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immunosuppression and that we do not know whether ICAare useful for monitoring the efficacy of immunosuppressionor whether the appearance of ICA predicts clinical relapse.

We thank Mrs Anne Rafn and Mrs Helle Espersen for preparation of themanuscript. This study was supported by grants from the Medical ResearchCouncil of Canada, Sandoz Corporation (Basle), and the Transplant Fund,Centre for Transplant Studies, University Hospital, London, Ontario,Canada. T. M. P. is the recipient of a fellowship from the MichaelsenFoundation, Denmark.

Correspondence should be addressed to J. N., Steno Memorial Hospital,Niels Steensensvej 2, DK 2820 Gentofte, Denmark, or C. R. S., UniversityHospital, London, Ontario, Canada. a

REFERENCES

1. Nerup J, Lernmark A, Scott J. Autoimmunity. In: Gupta S, ed. Immunology of clinicaland experimental diabetes. New York and London: Plenum, 1984: 351-67.

2. MacCuish AC, Irvine WJ. Autoimmunological aspects of diabetes mellitus Clin

Endocrinol Metab 1975; 4: 435-71.3. Nerup J, Platz P, Andersen OO, et al. HLA antigens and diabetes mellitus. Lancet

1974; ii: 864-664. Leslie RDG, Pyke DA. Immunosuppression of acute insulin-dependent diabetics. In:

Irvine WJ, ed. Immunology of diabetes. Edinburgh: Teviot Scientific PublicationsLtd, 1980: 345-47.

5. Elliott RB, Crossby JR, Berryman CC, James AG. Partial preservation of pancreatic&bgr;-cell function in children with diabetes Lancet 1981; ii: 1-4.

6. Jackson RA, Morris MA, Haynes BF, Eisenbarth GS. Increased circulating Ia-antigen-bearing T-cells in type I diabetes mellitus. N Engl J Med 1982; 306: 785-88.

7. Cobb WE, Motlich M, Reichlin S. Levamisole in insulin-dependent diabetes mellitus.N Engl J Med 1980, 303: 1065-66.

8. Ludvigsson J, Heding L, Liedén G, Marner B, Lernmark Å. Plasmapheresis in theinitial treatment of insulin-dependent diabetes mellitus in children. Br Med J 1983;286: 176-78.

9. Bendtzen K, Petersen J. Effect of cyclosporin A (CyA) on the immune response. CyAcompetitively inhibits the function of monocyte/macrophage-derivedT-lymphocyte-activating factors. Immunol Lett 1982; 5: 331-36

10. Stiller CR, Keown P. Cyclosporine therapy in perspectives. In: McLeod C, Hunter S,eds Progress in organ transplantation. Harlow: Tilney and Morris (Longman),1983: 11-45

11. Krönke M, Leonard WJ, Depper JM, et al. Cyclosporin A inhibits T-cell growth factorgene expression at the level of mRNA transcription. Proc Natl Acad Sci USA 1984;81: 5214-18.

12. Laupacis A, Stiller CR, Gardell C, et al. Cyclosporin prevents diabetes in BB Wistarrats. Lancet 1983; i: 10-12.

13. Like AA, Anthony M, Gubershi DL, Rossini AA. Spontaneous diabetes mellitus in theBB/W rat. Effects of glucocorticoids, cyclosporin A, and antiserum to rat

lymphocytes. Diabetes 1984; 32: 326-30.14. Stiller CR, Dupré J, Gent M, et al. Effects of cyclosporine immunosuppression in

insulin-dependent diabetes mellitus of recent onset. Science 1984: 223: 1362-6715. Madsbad S, Krarup T, McNair P, et al. Practical clinical value of the C-peptide

response to glucagon stimulation in the choice of treatment in diabetes mellitus.Acta Med Scand 1981; 210: 153-56.

