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Antiepithelial cell antibodies do not impair paediatric renal allograft survival but appear to be associated with acute viral infections
Trunsplnnt I?nmunolo,q
1996; 4: 19-22
Antiepithelial cell antibodies do not impair paediatric renal allograft survival but appear to be associated with acute viral infections Susan E Spencer”, Noel McCarthyb, B Hannigand, Denis Gill”, Mervyn R Taylor”, Denis Murphy’ and J Joseph Walsheb “Transplant Immunology, bDepartnaent of Nephrology and ‘Department of Transplant Surgery, Beaumont Hospital, Dublin, ‘Universifv of Ulster at Coleraine, Co. Londonderry and “National Children’s Hospital, Harcourt Street, Dublin Received
31 May
1995: rcviscd manuscript
Abstract:
accepted 29 August
1995
There is a reported association between antiepithclial
allograft loss in paediatric
recipients. Our unit experienced
ccl1 (AEC)
antibodies and incrcascd renal
a dramatic fall in I -year graft survival so we
undertook a study to determine if AEC antibodies could account for such loses. children and adults as well as a group of individuals with serologically to determine antibodies
the prevalence
and possible aetiology
in a microcytotoxicity
of these antibodies in our paediatric correlation
of these antibodies.
test win g a lung epithelial recipients
bctwcen the prcscncc of AEC
found the antibody was more prevalent
antibody and allo@i
higher prcvalcncc
than in our healthy control groups (p = 0.00003). respiratory
syncytial virus and AEC
but we found no
loss. Within the control populations,
in children than in adults (/I < 0.0001).
antibody had a significantly
as target. The prevalence
was similar to that reported elsewhere
age banding pattern, with antibody being present in 50%~ of children
AEC
Set-a were screened for AEC
cell line (AS49)
increasing age. so that by the age of I6 years the sewprevalence Howcvcr.
We also studied healthy
proven viral infection in an attempt
we
We also found a strong
under IO years and declining
with
was similar to that found in our adults. in individuals
with active viral infection
A positive association uas noted bctwccn rubella and
antibody presence and a negative nasociatton v+ith varicella
Lostcr.
We conclude that AEC antibodies do not correlate with incrcaacd pacdiatric renal allopraft loss but appear to be linked to certain viral infections.
Introduction Recent reports have associated a novel antibody with early graft loss in paediatric recipients.” In one such study, of the eight children whose grafts were lost. six were found to have an IgM antibody directed against an epithelial cell line, while none of those with functioning grafts had this antibody.’ Because we also noted a significant fall in our l-year paediAddress for correspondence: J Joseph Walshe. Department of Nephrology, Beamnont Hospital. PO Box 1297. Dublin 9, Ireland. 0
Arnold
I996
atric graft survival from 847r to 64%. we elected to ascertain if antiepithelial cell (AEC) antibodies correlated with graft loss in our patients. We also decided to study the prevalence of this antibody in the general population and see if it might be related to viral infection as postulated by others.’
Objectives To determine if AEC antibodies were present in our paediatric transplant patients and, if so. whether their presence correlated
20
SE Spencer
et al.
with graft loss. To determine their prevalence in healthy individuals. TO ascertain if certain viral infections may be associated with the production of AEC antibody.
