DEPLETION OF T LYMPHOCYTES IN DONOR MARROW PREVENTS SIGNIFICANT GRAFT-VERSUS-HOST DISEASE IN MATCHED ALLOGENEIC LEUKAEMIC MARROW TRANSPLANT RECIPIENTS

472

DEPLETION OF T LYMPHOCYTES IN DONOR MARROW PREVENTS SIGNIFICANT GRAFT-VERSUS-HOST DISEASE IN MATCHED ALLOGENEIC LEUKAEMIC MARROW TRANSPLANT RECIPIENTS H. G. PRENTICE G. JANOSSY L. PRICE-JONES L. K. TREJDOSIEWICZ D. PANJWANI S. GRAPHAKOS K. IVORY

H. A. BLACKLOCK M. J. M. L. GILMORE N. TIDMAN D. B. L. SKEGGS S. BALL

J. PATTERSON A. V. HOFFBRAND

Departments of Haematology, Immunology, and Radiotherapy, Royal Free Hospital, and School of Medicine, London For more than 15 years preclinical studies have suggested that acute graft-versus-host disease (aGvHD) might be prevented by the removal of immunocompetent T lymphocytes from the donor marrow inoculum. To test this observation in man 14 patients were depleted of identifiable given marrows virtually (>99%) donor marrow T lymphocytes by the use of a "cocktail" of specific anti-T-cell monoclonal antibodies (MBG6 and RFT8) and rabbit complement. Patients were not given immunosuppressive prophylaxis after bone-marrow transplantation. Moderate to severe (grades II—IV) GvHD was totally prevented. 2 of 13 evaluable patients showed mild (grade I) skin GvHD only. Although peripheral blood recovery was slower than that obtained with other forms of GvHD prophylaxis, no fatal infections occurred. All patients survived the early post-transplant period.

Summary

Introduction

graft-versus-host disease (aGvHD), a clinical first described in the chicken,’ is characterised in syndrome man by an exfoliative skin rash, severe diarrhoea, and hepatitis.2 In recent years the incidence of aGvHD after major histocompatibility matched (MHC) allogeneic boneACUTE

transplantation (BMT) has declined,3 probably because of the selection of patients in better general health who are overall less at risk of developing infection, a complication which sometimes triggers off aGvHD.4 Still, aGvHD may develop in more than 50% of allogeneic matched BMT recipients, and it may be fatal in 25% of those marrow

affected.3-5 In the early part of the past decade methotrexate (MTX) introduced as an immunosuppressive prophylactic, following preclinical studies in dogs,6 and since then this drug has been widely used as a standard prophylactic. was

A. H. FILIPOVICH AND OTHERS: REFERENCES—continued RJ, Neville DM Jr. Anti-Thy 1·2 monoclonal antibody hybrid utilized as a tumor suppressant. US patents 4,359,457. (Related US patents pending SN 399257 and SN456,401). 18. LeBien TW, Kersey JH. Monoclonal antibody (TA-1) reactive with human T lymphocytes and monocytes. J Immunol 1980; 125: 2208-14. 19. Royston I, Majda A, Baird SM, Meserve BL, Griffiths JC. Monoclonal antibody specific for human T lymphocyte-identification of normal and malignant T-cells. Blood 1979; 54 (suppl 1): A106. 20. Beverley PCL, Callard RE. Distinctive functional characteristics of human T lymphocytes defined by E rosetting or a monoclonal anti T cell antibody. Eur J Immunol 1981; 11: 34. 21. Vallera DA, Ash RC, Zanjani ED, et al. Anti-T-cell reagents for human bone marrow transplantation: ricin linked to three monoclonal antibodies. Science 1983; 222: 512-14. 22. Ash RC, Retrick RA, Zanjani ED. Studies of human pluripotential hematopoietic stem cells (CFU-GEMM) in vitro. Blood 1981; 58: 309-16. 23. Quinones RR, Youle RJ, Kersey JH, et al. Anti-T cell monoclonal antibodies conjugated to ricin as potential reagents for human GvHD prophylaxis: effect on the generation of cytotoxic T cells in both peripheral blood and bone marrow. J Immunol 1984; 132: 678-83. 17. Youle