16. Madsbad S, Bottazzo GF, Cudworth AG, Dean B, Faber OK, Binder C. Islet cellantibodies and beta-cell function in insulin-dependent diabetics. Diabetologia 1980;18: 45-47.

17. Crossby JR, James AG, Elliott RB, Berryman CC, Edgar BW. Residual B-cell functionand islet cell antibodies in diabetic children Pediatr Res 1981; 15: 62-65.

18. Rubenstein AG, Goren B, Irvine WJ, et al. Beta cell function in diabetes mellitus In:

Irvine WJ, ed. Immunology of diabetes. Edinburgh: Teviot Scientific PublicationsLtd, 1980: 267-73

19. Theophanides CG, Pyke DA, Watkins TJ Islet cell function in diabetes with

persistent islet cell antibodies. Diabetes 1978; 27 (suppl 1): 265-6620 Marner B, Agner T, Binder C, Lernmark A, Nerup J. Prospective analysis of islet cell

antibody and C-peptide levels during the first 24 months of insulin-dependentdiabetes. Diabetologia 1983; 25: 179 A.

21. Mustonen Å, Kemp M, Ákerbolm HK. An association between complement fixingcytoplasmic islet cell antibodies and endogenous insulin secretion in children withinsulin-dependent diabetes mellitus. Diabetes 1983; 32: 743-47.

22. Irvine WJ, Gray RS, McCallum CJ Pancreatic islet-cell antibody as a marker forasymptomatic and latent diabetes and prediabetes. Lancet 1976; ii: 1097-102.

23. Bottazzo GF, Dean BM, Gorsuch AN, Cudworth AG, Doniach D. Complement fixingislet cell antibodies in type 1 diabetes; possible monitors of active beta-cell damage.Lancet 1980, i: 668-72.

24. Gorsuch AN, Spencer KM, Lister J et al. Evidence for a long prediabetic period in type1 (insulin-dependent) diabetes mellitus. Lancet 1981; ii: 1363-65

25. Srikanta S, Ganda OP, Eisenbarth GS, Soeldner JS. Islet-cell antibodies and beta-cellfunction in monozygotic triplets and twins initially discordant for type 1 diabetesmellitus. N Engl J Med 1983; 308: 322-25.

26. Marner B, Agner T, Binder C, et al. Islet cell antibodies predict loss of fastingC-peptide Diabetologia 1987; 27: 307A.

27. WHO Expert Committee on Diabetes Mellitus. Second Report. World HealthOrganization Technical Report Series 646, p 8-14. Geneva World Health

Organisation 1980.28. Marner B, Lernmark A, Nerup J, Molenaar JL, Tuk CW, Bruining GJ. Analysis of islet

cell antibodies on frozen sections of human pancreas. Diabetologia 1983; 25: 93-96.29. Faber OK, Binder C C-peptide response to glucagon. Test for the residual &bgr;-cell

function in diabetes mellitus Diabetes 1977, 26: 605-10.

30. Papadopoulos G, Petersen J, Andersen V, et al. Spontaneous in vitro immunoglobulinsecretion at the diagnosis of insulin-dependent diabetes. Acta Endocrinol 1984, 105:521-27.

31. Sharon E, Kaplan D, Diamond HS. Exacerbation of systemic lupus erythematosusafter withdrawal of azathioprine therapy. N Engl J Med 1973; 288: 122-24

32. Spencer KM, Tarn A, Dean BM, Lister J, Bottazzo GF. Fluctuating islet-cell

autoimmunity in unaffected relatives of patients with insulin-dependent diabetesLancet 1984, i: 764-66.

33. Srikanta S, Eisenbarth GS. Disappearing anti-islet antibodies? Lancet 1984, i:1176-77.

34. Riley W, MacLaren N Islet cell antibodies are seldom transient. Lancet 1984. i

1351-52.35. Keown P, Stiller CR, Ulan RA, et al Immunological and pharmacological monitoring

in the clinical use of cyclosporin A Lancet 1981; i: 686-89.36 Keown P, Stiller CR, Sinclair NK, et al. The clinical relevance of cyclosporine blood

levels as measured by radioimmunoassay. Transpl Proc 1983; 15: 2438-41.

