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ABSTRACT: We evaluated the potential of lung-directed gene therapy for alpha1-antitrypsin (AAT) deficiency using an adeno-associated virus type 6 (AAV6) vector containing a human AAT (hAAT) complementary DNA (cDNA) delivered to the lungs of mice and dogs. The results in normal and immune-deficient mice showed that hAAT concentrations were much higher in lung fluid than in plasma, and therapeutic levels were obtained even in normal mice. However, in normal mice an immune response against the vector and/or transgene limited long-term gene expression. An AAV6 vector expressing a marker protein verified that AAV6 vectors efficiently transduced lung cells in dogs. Delivery of AAV6-hAAT resulted in low levels of hAAT in dog serum but therapeutic levels in the lung that persisted for at least 58 days to 4 months in three immunosuppressed dogs. Expression in the serum was not detectable after 45 days in one nonimmune suppressed dog. A lymphoproliferative response to AAV capsid but not to hAAT was detected even after immunosuppression. These results in mice and dogs show the feasibility of expression of therapeutic levels of AAT in the lungs after AAV vector delivery, and advocate for approaches to prevent cellular immune responses to AAV capsid proteins for persistence of gene expression in humans.
Molecular Therapy 04/2010; 18(6):1165-72. · 6.87 Impact Factor
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Peter Chen,
John K McGuire,
Robert C Hackman,
Kyoung-Hee Kim,
Roy A Black,
Kurt Poindexter,
Wei Yan,
Phillip Liu,
Ann J Chen,
William C Parks, David K Madtes
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ABSTRACT: Obliterative bronchiolitis (OB) is the histopathological finding in chronic lung allograft rejection. Mounting evidence suggests that epithelial damage drives the development of airway fibrosis in OB. Tissue inhibitor of metalloproteinase (TIMP)-1 expression increases in lung allografts and is associated with the onset of allograft rejection. Furthermore, in a mouse model of OB, airway obliteration is reduced in TIMP-1-deficient mice. Matrilysin (matrix metallproteinase-7) is essential for airway epithelial repair and is required for the re-epithelialization of airway wounds by facilitating cell migration; therefore, the goal of this study was to determine whether TIMP-1 inhibits re-epithelialization through matrilysin. We found that TIMP-1 and matrilysin co-localized in the epithelium of human lungs with OB and both co-localized and co-immunoprecipitated in wounded primary airway epithelial cultures. TIMP-1-deficient cultures migrated faster, and epithelial cells spread to a greater extent compared with wild-type cultures. TIMP-1 also inhibited matrilysin-mediated cell migration and spreading in vitro. In vivo, TIMP-1 deficiency enhanced airway re-epithelialization after naphthalene injury. Furthermore, TIMP-1 and matrilysin co-localized in airway epithelial cells adjacent to the wound edge. Our data demonstrate that TIMP-1 interacts with matrix metalloproteinases and regulates matrilysin activity during airway epithelial repair. Furthermore, we speculate that TIMP-1 overexpression restricts airway re-epithelialization by inhibiting matrilysin activity, contributing to a stereotypic injury response that promotes airway fibrosis via bronchiole airway epithelial damage and obliteration.
American Journal Of Pathology 06/2008; 172(5):1256-70. · 4.89 Impact Factor
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ABSTRACT: Currently, statistical techniques for analysis of microarray-generated data sets have deficiencies due to limited understanding of errors inherent in the data. A generalized likelihood ratio (GLR) test based on an error model has been recently proposed to identify differentially expressed genes from microarray experiments. However, the use of different error structures under the GLR test has not been evaluated, nor has this method been compared to commonly used statistical tests such as the parametric t-test. The concomitant effects of varying data signal-to-noise ratio and replication number on the performance of statistical tests also remain largely unexplored. In this study, we compared the effects of different underlying statistical error structures on the GLR test's power in identifying differentially expressed genes in microarray data. We evaluated such variants of the GLR test as well as the one sample t-test based on simulated data by means of receiver operating characteristic (ROC) curves. Further, we used bootstrapping of ROC curves to assess statistical significance of differences between the areas under the curves. Our results showed that i) the GLR tests outperformed the t-test for detecting differential gene expression, ii) the identity of the underlying error structure was important in determining the GLR tests' performance, and iii) signal-to-noise ratio was a more important contributor than sample replication in identifying statistically significant differential gene expression.
