Acute Rejection Group Research Articles

LOW DOSE MYCOPHENOLATE MOFETIL ACCELERATES STEROID WITHDRAWAL ADULT LIVING DONOR LIVER TRANSPLANTATION

A164 Aims: Adult LDLT, explosively increasing to compensate cadaveric donor shortage, has an inevitable and unique complication named small for size syndrome (SFS) composed of hypersplenism, sustained jaundice etc. SFS dose not only deteriorate early clinical outcome of adult LDLT but also affects on the postoperative immnosuppression protocol. We investigated whether Mycophenolate mofetil (MMF) has beneficial effects as a less bone-marrow suppressive agent on adult LDLT. Methods: From August 1996 to July 2003, fifty two patients of terminal liver diseases received adult LDLT. Four recipients died of operation-associated death (within a month after LDLT) were excluded. Forty eight recipients, who could be followed more than 1 year, were studied. The recipients divided into two groups by administration of MMF. Group 1 (n=23): Calceneurin inhibitor (CNI) + Steroids + Azathioprine (AZ), including 11 patients who were started in double regimen protocol (CNI + Steroids). Group 2 (n=21): CNI + Steroids + MMF, low dose MMF (20mg/kg/day) was started simultaneous to per os. Results: Most of the recipients maintained by Tacrolimus as basic CNI (Group 1: 100%, Group 2: 85.7%). There was no significant difference in mean trough levels of Tacrolimus in both groups. One-year surviving rate and rejection rate in Group 1 were 87.0% and 39.1%. In three lost recipients, one died of recurrence of PSC-associated bile duct cancer and two died of fungal infection. In Group 2, one-year surviving rate was significantly higher (95.2%) but rejection rate did not have any significant difference(42.9%) compared to Group 1. In Group 2, one recipient died of fibrous cytolytic hepatitis C and the other had recurrent hepatocellular carcinomas. There was no significant difference in rejection activity index of liver biopsy in both groups (3.6±2.8 vs. 4.0±1.12), however incidence of delayed acute rejection after 6 months was significantly higher in Group 1 (3/23 vs. 0/21). None of the recipient freed from steroids experienced delayed and/or refractory acute rejection in Group 2. Steroid withdrawal rates at 12 and 18 months after LDLT in Group 2 were significantly higher than those in Group 1 (Group 1 : 21.7%, 34.8% vs. Group 2 : 42.9%, 66.7%, p<0.05). In Group 1, AZ was discontinued in 5 cases thrombo- and/or leukocytopenia derived from SFS whereas no recipient dropped out from MMF protocol in Group 2. Bone marrow suppression of AZ, synergistically enhanced by SFS-associated hypersplenism, thought to be one of the major causes preventing successful withdrawal from steroids in conventional triple regimen of adult LDLT. Conclusions: Triple regimen including low dose MMF, safely administered to adult LDLT recipients with SFS, induces earlier withdrawal of steroids and improves clinical outcome of the adult LDLT recipients.

