 Hendriksen
(NIEHS) and the
CFM, Gun JWvd, Nagel J, Kreeftenberg JG. The
toxin
binding
inhibition test as a reliable in vitro alternative to the toxin neutralization test in mice for the estimation of tetanus antitoxin in human sera J Biol Standard 1988; 16: 287-97. Altman DG, Bland JM. Measurement m medicine: the analysis of method comparison studies. Statistician 1983; 32: 307-17. Knight PA, Tilleray J, Queminet J Studies on the correlaton of a range of immunoassays for diphtheria antitoxin with the guinea-pig intradermal test. Develop Biol Standard 1986; 64: 25-32. Melville-Smith M, Balfour A Estimation of Corynebacterium diphtheriae antitoxin in human sera a comparison of an enzyme-linked immunosorbent assay with the toxin neutralisation test. J Med Microbiol 1988; 25: 279-83. Perera VY, Corbel MJ. Human antibody response to fragments A and B of diphtheria toxin and a synthetic peptide of amino acid residues 141-157 of fragment A. Epidemiol Infect 1990; 105: 457-68. Triebel F, Autran B, De Roquefeuil S, Falmagne P, Debré P Immune response to diphtheria toxin and to different CNBr fragments evidence for different B and T cell reactivities Eur J Immunol 1986; 16: 47-53. Sesardic D, Khan V, Corbel MJ Protective epitopes on diphtheria toxin In Witholt B, Alouf JE, Boulnois GJ, et at, eds. Bacterial protein toxins Jena: Gustav Fischer Press, 1992; 23 (suppl). 355-56. Padovan E, Papini E, Rappuoli R, Montecucco C Determination of diphtheria toxin neutralizing antibody titres with a cell protein synthesis inhibition assay Med Microbiol Immunol 1991, 180: 29-35.
Lymphocyte &bgr;-adrenoceptors, asthma, and ethnicity SIR,-Asthma prevalence and mortality is higher in blacks than in whites.12 While low income and associated conditions may underlie some of this differencethese socioeconomic factors do not explain all the differences in asthma mortality.4 There are also ethnic differences in the response to certain drugs,’ particularly &bgr;-adrenergic agonists such as isoprenaline.6 Although the pharmacokinetics may be different, a pharmacodynamic component, such as differences in number and/or function of &bgr;-adrenoceptors, may also be involved. &bgr;-lymphocytes are often used as indicators of &bgr;-adrenoceptors in asthmatic conditions, so we tested the hypothesis that lymphocytes from asthmatic blacks may have lower &bgr;-adrenoceptors binding than those from white patients. All subjects (age 18-37) had clinically diagnosed asthma and a positive response to a standardised methacholine challenge.’ The black group had 7 men and 1 woman, the white group had 4 men and 4 women. Subjects were asked to give a 20 ml blood sample. The technician who analysed the blood was blind to the ethnicity of
5. 6
7.
8.
9
W, Polis MA, Kaslow RA. Occurrence, predictors and consequences of adult asthma in NHANES I and follow-up survey. Am Rev Respir Dis 1989; 139: 721-24 Schwartz J, Gold D, Dockery DW, Weiss ST, Speizer FE. Predictors of asthma and persistent wheeze in a national sample of children in the United States associations with social class, perinatal events and race. Am Rev Respir Dis 1990; 142: 555-62. Wood AJJ, Zhou AA Ethnic differences in drug disposition and responsiveness Clin Pharmacokinet 1991, 20: 350-73. Rutledge DR, Wallace A, Steinberg JD, Cardozo L, Lavine SJ Racial differences in drug response: isoproterenol effects before and after propranolol. Pharm Res 1991, 8: 754-57. Chai H, Farr RS, Froehlich LA and Committee Standardization of bronchial challenge procedures. J Allergy Clin Immunol 1975; 56: 323-27. Coccini T, Manzo L, Costa LG. 3H - spiperone labels sigma receptors, not dopamine D2 receptors, in rat and human lymphocytes Immunopharmacology 1991; 22: 93-106. Meurs H, Koeter GH, deVnes K, Kauffman HF. The beta-adrenergic system and allergic bronchial asthma changes in lymphocyte beta-adrenergic receptor number and adenylate cyclase activity after an allergen-induced asthmatic attack. J Allergy Clin Immunol 1982; 70: 272-80.
