- Email: [email protected]
ATTEMPT TO ISOLATE INFECTIOUS AGENT FROM BONE-MARROW OF PATIENTS WITH MULTIPLE SCLEROSIS
414 h both her serum-calcium and ECG had returned to normal. Tamoxifen without steroid cover was reintroduced after a further 8 days, the serum-calcium remaining normal throughout this period. A subsequent rise in the serum-calcium (3-03 mmol/1) within 72 h of reintroducing tamoxifen preceded a progressive onset of confusion and drowsiness which improved with discontinuation of tamoxifen and fluid replacement. A 41-year-old woman, who had had a therapeutic oophorectomy in 1976 after right radical mastectomy in 1973, was prescribed tamoxifen 10 mg for uncontrolled disseminated soft tissue and osseous metastatic disease. 16 days later she was admitted with a serum-calcium of 4.36 mmol/1 (pre-treatment 2-6mmol/1) which responded to rehydration, diuretics, and steroids, returning to normal within 72 h. Tamoxifen in the same dosage was reintroduced together with prednisolone 10 mg daily (which has since been stopped) without further hypercalcaemia. She remains well and pain-free on the tamoxifen alone 4 months later. Hormone-receptor assays were not done in either case. These two case-reports suggest that tamoxifen may, like oestrogen, occasionally precipitate serious and potentially lifethreatening hypercalcxmia in the early stages of treatment, especially in patients with osseous metastases. It has been suggested that such an early untoward response for oestrogen9 therapy may predict long-term objective tumour regression and the second case described here suggests that the same may sometimes be true with tamoxifen. We suggest that the serumcalcium should be closely monitored during the first 2 weeks of tamoxifen therapy in patients with known bony metastases. Tamoxifen-induced hypercalcaemia should be treated by stopping tamoxifen, rehydration, steroids and, if necessary, mithramycin. Once this has been controlled tamoxifen should be reintroduced under initial steroid cover, with careful monitoring of serum-calcium and subsequent responses may be seen. We thank Dr V. M. Dalley and Dr I. E. Smith for these natients under their care.
permitting us
to renort
D. SPOONER B. D. EVANS
Royal Marsden Hospital, London SW3 6JJ
ATTEMPT TO ISOLATE INFECTIOUS AGENT FROM BONE-MARROW OF PATIENTS WITH MULTIPLE
SCLEROSIS
SiR,-An infectious aetiology for multiple sclerosis (MS) has been postulated but none of the agents putatively associated with the disease have, as yet, been proven to be causative.1-4 The reportedisolation of an infectious agent from bone-marrow aspiration of patients with MS prompted us to attempt recovery of an agent from our MS patients. We studied samples of bone-marrow from 2 male and 5 female MS patients all meeting the accepted criteria for the diagnosis, and five volunteer controls (2 men, 3 women, 2 normal, 2 with seizure disorder, and 1 with an emotional problem) kindly obtained with permission of Dr K. Engel and Dr R. Porter of the N.I.H. Bone-marrow was aspirated into 10 ml Eagle’s minimal essential medium (EMEM) containing preservative-free heparin. After washing, the samples were divided into four parts; A for tube cultures, B for coverslip cultures in multidish wells; C was placed on a 9. Hall CT, Dederick MM, Nevinney HB. Cancer Chemother Rep 1963; 30: 21. 10. Teatle R, Mendelssohn J, Green MR. Ann Intern Med 1978; 88: 287. 1. Brody J, Sever JL, Edgar A, McNew J. Measles antibody titers of multiple sclerosis patients and their siblings. Neurology 1972; 22: 492, 495-99. 2. Koldovsky U, Koldovsky P, Henle G, et al. Multiple sclerosis associated agent: transmission to animals and some properties of the agent. Infect Immun 1975; 12: 1355-66. 3. Carp RI, Licursi PC, Merz PA, Merz GS. Decreased percentage of polymorphonuclear neutrophils in mouse peripheral blood after inoculation material from multiple sclerosis patients.J Exp Med 1972; 136: 618-29. 4. Pertschuck LP, Cook AW, Gupta J. Measles antigen in multiple sclerosis: identification in the jejunum by immunofluorescence. Life Sci 1976; 19: 1603-08.