CLEARANCE OF TECHNETIUM-99M MERCAPTOACETYLTRIGLYCINE IN THE ASSESSMENT OF TRANSPLANTED KIDNEY FUNCTION

P405 Aims: Dynamic renal scintigraphy with Technetium-99m Mercaptoacetyltriglycine (99mTc-MAG3) has gained wide acceptance in assessing kidney graft function after renal transplantation, due to the possibility of simultaneous monitoring of kidney perfusion, functional parenchyma and collecting system. Data acquired during dynamic scintigraphy can be used for the quantification of the clearance of 99mTc-MAG3, which is proportional to effective renal plasma flow and reflects tubular function of the kidney. Our study aimed at evaluating the sensitivity of two methods for MAG3 clearance determination in patients with transplanted kidney. The first method used a single blood sample obtained over a continuous time interval, whereas the second method evaluates 99mTc-MAG3 clearance without blood sampling. Methods: The investigation was carried out in 62 patients, who were divided into three groups: group A consisted of 22 patients with well-functioning grafts, group B of 20 patients with suspected acute rejection and control group (C) of 20 healthy potential kidney donors. Renal scintigraphy was done 20 minutes after injection of 99mTc-MAG3. For the calculation of clearance by single-sample method (CL), the following formula was used:FigureThe values of CL were normalized to body surface area and expressed in physiological units of ml · min-1. The second method for clearance calculation used the counting rates acquired from kidney region during the second minute of the study. The obtained value is expressed as clearance index (CI):FigureResults: Mean values of CL were as follows: 187.8±39.2 ml · min-1 (group A), 64.3±22.6 ml · min-1 (group B) and 361.7±38.4 ml · min-1 (group C). The difference between groups A and B was statistically significant (p<0.05). There was a slight overlapping between these two groups in results of CL. Both groups of patients with transplanted kidney showed significantly lower values of CL in comparison with control group. The results of CI were: 50.9±15.6 (group A), 27.9±7.9 (group B) and 75.2±9.5 (group C). The difference between three groups was statistically significant, but overlapping of CI values between groups A and B was obvious. Correlation between CL and CI was statistically significant (r = 0.73; y=20.56+0.18x). Results of CL also correlated well with biochemical parameters of renal function: blood urea nitrogen (BUN), serum creatinine (Cr) and creatinine clearance (CCr): BUN/CL r = -0.78; Cr/CL r = -0.69; CCr/CL r = 0.86. Significant linear correlation between CI and kidney lab was not obtained. Conclusion: Results of our study confirmed the validity of single-sample method for estimation of 99mTc-MAG3 clearance. CL was shown to be a sensitive quantitative parameter of renal transplant function. CI could also reflect changes in kidney graft function, but is less sensitive in comparison with CL. Nevertheless, due to the simplicity of its calculation, CI merits further clinical evaluation as potential parameter of transplanted kidney function.

Pathological study on the relationship between C4d, CD59 and C5b-9 in acute renal allograft rejection.

In order to evaluate the activation or inhibition of the later phases of classical complement cascade in renal allograft presenting with acute rejection, particularly with C4d deposition on the peritubular capillary (PTC), we observed the expression of CD59 and C5b-9 on the PTC. Subjective cases were divided into two groups, an acute rejection group, of 4 males and 6 females, and a normal donor group, of 5 males and 5 females. Renal biopsies were performed at the onset of acute rejection and at the transplant operation, before reperfusion. C4d deposition on PTC was found in three of 10 cases (30%) with biopsy proven acute rejection, whereas CD59 on PTC was positively expressed in all of the rejection cases. Although C5b-9 was not observed on PTC in the acute rejection group, it was intensively deposited on the tubular basement membrane (TBM) in five cases, including the three with positive C4d on PTC. In the normal donor group, CD59 on PTC was intensively observed, whereas C5b-9 was weakly expressed on TBM. CD59, a complement regulatory factor, works as an inhibitory factor against the formation of C5b-9, a membrane attack complex. From our data, we noted the dissociation between the depositions of C4d and C5b-9 on PTC. The substantially expressed CD59 on PTC may affect this dissociation between C4d and C5b-9 on PTC. The intensive deposition of C5b-9 on TBM in acute rejection cases may suggest an independent immunological injury attacking tubular cells.

Evolving practice patterns in heart transplantation: a single-center experience over 15 years

This analysis is a retrospective characterization of evolving patterns in donor and recipient risk factors for early and late outcomes (survival and freedom from rejection) along with determinants of hospital and 1-year mortality after heart transplantation over a 15-year experience in a single center. Profiles and outcomes were evaluated for procedures performed between 1988 and 1995 (group A, n = 105) versus 1996 and 2003 (group B, n = 218). The following parameters were considered: pretransplant diagnosis, recipient age UNOS status, donor age, total postretrieval ischemic time, donor/recipient size match, and degree of myocardial necrosis at biopsy. Recipients in group B were significantly more compromised as demonstrated by UNOS status (11.4% vs 19.3%; P = .05) and pretransplant pulmonary vascular resistance (2.3 ± 1.5 vs 3.1 ± 1.5; P = .04). Marginal donors were more frequently used for group B procedures (21.9% vs 47.7%; P < .0001). Outcomes were significantly more favorable among group B patients in terms of hospital mortality (18.1% vs 10.6%; P = .046), and 1- and 5-year actuarial survival (72.4% vs 83.4%, 60% vs 73.3%, respectively; P = .006). Analysis of the causes of death disclosed a significant reduction in fatal events due to graft failure and acute rejection in group B. No difference emerged with regard to actual freedom from acute rejection. Determinants of hospital mortality were pretransplant diagnosis, UNOS status, donor age, and cardioplegic solution. Transplant era, recipient age, infectious episodes, and ischemic necrosis at biopsy were risk factors for 1-year mortality. We conclude that despite extensive usage of marginal donors and selection of worse candidates, significantly better outcomes were achieved due to improvements in global management strategies.

