ISOLATION OF AN INFECTIOUS AGENT FROM BONE-MARROWS OF PATIENTS WITH MULTIPLE SCLEROSIS

• Saturday 19 August 1978

ISOLATION OF AN INFECTIOUS AGENT FROM BONE-MARROWS OF PATIENTS WITH MULTIPLE SCLEROSIS

may harbour an infective agent concerned in the genesis of the disease.

patho-

Patients and Methods D. N. MITCHELL

Patients

Medical Research Council Tuberculosis and Chest Diseases Unit, Brompton Hospital, Fulham Road, London SW3 6HP

J.

S. PORTERFIELD

R. MICHELETTI

Sir William Dunn School of Pathology,

Oxford University

L. S. LANGE Central Middlesex Hospital, London NW10, and Mount Vernon Hospital, Northwood, Middlesex

K. K. A. GOSWAMI

P. TAYLOR

Brompton Hospital,

Methods

London SW3 6HP

D.

J. P. JACOBS

J. HOCKLEY

National Institute for Biological Standards and Control, Hill, London NW3 6RB

Holly

A. J. SALSBURY Brompton Hospital and Cardiothoracic Institute,

London

SW3 6HP

Bone-marrow aspirates from 5 patients with recent episodes of multiple sclerosis (M.s.) contained increased numbers of plasma cells, and aspirates from 3 of these patients contained atypical reticulum cells. When bone-marrow from 4 of the patients was inoculated into cell cultures a cytopathic effect (C.P.E.) was observed, and this effect could be serially passaged to further cultures. The C.P.E. was not observed when ether-treated extracts of cells showing C.P.E. were used for passage or when the passage material was filtered through a 100 nm filter. Appearance of C.P.E. was delayed in cultures inoculated with 220 nm filtrates of the passage material, but there was no delay when the inoculum was passed through filters of greater pore size. No C.P.E. was observed after culture of bonemarrow aspirated from each of 7 patients under clinical investigation for disorders other than M.S.

Summary

Introduction

WE have studied the morphology of cells in bone-maraspirates from patients with multiple sclerosis (M.S.) and have investigated the possibility that bone-marrow

row

was aspirated from 5 patients with M.S. (4 23-37 and 1 man aged 37). The mean duration aged of symptoms in the women was 5 y, and the man had had symptoms for 13 y. All patients satisfied accepted criteria for the diagnosis of definite ivt.s.’ The mean interval between the onset of the most recent clinical relapse and bone-marrow aspiration was 4 weeks. Aspiration was done before any treatment with corticosteroids. As controls, bone-marrow was aspirated from 7 patients under investigation for disorders other than M.S. (1 woman aged 46 and 6 male patients aged 14-67). 6 of these patients had no neurological disease, and 1 presented with cerebellar ataxia.

Bone-marrow

women

Bone-marrow was aspirated into preservative-free heparin saline (5 units/ml). Smears of the material were examined by light microscopy, and a differential count of 500 nucleated cells was made. Samples of bone-marrow suspension from M.S. patients 1, 2, and 3 and control donor 1 were treated with lysolecithin.2 0.2 ml volumes of 1:20 dilution of lysolecithin preparations were inoculated into suspensions and monolayers of MRC-5 cells ( a diploid human cell line), HEp-2 cells (transformed human cells), Vero cells (transformed monkey kidney cells), and PS cells (continuous pig kidney cells) in tube cultures and disposable plastic plates.4 Concurrently, 0.2 ml volumes of the same material, not treated with lysolecithin, were incubated with suspensions and monolayers of MRC-5, HEp-2, and Vero cells. Bone-marrow cells from M.S. patient 4 and control donors 2-6 were co-cultivated with suspensions of these three cell types. Eagle’s minimal essential medium (M.E.M.) buffered with bicarbonate was used as medium. Penicillin (100 units/ml), streptomycin (100 g/ml), and 1% of 200 mmol/1 L-glutamine were added to the medium. Fetal-calf serum, 10% in growth medium and 2% in maintenance medium, was used. Cells were harvested when a cytopathic effect (C.P.E.) was observed, and cells or cell-free supernatant fluids from frozen or thawed cultures, with or without filtration, were used for further passages. For ultrastructural studies two cultures of HEp-2 cells were inoculated with the filtrate (220 nm filter) from 10th-passage cultures which originated from patient 1. These cultures, together with four uninoculated cultures, were fixed in situ with 2.5% cacodylate-buffered glutaraldehyde. The cells were subsequently detached, pelleted, and post-fixed in 1% s-collidine-buffered osmium tetroxide and 0.5% aqueous ùranyl acetate. The pellets were dehydrated in ethanol and embedded in ’Araldite’, andthin sections were prepared for examination with the electron microscope. Neutralisation experiments were conducted according to the