-

Considerable enthusiasm was generated by the preliminary results of therapy’ and prophylaxis8 of aGvHD with cyclosporin A (CSA), but although the incidence of aGvHD is slightly less after CSA than after MTX, CSA does not 9 improve survival. Another promising prophylactic immunosuppressive regimen is the combination of steroid and antithymocyte globulin prophylaxis with MTX.1O Another approach to prevention of aGvHD is the elimination of immunocompetent T lymphocytes from donor marrow. The observation was originally made in murine transplants," and it has been confirmed in other animals ranging from mice to monkeys, 12-1 both after MHC identical and mismatched transplantation. The only differing viewpoint is that of Muller-Ruchholtz, whose work in rats suggests that the removal of a hypothetical pre-thymic bonemarrow cell population may also be necessary for preventing aGvHD in mismatched rodents.16 Preliminary data in man suggested that incubation of donor marrow with highly absorbed rabbit antihuman T-cell sera before infusion reduced the incidence and severity of aGvHD.’ By using a murine monoclonal antibody (OKT3) and MTX prophylaxis we obtained a lower overall incidence and severity of aGvHD than in historical controls (MTX prophylaxis), but we did not completely prevent GvHD.18 Later we attempted in-vitro removal of T lymphocytes with OKT3 and one round of complement, but the lysis was incomplete (mean 73%) and aGvHD was not prevented. 19 An alternative, although elaborate and time-consuming, technique for eliminating T lymphocytes from donor marrow is the use of lectin separation plus E rosetting." In mismatched BMT in children with severe combined immune deficiency (SCID) this gave encouraging results.21 The current study reported here examined the effect of complete T-cell depletion (>99% lysis) of donor marrow, achieved by the use of a selected "cocktail" of two T-cellspecific monoclonal antibodies (MBG6 and RFT8) with rabbit complement. Patients and Methods The safety of the procedure was tested in 1 patient (UPN 73). When safety was established all patients aged <45 years with poor prognosis leukaemiawho were in complete remission (1st or 2nd) or in early relapse (marrow blasts <20%) and who had MHC-identical sibling donors were admitted to the study (table I). 14 (including UPN 73) satisfied the criteria for inclusion in the study. Patient management, including supportive care, has been described before. 17,22 Antileukaemic conditioning therapy consisted of cyclophosphamide (60 mg/kg) and mesna (160% v/v) on 2 consecutive days (days -4, -3) to prevent haemorrhagic cystitis,23 followed by total body irradiation (TBI) on day 0 and five fortnightly doses of intrathecal MTX, beginning on day +32. TBI was administered by a Phillips 8 MeV linear accelerator at 26 cGy/min in air, to a planned maximum dose of 7 - 5 Gy to the lungs. Patients were nursed in reverse-barrier isolation and received prophylactic ’Septrin’ from day 0 until +66 months, prophylactic acyclovir24 (intravenously and then orally) until +4to +6weeks (if seropositive pre-BMT), and prophylactic ketoconazole until +6 months. 25 All blood products were irradiated. Marrow was harvested from anaesthetised donors, who were

given preservative-free heparin(100 u/kg). Small volumes (5-10 ml) of marrow were aspirated from the anterior and posterior pelvis into’ heparinised syringes until an adequate cell count was achieved. It was not always possible to harvest more than 2 x 108 nucleated cells per kg recipient weight (table n) because of discrepancies in donor/recipient size. The collected cells were transferred via a standard blood transfusion filter to a sterile bag containing acid citrate dextrose (ACD, National Institutes of Health formula A). The volume of marrow aspirated ranged from 456 ml to 902 ml (mean 630 ml). Whole marrow was transferred to the laboratory,

473 marrow cells were infused into the irradiated recipient without further filtration. Both monoclonal antibodies used were of the IgM class. MBG6 is a pan-peripheral T-cell antibody reacting with >95% of T cells (which carry an antigen of 100000 MW).27 RFT8 reacts with a subset of T cells (T8 lymphocytes of suppressor cytotoxic type). Individually, the antibodies lysed 90-95% of reactive cells when used with rabbit complement. Together, they destroyed >99% of T lymphocytes. The lytic efficacy of the antibodies was monitored by testing the residual T cells with an independent marker (OKT3) in an assay using double labelling for fluoresceinated antibody (FITC) and ethidium bromide (red channel, positive on dead cells). Rabbit complement was supplied by Pelfreeze Ltd, and was selected at the Royal Free Hospital (RFH) from several batches on the basis of lytic efficacy when used with the relevant antibodies, and a lack of toxicity in BM progenitor assay studies. All material used was sterile filtered, tested for pyrogenicity, and screened for viral contamination. Patients were examined daily for signs of infection, bleeding, and aGvHD. Assessment of marrow engraftment was based on a marrow aspiration between days 10 and 14 (with or without trephine biopsy) and the time taken to achieve a stable white blood cell (WBC) count of > 1x10/1, a neutrophil count >0-5xl0"/l, and platelets

TABLE I-CLINICAL DETAILS AND DISEASE STATUS

UPN=Unique patient number; KS=Karnofsky score.30 M=male; F=female; NE=not yet available; CRl 1st complete remission; CR2=2nd complete remission. T-ALL=thymic acute lymphoblastic leukaemia; C-ALL=common acute lymphoblastic leukaemia; AML = acute myeloblastic leukaemia; CGL = chronic granulocytic leukaemia; NULL-ALL = non B, non =

109/1.

