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Effects of FK506 inhalant on the suppression of acute rejection in lung transplantation: use of rat orthotopic lung transplantation model
The Journal of Heart and Lung Transplantation Volume 22, Number 1S group. Adhesion molecule upregulation, as seen on the third postoperative day in the control group was reduced with the use of FK778. ICAM-1, as well as VCAM-1-upregulation on coronary endothelial cells was significantly attenuated in the study group. Conclusion: FK778 treatment after cardiac transplantation reduces early mononuclear graft infiltration and attenuates adhesion molecule upregulation. The cellular rejection process has been effectively suppressed. 65 REDUCTION OF PECAM-1 AT GRAFT ENDOTHELIUM AFTER CARDIAC TRANSPLANTATION IS ACCOMPANIED BY THE REDUCTION OF LEUKOCYTE INFILTRATION INTO THE PERIVASCULAR SPACE OF ARTERIES. A COMPARATIVE STUDY WITH CSA, FK506 AND MMF M.H.C. Richter,1 K. Kafchitsas,2 H.R. Richter,2 J.F. Gummert,1 F.W. Mohr,1 1Cardiac Surgery, Heartcenter, Leipzig, Germany; 2 Experimental Heart Transplant Group, University of Frankfurt, Frankfurt a.M., Germany Platelet endothelial cell adhesion molecule (PECAM)-1, a member of the Ig superfamily, is expressed under inflammatory circumstances at the endothelium of vessels. PECAM-1 is a multifunctional molecule, it has been implicated in T-cell activation and transendothelial migration of leukocytes into the perivascular space or into the myocardium, a common phenomenon in transplant rejection. We evaluated the effects of CsA, FK506 and MMF on the expression of PECAM-1 on vascular endothelium in an experimental model. After cardiac transplants (Lewis to Fisher rats) animals were divided into 4 groups: CSA 3mg/kg/d (n⫽74), MMF 40mg/kg/d (n⫽96), FK506 0.3mg/kg/d (n⫽96) and control (no therapy, n⫽74). 3-4 animals of each group were sacrificed in intervals of 1-4 days up to day 60. Using immunohistochemistry we investigated the quote of positive stained vessels, the intensity of staining and the geometric distribution of positive staining for PECAM-1 and analysed CD4, CD8, CD11a and CD18 positive leukocytes in the perivascular space. In controls we found an intensive and nearly circumferent staining of PECAM-1 in intra- and epicardial arteries. All used immunosuppressiva reduced the intensity of staining, the quote of stained vessels as well as the geometry of staining. Comparing CsA and FK506, we did not find significant differences. In MMF treated animals stained arteries, intensity and geometry of staining were significantly reduced compared to both calcineurin inhibitors. When leukocyte infiltration into the perivascular space of arteries and into the myocardium was analysed we found a significant correlation between the intensity of PECAM-1 staining and leukocyte infiltration. MMF therapy reduced PECAM-1 staining in graft endothelium as well as the amount of leukocytes in the perivascular space at intra- and epicardial arteries. 66 DIFFERENT CYCLOSPORINE REGIMENS AND THEIR INFLUENCES ON TRANSPLANT VASCULOPATHY M.H.C. Richter,12 D. Schramm,2 H.R. Richter,2 1Cardiac Surgery, Heartcenter, Leipzig, Germany; 2Experimental Heart Transplant Group, Goethe-University, Frankfurt a.M., Germany Cyclosporine A (CsA) is linked with the development of transplant vasculopathy (TVP). We investigated the influence of different CsA regimens on TVP after rat heart transplantation. After heterotopic cardiac transplantation (Lewis to Fisher) animals were divided into 3
