9 Barker D, Osmond C, Simmonds S, Wield G. The relation of small head circumference and thinness at birth to death from cardiovascular disease in adult life. BMJ 1993; 306: 422-26. 10 Barker D. The intrauterine origins of cardiovascular and obstructive lung disease in adult life: the Marc Danields Lecture 1990. J Roy Coil Physicians Lond 1991; 25: 129-33. 11 Ben-Shlomo Y, Davey Smith G. Deprivation in infancy or in adult life: which is more important for mortality risk? Lancet 1991; 337: 530-34. 12 Barker D, Osmond C, Golding J. Height and mortality in the counties of England and Wales. Ann Hum Biol 1990; 17: 1-6. 13 Nyström Peck M, Vågerö D: Adult body height, self-perceived health and mortality in the Swedish population. J Epidem Comm Health 1989; 43: 380-84. 14 Notkola V. Living conditions in childhood and coronary heart disease in adulthood. A mortality and morbidity study in two areas of Finland. Helsinki: The Finnish Society of Sciences and Letters, 1985. 15 Cederlöf R. The twin method in epidemiological studies on chronic disease. Stockholm: Karolonska Institute, 1966. 16 Cederlöf R, Friberg L, Lundman T. The interaction of smoking, environment and heredity and their implications for disease etiology. A report of epidemiological studies on the Swedish twin registries. Acta Med Scand 1977; 202: S49-51. 17 Vågerö D, Lundberg O. Socio-economic differentials among adults in Sweden-towards an explanation. In: Lopez A, Valkonen T,
Short
18
19 20 21 22
23
24 25 26
Casseli G, eds. Premature adult mortality in developed countries. Oxford: Oxford University Press, 1994 (in press). Fischbein S, Alin Åkerman B. Tvillingar Research Report no 29 from the Dept of Sociology. Stockholm: University of Stockholm, 1990. Wilson R. Measures of birth size at different gestational ages. Ann Hum Biol 1974; 1: 57-64. Marmot M, Shipley M, Rose G. Inequalities in death-specific explanations or a general pattern? Lancet 1984; i: 1003-06. Waaler H. Height, weight and mortality. The Norwegian experience. Acta Med Scand 1984; suppl 679. Moilanen I, Rantakallio P. The growth, development and education of Finnish twins: longitudinal follow-up study in a birth cohort from pregnancy to nineteen years of age. Growth Devel Aging 1989; 53: 145-50. Kuh D, Wadsworth M. Parental height; childhood environment and subsequent adult height in a national birth cohort. Int J Epidemiol 1989; 18: 663-68. Wilson R. Twin growth: Initial deficit, recovery, and trends in concordance from birth to nine years. Ann Hum Biol 1979; 6: 205-20. Illsley R. Social class selection and class differences in relation to stillbirths and infant deaths. BMJ 1955; ii: 1520-24. Macintyre S. A review of the social patterning and significance of measures of height, weight, blood pressure and respiratory function. Soc Sci Med 1988; 27: 327-37.
reports
Donor-specific bone marrow infusion after orthotopic liver transplantation
Donor-specific bone
marrow
infusion after organ
grafting can
induce tolerance in animals. In this randomised controlled
study we show it has no benefit in patients undergoing liver transplantation. Of 25 patients, 9 received bone marrow 5 days after a 10 day course of antithymocyte globulin. Immunosuppression was maintained with cyclosporin only. An average of z 0 rejection episodes per patient was seen in the bone marrow group compared to 3 1 in the controls. Chimerism was not found in peripheral blood or bone marrow of recipients using erythrocyte antigen markers, PCR for donor class II DNA or Y-probe in-situ hybridisation in one female
recipient of male
liver and bone
marrow.