8086

388 of Lennette and Schmidt.5 Before testing, sera were heated at 560C for 30 min. Dilutions of the sera were then mixed with equal volumes of harvests of the cultures showing C.P.E. The mixtures were allowed to stand for 2 h at room temperature before being inoculated into cell cultures. The cell cultures were then incubated at room temperature for 30 min, after which time culture medium was added. The cultures were then incubated at 370C in the stationary position and were observed for C.P.E. The endpoint of the titration was taken as the highest dilution of antiserum inhibiting the development of C.P.E. at the time when cultures incubated with material from an M.S. patient alone showed advanced C.P.E. The presence of C.P.E. was assessed blindly in coded material. Some neutralisation tests were also carried out with canine-distemper-virus (c.D.v.) antiserum. An antiserum prepared by inoculating a rabbit with intravenous injections of M.S. material from donor 1 at passage 3 in PS cells at three sequential intervals of 7 days was used in the neutralisation tests. The rabbit was bled before inoculation and on the 7th, 14th, and 21st days after inoculation.

technique

2-C.P.E. on Vero cells 4 days after inoculation with passage (220 nm filtrate) from M.S. patient 1 showing cell formation. (x150.)

Fig.

a

13th

giant-

Results

cell-count of the suspensions of bone-marrow aspirates from M.S. was 4.2 x 106/ml. In the bonemarrow samples from 3 of the M.S. patients reticulum cells were increased in number and included primitive forms and occasional large atypical phagocytic cells: some were binucleate cells virtually indistinguishable from Sternberg-Reed cells (fig. 1). The reticulum cells resembled the cells found in granulomatous involvement of the bone-marrow (as occasionally seen in tuberculosis or sarcoidosis). In all 5 marrows, plasma cells were increased in number; in 4 of the marrows lymphocytes, monocytes, and eosinophils were also prominent. A C.P.E. was observed in each of the MRC-5 cultures inoculated with bone-marrow from M.S. patients after incubation at 37°C. Lysolecithin-treated material produced a C.P.E. after 4-5 days, whereas cultures inoculated with material not treated with lysolecithin showed a C.P.E. only after 7 days. No C.P.E. was observed in the uninoculated control cultures or in MRC-5 cultures inoculated with bone-marrow from control patient 1, whether treated with lysolecithin or not. Nor was a C.P.E. observed after 3 passages of these preparations. Harvests from MRC-5 cultures inoculated with bonemarrow from M.S. patients 1 and 2 were passed through a 220 nm filter, and the filtrates were sequentially passaged in serial dilutions (10-’ to 10-6) on MRC-5, HEp-2, and Vero cell monolayers. A,C.P.E. in which the cells initially had a rounded appearance and later The

mean

Fig. I-Bone-marrow smear showing binucleate reticulum cell resembling a Sternberg-Reed cell (A) and phagocytic reticuloendothelial cell (B). (May-Grunwald/Giemsa, x 500.)