Proteomic-based detection of urine proteins associated with acute renal allograft rejection.

At present, the diagnosis of renal allograft rejection requires a renal biopsy. Clinical management of renal transplant patients would be improved by the development of non-invasive markers of rejection that can be measured frequently. This study sought to determine whether such candidate proteins can be detected in urine using mass spectrometry. Four patient groups were rigidly defined on the basis of allograft function, clinical course, and allograft biopsy result: acute clinical rejection group (n = 18), stable transplant group (n = 22), acute tubular necrosis group (n = 5), and recurrent (or de novo) glomerulopathy group (n = 5). Urines collected the day of the allograft biopsy were analyzed by mass spectrometry. As a normal control group, 28 urines from healthy individuals were analyzed the identical manner, as well as 5 urines from non-transplanted patients with lower urinary tract infection. Furthermore, sequential urine analysis was performed in patients in the acute clinical rejection and the stable transplant group. Three prominent peak clusters were found in 17 of 18 patients (94%) with acute rejection episodes, but only in 4 of 22 patients (18%) without clinical and histologic evidence for rejection and in 0 of 28 normal controls (P < 0.001). In addition, the presence or absence of these peak clusters correlated with the clinicopathologic course in most patients. Acute tubular necrosis, glomerulopathies, lower urinary tract infection, and cytomegalovirus viremia were not confounding variables. In conclusion, proteomic technology together with stringent definition of patient groups can detect urine proteins associated with acute renal allograft rejection. Identification of these proteins may prove useful as non-invasive diagnostic markers for rejection and the development of novel therapeutic agents.

Influence of Th1, Th2, and Th3 cytokines during the early phase after liver transplantation

To investigate the change of Th1, Th2, Th3 cytokines during early liver transplantation. IFN-r, IL-4, TGF-β production by CD4 + T cells were determined by fluorescence activated cell sorter analysis. Comparing the acute rejection with the non-acute rejection groups on the 7th, 14th, and 28th day showed that high interferon-γ production associated with acute rejection in the early posttransplant period. There was no evidence of significant changes in interleukin-4 and transforming growth factor β (TGF-β) levels between non-acute rejection groups with acute rejection groups. Th1 cytokine high production is significantly associated with acute rejection in liver transplant recipients.

Study 2: BK polyomavirus nephritis and acute rejection tend to affect distinct groups of renal allograft recipients: a role for gender, race, and tacrolimus levels

B K polyomavirus nephritis (BKN) has been linked to the newer immunosuppressants tacrolimus (TAC) and mycophenolate mofetil (MMF) even though risk factors for the disease have not been well defined. A recent study, however, suggested that BKN occurs as a complication of the treatment of acute rejection with methylprednisolone. To further examine the relationship between the treatment of acute rejection and BKN, we analyzed all kidney transplants performed at our center between January 1999 and August 2001 (n 286). After a mean follow-up of 622 63 days, we identified 9 cases of BKN (3.1%) using urine electron microscopy. Of these, 8 patients also underwent allograft biopsy, which confirmed the diagnosis. The mean time to diagnosis of BKN was 326 56 days. No patient with BKN had a history of acute rejection. During the same period, 59 (21%) patients were diagnosed with acute rejection and treated with methylprednisolone. The mean time to diagnosis of acute rejection was 197 40 days (P .01, vs time to diagnosis of BKN). None of these patients had subsequent development of BKN during the follow-up period. We compared the characteristics of patients with development of BKN with those of patients who had acute rejection and found that patients with BKN were more likely to be male (89% vs 53%, P .04) and white (78% vs 44%, P .05). Moreover, the mean TAC levels from the time of transplant to the time of diagnosis were higher in the BKN group than in the acute rejection group (12 1 vs 7 1 ng/mL, P .001). The treatment of BKN consisted mainly of a reduction in immunosuppression. MMF was discontinued in 7 of 9 patients, all of whom experienced disappearance of viruria (median time to negative result of urine electron microscopy, 147 days; range, 29-257) and remained independent of dialysis at last follow-up. In 2 of 9 BKN patients, MMF doses were only reduced. Neither patient had clearance of the viruria, and 1 ultimately lost the graft because of the viral infection. Conclusion: Our study does not show an association between acute rejection or its treatment and the development of BKN. Our findings suggest that these conditions tend to affect distinct groups of renal allograft recipients. Although there may be a potential relationship between high TAC levels and BKN, discontinuation of MMF was associated with clearance of viruria and preservation of renal function. From the Departments of Medicine, Nephrology, and Pathology, Duke University Medical Center, Durham, NC. © 2003 Elsevier Inc. All rights reserved. 0955-470X/03/1704-0000$30.00/0 doi:10.1016/j.trre.2003.10.019

