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Memorable responses
Parasitology Today, vol. 7, no. 5, 1991 10 Helsby, N.A. et al. (1990) Br.]. Gin. Pharmacol. 30, 593-598 I I Watkins, W.M. et al. (1990) Trans. R. Sac. Trap. Med. Hyg. 84,492-495 12 Powell, R.D. and Brewer, G.J. (1967) Am. ]. Trap.Med. Hyg. 16, 693~98 13 Vessell, E.S.(1973)Clin. Pharmacol. Ther. 220, 659-679 14 Mahgoub,A. et aL(1977) Lancetii, 584-586 15 Kupfer, A. and Preisig, R. (I 984) Eur. J. Gin. Pharmacol.26,753-759 16 Wedlund, P.J.et aL(1984) Clin. Pharmacol.Ther. 36,773-780 17 Eichelbaum,M. and Woolhouse, N.M. (1985) Eur.]. Clin.Pharmacol.28, 79-83 18 Mbanfeno, C. et al. (1980) Xenobiotica 10, 811-818 19 Nakamura, K. et al. (I 985) Clin. Pharmacol. Ther. 38,402-408 20 Lee, E.D.J., Nam, Y.P. and Flee, G.N. (1988)
Memorable Responses In the editorial 'Memorable Responses' (Parasitology Today, January 1991) there is reference to a reported association between human T-cell immune responses to immunodominant peptides of the circumsporozoite protein of Piasmodium falciparum and HLA class II I , The particular association quoted, between peptide 24 and HLA-DRw 13, is presented in detail in another paper by De Groat et al.2 Close examination of the statistical analysis of the data presented in this paper reveals a number of methodological problems that render the conclusions invalid (see Box I). First, the number of observations analysed is six times the number of different genotypes, far greater than the true number of independent observations (ten individuals). Second, each observation pools data from all epitopes in both regions and from all HLA specificities. Responses to epitopes are positively correlated, and this pooling therefore magnifies any association. Third, the test statistic used for the analysis, by its very definition, increases in magnitude with the frequency of the genotype. It is hardly surprising, therefore, that the most common allele, DRw 13, is found to be associated with a positive response. Given the two former points, it is not surprising that the results appear to be significant. In fact, there is no justification using the data given for the claims made in this paper. Zevering et al. i also report,;ignificant associations between HLA-DR4 and peptide 21, and between HLA-DQw3 and peptide 16. The method of statistical analysis used is not described but the appropriate method would appear to be Fisher's exact test, since the numbers are small. Using this, or a standard X 2 test, gives in each caseP values that are much larger and quite nonsignificant; for example, I obtained quite different P values (two-sided) from those given on p953.
123 C/in.Exp.Pharmacol.Physiol. 15, 889-89 I 21 Helsby, N.A. et al. (1990)Br.]. Clin. Pharmacol. 30, 287-29 I 22 Ward, S.A.etal. Br.]. Clin.Pharmacol.(in press) 23 Estabrook, R.W. (1984) in Drug Metabolism and Drug Toxicity (Mitchell, J.R. and Horning, M.G., eds),pp I-2 I, RavenPress 24 Van Wauwe, J.P. and Janssen, P.A.J. (1989) ]. Meal.Chem.32, 2231-2239 25 Nebert, D.W. etal. (1989)DNA 9, I-I 3 26 Guengerich, F.P.(I 989) TrendsPharmacol. I O, 107-109 27 Ward, S.A. et al. (1987) Clin. Pharmacol. Ther. 42, 96-99 28 Meier, U.T., Dayer, P. and Male, P-J. (1985) Clin. Pharmacol.Ther.38,488-494 29 Wilkinson, G.R., Guengerich, F.P. and Branch, R.A. (1989) Pharmacal.Rev.43, 57-76 30 Okino, S.T. et al. (1987)J. Biol. Chem. 262, 16072-16079
31 Meehan, R.R. etal. (1988)Am.]. Hum. denet. 42, 26-37
Nuala Helsby and Stephen Ward are both at the Department of Pharmacology and Therapeutics, University of Liverpool, New Medical Building, Ashton Street, PO Box 147, Liverpool L69 3BX, UK and the Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. William Watkins and Edward Mberu are both at the Wellcome Trust Research Laboratories, PO Box 43640, Nairobi, Kenya and the Clinical Research Centre, KEMRI, Nairobi, Kenya. William Watkins is also at the Department of Parasitology, Liverpool School of Tropical M edicine, Pembroke Place, Liverpool L3 5QA, UK.