Materials and methods Twenty-three patients under the age of 15 years at the time of transplant and who received 27 transplants were included in the study. One transplant was from a haploidentical parent and the remainder were of cadaveric origin. The mean age of our recipients was 10 years (range 3-15 years); 16 were male and seven female. Two of the patients had had a previous transplant at the time the study period commenced. Both of these were parent to child transplants that had failed at 3 and 5 years, respectively, as a result of chronic rejection. Four children received two renal transplants during the study period. Serum samples for screening Sera were tested at three time intervals: the time of transplant; the time of graft failure or within 1 month of transplantation whichever was appropriate; during the period of the current study. Blood transfusions and HL.A antibodies Prior to transplantation, all but three children had received blood transfusions (mean, 5 units) and 21 of 24 had no detectable clinically significant anti-HLA antibodies as determined by screening against a lymphocyte panel in the presence and absence of the reducing agent dithiothreitol (DTT).4 None of the three sensitized patients was highly sensitized (> 85% panel reactive antibodies (PRA); the range of PRA was lO-50%). Immunosuppression The immunosuppression protocol was cyclosporine based, and used in conjunction with either steroids (dual therapy) or azathioprine and prednisone (triple therapy). In 12 transplants dual therapy was used and triple therapy was used in another 12. There were three cases where azathioprine and prednisone only were used as these grafts never achieved sufficient function for cyclosporine therapy to be introduced. In one case, Orthoclone OKT3 was used for the treatment of severe cellular rejection. Donor kidney allocation The minimum age for acceptance as a donor was 3 years and 60% of the donors in the group were under the age of 15 years. Where a donor was aged 11 years or under, kidneys were allocated on the basis of recipient age rather than by HLA matching and priority was given to recipients aged less than 15 years. This was in keeping with the policy in operation at the time within the UK Transplant Service. Over half of the kidneys were received through this organ exchange network and the mean number of HLA mismatches was 2.7. There was no difference in the mean number of mismatches betwen those receiving imported kidneys and locally retrieved kidneys (2.8 and 2.5, respectively). Control populations Blood donors were taken as representative of a healthy adult population and aliquots of serum from 290 blood donations from the National Blood Transfusion Service, Dublin, were Transplant Immur101ogy 1996; 4: 19-22
collected for testing. Samples were also taken from 950 school children aged between 4 and 18 years. The schools were from the greater Dublin area as well as from outlying districts so that both rural and urban areas were covered. All samples were taken between April and September 1992 inclusive as part of an existing study of the prevalence of Toxicaru in school children.’ Sera were also obtained from the National Virus Reference Laboratory representing a range of ten viral infections. Serum from 49 individuals who had serological evidence of viral infection were submitted for AEC antibody screening. These samples covered a range of viral infections (see Table 4). Seventeen of these individuals were adults and 32 were aged 15 years or younger. All samples were tested in a blind fashion with only a unique number used to identify each specimen. Cytotoxicity test using A549 cell line Screening was carried out using the microcytotoxicity test reported by Martin et aL3 Epithelial cells of the A549 cell line (CCL185) were maintained in culture at 37°C in 5% CO2 in DMEM/F12 nutrient mix containing 10% fetal calf serum (FCS), L-glutamine (200 mM) and penicillin/streptomycin solution (10 000 IU/ml). A confluent growth of cells was achieved after 2-3 days of incubation. Adherent cells were remove from the flask by addition of 3 ml of 0.5% (w/v) trypsin + 0.2% EDTA and incubation at 37°C for 5 min. Cells were suspended in DMEM/F12 + 10% FCS and centrifuged at 300 x g for 5 min. The cell pellet was resuspended in DMElWF12 + 10% FCS and the cell count adjusted to 0.54.7 x lO’/ml. Terasaki trays were soaked in 70% methanol for 30 min, drained and allowed to air-dry. Then 10 pl of the cells were plated onto the trays and allowed to adhere by overnight incubation in a humidified box in the CO2 incubator. Supernatant medium was removed by vigorous flicking of the plate. Serum (2 ~1) was added to the cells and incubated at 4°C for 1 h; 5 p,l of rabbit complement then added and the plates incubated for a further 1 h at 22’C. Cells were visualized with a staining cocktail of ethidium bromide (1 mg/ml), acridine orange (0.3 mg/ml) in phosphate-buffered saline/bovine serum albumin and calligraphy ink. Cell viability was assessed and graded, with cell death greater than 10% being taken as positive. Dithiothreitol treatment of serum Positive sera were rescreened using 0.01 M dithiothreitol (DTT) added to sera (1:9) prior to the addition of serum to the epithelial cells. Antibodies digested by DTT treatment were designated IgM class.4 Statistics All assays were carried out in sextuplicate and results from the four centre wells were recorded. Statistical analysis was carried out using Fisher’s exact p; a p-value < 0.05 was taken as significant.
Results Paediatric transplant recipients Ten of the 23 children (43%) were positive for AEC antibody at some time during the study period. Six (26%) were positive at the time of transplant, and four seroconverted subsequently. Three of the patients who converted did so within 6 weeks of
Antiepithelial
Table 1 Preformed
AK
antibodies
in relation to graft outcome Graft functioning
AEC positi\#e at transplant AEC negative
at transplant
cell antibodies
Graft failed
3
3
I4
I
,I = NS.
