Journal of the Neurological Sciences, 1978, 35:375-379 © Elsevier/North-Holland Biomedical Press
375
HERPES SIMPLEX VIRUS ANTIBODIES IN PATIENTS W I T H P A R K I N S O N ' S DISEASE
REIJO J. MARTTILA and URPO K. RINNE Departments of Neurology and Virology, University of Turku, SF-20520 Turku 52 (Finland)
(Received 4 August, 1977)
SUMMARY Sera from 40 patients with idiopathic Parkinson's disease and their age- and sex-matched controls were assayed for immunoglobulin G (lgG) antibodies against herpes simplex virus (HSV) type 1-induced cell surface antigens with the indirect immunofluorescent test. An increased level of HSV antibodies was found among the patients with Parkinson's disease when the distribution of titres (P < 0.001), the mean titres (P < 0.001), or the mean paired titre difference (P < 0.001) was compared with the controls. This may suggest a part of HSV in the pathogenesis of Parkinson's disease. Alternatively, the increased HSV antibody response might be an epiphenomenon indirectly associated with Parkinson's disease.
INTRODUCTION Postencephalitic Parkinson's disease is known to have been a common outcome of an encephalitis with a probable viral etiology, i.e., lethargic encephalitis (Duvoisin and Yahr 1965). Furthermore, suggestions have been presented that even idiopathic Parkinson's disease should be considered as a potential slow virus infection of the central nervous system (Eadie, Sutherland and Doherty 1965; Poser, Huntley and Poland 1969). Despite this, only a few serological studies have been carried out comparing viral antibodies in patients with Parkinson's disease and control subjects. These few studies deal with postencephalitic patients (Andrewes and Carmichael 1930; Gay and Holden 1931 ; Eadie et al. 1965) and one study with Parkinson-dementia disease of Guam (Elizan, Hirano, Bernard, Need, van Nuis and Kurland 1966). However, in our recent study of several viral antibodies in a large population of patients with Correspondence to: R. J. Marttila, Department of Neurology, University of Turku, SF-20520 Turku 52, Finland.
376 Parkinson's disease, a slight but clearly significant increase was found in complementfixing antibodies against HSV when compared with healthy, matched control subject~ (Marttila, Arstila, Nikoskelainen, Halonen and Rinne 1977). To find out whether the observed increase of HSV antibodies in Parkinsonian patients is reproducible with more sensitive serological techniques than complement fixation, we have assayed a random sample of sera from patients with idiopathic Parkinson's disease and their matched controls with the indirect immunofluorescent technique developed to detect lgG class antibodies against HSV-induced cell surface antigens, analogous to HSV envelope components (Roizman 1970, 1971). PATIENTS AND METHODS The study group comprised 40 patients with Parkinson's disease randomly selected from among the 421 patients with idiopathic Parkinson's disease originally found in an epidemiological study (Marttila and Rinne 1976). The diagnostic criteria are described elsewhere in detail (Marttila and Rinne 1976). The main clinical features of the patients are shown in Table 1. The control persons, matched for sex and age and free of extrapyramidal disease, were selected from among the general population residing in the same area as previously described (Marttila et al. 1977). The patient and control sera were collected during 1972-1973, divided into small aliquots and stored at - - 2 5 °C until tested. The indirect immunofluorescent test for detection of I g G antibodies against HSV-induced cell surface antigens has been previously described in detail (Marttila and Kalimo 1977). In brief, H e L a cells grown on coverslips were infected with the VR strain of HSV type 1 at a multiplicity of 5-10 plaque-forming units per cell, incubated for 6 hr at 37 °C and washed. The excessive water on the coverslips was allowed to evaporate at room temperature for 5-10 rain and the cells were treated while still moist with serial 2-fold heat-inactivated serum dilutions and incubated in a moist chamber at 37 °C for 45 min. After washing, the FITC-conjugated sheep anti-human I g G (Statens Bakteriologiska Laboratorium, Stockholm, Sweden) was applied. The slides were incubated at 37 °C for 45 min and thoroughly washed. The specimens were TABLE 1 MAIN CLINICAL FEATURES OF THE PATIENTS WITH PARKINSON'S DISEASE Total Women Men Age (years)
Patients 40 Controls 40
22 22
18 18
63.9 d: 6.4 b 64.1 i 7.4
According to Hoehn and Yahr (1967). b Mean + SD. e Number of patients.