became

syncytial

was

observed after 9-15

days

on

MRC-5, 3-4 days on HEp-2, and after 4 days on Vero cells, some of which cultures showed giant cells (fig. 2). The limit of dilution at which C.P.E. was observed was 10-4 for MRC-5 and HEp-2 cell cultures and 10-5 for Vero cell cultures. A C.P.E. was also obtained after co-cultivation of bone-marrow cells from M.S. patient 4 with HEp-2 and with Vero cell suspensions and when harvests from these cultures were serially passaged on Vero cell cultures. C.P.E. similar to that described above was observed in passage cultures. No C.P.E. was produced after co-cultivation of bone-marrow cells from control patients 2-7 with HEp-2 or Vero cells, or after three subsequent passages from each harvest on to Vero cells. Initial attempts to produce cytopathic changes in another laboratory (J.S.P.) with material from M.S. patient 1 which had been passaged nine times in MRC-5 cells (D.N.M.) were unsuccessful when MRC-5 cells were used. However, this material produced plaques in monolayer cultures of PS and Vero cells overlaid with carboxymethyl cellulose.4 In PS cells infected with serial dilutions of material from M.S. patient 1 (MRC-5 passage-9 material), microplaques were visible under low-power microscopy on day 5 in wells treated with undiluted and 1:10 inocula. When these cultures were stained on the 7th day after inoculation, semiconfluent plaques were present inwells inoculated with undiluted material, about 100 plaques developed per O’l1 ml of 10-1 dilutions, and a mean of 16 plaques developed for each 01ml of 10-2 dilution. The plaques were under 1 mm in diameter but were clearly defined and were totally absent from control wells. Pooled supernatant fluids from wells infected with undiluted and 10-1 dilution had an infectivity titre of about 10 plaque-forming units (p.f.u.) /ml when titrated in PS cells and stained on day 6 (fig. 3). When the same material was titrated in Vero cells, plaques were somewhat larger but less clearly defined; the sensitivity of the two cell lines was similar.’ Second-passage PS material was titrated before and after filtration: the unfiltered material and 650 nm filtrates both titrated at about 105 p.f.u./ml and the 450 nm filtrate at about 104 p.f.u./ml. No plaques developed in wells inoculated with 220 nm filtrate, but a bottle of PS cells inoculated with this filtrate developed a c.P.E.

389

,

cells stained on day 6 after inoculation with tenfold dilutions of material from M.S. patient 1 which had been passaged 9 times in MRC-5 cells and once in PS cells.

Fig. 3-Pig kidney (PS)

Rows A, B, C, and D are replicates; column 1, undiluted; 2-5, 10-1 to 10’’*; column 6, uninoculated controls.

on

day 7, and

successful passages

were

columns

made from this

material.

Initially, neutralisation tests were done on HEp-2 cell monolayers in tubes (D.N.M.). The C.P.E. induced by material from patients 1 and 2 which had been passaged 9 times in MRC-5 cells was not observed when the passage material was treated with 1:1, 1:2, and 1 :5dilutions of each patient’s serum. Inhibition of C.P.E. was also obtained with similar and higher dilutions (1:10) of sera from each of 5 additional :s. patients. The C.P.E. was not inhibited by sera in similar dilutions from each of 5 healthy subjects. When these neutralisation experiments were repeated with a 15th-passage material from M.S. patient 1 on Vero cells the C.P.E. was inhibited only by treatment of the material with a 1:1 and 1:2 dilution of the serum.

Fig. 4--Neutralisation of virus from M.S. patient 1 by antiserum prepared in rabbit. Rows A and B preinoculation serum; rows C and D, day 7; rows E and F, day 14; rows G and H, day-21 bleedings. Column 1, serum 1:10 without virus; columns 2::-9, serial twofold dilutions of sera 1:10-1:1280. One volume (50 1) of serum dilutions was mixed with one volume of virus dilution (10-3), and the mixtures were held at 4C for 2 h. One volume of Vero cells (3 x 105/ml) was then added and the plate was incubated for 6 days and then stained. Virus controls are in columns 10 and 11, row 1 10-2, row 3 10-1, row 2 10-3, row 4 = 10-4, row 5 10- 5 row 6 10-6. The remaimng wells are cell controls. =