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groups: controls (no therapy, n⫽20), 3mg-CsA (3mg/kg/d/s.c., n⫽50), 12mg-CsA (12mg/kg/d/s.c., n⫽50). In group 2 and 3 CsA treatment ended either at day 20, 40, 60 or 80. 5 animals each were killed 20, 40, 60 and 80 days after transplantation with the following distribution: no therapy, treatment until sacrification, treatment ending 20, 40 or 60 days before sacrification. CsA levels were determined every ten days and ten days after withdrawal. TVP was assessed by digitizing morphometry and expressed as mean vessel occlusion (mvo). Acute rejection was scored (ISHLT criteria). In 12mg-CsA animals CsA level was 5 to10 fold higher than in 3mg-CsA animals and undetectable 10 days after withdrawal in both groups. In control animals we found a score IV of acute rejection, in continuously treated animals IIa in 12mg-CsA and IIIa in 3mg-CsA animals. But already 20 days after the withdrawal of CsA no significance was found when compared to controls and there was no difference at the end of the study when a 20, 40 or 60-day course of CsA application (group 2 and 3) was compared with untreated controls. Both continuous CsA regimens reduced the mvo significantly when compared with controls. We found no significant difference between 12mg and 3mg. The withdrawal of CsA therapy lead to an increase of mvo to levels with no significant difference to control animals. Despite the excessive increase of CsA blood levels we found neither a further reduction nor an increase of mvo in the high dose group compared with the low dose regimen. Our data suggest that CsA is not responsible for the development of TVP and that high CsA-levels lead to the same extent of TVP as low levels do. 67 EFFECTS OF FK506 INHALANT ON THE SUPPRESSION OF ACUTE REJECTION IN LUNG TRANSPLANTATION: USE OF RAT ORTHOTOPIC LUNG TRANSPLANTATION MODEL A. Ingu,1 K. Komatsu,1 M. Morikawa,1 T. Abe,1 S. Ichimiya,2 Y. Hirayama,3 1Thoracic and Cardiovascular Surgery, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan; 2Pathology, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan; 3Immunology and Inflammation Medical Biology Reserch Laboratories, Kashima Institution, Osaka, Osaka, Japan Background: We investigated the acute rejection suppression effect of FK506 0.1% inhalant developed for localized administration, and examined its immunosuppressant effect at a low blood concentration compared to systemic administration by intramuscular injection. Methods: We performed orthotopic left lung transplantations using the conventional cuff technique. Non-anesthetized rats were each stabilized inside a model P (Boxco Electronics, Inc.) and subjected to inhalation. M-1 (1.0 mg/kg), M-0.1 (0.1 mg/kg), M-0.05 (0.05 mg/kg) groups given intramuscular injection, and A-20 (20 puffs), A-10 (10 puffs), A-5 (5 puffs) groups given inhalation. In terms of the acceptance and lengthening effects, firstly, x-ray images were classified. Secondly, the transplanted lungs were extracted 6 after transplantation, and fixed 10% formalin solution and stained with hematoxylin-eosin. Samples were then histologically categorized. Thirdly, FK506 concentrations in the blood was calculated using ERISA and, fourthly, the expressions of IL-2, IFN-r, and TNF-a were examined by PCR. Results: 1) In all six groups that received treatment, the graft was seen to be accepted and lengthen, but lengthening after 14 days was only seen in four groups (M-1, M-0.1, A-20, and A-10 groups). 2) In the A-10 group perivascular edema was milder than in the M-0.1 group. 3) In the A-10 group, FK506 was not detected in the blood. In the A-10 group, tissue concentrations were about 1/10 lower than in the M-0.1 group. 4) Regarding cytokine suppression, IL-2, IFN-r, and TNF-a were all similarly suppressed in both the M-0.1 and A-10 groups.