Table 2 AEC antibodies
at any time period in relation to graft outcome Graft functioning
AEC pre- or post transplant AEC negative
Graft failed
5
3
I2
5
Note: Grafts that had already failed prior to the formation of AEC antibody were excluded IIWO ca\es). ,I = NS.
transplantation. and the fourth after 8 months. Of the six patients positive for AEC at the time of transplant, three lost their grafts and three grafts are still functioning (JJ = 0.38) (see Table I ). Of the four patients who became AEC antibody positive post-transplant, two conversions occurred 4-6 weeks after grafts had already failed, and the remaining two have stable renal function 4 years post-transplant. Analysis of the paediatric data found no difference in graft survival associated with the presence of AEC antibody formed at any time (p = 0.51) (see Table 2). Control groups AEC antibody was detected in the serum of 319 of the 950 healthy school children, giving a prevalence of 33.5% in this group. Of the 290 blood donors tested, 42 (14%) were positive, giving a highly significant difference in prevalence of the antibody in children compared to an adult population @ = 0.00000 I ). There was no difference in prevalence of AEC antibody between the healthy school children and our paediatric recipient population @ = 0.2). Analysis of the influence of age on the presence of AEC antibody showed a strong age banding pattern. with the percentage of children positive for the antibody peaking by the age of IO years and falling off with increasing age. After the age of 15 years. the prevalence of antibody is identical to that seen in our adult control group (I 3.5% vs 14%) (see Table 3). Screening of serum samples from all patients with serologitally proven viral infections showed that 29 of 49 (58%) were AEC antibody positive compared to 28% of the combined
Table 3 Prevalence
of AEC antibody
in relation Age froupa
in renal allograft
survival
and
viral
infection
adult and child controls (JI = 0.00003). When these results are analysed on the basis of age (< I5 years vs > 15 years), 55% (18 of 32) of children with viral infections are positive for AEC antibody compared 33.5% in healthy controls (p = 0.01) and 65% of adults (11 of 17) compared with 14% in the blood donor population (p = 0.000009). Statistical analysis of the virology group as a whole also showed that respiratory syncytial virus (RSV) and rubella infections were positively associated with the presence of AEC antibodies 07 = 0.03). Whilst cytomegalovirus (CMV) did not appear to be linked with AEC antibody when looked at overall. if the group is divided into adults (six cases) and children (one case) then an association with AEC antibody can be found. Five of the six adults who had a proven IgM anti-CMV response had AEC antibody and this was significantly different to that seen in the adult control group (~7= 0.0004). There was a negative association between infection with varicella roster (VZV) and AEC antibody with only one of eight individuals positive for VZV being positive for AEC (p = 0.004). The patient numbers and results of AEC screening are given in Table 4.
Discussion Our paediatric transplant recipients were found to have AEC with a seroprevalence similar to that previously reported.‘,’ The presence of preformed AEC antibodies in the children or their production post-transplantation was not. however, associated with increased renal allograft loss. unlike the findings of both Harmer and Martin.‘.‘.7.x Our results are, on the other hand. in agreement with those of Nauta et ul.’ in their random study of a group of children who were transplanted in The Netherlands. The paediatric control consisted of 950 school children with an overall prevalence of AEC antibody of 33.5%. This is sig-
Table 4 Presence of AEC antibody Virus
Herpes
simplex
in association
No. of patients
AEC positive
with viral infections
AEC negative
p-value
7
4
3
NS
Measles CMV vzv
4 7 8
2 5
I
2 2 7
NS NS 0.005
Mumps Rubella Parve B 19 RSV
6 6 2 9
3 6 0 8
3 0 2 I
NS 0.03 NS 0.04
NS. not significant.
to age group (years) 4-S
6-10
I I-15
16-18
> 18 290
Number per age group
38
470
367
7s
Number of AK
24
234
I38
IO
42
50
50
37
14
I4
Pcrcentape
Tnmsplar~t
positr\e
AEC positive per age group
Irnm~mlo,~y
1996: 4: 19-22
21
22
SE Spencer et al.
