/. Periodontal Res. 13: 120-126, 1978
The effect of age on the cellular immune response to dento-gingival plaque extract HILARY CHURCH AND A. E. DOLBY
Dental School, Welsh National School of Medicine, Heath Park, Cardiff The relationship between the cellular immune response to dento-gingival plaque extract and age was examined in 28 individuals (age range 21-47 for 27 subjects, one subject aged 64). There was a decrease, with age, in the response in a sub-group of individuals with a severity of disease ranging from PI 4.1-7.3. There was also a correlation between the degree of disease (range PI 0.4-7.3) and the product of the numerical indicator of the response and the age of the 28 individuals studied. The significance of these findings in relation to the history of the disease is discussed. (Accepted for publication April 15, 1977)
introduction
The cellular immune response to dentogingival plaque (DGP) extract increases significantly with increasing degrees of periodontal disease (Ivanyi & Lehner 1970, Horton, Leikin & Oppenheim 1972). This relationship is linear up to the level of Periodontal Index 4.0 (PI - Russell 1956): with increasing severity of the disease the response tends to decrease to normal or near normal levels (Ivanyi & Lehner 1970). This non-linearity of response over the entire range of PI is due apparently to the inhibition of the initial antigen driven reaction by blocking antibodies present in the patient's serum (Ivanyi & Lehner 1971). The cellular immune response to DGP appears also to reflect the activity of polyclonal activators present in the DGP (Ivanyi & Lehner 1974). There is an alteration in the lymphon with increasing age, manifested by a reduc-
tion in the absolute and percentage T cell counts (Augener et al. 1974, Carosella, Mochanko & Braun 1974), a reduced response to phytohaemagglutinin (Pisciotta et al. 1967, Hallgren et al. 1973) and pokeweed mitogen (Teasdale et al. 1976) and a reduction in the T cell dependent late IgG response to monomeric flagcllin (RobertsThomson et al. 1974). Since there is a positive correlation between the degree of periodontal disease and age (Loe 1963) it would appear pertinent to determine the effect of age upon the cellular immune response to DGP extract. Subjects, Materiais and Methods
Subjects The subjects examined were the first 22 patients, with at least 20 standing teeth, drawn from a list of patients referred by General Dental Practitioners for periodontal therapy
O E L L U L A R
I M M U N I T Y
IN
P E R I O D O N T A L
D I S E A S E
121
Tabie 1
The maximal stimulation indices for the subjects arranged in order of increasing values of periodontal index and total score together with the sex and age Subject
P. 1.
P. t.. (V) M. A. (V) J. A. (V) C. M. (V) L. P. (V) J. R. (V) R. K.
0.4 0.4 0.5 0.9 1.2 1.4 2.2 2.4 2.5 3.5 3.6 3.9 4.1 4.2 4.8 4.9 5.0 5.1 5.4 5.5
P. D. L. E.
S. H. A. H. M. T. K. A. R. F. A. F. D. M. A. M. A. P. V. W. J. D. G. A. W. H. J. J. B. T. V. P. M. N. D. C. 3. C.
5.7
5.8 5.8 5.9
6.0 6.4 6.7 7.3 AS FCS * .
PI Total
Sex
Age
12
F M
F
21 28 21 23 22 43 34 35 28 25 22 36 34 21 38 41 33 36 41
M F
28
M
39
F M
29 38 29 47 64 39
16 12 26 31 29 57 59 79 91 109 114 76 128 142 132 101
M M
149
F
147 110 154 134 150 106 162 102 162 184
F F
F M F
M F M F F M M F
F F
F F
42
SI/AS 1.06 1.01 1.18 1.46 1.19 1.79 0.49 1.87 2.69 1.01 2.04 0.86 1.19 4.65 1.96 3.23 5.26 1.28 1.60 2.69 3.39 2.10 2.21 4.46 4.13 2.32 2.06 3.22
SI/FCS
+ 0.57 + ± ± ±
0.280.67* 0.21* 0.78-
1.27 — 0.99 1.03 0.93 1.16 3.38 1.40 2.43 4.08 1.26 1.40 2.81 — 1.10 — 2.61 0.75 1.14 1.81 1.34 . 1.74 3.47 3.50 1.14 1.94 4.91
± 0.36 ± ± ± +
0.6 0.56 0.36 0.07
-
Autologous serum Foetal calf serum Mean of 3 recordings Mean of 2 recordings Unsatisfactory results (V) - Volunteer Subject
and 6 volunteer members of staff (Table 1). The Periodontal Index (Russell 1956) ranged from 0.4-7.3, the ages from 21 to 64.