with
CLINICAL STATUS OF MS PATIENTS USED FOR ISOLATION ATTEMPTS
* RR=recurrent and remitting, C=chronic; P=probably MS, D=Definite MS t N=normal, +=elevated IgG and oligoclonal elevated IgG and oligoclonal bands.
bands, +=borderline
’Ficoll’-’Hypaque’ gradient to obtain free mononuclear cells, and D frozen in liquid nitrogen in cryopreservative medium containing 7% dimethyl sulphoxide. After separation of the mononuclear cells, half of sample C was incubated with 1 Rg of phytoheemagglutinin for 72 h to obtain mitogen-activated lymphocytes (C’). 0.2 ml samples from A were placed in triplicate in tube cultures. Six replicate multidish plate wells containing cells grown on coverslips were seeded with 0-2ml aliquots of test cells from groups B, C, and C’. The cell lines employed to rescue the agent included Flow 7000 (a diploid human fibroblast cell line), HEP-2 (transformed human epithelial cells), Vero cells (transformed African green monkey kidney cells), PRK (primary rabbit kidney cells), and three separate lines of Hela cells (a transformed human cervical carcinoma cell line). These were grown initially in EMEM supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine (200 mmol/1). Bone-marrow samples were incubated on the monolayer cells for 2 h. The cultures were then fed with a maintenance medium (EMEM with 2% FBS) and were observed intermittently for two weeks. Fluid containing cells was blindly passaged twice to new tube or coverslip cultures containing the corresponding monolayer cells. Each culture was maintained for six to eight weeks before termination. Any cultures showing cytotoxic or cytopathic effects were scraped and transferred to large flasks (150 cm2) containing freshly planted corresponding monolayer cells. These flask cultures were maintained for two additional weeks and then blindly passaged twice. For ultrastructural studies, the cells grown in large flasks were ’scraped off, centrifuged, and fixed in 4% cacodylate-buffered glutaraidehyde. After 120 min, the cells were postfixed in 1% osmium tetroxide, dehydrated with ethanol and embedded. Thin sections were then prepared for electronmicroscopic examination. was
No infectious agent
was
recovered from the bone-marrow
samples from either the MS patients or the controls. Samples inoculated directly into tube and coverslip cultures of Vero, Flow 7000, PRK, and HEP-2 showed no cytopathic effect. However, Hela cells inoculated with samples from six of seven MS patients and three of the five controls rapidly formed syncytia within 24 to 48 h which frequently contained up to 25 nuclei. This happened to two of the three Hela cell lines. Careful re-examination of the three Hela cell lines revealed an occasional syncytium with 3-8 nuclei in the control cultures of two of these cell lines. When fluid and cells from the cultures (showing syncytial formation) with the MS patients and controls were passaged to the Hela cell line not forming spontaneous syncytia, occasional syncytia formed for a short period of time. In each case, the capacity to form syncytia could be passaged. Ultrastructural examination of cultures with syncytium revealed small, spiked, viral-like particles in the Hela cells resembling a foamy virus. No attempt was made to identify this agent further because extensive EM examination of original uninfected cultures revealed a similar type of agent. The passage of fluid from uninoculated control cells spontaneously showing syncytial formation did not enhance this effect in fresh cultures in contrast to the passage from bone-marrow cultures. Direct inoculation of bone-marrow cells from MS or controls on to the third Hela cell line did not result in syncytial formation. Therefore, we believe that a cell-associated syncytial forming agent pre-existed in two of the Hela cell lines and was activated when these cells were cultured with bone-marrow cells from some of the patients or controls.