Lancet 1994; 343: 263-65
Tolerance has been induced in a wide variety of animal models with radiotherapy, polyclonal or monoclonal
antilymphocyte
sera,
or
non-specific immunosuppressive
agents. Tolerance induction was first described in mice with skin allografts treated with a short course of antilymphocyte serum and an infusion of donor-specific bone marrow. Over the next two decades, there were reports of successful tolerance induction to renal allografts in dogs2 and in rhesus monkeys.3 Clonal deletion, clonal anergy, or active immunological suppression by T cells, natural suppressor cells, or veto cells have all been suggested as the mechanism by which marrow infusion confers tolerance. However, data from different models conflict. More recently both acute and chronic graft rejection have been significantly reduced in those kidney recipients given
post-transplant infusion of donor-specific bone marrow.4 We believe the patients described here are the first to receive donor-specific bone marrow infusions after orthotopic liver transplantation. a
Between October, 1991, and July, 1992, 25 patients had elective orthotopic liver transplantation for end-stage chronic liver disease. All patients received a postoperative 10-day course of intravenous polyclonal anti-thymocyte globulin (ATG, Merieux) at 25 mg/kg per day starting 6-18 hours after the surgical procedure. Cyclosporin was started on the fifth postoperative day at 4 mg/kg intravenously or 10 mg/kg orally. Blood cyclosporin concentration was maintained between 50 to 150 ng/mL. Maintenance prednisolone and azathioprine were not given. 9 out of 25 randomly selected patients received an infusion of cryopreserved donor bone marrow. Bone marrow was harvested immediately before liver retrieval from the specific liver donor. The red-cell depleted bone marrow was infused 5 days after the last dose of ATG at 2-3 x 108 nucleated cells per kg. Patients were randomised and consent from the donor’s family was given for bone marrow harvesting. Biopsy-proven rejection episodes were treated with three 1 g doses of intravenous methylprednisolone on 3 consecutive days. Steroid-resistant rejection was defined as continuing histological of evidence rejection after two 3-day courses of methylprednisolone and was treated with OKT3 (Cilag Biotech) 5 mg/kg per day for 5 days. Liver biopsies and blood samples were obtained on days 0, 5, 15, and 25. Additional biopsies were performed when clinically indicated. All patients were monitored clinically, haematologically, and biochemically daily when in hospital and three times a week after discharge. Recipient peripheral blood lymphocytes (PBL) were collected and stored for subsequent immunological testing. Recipient bone marrow was harvested and stored at 3 months after
transplantation. 6-month survival in the group receiving donor-specific bone marrow was 89% (8 out of 9). 1 patient died 43 days after transplantation as a result of massive intracerebral haemorrhage; liver function, coagulation profile, and platelet count were normal at the time of death, which did not appear to be related to donor bone marrow infusion. 75% (12 out of 16) of the control group survived up to 6 months. The deaths were due to sepsis, renal failure and rejection (day 25), myocardial infarction (day 33), 263
cytomegalovirus pneumonitis (day 48), and fulminant hepatitis B recurrence (day 94). In the donor bone marrow group, 1 patient developed ductopenic rejection requiring retransplantation at 16 months,1 patient died of myocardial infarction at 7 months, and 1 patient died of cryptococcal meningitis at 9 months. The surviving 6 patients were followed up for between 16 and 24 months. In the control group, 1 patient died of recurrent hepatitis C at 12 months and another of undiagnosed hepatitis at 15 months. The remaining 10 patients were also followed up for 16 to 24 months. Of the 25 patients reported, only 1 (who received bone marrow) did not have postoperative cellular rejection. In the bone marrow group, a total of 27 rejection episodes were recorded, of which 20 occurred in the first 4 postoperative weeks (3 rejection episodes per patient). Pulse doses of methylprednisolone were used in 18 cases and OKT3 was used in 2 cases. 15 rejection episodes occurred before infusion of bone marrow and were graded mild (2), moderate (10), and severe (3). 12 rejection episodes occurred after bone marrow infusion (mild 7 and moderate 5). In the control group we recorded 50 rejection episodes (3-125 per patient); 45 occurred in the first 4 postoperative weeks. 16 rejection episodes were mild, 24 were moderate and 1 was severe. Methylprednisolone pulse therapy was used in 41 cases and OKT3 was used in 2. Flow cytometry to demonstrate red-cell antigen differences between donor and recipient cells did not show any donor red cells among 30 000 immunophenotyped recipient red cells from peripheral blood between 20 and 100 days after transplantation in any of the 25 patients in this study. The sensitivity of this technique detected 1 donor cell among 200 recipients. Fluorescent in-situ hybridisation for the Y chromosome probe was used in a female recipient. No circulating male cells were seen among 3000 nucleated female cells (recipient) from peripheral blood between 30 to 80 days postoperatively or in recipient bone marrow harvested at day 90 after the transplantation (sensitivity 1 in 30). Nested PCR with sequence-specific primers for HLA class 2 determinants was used to look for evidence of donor engraftment (haematolymphoid chimerism). This technique has a sensitivity of at least 1 in 10.5 With the exception of 1 patient who showed transient evidence of donor lymphoid cells in the peripheral blood 2 days after bone marrow (by nested PCR), all other tests were negative. There was no evidence of donor-specific DNA in the peripheral blood of any patient between 32 and 100 days post-transplant or in bone marrow harvested at 90 days
post-transplant. Dual-colour flow cytometry showed that the PBL of patients undergoing orthotopic liver transplantation with ATG contained a transient population of cells in the peripheral blood which have the morphological appearance of large granular lymphocytes and are CD3- and CD56.