dialysate was used in the cultures described below at concentrations of 1:10, 1:100, 1:1000.
Dento-gingival plaque extract DGP was collected from the subjects into 0.9 % saline in tared, cooled, plastic bottles and the wet weight of DGP determined. The contents were ultrasonicated at 8 [^i for 15 minutes in an MSE ultrasonicator with miniature probe tip. After centrifugation at 20,000 g for 20 minutes the supernatant was dialysed against 0.9 % saline overnight. The
Lymphocyte culture Lymphocytes were separated from peripheral blood by the gelatin separation method (Coulson & Chalmers 1964) and cultured in microculture trays (Dynatech Laboratories Billingshurst, Kent) at 2 X 10^ cells/well. Triplicate cultures in TC199 and 10 % foetal calf scrum (FCS) and 10 % autologous serum (AS) were made for the three
122
C H U R O H
GHOIIP [ 0-4 - 10
GHOUP II 1-2 - 4-0
Fig. 1I. The maximal stimulation indices for the subjects arranged in groups of differing periodontal status
A N D
D O L B Y
concentrations of DGP dialysate and saline in a randomised, coded manner. Phytohaemagglutinin (Burroughs Wellcome, Beckenham, Kent, 5 ml reconstituted used at 1:10 dilution) was employed as an indicator of satisfactory cultural conditions. The culture trays were maintained for 6 days in an atmosphere of 5 % COo/air at 37 °C after which 0.1 [iCi Tritiated thy midine (Radiochemical Centre, Amersham, Bucks, specific activity 23 Ci/m mol) was added to those wells where assessment of lymphocyte response was to be determined. After further incubation for 4 hours Trichloracetic acid precipitable material from the cultures was washed with saline and 95 % alcohol on Whatman GF/C filter
o
t80 STIMULATION INDEX X AGE
Fig. 2. Relationship between the Periodontal Index and (stimulation index X age).
C E L L U L A R
I M M U N I T Y
IN P E R I O D O N T A L
D I S E A S E
123
200
s
150
X
g
4), high values of background subtracted, the means derived SI O 3) do not occur in lower values of and the ratios of stimulated to unstimulatcd PI « 4 ) . The wide range of SI in Group cultures (stimulation index - SI) calculated 111 indicates that high values of PI in part by use of an Intertechnique multi 8 mini influence the high values in SI (possibly in computer. Viability of the cells was assessed the presence of another variable). by exclusion of trypan blue at the beginning There was no correlation between age and end of each culture. and peak St with either AS or FCS. HowFor 5 of the subjects the response was ever, when the relationship of PI and the measured on more than one occasion. product of the variable (SI X age) was examined, an appreciable positive correlation was found (r = 0.70) (Fig. 2). There was Results a weaker correlation (r = 0.54) between The peak Si's recorded with DGP using (SI X age) and the total Periodontal Index AS and FCS are shown in Table 1, together scores (the sum cf the individual tooth with the standard deviation in the 5 subjects scores) for the subjects (Fig. 3). For the
124
9
C H U R O H
A N D
D O L B Y
3-0
1-0
20
30
40
50
60
A G E
Fig. 4. Relationship between (stimulation index and age) for those subjects in the group who possess a Perio dontal Index of greater than 4.