415
separated from bone-marrow samples by ficoll-hypaque density gradient centrifugation with or without phytohaemagglutinin treatment were seeded at 1 x 106 cells/0-22 ml on to wells of the indicator cell lines in triplicate. Syncytial Mononuclear cells
formation with Hela cells similar to that observed after direct inoculation of bone-marrow was seen but was less frequent. In addition, samples from two of the six MS patients cultured in Vero cells resulted in the formation of small syncytia containing 8-12 nuclei. However, in neither case could transfer of fluid or cells from the affected cultures reproduce this activity nor was any activity noted from any of the controls. In the previous investigation reporting the isolation of an infectious agent from MS bone-marrow, the agent(s) could be readily isolated from MS patients (5 of 5) but not from controls (0 of 7).5 Successful isolation was obtained employing MRC-5 (human diploid cells), HEP-2, and Vero cells. Lysolecithin was found to enhance the recovery rate of the agent(s), although it was apparently not necessary for primary isolation. We were unable to isolate an agent from seven MS patients with similar cell lines and procedures. There is no apparent reason for our inability to do so. However, we did find an adventitious agent(s) in two commercial sources of Hela cells. Although not initially apparent, this agent(s) became active upon incubation with either MS-patient or control bone-marrow cells and induced large syncytia when passed to other Hela cells.
National Institutes of Health, National Institute of Neurological and Communicative Disorders and Stroke,
Building 36, Room 5D06, Bethesda, Maryland 20205, U.S.A.
WILLIAM C. WALLEN JOHN L. SEVER DALE E. MCFARLIN HENRY F. MCFARLAND RENÉE G. TRAUB BERNARD RENTIER JEFFREY I. GREENSTEIN PATRICIA M. MOORE
FAILURE TO ISOLATE A TRANSMISSIBLE AGENT FROM THE BONE-MARROW OF PATIENTS WITH MULTIPLE SCLEROSIS
SIR,-It has been postulated that multiple sclerosis (MS) is causally related to either a virus infection or to an aberrant imvirus infection.1,2 Hence, the recent report by Mitchell et awl. of the isolation of a transmissible agent from the bone-marrow of multiple sclerosis patients is of considerable importance. In an attempt to confirm this report we studied bone-marrow samples aspirated from 7 patients with MS and 1 normal subject. All 7 patients fulfilled the criteria of clinically definite MS4 and all were experiencing an acute attack of MS when the sample was taken. 6 of the 7 patients and the normal subject were female, aged 23-44. mune
response which may be associated with
a
carcinoma cell line) VERO (a transformed African Green monkey kidney cell line), WI-38 (a human diploid fibroblast line), and CV-1 (a fibroblast line derived from African green monkey kidney). All of the cultures were refed (MEM used for all manipulations) after a week. After 2 weeks the supernatants from each culture were passed to a fresh monolayer and the original monolayer was subcultured. All of the monolayers were carried in this manner for a minimum of 10 weeks. Monolayers demonstrating a cytopathic effect (CPE) were subcultured onto fresh monolayers of the corresponding cell line. Also, supernatants from these monolayers were passed through Millipore filters and then inoculated on to fresh monolayers. Syncytia observed in association with CPE were tested for hmmadsorption with washed rhesus-monkey erythrocytes and were fixed in situ with 2.5% glutaraldehyde for electronmicroscopic examination.
We were unable to detect a transmissible agent in samples from 7 patients and 1 normal subject. VERO, Wl-38, and CV-1 lines co-cultured with bone-marrow samples grew normally and failed to demonstrate CPE. HeLa cultures inoculated with samples from 6 of the 7 patients and the normal subject developed significant CPE accompanied by syncytial formation after 7 to 10 days of incubation but syncytia did not demonstrate haemadsorption, and electronmicroscopic examination of the syncytia fixed in situ failed to reveal virus-like structures. Filtration of the culture supernatants and low serial dilutions of the positive cultures resulted in eventual loss of the CPE and syncytium-forming capacity. In most, fluids diluted more than 1/2 and filtered failed to elicit syncytium formation on HeLa cells. Fluids which contained lymphoblasts initiated syncytial formation. Thus, the development of the CPE was associated with the appearance of lymphoblasts in the cultures while their removal was associated with the loss of CPE. Mitchell et al. reported the isolation of an infectious agent from bone-marrow of 5 consecutive MS patients, but not from bone-marrow of normal subjects. The cell lines used in their isolation were MRC-5 (human diploid cells), HEP-2, and VERO lines. Although lysolecithin was used with some of their material they reported that it was not necessary for isolation. Using material from patients with a similar clinical state, and employing similar cell lines and procedures, we were unable to isolate a replicating agent in 7 MS patients experiencing acute relapse. The syncytium formation we observed was closely related to activated mononuclear cells. This is publication no. 1869 from Scripps Clinic and Research Foundation, La Jolla, CA 92037.