Duration of the disease (years)
Disability a 1
2
3
4
5
6.2 4- 5.2
3e
19
15
3
--
377 TABLE 2 ANTIBODIES AGAINST HSV TYPE 1-INDUCED CELL SURFACE ANTIGENS IN PATIENTS WITH PARKINSON'S DISEASE IN COMPARISON WITH MATCHED CONTROLS Per cent positive Patients (n = 40) Controls (n = 40)
100 100
Titre (log2) _10
mean ~SD
-6
8• 17
16 14
16 3b
9.2 ± 0.8 8.3 ± 1.0c
Geometric mean titre 1:586 1:315
Number of sera with indicated titre. b Difference between the distribution of titres, P < 0.001. e Difference between the mean titres, P < 0.001. m o u n t e d with buffered glycerol and examined with a fluorescence microscope with incident light. The end-point titre o f a serum specimen was defined as that serum dilution, which still showed a definite, specific m e m b r a n e fluorescence. The titres were expressed as log~ values o f the reciprocals o f the serum dilutions. Each patient and respective control serum were assayed on the same day, with the tests read blindly. RESULTS The antibody assay results are shown in Table 2. All sera examined were HSV antibody positive. A comparison o f the distribution o f H S V antibody titres showed a significant difference between the patients with Parkinson's disease and the control subjects (P < 0.001, 2 × k contingency table ;t 2 test): the patients having high titres more often than the controls and the controls low titres more often than the patients. The patients' mean H S V antibody titre was almost double that o f the controls. The difference between the mean titres was statistically significant (P < 0.001, Student's t test). Even the mean paired titre difference (0.9 log2) between the patients and controls was significant (P < 0.001, paired t test). A t t e m p t s to demonstrate significant correlations o f the HSV antibody titres with the patients' age, duration o f the disease or the disability o f the patients, were negative. DISCUSSION This study is an extension o f our previous investigation (Marttila et al. 1977). Both studies show an increased level o f H S V antibodies in the sera of patients with idiopathic Parkinson's disease in comparison with healthy, matched controls. Our previous study was carried out with a relatively low sensitive complement fixation method. The present immunofluorescent antibody detection, however, is more sensitive, having also a greater discrimination capacity as well as being more suitable in comparative serological studies than the complement fixation test. In most currently available serological tests for the determining o f H S V antibodies, a cross-reacting antibody activity is observed with HSV type 1 and 2 antigens,
378 which is also the shortcoming of the present assay method (Marttila and Kalimo 1977). Therefore, a direct typing of this increased HSV antibody response was not possible. On the other hand, results from the indirect haemagglutination test allowing type-specific HSV antibody detection (Fuccillo, Moder, Catalano, Vincent and Sever 1969) show that the increased HSV antibody response in patients with Parkinson'~ disease is predominantly directed against HSV type 1 antigens (Marttila et al. unpublished). Regarding the relevance of the increased HSV antibody response in patients with Parkinson's disease, there are 3 main possibilities. First, it might suggest that HSV is connected with the aetiopathogenesis of Parkinson's disease. The peculiar behaviour of HSV in relation to the nervous tissue, i.e., its capability of remaining latent in the nerve cells of the trigeminal ganglion (Stevens 1975), and in experimental models also inside the central nervous system of rabbits (Knotts, Cook and Stevens 1973) as well as its affinity to alter particularly the brain dopamine metabolism in acute experimental encephalitis in mice (Lycke, Modigh and Roos 1970; Lycke and Roos 1972) are of most interest in this context. Second, the increase of HSV antibodies depends on a factor involved in Parkinson's disease even though the increased antibody response itself is not in direct relationship with the aetiology of Parkinson's disease. This kind of phenomenon could conceivably be related to factors connected with the regulation of the immune response of the host and, on the other hand, with the susceptibility of the host to a certain disease, e.g., the histocompatibility system (Lehrich and Arnason 1976). Another explanation is that during the disease process of Parkinson's disease, the antigenic determinants of latent HSV were secondarily exposed or expressed resuiting in increased antigenic stimulation. Third, the increased HSV antibody response found is merely a coincidental finding and not connected with Parkinson's disease_ For the time being, the above contingencies remain unanswered, but nevertheless suggest further studies to investigate more fully the antibody response against HSV in patients with Parkinson's disease. ACKNOWLEDGEMENTS This study was supported by the Academy of Finland, Medical Research Council and the Sigrid Juselius Foundation.