Steroid anaesthetic agents SIR,- With respect to your July 11editorial, when I joined Hans Selye’s laboratory more than 41 years ago, my first assignment was to investigate the relative anaesthetic potency of various steroids to see if certain characteristics of the molecule facilitated this response. A C-17 side chain seemed important, and the most potent steroids were those oxygenated only at the two extreme ends of the molecule. When steroids were arranged in terms of decreasing folliculoid activity, with oestradiol followed by testosterone at the top, and desoxycorticosterone preceded by progesterone at the bottom, that order reflected a corresponding increase in anaesthetic potency. Previous research had also shown that desoxycorticosterone could dampen the excitability of the central nervous system and even produce a cataleptic state.1,2 However, even hormonally inactive steroid precursors or degradation products such as pregnanediol and etiocholanolone could produce anaesthesia. This fmding can be demonstrated in pigeons, monkeys, cats, dogs, as well as rodents, Fish are especially sensitive, and can be anaesthetised merely by adding a few crystals of desoxycorticosterone to their swimming water, with quick revival after placement in fresh water. As with barbiturates, there is a preliminary stage of excitation, followed by complete anaesthesia, and I have operated on rats under deep anaesthesia after intraperitoneal administration of both progesterone and desoxycorticosterone. When subanaesthetic doses of steroids and
739
barbiturates are combined there is an effective synergistic response. Analeptics such as picrotoxin and metrazol, which reverse the effects of barbiturates, similarly interrupt steroid anaesthesia. These unpublished observations may be of interest in the development of steroids that can produce safe and effective anaesthesia. American Institute of Stress, 124 Park Avenue, Yonkers, New York 10703, USA
PAUL J. ROSCH
Woodbury DM, Davenport VD Brain and plasma cartions and experimental seizures with normal and esoxycorticosterone treated rats Am J Physiol 1949, 157: 234-37. 2. Kerman EF. Experimental catelepsy in the rat produced by steroid hormone: desoxycorticosterone acetate and antagonistic effect of pentamethylenetetrazol (metrazol). Dis Nerv Sys 1947; 8: 313-15. 1.
Indoor radon exposure and cytogenetic
damage SIR,-Radon contributes to pulmonary carcinogenesis in uranium miners and is an important lung cancer risk in the general population. Correlation studies indicate an additional role for indoor radon exposure in the development of extrapulmonary cancers, suggesting that carcinogenic effects may not be restricted to lung epithelium.1.2 Studies in populations living and/or working in areas with increased background radiation have shown higher frequencies of peripheral lymphocyte chromosome aberrations, or hypoxanthine guanine phosphoribosyl transferase-locus (hprt) mutations.3Genetic effects in peripheral lymphocytes of radonexposed individuals may also indicate extrapulmonary distribution
of genotoxic particles. Our pilot study was designed to test the feasibility of multiple genetic marker analysis (sister chromatid exchanges, hprt mutadons, micronuclei, and chromosome aberrations in peripheral lymphocytes from subjects in the Netherlands and Belgium exposed to indoor radon concentrations in their dwellings (16-713
Bq/m3).