RECOMBINANT HUMAN INTERFERON ALFA-ASUPPRESSES HTLV-III REPLICATION IN VITRO

DAVID D. HOTERESA R. ROTA

JOAN C. KAPLAN

KEVAN L. HARTSHORNCHARLA A. ANDREWSROBERT T. SCHOOLEY

MARTIN S. HIRSCH

Infectious Disease Unit, Massachusetts General Hospital, HarvardMedical School, Boston, Massachusetts, USA

Summary Recombinant human interferon alfa-A

(rIFN&agr;A) had a dose-related suppressiveeffect on human T lymphotropic virus type III (HTLV-III)replication in vitro in normal peripheral-blood mononuclearcells (PBMC). Both single-dose and multiple-dose regimenswere inhibitory. Such inhibitory concentrations (4-1024units/ml) were not toxic to PBMC in culture, and were withinthe ranges achievable in blood after injection. These studiessuggest that clinical trials of rIFN&agr;A in early HTLV-IIIinfection are warranted.

Introduction

ACQUIRED immune deficiency syndrome (AIDS) is a

retrovirus-induced disorder with an extraordinarily highmortality. Increasing evidence indicates that human

T-lymphotropic virus type III (HTLV-III),1-3 which issimilar to or identical with the lymphadenopathy-associatedvirus (LAV)4 or AIDS-associated retrovirus (ARV),s is theaetiological agent of AIDS. Since interferons are known tosuppress the replication of many animal retroviruses in vitroand in vivo,6-10 we studied the effects of recombinant humaninterferon alfa-A (rIFNaA) on HTLV-III infection ofnormal peripheral-blood mononuclear cells (PBMC) in vitro.

Materials and Methods

Cells.-PBMC, obtained by’Ficoll-Hypaque’ separation of blooddonated by healthy HTLV-III-seronegative individuals, were usedas target cells in these experiments.

M.—HTLV-IILg;, provided by M. Popovic and R. Gallo, wasgrown in H9 cellsl (0 - 5-1- 0 x 106 cells/ml) in RPMI 1640 mediumsupplemented with fetal calf serum (FCS, 20%), HEPES (10mmol/1), penicillin (250 u/ml), streptomycin (250 g/ml), andL-glutamine (2 mmol/1). Cell-free supernatant fluid was harvestedwhen the cell density and viability were approximately 1 - 0 X 106/Mland 90%, respectively. Titration of this virus preparation on normalPBMC demonstrated a 50% tissue culture infective dose (TCIDsü)of 104/ml.

Interferon. -Recombinant human interferon alfa-A was

provided by Hoffmann-La Roche (Nutley, NJ [lot C114941-03])and assayed by a cytopathic effect reduction method against

603

Fig 1—HTLV-111 immunofluorescence results from days 7 and 10 forsingle and multiple dose rIFNaA treatment.

Slides were coded, read by two investigators, and scored as percentage(±1 SD) of cells expressing HTLV-111 antigens.

vesicular stomatitis virus in GM 2504 cells (Human GeneticMutant Cell Repository, Camden, NJ). National Institutes ofHealth interferon preparation G023-901-527 was used as thestandard.