REFERENCES Andrewes, C. H. and E. A. Carmiehael (1930) A n o t e o n the presence of antibodies to herpes virus in postencephalitic and other human sera, Lancet, 1 : 857-858. Duvoisin, R. C. and M. D. Yahr (1965) Encephalitis and parkinsonism, Arch. Neurol. (Chic.), 12: 227-239. Eadie, M. J., J. M. Sutherland and R. L. Doherty (1965) Encephalitis in etiolol~y of parkinsonism in Australia, Arch. Neuroi. (Chic.), 12: 240-245. Eliza_q, T. S., A. Hirano, M. A. Bernard, R. I. Need, C. van Nuis and L. T. Kurland (1966) Amyotrophic lateral sclerosis and parkinsonism-dementia complex of Guam, Arch. Neurol. (Chic.), 14: 356-368.
379 Fuccillo, D. A., F. L. Moder, L. W. Catalano, M. M. Vincent and J. L. Sever (1969) Herpes virus hominis types I and II: a specific microindirect hemagglutination test, Proc. Soc. exp. Biol. (N. Y.), 133: 735-739. Gay, F. P. and M. Holden (1931) Loss of v~ricidal property in serums from patients with herpes and encephalitis, J. ,4mer. reed. Ass., 96: 2028-2029. Hoehn, M. M. and M_ D. Yahr (1967) Parkinsonism: onset, progression, and mortality, Neurology (Minneap.), 17 : 427-442. Knotts, F. B., M. L_ Cook and J_ G. Stevens (1973) Latent herpes simplex virus in the central nervous system of rabbits and mice, J. exp. Med_, 138: 740-744. Lehrich, J. R. and B. G. W. Arnason (1976) Histocompatibility types and viral antibodies, ,4rch. Neurol. (Chic.), 33- 404-405. Lycke, E. and B. E. Roos (1972) The monoamine metabolism in viral encephahtides of the mouse. II_ Turnover of monoamines in mice infected with herpes simplex virus, Brain Res., 44: 603-613. Lycke, E., K. Modigh and B. E. Roos (1970) The monoamine metabolism in viral encephalitides of the mouse. I. Virological and biochemical results, Brain Res., 23: 235-246. Marttila, R. J. and K. O. K. Kalimo (1977) Indirect immunofluorescence detection of human IgM and IgG avtibodies against herpes simplex virus type 1 induced cell surface antigens, Acta path. microbiol, scand., Sect. B, 85: 195-200. Marttila, R. J. and U. K. Rinne (1976) Epidemiology of Parkinson's disease in Finland, ,4cta neurol. scand., 53: 81-102. Marttlla, R. J., P_ Arstila, J. Nikoskelainen, P. Halonen and U. K. Rinne (1977) Viral antibodies in the sera from patients with Parkinson disease, Europ. NeuroL, 15: 25-33. Poser, C. M., C. J. Huntley and J. D. Poland (1969) Para-encephalitic parkinsonism, Acta neurol. scand., 45: 199-215. Roizman, B. (1970) Herpes viruses, membranes, and the social behavior of infected cells. In: M. Sanders (Ed_), Viruses Affecting Man and Animals, W. H. Green, St. Louis, Mo., pp. 37-72. Roizman, B. (1971) Biochemical features of herpes virus infected cells. In: W. Nakahara, K. Nishioka, T. Hirayma and Y. Ito (Eds.), Recent ,4dvances in Human Tumor Virology and Immunology (Proc. 1st Int. Symp_ Princess Takamatsu Cancer Res. Fund.), University of Tokyo Press,Tokyo, pp. 109-138. Stevens, J. G. (1975) Latent herpe3 simplex virus and the nervous system, Curr. Top. Microbiol. Immunol., 70: 31-50.