>50x

Acute GvHD was defined as that which began <60 days after BMT. The day of onset was taken as the post-transplant day on which the signs of GvHD appeared. Severity was defined according to the Seattle criteria.29 To compare these results with other forms of GvHD prophylaxis, a simple scoring system (RF GvHD score) was devised. The cumulative maximum grade of GvHD observed in the patients is divided by the cumulative maximum grade possible (ie, number of patients x 4). Thus a score of 0 - 25 represents a mean GvHD grade of 1 in the patient group. The current condition of the patients is indicated by the Karnofsky score.0

acute lymphoblastic leukaemia. Patients not rncluded: UPN 74-76 were patients not given T-cell depleted marrows for aGvHD prophylaxis because the safety of the procedure, tested on patient 73, had not been established (MTX prophylaxis was used); UPN 81, 88, and 90 are mismatched cases given T-cell depleted marrows (1of them is alive and well), and will be reported separately; UPN 77 and 86 were transplanted in advanced relapse and were not given T-cell depleted marrows

T

*

physical

(MTX prophylaxis). t Major ABO mismatch. At present in relapse; all other patients alive and well.

Results

TABLE II-ENGRAFTMENT DATA

Patients 13 of the 14 patients have now been followed up for at least 60 days. The other one is at day 55 from BMT. All patients are

alive and well (except for UPN 73, who died from leukaemic relapse, and UPN 84, who is in leukaemic relapse (table 1). In-vitro T-edl Depletion The

mean

number of nucleated cells harvested

3-3xl0/kg recipients’ weight (table n) containing,

was

after

Ficoll separation, 24% (mean) of T lymphocytes (table III). One round of complement lysis produced incomplete T lysis, but after the second exposure >99% of T cells were killed in TABLE III-% VIABLE T CELLS BEFORE AND AFTER FIRST AND SECOND ROUNDS OF COMPLEMENT LYSIS

where most subsequent procedures were done in a laminar air-flow cabinet. After samples were taken for counts, viability, and sterility the buffy coat fraction of the BM was prepared with the IBM 2991 blood-cell washer.26 The mononuclear fraction of marrow cells left at the interface was collected, sampled as above, washed, and resuspended in Hanks balanced salt solution (HBSS) containing Ca + and Mg++. Purified murine monoclonal anti-T cell antibodies MBG6 and RFT8 (1 mg each) were added. After incubation of the mixture at room temperature (RT) for 30 min an equal volume of rabbit complement was added, and the BM was incubated at 37°C for 30 min with gentle agitation. After the BM mixture had been washed with HBSS (with Ca + and Mg ’), a further equal volume of rabbit complement was added and the incubation repeated. After each round of lysis, the cells were washed twice (or more recently once) in 507o human serum albumin in saline, then sampled for counts, viability, sterility, and CFU-assays. The

Cl

and C2 = 1st and 2nd rounds of complement lysis, respectively. < I in 500 are positive for T-cell antigen when studied with an independent marker (T3). tRFT8+MBG6+RFT2. The rest of the cases: RFT8+MBG6. Murine monoclonal anti-T cell antibodies. *

474 TABLE IV-COMPARISON OF aGvHD INCIDENCE BETWEEN DIFFERENT PROPHYLACTIC REGIMENS (MATCHED DONORS, LEUKAEMIC CASES IN

REMISSION/EARLY RELAPSE

ONLY)

number of

cells and his clinical course mild CMV infection (see below).

marrow

complicated by

a

cGvHD Evidence of cGvHD

developed in 1 patient (UPN 73). He

had modest lichenoid lesions in his mouth and

MTX=methotrexate; P=prednisolone; A TG=antithymocyte globulin; CSA=cyclosponn; Cy=cyclophosphamide; OKT3=OKT3 incubation; RC=rabbit complement. *

minimum

score

from data.

tIyr survival.

all but one patient (UPN 84). CFU-GM assays before and after complement lysis indicate that these in-vitro manipulations did not result in significant stem-cell loss

(table II). Patient Toxicity The only side-effects of the marrow infusion were moderate transient fevers (n = 5) and mild rigors (n = 6). In no patient did an infection attributable to marrow handling develop, and -