Acute liver transplant rejection: incidence and the role of high-doses steroids

The aim of this study was to assess the incidence of acute rejection (AR), and the efficacy of high doses of steroids during induction of immunosuppression for AR treatment. Fifty-five patients (33.5%) experienced AR episodes in our series; but, there were no deaths or retransplantations related to AR. The median time from liver transplantation to AR was 18.5 days (range, 2–351 days). In the group with the initial dose of methylprednisolone (MP) ≤0.75 g, AR occurred in 32.9% of patients; and in the group with higher dose of MP, 43.6% ( P > 0.05). After 1-year observation, liver function tests were similar in both AR and non-AR groups. The only biochemical parameter that was significantly lower in the non-AR group was the aspartate aminotransferase (AST). Liver function tests determined after 1-year follow-up were not significantly different between the groups with AR treated with doses of MP lower versus higher than 1.25 g. However, liver function tests in the group treated for AR with higher doses of MP were slightly better than in the remaining subjects. Recurrence of AR occurred in 5 cases in the group with lower doses of MP (≤1.25 g), and in 2 cases in the group with higher doses of MP (>1.25 g). A relatively low dose of MP was effective to treat AR. The tendency of AR patients treated with higher dose of MP to display better liver function needs further investigation. However, AR does not seem to affect later liver function.

Impact of the delayed graft function in hypersensitized kidney transplant patients

Aim The aim was to study the incidence, impact, and association of pretransplantation anti-HLA antibodies and delayed graft function (DGF) on the outcome of cadaver kidney transplants independent of the immunosuppressive therapy. Methods Data from 1325 cadaver donor kidneys (February 1975 to December 2002) included the variables of current and peak anti-HLA antibodies, presence of DGF, acute rejection (AR) episodes, patient survival, and graft half-life. Results DGF (need for dialysis in the first week posttransplantation) ranged between 15% and 40% with a mean of 30% in last 5 years. Eighty-five percent of the candidates on the waiting list for kidney transplants displayed <25% panel reactive antibody (PRA) at transplantation with 4.6% between 26% and 50%, 7.7% between 51% and 75%, and 1.5% > 75%. Among the patients who developed DGF, 47% displayed AR compared an incidence of 30% among patients without DGF ( P = .0026). The patients displaying >50% PRA (either current or maximum) showed a worse graft survival compared with patients with <50% PRA (log rank, P = .0000). DGF reduced graft survival ( P = .04), the difference appearing in the early phase after transplantation. The best outcome was observed in the no DGF-no AR group (half-life, 11.6 years) and the worst results were in the hypersensitized patient groups: peak and current PRA > 50% (half-lives of 2.4 and 2.2 years). A multivariate analysis showed that the presence of peak or current PRA >50% is the most important risk factor for graft loss. Conclusion Sensitization is the key factor in graft outcome. Presensitization increases the risk of DGF and DGF increases the incidence of AR and both together produce the worst graft survivals.

Histological evaluation of renal allograft protocol biopsies in the early period and 1 year after transplantation.

We histologically evaluated protocol biopsy specimens of renal allografts obtained in the early period and 1 year after transplantation. The patients were divided into those with at least one history of acute rejection (AR group) and no history of rejection (NAR group), and the histopathological features in the two groups were compared. A total of 45 early protocol biopsy specimens were obtained from 40 patients, and 31 1-year biopsy specimens were obtained from 30 patients. Acute rejection (AR) or borderline change was observed in the early protocol biopsy specimens from 19 (45.2%) cases. AR or borderline change was observed in 12 of 19 (63.2%) in the AR group, and in 7/26 cases (26.9%) in the NAR group. The incidence of AR or borderline change in the AR group was higher than in the NAR group. Toxic tubulopathy was found in the early protocol biopsy in 16 cases (35.6%). The 1-year biopsies tended to reveal more complicated findings. Chronic rejection (CR) was seen in 8/16 cases (50.0%) in the AR group, and it was more frequent than NAR group (two cases, 13.3%). In conclusion, the incidences of both AR and CR were higher in the cases with a previous episode of AR. The early protocol biopsy was useful in screening for subclinical AR and toxic tubulopathy. The 1-year biopsy was useful for evaluating various types of chronic graft damage. We expect that adequate treatment based on protocol biopsy findings in each patient will lead to better graft survival.