Experiment 1 (single-dose rIFNaA).-On day -1, normal PBMCwere stimulated with phytohaemagglutinin-P (Sigma, 10 µg/ml) anddistributed (2 x 106 cells per culture) in 25 cm2 flask containing 5 mlRPMI 1640 medium supplemented with HEPES, penicillin,streptomycin, L-glutamine, and 20% FCS. Duplicate cultures werethen exposed to rIFNaA at concentrations of 0, 4, 16, 64, 256, and1024 units/ml. After 18 h of interferon treatment (day 0), eachculture was inoculated with 25 TCIDso of cell-free HTLV-IIIB andmaintained on 10% interleukin-2 (Electronucleonics, Bethesda,MD). Fluid was changed on days 4 and 7. On days 7 and 10 viablecell numbers were counted, HTLV-III antigen expression wasdetermined by indirect immunofluorescence testing with an

HTLV-III-seropositive human serum, II and reverse transcriptase(RT) activity was measured. 11,12

Experiment 2 (multiple-dose rIFNaA).- The experiment wascarried out as outlined in experiment 1 with the followingexceptions: (1) rIFNaA concentrations were 16, 64, and 256units/ml, and (2) rIFNaA doses were repeated on days 4 and 7 withthe fluid changes.

Results

Experiment 1.—Single-dose rIFNaA had no anti-

proliferative effect on PBMC even at a concentration of 1024units/ml (table). However, rIFNaA had a dose-related

suppressive effect on HTLV-III replication, as demonstratedby the reduction in the number of cells expressing HTLV-IIIantigens and in RT activity (figs 1 and 2). Inhibition wasobserved at doses as low as 4 units/ml, and was pronounced at

Fig 2-Reverse transcriptase activities from days 7 and 10 for singleand multiple dose rIFNaA treatment.

Results are expressed as counts per minute per ml of culture medium(±1SD).

64 units/ml. Complete inhibition was achieved with 1024units/ml. Three additional experiments (data not shown)with PMBC from other normal donors confirmed the above

findings. -

Experiment 2.-Multiple doses of rIFNaA, had no

antiproliferative effect on PBMC (table). Low concentrationsof rIFNaA reduced antigen positivity and RT activity and256 units/ml completely suppressed HTLV-111 replication(figs 1 and 2).

Discussion

Approximately 8500 persons in the USA have been

reported to have AIDS, a syndrome with a 3-year mortalityrate approaching 100%. The number of cases continues toincrease and new cases are appearing on six continents. Manymore individuals have or are developing AIDS-relatedcomplex (ARC), a prodromal or less virulent form of AIDS.Furthermore, as many as 65% of the urban male homosexualpopulation and 94% of haemophiliacs in the USA have beeninfected by HTLV-III according to recent serologicalstudies.13,14 These antibody-positive persons are not only atrisk for possible subsequent development of AIDS, but somemay be virus carriers and potential reservoirs for the

dissemination of HTL V-III.11,15 Therefore, in addition tovaccine development, an effective treatment for AIDS andHTLV-III infection is urgently needed. Recent studies

suggest that suramin16 and ribavirin17 have suppressiveeffects on the in vitro replication of HTLV-III and LAV,respectively.Our results demonstrate that rIFNaA also has dose-related,

inhibitory activity on HTLV-III replication. Virus-

inhibitory effects are seen at doses between 4 and 64 units/ml,and complete inhibition is possible between 256 and 1024units/ml, depending on the schedule of drug administration.These doses do not have toxic effects on the target cells. Peakserum concentrations of 50-300 units/ml can be achievedwith parenteral doses of 10-100 million units of rIFNaA.18Interferons have clinical activity in various human viralinfections including those caused by herpes simplex,19cytomegalovirus, 20 and varicella-zoster.21 It is clear from

604

these studies that prophylaxis or early therapy is morebeneficial than late treatment. Clinical trials of interferon fortreatment of AIDS are already in progress at many centres.However, these trials are generally on late disease, wheresignificant antiviral effects may be difficult to demonstrate.Trials in early infection may be required for thedemonstration of in vivo antiviral activity.

Interferons can inhibit many animal retroviruses,including murine leukaemia viruses, murine sarcoma virus,feline leukaemia virus, and Mason-Pfizer monkey virus.6-8These effects are believed to be mediated in part by criticalalterations in viral assembly and release.6,7 Our current studydoes not address the question of how rIFNaA suppressesHTLV-111 replication, although such studies are in progress.We are currently testing the in-vitro efficacy of various

other interferon preparations (aD, /?, y) against severalHTLV-III isolates. Synergistic studies of different interferonpreparations and antiviral agents, such as suramin and

ribavirin, are also indicated.