nificantly different fp = 0.0002) to that found by Deal et al. in their study of a small group of 40 school children and 35 children attending the paediatric Accident and Emergency Dep~ment in Guy’s Hospital in which they found the prevalence of AEC to be 67% and 62% respectively.* In the adult control group we found the seroprevalence to be significantly lower than that seen in children. We also noted a very strong association between age and antibody prevalence. The virology positive samples, al~ough a relatively small and heterogeneous group, provide data suggesting an association between AEC antibody and infection with certain viral agents. RSV and rubella were positively associated with AEC antibody while VZV was negatively associated. The association of AEC with viral infections was more marked in adults, with nearly 60% of adults with infection being AEC positive compared to only 14% in healthy adults. These are new findings with no equivatent data reported in the literature. Although the technique we used was a cytotoxicity test, while Harmer applied a flow cytome~c method, prelimina~ results of a collaborative study of AEC antibodies in six renal transplant units within the UK and IrelandlO showed 80% concordance overall between the two methods. The cytotoxicity test is more sensitive than flow cytometry and allowing for this increased sensitivity there was only a 3% discrepancy rate in that study. lo In addition, our samples were tested independently in two centres and showed excellent concordance. The timing of serum samples could be another factor affecting the results. Deal et al. have reported that the timing of samples in respect to ~~spl~tation is impo~~t, and that the failure of Nauta et al. to show an association between AEC and graft rejection was related to both the timing of samples and the fact that many of the children tested had stable grafts.’ However, all our patients were tested on samples taken on the day of transplant so that we can assess the impact of AEC antibody present in the circulation at the time of transplant and, furthermore, all grafts were followed from time of transplant irrespective of whether the graft was functioning or had failed. In view of the observation made by Harmer et al. that the dis~ibution of AEC antibody within families may be linked to a possible viral aetiology,* and our finding of an association with rubella and RSV one possible difference between the two populations could be the time interval from viral infection to screening for AEC antibody. If AEC antibody is associated with active viral infection, it may be that the London patients had very recent infection and ours may have been more remote. Harmer et al. found five nontransplanted children with AEC antibody who had epithelial skin lesions,’ which would support the idea of active viralinfection. Our findings show that AEC occurs in a large percentage of normal healthy children and its presence may be linked to rubella and RSV infection or other viruses. It may be that the antibody detected using A549 cells is, in fact, antibody directed against a viral protein present on the A549 used as the target cell and, in the light of our findings of an association
Transport
Immunology
1996; 4: 19-22
with more than one virus, the antibody may be against a protein common to a host of viruses. Our virology positive group was relatively small and it is possible that more extensive screening of such samples would find other viruses associated with AEC seroconversion. In our paediatric recipient group we found that one child whose sera were screened sequentially over a prolonged period became seropositive for AEC antibody at the time of developing IgM anti-CMV antibody. This, added to the finding of a positive association with CMV in our adult population, suggests that anti-CMV antibodies may also be implicated in the AEC antibodies detected in the assay. As a result of this study, over the past 3 years it has been our policy not to take the AEC antibody status into account when selecting recipients for ~ansplantation and during this time period the l-year paediatric graft survival has been 84%. In conclusion, we believe a decision not to transplant in cases where a patient is AEC positive is unjustified and that these antibodies are in all probability an epiphenomenon. Acknowledgement This work has been funded in part by the Kidney Transplant Foundation of Ireland.
References 1 Harmer A, Rigden S, Koffman C, Welsh K. Preliminary report: dramatic rise in renal allograft failure rate. L.uncet 1990; 335: 1184. 2 Harmer AW, Haskard D, Koffman G, Welshe KI. Novel antibodies associated with unexplained loss of renal allografts. TransplantInt 1990; 3: 6669. 3 Martin S, Brenchley P, Postlethwaite R, Johnston R, Dyer P. Detection of anti-epithelial cell antibodies in association with paediatric renal transplant failure using a novel microcytotoxicity assay. Tissue Antigens 1991; 37: 152-53. 4 Chapman JR, Taylor C, Ting A, Morris PJ. Immunoglobulin class and specificity of antibodies causing positive crossmatches: relationship with renal transplant outcome. ~r~~~~zff~fa~~~~1986; 42: 608. 5 Holland C, O’Lorcain P, Taylor MRH, Kelly A. Seroepidemiology of toxicariasis in school children. Parasitolugy f995 (in press). 6 Deal JE, Harmer A, Welsh KI, Rigden SPA. Seroprevalence of an anti-epithelial cell antibody in normal children and children with end-stage renal disease (ESRD). Puediatr Nephrul 1991; 5: C50. 7 Deal JE, Rigden SPA, Harmer A, Koffman CG, Welsh IU. Renal allograft failure and antibodies to epithelial cells. Lartcet 1992; 339: 941. 8 Harmer A, Rigden SPA, Hartley B, Koffman CG, Welsh KI. Close association of renal allograft failure and antibody directed against epithelial cells. Transplant Proc 1991; 23: 412-13. 9 Nauta J, Wolffe ED, Claas FHJ, Schroder CH, Donckerwolcke RA. Renal allograft rejection and antibodies to epithelial cells. I.uncet 1992; 339: 503. 10 Harmer A, Liggett H, Norman PJ, Martin S. Microcytotoxicity and flow cytometry assays for anti-epi~elial cell antibodies compared in a national collaborative study of paediatric renal transplants. Eur J lmm~ogenet 1993; 20: 447.