Gene regulation and systems biology 02/2008; 2:125-139.
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ABSTRACT: An imbalance between the proteolytic activity of matrix metalloproteinases (MMPs) and the activity of tissue inhibitors of metalloproteinases (TIMPs) is implicated in tissue injury during inflammation. The N-terminal cysteine of TIMP-1 plays a key role in the inhibitory activity of the protein because it coordinates the essential catalytic Zn2+ of the MMP, preventing the metal ion from functioning. An important mechanism for controlling the interaction of TIMPs with MMPs might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase (MPO) system of phagocytes. Here, we show that HOCl generated by the MPO-H2O2-chloride system inactivates TIMP-1 by oxidizing its N-terminal cysteine. The product is a novel 2-oxo acid. Liquid chromatography-mass spectrometry and tandem mass spectrometry analyses demonstrated that methionine and N-terminal cysteine residues were rapidly oxidized by MPO-derived HOCl but only oxidation of the N-terminal cysteine of TIMP-1 correlated well with loss of inhibitory activity. Importantly, we detected the signature 2-oxo-acid N-terminal peptide in tryptic digests of bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome, demonstrating that TIMP-1 oxidation occurs in vivo. Loss of the N-terminal amino group and disulfide structure are crucial for preventing TIMP-1 from inhibiting MMPs. Our findings suggest that pericellular production of HOCl by phagocytes is a pathogenic mechanism for impairing TIMP-1 activity during inflammation.
Journal of Biological Chemistry 12/2007; 282(44):31826-34. · 4.77 Impact Factor
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ABSTRACT: An imbalance between the proteolytic activity of matrix metalloproteinases (MMPs) and the activity of tissue inhibitors of
metalloproteinases (TIMPs) is implicated in tissue injury during inflammation. The N-terminal cysteine of TIMP-1 plays a key
role in the inhibitory activity of the protein because it coordinates the essential catalytic Zn2+ of the MMP, preventing the metal ion from functioning. An important mechanism for controlling the interaction of TIMPs with
MMPs might involve hypochlorous acid (HOCl), a potent oxidant produced by the myeloperoxidase (MPO) system of phagocytes.
Here, we show that HOCl generated by the MPO-H2O2-chloride system inactivates TIMP-1 by oxidizing its N-terminal cysteine. The product is a novel 2-oxo acid. Liquid chromatography-mass
spectrometry and tandem mass spectrometry analyses demonstrated that methionine and N-terminal cysteine residues were rapidly
oxidized by MPO-derived HOCl but only oxidation of the N-terminal cysteine of TIMP-1 correlated well with loss of inhibitory
activity. Importantly, we detected the signature 2-oxo-acid N-terminal peptide in tryptic digests of bronchoalveolar lavage
fluid from patients with acute respiratory distress syndrome, demonstrating that TIMP-1 oxidation occurs in vivo. Loss of the N-terminal amino group and disulfide structure are crucial for preventing TIMP-1 from inhibiting MMPs. Our findings
suggest that pericellular production of HOCl by phagocytes is a pathogenic mechanism for impairing TIMP-1 activity during
inflammation.
Journal of Biological Chemistry 11/2007; 282(44):31826-31834. · 4.77 Impact Factor
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ABSTRACT: Acute lung injury (ALI) is characterized by an early inflammatory response followed by a late fibroproliferative phase, and by an increase in the bronchoalveolar lavage fluid (BALF) concentrations of bioactive soluble FasL (sFasL). Activation of Fas (CD95) has been associated with the development of lung fibrosis in mice. The goal of this study was to determine the mechanisms that link Fas activation with the development of fibrosis in the lungs. We treated mice with three daily intratracheal instillations of a Fas-activating monoclonal antibody (Jo2) or a control IgG, and studied the animals at sequential times. Mice treated with Jo2 had increased caspase-3 activation in alveolar wall cells on Days 2, 4, and 7; an inflammatory response peaking on Day 7, and increased total lung collagen on Day 21. Gene expression profiling performed on Days 2, 4, and 7 showed sequential activation of co-regulated profibrotic genes, including marked up-regulation of matrix metalloproteinase 12 (MMP-12). Targeted deletion of MMP-12 protected mice from Fas-induced pulmonary fibrosis, even though the inflammatory responses in the lungs were similar to those of wild-type mice. Compared with wild-type mice, the mmp12(-/-) mice showed decreased expression of the profibrotic genes egr1 and cyr61. We conclude that Fas activation in the lungs induces a complex response that includes apoptosis, inflammation, and eventually fibrosis, and that MMP-12 is essential for the fibrotic phenotype. We speculate that MMP-12 activity is required for activation of the profibrotic genes egr1 and cyr61.