Departments of Immunopathology and Cellular and Developmental Immunology, Scripps Clinic and Research Foundation, La Jolla, California and Department of Pathology, University of California at San Diego
JOHN R. HUDDLESTONE JACK SIPE SHERRY BRAHENY
FRED C. JENSEN ROBERT McMILLAN PETER LAMPERT MICHAEL B. A. OLDSTONE
’
Bone-marrow aspirtates (for 3 of the 7 patients, mononuclear cells obtained from the bone-marrow aspirate by centrifugation on a ’Ficoll-Hypaque’ gradient) were placed in 15 ml of Eagle’s minimal essential medium containing 10% heat-inactivated fetal calf serum, 1% penicillin, streptomycin, and glutamine (MEM) plus heparin. 1 ml samples of the aspirate were fixed in 2.5% glutaraldehyde for electronmicroscopic examination. 1 ml of each sample was incubated for an hour on monolayers of each of four cell lines: HeLa (a human cervical were
5. Mitchell DN, Porterfield JS, Micheletti R, et al. Isolation of an infectious agent from bone-marrows of patients with multiple sclerosis. Lancet 1978;
ii: 387-90. 1. Report and Recommendations: National Advisory Commission on Multiple Sclerosis. Washington, D.C.: U.S. Government Printing Office, 1974. DHEW Pub. No. 74-534. 2. Maughn T. Multiple sclerosis: genetic link, viruses suspected. Science 1977;
195: 667-89, 768-71, 969-71. 3. Mitchell DN, Porterfield JS, Micheletti R, et al. Isolation of an infectious agent from bone-marrows of patients with multiple sclerosis. Lancet 1978;
ii: 387-90. 4. McDonald WI, Halliday AM. Diagnosis and classification of osis.Br Med Bull 1977; 33: no. 1:4-8.
multiple
scler-
SIR,-We have read with interest the articles by Dr Tyrrell and his colleagues’.2 and welcome this opportunity to comment on their findings in relation to our published’ and subsequent investigations on the isolation of an infectious agent from bone-marrows of patients with multiple sclerosis (MS). Tyrrell et al. found that cerebrospinal fluid from some schizophrenics and from two of four MS patients had a cytopathic effect (CPE) on cultures of MRC5 cells and concluded that the causative agent was not a mycoplasma; they isolated Mycoplasma orale from two MRC5 cultures which showed extensive CPEs following incubation with cerebrospinal fluids but they Tyrrell DAJ, Perry RP, Crow TJ, et al. Possible virus in schizophrenia and some neurological disorders. Lancet 1979; i: 839-41. 2. Crow TJ, Ferrier IN, Johnstone EC, et al. Characteristics of patients with schizophrenia or neurological disorder and virus-like agent in cerebrospinal fluid. Lancet1979;i: 842-44. 3. Mitchell DN, Porterfield JS, Micheletti R, et al. Isolation of an infectious agent from bone-marrows of patients with multiple sclerosis. Lancet 1978; 1.
ii: 387-90.