A Monocyte chemoattractant protein-1 (MCP-1) polymorphism and outcome after renal transplantation.

Among the factors modulating transplant rejection and cardiovascular disease, chemokines and their respective receptors deserve special attention. In this respect, increased expression of MCP-1 and the corresponding receptor CCR2 have been demonstrated in renal transplant rejection and coronary artery disease. The impact of the MCP-1-2518G and CCR2-64I genotypes on renal allograft function was investigated in 232 patients who underwent transplantation over an 11-yr period. Genomic DNA was genotyped using PCR with sequence-specific primers followed by restriction fragment length polymorphism analysis. Eighteen (7.8%) patients were homozygous for the MCP-1-2518G mutation. The G/G allele of MCP-1 -2518 behaved as a determinant for long-term allograft survival and resulted in reduction of the mean graft survival, as compared with the heterozygous (A/G) or wild-type (A/A) allele (67 +/- 14 versus 95 +/- 4 mo; Log rank P = 0.0052). The 64I mutation of CCR2 had no effect on kidney graft failure (93 +/- 6 and 91 +/- 5 mo, respectively; P = 0.81). None of the investigated polymorphisms showed a significant shift in gene frequency in acute rejection and rejection-free groups. In conjunction with these findings, peripheral blood mononuclear cells from kidney transplant recipients carrying the G-allele were characterized by a 2.5-fold higher MCP-1 secretion (P < 0.05). In conclusion, recipients of renal transplants homozygous for the -2518 G mutation of the MCP-1 gene are at risk for premature kidney graft failure. This variant of MCP-1 may be a future predictor for long-term kidney graft failure.

The Impact of Polymorphisms in Chemokine and Chemokine Receptors on Outcomes in Liver Transplantation

Chemokines and their corresponding receptors likely play a central role in directing mononuclear cells to the graft sites during rejection. Genes for the chemokine stromal derived factor-1 (SDF1) and CC chemokine receptors CCR2 and CCR5 are characterized by polymorphisms which alter their function. We genotyped DNA of 207 liver transplant recipients by PCR or PCR-RFLP for CCR2-641, CCR5delta32, and SDF1-3'A polymorphisms, and examined their association on outcomes in liver allograft recipients. Due to the low number of patients homozygous for CCR2-641 and CCR5delta32, only the effects of their heterozygous variants were addressed in this study. None of the investigated polymorphisms showed a significant shift in gene frequency in acute rejection and rejection-free groups, or for graft survival. The gene frequency of the SDF1-3'A allele was significantly (p = 0.034) higher in patients who died (29.0%, n = 31) compared to recipients still alive (17.1%, n = 172). The mean patient survival time post transplant was 134 months in patients with SDF1 wild-type, significantly (log rank p = 0.014) longer than 98 months in patients with at least one SDF1-3'A allele. The CCR2 and CCR5 polymorphisms were not associated with significant differences in mortality rate. In conclusion, CCR2-641, CCR5delta32, and SDF1-3'A genotypes did not influence the risk for acute rejection or graft survival. However, in liver allograft recipients SDF1-3'A is significantly associated with higher mortality.

Determinants of hypofiltration during acute renal allograft rejection.

This study sought to determine the extent to which GFR is decreased during acute renal allograft rejection in human subjects and to determine the mechanism of the decrease in GFR. Eight patients with biopsy-proven acute rejection were compared with 18 recipients of optimally functioning renal allografts. GFR and renal plasma flow (RPF) were measured as the clearance of inulin and para-aminohippuric acid, respectively. Arterial BP was determined, blood was sampled, and plasma oncotic pressure (pi(A)) was measured. Glomeruli obtained by biopsy during rejection were subjected to morphometric analysis, for determination of K(f). Control morphometric values for healthy glomeruli were provided by 10 living donors from whom biopsies were obtained at the time of organ donation. The subjects in the acute rejection group exhibited a significantly reduced GFR of 17 +/- 4 ml/min per 1.73 m(2), compared with 72 +/- 4 ml/min per 1.73 m(2) for control subjects (P < 0.001). With the use of a sensitivity analysis to take into account the unknown para-aminohippuric acid extraction ratio, the RPF rate was calculated to have likely been significantly decreased, by 45 to 70%, in the acute rejection group. Neither the plasma oncotic pressure nor the mean arterial pressure differed between the two groups. Morphometric analysis revealed no difference in the single-nephron K(f) values for the acute rejection group, compared with the control group. These results indicate that acute renal allograft rejection causes a profound decrease in GFR, which is attributable to a decrease in RPF alone or in combination with a decrease in the glomerular transcapillary hydraulic pressure gradient (DeltaP).

