- Email: [email protected]
Zinc and thymic hormone-dependent immunity in normal ageing and in patients with senile dementia of the Alzheimer type
Journal of Neuroimmunology, 27 (1990) 201-208
201
Elsevier JNI 00929
Zinc and thymic hormone-dependent immunity in normal ageing and in patients with senile dementia of the Alzheimer type Federico Licastro 1, Giancarlo Savorani 2, Gabriele Sarti 2 Afro Salsi 2, Francesco Cavazzuti 2, Lucio Zanichelli 2, Giuseppe Tucci z, Eugenio Mocchegiani 3 and Nicola Fabris 3 Department of Experimental Pathology, University of Bologna, Bologna, Italy, 2 Malpighi Hospital USL-28, Bologna, Italy, and 3 INRCA, Department of Immunogerontology, Ancona, Italy (Received 31 August 1989) (Revised, received 20 October 1989) (Accepted 20 October 1989)
Key words: Thymic hormone; Thymulin; Zinc; T cell proliferation; Ageing; Dementia
Summary Plasma zinc levels were measured in young controls, aged controls, patients with dementia of the Alzheimer type and patients with non-Alzheimer type dementia. Zinc levels decreased with age; however, no difference was found between patients with dementia and age-matched controls. Plasma levels of active or inactive thymulin, a nonapeptide produced and released by the thymus gland, were also determined in young controls, aged controls, patients with dementia of the Alzheimer type and patients with non-Alzheimer type dementia. Basal levels of active thymulin were decreased in aged controls and in patients with dementia. In vitro reactivation of thymulin after zinc addition to plasma samples was decreased in aged controls. A further impairment of thymulin reactivation was present in patients with dementia. A significant age-dependent decrease in lymphocyte proliferation after mitogen stimulation was found; however, no difference was present between aged controls and patients with dementia of the Alzheimer type. Interleukin-2-induced cell activation and its effect on mitogen-induced proliferation were also measured; once again only an age-associated decrease was found. The endocrine function of the thymus of patients with dementia appears to be more compromised than that from aged controls.
Introduction
Alzheimer's disease (AD) is a primary degenerative and progressive form of dementia with an
Address for correspondence: Prof. Federico Licastro, Dipartimento di Patologia Sperimentale, Via S. Giacomo 14, 40126 Bologna, Italy.
insidious onset (Amaducci and Sorbi, 1985; Gottfries, 1985). In industrialized countries, approximately 6% of people over 65 years, 22% of those over 80 years and 40% of the elderly over 90 years are affected by this debilitating illness (Henderson, 1986). In Italy, according to a recent survey, an increased prevalence of the disease of about 40% has been calculated for the next 20 years (Amaducci et al., 1986). In Japan, projections for
0165-5728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
202 the increment of AD prevalence are even more dramatic, reaching 72% (Amaducci and Sorbi, 1985). These figures have complex and serious social implications, since dementia is the main cause of disability in old age. In fact, patients with AD show a gradual memory loss and intellectual failure with cognitive impairment, along with changes of personality and behavior which interfere with previous social and working activities (McKhann et al., 1984). Clinical diagnosis of AD is very difficult, and only presumptive in vivo (Hollander et al., 1986; Roth, 1986). However, the accuracy in diagnosis has considerably increased after the introduction of more sensitive screening protocols (Nerl et al., 1984; Amaducci and Sorbi, 1985; Wade et al., 1987). The phenotypic heterogeneity of this disease makes diagnosis difficult. In fact, AD most probably cannot be considered a single clinical entity, but it is a complex syndrome that can be divided into different subtypes showing different clinical, neurophysiological, histological and neurochemical features (Rossor et al., 1984; Mayeux et al., 1985; Roth, 1986). Some alterations of the immune system have been reported in AD (Tavolato and Argentiero, 1980; MacDonald et al., 1982; Pouplard et al., 1983; Savorani et al., 1986; Gaskin et al., 1987). However, data on the immune status of AD patients are discordant, and several contradictory results are on record (Miller et al., 1981; Skias et al., 1985). In this report we present data regarding some immune functions of the thymus branch of the immune system in a group of non-familial AD patients, and in age- and sex-matched controls.
Materials and methods
Subjects Forty patients with senile dementia of the Alzheimer type (mean age 73 + 1 years) and 43 ageand sex-matched controls (mean age 74 + 1 years) were studied. Young controls were healthy hospital and laboratory personnel (mean age 30 + 5 years). Age-matched controls consisted of either healthy
elderly living in a retirement home, or elderly hospitalized with minor pathological conditions which did not interfere with brain or immune functions. Control patients consisted of four subjects with mental impairment of the non-Alzheimer type. Clinical diagnosis Diagnosis of dementia of the Alzheimer type was carried out after careful analysis of the patient's personal and family records, and clinical examination according to the recommendations of DSM III. Biochemical and metabolic parameters such as red blood cell count (RBC), white blood cell count (WBC), hemoglobin (Hb), platelet number, blood urea nitrogen (BUN), glucose, uric acid, creatine, cholesterol, triglycerides, Na +, K +, CI-, total protein, bilirubin, glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and alkaline phosphatase were evaluated. Patients were also monitored by electrocardiogram (ECG), chest X-ray and computed tomography (CT) brain scanning. Hormonal status was evaluated by measuring thyroid-stimulating hormone (TSH), triidothyronine (T3) , thyroxine (T4), oestradiol (OD), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) by commercial radioimmunoassay (RIA) (ARE, Serono Diagnostica, Italy). Intellectual performance tests AD patients were examined by performing the Mini Mental State (MMS) test (Folstein et al., 1975). Memory and attention were also evaluated. Plasma zinc levels Venous blood was collected in fluorinated plastic tubes in the presence of EDTA. After centrifugation at 4 ° C and 600 × g, plasma was collected and samples stored at - 80 o C. Zinc concentration was measured by atomic adsorption as previously described (Fabris et al., 1984). Plasma thymulin levels Thymulin concentration in the plasma was determined by a bioassay as previously described (Fabris et al., 1984). Levels of active and inactive thymulin were distinguished according to the
203 thymic hormone (TH) bioassay after the addition of exogenous zinc to plasma samples as described elsewhere (Fabris et al., 1984, 1986).
Purification of peripheral blood lymphocytes and lymphocyte microcultures Venous blood was collected in plastic tubes containing EDTA. Peripheral blood lymphocytes (PBL) were purified by Ficoll gradient centrifugation (Boyum, 1968). PBL cultures were performed in quadruplicate as previously described (Licastro et al,, 1983). Briefly, 1 × 105 lymphocytes were cultured in complete medium ( R P M I 1640 containing 10% heat-inactivated pooled AB human serum, 100 U penicillin, 100 # g / m l streptomycin, and 2 m M glutamine; Flow Lab., Italy). Stimulated lymphocytes were activated by phytohemagglutinin (PHA-P) (Difco; final concentration in complete medium 0.1 or 1 # l / m l ) and cultured in a CO 2 incubator for 3 or 4 days. Some lymphocyte cultures were treated with human recombinant interleukin 2 (IL-2) (Boehringwerke; final concentration in complete medium 20 U / m l ) at the beginning of the culture period with or without the addition of PHA. Cell proliferation was assessed by pulsing lymphocytes with tritiated thymidine ([3H]methylthymidine, [3H]TdR) (Amersham, U.K.; spec. act. 5 C i / m m o l ) for the last 6 h of culture.