permitting us
to renort
D. SPOONER B. D. EVANS
Royal Marsden Hospital, London SW3 6JJ
ATTEMPT TO ISOLATE INFECTIOUS AGENT FROM BONE-MARROW OF PATIENTS WITH MULTIPLE
SCLEROSIS
SiR,-An infectious aetiology for multiple sclerosis (MS) has been postulated but none of the agents putatively associated with the disease have, as yet, been proven to be causative.1-4 The reportedisolation of an infectious agent from bone-marrow aspiration of patients with MS prompted us to attempt recovery of an agent from our MS patients. We studied samples of bone-marrow from 2 male and 5 female MS patients all meeting the accepted criteria for the diagnosis, and five volunteer controls (2 men, 3 women, 2 normal, 2 with seizure disorder, and 1 with an emotional problem) kindly obtained with permission of Dr K. Engel and Dr R. Porter of the N.I.H. Bone-marrow was aspirated into 10 ml Eagle’s minimal essential medium (EMEM) containing preservative-free heparin. After washing, the samples were divided into four parts; A for tube cultures, B for coverslip cultures in multidish wells; C was placed on a 9. Hall CT, Dederick MM, Nevinney HB. Cancer Chemother Rep 1963; 30: 21. 10. Teatle R, Mendelssohn J, Green MR. Ann Intern Med 1978; 88: 287. 1. Brody J, Sever JL, Edgar A, McNew J. Measles antibody titers of multiple sclerosis patients and their siblings. Neurology 1972; 22: 492, 495-99. 2. Koldovsky U, Koldovsky P, Henle G, et al. Multiple sclerosis associated agent: transmission to animals and some properties of the agent. Infect Immun 1975; 12: 1355-66. 3. Carp RI, Licursi PC, Merz PA, Merz GS. Decreased percentage of polymorphonuclear neutrophils in mouse peripheral blood after inoculation material from multiple sclerosis patients.J Exp Med 1972; 136: 618-29. 4. Pertschuck LP, Cook AW, Gupta J. Measles antigen in multiple sclerosis: identification in the jejunum by immunofluorescence. Life Sci 1976; 19: 1603-08.
with
CLINICAL STATUS OF MS PATIENTS USED FOR ISOLATION ATTEMPTS
* RR=recurrent and remitting, C=chronic; P=probably MS, D=Definite MS t N=normal, +=elevated IgG and oligoclonal elevated IgG and oligoclonal bands.
bands, +=borderline
’Ficoll’-’Hypaque’ gradient to obtain free mononuclear cells, and D frozen in liquid nitrogen in cryopreservative medium containing 7% dimethyl sulphoxide. After separation of the mononuclear cells, half of sample C was incubated with 1 Rg of phytoheemagglutinin for 72 h to obtain mitogen-activated lymphocytes (C’). 0.2 ml samples from A were placed in triplicate in tube cultures. Six replicate multidish plate wells containing cells grown on coverslips were seeded with 0-2ml aliquots of test cells from groups B, C, and C’. The cell lines employed to rescue the agent included Flow 7000 (a diploid human fibroblast cell line), HEP-2 (transformed human epithelial cells), Vero cells (transformed African green monkey kidney cells), PRK (primary rabbit kidney cells), and three separate lines of Hela cells (a transformed human cervical carcinoma cell line). These were grown initially in EMEM supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine (200 mmol/1). Bone-marrow samples were incubated on the monolayer cells for 2 h. The cultures were then fed with a maintenance medium (EMEM with 2% FBS) and were observed intermittently for two weeks. Fluid containing cells was blindly passaged twice to new tube or coverslip cultures containing the corresponding monolayer cells. Each culture was maintained for six to eight weeks before termination. Any cultures showing cytotoxic or cytopathic effects were scraped and transferred to large flasks (150 cm2) containing freshly planted corresponding monolayer cells. These flask cultures were maintained for two additional weeks and then blindly passaged twice. For ultrastructural studies, the cells grown in large flasks were ’scraped off, centrifuged, and fixed in 4% cacodylate-buffered glutaraidehyde. After 120 min, the cells were postfixed in 1% osmium tetroxide, dehydrated with ethanol and embedded. Thin sections were then prepared for electronmicroscopic examination. was
No infectious agent
was
recovered from the bone-marrow
samples from either the MS patients or the controls. Samples inoculated directly into tube and coverslip cultures of Vero, Flow 7000, PRK, and HEP-2 showed no cytopathic effect. However, Hela cells inoculated with samples from six of seven MS patients and three of the five controls rapidly formed syncytia within 24 to 48 h which frequently contained up to 25 nuclei. This happened to two of the three Hela cell lines. Careful re-examination of the three Hela cell lines revealed an occasional syncytium with 3-8 nuclei in the control cultures of two of these cell lines. When fluid and cells from the cultures (showing syncytial formation) with the MS patients and controls were passaged to the Hela cell line not forming spontaneous syncytia, occasional syncytia formed for a short period of time. In each case, the capacity to form syncytia could be passaged. Ultrastructural examination of cultures with syncytium revealed small, spiked, viral-like particles in the Hela cells resembling a foamy virus. No attempt was made to identify this agent further because extensive EM examination of original uninfected cultures revealed a similar type of agent. The passage of fluid from uninoculated control cells spontaneously showing syncytial formation did not enhance this effect in fresh cultures in contrast to the passage from bone-marrow cultures. Direct inoculation of bone-marrow cells from MS or controls on to the third Hela cell line did not result in syncytial formation. Therefore, we believe that a cell-associated syncytial forming agent pre-existed in two of the Hela cell lines and was activated when these cells were cultured with bone-marrow cells from some of the patients or controls.