Although not seen in the first 5 days post-transplant, they were identified regularly up to 21 days after transplant. No patient of either group displayed any evidence of GVHD at any time after transplantation. The preliminary results of our study do not agree with reports regarding this approach to chimerism and ultimately, immunological tolerance.l-4 There was no difference in the short-term result of liver transplantation between those who received a donor-specific bone marrow infusion and those who did
264
not.
The frequency and histological severity of acute rejection episodes were similar in both groups. Only one graft was lost because of acute rejection in the control group, which suggests that the basic immunosuppressive regimen was adequate. 1 patient in each of the study groups developed histological changes compatible with chronic rejection. There was no evidence of circulating donor-specific haematopoietic products or bone marrow chimerism with erythrocyte antigen typing or Y-probe in-situ hybridisation. PCR studies for class II DNA showed donor DNA circulating only very briefly in 1 recipient of bone 2 days after the infusion. ’We do not know why our study has yielded such consistently negative results in terms of peripheral blood and bone marrow chimerism. We cannot confirm the work of Barber et al4 who, with a similar protocol in cadaver kidney grafts, reported evidence of chimerism in the peripheral blood of bone marrow recipients and controls on PCR. Differences in detail may account for our disagreement with other published work. Previous researchers have emphasised the critical timing of the bone marrow infusion with respect to the last dose of antilymphocyte globulin (ALG) and have suggested that this interval may be species-specific. In this study, bone marrow was infused 5 days after the last dose of ATG compared with 7 days in Barber’s study. Other factors are the choice of anti-lymphocyte preparation; Merieux ATG in this study differed from that used by Barber (Minnesota ALG). The cryopreservation and red-cell depletion of the bone marrow used here is a conventional method of harvesting and preparation identical to that used in our large clinical bone maikrow transplant programme. Transient, peripheral blood, circulating chimerism may have been missed in our studies because of the timing of our
marrow
immunological monitoring. Donor cells have been reported in recipient tissues many years after liver allografting without bone marrow innoculation.5 This implies a state of stable chimerism and early cell migration of donor cells from the grafted organ may be responsible. We have not yet examined skin or lymph-node tissues from any of our recipients for donortype cells. The exact significance of chimerism in this setting has yet to be elucidated. Whether chimerism is a marker of profound donor-specific unresponsiveness, or a marker of a more fragile specific or non-specific immunological equilibrium, or even simply recipient immunological anergy produced by the cumulative effects of years of non-specific immunosuppressive drugs has yet be determined. The transient population of CD3- and CD56+ large granular lymphocytes seen in both bone marrow recipients and controls early post-operatively is obscure. Such cells are frequently seen after intensive myeloblative therapy. The cells were seen in controls and bone marrow recipients at times after operation but before bone marrow infusion. This suggests they are unrelated to the donor bone marrow but are more likely to be re-emergent recipient cells or possibly donor cells derived from the liver graft itself. Our results do not encourage or justify the eventual withdrawal of immunosuppressive therapy in the hope that donor-specific unresponsiveness has been achieved.
to
References 1
Wood ML, Monaco AP, Gozzo JJ, Liegeois A. Use of allogeneic bone marrow for induction of tolerance with ALS I dose and timing. Transplant Proc 1971; 3: 676.
Caridis DT, Liegeosis A, Barrett I, Monaco AP. Enhanced survival of canine renal allografts of ALS treated dogs given bone marrow. Transplant Proc 1973; 5: 671. 3 Thomas FT, Carver FM, Foil MB, et al. Long term incompatible kidney survival in outbred higher primates without chronic 2
4
5
immunosuppression. Ann Surg 1983; 198: : 370. Barber WM, Mankin JA, Laskow DA, et al. Long term results of a controlled prospective study with transfusion of donor specific bone marrow in 57 cadaver renal allograft recipients. Transplantation 1991; 51: 70. Starzl TE, Demetris AJ, Murase N, et al. Cell migration, chimerism, and graft acceptance. Lancet 1992; 339: 1579-82.
University Department of Surgery, Royal Free Hospital, Pond Street, London NW3 2QG, UK (K Rolles FRCS, A K Burroughs FRCP, B R Davidson FRCS, Prof H G Prentice FRCPath, M D Hamon NRCPath) Correspondence to: Mr K Rolles
Epstein-Barr virus in Hodgkin’s disease and site of origin of tumour
Epstein-Barr virus (EBV) may be involved in the pathogenesis Hodgkin’s disease. We investigated whether EBV in Hodgkin’s disease is related to the site of origin of the tumour. In 40 patients with stageI disease, there was a significant association between EBV latent membrane protein (LMP-1) expression and presentation in neck lymph nodes. There was no association in stage II-IV disease (57 cases). Nodular sclerosing subtype was rarely associated with LMP-1 expression. In some cases of Hodgkin’s disease of mixed cellularity or lymphocyte predominant subtype originating in the neck, EBV may be an important aetiological co-factor. Lancet 1994; 343: 265-66
of
I I)IU UI cases.