375
HERPES SIMPLEX VIRUS ANTIBODIES IN PATIENTS W I T H P A R K I N S O N ' S DISEASE
REIJO J. MARTTILA and URPO K. RINNE Departments of Neurology and Virology, University of Turku, SF-20520 Turku 52 (Finland)
(Received 4 August, 1977)
SUMMARY Sera from 40 patients with idiopathic Parkinson's disease and their age- and sex-matched controls were assayed for immunoglobulin G (lgG) antibodies against herpes simplex virus (HSV) type 1-induced cell surface antigens with the indirect immunofluorescent test. An increased level of HSV antibodies was found among the patients with Parkinson's disease when the distribution of titres (P < 0.001), the mean titres (P < 0.001), or the mean paired titre difference (P < 0.001) was compared with the controls. This may suggest a part of HSV in the pathogenesis of Parkinson's disease. Alternatively, the increased HSV antibody response might be an epiphenomenon indirectly associated with Parkinson's disease.
INTRODUCTION Postencephalitic Parkinson's disease is known to have been a common outcome of an encephalitis with a probable viral etiology, i.e., lethargic encephalitis (Duvoisin and Yahr 1965). Furthermore, suggestions have been presented that even idiopathic Parkinson's disease should be considered as a potential slow virus infection of the central nervous system (Eadie, Sutherland and Doherty 1965; Poser, Huntley and Poland 1969). Despite this, only a few serological studies have been carried out comparing viral antibodies in patients with Parkinson's disease and control subjects. These few studies deal with postencephalitic patients (Andrewes and Carmichael 1930; Gay and Holden 1931 ; Eadie et al. 1965) and one study with Parkinson-dementia disease of Guam (Elizan, Hirano, Bernard, Need, van Nuis and Kurland 1966). However, in our recent study of several viral antibodies in a large population of patients with Correspondence to: R. J. Marttila, Department of Neurology, University of Turku, SF-20520 Turku 52, Finland.
376 Parkinson's disease, a slight but clearly significant increase was found in complementfixing antibodies against HSV when compared with healthy, matched control subject~ (Marttila, Arstila, Nikoskelainen, Halonen and Rinne 1977). To find out whether the observed increase of HSV antibodies in Parkinsonian patients is reproducible with more sensitive serological techniques than complement fixation, we have assayed a random sample of sera from patients with idiopathic Parkinson's disease and their matched controls with the indirect immunofluorescent technique developed to detect lgG class antibodies against HSV-induced cell surface antigens, analogous to HSV envelope components (Roizman 1970, 1971). PATIENTS AND METHODS The study group comprised 40 patients with Parkinson's disease randomly selected from among the 421 patients with idiopathic Parkinson's disease originally found in an epidemiological study (Marttila and Rinne 1976). The diagnostic criteria are described elsewhere in detail (Marttila and Rinne 1976). The main clinical features of the patients are shown in Table 1. The control persons, matched for sex and age and free of extrapyramidal disease, were selected from among the general population residing in the same area as previously described (Marttila et al. 1977). The patient and control sera were collected during 1972-1973, divided into small aliquots and stored at - - 2 5 °C until tested. The indirect immunofluorescent test for detection of I g G antibodies against HSV-induced cell surface antigens has been previously described in detail (Marttila and Kalimo 1977). In brief, H e L a cells grown on coverslips were infected with the VR strain of HSV type 1 at a multiplicity of 5-10 plaque-forming units per cell, incubated for 6 hr at 37 °C and washed. The excessive water on the coverslips was allowed to evaporate at room temperature for 5-10 rain and the cells were treated while still moist with serial 2-fold heat-inactivated serum dilutions and incubated in a moist chamber at 37 °C for 45 min. After washing, the FITC-conjugated sheep anti-human I g G (Statens Bakteriologiska Laboratorium, Stockholm, Sweden) was applied. The slides were incubated at 37 °C for 45 min and thoroughly washed. The specimens were TABLE 1 MAIN CLINICAL FEATURES OF THE PATIENTS WITH PARKINSON'S DISEASE Total Women Men Age (years)
Patients 40 Controls 40
22 22
18 18
63.9 d: 6.4 b 64.1 i 7.4
According to Hoehn and Yahr (1967). b Mean + SD. e Number of patients.