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Conclusions: FK506 by inhalation was also effective. Despite blood concentrations being significantly lower in the inhalation groups compared to the intramuscukar injection groups, there were no differences in acceptance and lengthening effects and cytokine suppression. 68 TACROLIMUS: IN VITRO EFFECTS ON NEUTROPHIL PRODUCTION AND APOPTOSIS J.M. Koenig, N. Matharoo, J.J. Stegner, K.O. Schowengerdt, Pediatrics, University of Florida, Gainesville, FL Purpose: Tacrolimus is a common component of multi-drug immunosuppressive regimens used for the prevention of rejection in transplant recipients. Anecdotal observations and recent evidence suggest an association between tacrolimus therapy in children and the development of neutropenia. We hypothesized that this variety of neutropenia might be related to a negative effect of tacrolimus on neutrophil (PMN) production and/or survival. Methods: We designed in vitro studies to determine the dose-dependent effects of tacrolimus on PMN production and/or apoptosis. PMN and CD34⫹ cells isolated from umbilical cord blood of term gestations were cultured in the presence of tacrolimus (0 - 1000 ng/mL). To evaluate apoptosis, cells cultured for 24 h were stained with Annexin-V-FITC/ AAD and analyzed by flow cytometry. For clonal analysis, CD34⫹ cells cultured for 10 d in cytokine-enhanced semi-solid media were scored for their myeloid/erythroid (CFU-Mix), myeloid (CFU-GM), and erythroid (BFU-E) progenitor cell contents. Results: We observed that tacrolimus induced a dose-dependent enhancement of myeloid clonogenesis of CD34⫹ cells that peaked at 10 ng/mL (mean ⫾ SD, colonies/1000 plated cells): CFU-Mix, 14 ⫾ 2 (0 ng/mL) vs. 30 ⫾ 12 (p ⬍ 0.05); CFU-GM, 61 ⫾ 4 (0 ng/mL) vs. 160 ⫾ 6 (p ⬍ 0.01). Tacrolimus at these doses did not consistently decrease the survival of either CD34⫹ cells or PMNs. Conclusions: In contrast to our initial hypothesis, we observed that tacrolimus at clinically relevant concentrations enhanced clonogenesis of PMN progenitors and inconsistently affected cell survival. Tacrolimus is generally administered in combination with other immunosuppressive drugs after solid organ transplantation. Our data suggest that tacrolimus alone is unlikely to cause the neutropenia observed in this setting. 69 INTER-READER AND INTRA-READER AGREEMENT FOR GRADING ACUTE REJECTION AND AIRWAY INFLAMMATION AFTER LUNG TRANSPLANTATION, BASED ON THE LUNG REJECTION STUDY GROUP REVISED WORKING FORMULATION M.M. Chakinala,1 J. Ritter,2 B.F. Gage,1 A.A. Aloush,1 E.P. Trulock,1 1 Internal Medicine, Washington University School of Medicine, Saint Louis, MO; 2Pathology, Washington University School of Medicine, Saint Louis, MO The Lung Rejection Study Group standardized grading of acute rejection after transplantation by the degree of perivascular inflammation (A grade). They also emphasized the degree of airway inflammation (B grade), a likely precursor to bronchiolitis obliterans and chronic rejection. We determined the inter-reader and intra-reader agreements for both categories from transbronchial biopsies. Methods: While blinded to original interpretations and after excluding Cytomegalovirus pneumonitis, a single pathologist (JR) assigned A and B grades. When original and repeat grades were available, a weighted
The Journal of Heart and Lung Transplantation January 2003 Kappa [(w)] was calculated [range of -1.0 (total disagreement) to 1.0 (total agreement)]. Biopsies originally read by JR were used for intra-reader determinations. Because minimal airway inflammation (B1) is often non-specific, B grades were further dichotomized (0/1 or 2/3/4) and simple Kappa [(s)] calculated. And, since asymptomatic rejection is often treated when ⬎A1, A grades from surveillance biopsies were similarly dichotomized (0/1 or 2/3/4) and (s) calculated. Results: We reviewed 626 transbronchial biopsies from 207 recipients, obtained between 1/96 and 12/00. Weighted Kappa - K(w)
A score
B score
Inter-reader Intra-reader
0.65a (n ⫽ 529) 0.65a (n ⫽ 97)
0.28b (n ⫽ 164) 0.33c (n ⫽ 58)
a
p ⬍ 0.001; bp ⫽ .005; cp ⫽ .008.
Inter-reader (s) for dichotomized B grades was 0.25; p⫽.001. Interreader (s) for dichotomized A grades on surveillance biopsies (n⫽273) was 0.65; p⬍.001. Conclusion: Inter-reader and intra-reader agreements for A grades are good, while agreements for B grades are only fair. Because airway inflammation is emphasized as a potential precursor for chronic rejection, better agreement of B grading is necessary to incorporate airway pathology into clinical decision-making.