We thank Dr M. Popovic and Dr R. Gallo for H9 cells and HTLV-IIIg, DrP. Trown for recombinant human interferon alfa-A, and Ms J. Steele forassistance in preparing the paper. D. D. H. is a fellow of the American CancerSociety, Massachusetts Division. This research was supported in part bygrants CA12464 and CA37461 from the National Cancer Institute,Massachusetts Department of Public Health Contract 349, and the Mashud A.Mezerhane B. Fund.

Correspondence should be addressed to D. D. H., Infectious Disease Unit,Massachusetts General Hospital, Boston, Massachusetts, USA.

REFERENCES

1. Popovic M, Sarngadharan MG, Read E, Gallo RC. Detection, isolation, andcontinuous production of cytopathic retroviruses (HTLV-III) from patients withAIDS and pre-AIDS. Science 1984; 224: 497-500.

2. Gallo RC, Salahuddin SZ, Popovic M, et al. Frequent detection and isolation ofcytopathic retroviruses (HTLV-III) from patients with AIDS and at risk for AIDS.Science 1984; 224: 500-03.

3. Sarngadharan MG, Popovic M, Bruch L, et al. Antibodies reactive with human

T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDSScience 1984; 224: 506-08.

4. Barré-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a T-lymphotropic retrovirusfrom a patient at risk for acquired immune deficiency syndrome (AIDS). Science1983, 220: 868-71.

5. Levy JA, Hoffman AD, Kramer S, et al. Isolation of lymphocytopathic retrovirusesfrom San Francisco patients with AIDS. Science 1984; 225: 840-42.

6. Pitha PM, Bilello JA, Riggin CH. Effect of interferon on retrovirus replication. TexasRep Biol Med 1982; 41: 603-09.

7. Aboud M, Hassan Y. Accumulation and breakdown of RNA-deficient intracellularvirus particles in interferon-treated NIH 3T3 cells chronically producing Moloneymurine leukemia virus. J Virol 1983; 45: 489-95.

8. Sen GC, Herz R, Davatelis V, Pestka S. Antiviral and protein-inducing activities ofrecombinant human leukocyte interferons and their hybrids. J Virol 1984; 50:445-50.

9. Hirsch MS, Ellis DA, Profitt MR, Black P. Effects of interferon on leukaemia virusactivation in graft versus host disease. Nature New Biol 1973; 244: 1-6.

10. Gresser I, Coppey J, Chantal B. Interferon and murine leukemia. VI. Effect ofinterferon preparations on the lymphoid leukemia of AKR mice. J Natl Cancer Inst1969; 43: 1083-89.

11. Ho DD, Schooley RT, Rota TR, et al. HTLV-III in the semen and blood of a healthyhomosexual man. Science 1984; 226: 451-53.

12. Ho DD, Rota TR, Hirsch MS. Infection of human endothelial cells by humanT-lymphotropic virus type I. Proc Natl Acad Sci USA 1984; 81: 7588-90.

13. DeJarlais DC, Marmor M, Cohen H, et al. Antibodies to a retrovirus etiologicallyassociated with acquired immunodeficiency syndrome (AIDS) in populations withincreased incidences of the syndrome. Morbid Mortal Weekly Rep 1984; 33: 377-79.

14. Kitchen LW, Barin F, Sullivan JL, et al. Aetiology of AIDS-antibodies to humanT-cell leukaemia virus (type III) in haemophiliacs. Nature 1984; 312: 367-69.

15. Groopman JE, Salahuddin SZ, Sarngadharan MG, et al. Virologic studies in a case oftransfusion-associated AIDS. N Engl J Med 1984; 311: 1419-22.