Role of macrophages and lymphocytes in the induction of neovascularization in renal allograft rejection.

The aim of this study was to clarify the relationship between angiogenesis and mononuclear cell infiltration in renal allografts. Kidney biopsies from 70 renal transplant recipients were evaluated. The density of CD68, CD3, and HLA-DR-positive infiltrating cells were semiquantitatively assessed by immunohistochemistry. Microvessels were highlighted by immunostaining endothelial cells for factor VIII-RA. Of the 70 patients, 38 showed acute rejection (AR), and 32 showed chronic rejection (CR). The mean microvessel density (MVD) in the AR cases was 931.6 +/- 82 and 481.6 +/- 43.6 in the CR cases. MVD in the controls was 210.4 +/- 21.7. A significant difference was found between the 3 groups (P < 0.001). There were significant positive correlations between MVD and the proportions of the various types of mononuclear cells in the interstitial tissue in both the AR (P < 0.001) and CR (P < 0.001) groups. In the CR group, mean MVD increased in parallel with increasing interstitial fibrosis (P < 0.001). In follow-up biopsies, 23 of 38 patients with AR showed variable degrees of interstitial fibrosis. The mean MVD in the initial biopsy was 537.2 +/- 46.8 in cases that showed no fibrosis in follow-up biopsies, whereas mean MVD in the initial biopsy was 1,196 +/- 98.7 in 23 patients who showed interstitial fibrosis in follow-up biopsies. The difference between these values was significant (P < 0.001). Patients with AR whose initial biopsies showed high MVD developed interstitial fibrosis earlier and had poorer graft outcome than those whose initial biopsies showed low MVD. The results suggest that advanced tubulointerstitial injury and mononuclear cell infiltration may play an important role in the induction of angiogenesis. Mononuclear cells may potentiate interstitial fibrosis in vivo by stimulating neovascularization, which leads to early fibrotic changes and poor outcome.

Patterns and significance of exhaled-breath biomarkers in lung transplant recipients with acute allograft rejection

Background Obliterative bronchiolitis (OB) remains one of the leading causes of death in lung transplant recipients after 2 years, and acute rejection (AR) of lung allograft is a major risk factor for OB. Treatment of AR may reduce the incidence of OB, although diagnosis of AR often requires bronchoscopic lung biopsy. In this study, we evaluated the utility of exhaled-breath biomarkers for the non-invasive diagnosis of AR. Methods We obtained breath samples from 44 consecutive lung transplant recipients who attended ambulatory follow-up visits for the Johns Hopkins Lung Transplant Program. Bronchoscopy within 7 days of their breath samples showed histopathology in 21of these patients, and we included them in our analysis. We measured hydrocarbon markers of pro-oxidant events (ethane and 1-pentane), isoprene, acetone, and sulfur-containing compounds (hydrogen sulfide and carbonyl sulfide) in exhaled breath and compared their levels to the lung histopathology, graded as stable (non-rejection) or AR. None of the study subjects were diagnosed with OB or infection at the time of the clinical bronchoscopy. Results We found no significant difference in exhaled levels of hydrocarbons, acetone, or hydrogen sulfide between the stable and AR groups. However, we did find significant increase in exhaled carbonyl sulfide (COS) levels in AR subjects compared with stable subjects. We also observed a trend in 7 of 8 patients who had serial sets of breath and histopathology data that supported a role for COS as a breath biomarker of AR. Conclusions This study demonstrated elevations in exhaled COS levels in subjects with AR compared with stable subjects, suggesting a diagnostic role for this non-invasive biomarker. Further exploration of breath analysis in lung transplant recipients is warranted to complement fiberoptic bronchoscopy and obviate the need for this procedure in some patients.

An increase in myeloid-related protein serum levels precedes acute renal allograft rejection.