Box I. H u m a n T-cell Recognition by Polymorphic Epitopes De Groot et al. 2 assess the association between HLA type and response to various epitopes by measuring the relative frequency of concordant events. This appears to involve creating the value R(i,j, k), given by (no. personswith DR = i and responseto j, k) + (no. personswith DR = i and no response to j, k) total no. of persons where region = j(= I, 2) and epitope = k(= I, 2, 3). This is then analysed by a three-way analysis of variance using region, epitope and HLA type. The expected value of R can be calculated, under the null hypothesis (H0) of no association of response with DR type, as follows: the overall proportion of responders is 31 - 56 = 0.554 (excluding four 'not determined'); thus under H0 one would expect 5.54 out of I 0 persons to respond to each epitope. The expected value E(R) of R is given by (the sum of the expected number Y, + and those N , - ) - I 0, and, as shown by the following tables, depends on the overall frequency of DR genotype i: DR=i
DR=i
Y
DR=/
Y
N
Resp.(+) 0.55
4.99
5.54
(-)
4.01
4.46
9
10
0.45 I
E(R) =
Resp.
I0 =
Y
(+) 2.77
2.77
(-)
2.23
4.46
5
10
2.23 5
(0.55 + 4.01) -
N 5.54
N
(+) 4.99
0.55
5.5
(-)
4.01
0.45
4.4
9
I
10
Resp.
E(R) = 0.500
E(R) : 0.544
0.456
Thus even if there is no association, R inevitably increases with the frequency of the genotype. If one looks at each column of Table II separately, a positive association of DRw 13 with response is shown only for Th2R variant 7G8 and Th 3R variant LE5; a negative association is shown with Th3PJWEL, and absolute identity with the other three epitopes. The very small sample size means that none of these associations is anywhere near significance.
In sequence, these were: P--0.17 (instead of 0.059), P= 0. I 0 (instead of P<0,022) and P=0.13 (instead of 0.033). This is without making any allowance for the fact that these were hypotheses generated by the data out of a very large number of possible associations that could have been investigated. Thus, as discussed in the review article 'Immune Recognition of Malaria Antigens '3 in the same edition of Parasitology Today, these studies do not offer any evidence that human immune responses to particular epitopes of malaria antigens are restricted by HLA class II phenotype. Much larger
immunoepidemiological studies are needed before we can draw any conclusions about the possible importance of genetic restriction of immune responses to malaria vaccines. Steve Bennett
London Schoolof Hygieneand Tropical Medicine KeppelStreet London WC I E7HT, UK References
I Zevering, Y. et al. (1990) Int. Immunol.2, 945-955 2 De Groat, A.S. et al. (1989)j. Immunol. 142, 4000-4005 3 Riley, E.M. et al. ( 1991) Parasitology Today7, 5-11
Memorable Responses In the editorial 'Memorable Responses' (Parasitology Today, January 1991) there is reference to a reported association between human T-cell immune responses to immunodominant peptides of the circumsporozoite protein of Piasmodium falciparum and HLA class II I , The particular association quoted, between peptide 24 and HLA-DRw 13, is presented in detail in another paper by De Groat et al.2 Close examination of the statistical analysis of the data presented in this paper reveals a number of methodological problems that render the conclusions invalid (see Box I). First, the number of observations analysed is six times the number of different genotypes, far greater than the true number of independent observations (ten individuals). Second, each observation pools data from all epitopes in both regions and from all HLA specificities. Responses to epitopes are positively correlated, and this pooling therefore magnifies any association. Third, the test statistic used for the analysis, by its very definition, increases in magnitude with the frequency of the genotype. It is hardly surprising, therefore, that the most common allele, DRw 13, is found to be associated with a positive response. Given the two former points, it is not surprising that the results appear to be significant. In fact, there is no justification using the data given for the claims made in this paper. Zevering et al. i also report,;ignificant associations between HLA-DR4 and peptide 21, and between HLA-DQw3 and peptide 16. The method of statistical analysis used is not described but the appropriate method would appear to be Fisher's exact test, since the numbers are small. Using this, or a standard X 2 test, gives in each caseP values that are much larger and quite nonsignificant; for example, I obtained quite different P values (two-sided) from those given on p953.