American Journal of Respiratory Cell and Molecular Biology 09/2007; 37(2):210-21. · 5.13 Impact Factor
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ABSTRACT: The purpose of this study was to determine the prevalence of pulmonary dysfunction in pediatric hematopoietic cell transplant (HCT) survivors and to identify associated risk factors.
In a cross-sectional study, patients surviving at least 5 years after pediatric HCT were requested to undergo pulmonary function testing (PFT). Risk factors for restrictive lung disease (RLD) and obstructive lung disease (OLD) were analyzed using multivariate analysis.
Among 472 patients contacted, 260 (55%) participated and 215 were selected for analysis. These patients were transplanted at a median age of 8.3 (0.3-18.0) years; 175 for hematologic malignancies and 40 for non-malignant diseases. The preparative regimens for 133 patients included fractionated TBI (FTBI), 29 single-fraction TBI (SFTBI), and 53 non-TBI regimens. PFT was performed at a median of 10 (5.0-27.5) years after HCT. Forty percent of patients had either RLD or OLD (28% RLD, 9% OLD, 3% mixed RLD/OLD) and at least 15% had an isolated low-DLCO. Moderate-to-severe impairment was present in 45% of patients with RLD or OLD. In multivariate analysis, risk factors associated with RLD included transplant regimen, transplant diagnosis, scleroderma/contracture, and donor relation. Patients treated with SFTBI had the highest risk of RLD. Risk factors for OLD included chronic graft-versus-host disease, transplant regimen, and time after HCT. Patients surviving 20 or more years after HCT had the highest risk of OLD.
Fifty-five percent of long-term pediatric HCT survivors had pulmonary dysfunction. These findings stress the need for long-term follow-up to detect pulmonary dysfunction.
Pediatric Blood & Cancer 11/2006; 47(5):594-606. · 1.89 Impact Factor
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ABSTRACT: Obliterative bronchiolitis (OB) is a major cause of allograft dysfunction after lung transplantation and is thought to result from immunologically mediated airway epithelial destruction and luminal fibrosis. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) have been implicated in the regulation of lung inflammation, airway epithelial repair, and extracellular matrix remodeling and therefore may participate in the pathogenesis of OB. The goals of this study were to determine the expression profiles of MMPs and TIMPs and the role of TIMP-1 in the development of airway obliteration using the murine heterotopic tracheal transplant model of OB. We demonstrate the selective induction of MMP-3, MMP-9, MMP-12, and TIMP-1 in a temporally restricted manner in tracheal allografts compared with isografts. In contrast, the expression of MMP-7, TIMP-2, and TIMP-3 was decreased in allografts relative to isografts during the period of graft rejection. TIMP-1 protein localized to epithelial, mesenchymal, and inflammatory cells in the tracheal grafts in a temporally and spatially restricted manner. Using TIMP-1-deficient mice, we demonstrate that the absence of TIMP-1 in the donor trachea or the allograft recipient reduced luminal obliteration and increased re-epithelialization in the allograft compared with wild-type control at 28 d after transplantation. Our findings provide direct evidence that TIMP-1 contributes to the development of airway fibrosis in the heterotopic tracheal transplant model, and suggest a potential role for this proteinase inhibitor in the pathogenesis of OB in patients with lung transplant.
American Journal of Respiratory Cell and Molecular Biology 05/2006; 34(4):464-72. · 5.13 Impact Factor
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ABSTRACT: Bleomycin-induced lung injury triggers a profound and durable increase in tissue inhibitor of metalloproteinase (TIMP)-1 expression, suggesting a potential role for this antiproteinase in the regulation of lung inflammation and fibrosis. TIMP-1 protein induction is spatially restricted to areas of lung injury as determined by immunohistochemistry. Using TIMP-1 null mutation mice, we demonstrate that TIMP-1 deficiency amplifies acute lung injury as determined by exaggerated pulmonary neutrophilia, hemorrhage, and vascular permeability compared with wild-type littermates after bleomycin exposure. The augmented pulmonary neutrophilia observed in TIMP-1-deficient animals was not found in similarly treated TIMP-2-deficient mice. