1282

Cytomegalovirus and Rasmussen’s encephalitis

hybridisation with a biotinylated cytomegalovirus (CMV) DNA probe was done on brain biopsy specimens from 10 patients with Rasmussen’s encephalitis (RE) and 46 agematched control patients with other neurological diseases. All 10 patients with RE had intractable epilepsy and focal neurological deficits, and there was perivascular cuffing, microglial nodules, astrogliosis, and neuronal loss. CMV genomic material was demonstrated in 7 of the 10 patients with RE (in neurons, astrocytes, oligodendrocytes, and endothelial cells) and in 2 of the 46 control patients. Probes for herpes simplex virus and hepatitis B virus were negative in all patients and in In-situ

fibroblast controls. The results suggest that CMV is a likely cause of Rasmussen’s encephalitis.

Introduction The features of Rasmussen’s encephalitis (RE)1 include intractable partial epilepsy and focal neurological deficits with focal inflammation in cerebral tissue.2-4 An agent has not yet been isolated, although a viral cause has been suspected This rare illness commonly begins in childhood with a "viral" syndrome. Microglial nodules are common pathological features. Cytomegalovirus (CMV) is an uncommon brain pathogen, but CMV infection is being seen with increasing frequency in patients with AIDS, or in allograft recipients as a subacute encephalopathy.8Like RE, the virus infection is often acquired in infancy or childhood, and a primary encephalitis occurs typically with microglial nodules. Thus, we hypothesise that CMV has a role in the pathogenesis of RE.

Patients and methods Between 1972 and 1989, 365 patients had surgery for intractable epilepsy in the Epilepsy Unit, University Hospital, London, Canada. Between 1974 and 1989, 10 of these patients were found to have chronic progressive neurological features with inflammatory changes. Selection criteria for RE included chronic epilepsy with intractable focal seizures and neurological signs. Patients were excluded if they had no brain inflammation or if a specific cause was identified (eg, herpes simplex virus, measles). Clinical records and pathological samples were reviewed in all cases. Brain biopsy specimens from all cases of RE and from 46 age-matched controls with other neurological diseases who died

acutely (trauma 18, hypoxic-ischaemic encephalopathy 10, Alzheimer’s disease 1, subarachnoid haemorrhage 1, and HTLV-1associated myelopathy 1) or had surgery for intractable epilepsy (15 due to mesial temporal sclerosis) were probed for CMV genomic material by in-situ hybridisation. In-situ hybridisation was done with a biotinylated CMV DNA probe (Enzo Biochem, New York). Details of the hybridisation technique are described elsewhere. 10 Controls for the probe included CMV-infected and CMV-noninfected human fetal fibroblasts. The CMV probe consisted of two fragments (17and 25-2 kb) of CMV DNA cloned into the Bam HI1 site of pBR322; it represents 17% of the total CMV genome. This probe has been shown to specifically label CMV-infected cells in

paraffin embedded tissues.’1 Biotinylated herpes simplex virus and hepatitis B virus specific probes (Enzo Biochem) were also tested simultaneously with the CMV probe both in control patients and in patients with RE. A slide from two tissue blocks from each patient was probed. Slides were judged to be positive if more than 10 cells were seen per slide. Two without microscopists, knowledge of details of the case, interpreted each slide. A statistical analysis (Fisher’s exact ratio) was used to compare the proportion of CMV-positive specimens in patients with RE and in controls.

infected

probe-positive

Results Clinical features

(table)

There were 5 females and 5 males with RE (mean age 16 years, range 6-5-38 years). Mean duration of seizures before surgery was 6 years. 5 patients had a viral illness preceding their epilepsy. 8 were hemiparetic and 5 had subnormal intellectual function at surgery. 2 patients had subnormal intellect at the onset of their epilepsy. Partial seizures were seen in all cases (simple partial 7, chronic progressive epilepsia partialis continua 3, complex partial 5) while secondarily generalised seizures were present in 8 cases. All patients had focal epileptiform activity (spikes and/or periodic lateralised epileptiform discharges) with focal (5)

and generalised (5) the time of surgery.

slowing on electroencephalography at Radiologically (computed tomography and/or magnetic resonance imaging), focal atrophy in the epileptogenic area was the common feature, only 1 patient had any systemic illness (patient 9 had an immune mediated thrombocytopenia).