123 C/in.Exp.Pharmacol.Physiol. 15, 889-89 I 21 Helsby, N.A. et al. (1990)Br.]. Clin. Pharmacol. 30, 287-29 I 22 Ward, S.A.etal. Br.]. Clin.Pharmacol.(in press) 23 Estabrook, R.W. (1984) in Drug Metabolism and Drug Toxicity (Mitchell, J.R. and Horning, M.G., eds),pp I-2 I, RavenPress 24 Van Wauwe, J.P. and Janssen, P.A.J. (1989) ]. Meal.Chem.32, 2231-2239 25 Nebert, D.W. etal. (1989)DNA 9, I-I 3 26 Guengerich, F.P.(I 989) TrendsPharmacol. I O, 107-109 27 Ward, S.A. et al. (1987) Clin. Pharmacol. Ther. 42, 96-99 28 Meier, U.T., Dayer, P. and Male, P-J. (1985) Clin. Pharmacol.Ther.38,488-494 29 Wilkinson, G.R., Guengerich, F.P. and Branch, R.A. (1989) Pharmacal.Rev.43, 57-76 30 Okino, S.T. et al. (1987)J. Biol. Chem. 262, 16072-16079
31 Meehan, R.R. etal. (1988)Am.]. Hum. denet. 42, 26-37
Nuala Helsby and Stephen Ward are both at the Department of Pharmacology and Therapeutics, University of Liverpool, New Medical Building, Ashton Street, PO Box 147, Liverpool L69 3BX, UK and the Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK. William Watkins and Edward Mberu are both at the Wellcome Trust Research Laboratories, PO Box 43640, Nairobi, Kenya and the Clinical Research Centre, KEMRI, Nairobi, Kenya. William Watkins is also at the Department of Parasitology, Liverpool School of Tropical M edicine, Pembroke Place, Liverpool L3 5QA, UK.
Box I. H u m a n T-cell Recognition by Polymorphic Epitopes De Groot et al. 2 assess the association between HLA type and response to various epitopes by measuring the relative frequency of concordant events. This appears to involve creating the value R(i,j, k), given by (no. personswith DR = i and responseto j, k) + (no. personswith DR = i and no response to j, k) total no. of persons where region = j(= I, 2) and epitope = k(= I, 2, 3). This is then analysed by a three-way analysis of variance using region, epitope and HLA type. The expected value of R can be calculated, under the null hypothesis (H0) of no association of response with DR type, as follows: the overall proportion of responders is 31 - 56 = 0.554 (excluding four 'not determined'); thus under H0 one would expect 5.54 out of I 0 persons to respond to each epitope. The expected value E(R) of R is given by (the sum of the expected number Y, + and those N , - ) - I 0, and, as shown by the following tables, depends on the overall frequency of DR genotype i: DR=i
DR=i
Y
DR=/
Y
N
Resp.(+) 0.55
4.99
5.54
(-)
4.01
4.46
9
10
0.45 I
E(R) =
Resp.
I0 =
Y
(+) 2.77
2.77
(-)
2.23
4.46
5
10
2.23 5
(0.55 + 4.01) -
N 5.54
N
(+) 4.99
0.55
5.5
(-)
4.01
0.45
4.4
9
I
10
Resp.
E(R) = 0.500
E(R) : 0.544
0.456
Thus even if there is no association, R inevitably increases with the frequency of the genotype. If one looks at each column of Table II separately, a positive association of DRw 13 with response is shown only for Th2R variant 7G8 and Th 3R variant LE5; a negative association is shown with Th3PJWEL, and absolute identity with the other three epitopes. The very small sample size means that none of these associations is anywhere near significance.
In sequence, these were: P--0.17 (instead of 0.059), P= 0. I 0 (instead of P<0,022) and P=0.13 (instead of 0.033). This is without making any allowance for the fact that these were hypotheses generated by the data out of a very large number of possible associations that could have been investigated. Thus, as discussed in the review article 'Immune Recognition of Malaria Antigens '3 in the same edition of Parasitology Today, these studies do not offer any evidence that human immune responses to particular epitopes of malaria antigens are restricted by HLA class II phenotype. Much larger
immunoepidemiological studies are needed before we can draw any conclusions about the possible importance of genetic restriction of immune responses to malaria vaccines. Steve Bennett
London Schoolof Hygieneand Tropical Medicine KeppelStreet London WC I E7HT, UK References
I Zevering, Y. et al. (1990) Int. Immunol.2, 945-955 2 De Groat, A.S. et al. (1989)j. Immunol. 142, 4000-4005 3 Riley, E.M. et al. ( 1991) Parasitology Today7, 5-11