Table : Site-related expression of LMP-1 in Hodgkin’s disease*
(CSI-4) against LMP-1. Site-related expression of LMP-1 was analysed separately in stage I disease and stage II-IV disease because only in stage I disease could the site of origin of the tumour be determined with certainty. The X2 test with Yates’ correction and multiple logistic regression analysis were used. For stage I disease LMP-1 expression by ReedSternberg cells was significantly associated with presentation in neck nodes compared with non-neck nodes (p<0-01) (table). The odds ratio was 17-7 (95% CI 2-01-156-5). For stage II-IV disease, there was no such association. Nodular sclerosing subtype was rarely associated with LMP-1 expression (p < 0-001 for stage I and p < 0.01for stages II-IV). In stage I disease female gender but this is was related to absence of LMP-1 (p<0-01), to number of women with attributable the probably large nodular sclerosing disease. LMP-1 expression was not associated with gender or age in stage 11-IV disease. For stage I disease, the association between site and LMP-1 status remained statistically significant after multiple logistic regression analysis adjusting for sex, age, andhistological subtype (p < 0 05, odds ratio 51 -94,95% CI 2-09-1291). Site, sex, and histological subtype were all significant independent predictors of LMP-1 status for stage I disease. In common with other studies,1-3 we found that LMP-11 was expressed more in mixed cellularity than in other subtypes, and our percentage of positive mixed cellularity cases was
Epstein-Barr virus (EBV) may be involved in the pathogenesis of Hodgkin’s disease.1-3 EBV replicates in the epithelium of the oropharnyx with shedding of virus particles into the saliva. B lymphocytes are infected as they pass through lymphoid tissue in this area. Most normal seropositive individuals shed virus continuously at a constant rate that correlates with the numbers of circulating infected B cells.4 In infectious mononucleosis, the tonsils and cervical lymph nodes, but not inguinal lymph nodes and spleen, contain many Reed-Sternberg-like cells showing strong latent membrane protein (LMP-1) expression.5 Detection of EBV in Hodgkin’s disease may be related to presentation in nodes draining tonsillar or oropharyngeal lympho-epithelial tissue. We investigated whether the presence of EBV in Hodgkin’s disease is related to the site of origin of the tumour and to other factors. 40
patients with stage I (28 males) and 57 with stage II-IV (35 males) Hodgkin’s disease were selected from pathology department files. All cases had been fully staged with the modified Ann-Arbor system. The stage I cases represented all such cases diagnosed in our department during 1979-90 in which tissue was still available for analysis. Cases of stage II-IV disease were chosen to give a similar distribution of sites and subtypes to the stage I data. Clinical data were obtained from the files of the Scotland and Newcastle Lymphoma Group and patient’s notes. We analysed site of biopsy, stage, histological subtype, age, and sex. Presence of EBV in Reed-Sternberg cells was assessed by immunohistological labelling of the first diagnostic biopsy specimen with antibodies
similar. The
proportion of nodular sclerosing
that were LMP-1positive in our study was lower than that reported,1-3 which may reflect selection for and over-representation of the rare stage I, nodular sclerosing cases that were all LMP-1 negative. When our results were analysed with the exclusion of stage I disease, the proportion of EBV-positive, nodular sclerosing cases was similar to that in other studies. Other factors, such as differences in classification, may also play a part in explaining these differences. Not all cases arising in neck nodes expressed LMP-1, which may be explained by the fact that not all neck nodes drain the oropharynx. The supraclavicular nodes do not drain the oropharynx directly, yet are a common site of origin of nodular sclerosing Hodgkin’s disease. ReedSternberg cells in this subtype rarely express LMP-1. We could not fully address this question because of lack of information about the exact group of cervical nodes cases
sampled by biopsy. No correlation between site of biopsy and LMP-1 status could be detected in stage II-IV disease, possibly because biopsy site does not necessarily indicate site of origin of disease in these cases, and because Hodgkin’s disease patients retain the EBV status of Reed-Sternberg cells as the tumour spreads throughout the body.6 We propose that Hodgkin’s disease associated with EBV more commonly arises in the neck, as supported by our stage I data. On spread of disease to other sites, the tumour cells 265