Neuropathology of the RE patients 10 patients had perivascular lymphocytic cuffing and 9 had microglial nodules. Astrogliosis, neuronal loss, and leptomeningeal inflammation was seen in 8, 8, and 7 patients, respectively. Viral inclusions were not seen by light microscopy or electronmicroscopy in any patient. Immunocytochemistry for CMV, herpes simplex virus, Epstein-Barr virus, and measles virus was negative in 6 patients and not done in the remaining patients. In-situ hybridisation 7 of 10 RE patients and 2 of 46 control patients were positive for CMV (p < 0-0001). The probe hybridised to the

nuclei of the cells within the cortex and subcortical white with clustering in the perivascular regions. Staining was concentrated in neurons, oligodendrocytes, astrocytes, and in endothelial and perivascular cells. In 2 of the 3 negative RE cases, CMV-positive cells were seen but there matter

ADDRESS: Department of Clinical Neurological Sciences, University of Western Ontario, London, Ontario, Canada (C. Power, MD, S. D Poland, BSc, Prof W T. Blume, FRCP, Prof J P Girvin, FRCSC, G. P A. Rice, FRCPC) Correspondence to DrG P.A. Rice, Department of Clinical Neurological Sciences, University Hospital, 339 Windermere Road, London, Ontario, Canada N6A 5A5.

1283

CLINICAL FEATURES OF PATIENTS WITH RE

I

I

I

i

VPA=valproate, MR= mental retardation, PTN=phenytO!n, Pb=phenobarbitone, CLON—c!onazepam, CLOB=clobazam, PR I= primidone, CBZ=carbamazep!ne

less than 10 cells per slide in total. A repeat biopsy sample of RE patient 3 (who was initially CMV positive) was negative; there was only neuronal loss and astrogliosis with neither perivascular cuffmg nor microglial nodules. Of the 2 CMV-positive patients with other neurological diseases, 1 had intractable epilepsy secondary to mesial temporal sclerosis and the other, who had HTLV-1myelopathy, died with metastatic adenocarcinoma. The hepatitis B virus probe and herpes simplex virus probe were negative in all patients and fetal fibroblasts. were

Discussion These 10

of intractable partial epilepsy and focal deficits resemble the classic descriptions of neurological Rasmussen’s (chronic-focal) encephalitis.1 Even though our patients were not clinically immunocompromised, they had a microglial nodule encephalitis reminiscent of a viral illness. The fmding of CMV in brain tissue in most of our patients with RE supports our hypothesis that CMV is a likely aetiological agent for the disease. Two other results accord with this notion. First, that the second cortical resection of patient 3 showed no CMV hybridisation concomitantly with a lack of vigorous inflammation suggests that active CMV infection is required for an inflammatory response. Secondly, in 2 of the 3 CMV-negative RE patients (patients 4 and 6) there was some CMV hybridisation (albeit fewer than the critical 10 cells per slide). The interval between onset of disease and time of surgery was 4 months (patient 6) and 6 years (patient 4), whereas in patient 5 the interval was 20 years. Although the negative result for patients 4 and 6 may reflect a tissue sampling error, the lack of hybridisation in patient 5 could be because another unrecognised pathogen was implicated or because the initial CMV infection had been replaced by a secondary autoimmune response. Walter and Renella12 reported 2 cases of chronic encephalitis that were positive for Epstein-Barr virus by DNA probe hybridisation, but these workers did not indicate which other viruses were studied. Like many herpesvirus infections, the encephalitis described by Rasmussen is regionally accentuated. We suggest that a chronic regionally accentuated electrical disturbance, such as periodic lateralised epileptic discharges or a regionally concentrated spike focus, might change the neuronal or perineuronal milieu to promote regional accentuation of cases