1996; 4: 19-22
Antiepithelial cell antibodies do not impair paediatric renal allograft survival but appear to be associated with acute viral infections Susan E Spencer”, Noel McCarthyb, B Hannigand, Denis Gill”, Mervyn R Taylor”, Denis Murphy’ and J Joseph Walsheb “Transplant Immunology, bDepartnaent of Nephrology and ‘Department of Transplant Surgery, Beaumont Hospital, Dublin, ‘Universifv of Ulster at Coleraine, Co. Londonderry and “National Children’s Hospital, Harcourt Street, Dublin Received
31 May
1995: rcviscd manuscript
Abstract:
accepted 29 August
1995
There is a reported association between antiepithclial
allograft loss in paediatric
recipients. Our unit experienced
ccl1 (AEC)
antibodies and incrcascd renal
a dramatic fall in I -year graft survival so we
undertook a study to determine if AEC antibodies could account for such loses. children and adults as well as a group of individuals with serologically to determine antibodies
the prevalence
and possible aetiology
in a microcytotoxicity
of these antibodies in our paediatric correlation
of these antibodies.
test win g a lung epithelial recipients
bctwcen the prcscncc of AEC
found the antibody was more prevalent
antibody and allo@i
higher prcvalcncc
than in our healthy control groups (p = 0.00003). respiratory
syncytial virus and AEC
but we found no
loss. Within the control populations,
in children than in adults (/I < 0.0001).
antibody had a significantly
as target. The prevalence
was similar to that reported elsewhere
age banding pattern, with antibody being present in 50%~ of children
AEC
Set-a were screened for AEC
cell line (AS49)
increasing age. so that by the age of I6 years the sewprevalence Howcvcr.
We also studied healthy
proven viral infection in an attempt
we
We also found a strong
under IO years and declining
with
was similar to that found in our adults. in individuals
with active viral infection
A positive association uas noted bctwccn rubella and
antibody presence and a negative nasociatton v+ith varicella
Lostcr.
We conclude that AEC antibodies do not correlate with incrcaacd pacdiatric renal allopraft loss but appear to be linked to certain viral infections.
Introduction Recent reports have associated a novel antibody with early graft loss in paediatric recipients.” In one such study, of the eight children whose grafts were lost. six were found to have an IgM antibody directed against an epithelial cell line, while none of those with functioning grafts had this antibody.’ Because we also noted a significant fall in our l-year paediAddress for correspondence: J Joseph Walshe. Department of Nephrology, Beamnont Hospital. PO Box 1297. Dublin 9, Ireland. 0
Arnold
I996
atric graft survival from 847r to 64%. we elected to ascertain if antiepithelial cell (AEC) antibodies correlated with graft loss in our patients. We also decided to study the prevalence of this antibody in the general population and see if it might be related to viral infection as postulated by others.’
Objectives To determine if AEC antibodies were present in our paediatric transplant patients and, if so. whether their presence correlated
20
SE Spencer
et al.
with graft loss. To determine their prevalence in healthy individuals. TO ascertain if certain viral infections may be associated with the production of AEC antibody.