70 INTER-OBSERVER VARIABILITY IN GRADING ACUTE REJECTION IN ENDOMYOCARDIAL BIOPSIES G.B.M. Lindop, M.M. Burke, S. Ogston, P. Bishop, C. Corbishley, M. Goddard, R. Harrison, C. Kjellstrom, A. McPhaden, M. Malone, D. Parums, K. Suvarna, S. Stewart, UK Heart Lung Transplant Pathology Group, Papworth Hospital, Cambs, United Kingdom The ISHLT Working Formulation (WF) for the grading of acute cardiac allograft rejection is used worldwide for the management of transplant recipients and multicentre evaluation of antirejection therapies. Difficulties in interpretation have arisen because of its complexity, the questionable clinical significance of focal moderate rejection (grade 2) and local variations in laboratory protocols. We therefore assessed reproducibility of the WF amongst a group of cardiac transplant pathologists in 10 UK cardiac transplant centres in 1999. Materials and Methods: Each centre contributed 10 cases. At least one case covered each of the WF grades of rejection. Remaining biopsies were from unselected sequential cases. Slides were randomised, coded and circulated. The only clinical information provided was the time interval post transplantation. Participants assessed biopsy adequacy, rejection grade and presence of Quilty lesions. Results were analysed using Kappa statistics to measure the level of agreement between observers. Results: 972 proformas were returned for analysis. The panel could reliably assess adequacy of the biopsy (K⫽0.51) and the presence of a Quilty lesion (K-0.54). Crude kappa value for Grade 0 rejection vs the rest was 0.49 (range 0.30-0.52). Weighted kappa values were 0.54 (0.31-0.60) for distinguishing Grades 0-1B from Grades 2-4 and 0.53 (0.38-0.63) for distinguishing Grades 0-2 from Grades 3A - 4. Kappa values for individual grades were: Grade 0-0.45, 1A-0.13, 1B-0.18, 2-0.15, 3A-0.31, 3B-0.30, 4-0.16. Conclusions: There is good agreement on biopsy grades 0,3A and 3B rejection, biopsy adequacy and Quilty lesions. In contrast there is poor agreement on biopsy grades 1A,1B and 4. We suggest that a simpler grading system would improve reproducibility and identify patients for augmented immunosuppression.
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groups: controls (no therapy, n⫽20), 3mg-CsA (3mg/kg/d/s.c., n⫽50), 12mg-CsA (12mg/kg/d/s.c., n⫽50). In group 2 and 3 CsA treatment ended either at day 20, 40, 60 or 80. 5 animals each were killed 20, 40, 60 and 80 days after transplantation with the following distribution: no therapy, treatment until sacrification, treatment ending 20, 40 or 60 days before sacrification. CsA levels were determined every ten days and ten days after withdrawal. TVP was assessed by digitizing morphometry and expressed as mean vessel occlusion (mvo). Acute rejection was scored (ISHLT criteria). In 12mg-CsA animals CsA level was 5 to10 fold higher than in 3mg-CsA animals and undetectable 10 days after withdrawal in both groups. In control animals we found a score IV of acute rejection, in continuously treated animals IIa in 12mg-CsA and IIIa in 3mg-CsA animals. But already 20 days after the withdrawal of CsA no significance was found when compared to controls and there was no difference at the end of the study when a 20, 40 or 60-day course of CsA application (group 2 and 3) was compared with untreated controls. Both continuous CsA regimens reduced the mvo significantly when compared with controls. We found no significant difference between 12mg and 3mg. The withdrawal of CsA therapy lead to an increase of mvo to levels with no significant difference to control animals. Despite the excessive increase of CsA blood levels we found neither a further reduction nor an increase of mvo in the high dose group compared with the low dose regimen. Our data suggest that CsA is not responsible for the development of TVP and that high CsA-levels lead to the same extent of TVP as low levels do. 67 EFFECTS OF FK506 INHALANT ON THE SUPPRESSION OF ACUTE REJECTION IN LUNG TRANSPLANTATION: USE OF RAT ORTHOTOPIC LUNG TRANSPLANTATION MODEL A. Ingu,1 K. Komatsu,1 M. Morikawa,1 T. Abe,1 S. Ichimiya,2 Y. Hirayama,3 1Thoracic and Cardiovascular Surgery, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan; 2Pathology, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan; 