=

=

=

=

The C.P.E. observed in Vero-cell cultures in tubes with material from M.S. patient 1, passage 13, was delayed in its formation and partially inhibited when the passage material was treated with 1:10, 1 :50, 1 :500, and 1:1000 dilutions of immune horse serum to canine distemper virus (C.D.V.). Similarly, partial inhibition of the C.P.E. in Vero cells was obtained with a 1:10 dilution of a human convalescent measles antiserum, but the C.P.E. was not affected by immune sheep serum, to human measles virus or by a bovine rinderpest antiserum. Undiluted serum from M.S. patients 1 and 2 inhibited the development of a c.P.E. in cultures of Vero cells inoculated with C.D.V. (10-5 p.f.u./ml). Attempts to corroborate these neutralisation-test results with plaque assays in PS and Vero cells were largely unsuccessful (J.S.P.). The occurrence of C.P.E. on HEp-2 or Vero cells was not inhibited by antisera to echovirus 1-10, Coxsackie B 1-6, poliovirus 1-3, herpes-simplex virus, or adenovirus types 1, 2, 5, 6, 3, 4, 7, 14. An antiserum prepared by inoculating a rabbit with material from M.S. donor 1 at passage 3 on PS cells gave complete plaque inhibition of the homologous agent at serum dilutions of 1:20 on day 7 and at 1:40 on days 14 and 21 (fig. 4). The 21-day rabbit antiserum also neutralised the C.P.E. from M.S. patient 2 (MRC-5, 7 passes; Vero 4). The pre-inoculation sample did not neutralise plaque formation. The rabbit antiserum failed to neutralise c.D.v., and C.D.V. antiserum did not neutralise plaque formation by material from M.S. patient 1, except partially at 1:4 dilution. Six sera from M.S. subjects failed to neutralise 10-100 p.f.u. of the agent from M.S. patient 1 when tested on both PS and Vero cell cultures. Additional experiments have shown that the C.P.E. produced in tube cultures by inoculating material from M.S. bone-marrow into MRC-5 cells is unaffected by subjecting material to acid treatment (pH 2.0). Conversely, the C.P.E. and plaques produced by inoculating 14th-passage material from M.S. patient 1 on to Vero cells were completely inhibited by treating this material with 20% ether. The filtrate produced by passing the 14th-passage material through a 100 nm filter showed no evidence of C.P.E. when added to cultures of Vero cells. Electron microscopy of HEp-2 cells from the two cultures inoculated with lOth-passage material revealed aggregations of hexagonal crystalline filaments in the cell cytoplasm (fig. 5). No other unusual structures were seen in these cells. The ultrastructural morphology of cells from three uninoculated control cultures was entirely normal, and no crystalline inclusions were seen. A fourth control culture, however, handled in the same laboratory as the M.S. material, did contain cells with cytoplasmic crystalline inclusions. This finding is at present unexplained, and further studies are in progress to determine if the crystalline inclusions are truly related to inoculation with M.S. material.

Discussion

,

3 of the 5 patients with M.S. had morphologically abnormal reticulum cells in bone-marrow smears. Schmidt6 also reported an increase in reticular elements in the bone-marrow of M.S. patients, but Plum and Fog’ were unable to confirm this observation. In the present study, the 3 patients with abnormal cells in bone-mar-

390 also had electron-dense bodies in the macrophages of the jejunal mucosa.8 The significance of this finding is unknown, and its relevance to reports of measles antigen in the jejunal mucosa is likewise uncertain.9 According to Fraser, 10 "Any virus found to be responsible for the lesions of M.S. is likely to be present throughout the course of the disease". Thus, it may be relevant that a possible viral agent has been isolated after the culture of bone-marrow aspirates from each of the 4 patients with M.S. thus far examined. In both laboratories early subcultures sometimes failed to show C.P.E. Likewise, there were inconsistencies in the nature of the C.P.E. encountered and in the time taken for the C.P.E. to appear. The reasons for these anomalies remain undefined, but delay in the addition of medium to the cells for some 4 h after inoculation, a lower concentration (1%) of fetal-calf serum in the cultures, and static rather than rotary incubation at 370C all seemed to aid more consistent passage. The C.P.E. obtained from early passages of bone-marrow aspirates was inhibited only by the patient’s own or other M.S. sera in dilutions not exceeding 1:10. The effect of these sera was less apparent when the neutralisation experiments were repeated later in tubes and row

Fig. 5-Aggregations of hexagonal crystalline filaments in cytoplasm patient 1. (Electron micrograph, reduced by a sixth from x 41 100.)

when

they showed

no

significant

neutralisation in

pla-

que assays.