Materials and methods Twenty-three patients under the age of 15 years at the time of transplant and who received 27 transplants were included in the study. One transplant was from a haploidentical parent and the remainder were of cadaveric origin. The mean age of our recipients was 10 years (range 3-15 years); 16 were male and seven female. Two of the patients had had a previous transplant at the time the study period commenced. Both of these were parent to child transplants that had failed at 3 and 5 years, respectively, as a result of chronic rejection. Four children received two renal transplants during the study period. Serum samples for screening Sera were tested at three time intervals: the time of transplant; the time of graft failure or within 1 month of transplantation whichever was appropriate; during the period of the current study. Blood transfusions and HL.A antibodies Prior to transplantation, all but three children had received blood transfusions (mean, 5 units) and 21 of 24 had no detectable clinically significant anti-HLA antibodies as determined by screening against a lymphocyte panel in the presence and absence of the reducing agent dithiothreitol (DTT).4 None of the three sensitized patients was highly sensitized (> 85% panel reactive antibodies (PRA); the range of PRA was lO-50%). Immunosuppression The immunosuppression protocol was cyclosporine based, and used in conjunction with either steroids (dual therapy) or azathioprine and prednisone (triple therapy). In 12 transplants dual therapy was used and triple therapy was used in another 12. There were three cases where azathioprine and prednisone only were used as these grafts never achieved sufficient function for cyclosporine therapy to be introduced. In one case, Orthoclone OKT3 was used for the treatment of severe cellular rejection. Donor kidney allocation The minimum age for acceptance as a donor was 3 years and 60% of the donors in the group were under the age of 15 years. Where a donor was aged 11 years or under, kidneys were allocated on the basis of recipient age rather than by HLA matching and priority was given to recipients aged less than 15 years. This was in keeping with the policy in operation at the time within the UK Transplant Service. Over half of the kidneys were received through this organ exchange network and the mean number of HLA mismatches was 2.7. There was no difference in the mean number of mismatches betwen those receiving imported kidneys and locally retrieved kidneys (2.8 and 2.5, respectively). Control populations Blood donors were taken as representative of a healthy adult population and aliquots of serum from 290 blood donations from the National Blood Transfusion Service, Dublin, were Transplant Immur101ogy 1996; 4: 19-22
collected for testing. Samples were also taken from 950 school children aged between 4 and 18 years. The schools were from the greater Dublin area as well as from outlying districts so that both rural and urban areas were covered. All samples were taken between April and September 1992 inclusive as part of an existing study of the prevalence of Toxicaru in school children.’ Sera were also obtained from the National Virus Reference Laboratory representing a range of ten viral infections. Serum from 49 individuals who had serological evidence of viral infection were submitted for AEC antibody screening. These samples covered a range of viral infections (see Table 4). Seventeen of these individuals were adults and 32 were aged 15 years or younger. All samples were tested in a blind fashion with only a unique number used to identify each specimen. Cytotoxicity test using A549 cell line Screening was carried out using the microcytotoxicity test reported by Martin et aL3 Epithelial cells of the A549 cell line (CCL185) were maintained in culture at 37°C in 5% CO2 in DMEM/F12 nutrient mix containing 10% fetal calf serum (FCS), L-glutamine (200 mM) and penicillin/streptomycin solution (10 000 IU/ml). A confluent growth of cells was achieved after 2-3 days of incubation. Adherent cells were remove from the flask by addition of 3 ml of 0.5% (w/v) trypsin + 0.2% EDTA and incubation at 37°C for 5 min. Cells were suspended in DMEM/F12 + 10% FCS and centrifuged at 300 x g for 5 min. The cell pellet was resuspended in DMElWF12 + 10% FCS and the cell count adjusted to 0.54.7 x lO’/ml. Terasaki trays were soaked in 70% methanol for 30 min, drained and allowed to air-dry. Then 10 pl of the cells were plated onto the trays and allowed to adhere by overnight incubation in a humidified box in the CO2 incubator. Supernatant medium was removed by vigorous flicking of the plate. Serum (2 ~1) was added to the cells and incubated at 4°C for 1 h; 5 p,l of rabbit complement then added and the plates incubated for a further 1 h at 22’C. Cells were visualized with a staining cocktail of ethidium bromide (1 mg/ml), acridine orange (0.3 mg/ml) in phosphate-buffered saline/bovine serum albumin and calligraphy ink. Cell viability was assessed and graded, with cell death greater than 10% being taken as positive. Dithiothreitol treatment of serum Positive sera were rescreened using 0.01 M dithiothreitol (DTT) added to sera (1:9) prior to the addition of serum to the epithelial cells. Antibodies digested by DTT treatment were designated IgM class.4 Statistics All assays were carried out in sextuplicate and results from the four centre wells were recorded. Statistical analysis was carried out using Fisher’s exact p; a p-value < 0.05 was taken as significant.
Results Paediatric transplant recipients Ten of the 23 children (43%) were positive for AEC antibody at some time during the study period. Six (26%) were positive at the time of transplant, and four seroconverted subsequently. Three of the patients who converted did so within 6 weeks of
Antiepithelial
Table 1 Preformed
AK
antibodies
in relation to graft outcome Graft functioning
AEC positi\#e at transplant AEC negative
at transplant
cell antibodies
Graft failed
3
3
I4
I
,I = NS.