.

there

was no serum

sickness.

aGvHD Incidence 11 of the 13 evaluable patients did not have aGvHD. The other 2 patients had grade I (skin only) aGvHD. In each the of the body surface area. The area of skin involved was <25% RF GvH score in our group is therefore 0 - 04 (see table IV). In 1 of these 2 patients (UPN 73) a mild erythematous rash developed on day + 56 on the upper trunk and face after a clinically mild reactivation of cytomegalovirus (CMV) infection (virus isolation from saliva and urine in association with mild hepatocellular dysfunction). In the second patient (UPN 91) a rash developed (day + 16) on the dorsum of both hands. Both rapidly responded to low doses of steroids. The histological grade was II (with possible areas of III) in both cases.

patients received any form of immunosuppression during the study period (except None

was

of the other

intrathecal methotrexate for central nervous system leukaemic prophylaxis). No patient has had significant diarrhoea since BMT, and no patient has had hepatocellular dysfunction to fulfill the criteria for liver GvHD. The RF GvHD score was used to compare effects of different methods of GvH prophylaxis data in table IV. Complete T-cell lysis reduced the aGvHD score

dramatically. Engraftment The time to recovery to 1 x 109/1 WBC was 15-36 days (mean 26) and to >0’5x 109/1 neutrophils (table n). 16-72 days (mean 31 - 5). The patient (UPN 79) with the slowest recovery of neutrophils and platelets received the lowest

on

his hands

only. Infections No patient in this group died from infections. The number of days on which patients had a fever (>38°C) during the 60 day aGvHD study period ranged from 0-16 (median 7). Bacterial infections were eradicated with appropriate systemic antibiotics. Of the 8 patients who had serological evidence of prior CMV infection before transplantation, 2 (UPN 73 and 94) had laboratory and/or clinical evidence of CMV reactivation. UPN 94 developed interstitial pneumonitis due to CMV (resolving, as at Jan 28). Of the 6 patients seronegative (by RIA) at BMT, only 1 (UPN 79) had a mild new infection (temporary fever, positive saliva and urine cultures, and prolonged thrombocytopenia). 2 patients with previous fungal infections (UPN 84 with Candida tropicalis oesophagitis; UPN 85 with pulmonary aspergilloma resected 3 weeks before transplantation) were treated early and successfully with intravenous amphotericin for recurrence after BMT (UPN 84 clinical signs and positive culture; UPN 85 fever unresponsive to antibiotics). None of the patients had viral enteritis, or hepatitis. relapse 2 of the 4 patients transplanted while in early relapse (UPN 73 and 84) went into remission after BMT but subsequently relapsed, and 1 of these 2 (UPN 73) died. To date the other 12 patients are in remission (table I). Discussion This study aimed to eliminate aGvHD in patients receiving fully matched allogeneic bone marrow by the use of specific anti-T-cell monoclonal antibodies to eliminate T lymphocytes from donor marrow. For years animal studies have suggested that immunocompetent T lymphocytes in the donor marrow inoculum are required for aGvHD.11-1The early clinical trials with T-cell-specific heteroantiseral7 and monoclonal antibodiesl8°3i were performed by "coating" (opsonising) the T cells. Since these trials were not fully effective in preventing aGvHD, it was difficult to know whether this failure was due to the inefficiency of T-cell removal or the participation of pre- TI6 and/or other cell types in aGvHD. Our study shows that donor marrow T-cell removal by a combination of monoclonal antibodies selected on the basis of >99% lysis against T cells is the most efficient technique for preventing aGvHD in man (RFH score 0 -04; table iv)—only 2 out of 13 patients had a mild aGvHD (grade I), which responded to low doses of steroids. The next best result, reported by the Minnesota group (RFH score 0-09), was obtained at the expense of considerable immunosuppression post BMT.’° Only 1 of our patients (UPN 73) had mild chronic graft-versus-host disease after his episode of aGvHD, a known major risk factor for chronic GvHD.3z To achieve T-cell-specific lysis the bone-marrow inocula were rapidly concentrated on a Ficoll gradient with the IBM 2991 cell processor6 and treated with two courses of previously tested cytolytic rabbit complement. This procedure can be completed within 3 h and is more convenient and suitable for large-scale preparation than the lectin-separation method combined with sheep erythrocyte