Lymphocyte harvesting and radioactivity measurement
TABLE 1 ZINC PLASMA LEVELS IN YC, OC, AD, AND CD Data are presented as mean_+1 SE. Groups (n)
Zinc (#g/dl)
YC (15) OC (43) AD patients (40) CD patients (4)
105 _+2 83 _+3 77 + 3 80 _+8
Statistical differences: YC versus OC, P < 0.0001; YC versus AD, P < 0.0001; YC versus CD, P < 0.001. No statistically significant difference was present among OC, AD and CD.
of circulating TH, measured in 15 YC, 13 OC, 16 A D patients and four C D patients, are shown in Fig. 1. OC, A D and C D patients showed significantly lower levels of active T H than YC ( P < 0.05). In Fig. 2, levels of inactive T H are shown as determined by performing the T H bioassay after in vitro addition of zinc. A partial recovery of T H activity was found in the plasma from OC. The extent of T H reactivation in plasma from A D and C D was less than that observed in plasma from OC. Differences observed between OC and A D or C D were statistically significant ( P < 0.05). N o difference in T H reactivation was detected between A D and CD patients. The proliferative capacity of peripheral blood lymphocytes after P H A activation for 3 and 4 days of culture in YC, OC, and A D patients was also studied (Fig. 3A and B). Using a suboptimal
At the end of the culture period, lymphocyte cultures were harvested, washed on glass fiber filters with the aid of a multiple cell harvester (Skatron), and [3H]TdR incorporation measured by liquid scintillation counting as previously described (Franceschi et al., 1981).
[] YC
[] ~
[ ] AD
[] CD
QQ Q I
o'
Results
Plasma zinc levels were measured in 15 young controls (YC) and 43 aged controls (OC), 40 A D patients and four subjects with dementia of the non-Alzheimer type (control disease; CD) (Table 1). Zinc levels were significantly lower in OC, A D and C D than in YC. Differences between OC and A D were not statistically significant. Basal levels
g
O
Fig. 1. Basal levels of active TH in plasma from YC, OC, AD. and CD. Black circles refer to individual data; histograms represent mean values ( + SE) for each group.
204 150
D~
[] YC
eel
°I",,"
[] At)
[ ] YC [ ] oc
°
OO0
4
[ ] CD
•
01
[] AD
'7 100 o x
o,
I[11
o. u
150
Fig. 2. Levels of active TH after the in vitro addition of zinc to plasma samples from YC, OC, AD, and CD. Black circles refer to individual data; histograms represent mean values ( 5:1 SE) for each group.
O:O
•
5O
=:
,
,,,
|
"
B
[ ] YC ,~ oc o
10o
~] AO
x o. u
150 [ ] YC
S0
•
[] oc
~, 100 o
AD
x
It'2 (20p/rnl)
.....
0.1"HL-2
I+IL-Z
PHA (pl/rnl)
a.
Fig. 4. Incorporation of [3H]TdR in cultures of peripheral blood lymphocytes from YC, OC, and AD after stimulation with PHA in the presence of human recombinant IL-2 for 3 (A) or 4 (B) days. Black circles refer to individual data; histograms represent mean values ( 5:1 SE) for each group.
50
""
N~
0 150 YC
'7 100
•
E3oc
o
AD
x
i~ •
o. o
5O
o
0.1
1
PHA (.pt/ml)
Fig. 3. Incorporation of [3H]TdR in cultures of peripheral blood lymphocytes from YC, OC, and AD after stimulation with PHA for 3 (A) or 4 (B) days, Black circles refer to individual data; histograms represent mean values ( 5:1 SE) for each group.
PHA concentration (0.1/~l/ml), lymphocytes from OC and AD patients proliferated significantly less (P < 0.03) than those from YC after 3 days of culture. However, no difference was present after 4 days of culture. Using an optimal PHA concentration (1 ~l/ml), proliferation of lymphocytes from both OC and AD patients was significantly lower (P < 0.04) than that of YC after 3 or 4 days of culture. No statistically significant difference was detectable between OC and AD. Modulation by exogenous human recombinant IL-2 upon PHA-induced proliferation of parallel lymphocyte cultures from YC, OC and AD after 3 or 4 days of culture was also investigated (Fig. 4A and B). IL-2 significantly increased only suboptimal PHA lymphocyte responsiveness in YC, OC and AD patients. Differences in proliferative re-
205 sponse after IL-2 addition among these three groups were still present after 3 days of culture (P < 0.03). In contrast, after 4 days of culture in the presence of IL-2, no difference in lymphocyte proliferative capacity was detected among the three groups studied. IL-2 per se was able to induce a low level of proliferation in lymphocyte cultures (Fig. 4A and B). In these conditions, the proliferative activity of lymphocytes from OC and AD patients was comparable to that observed in YC.