Duration of the disease (years)
Disability a 1
2
3
4
5
6.2 4- 5.2
3e
19
15
3
--
377 TABLE 2 ANTIBODIES AGAINST HSV TYPE 1-INDUCED CELL SURFACE ANTIGENS IN PATIENTS WITH PARKINSON'S DISEASE IN COMPARISON WITH MATCHED CONTROLS Per cent positive Patients (n = 40) Controls (n = 40)
100 100
Titre (log2) _10
mean ~SD
-6
8• 17
16 14
16 3b
9.2 ± 0.8 8.3 ± 1.0c
Geometric mean titre 1:586 1:315
Number of sera with indicated titre. b Difference between the distribution of titres, P < 0.001. e Difference between the mean titres, P < 0.001. m o u n t e d with buffered glycerol and examined with a fluorescence microscope with incident light. The end-point titre o f a serum specimen was defined as that serum dilution, which still showed a definite, specific m e m b r a n e fluorescence. The titres were expressed as log~ values o f the reciprocals o f the serum dilutions. Each patient and respective control serum were assayed on the same day, with the tests read blindly. RESULTS The antibody assay results are shown in Table 2. All sera examined were HSV antibody positive. A comparison o f the distribution o f H S V antibody titres showed a significant difference between the patients with Parkinson's disease and the control subjects (P < 0.001, 2 × k contingency table ;t 2 test): the patients having high titres more often than the controls and the controls low titres more often than the patients. The patients' mean H S V antibody titre was almost double that o f the controls. The difference between the mean titres was statistically significant (P < 0.001, Student's t test). Even the mean paired titre difference (0.9 log2) between the patients and controls was significant (P < 0.001, paired t test). A t t e m p t s to demonstrate significant correlations o f the HSV antibody titres with the patients' age, duration o f the disease or the disability o f the patients, were negative. DISCUSSION This study is an extension o f our previous investigation (Marttila et al. 1977). Both studies show an increased level o f H S V antibodies in the sera of patients with idiopathic Parkinson's disease in comparison with healthy, matched controls. Our previous study was carried out with a relatively low sensitive complement fixation method. The present immunofluorescent antibody detection, however, is more sensitive, having also a greater discrimination capacity as well as being more suitable in comparative serological studies than the complement fixation test. In most currently available serological tests for the determining o f H S V antibodies, a cross-reacting antibody activity is observed with HSV type 1 and 2 antigens,
378 which is also the shortcoming of the present assay method (Marttila and Kalimo 1977). Therefore, a direct typing of this increased HSV antibody response was not possible. On the other hand, results from the indirect haemagglutination test allowing type-specific HSV antibody detection (Fuccillo, Moder, Catalano, Vincent and Sever 1969) show that the increased HSV antibody response in patients with Parkinson'~ disease is predominantly directed against HSV type 1 antigens (Marttila et al. unpublished). Regarding the relevance of the increased HSV antibody response in patients with Parkinson's disease, there are 3 main possibilities. First, it might suggest that HSV is connected with the aetiopathogenesis of Parkinson's disease. The peculiar behaviour of HSV in relation to the nervous tissue, i.e., its capability of remaining latent in the nerve cells of the trigeminal ganglion (Stevens 1975), and in experimental models also inside the central nervous system of rabbits (Knotts, Cook and Stevens 1973) as well as its affinity to alter particularly the brain dopamine metabolism in acute experimental encephalitis in mice (Lycke, Modigh and Roos 1970; Lycke and Roos 1972) are of most interest in this context. Second, the increase of HSV antibodies depends on a factor involved in Parkinson's disease even though the increased antibody response itself is not in direct relationship with the aetiology of Parkinson's disease. This kind of phenomenon could conceivably be related to factors connected with the regulation of the immune response of the host and, on the other hand, with the susceptibility of the host to a certain disease, e.g., the histocompatibility system (Lehrich and Arnason 1976). Another explanation is that during the disease process of Parkinson's disease, the antigenic determinants of latent HSV were secondarily exposed or expressed resuiting in increased antigenic stimulation. Third, the increased HSV antibody response found is merely a coincidental finding and not connected with Parkinson's disease_ For the time being, the above contingencies remain unanswered, but nevertheless suggest further studies to investigate more fully the antibody response against HSV in patients with Parkinson's disease. ACKNOWLEDGEMENTS This study was supported by the Academy of Finland, Medical Research Council and the Sigrid Juselius Foundation.