subjects with severe periodontal disease (PI > 4) it was found that there was a significant negative correlation between age and SI (r = 0.41) (Fig. 4). Discussion
In the group of individuals studied lymphocyte stimulation indices with DGP extract, in the presence of autologous serum, tended to correlate with the degree of periodontal disease (Fig. 1), a finding previously reported (Ivanyi & Lehner 1970, Horton, Leikin & Oppenheim 1972). Although the degree of periodontal disease tended to increase with the increasing age throughout the group (Fig. 5) the stimulation index by itself was unrelated to age. However, the use of the multiplied variables (SI X age) provided a good correlation with PI (Fig. 2). This positive correlation between PI and the multiplied variables (SI X age) implies
that high values of PI can occur in combinations of (i) higher values of SI at lower ages (ii) lower values of SI at higher ages. The latter implication is suggested in our Group III patients (15 subjects age range 21-47 years one subject aged 64) with high values of PI (Fig. 4) showing a negative correlation between SI and age. There are significant differences in the composition and reactivity of the immune system in older individuals (Teasdale et al. 1976) and this may in part account for the findings recorded here. Thus the adjuvant activity of B cell mitogens such as lipopoly. saccharide, would appear to contribute to the potentiation of the cellular immune response to antigens of the gingival crevice (Lehner et al. 1974). This adjuvant effect has been related to the mitogenic effect (Diamanstein 1976) so that it is conceivable that, like the mitogenic effect (Teasdale et al. 1976) it declines with increasing age.
C E L L U L A R
I M M U N I T Y
IN P E R I O D O N T A L
D I S E A S E
125
7-0
X
u
Q
2
o Q O
2-0 -
40
A G E
Fig. 5. Relationship between the Periodontal Index and age.
In this investigation a 'blocking' of the cellular immune response to DGP with severe periodontal disease was not observed and differs therefore from a previous observation (Ivanyi & Lehner 1970). However, the assay systems in the two investigations differ both in respect to the cell culture system and the duration of culture. Thus there was a greater variability in results observed with FCS than with AS (see Table 1) suggesting that the cultural conditions were less satisfactory where FCS was employed. In addition the findings are drawn from a relatively small group (28) of individuals over a narrow age range (27 subjects ranging from 21 years to 47 years, one subject aged 64) and need to be extended.
It has been suggested that the cellular immune response to DGP may play a significant role in the tissue destruction which occurs in periodontal disease (Horton, Oppenheim & Mergenhagen 1974). If such is the case, then a possible decline in this response with increasing age would appear to be of considerable significance in relation to the natural history and treatment of the disease. Acknowledgments
We are grateful to Dr. T. Khosla, Senior Lecturer in Medical Statistics, Welsh National School of Medicine, for help with the statistical analysis. This work was supported in part by a grant from the Medical Research Council.
126
O H U R C H
References Augener, W., Cohnen, G., Reuter, A. & Biittinger, G. 1974. Decrease of T lymphocytes during ageing. Lancet 1: 1164. Carosella, E. D., Mochanko, K. & Braun, M. 1974. Rosette-forming T cells in human peripheral blood at different ages. Cell Immunology 12: 323-325. Coulson, A. S. & Chalmers, D. G. 1964. Separation of viable lymphocytes from human blood. Lancet 1: 468-469. Diamanstein, T., Keppler, W. & Blitstein-Willinger, Eveline 1976. Suppression of the primary immune response in vivo to sheep red blood cells by B-cell mitogens. Immunology, 30: 401-407. Hallgren, H.M., Buckley, C. E. Ill, Gilbertsen, V. A. & Yunes, E. I. 1973. Lymphocyte phytohaemagglutinin responsiveness immunoglobulins and auto antibodies in ageing humans. J. Immunology, 111: 1101-1107. Horton, J.E., Leikin, S. & Oppenheim, J. J. 1972. Human lymphoproliferative reaction to saliva and dental plaque deposits: An in vitro correlation with periodontal disease. ./. Periodontol 43: 522-527. Horton, 1. E., Oppenheim, 1. J. & Mergenhagen, S. E. 1974. A role for cell-mediated immunity in the pathogenesis of periodontal disease. J. Periodontol. 45: 351-360. Ivanyi, L. & Lehner, T. 1970. Stimulation of lymphocyte transformation by bacterial antigen in patients with periodontal disease. Arch. Oral Biol. 15: 1089-1096. Ivanyi, L. & Lehner, T. 1971. The significance of serum factors in stimulation of lymphocytes from patients with periodontal disease