415
separated from bone-marrow samples by ficoll-hypaque density gradient centrifugation with or without phytohaemagglutinin treatment were seeded at 1 x 106 cells/0-22 ml on to wells of the indicator cell lines in triplicate. Syncytial Mononuclear cells
formation with Hela cells similar to that observed after direct inoculation of bone-marrow was seen but was less frequent. In addition, samples from two of the six MS patients cultured in Vero cells resulted in the formation of small syncytia containing 8-12 nuclei. However, in neither case could transfer of fluid or cells from the affected cultures reproduce this activity nor was any activity noted from any of the controls. In the previous investigation reporting the isolation of an infectious agent from MS bone-marrow, the agent(s) could be readily isolated from MS patients (5 of 5) but not from controls (0 of 7).5 Successful isolation was obtained employing MRC-5 (human diploid cells), HEP-2, and Vero cells. Lysolecithin was found to enhance the recovery rate of the agent(s), although it was apparently not necessary for primary isolation. We were unable to isolate an agent from seven MS patients with similar cell lines and procedures. There is no apparent reason for our inability to do so. However, we did find an adventitious agent(s) in two commercial sources of Hela cells. Although not initially apparent, this agent(s) became active upon incubation with either MS-patient or control bone-marrow cells and induced large syncytia when passed to other Hela cells.
National Institutes of Health, National Institute of Neurological and Communicative Disorders and Stroke,
Building 36, Room 5D06, Bethesda, Maryland 20205, U.S.A.
WILLIAM C. WALLEN JOHN L. SEVER DALE E. MCFARLIN HENRY F. MCFARLAND RENÉE G. TRAUB BERNARD RENTIER JEFFREY I. GREENSTEIN PATRICIA M. MOORE
FAILURE TO ISOLATE A TRANSMISSIBLE AGENT FROM THE BONE-MARROW OF PATIENTS WITH MULTIPLE SCLEROSIS
SIR,-It has been postulated that multiple sclerosis (MS) is causally related to either a virus infection or to an aberrant imvirus infection.1,2 Hence, the recent report by Mitchell et awl. of the isolation of a transmissible agent from the bone-marrow of multiple sclerosis patients is of considerable importance. In an attempt to confirm this report we studied bone-marrow samples aspirated from 7 patients with MS and 1 normal subject. All 7 patients fulfilled the criteria of clinically definite MS4 and all were experiencing an acute attack of MS when the sample was taken. 6 of the 7 patients and the normal subject were female, aged 23-44. mune
response which may be associated with
a
carcinoma cell line) VERO (a transformed African Green monkey kidney cell line), WI-38 (a human diploid fibroblast line), and CV-1 (a fibroblast line derived from African green monkey kidney). All of the cultures were refed (MEM used for all manipulations) after a week. After 2 weeks the supernatants from each culture were passed to a fresh monolayer and the original monolayer was subcultured. All of the monolayers were carried in this manner for a minimum of 10 weeks. Monolayers demonstrating a cytopathic effect (CPE) were subcultured onto fresh monolayers of the corresponding cell line. Also, supernatants from these monolayers were passed through Millipore filters and then inoculated on to fresh monolayers. Syncytia observed in association with CPE were tested for hmmadsorption with washed rhesus-monkey erythrocytes and were fixed in situ with 2.5% glutaraldehyde for electronmicroscopic examination.