The table surnmarises indoor radon exposure and
cytogenetic indices in the 11 participants. With or without correction for confounding factors, such as passive smoking and age, there
was no
relation between the level of exposure and
of
micronuclei, chromosome aberrations, and sister chromatid exchanges in peripheral lymphocytes of exposed individuals, as assessed by standard procedures.7,8 For hprt mutations, we detected 6-thioguanine-resistant lymphocytes with 5-bromodeoxyuridine labelling and immunochemical staining. These mutations were inversely related to the level of exposure by linear regression (R = — 0-632, p < 0-05). After adjustment for age and peripheral lymphocyte proliferation rate by multiple regression, the relation between individual hprt mutation and occurrence
SISTER CHROMATID EXCHANGES (SCE) hprt MUTATIONS (MF), MICRONUCLEI (MN), AND CHROMOSOME ABERATIONS (CA) IN PERIPHERAL LYMPHOCYTES OF 11 SUBJECTS EXPOSED TO INDOOR RADON
Indoor radon concentrations were initially measured for at least 3 months by x tract detector in 1987-905,6 Radon concentration is shown as reassessed in 1991 by charcoal canister for 24 h, correlation between these measurements was significant SCE frequencies significantly correlated with exposure to (R=088, p
assay, but only 2 of these gave the
same
value in ELI SA. Of the 51 24 (47%) had ELISA
ELISA-positive, non-neutralising samples,
values of more than 0-01 but less than 01 IU/ml; 27 (53%) had ELISA values greater than 0’ 1 IU/ml. None of the samples with an ELISA value of less than 0-01 IU/ml had a higher value in the tissue culture assay. The implication of these findings is that, even assuming that an ELISA value of 0. 1 IU/ml or more is necessary to confer protection, 23% of subjects assessed by this method would be wrongly assumed not to need immunisation. Thus, ELISA estimation of antitoxin to diphtheria seems to detect a high proportion of specific IgG that cannot neutralise the toxin. In our studies with purified fragments of diphtheria toxin we have shown that fragment A is very immunogenic in several species (unpublished) including man,s and with the intact toxin this fragment contains the immunodominant epitopes.6 Since antibodies to fragment A play no part in protection, it is perhaps not surprising that there is so little correlation between ELISA and toxin neutralisation assays. In contrast to fragment A, antibodies to fragment B can neutralise the toxin. The monoclonal antibodies against the intact toxin that are neutralising are directed against fragment B or the intact toxin, but not fragment A.7 Thus, an ELISA with purified fragment B as antigen might provide a better correlation with neutralising activity. It should be emphasised, however, that most neutralising antibodies will probably be directed against the intact toxin, and clearly the best way to detect these would be to use a functional toxin neutralisation assay. Such assays correlate very well with toxin neutralisation in vivo for
subject. Lymphocytes were isolated by standard methods$ with 3Hwas assessed9 binding &bgr;-adrenoceptor dihydroalprenolol as a ligand at the saturating concentration of the
10
nmol/l.
Lymphocytes from black subjects with asthma had significantly lower binding (fmol/106 cells) than those from whites with asthma (p = 0-03, unpaired t test):
These data suggest that differences in &bgr;-receptors may be a factor in the differences in asthma prevalence and mortality between black and white subjects. Although the results need to be expanded to include more complete binding and functional studies, they pose the intriguing question of possible ethnically determined biochemical differences underlying susceptibility to, and outcome of, asthma, as well as differences in response to standard treatments.
Support from grants ES-04696 (NIEHS), ES-02366 Charles A. Dana Foundation is acknowledged. Department of Environmental Health, SC-34, University of Washington, Seattle, Washington 98195, USA
diphtheria antitoxins.8
1. Weiss
Division of Bacteriology, National Institute for Biological Standards and Control, South Mimms, Hertfordshire EN6 3QG, UK
2
2 3.
4.
5.
6.
7
8.
JANE Q. KOENIG LUCIO G. COSTA GRACE KAYLOR
KB, Wagener DK. Changing patterns of asthma mortality: identifying target populations at risk. JAMA 1990; 264: 1683-87. Sly M, O’Donnell R Regional distribution of deaths from asthma Ann Allergy 1989; 62: 347-54.