475

rosetting.21 Our procedure did not damage the performance of the infused bone marrow, as indicated by results of the functional CFU-GM (table II) and the mixed myeloid progenitor (CFU-GEMM) assays (data not shown). Despite these CFU findings, the mean peripheral blood recovery was moderately delayed (table II). The >1 x 109/1 WBC count was reached on day + 26 (mean), instead of the usual recovery observed at day + 22 (mean) in patients receiving methotrexate prophylaxis.33 The finding that T cells, most probably T8+ lymphocytes, are involved in granulocyte differentition34 may explain the delay in recovery of the neutrophils (table II). Somewhat surprisingly, despite prolongation of the neutropenia period, these patients did not show a striking increase in incidence of infections-viral, bacterial, or fungal-even though they were not kept in a strictly protected environment.4Admittedly, some of the patients received prophylactic acyclovir, a drug effective against herpes infections,2’1 as well as antifungal agents.2s According to most reports, acyclovir is ineffective against CMV,24,35 and it was given for only 6 weeks immediately after BMT, which is not the usual time of onset of post-BMT CMV infection.36 Yet CMV infection occurred only in 3 patients. None of the 3 patients was receiving acyclovir at the time ofCMV infection. It may be relevant that our patients showed regeneration of the T-cell system, although the follow-up period is too short for a detailed account. It is also important that during the whole follow-up period no prophylactic immunosuppressive steroids; cyclosporin A, or agent (methotrexate, was given and this may contribute to antithymocyte globulin) the lack of infectious complications. Furthermore, aGvHD and (viral) infections represent a vicious circle, both aggravating an abnormal T-cell system by the overproduction of suppressor/cytotoxic type of T cells.37,38 In conclusion, our study provides the first clear evidence that T lymphocytes recognise not only the foreign major histocompatibility antigens in the animal models of aGvHD,II-15 but also seem to be the primary cause of aGvHD in man following transplantation of allogeneic bonemarrow fully matched for the major histocompatibility antigens. The study offers a more logical, yet simple, approach to aGvHD prevention than do protocols of severe immunosuppressive regimens given during the regeneration phase of BMT (table IV). In our view elimination of T lymphocytes from donor marrow will be helpful for investigating whether the effects of graft against leukaemia40 is maintained

enhanced - in the absence of immunosuppressive therapy. Perhaps the most important practical outcome of our work is that it may prevent the fatal complications associated with GvHD-a factor which may reduce the ethical problems of transplanting patients with matched allogeneic bone marrow for other malignant and non-malignant haematological diseases. It remains to be seen how the new techniques can be used, perhaps in a modified form, for preventing aGvHD-associated problems during the transplantation of haploidentical (mismatched) bone-marrow without endangering marrow engraftment. or

even

We thank the Leukaemia Research Fund of Great

Britain,

A. Onassis

Foundation, and Lady Tata Memorial Trust for their support; Mrs Megan Evans for typing this manuscript; ProfA. McMichael for the original clone of MBG6; and nurses of Riddell and Garrett Anderson wards, medical staff of our department, members of the radiotherapy department, technical staff of the haematology department and the referring physicians for their help; and

Z.Varghese and Miss C. Lang for tissue typing. Correspondence should be addressed to H. G. P., Department of Haematology, Royal Free Hospital, Pond Street, Hampstead, London NW3

Dr

2QG.

REFERENCES 1. Simonsen M. The impact on the developing embryo and newborn animal of adult homologous cells. Acta Path Microbiol Scand 1957; 40: 480-500. 2. Thomas ED, Storb R, Clift RA, et al. BMT transplantation. N Engl J Med 1975; 292: 832-95. 3. Weiden PL and the Seattle Marrow Transplant Team. Graft-vs.-host disease in allogeneic marrow transplantation. In: Gale RP, Fox CF, eds. Biology of bone marrow transplantation. New York: Academic Press, 1980. 4. Storb R, Prentice RL, Buckner CD, et al. Graft versus host disease and survival in patients with aplastic anemia treated by marrow grafts from HLA-identical siblings. Beneficial effect of a protective environment. N Engl J Med 1983; 308: 302-07. 5. Gale RP, Kersey JH, Bortin MM, Dicke KA, Good RA, Zwaan FE. Bone marrow transplantation for acute lymphoblastic leukaemia. Lancet 1983; ii: 639-42. 6. Storb R, Epstein RB, Graham TC, Thomas ED. Methotrexate regimens for control of graft-versus-host disease in dogs with allogeneic marrow grafts. Transplantation 1980; 9: 240-46. 7. Powles RL, Clink H, Sloane J, Barrett AJ, Kay HE, McElwam TJ Cyclosporin A for the treatment of graft versus host disease in man Lancet 1978; ii: 1327-31. 8. Powles RL, Clink HM, Spence D, et al. Cyclosporin A to prevent graft versus host disease in man after allogeneic bone marrow transplantation. Lancet 1980; ii:

327-29.