Upon interaction with activated endothelium, monocytes and neutrophils form complexes of myeloid-related protein 8 (MRP8) (S100A8) and MRP14 (S100A9), two members of the calcium-binding S100 family that are secreted during transendothelial migration. In a pilot study of 20 renal transplant recipients and a validation study of 36 renal transplant recipients, MRP8/14 serum levels were measured with an enzyme-linked immunosorbent assay for 28 d, associated with C-reactive protein and creatinine serum levels, and grouped according to biopsy-proven acute rejection. Serum levels of MRP8/14 but not C-reactive protein were significantly increased for several days during the first 2 wk for the acute rejection groups in both studies (P < 0.005, on day 6 after transplantation). As determined by using receiver operating characteristic curves, the optimal cutoff for 100% specificity and high sensitivity (67%) for acute rejection on day 6 after transplantation was calculated to be 4.2 microg/ml for MRP8/14 in the pilot study; this value was confirmed in the validation study. Positive MRP8/14 serum levels preceded acute rejection episodes by a median of 5 d. A 3-d course of intravenous methylprednisolone therapy reduced prerejection MRP8/14 serum levels from 5.7 microg/ml to 3.3 microg/ml (P < 0.05). All MRP8/14 serum levels were below the cutoff during urinary tract infections, delayed graft function, or cytomegalovirus infections, and these values did not differ significantly from control values. It is concluded that the MRP8/14 complex is a very early serum marker suitable for monitoring of acute rejection with high sensitivity and specificity.

Granzyme B and TIA-1 expression in chronic and acute on chronic renal allograft rejection.

Although active inflammation may be deleterious and indicate immunologic activation in chronically rejected grafts, the underlying mechanism of tissue destruction has been little studied. Twenty-four cases of chronic rejection (CR) with or without acute rejection (AR) were stained with antibodies against CD3, CD8, CD68, granzyme B and TIA-1, and the number of positive cells were counted. Eleven cases of AR served as controls. The number of CD3 and CD8 positive cells increased in the acute on CR group compared to the CR group. About a half of CD3 positive T cells were CD8 positive in both groups, however, the proportion of TIA-1 or granzyme B positive cells was higher in the acute on CR group. The numbers of CD3, CD68, granzyme B and TIA-1 positive cells were higher in the AR group than the acute on CR group, however, no significant difference was found between the two groups. Serum creatinine level and proteinuria at the time of biopsy and the percentages of late onset AR and graft failure rate were higher in the acute on CR group than the CR group. Summarizing, these results suggest that infiltration of activated T cells containing cytotoxic granules plays a role in graft destruction in acute on CR.

LONG-TERM RESULTS AFTER CONVERSION FROM CYCLOSPORINE TO TACROLIMUS IN PEDIATRIC LIVER TRANSPLANTATION FOR ACUTE AND CHRONIC REJECTION

Tacrolimus is beneficial in liver transplantation for reversing steroid-resistant acute rejection, and for controlling the process of chronic rejection in allograft recipients receiving Cyclosporine- (CyA) based regimens. Very little is known about the long-term efficacy of tacrolimus in pediatric transplantation after conversion from CyA. Our study examines the long-term outcome after conversion to tacrolimus for acute or chronic rejection in pediatric liver transplant (LTx) recipients. Seventy-three children (age < 18 years) receiving their primary LTx under CyA between August 1989 and April 1996 were converted to tacrolimus for ongoing acute rejection (n=22, group I) or chronic rejection (n=51, group II). Mean age at the time of conversion was 10.2+/-5.5 years with a mean interval from LTx to conversion of 3.5+/-2.9 (range 0.5-10.1 years). There were 33 boys and 40 girls. All patients were followed until June 1999. Mean follow-up was 97.3+/-17.4 months (range 62.4-118.9 months). Overall 5-year actual patient survival was 78.1% and 8-year actuarial survival was 74.6%. Patients converted to tacrolimus therapy to resolve acute rejection (group I) experience significantly better patient and graft survival at 5 and 8 years than those converted to resolve chronic rejection (group II). Eight-year patient survival and graft survival was 95.5 and 90.9% for group I compared to 74.6 and 53.5% for group II, respectively (long rank P=0.035 and 0.01, respectively). Nearly 75% of children were weaned off steroids after conversion. There was a marked improvement in hypertension, gum hyperplasia, hirsutism, and cushingoid appearance. One child in group I (4.5%) and four children in group II (7.8%) developed posttransplant lymphoproliferative disorder after conversion. There was an improvement in growth in children who were less than the age of 12 years at the time of conversion and who were weaned off steroids; more significantly girls responded more favorably than boys. The benefit of transplantation is maintained long-term after conversion to tacrolimus for acute or chronic rejection. The response rate was significantly better in group I as compared with group 11. Marked improvement in growth, hypertension, and reversal of the brutalizing effects of CyA was noted after conversion to tacrolimus. The results suggest that early conversion of pediatric liver transplant patients is warranted for the treatment of acute and chronic rejection, and for improvements in quality of life.