D. SESARDIC M. J. CORBEL
3. McWorter
4. 1 Hendriksen
(NIEHS) and the
CFM, Gun JWvd, Nagel J, Kreeftenberg JG. The
toxin
binding
inhibition test as a reliable in vitro alternative to the toxin neutralization test in mice for the estimation of tetanus antitoxin in human sera J Biol Standard 1988; 16: 287-97. Altman DG, Bland JM. Measurement m medicine: the analysis of method comparison studies. Statistician 1983; 32: 307-17. Knight PA, Tilleray J, Queminet J Studies on the correlaton of a range of immunoassays for diphtheria antitoxin with the guinea-pig intradermal test. Develop Biol Standard 1986; 64: 25-32. Melville-Smith M, Balfour A Estimation of Corynebacterium diphtheriae antitoxin in human sera a comparison of an enzyme-linked immunosorbent assay with the toxin neutralisation test. J Med Microbiol 1988; 25: 279-83. Perera VY, Corbel MJ. Human antibody response to fragments A and B of diphtheria toxin and a synthetic peptide of amino acid residues 141-157 of fragment A. Epidemiol Infect 1990; 105: 457-68. Triebel F, Autran B, De Roquefeuil S, Falmagne P, Debré P Immune response to diphtheria toxin and to different CNBr fragments evidence for different B and T cell reactivities Eur J Immunol 1986; 16: 47-53. Sesardic D, Khan V, Corbel MJ Protective epitopes on diphtheria toxin In Witholt B, Alouf JE, Boulnois GJ, et at, eds. Bacterial protein toxins Jena: Gustav Fischer Press, 1992; 23 (suppl). 355-56. Padovan E, Papini E, Rappuoli R, Montecucco C Determination of diphtheria toxin neutralizing antibody titres with a cell protein synthesis inhibition assay Med Microbiol Immunol 1991, 180: 29-35.
Lymphocyte &bgr;-adrenoceptors, asthma, and ethnicity SIR,-Asthma prevalence and mortality is higher in blacks than in whites.12 While low income and associated conditions may underlie some of this differencethese socioeconomic factors do not explain all the differences in asthma mortality.4 There are also ethnic differences in the response to certain drugs,’ particularly &bgr;-adrenergic agonists such as isoprenaline.6 Although the pharmacokinetics may be different, a pharmacodynamic component, such as differences in number and/or function of &bgr;-adrenoceptors, may also be involved. &bgr;-lymphocytes are often used as indicators of &bgr;-adrenoceptors in asthmatic conditions, so we tested the hypothesis that lymphocytes from asthmatic blacks may have lower &bgr;-adrenoceptors binding than those from white patients. All subjects (age 18-37) had clinically diagnosed asthma and a positive response to a standardised methacholine challenge.’ The black group had 7 men and 1 woman, the white group had 4 men and 4 women. Subjects were asked to give a 20 ml blood sample. The technician who analysed the blood was blind to the ethnicity of
5. 6
7.
8.
9
W, Polis MA, Kaslow RA. Occurrence, predictors and consequences of adult asthma in NHANES I and follow-up survey. Am Rev Respir Dis 1989; 139: 721-24 Schwartz J, Gold D, Dockery DW, Weiss ST, Speizer FE. Predictors of asthma and persistent wheeze in a national sample of children in the United States associations with social class, perinatal events and race. Am Rev Respir Dis 1990; 142: 555-62. Wood AJJ, Zhou AA Ethnic differences in drug disposition and responsiveness Clin Pharmacokinet 1991, 20: 350-73. Rutledge DR, Wallace A, Steinberg JD, Cardozo L, Lavine SJ Racial differences in drug response: isoproterenol effects before and after propranolol. Pharm Res 1991, 8: 754-57. Chai H, Farr RS, Froehlich LA and Committee Standardization of bronchial challenge procedures. J Allergy Clin Immunol 1975; 56: 323-27. Coccini T, Manzo L, Costa LG. 3H - spiperone labels sigma receptors, not dopamine D2 receptors, in rat and human lymphocytes Immunopharmacology 1991; 22: 93-106. Meurs H, Koeter GH, deVnes K, Kauffman HF. The beta-adrenergic system and allergic bronchial asthma changes in lymphocyte beta-adrenergic receptor number and adenylate cyclase activity after an allergen-induced asthmatic attack. J Allergy Clin Immunol 1982; 70: 272-80.