Deeg HJ, Storb R, Thomas ED, et al. Marrow transplantation for acute, nonlymphoblastic leukaemia in first remission: Preliminary results of a randomised trial comparing cyclosporin and methotrexate for the prophylaxis of graft-versus-host disease. Transplant Proc 1983; 15: 1385-88. 10. Ramsay N, Kersey JH, Robinson LL, et al. A randomised study of the prevention of acute graft versus host disease. N Engl J Med 1982; 306: 392-97. 11. Dicke KA, Van Hooft J, Van Bekkum DW. The selective elimination of immunologically competent cells from bone marrow and lymphatic cell mixtures. Transplantation 1968; 6: 562-70. 12. Rodt H, Thierfelder S, Eulitz M. Anti-lymphocytic antibodies and marrow transplantation. III. Effect ofheterologous anti-brain antibodies on acute secondary disease in mice. Eur J Immunol 1974; 4: 25-29. 13. Korbling M, Fliedner TM, Calvo W, Ross WM, Northdurft W, Steinbach F. Albumin density gradient purification of canine hemopoietic blood cells (HBSC): Long-term allogeneic engraftment without GvH-reaction. Exp Hematol 1979; 7: 277-88 14. Wagemaker G, Vriesendorp HM, Van Bekkum DW. Successful bone marrow transplantation across major histocompatibility barriers in rhesus monkeys. Transplant Proc 1981; 13: 875-80 15. Yunis EJ, Fernandes G, Smith J, Good RA. Long survival and immunologic reconstitution following transplantation with syngeneic or allogeneic fetal liver and neonatal spleen cells. In: Dupont B, Good RA, eds. Immunobiology of bone marrow transplantiation, Vol 1. New York: Grune and Stratton, 1976: 173-77. 16. Muller-Ruchholtz W, Wottge HU, Muller-Hermelink HK. Bone marrow transplantation in rats across strong histocompatibility barriers by selective elimination of lymphoid cells in donor marrow. Transplant Proc 1976; 8: 537-41. 17. Rodt H, Kolb HJ, Netzel B, et al. Effect of anti-T cell globulin on GvHD in leukaemic patients treated with BMT. Transplant Proc 1981; 13: 257-61. 18. Prentice HG, Blacklock HA, Janossy G. Use of the anti-T monoclonal antibody OKT3 for the prevention of acute graft versus host disease (GvHD) in allogeneic bone marrow transplantation for acute leukaemia. Lancet 1982; i: 700-03. 19. Blacklock HA, Prentice HG, Gilmore MJML. Attempts at T cell depletion using OKT3 and rabbit complement to prevent aGvHD in allogeneic BMT. Exp Hematol 1983; 11 (suppl 13): 37-39. 20. Reisner Y, Kapoor N, O’Reilly RJ, Good RA. Allogeneic bone marrow transplantation using stem cells fractionated by lectins. VI. in vitro analysis of human and monkey bone marrow cells fractionated by sheep red blood cells and soybean agglutinin. 9.

Lancet 1980; ii: 1320-24 21. Reisner Y, Kapoor N, Kirkpatrick D, et al. Transplantation for acute leukaemia with HLA-A and B non-identical parental marrow cells fractionated with soybean agglutinin and sheep red blood cells. Lancet 1981; ii: 327-31. 22. Prentice HG. A review of the current status and techniques of allogeneic BMT for treatment of leukaemia. J Clin Pathol 1983; 36: 1207-14. 23. Blacklock H, Ball L, Knight C, Schey S, Prentice G Experience with mesna in patients receiving allogeneic bone marrow transplants for poor prognostic leukaemia. Cancer Treat Rev 1983; 10 (suppl A): 45-52. 24. Hann IM, Prentice HG, Blackclock HA, et al. Acyclovir prophylaxis against herpes

virus infections

in severely immunocompromised patients: randomised double Br Med J 1983; 287: 384-88. Hann IM, Prentice HG, Corringham R, et al. Ketoconazole versus nystatin plus amphotericin B for fungal prophylaxis in severely immunocompromised patients. Lancet 1982; i: 826-29. Gilmore MJML, Prentice HG, Blacklock HA, Ma DDF, Janossy G, Hoffbrand AV. A technique for rapid isolation of bone marrow mononuclear cells using FicollMetrizoate and the IBM 2991 blood cell processor. Br J Haematol 1982; 50:

blind trial.

25.

26.