3Immunology and Inflammation Medical Biology Reserch Laboratories, Kashima Institution, Osaka, Osaka, Japan Background: We investigated the acute rejection suppression effect of FK506 0.1% inhalant developed for localized administration, and examined its immunosuppressant effect at a low blood concentration compared to systemic administration by intramuscular injection. Methods: We performed orthotopic left lung transplantations using the conventional cuff technique. Non-anesthetized rats were each stabilized inside a model P (Boxco Electronics, Inc.) and subjected to inhalation. M-1 (1.0 mg/kg), M-0.1 (0.1 mg/kg), M-0.05 (0.05 mg/kg) groups given intramuscular injection, and A-20 (20 puffs), A-10 (10 puffs), A-5 (5 puffs) groups given inhalation. In terms of the acceptance and lengthening effects, firstly, x-ray images were classified. Secondly, the transplanted lungs were extracted 6 after transplantation, and fixed 10% formalin solution and stained with hematoxylin-eosin. Samples were then histologically categorized. Thirdly, FK506 concentrations in the blood was calculated using ERISA and, fourthly, the expressions of IL-2, IFN-r, and TNF-a were examined by PCR. Results: 1) In all six groups that received treatment, the graft was seen to be accepted and lengthen, but lengthening after 14 days was only seen in four groups (M-1, M-0.1, A-20, and A-10 groups). 2) In the A-10 group perivascular edema was milder than in the M-0.1 group. 3) In the A-10 group, FK506 was not detected in the blood. In the A-10 group, tissue concentrations were about 1/10 lower than in the M-0.1 group. 4) Regarding cytokine suppression, IL-2, IFN-r, and TNF-a were all similarly suppressed in both the M-0.1 and A-10 groups.
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Conclusions: FK506 by inhalation was also effective. Despite blood concentrations being significantly lower in the inhalation groups compared to the intramuscukar injection groups, there were no differences in acceptance and lengthening effects and cytokine suppression. 68 TACROLIMUS: IN VITRO EFFECTS ON NEUTROPHIL PRODUCTION AND APOPTOSIS J.M. Koenig, N. Matharoo, J.J. Stegner, K.O. Schowengerdt, Pediatrics, University of Florida, Gainesville, FL Purpose: Tacrolimus is a common component of multi-drug immunosuppressive regimens used for the prevention of rejection in transplant recipients. Anecdotal observations and recent evidence suggest an association between tacrolimus therapy in children and the development of neutropenia. We hypothesized that this variety of neutropenia might be related to a negative effect of tacrolimus on neutrophil (PMN) production and/or survival. Methods: We designed in vitro studies to determine the dose-dependent effects of tacrolimus on PMN production and/or apoptosis. PMN and CD34⫹ cells isolated from umbilical cord blood of term gestations were cultured in the presence of tacrolimus (0 - 1000 ng/mL). To evaluate apoptosis, cells cultured for 24 h were stained with Annexin-V-FITC/ AAD and analyzed by flow cytometry. For clonal analysis, CD34⫹ cells cultured for 10 d in cytokine-enhanced semi-solid media were scored for their myeloid/erythroid (CFU-Mix), myeloid (CFU-GM), and erythroid (BFU-E) progenitor cell contents. Results: We observed that tacrolimus induced a dose-dependent enhancement of myeloid clonogenesis of CD34⫹ cells that peaked at 10 ng/mL (mean ⫾ SD, colonies/1000 plated cells): CFU-Mix, 14 ⫾ 2 (0 ng/mL) vs. 30 ⫾ 12 (p ⬍ 0.05); CFU-GM, 61 ⫾ 4 (0 ng/mL) vs. 160 ⫾ 6 (p ⬍ 0.01). Tacrolimus at these doses did not consistently decrease the survival of either CD34⫹ cells or PMNs. Conclusions: In contrast to our initial hypothesis, we observed that tacrolimus at clinically relevant concentrations enhanced clonogenesis of PMN progenitors and inconsistently affected cell survival. Tacrolimus is generally administered in combination with other immunosuppressive drugs after solid organ transplantation. Our data suggest that tacrolimus alone is unlikely to cause the neutropenia observed in this setting. 