Steroid anaesthetic agents SIR,- With respect to your July 11editorial, when I joined Hans Selye’s laboratory more than 41 years ago, my first assignment was to investigate the relative anaesthetic potency of various steroids to see if certain characteristics of the molecule facilitated this response. A C-17 side chain seemed important, and the most potent steroids were those oxygenated only at the two extreme ends of the molecule. When steroids were arranged in terms of decreasing folliculoid activity, with oestradiol followed by testosterone at the top, and desoxycorticosterone preceded by progesterone at the bottom, that order reflected a corresponding increase in anaesthetic potency. Previous research had also shown that desoxycorticosterone could dampen the excitability of the central nervous system and even produce a cataleptic state.1,2 However, even hormonally inactive steroid precursors or degradation products such as pregnanediol and etiocholanolone could produce anaesthesia. This fmding can be demonstrated in pigeons, monkeys, cats, dogs, as well as rodents, Fish are especially sensitive, and can be anaesthetised merely by adding a few crystals of desoxycorticosterone to their swimming water, with quick revival after placement in fresh water. As with barbiturates, there is a preliminary stage of excitation, followed by complete anaesthesia, and I have operated on rats under deep anaesthesia after intraperitoneal administration of both progesterone and desoxycorticosterone. When subanaesthetic doses of steroids and
739
barbiturates are combined there is an effective synergistic response. Analeptics such as picrotoxin and metrazol, which reverse the effects of barbiturates, similarly interrupt steroid anaesthesia. These unpublished observations may be of interest in the development of steroids that can produce safe and effective anaesthesia. American Institute of Stress, 124 Park Avenue, Yonkers, New York 10703, USA
PAUL J. ROSCH
Woodbury DM, Davenport VD Brain and plasma cartions and experimental seizures with normal and esoxycorticosterone treated rats Am J Physiol 1949, 157: 234-37. 2. Kerman EF. Experimental catelepsy in the rat produced by steroid hormone: desoxycorticosterone acetate and antagonistic effect of pentamethylenetetrazol (metrazol). Dis Nerv Sys 1947; 8: 313-15. 1.
Indoor radon exposure and cytogenetic
damage SIR,-Radon contributes to pulmonary carcinogenesis in uranium miners and is an important lung cancer risk in the general population. Correlation studies indicate an additional role for indoor radon exposure in the development of extrapulmonary cancers, suggesting that carcinogenic effects may not be restricted to lung epithelium.1.2 Studies in populations living and/or working in areas with increased background radiation have shown higher frequencies of peripheral lymphocyte chromosome aberrations, or hypoxanthine guanine phosphoribosyl transferase-locus (hprt) mutations.3Genetic effects in peripheral lymphocytes of radonexposed individuals may also indicate extrapulmonary distribution
of genotoxic particles. Our pilot study was designed to test the feasibility of multiple genetic marker analysis (sister chromatid exchanges, hprt mutadons, micronuclei, and chromosome aberrations in peripheral lymphocytes from subjects in the Netherlands and Belgium exposed to indoor radon concentrations in their dwellings (16-713
Bq/m3).
The table surnmarises indoor radon exposure and
cytogenetic indices in the 11 participants. With or without correction for confounding factors, such as passive smoking and age, there
was no
relation between the level of exposure and
of
micronuclei, chromosome aberrations, and sister chromatid exchanges in peripheral lymphocytes of exposed individuals, as assessed by standard procedures.7,8 For hprt mutations, we detected 6-thioguanine-resistant lymphocytes with 5-bromodeoxyuridine labelling and immunochemical staining. These mutations were inversely related to the level of exposure by linear regression (R = — 0-632, p < 0-05). After adjustment for age and peripheral lymphocyte proliferation rate by multiple regression, the relation between individual hprt mutation and occurrence
SISTER CHROMATID EXCHANGES (SCE) hprt MUTATIONS (MF), MICRONUCLEI (MN), AND CHROMOSOME ABERATIONS (CA) IN PERIPHERAL LYMPHOCYTES OF 11 SUBJECTS EXPOSED TO INDOOR RADON
Indoor radon concentrations were initially measured for at least 3 months by x tract detector in 1987-905,6 Radon concentration is shown as reassessed in 1991 by charcoal canister for 24 h, correlation between these measurements was significant SCE frequencies significantly correlated with exposure to (R=088, p