619-26. 27. Bastin JM, Granger S, Tidman N, Janossy G, McMichael AJ. Recognition ofa human T lymphocyte differentiation antigen by an IgM monoclonal antibody. Clin Exp Immunol 1981; 46: 597-606. 28. Granger S, Janossy G, Francis G, Blacklock H, Poulter LW, Hoffbrand AV. Elimination of T lymphocytes from human bone marrow with monoclonal T antibodies and cytoclytic complement Br J Haematol 1982; 50: 367-74. 29. Glucksberg H, Storb R, Fefer Q, et al. Clinical manifestations of graft-versus-host disease in human recipients of marrow from HLA-matched sibling donors. Transplantation 1974; 18: 295-304. 30. Karnofsky DA. Meaningful clinical classification of therapeutic response to anti-cancer drugs. Clin Pharmacol Ther 1961; 2: 709-12. 31. Filipovitch AH, McGlave PB, Ramsey NK, Goldstein G, Warkentin PF, Kersey JH. Pretreatment of donor bone marrow with monoclonal antibody OKT3 for prevention of acute graft versus host disease in allogeneic histocompatible bone marrow transplantation. Lancet 1982; i: 1266-69.

476

CONTROLLED TRIAL OF ARTIFICIAL SURFACTANT TO PREVENT RESPIRATORY DISTRESS SYNDROME HENRY L. HALLIDAY GARTH MCCLURE MARK MCC. REID TERENCE R. J. LAPPIN COWAN MEBAN PAUL S. THOMAS

Departments of Child Health, Haematology, Anatomy, and Radiology, The Queen’s University of Belfast, and Royal Maternity Hospital, Belfast In a randomised, controlled trial of the Summary effectiveness of artificial surfactant

therapy

100 babies of <34 weeks’ gestation were intubated at birth and received manual ventilation. The 49 babies in the treated group also received 30 mg dipalmitoylphosphatidylcholine and 3 mg high-density lipoprotein in a 5 ml suspension. There were no significant differences in mortality or in the incidence or severity of respiratory distress syndrome between the surfactant-treated and control groups as a whole or between subgroups divided on the basis of sex or

pulmonary maturity (as assessed by the lecithinsphingomyelin ratio). However, there appeared to be a trend towards improved survival in treated babies of 27-29 weeks’ gestation. Introduction THE respiratory distress syndrome remains a major cause of neonatal mortality and morbidity, accounting for some 1500 deaths each year in the United Kingdoml and up to 12 000 in the United States.2 Soon after Pattle’s discovery of surfactant in 19553 Avery and Mead suggested that its deficiency was the cause of neonatal respiratory distress syndrome.4 Since then, many attempts have been made to treat respiratory distress syndrome by administering surfactant to preterm babies. Most studies have used artificial surfactant5-9 but the results have not been satisfactory. Only one study was controlled9 but since the controls were not enrolled randomly, the results cannot be considered to be conclusive. Natural surfactant has been used only recently’o>m because of the risks of antigenic sensitisation. We now report a randomised, controlled study to test the effects of administering artificial surfactant to preterm babies of less than 34 weeks’ gestation. 32. Storb R, Prentice RL, Sullivan KM, et al. Predictive factors in chronic graft-versus host disease in patients with aplastic anaemia treated by marrow transplantation from HLA-identical siblings. Ann Intern Med 1983; 98: 461-66. 33. Thomas ED, Storb R, Clift RA, et al. BMT transplantation. N Engl J Med 1975; 292:

895-902. al. T-Lymphocyte subsets and partial uncoupling of granulocyte-macrophage progenitor cell differentiation and proliferation in normal cells. Exp Hematol 1982; suppl 10: 179-93. 35. Tyms AS, Scamans EM, Naim HM In-vitro activity of acyclovir and related compounds against cytomegalovirus infections. J Antimicrob Chemother 1981; 8: 65-72. 36 Buckner CD, Clift RA, Fefer A, et al. Bone marrow transplantation. In: Hoffbrand AV, ed. Recent advances in haematology. Edinburgh: Churchill Livingstone, 1982: 34. Francis