16. Mitsuya H, Popovic M, Yarchoan R, et al. Suramin protection of T-cells in-vitroagainst infectivity and cytopathic effect of HTLV-III. Science 1984; 226: 172-74.

17. McCormick JB, Getchell JP, Mitchell SW, Hicks DR. Ribavirin suppresses replicationof lymphadenopathy-associated virus in cultures of human adult T lymphocytes.Lancet 1984; ii: 1367-69.

18. Gutterman JU, Fine S, Quesada J, et al. Recombinant leukocyte A interferon:pharmacokinetics, single-dose tolerance, and biologic effects in cancer patients. AnnIntern Med 1982; 96: 549-56.

19. Pazin GJ, Armstrong JA, Lam MT et al. Prevention of reactivated herpes simplexinfection by human leukocyte interferon after operation on the trigeminal root. NEngl J Med 1979; 301: 225-30.

20. Hirsch MS, Schooley RT, Cosimi B, et al. Effects of interferon-alpha oncytomegalovirus reactivation syndromes in renal transplant recipients. N Engl JMed 1983; 308: 1489-93

21. Merigan TC, Rand KH, Pollard RB, et al. Human leukocyte interferon for thetreatment of herpes zoster in patients with cancer. N Engl J Med 1978; 298: 981-87.

REGULATION OF CEREBRAL BLOOD FLOWIN RESPONSE TO CHANGES IN BLOOD

VISCOSITY

MARTIN M. BROWN JOHN MARSHALL

Institute of Neurology, National Hospitals for Nervous Diseases,London

Summary Cerebral blood flow (CBF) was measured bythe non-invasive xenon-133 technique in

patients with increased blood viscosity as a result of

paraproteinaemia or leukaemia. A highly significant inverserelation was found between CBF and arterial oxygen contentin 59 paraproteinaemic patients. There was no significantcorrelation between CBF and whole blood viscosity, and nosignificant difference between CBF in paraproteinaemicpatients and a matched group of anaemic patients. 7leukaemic patients with up to threefold increases in wholeblood viscosity were also found to have CBF appropriate totheir degree of anaemia. The effects of treatment to reduceblood viscosity were studied in 7 paraproteinaemic and 5leukaemic patients; changes in CBF were significantly relatedto changes in arterial oxygen content but not to changes inblood viscosity. These studies confirm the importance ofarterial oxygen content in the determination of CBF, anddemonstrate that regulatory mechanisms can maintainnormal cerebral oxygen transport despite increased plasmaand whole blood viscosity.

Introduction

UNDER certain conditions, flow of a liquid in a narrow tubeis inversely proportional to the viscosity of the liquid,according to Poiseuille’s law. Conditions in the cerebralmicrocirculation are suitable for viscous forces to dominateflow, and cerebral blood flow (CBF) might therefore beexpected to obey Poiseuille’s law. Reduction in CBF as aresult of increased blood viscosity may explain the

neurological symptoms of haematological conditions such aspolycythaemia, paraproteinaemia, and leukaemia. Thismechanism may also be important in the development ofischaemic stroke.2,3 Furthermore, lowering of blood viscosityhas been proposed as a treatment for acute stroke, in order toimprove CBF.4,5The assumption that CBF will be affected by changes in

blood viscosity has been challenged on the grounds that itignores the vascular regulatory mechanisms of the brain.6,7Earlier work on the effects of raised blood viscosity on CBFconcentrated on polycythaemic patients because packed cellvolume (PCV) is a major determinant of blood viscosity asmeasured in vitro.8 CBF is low in polycythaemia9,lo but themechanism of this reduction has been disputed. Thomas etal11,12 found that venesection reduced blood viscosity andincreased CBF in polycythaemic patients and in subjects withPCV at the upper end of the normal range, while Humphreyet al13 showed an inverse relation between CBF and bloodviscosity in patients with high PCV. These findings appearedto support the suggestion that blood viscosity is an important