A N D DOLBY
by veillonella alcalescens. Int. Arch. Allergy 41: 620-627. Ivanyi, L. & Lehner, T. 1974. Stimulation of human lymphocytes by B-cell mitogens. Clin. Exp. Immunol. 18: 347-356. Lebner, T., Wilton, ]. M. A., Challacombe, S. J. & Ivanyi, L. 1974. Sequential cell-mediated immune responses in experimental gingivitis in man. Clin. Exp. Imnjunol. 16: 481-492. Loe, H. 1963. Epidemiology of periodontal disease. An evaluation of the relative significance of the aetiological factors in tbe light of recent epidemiological research. Odont. T. 71: 479-503. Pisciotta, A. V., Westring, D. W., DePrey, C. & Walsh, B. 1967. Mitogenic effect of phytohaemagglutinin at different ages. Nature 215193-194. Roberts-Thomson, 1. C, Whittingham, S., Youngchaiyud, U. & Mackay, I. R. 1974. Ageing, immune response and mortality. Lancet 2: 368-370. Russell, A. L. 1956. A system of classification and scoring for prevalence surveys of periodontal disease. 1. Dent. Res. 35: 350-358. Teasdale, C, Thatcher, 1., Whitehead, R. H. H. & Hughes, L. E. 1976 Age dependence of T lymphocytes. Lancet I: 1410-1411. Address: Professor A. E. Dolby Department of Periodontology Dental School Welsh National School of Medicine Heath Park Cardiff, South Glamorgan Great Britain
The effect of age on the cellular immune response to dento-gingival plaque extract HILARY CHURCH AND A. E. DOLBY
Dental School, Welsh National School of Medicine, Heath Park, Cardiff The relationship between the cellular immune response to dento-gingival plaque extract and age was examined in 28 individuals (age range 21-47 for 27 subjects, one subject aged 64). There was a decrease, with age, in the response in a sub-group of individuals with a severity of disease ranging from PI 4.1-7.3. There was also a correlation between the degree of disease (range PI 0.4-7.3) and the product of the numerical indicator of the response and the age of the 28 individuals studied. The significance of these findings in relation to the history of the disease is discussed. (Accepted for publication April 15, 1977)
introduction
The cellular immune response to dentogingival plaque (DGP) extract increases significantly with increasing degrees of periodontal disease (Ivanyi & Lehner 1970, Horton, Leikin & Oppenheim 1972). This relationship is linear up to the level of Periodontal Index 4.0 (PI - Russell 1956): with increasing severity of the disease the response tends to decrease to normal or near normal levels (Ivanyi & Lehner 1970). This non-linearity of response over the entire range of PI is due apparently to the inhibition of the initial antigen driven reaction by blocking antibodies present in the patient's serum (Ivanyi & Lehner 1971). The cellular immune response to DGP appears also to reflect the activity of polyclonal activators present in the DGP (Ivanyi & Lehner 1974). There is an alteration in the lymphon with increasing age, manifested by a reduc-
tion in the absolute and percentage T cell counts (Augener et al. 1974, Carosella, Mochanko & Braun 1974), a reduced response to phytohaemagglutinin (Pisciotta et al. 1967, Hallgren et al. 1973) and pokeweed mitogen (Teasdale et al. 1976) and a reduction in the T cell dependent late IgG response to monomeric flagcllin (RobertsThomson et al. 1974). Since there is a positive correlation between the degree of periodontal disease and age (Loe 1963) it would appear pertinent to determine the effect of age upon the cellular immune response to DGP extract. Subjects, Materiais and Methods
Subjects The subjects examined were the first 22 patients, with at least 20 standing teeth, drawn from a list of patients referred by General Dental Practitioners for periodontal therapy
O E L L U L A R
I M M U N I T Y
IN
P E R I O D O N T A L
D I S E A S E
121
Tabie 1
The maximal stimulation indices for the subjects arranged in order of increasing values of periodontal index and total score together with the sex and age Subject