The

finding of a viral agent in the haemopoietic tissues patients with M.S. is not necessarily related to the aetiology of the disease; herpes-simplex virus has been isolated from human trigeminal ganglia, including ganglia from 1 patient with M.S. 11I Cook and others12 have summarised epidemiological observations which are in keeping with a possible relationship between C.D.V. infection and M.S. Krakowa and Koestner13 found higher titres of M.v. neutralising antibody in M.S. patients than in controls, but they observed no significant differences in mean c.D.v. neutralisingantibody titres between these same M.S. patients and controls. In the present experiments there was only partial inhibition and retardation of the C.P.E. and virtually no reduction in plaque formation when the passage of

material from M.S. donor 1 was neutralised with C.D.V. antisera at dilutions up to 1:10. C.D.v. antisera in dilutions up to 1:1000 gave complete inhibition of the C.P.E. produced by c.D.v. in high titre. 14 None the less, the difficulties encountered in the cultures of early passages, the finding that the isolates pass with difficulty through a 220 nm filter, their ether lability, the suggestion from

of

HEp-2

cells inoculated with

10th-passage material from M.S.

391

of a lesser

possible antigenic relaextent to measles virus, tionship and the preliminary electron-microscopy findings suggest that the agent might be related in some way to the paramyxoviruses. The finding that material cultured neutralisation

experiments

to c.D.v.

and

to a

bone-marrow can be used to prepare a neutrain the rabbit may be useful in further antiserum lising to characterise the agent. attempts Bone-marrow from a further M.S. patient was cultured in Oxford without prior passage in London. Inconsistency in early subcultures was again encountered; cocultivation with Vero cells yielded a serially transmissible c.p.E. similar to that described above.

from

M.S.

Part of this work was supported by a grant from The Multiple Sclerosis Society (A.J.S.). Dr D. M. Burley, of Ciba Laboratories, Horsham, kindly arranged additional financial support. We thank Dr J. B. Rooksby for rinderpest antiserum; Dr W. C. Russell and Dr P. B. Stones for canine distemper antisera; and Prof. H. Harris, Dr W. C. Russell, Dr R. J. W. Rees, and Dr G. C. Schild for much valuable help and advice. We thank Miss Brenda Moore for secretarial assistance.

Requests for reprints should be addressed to D.N.M. REFERENCES 1. McDonald, W. I., Halliday, A. M. Br. med. Bull. 1977, 33, 4. 2. Clarke, M. C., Millson, G. C. Nature, 1976, 261, 144. 3. Jacobs, J. P. J. biol. Standardisation, 1976, 4, 97. 4. de Madrid, A. T., Porterfield, J. S. Bull. Wld Hlth Org. 1969, 40, 113. 5. Lennette, E. H. Schmidt, N. J. Diagnostic Procedures for Viral and Rickettsial Infections; p. 43. New York, 1969. 6. Schmidt, R.M. Medsche Mschr., Stuttg. 1955, 10, 661. 7. Plum, C. M., Fog, T. Acta psychiat. neurol. scand. 1959, suppl. 128, 34, 1. 8. Lange, L. S., Shiner, M. Lancet, 1976, ii, 1319. 9. Pertschuk, L. P., Cook, A. W., Gupta, J. K., et al. ibid. 1977, i, 1119. 10. Fraser, K. B. Br. med. Bull. 1977, 33, 34. 11. Warren, K. G., Devlin, M., Gilden, D. H., Wroblewska, Z., Brown, S. M., Subak-Sharpe, J., Koprowski, H. Lancet, 1977, ii, 637. 12. Cooke, S. D., Dowling, P. C., Russell, W. C. ibid. 1978, i, 605. 13. Krakowa, S., Koestner, A. ibid. p. 1127. 14. Russell, W. C. Personal communication.

PLASMA-TRIGLYCERIDES IN REGULATION OF H.D.L.-CHOLESTEROL LEVELS E. J. SCHAEFER D. W. ANDERSON H. B. BREWER, JR

R. I. LEVY R. N. DANNER W. C. BLACKWELDER

Molecular Disease Branch, National Heart, Lung, and Blood Institute, and Division of Computer Research and Technology, National Institutes of Health, Bethesda, MD, U.S.A.