69 INTER-READER AND INTRA-READER AGREEMENT FOR GRADING ACUTE REJECTION AND AIRWAY INFLAMMATION AFTER LUNG TRANSPLANTATION, BASED ON THE LUNG REJECTION STUDY GROUP REVISED WORKING FORMULATION M.M. Chakinala,1 J. Ritter,2 B.F. Gage,1 A.A. Aloush,1 E.P. Trulock,1 1 Internal Medicine, Washington University School of Medicine, Saint Louis, MO; 2Pathology, Washington University School of Medicine, Saint Louis, MO The Lung Rejection Study Group standardized grading of acute rejection after transplantation by the degree of perivascular inflammation (A grade). They also emphasized the degree of airway inflammation (B grade), a likely precursor to bronchiolitis obliterans and chronic rejection. We determined the inter-reader and intra-reader agreements for both categories from transbronchial biopsies. Methods: While blinded to original interpretations and after excluding Cytomegalovirus pneumonitis, a single pathologist (JR) assigned A and B grades. When original and repeat grades were available, a weighted
The Journal of Heart and Lung Transplantation January 2003 Kappa [(w)] was calculated [range of -1.0 (total disagreement) to 1.0 (total agreement)]. Biopsies originally read by JR were used for intra-reader determinations. Because minimal airway inflammation (B1) is often non-specific, B grades were further dichotomized (0/1 or 2/3/4) and simple Kappa [(s)] calculated. And, since asymptomatic rejection is often treated when ⬎A1, A grades from surveillance biopsies were similarly dichotomized (0/1 or 2/3/4) and (s) calculated. Results: We reviewed 626 transbronchial biopsies from 207 recipients, obtained between 1/96 and 12/00. Weighted Kappa - K(w)
A score
B score
Inter-reader Intra-reader
0.65a (n ⫽ 529) 0.65a (n ⫽ 97)
0.28b (n ⫽ 164) 0.33c (n ⫽ 58)
a
p ⬍ 0.001; bp ⫽ .005; cp ⫽ .008.
Inter-reader (s) for dichotomized B grades was 0.25; p⫽.001. Interreader (s) for dichotomized A grades on surveillance biopsies (n⫽273) was 0.65; p⬍.001. Conclusion: Inter-reader and intra-reader agreements for A grades are good, while agreements for B grades are only fair. Because airway inflammation is emphasized as a potential precursor for chronic rejection, better agreement of B grading is necessary to incorporate airway pathology into clinical decision-making.
70 INTER-OBSERVER VARIABILITY IN GRADING ACUTE REJECTION IN ENDOMYOCARDIAL BIOPSIES G.B.M. Lindop, M.M. Burke, S. Ogston, P. Bishop, C. Corbishley, M. Goddard, R. Harrison, C. Kjellstrom, A. McPhaden, M. Malone, D. Parums, K. Suvarna, S. Stewart, UK Heart Lung Transplant Pathology Group, Papworth Hospital, Cambs, United Kingdom The ISHLT Working Formulation (WF) for the grading of acute cardiac allograft rejection is used worldwide for the management of transplant recipients and multicentre evaluation of antirejection therapies. Difficulties in interpretation have arisen because of its complexity, the questionable clinical significance of focal moderate rejection (grade 2) and local variations in laboratory protocols. We therefore assessed reproducibility of the WF amongst a group of cardiac transplant pathologists in 10 UK cardiac transplant centres in 1999. Materials and Methods: Each centre contributed 10 cases. At least one case covered each of the WF grades of rejection. Remaining biopsies were from unselected sequential cases. Slides were randomised, coded and circulated. The only clinical information provided was the time interval post transplantation. Participants assessed biopsy adequacy, rejection grade and presence of Quilty lesions. Results were analysed using Kappa statistics to measure the level of agreement between observers. Results: 972 proformas were returned for analysis. The panel could reliably assess adequacy of the biopsy (K⫽0.51) and the presence of a Quilty lesion (K-0.54). Crude kappa value for Grade 0 rejection vs the rest was 0.49 (range 0.30-0.52). Weighted kappa values were 0.54 (0.31-0.60) for distinguishing Grades 0-1B from Grades 2-4 and 0.53 (0.38-0.63) for distinguishing Grades 0-2 from Grades 3A - 4. Kappa values for individual grades were: Grade 0-0.45, 1A-0.13, 1B-0.18, 2-0.15, 3A-0.31, 3B-0.30, 4-0.16. Conclusions: There is good agreement on biopsy grades 0,3A and 3B rejection, biopsy adequacy and Quilty lesions. In contrast there is poor agreement on biopsy grades 1A,1B and 4. We suggest that a simpler grading system would improve reproducibility and identify patients for augmented immunosuppression.