Table 2 AEC antibodies
at any time period in relation to graft outcome Graft functioning
AEC pre- or post transplant AEC negative
Graft failed
5
3
I2
5
Note: Grafts that had already failed prior to the formation of AEC antibody were excluded IIWO ca\es). ,I = NS.
transplantation. and the fourth after 8 months. Of the six patients positive for AEC at the time of transplant, three lost their grafts and three grafts are still functioning (JJ = 0.38) (see Table I ). Of the four patients who became AEC antibody positive post-transplant, two conversions occurred 4-6 weeks after grafts had already failed, and the remaining two have stable renal function 4 years post-transplant. Analysis of the paediatric data found no difference in graft survival associated with the presence of AEC antibody formed at any time (p = 0.51) (see Table 2). Control groups AEC antibody was detected in the serum of 319 of the 950 healthy school children, giving a prevalence of 33.5% in this group. Of the 290 blood donors tested, 42 (14%) were positive, giving a highly significant difference in prevalence of the antibody in children compared to an adult population @ = 0.00000 I ). There was no difference in prevalence of AEC antibody between the healthy school children and our paediatric recipient population @ = 0.2). Analysis of the influence of age on the presence of AEC antibody showed a strong age banding pattern. with the percentage of children positive for the antibody peaking by the age of IO years and falling off with increasing age. After the age of 15 years. the prevalence of antibody is identical to that seen in our adult control group (I 3.5% vs 14%) (see Table 3). Screening of serum samples from all patients with serologitally proven viral infections showed that 29 of 49 (58%) were AEC antibody positive compared to 28% of the combined
Table 3 Prevalence
of AEC antibody
in relation Age froupa
in renal allograft
survival
and
viral
infection
adult and child controls (JI = 0.00003). When these results are analysed on the basis of age (< I5 years vs > 15 years), 55% (18 of 32) of children with viral infections are positive for AEC antibody compared 33.5% in healthy controls (p = 0.01) and 65% of adults (11 of 17) compared with 14% in the blood donor population (p = 0.000009). Statistical analysis of the virology group as a whole also showed that respiratory syncytial virus (RSV) and rubella infections were positively associated with the presence of AEC antibodies 07 = 0.03). Whilst cytomegalovirus (CMV) did not appear to be linked with AEC antibody when looked at overall. if the group is divided into adults (six cases) and children (one case) then an association with AEC antibody can be found. Five of the six adults who had a proven IgM anti-CMV response had AEC antibody and this was significantly different to that seen in the adult control group (~7= 0.0004). There was a negative association between infection with varicella roster (VZV) and AEC antibody with only one of eight individuals positive for VZV being positive for AEC (p = 0.004). The patient numbers and results of AEC screening are given in Table 4.
Discussion Our paediatric transplant recipients were found to have AEC with a seroprevalence similar to that previously reported.‘,’ The presence of preformed AEC antibodies in the children or their production post-transplantation was not. however, associated with increased renal allograft loss. unlike the findings of both Harmer and Martin.‘.‘.7.x Our results are, on the other hand. in agreement with those of Nauta et ul.’ in their random study of a group of children who were transplanted in The Netherlands. The paediatric control consisted of 950 school children with an overall prevalence of AEC antibody of 33.5%. This is sig-
Table 4 Presence of AEC antibody Virus
Herpes
simplex
in association
No. of patients
AEC positive
with viral infections
AEC negative
p-value
7
4
3
NS
Measles CMV vzv
4 7 8
2 5
I
2 2 7
NS NS 0.005
Mumps Rubella Parve B 19 RSV
6 6 2 9
3 6 0 8
3 0 2 I
NS 0.03 NS 0.04
NS. not significant.
to age group (years) 4-S
6-10
I I-15
16-18
> 18 290
Number per age group
38
470
367
7s
Number of AK
24
234
I38
IO
42
50
50
37
14
I4
Pcrcentape
Tnmsplar~t
positr\e
AEC positive per age group
Irnm~mlo,~y
1996: 4: 19-22
21
22
SE Spencer et al.
nificantly different fp = 0.0002) to that found by Deal et al. in their study of a small group of 40 school children and 35 children attending the paediatric Accident and Emergency Dep~ment in Guy’s Hospital in which they found the prevalence of AEC to be 67% and 62% respectively.* In the adult control group we found the seroprevalence to be significantly lower than that seen in children. We also noted a very strong association between age and antibody prevalence. The virology positive samples, al~ough a relatively small and heterogeneous group, provide data suggesting an association between AEC antibody and infection with certain viral agents. RSV and rubella were positively associated with AEC antibody while VZV was negatively associated. The association of AEC with viral infections was more marked in adults, with nearly 60% of adults with infection being AEC positive compared to only 14% in healthy adults. These are new findings with no equivatent data reported in the literature. Although the technique we used was a cytotoxicity test, while Harmer applied a flow cytome~c method, prelimina~ results of a collaborative study of AEC antibodies in six renal transplant units within the UK and IrelandlO showed 80% concordance overall between the two methods. The cytotoxicity test is more sensitive than flow cytometry and allowing for this increased sensitivity there was only a 3% discrepancy rate in that study. lo In addition, our samples were tested independently in two centres and showed excellent concordance. The timing of serum samples could be another factor affecting the results. Deal et al. have reported that the timing of samples in respect to ~~spl~tation is impo~~t, and that the failure of Nauta et al. to show an association between AEC and graft rejection was related to both the timing of samples and the fact that many of the children tested had stable grafts.’ However, all our patients were tested on samples taken on the day of transplant so that we can assess the impact of AEC antibody present in the circulation at the time of transplant and, furthermore, all grafts were followed from time of transplant irrespective of whether the graft was functioning or had failed. In view of the observation made by Harmer et al. that the dis~ibution of AEC antibody within families may be linked to a possible viral aetiology,* and our finding of an association with rubella and RSV one possible difference between the two populations could be the time interval from viral infection to screening for AEC antibody. If AEC antibody is associated with active viral infection, it may be that the London patients had very recent infection and ours may have been more remote. Harmer et al. found five nontransplanted children with AEC antibody who had epithelial skin lesions,’ which would support the idea of active viralinfection. Our findings show that AEC occurs in a large percentage of normal healthy children and its presence may be linked to rubella and RSV infection or other viruses. It may be that the antibody detected using A549 cells is, in fact, antibody directed against a viral protein present on the A549 used as the target cell and, in the light of our findings of an association
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with more than one virus, the antibody may be against a protein common to a host of viruses. Our virology positive group was relatively small and it is possible that more extensive screening of such samples would find other viruses associated with AEC seroconversion. In our paediatric recipient group we found that one child whose sera were screened sequentially over a prolonged period became seropositive for AEC antibody at the time of developing IgM anti-CMV antibody. This, added to the finding of a positive association with CMV in our adult population, suggests that anti-CMV antibodies may also be implicated in the AEC antibodies detected in the assay. As a result of this study, over the past 3 years it has been our policy not to take the AEC antibody status into account when selecting recipients for ~ansplantation and during this time period the l-year paediatric graft survival has been 84%. In conclusion, we believe a decision not to transplant in cases where a patient is AEC positive is unjustified and that these antibodies are in all probability an epiphenomenon. Acknowledgement This work has been funded in part by the Kidney Transplant Foundation of Ireland.
References 1 Harmer A, Rigden S, Koffman C, Welsh K. Preliminary report: dramatic rise in renal allograft failure rate. L.uncet 1990; 335: 1184. 2 Harmer AW, Haskard D, Koffman G, Welshe KI. Novel antibodies associated with unexplained loss of renal allografts. TransplantInt 1990; 3: 6669. 3 Martin S, Brenchley P, Postlethwaite R, Johnston R, Dyer P. Detection of anti-epithelial cell antibodies in association with paediatric renal transplant failure using a novel microcytotoxicity assay. Tissue Antigens 1991; 37: 152-53. 4 Chapman JR, Taylor C, Ting A, Morris PJ. Immunoglobulin class and specificity of antibodies causing positive crossmatches: relationship with renal transplant outcome. ~r~~~~zff~fa~~~~1986; 42: 608. 5 Holland C, O’Lorcain P, Taylor MRH, Kelly A. Seroepidemiology of toxicariasis in school children. Parasitolugy f995 (in press). 6 Deal JE, Harmer A, Welsh KI, Rigden SPA. Seroprevalence of an anti-epithelial cell antibody in normal children and children with end-stage renal disease (ESRD). Puediatr Nephrul 1991; 5: C50. 7 Deal JE, Rigden SPA, Harmer A, Koffman CG, Welsh IU. Renal allograft failure and antibodies to epithelial cells. Lartcet 1992; 339: 941. 8 Harmer A, Rigden SPA, Hartley B, Koffman CG, Welsh KI. Close association of renal allograft failure and antibody directed against epithelial cells. Transplant Proc 1991; 23: 412-13. 9 Nauta J, Wolffe ED, Claas FHJ, Schroder CH, Donckerwolcke RA. Renal allograft rejection and antibodies to epithelial cells. I.uncet 1992; 339: 503. 10 Harmer A, Liggett H, Norman PJ, Martin S. Microcytotoxicity and flow cytometry assays for anti-epi~elial cell antibodies compared in a national collaborative study of paediatric renal transplants. Eur J lmm~ogenet 1993; 20: 447.