Summary

Plasma-high-density-lipoprotein (H.D.L.)

cholesterol concentrations are lower in with patients coronary-artery disease than in control an In subjects. investigation of the relationship of H.D.L. cholesterol to other lipid and lipoprotein parameters in normal and hyperlipoproteinæmic subjects inverse correlations were found between H.D.L. cholesterol and verylow-density-lipoprotein (V.L.D.L.) cholesterol, and between H.D.L. cholesterol and plasma-triglyceride levels. Mean H.D.L.-cholesterol concentrations in normal subjects were 50 mg/dl, and in hyperlipoproteinæmic patients they were: type I, 17 mg/dl; type II, 44 mg/dl; type III, 38 mg/dl; type IV, 37 mg/dl; and type v, 27 mg/dl. H.D.L.-cholesterol levels were lowest in patients with fasting chylomicronæmia and were diminished in hyper-

triglyceridæmic subjects, suggesting a relationship between the metabolism of triglyceride-rich lipoproteins and H.D.L.

Introduction INTEREST in human high-density lipoproteins (H.D.L.) has grown largely because of the finding that plasmaH.D.L.-cholesterol levels are negatively associated with the incidence of coronary-artery disease in man. Epidemiological studies in Hawaii,! Framingham (U.S.A.),2 Norway,3 and Israel4 all support this concept, originally suggested by the studies of Barr et al. and Gofman et al. Furthermore, subjects with high plasma-H.D.L.-cholesterol concentrations appear to have increased longevity.7 It has been postulated that H.D.L. may be important in preventing cholesterol deposition or removing cholesterol from tissue, thereby reducing the amount of lipid deposition in the arterial wall.89 Whether H.D.L. is a primary factor in protection against coronary-artery disease or merely reflects an association with other factors remains to be determined. When H.D.L. is isolated in the density range (d) 1.063-1.21 g/ml from plasma by ultracentrifugation, its composition (weight %) is approximately 50% protein, 25% phospholipid, 20% cholesterol, and 5% triglyceride.1O Although H.D.L. has long been divided into two density classes, H.D.L.2 (d 1.063-1.125 g/ml) and H.D.L.3 (1.125-1.21 g/ml), recent investigations have characterised three or more density classes within H.D.L.ll 12 Population studies of healthy people suggest that variations in H.D.L. are largely due to changes in H.D.L./3 14 Thus the variable component ofnormolipaemic plasma-H.D.L.-cholesterol appears to be H.D.L.2 cholesterol. When measured directly by analytic ultracentrifugation, H.D.L.2 was negatively correlated with very-low-density lipoprotein (V.L.D.L.) in three normal populations. 6 14 11 Indirect corroboration of these findings comes from four epidemiological studies,1-3 16 in which H.D.L. cholesterol was negatively correlated with fasting plasma-triglyceride levels. Plasma-triglyceride concentrations are directly related to V.L.D.L. in the fasting state.15 Similar data have been summarised for single groups of healthy subjects in seven countries.8 In the present study we investigated the relationship between plasma cholesterol, triglyceride and lipoprotein-cholesterol levels in normal and hyperlipoproteinxmic sub-

jects.

Subjects

and Methods

Blood was obtained in 0-1% E.D.T.A. after an overnight fast (12-14 h), and the plasma was separated at 40C in a refrigerated centrifuge. All subjects were sampled while on an ad-libitum diet and were not receiving medication known to affect plasma-lipoproteins. Plasma cholesterol and triglyceride were measured with an AutoAnalyzer 11.17 H.D.L. cholesterol was measured after heparin-manganese precipitation of plasma or the 1.006 g/ml infranate.18 Plasma was ultracentrifuged at its own density (1.006 g/ml) for 18 h at 39 000 r.p.m. (4°C) in a Beckman 40.3 rotor (Beckman Instruments, Fullerton, CA), and the v.L.n.L. was separated from the other plasma-lipoproteins by tube slicing.19 The cholesterol concentration in the 1.006 g/ml infranate fraction was determined by these

methods, and

V.L.D.L.

Plasma and the 1.006

subjected ing.18

to

paper

and

L.D.L.

cholesterol

were

calculated. 18 also

g/ml supernate and infranate were

electrophoresis

for

lipoprotein phenotyp-

We sampled 1088 normal subjects and the following patients with hyperlipoproteinxmia: type i (1), type 11 (454), type in (66), type ly (229), and type v (95). Data were analysed at the National Institutes of Health, Division of Computer Research