Kidney Transplantation in Children Younger Than 1 Year Using Cyclosporine Immunosuppression

Objective The optimal age for transplantation in children with end-stage renal disease remains controversial. Supported by national data, many centers recommend dialysis until the child reaches a certain minimum age. The authors' policy, however, has been to encourage living donor (LD) transplants for young children, with no minimum age restriction. Methods Between January 1, 1984, and December 31, 1996, the authors performed 248 kidney transplants in children younger than age 13 years, using cyclosporine as the primary immunosuppressive agent. Recipients were analyzed in three age groups: group 1, younger than age 1 year (n = 26); group 2, age 1 through 4 (n = 92); and group 3, age 5 through 13 (n = 130). Almost all recipients in group 1 underwent a primary LD transplant. Therefore, to compare results more meaningfully among the three age groups, only primary LD transplants were analyzed (group 1, n = 25; group 2, n = 59; group 3, n = 58). Results In primary LD transplants, no significant difference was noted among the age groups in 1- and 5-year patient or graft survival rates. To date, all 25 recipients from group 1 are alive and well; 19 still have a functional original graft. Causes of graft loss in the remaining six recipients were chronic rejection (n = 3), vascular thrombosis (n = 2), and recurrent disease (n = 1). The incidence of acute rejection in group 1 recipients was lower than in the two older groups. However, the incidence of delayed graft function was slightly higher in the youngest group than in the two older groups. For recipients in group 1, growth (as measured by weight) improved significantly posttransplant: the mean standard deviation score rose from -2.8 pretransplant to -0.2 by age 5 and to +1.8 by age 10. The improvement in height was not as dramatic: the mean standard deviation score rose from -3.2 pretransplant to - 1.6 by age 5 and to -1.4 by age 10. Conclusions Kidney transplantation in young children, including those younger than 1 year old, can achieve results comparable to those in older children. As long as an adult LD is available, the timing of the transplant should be based on renal function rather than age.

EXPRESSION OF GRANULYSIN IN PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) CORRELATES WITH ACUTE RENAL ALLOGRAFT REJECTION IN CHILDREN

171 Introduction: Granulysin (GL) is expressed by CTL and NK cells and causes target cell death by granule mediated cytolysis and apoptosis. We have previously shown that is expressed in PBMC, mononuclear cells and eosinophils by tissue immunohistochemistry on renal transplant biopsy specimens. Methods: We looked for GL expression in PBMC of 20 pediatric renal transplant recipients with acute rejection (AR) and 7 graft dysfunction without acute rejection (GD). Expression analysis for GL was done using competitive reverse transcriptase polymerase chain reaction (RT-PCR) with message for glyceraldehyde phosphate dehydrogenase (GAPDH) serving as transcription control. We also collected urine during AR and in the peri-transplant period from 10 children and performed ELISA on centifuged sediment using murine anti-human monoclonal antibodies. Results: The GL/GAPDH was 0.099 in the AR vs 0.010 in the GD cohort (p< 0.005). There was a significant reduction in the GL/GAPDH value in the AR group, after treating AR with high dose steroids, suggesting that steroids may down-regulate GL expression in PBMC. To examine this further, we looked at GL expression in the peri-transplant period in 10 pediatric renal transplant recipients with living related transplant donation on our standard immunosuppression (Dacluzimab, Prednisone, CsA and Azathioprine). Blood was drawn on the day before transplant (D-1), day of transplant (D0) and days 1 and 7 post-transplant (D1 and D7). The mean GL/GAPDH values were 0.017 (D-1), 0.002 (D0), 0.008 (D1) and 0.015 (D7) suggesting that GL message is reduced with onset of high dose immunosuppression. Furthermore, in vitro FACS staining of PHA-induced PBMC GL expression was reduced after treatment with dexamethasone but not after treatment with CsA. GL was barely detectable in the urine prior to transplantation but increased significantly on D0 and D1 post transplant and with acute rejection episodes. Quantitative ELISA is being set up to investigate this as a non invasive marker of acute rejection. Conclusions: We have found that GL mRNA is significantly elevated in the PBMC of children with acute renal allograft rejection as compared to other causes of graft dysfunction. In addition, we have presented evidence, both in vitro and in vivo, that immunosuppression with steroids leads to a decreased GL expression. We continue to investigate the role of the novel molecule GL in the transplanted human kidney and its possible use as a marker for rejection.