Discussion
An involvement of the immune system in the pathogenesis of AD has been suggested (Fudenbergh et al., 1984), and a survey of AD mortality data has shown an increased incidence of infectious diseases in the terminal phase of the disease (Chandra et al., 1986). An imbalance of serum immunoglobulins has been observed in AD patients (Behan and Feldman, 1970; Kalter and Kelly, 1975; Tavolato and Argentiero, 1980), and a decreased responsiveness to polyclonal mitogens has also been reported (Miller et al., 1981; Singh et al., 1987). However, the latter findings have not been confirmed in other studies (Kalter and Kelly, 1975; MacDonald et al., 1982; Leffel et al., 1985). Contradictory results concerning also T suppressor cell activity have been found (Miller et al., 1981; Leffell et al., 1985; Skias et al., 1985). More recently, auto-antibodies specific for brain antigens have been found either in serum or in cerebrospinal fluid of AD patients (Pouplard and Vincent, 1983; Singh and Fudenberg, 1986; Gaskin et al., 1987). Interestingly, some of these antibodies appeared to react against cells of the pituitary gland (Pouplard and Vincent, 1983). Other studies, however, have not confirmed these latter data (Philpot et al., 1985; VanDorp et al., 1985). At present, the role of the immune system in the pathogenesis of Alzheimer type dementia is not clearly established and the presence of different clinical subgroups may imply a differential involvement of the immune system. Immune functions, particularly those belonging to the thymus branch of the immune system, decrease with advancing age in the normal elderly (Licastro et al., 1983; Fabris et al., 1984). This makes it more
difficult to identify an immune impairment specifically associated with AD. In the present report we have investigated the endocrine function of the thymus gland by studying the plasma levels of a zinc-dependent thymic hormone: a nonapeptide called thymulin (Dardenne et al., 1982). Thymulin has been shown to promote T cell differentiation and maturation (Dardenne et al., 1982). The active form of this hormone decreases dramatically in plasma of elderly people and in young subjects with Down's syndrome (Fabris et al., 1984). Interestingly, subjects with Down's syndrome over the age of 35 show an abnormal incidence of Alzheimer type dementia (Heston, 1976; Kay, 1986). Additionally, it has been shown that TH requires a zinc molecule to exert its biological activity on lymphocyte differentiation (Dardenne et al., 1982). Plasma zinc levels were lower in OC, AD patients and CD than in YC; however, no difference was found between OC and patients with dementia. In patients with Alzheimer type dementia the biological activity of TH after the addition of zinc in vitro increased less than in aged controls. In aged controls at least 50% of the circulating TH was deprived of zinc; however, the biological activity of the hormone was restored by addition of zinc in vitro. On the other hand, the addition of zinc in vitro did not significantly increase the basal activity of TH from plasma samples of AD. A decreased reactivation of TH was also found in patients with dementia of the non-Alzheimer type. These results suggest that, in patients with dementia, the thymus gland may release a reduced amount of TH in comparison to the normal elderly. However, if the relative increase in TH activity was calculated (i.e., TH activity after zinc add i t i o n / T H basal activity) a statistically significant difference (P < 0.05) was present between OC and AD, and also between AD and CD patients (relative increase 2.2, 1.2 and 2.1 in OC, AD and CD, respectively). These data suggest that an aliquot of TH molecules of AD patients might have a reduced capacity to bind a zinc atom. It has been shown that other metals can bind the TH molecule, and among these, there is aluminum, which can compete with zinc for the binding site of TH (Dardenne et al., 1982). It is
206 interesting to note that aluminum has been suggested to play a role in the pathogenesis of AD (Candy et al., 1986; Birchall and Chappel, 1988). Thus, a portion of T H molecules might bind aluminum and this latter metal might interfere with the normal functioning of the hormone. The responsiveness to PHA of lymphocytes from AD patients was within the normal age range. Lymphocytes from young and aged donors, and from AD patients were sensitive to the effect of IL-2 when a suboptimal concentration of PHA was used. Differences in proliferative activity among lymphocyte cultures from the three groups studied were not reduced after 3 days of incubation. However, after the addition of IL-2 and 4 days of culture, proliferation of lymphocytes from aged controls and patients with AD reached the values of that from young controls. Our data are partially at variance with those of Gillis et al. (1981) who found that lymphocytes from aged donors were not responsive to the addition of T cell growth factor (TCGF) present in supernatants of activated normal lymphocyte cultures. These discrepancies may be explained by the increased purity of IL-2 used by us, the length of the culture periods, and the dosages of PHA. Supernatants from cell cultures may also contain other lymphokines which might differently affect lymphocyte proliferation from donors of different ages. In our experimental conditions, IL-2 addition to PHA-stimulated cultures showed a positive effect on lymphocytes from either aged controis or AD patients after 4 days of culture, but only when a suboptimal mitogen dose was used. In conclusion, the impaired proliferation in AD patients appears to be more dependent upon the age than the disease. These data are at variance with other results showing a decrease in mitogen responsiveness in AD ,patients (Miller et al., 1981; Singh et al., 1987). Such a discrepancy may be partially related to the clinical heterogeneity of AD, i.e. only some clinical subgroups may show a further impairment of T cell proliferation after in vitro activation. Finally, it is important to bear in mind that AD patients studied by us were patients without signs of infectious diseases and that the immunological screening was performed early after the diagnosis. Some conditions such as: (a) the age of the
patient at the onset of the disease, (b) the time lag between the onset of the disease and the diagnosis, (c) the time lag between the onset of the disease, the time of the diagnosis, and the time of the immunological screening, which are not directly related to the biology of AD, may affect some immune parameters such as lymphocyte proliferation after polyclonal activation. A random distribution of patients according to the criteria listed above might at least in part explain contradictory results regarding some immune functions in AD. The impairment of the endocrine portion of the thymus gland might by an early sign of immune impairment in patients with dementia, since no other immunological dysfunctions were present. This immune alteration could be related to a disturbance of the neuroendocrine regulation of the thymus gland secondary to the mental impairment. Interestingly, patients with AD showed a further impairment of the thymus function, possibly related to the release of an altered T H molecule.
Acknowledgements This paper was supported by research grants from the Ministero della Pubblica lstruzione Italiana Fondi (60%), and partially from the Regione Emilia Romagna. We thank Dr. L.J. Davis for reviewing the manuscript and Dr. M. Chiricolo and Mr. A. DePietro for technical help.
References Amaducci, L. and Sorbi, S. (1985) L'invecchiamentocerebrale: definizioneed inquadramento nosologico. Giorn. Gerontol. 33, 1033-1054. Amaducci, L.A., Fratiglioni, L., Rocca, W., Fieschi, C., Livrea, P., Pedone, D., Bracco, L., Lippi, A., Gandolfo, C., Bino, G., Prencipe, M., Bonatti, M., Girotti, F., Carella, F., Tavolato, B., Ferla, S., Lenzi, G.L., Carolei, A., Gambi, A., Grigoletto, F. and Schoenberg, B. (1986) Risk factors for clinically diagnosed Alzheimer's disease: a case-control study for an Italian population. Neurology36, 922-931. Behan, P.O. and Feldman, R.G. (1970) Serum protein, amyloid and Alzheimer's disease. J. Am. Geriatr. Soc. 18, 792-797. Birchall, J.D. and Chappell, J.S. (1988) Aluminium, chemical physiology, and Alzheimer's disease. Lancet ii, 1008-1010.
207
Boyum, A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77-89. Candy, J.M., Klinowski, J., Perry, R.H., Perry, E.K., Fairbain, A., Oakley, A.E., Carpenter, T.A., Atack, J.R., Blessed, G. and Edwardson, J.A. (1986) Aluminosilicates and senile plaque formation in Alzheimer's disease. Lancet i, 354-357. Chandra, V., Bharucha, N.E. and Schoenberg, B.S. (1986) Conditions associated with Alzheimer's disease at death: case-control study. Neurology 36, 209-211. Dardenne, M., Pleau, J.M., Nabama, B., Lefancier, P., Denierr, P., Chosy, M. and Bach, J.F. (1982) Contributions of zinc and other metal to the biological activity of serum thymic factor. Proc. Natl. Acad. Sci. U.S.A. 79, 5370-5373. Fabris, N., Mocchegiani, E., Amadio, L., Zannotti, M., Licastro, F. and Franceschi, C. (1984) Thymic hormone deficiency in normal ageing and Down's syndrome: is there a primary failure of the thymus? Lancet i, 983-986. Fabris, N., Mocchegiani, E., Mariotti, S., Pacini, F. and Pinchera, A. (1896) Thyroid function modulates thymic endocrine activity. J. Clin. Endocrinol. Metab. 62, 474-478. Folstein, M., Folstein, S. and McHugh, P.R. (1975) Mini Mental State: a practical method for grading the cognitive state of patients for the clinician. J. Psychiatr. Res. 12, 189-198. Franceschi, C., Licastro, F., Chiricolo, M., Bonetti, F., Zannotti, M., Fabris, N., Mocchegiani, E., Fantini, M.P., Paolucci, P. and Masi, M. (1981) Deficiency of autologous mixed lymphocyte reactions and serum thymic factor level in Down's syndrome. J. Immunol. 126, 2161-2164. Fudenbergh, H.H., Whitten, H.D., Arnaud, P. and Khansari, N. (1984) Is Alzheimer's disease an immunological disorder? Observations and speculations. Clin. Immunol. Immunopathol. 32, 127-131. Gaskin, F., Kingsley, B.S. and Fu, S.M. (1987) Autoantibodies to neurofibrillary tangles and brain tissue in Alzheimer's disease. J. Exp. Med. 165, 245-250. Gillis, S., Kozak, R., Durante, M. and Weksler, M.A. (1981) Decreased production of and response to T cell growth factor by lymphocytes from aged humans. J. Clin. Invest. 67, 937-942. Gottfries, C.G. (1985) Definition of normal ageing senile dementia and Alzheimer's disease. In: G.G. Gottfries (Ed.), Normal Ageing Alzheimer's Disease and Senile Dementia, Editions Universit~ Bruxelles, Brussels, pp. 11-17. Henderson, A.S. (1986) The epidemiology of Alzheimer's disease. Br. Med. Bull. 42, 3-10. Heston, L.L. (1976) Alzheimer's disease, trisomy 21, and myeloproliferative disorders. Associations suggesting a genetic diathesis. Science 196, 322-323. Hollander, E., Mohs, R.C. and Davis, K.L. (1986) Antemortem markers of Alzheimer's disease. Neurobiol. Aging 7, 367387. Kalter, S. and Kelly, S. (1975) Alzheimer's disease: evaluation of immunological indices. N.Y. State J. Med. 75, 1222-1225. Kay, D.W.K. (1986) The genetic of Alzheimer's disease. Br. Med. Bull. 42, 19-23. Licastro, F., Chiricolo, M., Tabacchi, L., Barboni, F., Zannotti,
M. and Franceschi, C. (1983a) Enhancing effect of lithium and potassium ions on lectin-induced lymphocyte proliferation in ageing and Down's syndrome subjects. Cell. Immunol. 75, 111-121. Licastro, F., Tabacchi, L., Chiricolo, M., Parente, R., Cenci, M., Barboni, F. and Franceschi, C. (1983b) Defective selfrecognition in subjects of far advanced age. Gerontology 29, 64-72. Leffell, M.S., Lumsden, L. and Steiger, W.A. (1985) An analysis of T lymphocyte subpopulations in patients with Alzheimer's disease. J. Am. Geriatr. Soc. 33, 4-8. Macdonald, S.M., Goldstone, A.H., Morris, J.E., Exton-Smith, A.N. and Callard, R.E. (1982) Immunological parameters in the aged and in Alzheimer's disease. Clin. Exp. Immunol. 49, 123-128. Mayeux, R., Stem, Y. and Spaton, S. (1985) Heterogeneity in dementia of the Alzheimer type: evidence of subgroups. Neurology 35, 453-461. McKhann, G., Drachrnan, D., Folstein, M., Katzman, R., Price, D. and Stadlan, E. (1984) Clinical diagnosis of A1zheimer's disease: report of the NINCDS-ADRDA work group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology 34, 939-944. Miller, A.E., Neighbour, A., Katzman, R., Arouson, M. and Lipkowiz, R. (1981) Immunological studies in senile dementia of the Alzheimer type: evidence for enhanced suppressor cell activity. Ann. Neurol. 10, 506-510. Nerl, C., Mayeux, R. and O'Neill, G.J. (1984) HLA-linked complement markers in Alzheimer's and Parkinson's disease: Ca variant (C4B2) a possible marker for senile dementia of the Alzheimer type. Neurology 34, 310-314. Philpot, M., Colgan, J., Levy, R., Holland, A., Mirakian, R., Richardson, C.A. and Bottazzo, G.F. (1985) Prolactin cell autoantibodies and Alzheimer's disease. J. Neurol. Neurosurg. Psychiatry 48, 287-288. Pouplard, A., Emile, J. and Vincent-Pineau, F. (1983) Autoanticorps circulants anticellules h prolectine de l'hypophyse humaine et maladie d'Alzheimer. Rev. Neurol. (Paris) 139, 187-191. Rossor, M.N., Iversen, L.L., Reynolds, G.P., Mountjoy, C.P. and Roth, M. (1984) Neurochemical characteristics of early and late onset types of Alzheimer's disease. Br. Med. J. 288, 961-964. Roth, M. (1986) The association of clinical and neurological findings and its bearing on the classification and aetiology of Alzheimer's disease. Br. Med. Bull. 42(1), 42-50. Savorani, G., Salsi, A., De Vinci, C., Piazzi, S., Sarti, G., Corneli, M., Palmirani, R., Tupone, A., Tolomelli, M. and Cavazzuti, F. (1986) Studies on the possible role of zinc and immune response in Alzheimer's disease. In: J.J. Facchini, G. Haaijmann, and A. Lab6 (Eds.), Immunoregulation in Aging, Topics in Aging Research in Europe, 9, EURAGE, pp. 197-203. Singh, V.K., Fudenberg, H.H. and Brown, III, F.R. (1987) Immunologic dysfunction: simultaneous study of Alzheimer's and older Down's patients. Mech. Ageing Dev. 37, 257-264.
208 Skias, D., Bania, M., Reder, A.T., Luchins, D. and Antel, J.P. (1985) Senile dementia of Alzheimer type (SDAT): reduced T-cell-mediated suppressor activity. Neurology 35, 16351638. Tavolato, B. and Argentiero, V. (1980) Immunological indices in presenile Alzheimer's disease. J. Neurol. Sci. 46, 325-331. VanDorp, T.A., Endzt, L.J., "re Velde, J., Sleats, J.P.J. and
Reichgelt, J. (1985) Prolactin cell autoantibodies and Alzheimer's disease. J. Neurol. Neurosurg. Psychiatry 48, 1308-1309. Wade, J.P.H., Mirsen, T.R., Hachinski, V.C., Fisman, M., Lau, C. and Merskey, H. (1987) The clinical diagnosis of Alzheimer's disease. Arch. Neurol. 44, 24-29.
201
Elsevier JNI 00929
Zinc and thymic hormone-dependent immunity in normal ageing and in patients with senile dementia of the Alzheimer type Federico Licastro 1, Giancarlo Savorani 2, Gabriele Sarti 2 Afro Salsi 2, Francesco Cavazzuti 2, Lucio Zanichelli 2, Giuseppe Tucci z, Eugenio Mocchegiani 3 and Nicola Fabris 3 Department of Experimental Pathology, University of Bologna, Bologna, Italy, 2 Malpighi Hospital USL-28, Bologna, Italy, and 3 INRCA, Department of Immunogerontology, Ancona, Italy (Received 31 August 1989) (Revised, received 20 October 1989) (Accepted 20 October 1989)
Key words: Thymic hormone; Thymulin; Zinc; T cell proliferation; Ageing; Dementia
Summary Plasma zinc levels were measured in young controls, aged controls, patients with dementia of the Alzheimer type and patients with non-Alzheimer type dementia. Zinc levels decreased with age; however, no difference was found between patients with dementia and age-matched controls. Plasma levels of active or inactive thymulin, a nonapeptide produced and released by the thymus gland, were also determined in young controls, aged controls, patients with dementia of the Alzheimer type and patients with non-Alzheimer type dementia. Basal levels of active thymulin were decreased in aged controls and in patients with dementia. In vitro reactivation of thymulin after zinc addition to plasma samples was decreased in aged controls. A further impairment of thymulin reactivation was present in patients with dementia. A significant age-dependent decrease in lymphocyte proliferation after mitogen stimulation was found; however, no difference was present between aged controls and patients with dementia of the Alzheimer type. Interleukin-2-induced cell activation and its effect on mitogen-induced proliferation were also measured; once again only an age-associated decrease was found. The endocrine function of the thymus of patients with dementia appears to be more compromised than that from aged controls.
Introduction
Alzheimer's disease (AD) is a primary degenerative and progressive form of dementia with an
Address for correspondence: Prof. Federico Licastro, Dipartimento di Patologia Sperimentale, Via S. Giacomo 14, 40126 Bologna, Italy.
insidious onset (Amaducci and Sorbi, 1985; Gottfries, 1985). In industrialized countries, approximately 6% of people over 65 years, 22% of those over 80 years and 40% of the elderly over 90 years are affected by this debilitating illness (Henderson, 1986). In Italy, according to a recent survey, an increased prevalence of the disease of about 40% has been calculated for the next 20 years (Amaducci et al., 1986). In Japan, projections for
0165-5728/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
202 the increment of AD prevalence are even more dramatic, reaching 72% (Amaducci and Sorbi, 1985). These figures have complex and serious social implications, since dementia is the main cause of disability in old age. In fact, patients with AD show a gradual memory loss and intellectual failure with cognitive impairment, along with changes of personality and behavior which interfere with previous social and working activities (McKhann et al., 1984). Clinical diagnosis of AD is very difficult, and only presumptive in vivo (Hollander et al., 1986; Roth, 1986). However, the accuracy in diagnosis has considerably increased after the introduction of more sensitive screening protocols (Nerl et al., 1984; Amaducci and Sorbi, 1985; Wade et al., 1987). The phenotypic heterogeneity of this disease makes diagnosis difficult. In fact, AD most probably cannot be considered a single clinical entity, but it is a complex syndrome that can be divided into different subtypes showing different clinical, neurophysiological, histological and neurochemical features (Rossor et al., 1984; Mayeux et al., 1985; Roth, 1986). Some alterations of the immune system have been reported in AD (Tavolato and Argentiero, 1980; MacDonald et al., 1982; Pouplard et al., 1983; Savorani et al., 1986; Gaskin et al., 1987). However, data on the immune status of AD patients are discordant, and several contradictory results are on record (Miller et al., 1981; Skias et al., 1985). In this report we present data regarding some immune functions of the thymus branch of the immune system in a group of non-familial AD patients, and in age- and sex-matched controls.
Materials and methods
Subjects Forty patients with senile dementia of the Alzheimer type (mean age 73 + 1 years) and 43 ageand sex-matched controls (mean age 74 + 1 years) were studied. Young controls were healthy hospital and laboratory personnel (mean age 30 + 5 years). Age-matched controls consisted of either healthy
elderly living in a retirement home, or elderly hospitalized with minor pathological conditions which did not interfere with brain or immune functions. Control patients consisted of four subjects with mental impairment of the non-Alzheimer type. Clinical diagnosis Diagnosis of dementia of the Alzheimer type was carried out after careful analysis of the patient's personal and family records, and clinical examination according to the recommendations of DSM III. Biochemical and metabolic parameters such as red blood cell count (RBC), white blood cell count (WBC), hemoglobin (Hb), platelet number, blood urea nitrogen (BUN), glucose, uric acid, creatine, cholesterol, triglycerides, Na +, K +, CI-, total protein, bilirubin, glutamic-oxalacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and alkaline phosphatase were evaluated. Patients were also monitored by electrocardiogram (ECG), chest X-ray and computed tomography (CT) brain scanning. Hormonal status was evaluated by measuring thyroid-stimulating hormone (TSH), triidothyronine (T3) , thyroxine (T4), oestradiol (OD), luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) by commercial radioimmunoassay (RIA) (ARE, Serono Diagnostica, Italy). Intellectual performance tests AD patients were examined by performing the Mini Mental State (MMS) test (Folstein et al., 1975). Memory and attention were also evaluated. Plasma zinc levels Venous blood was collected in fluorinated plastic tubes in the presence of EDTA. After centrifugation at 4 ° C and 600 × g, plasma was collected and samples stored at - 80 o C. Zinc concentration was measured by atomic adsorption as previously described (Fabris et al., 1984). Plasma thymulin levels Thymulin concentration in the plasma was determined by a bioassay as previously described (Fabris et al., 1984). Levels of active and inactive thymulin were distinguished according to the
203 thymic hormone (TH) bioassay after the addition of exogenous zinc to plasma samples as described elsewhere (Fabris et al., 1984, 1986).
Purification of peripheral blood lymphocytes and lymphocyte microcultures Venous blood was collected in plastic tubes containing EDTA. Peripheral blood lymphocytes (PBL) were purified by Ficoll gradient centrifugation (Boyum, 1968). PBL cultures were performed in quadruplicate as previously described (Licastro et al,, 1983). Briefly, 1 × 105 lymphocytes were cultured in complete medium ( R P M I 1640 containing 10% heat-inactivated pooled AB human serum, 100 U penicillin, 100 # g / m l streptomycin, and 2 m M glutamine; Flow Lab., Italy). Stimulated lymphocytes were activated by phytohemagglutinin (PHA-P) (Difco; final concentration in complete medium 0.1 or 1 # l / m l ) and cultured in a CO 2 incubator for 3 or 4 days. Some lymphocyte cultures were treated with human recombinant interleukin 2 (IL-2) (Boehringwerke; final concentration in complete medium 20 U / m l ) at the beginning of the culture period with or without the addition of PHA. Cell proliferation was assessed by pulsing lymphocytes with tritiated thymidine ([3H]methylthymidine, [3H]TdR) (Amersham, U.K.; spec. act. 5 C i / m m o l ) for the last 6 h of culture.
Lymphocyte harvesting and radioactivity measurement
TABLE 1 ZINC PLASMA LEVELS IN YC, OC, AD, AND CD Data are presented as mean_+1 SE. Groups (n)
Zinc (#g/dl)
YC (15) OC (43) AD patients (40) CD patients (4)
105 _+2 83 _+3 77 + 3 80 _+8
Statistical differences: YC versus OC, P < 0.0001; YC versus AD, P < 0.0001; YC versus CD, P < 0.001. No statistically significant difference was present among OC, AD and CD.
of circulating TH, measured in 15 YC, 13 OC, 16 A D patients and four C D patients, are shown in Fig. 1. OC, A D and C D patients showed significantly lower levels of active T H than YC ( P < 0.05). In Fig. 2, levels of inactive T H are shown as determined by performing the T H bioassay after in vitro addition of zinc. A partial recovery of T H activity was found in the plasma from OC. The extent of T H reactivation in plasma from A D and C D was less than that observed in plasma from OC. Differences observed between OC and A D or C D were statistically significant ( P < 0.05). N o difference in T H reactivation was detected between A D and CD patients. The proliferative capacity of peripheral blood lymphocytes after P H A activation for 3 and 4 days of culture in YC, OC, and A D patients was also studied (Fig. 3A and B). Using a suboptimal
At the end of the culture period, lymphocyte cultures were harvested, washed on glass fiber filters with the aid of a multiple cell harvester (Skatron), and [3H]TdR incorporation measured by liquid scintillation counting as previously described (Franceschi et al., 1981).
[] YC
[] ~
[ ] AD
[] CD
QQ Q I
o'
Results
Plasma zinc levels were measured in 15 young controls (YC) and 43 aged controls (OC), 40 A D patients and four subjects with dementia of the non-Alzheimer type (control disease; CD) (Table 1). Zinc levels were significantly lower in OC, A D and C D than in YC. Differences between OC and A D were not statistically significant. Basal levels
g
O
Fig. 1. Basal levels of active TH in plasma from YC, OC, AD. and CD. Black circles refer to individual data; histograms represent mean values ( + SE) for each group.
204 150
D~
[] YC
eel
°I",,"
[] At)
[ ] YC [ ] oc
°
OO0
4
[ ] CD
•
01
[] AD
'7 100 o x
o,
I[11
o. u
150
Fig. 2. Levels of active TH after the in vitro addition of zinc to plasma samples from YC, OC, AD, and CD. Black circles refer to individual data; histograms represent mean values ( 5:1 SE) for each group.
O:O
•
5O
=:
,
,,,
|
"
B
[ ] YC ,~ oc o
10o
~] AO
x o. u
150 [ ] YC
S0
•
[] oc
~, 100 o
AD
x
It'2 (20p/rnl)
.....
0.1"HL-2
I+IL-Z
PHA (pl/rnl)
a.
Fig. 4. Incorporation of [3H]TdR in cultures of peripheral blood lymphocytes from YC, OC, and AD after stimulation with PHA in the presence of human recombinant IL-2 for 3 (A) or 4 (B) days. Black circles refer to individual data; histograms represent mean values ( 5:1 SE) for each group.
50
""
N~
0 150 YC
'7 100
•
E3oc
o
AD
x
i~ •
o. o
5O
o
0.1
1
PHA (.pt/ml)
Fig. 3. Incorporation of [3H]TdR in cultures of peripheral blood lymphocytes from YC, OC, and AD after stimulation with PHA for 3 (A) or 4 (B) days, Black circles refer to individual data; histograms represent mean values ( 5:1 SE) for each group.
PHA concentration (0.1/~l/ml), lymphocytes from OC and AD patients proliferated significantly less (P < 0.03) than those from YC after 3 days of culture. However, no difference was present after 4 days of culture. Using an optimal PHA concentration (1 ~l/ml), proliferation of lymphocytes from both OC and AD patients was significantly lower (P < 0.04) than that of YC after 3 or 4 days of culture. No statistically significant difference was detectable between OC and AD. Modulation by exogenous human recombinant IL-2 upon PHA-induced proliferation of parallel lymphocyte cultures from YC, OC and AD after 3 or 4 days of culture was also investigated (Fig. 4A and B). IL-2 significantly increased only suboptimal PHA lymphocyte responsiveness in YC, OC and AD patients. Differences in proliferative re-
205 sponse after IL-2 addition among these three groups were still present after 3 days of culture (P < 0.03). In contrast, after 4 days of culture in the presence of IL-2, no difference in lymphocyte proliferative capacity was detected among the three groups studied. IL-2 per se was able to induce a low level of proliferation in lymphocyte cultures (Fig. 4A and B). In these conditions, the proliferative activity of lymphocytes from OC and AD patients was comparable to that observed in YC.
Discussion
An involvement of the immune system in the pathogenesis of AD has been suggested (Fudenbergh et al., 1984), and a survey of AD mortality data has shown an increased incidence of infectious diseases in the terminal phase of the disease (Chandra et al., 1986). An imbalance of serum immunoglobulins has been observed in AD patients (Behan and Feldman, 1970; Kalter and Kelly, 1975; Tavolato and Argentiero, 1980), and a decreased responsiveness to polyclonal mitogens has also been reported (Miller et al., 1981; Singh et al., 1987). However, the latter findings have not been confirmed in other studies (Kalter and Kelly, 1975; MacDonald et al., 1982; Leffel et al., 1985). Contradictory results concerning also T suppressor cell activity have been found (Miller et al., 1981; Leffell et al., 1985; Skias et al., 1985). More recently, auto-antibodies specific for brain antigens have been found either in serum or in cerebrospinal fluid of AD patients (Pouplard and Vincent, 1983; Singh and Fudenberg, 1986; Gaskin et al., 1987). Interestingly, some of these antibodies appeared to react against cells of the pituitary gland (Pouplard and Vincent, 1983). Other studies, however, have not confirmed these latter data (Philpot et al., 1985; VanDorp et al., 1985). At present, the role of the immune system in the pathogenesis of Alzheimer type dementia is not clearly established and the presence of different clinical subgroups may imply a differential involvement of the immune system. Immune functions, particularly those belonging to the thymus branch of the immune system, decrease with advancing age in the normal elderly (Licastro et al., 1983; Fabris et al., 1984). This makes it more
difficult to identify an immune impairment specifically associated with AD. In the present report we have investigated the endocrine function of the thymus gland by studying the plasma levels of a zinc-dependent thymic hormone: a nonapeptide called thymulin (Dardenne et al., 1982). Thymulin has been shown to promote T cell differentiation and maturation (Dardenne et al., 1982). The active form of this hormone decreases dramatically in plasma of elderly people and in young subjects with Down's syndrome (Fabris et al., 1984). Interestingly, subjects with Down's syndrome over the age of 35 show an abnormal incidence of Alzheimer type dementia (Heston, 1976; Kay, 1986). Additionally, it has been shown that TH requires a zinc molecule to exert its biological activity on lymphocyte differentiation (Dardenne et al., 1982). Plasma zinc levels were lower in OC, AD patients and CD than in YC; however, no difference was found between OC and patients with dementia. In patients with Alzheimer type dementia the biological activity of TH after the addition of zinc in vitro increased less than in aged controls. In aged controls at least 50% of the circulating TH was deprived of zinc; however, the biological activity of the hormone was restored by addition of zinc in vitro. On the other hand, the addition of zinc in vitro did not significantly increase the basal activity of TH from plasma samples of AD. A decreased reactivation of TH was also found in patients with dementia of the non-Alzheimer type. These results suggest that, in patients with dementia, the thymus gland may release a reduced amount of TH in comparison to the normal elderly. However, if the relative increase in TH activity was calculated (i.e., TH activity after zinc add i t i o n / T H basal activity) a statistically significant difference (P < 0.05) was present between OC and AD, and also between AD and CD patients (relative increase 2.2, 1.2 and 2.1 in OC, AD and CD, respectively). These data suggest that an aliquot of TH molecules of AD patients might have a reduced capacity to bind a zinc atom. It has been shown that other metals can bind the TH molecule, and among these, there is aluminum, which can compete with zinc for the binding site of TH (Dardenne et al., 1982). It is
206 interesting to note that aluminum has been suggested to play a role in the pathogenesis of AD (Candy et al., 1986; Birchall and Chappel, 1988). Thus, a portion of T H molecules might bind aluminum and this latter metal might interfere with the normal functioning of the hormone. The responsiveness to PHA of lymphocytes from AD patients was within the normal age range. Lymphocytes from young and aged donors, and from AD patients were sensitive to the effect of IL-2 when a suboptimal concentration of PHA was used. Differences in proliferative activity among lymphocyte cultures from the three groups studied were not reduced after 3 days of incubation. However, after the addition of IL-2 and 4 days of culture, proliferation of lymphocytes from aged controls and patients with AD reached the values of that from young controls. Our data are partially at variance with those of Gillis et al. (1981) who found that lymphocytes from aged donors were not responsive to the addition of T cell growth factor (TCGF) present in supernatants of activated normal lymphocyte cultures. These discrepancies may be explained by the increased purity of IL-2 used by us, the length of the culture periods, and the dosages of PHA. Supernatants from cell cultures may also contain other lymphokines which might differently affect lymphocyte proliferation from donors of different ages. In our experimental conditions, IL-2 addition to PHA-stimulated cultures showed a positive effect on lymphocytes from either aged controis or AD patients after 4 days of culture, but only when a suboptimal mitogen dose was used. In conclusion, the impaired proliferation in AD patients appears to be more dependent upon the age than the disease. These data are at variance with other results showing a decrease in mitogen responsiveness in AD ,patients (Miller et al., 1981; Singh et al., 1987). Such a discrepancy may be partially related to the clinical heterogeneity of AD, i.e. only some clinical subgroups may show a further impairment of T cell proliferation after in vitro activation. Finally, it is important to bear in mind that AD patients studied by us were patients without signs of infectious diseases and that the immunological screening was performed early after the diagnosis. Some conditions such as: (a) the age of the
patient at the onset of the disease, (b) the time lag between the onset of the disease and the diagnosis, (c) the time lag between the onset of the disease, the time of the diagnosis, and the time of the immunological screening, which are not directly related to the biology of AD, may affect some immune parameters such as lymphocyte proliferation after polyclonal activation. A random distribution of patients according to the criteria listed above might at least in part explain contradictory results regarding some immune functions in AD. The impairment of the endocrine portion of the thymus gland might by an early sign of immune impairment in patients with dementia, since no other immunological dysfunctions were present. This immune alteration could be related to a disturbance of the neuroendocrine regulation of the thymus gland secondary to the mental impairment. Interestingly, patients with AD showed a further impairment of the thymus function, possibly related to the release of an altered T H molecule.
Acknowledgements This paper was supported by research grants from the Ministero della Pubblica lstruzione Italiana Fondi (60%), and partially from the Regione Emilia Romagna. We thank Dr. L.J. Davis for reviewing the manuscript and Dr. M. Chiricolo and Mr. A. DePietro for technical help.
References Amaducci, L. and Sorbi, S. (1985) L'invecchiamentocerebrale: definizioneed inquadramento nosologico. Giorn. Gerontol. 33, 1033-1054. Amaducci, L.A., Fratiglioni, L., Rocca, W., Fieschi, C., Livrea, P., Pedone, D., Bracco, L., Lippi, A., Gandolfo, C., Bino, G., Prencipe, M., Bonatti, M., Girotti, F., Carella, F., Tavolato, B., Ferla, S., Lenzi, G.L., Carolei, A., Gambi, A., Grigoletto, F. and Schoenberg, B. (1986) Risk factors for clinically diagnosed Alzheimer's disease: a case-control study for an Italian population. Neurology36, 922-931. Behan, P.O. and Feldman, R.G. (1970) Serum protein, amyloid and Alzheimer's disease. J. Am. Geriatr. Soc. 18, 792-797. Birchall, J.D. and Chappell, J.S. (1988) Aluminium, chemical physiology, and Alzheimer's disease. Lancet ii, 1008-1010.
207
Boyum, A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77-89. Candy, J.M., Klinowski, J., Perry, R.H., Perry, E.K., Fairbain, A., Oakley, A.E., Carpenter, T.A., Atack, J.R., Blessed, G. and Edwardson, J.A. (1986) Aluminosilicates and senile plaque formation in Alzheimer's disease. Lancet i, 354-357. Chandra, V., Bharucha, N.E. and Schoenberg, B.S. (1986) Conditions associated with Alzheimer's disease at death: case-control study. Neurology 36, 209-211. Dardenne, M., Pleau, J.M., Nabama, B., Lefancier, P., Denierr, P., Chosy, M. and Bach, J.F. (1982) Contributions of zinc and other metal to the biological activity of serum thymic factor. Proc. Natl. Acad. Sci. U.S.A. 79, 5370-5373. Fabris, N., Mocchegiani, E., Amadio, L., Zannotti, M., Licastro, F. and Franceschi, C. (1984) Thymic hormone deficiency in normal ageing and Down's syndrome: is there a primary failure of the thymus? Lancet i, 983-986. Fabris, N., Mocchegiani, E., Mariotti, S., Pacini, F. and Pinchera, A. (1896) Thyroid function modulates thymic endocrine activity. J. Clin. Endocrinol. Metab. 62, 474-478. Folstein, M., Folstein, S. and McHugh, P.R. (1975) Mini Mental State: a practical method for grading the cognitive state of patients for the clinician. J. Psychiatr. Res. 12, 189-198. Franceschi, C., Licastro, F., Chiricolo, M., Bonetti, F., Zannotti, M., Fabris, N., Mocchegiani, E., Fantini, M.P., Paolucci, P. and Masi, M. (1981) Deficiency of autologous mixed lymphocyte reactions and serum thymic factor level in Down's syndrome. J. Immunol. 126, 2161-2164. Fudenbergh, H.H., Whitten, H.D., Arnaud, P. and Khansari, N. (1984) Is Alzheimer's disease an immunological disorder? Observations and speculations. Clin. Immunol. Immunopathol. 32, 127-131. Gaskin, F., Kingsley, B.S. and Fu, S.M. (1987) Autoantibodies to neurofibrillary tangles and brain tissue in Alzheimer's disease. J. Exp. Med. 165, 245-250. Gillis, S., Kozak, R., Durante, M. and Weksler, M.A. (1981) Decreased production of and response to T cell growth factor by lymphocytes from aged humans. J. Clin. Invest. 67, 937-942. Gottfries, C.G. (1985) Definition of normal ageing senile dementia and Alzheimer's disease. In: G.G. Gottfries (Ed.), Normal Ageing Alzheimer's Disease and Senile Dementia, Editions Universit~ Bruxelles, Brussels, pp. 11-17. Henderson, A.S. (1986) The epidemiology of Alzheimer's disease. Br. Med. Bull. 42, 3-10. Heston, L.L. (1976) Alzheimer's disease, trisomy 21, and myeloproliferative disorders. Associations suggesting a genetic diathesis. Science 196, 322-323. Hollander, E., Mohs, R.C. and Davis, K.L. (1986) Antemortem markers of Alzheimer's disease. Neurobiol. Aging 7, 367387. Kalter, S. and Kelly, S. (1975) Alzheimer's disease: evaluation of immunological indices. N.Y. State J. Med. 75, 1222-1225. Kay, D.W.K. (1986) The genetic of Alzheimer's disease. Br. Med. Bull. 42, 19-23. Licastro, F., Chiricolo, M., Tabacchi, L., Barboni, F., Zannotti,
M. and Franceschi, C. (1983a) Enhancing effect of lithium and potassium ions on lectin-induced lymphocyte proliferation in ageing and Down's syndrome subjects. Cell. Immunol. 75, 111-121. Licastro, F., Tabacchi, L., Chiricolo, M., Parente, R., Cenci, M., Barboni, F. and Franceschi, C. (1983b) Defective selfrecognition in subjects of far advanced age. Gerontology 29, 64-72. Leffell, M.S., Lumsden, L. and Steiger, W.A. (1985) An analysis of T lymphocyte subpopulations in patients with Alzheimer's disease. J. Am. Geriatr. Soc. 33, 4-8. Macdonald, S.M., Goldstone, A.H., Morris, J.E., Exton-Smith, A.N. and Callard, R.E. (1982) Immunological parameters in the aged and in Alzheimer's disease. Clin. Exp. Immunol. 49, 123-128. Mayeux, R., Stem, Y. and Spaton, S. (1985) Heterogeneity in dementia of the Alzheimer type: evidence of subgroups. Neurology 35, 453-461. McKhann, G., Drachrnan, D., Folstein, M., Katzman, R., Price, D. and Stadlan, E. (1984) Clinical diagnosis of A1zheimer's disease: report of the NINCDS-ADRDA work group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology 34, 939-944. Miller, A.E., Neighbour, A., Katzman, R., Arouson, M. and Lipkowiz, R. (1981) Immunological studies in senile dementia of the Alzheimer type: evidence for enhanced suppressor cell activity. Ann. Neurol. 10, 506-510. Nerl, C., Mayeux, R. and O'Neill, G.J. (1984) HLA-linked complement markers in Alzheimer's and Parkinson's disease: Ca variant (C4B2) a possible marker for senile dementia of the Alzheimer type. Neurology 34, 310-314. Philpot, M., Colgan, J., Levy, R., Holland, A., Mirakian, R., Richardson, C.A. and Bottazzo, G.F. (1985) Prolactin cell autoantibodies and Alzheimer's disease. J. Neurol. Neurosurg. Psychiatry 48, 287-288. Pouplard, A., Emile, J. and Vincent-Pineau, F. (1983) Autoanticorps circulants anticellules h prolectine de l'hypophyse humaine et maladie d'Alzheimer. Rev. Neurol. (Paris) 139, 187-191. Rossor, M.N., Iversen, L.L., Reynolds, G.P., Mountjoy, C.P. and Roth, M. (1984) Neurochemical characteristics of early and late onset types of Alzheimer's disease. Br. Med. J. 288, 961-964. Roth, M. (1986) The association of clinical and neurological findings and its bearing on the classification and aetiology of Alzheimer's disease. Br. Med. Bull. 42(1), 42-50. Savorani, G., Salsi, A., De Vinci, C., Piazzi, S., Sarti, G., Corneli, M., Palmirani, R., Tupone, A., Tolomelli, M. and Cavazzuti, F. (1986) Studies on the possible role of zinc and immune response in Alzheimer's disease. In: J.J. Facchini, G. Haaijmann, and A. Lab6 (Eds.), Immunoregulation in Aging, Topics in Aging Research in Europe, 9, EURAGE, pp. 197-203. Singh, V.K., Fudenberg, H.H. and Brown, III, F.R. (1987) Immunologic dysfunction: simultaneous study of Alzheimer's and older Down's patients. Mech. Ageing Dev. 37, 257-264.
208 Skias, D., Bania, M., Reder, A.T., Luchins, D. and Antel, J.P. (1985) Senile dementia of Alzheimer type (SDAT): reduced T-cell-mediated suppressor activity. Neurology 35, 16351638. Tavolato, B. and Argentiero, V. (1980) Immunological indices in presenile Alzheimer's disease. J. Neurol. Sci. 46, 325-331. VanDorp, T.A., Endzt, L.J., "re Velde, J., Sleats, J.P.J. and
Reichgelt, J. (1985) Prolactin cell autoantibodies and Alzheimer's disease. J. Neurol. Neurosurg. Psychiatry 48, 1308-1309. Wade, J.P.H., Mirsen, T.R., Hachinski, V.C., Fisman, M., Lau, C. and Merskey, H. (1987) The clinical diagnosis of Alzheimer's disease. Arch. Neurol. 44, 24-29.