We were unable to detect a transmissible agent in samples from 7 patients and 1 normal subject. VERO, Wl-38, and CV-1 lines co-cultured with bone-marrow samples grew normally and failed to demonstrate CPE. HeLa cultures inoculated with samples from 6 of the 7 patients and the normal subject developed significant CPE accompanied by syncytial formation after 7 to 10 days of incubation but syncytia did not demonstrate haemadsorption, and electronmicroscopic examination of the syncytia fixed in situ failed to reveal virus-like structures. Filtration of the culture supernatants and low serial dilutions of the positive cultures resulted in eventual loss of the CPE and syncytium-forming capacity. In most, fluids diluted more than 1/2 and filtered failed to elicit syncytium formation on HeLa cells. Fluids which contained lymphoblasts initiated syncytial formation. Thus, the development of the CPE was associated with the appearance of lymphoblasts in the cultures while their removal was associated with the loss of CPE. Mitchell et al. reported the isolation of an infectious agent from bone-marrow of 5 consecutive MS patients, but not from bone-marrow of normal subjects. The cell lines used in their isolation were MRC-5 (human diploid cells), HEP-2, and VERO lines. Although lysolecithin was used with some of their material they reported that it was not necessary for isolation. Using material from patients with a similar clinical state, and employing similar cell lines and procedures, we were unable to isolate a replicating agent in 7 MS patients experiencing acute relapse. The syncytium formation we observed was closely related to activated mononuclear cells. This is publication no. 1869 from Scripps Clinic and Research Foundation, La Jolla, CA 92037.
Departments of Immunopathology and Cellular and Developmental Immunology, Scripps Clinic and Research Foundation, La Jolla, California and Department of Pathology, University of California at San Diego
JOHN R. HUDDLESTONE JACK SIPE SHERRY BRAHENY
FRED C. JENSEN ROBERT McMILLAN PETER LAMPERT MICHAEL B. A. OLDSTONE
’
Bone-marrow aspirtates (for 3 of the 7 patients, mononuclear cells obtained from the bone-marrow aspirate by centrifugation on a ’Ficoll-Hypaque’ gradient) were placed in 15 ml of Eagle’s minimal essential medium containing 10% heat-inactivated fetal calf serum, 1% penicillin, streptomycin, and glutamine (MEM) plus heparin. 1 ml samples of the aspirate were fixed in 2.5% glutaraldehyde for electronmicroscopic examination. 1 ml of each sample was incubated for an hour on monolayers of each of four cell lines: HeLa (a human cervical were
5. Mitchell DN, Porterfield JS, Micheletti R, et al. Isolation of an infectious agent from bone-marrows of patients with multiple sclerosis. Lancet 1978;
ii: 387-90. 1. Report and Recommendations: National Advisory Commission on Multiple Sclerosis. Washington, D.C.: U.S. Government Printing Office, 1974. DHEW Pub. No. 74-534. 2. Maughn T. Multiple sclerosis: genetic link, viruses suspected. Science 1977;
195: 667-89, 768-71, 969-71. 3. Mitchell DN, Porterfield JS, Micheletti R, et al. Isolation of an infectious agent from bone-marrows of patients with multiple sclerosis. Lancet 1978;
ii: 387-90. 4. McDonald WI, Halliday AM. Diagnosis and classification of osis.Br Med Bull 1977; 33: no. 1:4-8.
multiple
scler-
SIR,-We have read with interest the articles by Dr Tyrrell and his colleagues’.2 and welcome this opportunity to comment on their findings in relation to our published’ and subsequent investigations on the isolation of an infectious agent from bone-marrows of patients with multiple sclerosis (MS). Tyrrell et al. found that cerebrospinal fluid from some schizophrenics and from two of four MS patients had a cytopathic effect (CPE) on cultures of MRC5 cells and concluded that the causative agent was not a mycoplasma; they isolated Mycoplasma orale from two MRC5 cultures which showed extensive CPEs following incubation with cerebrospinal fluids but they Tyrrell DAJ, Perry RP, Crow TJ, et al. Possible virus in schizophrenia and some neurological disorders. Lancet 1979; i: 839-41. 2. Crow TJ, Ferrier IN, Johnstone EC, et al. Characteristics of patients with schizophrenia or neurological disorder and virus-like agent in cerebrospinal fluid. Lancet1979;i: 842-44. 3. Mitchell DN, Porterfield JS, Micheletti R, et al. Isolation of an infectious agent from bone-marrows of patients with multiple sclerosis. Lancet 1978; 1.
ii: 387-90.