virus gene expression. Although we do not understand many of the factors involved in the regulatory control of CMV gene expression, two circumstances seem to be especially important-ie, the virus replicates in cells that are metabolically active (eg, rapidly dividing epithelial cells) and also has a predilection for complete gene expression in cells undergoing differentiation. Gonczol et al13 showed that human teratocarcinoma cells promote CMV gene expression only after chemically induced (retinoic acid) differentiation. In a study by Weinshenker and colleagues14 the monocyte line, THP-1, could be infected with a full cycle of CMV replication only after previous chemical treatment with 12-0-tetradecanoylphorbol- 13-acetate, which induced differentiation in the cell line into mature macrophages. The essential biochemical process is activation of protein kinase C, with some change in the intracellular metabolism of calcium. The cellular receptor for phorbol ester is protein kinase C.15 Activation of this enzyme can sequentially activate various enhancer elements, which directly promote transcription of some important viral regulatory genes. For example, phorbol esters induce a cellular protein known as NFkB (by post-translational activation); it functions as an enhancer binding protein, which in turn can upregulate various regulatory genes.16 This gene sequence is shared by mouse IgG kappa, Simian virus 40, CMV, HIV, and interleukin2.1’ Thus, activation of protein kinase C by physiological (calcium and or diacylglyceroh8) or by pharmacological means (phorbol esters) could change the cellular milieu to promote CMV expression. The biochemical factors which promote CMV replication are similar to changes that occur in the brain in the wake of the epileptic discharge. After the epileptic discharge, neuronal calcium metabolism is drastically altered. Intracellular calcium binding mechanisms are saturated after a seizure and the excess cation leads to activation of protein kinase C.19 Although this enzyme has pleiotropic effects and is probably involved in neuronal damage, it could promote virus replication. Our findings also have important therapeutic implications. That strains of CMV are sensitive to ganciclovir,2O indicates that medical treatment of RE is a future possibility. Further evidence of CMV as the cause of RE will depend on whether CMV-specific gene sequences

1284

be amplified with the polymerase chain reaction and whether the virus can be cultured from cerebral tissue of RE can

origin. and

thank Epilepsy Canada/Parke-Davis Incorporated for financial support.

We

Physicians Services

REFERENCES 1. Rasmussen T, Olszewski J, Lloyd-Smith D. Focal seizures due to chronic localized encephalitis. Neurology 1958; 8: 435-45. 2. Rasmussen T. Further observations on the syndrome of chronic encephalitis and epilepsy. Appl Neurophysiol 1978; 41: 1-12. 3. Gupta PC, Rapin I, Houroupian DS, et al. Smouldering encephalitis in

children.

Neuropediatrics 1984; 15: 191-97. JH, Hwang PA, Armstrong DC, et al. Chronic focal encephalitis (Rasmussen Syndrome): six cases. Epilepsia 1988; 29: 268-79. 5. Johnson RT, Herndon RM. Virologic studies of multiple sclerosis and other chronic and relapsing neurological diseases. Prog Med Virol 1974; 4. Pratt

18: 214-28.

Lyon G, Griscelli C, Fernandez-Alvarez E, et al. Chronic progressive encephalitis in children with X-linked hypogammaglobulinemia. Neuropaediatrie 1980; 11: 57-72. 7. Freedman H, Ch’ien L, Parham D. Virus in brain of child with hemiplegia, hemiconvulsions, and epilepsy. Lancet 1977; ii: 666. 8. Vinters HV, Kwok MK, Lo HN, et al. Cytomegalovirus in the nervous system of patients with acquired immune deficiency syndrome. Brain 6.

1989; 112: 127-39. C, Poland SD, Kassim KH,

9. Power

et

al.