REFERENCES Andrewes, C. H. and E. A. Carmiehael (1930) A n o t e o n the presence of antibodies to herpes virus in postencephalitic and other human sera, Lancet, 1 : 857-858. Duvoisin, R. C. and M. D. Yahr (1965) Encephalitis and parkinsonism, Arch. Neurol. (Chic.), 12: 227-239. Eadie, M. J., J. M. Sutherland and R. L. Doherty (1965) Encephalitis in etiolol~y of parkinsonism in Australia, Arch. Neuroi. (Chic.), 12: 240-245. Eliza_q, T. S., A. Hirano, M. A. Bernard, R. I. Need, C. van Nuis and L. T. Kurland (1966) Amyotrophic lateral sclerosis and parkinsonism-dementia complex of Guam, Arch. Neurol. (Chic.), 14: 356-368.
379 Fuccillo, D. A., F. L. Moder, L. W. Catalano, M. M. Vincent and J. L. Sever (1969) Herpes virus hominis types I and II: a specific microindirect hemagglutination test, Proc. Soc. exp. Biol. (N. Y.), 133: 735-739. Gay, F. P. and M. Holden (1931) Loss of v~ricidal property in serums from patients with herpes and encephalitis, J. ,4mer. reed. Ass., 96: 2028-2029. Hoehn, M. M. and M_ D. Yahr (1967) Parkinsonism: onset, progression, and mortality, Neurology (Minneap.), 17 : 427-442. Knotts, F. B., M. L_ Cook and J_ G. Stevens (1973) Latent herpes simplex virus in the central nervous system of rabbits and mice, J. exp. Med_, 138: 740-744. Lehrich, J. R. and B. G. W. Arnason (1976) Histocompatibility types and viral antibodies, ,4rch. Neurol. (Chic.), 33- 404-405. Lycke, E. and B. E. Roos (1972) The monoamine metabolism in viral encephahtides of the mouse. II_ Turnover of monoamines in mice infected with herpes simplex virus, Brain Res., 44: 603-613. Lycke, E., K. Modigh and B. E. Roos (1970) The monoamine metabolism in viral encephalitides of the mouse. I. Virological and biochemical results, Brain Res., 23: 235-246. Marttila, R. J. and K. O. K. Kalimo (1977) Indirect immunofluorescence detection of human IgM and IgG avtibodies against herpes simplex virus type 1 induced cell surface antigens, Acta path. microbiol, scand., Sect. B, 85: 195-200. Marttila, R. J. and U. K. Rinne (1976) Epidemiology of Parkinson's disease in Finland, ,4cta neurol. scand., 53: 81-102. Marttlla, R. J., P_ Arstila, J. Nikoskelainen, P. Halonen and U. K. Rinne (1977) Viral antibodies in the sera from patients with Parkinson disease, Europ. NeuroL, 15: 25-33. Poser, C. M., C. J. Huntley and J. D. Poland (1969) Para-encephalitic parkinsonism, Acta neurol. scand., 45: 199-215. Roizman, B. (1970) Herpes viruses, membranes, and the social behavior of infected cells. In: M. Sanders (Ed_), Viruses Affecting Man and Animals, W. H. Green, St. Louis, Mo., pp. 37-72. Roizman, B. (1971) Biochemical features of herpes virus infected cells. In: W. Nakahara, K. Nishioka, T. Hirayma and Y. Ito (Eds.), Recent ,4dvances in Human Tumor Virology and Immunology (Proc. 1st Int. Symp_ Princess Takamatsu Cancer Res. Fund.), University of Tokyo Press,Tokyo, pp. 109-138. Stevens, J. G. (1975) Latent herpe3 simplex virus and the nervous system, Curr. Top. Microbiol. Immunol., 70: 31-50.