P. 1.
P. t.. (V) M. A. (V) J. A. (V) C. M. (V) L. P. (V) J. R. (V) R. K.
0.4 0.4 0.5 0.9 1.2 1.4 2.2 2.4 2.5 3.5 3.6 3.9 4.1 4.2 4.8 4.9 5.0 5.1 5.4 5.5
P. D. L. E.
S. H. A. H. M. T. K. A. R. F. A. F. D. M. A. M. A. P. V. W. J. D. G. A. W. H. J. J. B. T. V. P. M. N. D. C. 3. C.
5.7
5.8 5.8 5.9
6.0 6.4 6.7 7.3 AS FCS * .
PI Total
Sex
Age
12
F M
F
21 28 21 23 22 43 34 35 28 25 22 36 34 21 38 41 33 36 41
M F
28
M
39
F M
29 38 29 47 64 39
16 12 26 31 29 57 59 79 91 109 114 76 128 142 132 101
M M
149
F
147 110 154 134 150 106 162 102 162 184
F F
F M F
M F M F F M M F
F F
F F
42
SI/AS 1.06 1.01 1.18 1.46 1.19 1.79 0.49 1.87 2.69 1.01 2.04 0.86 1.19 4.65 1.96 3.23 5.26 1.28 1.60 2.69 3.39 2.10 2.21 4.46 4.13 2.32 2.06 3.22
SI/FCS
+ 0.57 + ± ± ±
0.280.67* 0.21* 0.78-
1.27 — 0.99 1.03 0.93 1.16 3.38 1.40 2.43 4.08 1.26 1.40 2.81 — 1.10 — 2.61 0.75 1.14 1.81 1.34 . 1.74 3.47 3.50 1.14 1.94 4.91
± 0.36 ± ± ± +
0.6 0.56 0.36 0.07
-
Autologous serum Foetal calf serum Mean of 3 recordings Mean of 2 recordings Unsatisfactory results (V) - Volunteer Subject
and 6 volunteer members of staff (Table 1). The Periodontal Index (Russell 1956) ranged from 0.4-7.3, the ages from 21 to 64.
dialysate was used in the cultures described below at concentrations of 1:10, 1:100, 1:1000.
Dento-gingival plaque extract DGP was collected from the subjects into 0.9 % saline in tared, cooled, plastic bottles and the wet weight of DGP determined. The contents were ultrasonicated at 8 [^i for 15 minutes in an MSE ultrasonicator with miniature probe tip. After centrifugation at 20,000 g for 20 minutes the supernatant was dialysed against 0.9 % saline overnight. The
Lymphocyte culture Lymphocytes were separated from peripheral blood by the gelatin separation method (Coulson & Chalmers 1964) and cultured in microculture trays (Dynatech Laboratories Billingshurst, Kent) at 2 X 10^ cells/well. Triplicate cultures in TC199 and 10 % foetal calf scrum (FCS) and 10 % autologous serum (AS) were made for the three
122
C H U R O H
GHOIIP [ 0-4 - 10
GHOUP II 1-2 - 4-0
Fig. 1I. The maximal stimulation indices for the subjects arranged in groups of differing periodontal status
A N D
D O L B Y
concentrations of DGP dialysate and saline in a randomised, coded manner. Phytohaemagglutinin (Burroughs Wellcome, Beckenham, Kent, 5 ml reconstituted used at 1:10 dilution) was employed as an indicator of satisfactory cultural conditions. The culture trays were maintained for 6 days in an atmosphere of 5 % COo/air at 37 °C after which 0.1 [iCi Tritiated thy midine (Radiochemical Centre, Amersham, Bucks, specific activity 23 Ci/m mol) was added to those wells where assessment of lymphocyte response was to be determined. After further incubation for 4 hours Trichloracetic acid precipitable material from the cultures was washed with saline and 95 % alcohol on Whatman GF/C filter
o
t80 STIMULATION INDEX X AGE
Fig. 2. Relationship between the Periodontal Index and (stimulation index X age).
C E L L U L A R
I M M U N I T Y
IN P E R I O D O N T A L
D I S E A S E
123
200
s
150
X
g
4), high values of background subtracted, the means derived SI O 3) do not occur in lower values of and the ratios of stimulated to unstimulatcd PI « 4 ) . The wide range of SI in Group cultures (stimulation index - SI) calculated 111 indicates that high values of PI in part by use of an Intertechnique multi 8 mini influence the high values in SI (possibly in computer. Viability of the cells was assessed the presence of another variable). by exclusion of trypan blue at the beginning There was no correlation between age and end of each culture. and peak St with either AS or FCS. HowFor 5 of the subjects the response was ever, when the relationship of PI and the measured on more than one occasion. product of the variable (SI X age) was examined, an appreciable positive correlation was found (r = 0.70) (Fig. 2). There was Results a weaker correlation (r = 0.54) between The peak Si's recorded with DGP using (SI X age) and the total Periodontal Index AS and FCS are shown in Table 1, together scores (the sum cf the individual tooth with the standard deviation in the 5 subjects scores) for the subjects (Fig. 3). For the
124
9
C H U R O H
A N D
D O L B Y
3-0
1-0
20
30
40
50
60
A G E
Fig. 4. Relationship between (stimulation index and age) for those subjects in the group who possess a Perio dontal Index of greater than 4.
subjects with severe periodontal disease (PI > 4) it was found that there was a significant negative correlation between age and SI (r = 0.41) (Fig. 4). Discussion
In the group of individuals studied lymphocyte stimulation indices with DGP extract, in the presence of autologous serum, tended to correlate with the degree of periodontal disease (Fig. 1), a finding previously reported (Ivanyi & Lehner 1970, Horton, Leikin & Oppenheim 1972). Although the degree of periodontal disease tended to increase with the increasing age throughout the group (Fig. 5) the stimulation index by itself was unrelated to age. However, the use of the multiplied variables (SI X age) provided a good correlation with PI (Fig. 2). This positive correlation between PI and the multiplied variables (SI X age) implies
that high values of PI can occur in combinations of (i) higher values of SI at lower ages (ii) lower values of SI at higher ages. The latter implication is suggested in our Group III patients (15 subjects age range 21-47 years one subject aged 64) with high values of PI (Fig. 4) showing a negative correlation between SI and age. There are significant differences in the composition and reactivity of the immune system in older individuals (Teasdale et al. 1976) and this may in part account for the findings recorded here. Thus the adjuvant activity of B cell mitogens such as lipopoly. saccharide, would appear to contribute to the potentiation of the cellular immune response to antigens of the gingival crevice (Lehner et al. 1974). This adjuvant effect has been related to the mitogenic effect (Diamanstein 1976) so that it is conceivable that, like the mitogenic effect (Teasdale et al. 1976) it declines with increasing age.
C E L L U L A R
I M M U N I T Y
IN P E R I O D O N T A L
D I S E A S E
125
7-0
X
u
Q
2
o Q O
2-0 -
40
A G E
Fig. 5. Relationship between the Periodontal Index and age.
In this investigation a 'blocking' of the cellular immune response to DGP with severe periodontal disease was not observed and differs therefore from a previous observation (Ivanyi & Lehner 1970). However, the assay systems in the two investigations differ both in respect to the cell culture system and the duration of culture. Thus there was a greater variability in results observed with FCS than with AS (see Table 1) suggesting that the cultural conditions were less satisfactory where FCS was employed. In addition the findings are drawn from a relatively small group (28) of individuals over a narrow age range (27 subjects ranging from 21 years to 47 years, one subject aged 64) and need to be extended.
It has been suggested that the cellular immune response to DGP may play a significant role in the tissue destruction which occurs in periodontal disease (Horton, Oppenheim & Mergenhagen 1974). If such is the case, then a possible decline in this response with increasing age would appear to be of considerable significance in relation to the natural history and treatment of the disease. Acknowledgments
We are grateful to Dr. T. Khosla, Senior Lecturer in Medical Statistics, Welsh National School of Medicine, for help with the statistical analysis. This work was supported in part by a grant from the Medical Research Council.
126
O H U R C H
References Augener, W., Cohnen, G., Reuter, A. & Biittinger, G. 1974. Decrease of T lymphocytes during ageing. Lancet 1: 1164. Carosella, E. D., Mochanko, K. & Braun, M. 1974. Rosette-forming T cells in human peripheral blood at different ages. Cell Immunology 12: 323-325. Coulson, A. S. & Chalmers, D. G. 1964. Separation of viable lymphocytes from human blood. Lancet 1: 468-469. Diamanstein, T., Keppler, W. & Blitstein-Willinger, Eveline 1976. Suppression of the primary immune response in vivo to sheep red blood cells by B-cell mitogens. Immunology, 30: 401-407. Hallgren, H.M., Buckley, C. E. Ill, Gilbertsen, V. A. & Yunes, E. I. 1973. Lymphocyte phytohaemagglutinin responsiveness immunoglobulins and auto antibodies in ageing humans. J. Immunology, 111: 1101-1107. Horton, J.E., Leikin, S. & Oppenheim, J. J. 1972. Human lymphoproliferative reaction to saliva and dental plaque deposits: An in vitro correlation with periodontal disease. ./. Periodontol 43: 522-527. Horton, 1. E., Oppenheim, 1. J. & Mergenhagen, S. E. 1974. A role for cell-mediated immunity in the pathogenesis of periodontal disease. J. Periodontol. 45: 351-360. Ivanyi, L. & Lehner, T. 1970. Stimulation of lymphocyte transformation by bacterial antigen in patients with periodontal disease. Arch. Oral Biol. 15: 1089-1096. Ivanyi, L. & Lehner, T. 1971. The significance of serum factors in stimulation of lymphocytes from patients with periodontal disease
A N D DOLBY
by veillonella alcalescens. Int. Arch. Allergy 41: 620-627. Ivanyi, L. & Lehner, T. 1974. Stimulation of human lymphocytes by B-cell mitogens. Clin. Exp. Immunol. 18: 347-356. Lebner, T., Wilton, ]. M. A., Challacombe, S. J. & Ivanyi, L. 1974. Sequential cell-mediated immune responses in experimental gingivitis in man. Clin. Exp. Imnjunol. 16: 481-492. Loe, H. 1963. Epidemiology of periodontal disease. An evaluation of the relative significance of the aetiological factors in tbe light of recent epidemiological research. Odont. T. 71: 479-503. Pisciotta, A. V., Westring, D. W., DePrey, C. & Walsh, B. 1967. Mitogenic effect of phytohaemagglutinin at different ages. Nature 215193-194. Roberts-Thomson, 1. C, Whittingham, S., Youngchaiyud, U. & Mackay, I. R. 1974. Ageing, immune response and mortality. Lancet 2: 368-370. Russell, A. L. 1956. A system of classification and scoring for prevalence surveys of periodontal disease. 1. Dent. Res. 35: 350-358. Teasdale, C, Thatcher, 1., Whitehead, R. H. H. & Hughes, L. E. 1976 Age dependence of T lymphocytes. Lancet I: 1410-1411. Address: Professor A. E. Dolby Department of Periodontology Dental School Welsh National School of Medicine Heath Park Cardiff, South Glamorgan Great Britain
