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Tacrolimus: in vitro effects on neutrophil production and apoptosis
S96
Abstracts
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.
Abstracts
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.