Encephalopathy

in liver

transplantation: neuropathology and CMV infection. Can J Neurol Sci 1990; 17: 378-81. 10. Poland SD, Costello PC, Dekaban GA, Rice GPA. Cytomegalovirus in the brain: in vitro infection of human brain-derived cells. J Infect Dis (in press). 11. Wolber RA, Lloyd RC. Cytomegalovirus detection by nonisotopic in situ DNA hybridization and viral antigen immunostaining using a two-color technique. Hum Pathol 1988; 19: 736-41. 12. Walter GF, Renella RR. Epstein-Barr virus in brain and Rasmussen’s encephalitis. Lancet 1989; i: 279-80. 13. Gonczol L, Andrews PN, Plotkin SA. CMV replicates differentiated but not in undifferentiated cells. Science 1984; 224: 159-62. 14. Weinshenker BG, Wilton S, Rice GPA. Phorbol ester-induced differentiation permits productive human cytomegalovirus infection in a monocytic cell line. Immunology 1988; 140: 1625-31. 15. Parker PJ, Coussens L, Totty N, et al. The complete primary structure of protein kinase C the phorbol ester receptor. Science 1986; 233: 853-59. 16. Sen R, Baltimore D. Inducibility of k immunoglobulin enhancer binding protein NF-kB by a posttranslational mechanism. Cell 1986; 47: 921-28. 17. Leung K, Nabel GJ. HTLV-I transactivator induces interleukin 2 receptor expression through an NF-kB-like factor. Nature 1988; 333: 776-78. 18. Nishizuka Y. Studies and perspectives of protein kinase C. Science 1986; 233: 305-12. 19. Seisjo BB, Weiloch T. Epileptic brain damage: pathophysiology and neurochemical pathology. Adv Neurol 1986; 44: 813-47. 20. Fletcher CV, Balfour HH. Evaluation of gangiclovir for cytomegalovirus disease. DICP Ann Pharmacother 1989; 23: 5-12.

BOOKSHELF Bulimia Nervosa: Basic Research, Diagnosis, and

Therapy Edited by Manfred M. Fichter. Chichester/New York: Wiley & Sons. 1990. Pp 364. /:45/$90.60. ISBN 0-471924059.

Anorexia and Bulimia: Anatomy of

a

Social

Epidemic By Richard A. Gordon. Oxford: Basil Blackwell. 27.50 (hb), 9.95 (pb). ISBN 0-631148515.

1990.

Pp

174.

Bulimia Nervosa, with 22 chapters by authorities on eating disorders, summarises much of the work done in the decade since this disorder was recognised in 1979. In these ten years, disagreement over diagnostic criteria has been aired and largely resolved, instruments have been created to assess key clinical features, medical and psychological complications have been recognised, preliminary data have been gathered about course and outcome, and research into associated biological disturbances has begun. Perhaps most impressively, several effective treatment approaches, including forms of psychotherapy and the use of

antidepressant medication, have been developed. All these topics are concisely reviewed: in short, this book bears witness to substantial progress in clinically relevant knowledge about a newly recognised syndrome. But has all the easy work now been done? Missing from this volumeand, indeed, from all the published work-are answers to fundamental questions about bulimia nervosa. Although several putative risk factors have been identified, we do not know why only some women exposed to these risk factors get the disorder while most do not. We have no satisfying explanation for the impressive persistence of this disorder in many individuals despite their recognition of its malicious effects on their lives. And, although effective treatments have been developed, we cannot a priori match the patient to the "right" therapy, nor can we predict with any confidence

who will do well and who poorly. Fichter’s volume well describes the scientific basis for study of these important but troublesome questions in the next decade. Anorexia and Bulimia grapples with the "explosive increase" in the frequency of eating disorders in past decades. Gordon argues that anorexia nervosa and bulimia are usefully seen as "ethnic disorders". Two characteristics of an ethnic disorder are that it "expresses core conflicts and psychological tensions that are pervasive in the culture" and that its symptoms are "direct extensions and exaggerations of normal behaviors and attitudes within the culture, often including behaviors that are highly valued". He maintains that the radical change in social expectations for women since the mid-century-that they must now couple their traditional nurturing feminine role with competitiveness and achievement-is a critical element in the increased frequency of eating disorders. He suggests that women who get anorexia nervosa and bulimia are particularly vulnerable to these conflicts and resolve them by developing a disorder which expresses the contradictory meanings of female identity. Thus, the extreme thinness of individuals with anorexia nervosa both exaggerates the current cultural standard for female sexual attractiveness and implies a more assertive and asexual identity. Clinical experience strongly indicates that social factors are important in the development of eating disorders. However, it is difficult to demonstrate conclusively that such factors are critical and to discern which ones are most influential. There are few good cross-cultural studies of eating disorders, and even the basic epidemiology of anorexia nervosa and bulimia is relatively crude. For example, one of Gordon’s fundamental premises, that there has been a dramatic increase in the incidence of anorexia nervosa, has been seriously challenged (Lancet 1987; i: 205--07). This volume provides a useful and articulate discussion of the role of social factors in the development of eating disorders and adds the intriguing