GE, Guimaraes JJ, Berney JJ,

et

144-59 37 Favrot M, Janossy G, Tidman N, et al. T cell regeneration after allogeneic BMT. Clin Exp Immunol 1983; 54: 59-72. 38. Verdonck LF, deGast GC. Cytomegalovirus infection: an important cause of immune disturbances after autologous bone marrow transplantation. Exp Hematol 1983; 11 (suppl 14): 72 39 Weiden PL, Sullivan KM, Flournoy N, Storb R, Thomas ED. Anti-leukemic effect of chronic graft versus host disease. Contribution to improved survival after allogeneic marrow transplantation. N Engl J Med 1981; 304: 1529. 40. Thomas ED, Buckner CD, Clift RA, et al. Marrow transplantation for acute nonlymphoblastic leukaemia in first remission. N Engl J Med 1979; 301: 597-99. 41. Thomas ED, Sanders JE, Flournoy N, et al. Marrow transplantiation for patients with acute lymphoblastic leukaemia in remission. Blood 1979; 54: 468-76. 42. Blume KG, Beutler E, Bross KJ, et al. Bone marrow ablation and allogeneic marrow transplantation in acute leukaemia. N Engl J Med 1980; 302: 1041-46. 43. Santos GW, Tutschka PJ, Brookmeyer R, et al. Marrow transplantation for acute nonlymphocytic leukemia after treatment with busulphan and cyclophosphamide. N

Engl J Med 1983; 309: 1347-53.

Patients and Methods

preparation consisted of a mixture ofdipalmitoyl DL-a-phosphatidylcholine (DPPC; Sigma, London) and serum high-density lipoprotein (HDL, prepared by ultracentrifugation of The surfactant

human serum 2). 4 ml HDL and 1 g DPPC were added to 150 ml 0’ 9% sodium chloride. After mixing, the suspension was subjected to ultrasonication for approximately 10 min. 4-5 ml of the resulting colloidal suspension was placed in each of 30 plastic tubes, approximately 25 cm long. The tubing used was similar to that in transfusion giving sets. The tubes were sealed with transfusion sealer clips and sent for irradiation (Irradiated Products Ltd,

Swindon). A sample of each batch of the DPPC/HDL mixture was dispersed in saline by sonication and the adsorption of the surface film was studied by a previously described method.13 The half-adsorption times of the samples ranged from 0’ 9 to 1 -4min, and the static film tension from 36.00 to 36,8 mN/m. After cyclic compression and expansion of the film in a surface balance, the film tension fell to about 8 -5mN/m (at 80% area compression). Before administration the DPPC/HDL suspension was sonicated in a water bath sonicator (Dawe Sonicleaner Type 6443AB) for 10 min. Examination of this suspension under a polarising microscope with crossed Nicol prisms showed the presence of spherules (diameter 4-20 m) and occasional irregular aggregates. The spherules seemed to be liposomes-ie, smectic mesophases of the phospholipid DPPC. Lecithin-sphingomyelin ratios were measured in amniotic fluid or gastric aspirate by the method ofBlass et al.14 100 preterm babies of gestational age 25-33 weeks born in the Royal Maternity Hospital, Belfast, between January, 1982, and March, 1983, were enrolled in the study. Informed consent was obtained from the mother whenever possible and the study was approved by the ethical committee of the Queen’s University of Belfast. Reasons for non-enrolment were an antenatal lecithinsphingomyelin ratio 2-00 (9 babies), significant congenital abnormality (6 babies), or that no neonatologist was present at delivery to carry out endotracheal intubation (28 babies). Babies in the study were randomly allocated to control and treatment groups by means of sealed envelopes. The DPPC/HDL mixture was put into suspension by 5-10 min sonication just before the baby’s birth. All babies were intubated by a consultant neonatologist (H. L. H., G. McC., or M. McC. R.); the 51 babies in the control group received manual ventilation and the 49 in the treated group were given 3-5 ml DPPC/HDL suspension (30 mg DPPC, 3 mg HDL) by endotracheal tube, followed by manual ventilation. Gastric aspirate was obtained when possible for determination of the lecithin-sphingomyelin ratio before the surfactant was given. The babies were subsequently managed and assessed by a neonatologist unaware of the procedure at birth. The babies received uniform care after admission to the special-care baby unit and the guidelines for respiratory assistance were as previously

described. 15s Respiratory distress syndrome was diagnosed by standard clinical criteria:15,16 the severity was based upon maximum oxygen moderate 40-60%, and severe requirement-mild <40%, >60%-and the radiological severity was that adopted by Wesenberg. 17 Patent ductus arteriosus was diagnosed on clinical, radiological, and echographic findings and intraventricular haemorrhage on clinical and ultrasound evidence. Results The treated group were lighter and less mature than controls but the differences are not significant (table r). The median Apgar score at 1 min was similar in the two groups, but the median score at 5 min was significantly lower in the treated group. The difference between the groups in the lecithin-sphingomyelin ratio was not significant, but the proportion of babies with a ratio of <2’ 0 was significantly greater in the treated group (74% vs 51%). Mortality rates in the two groups were similar (table II). 67% of the treated group showed clinical evidence of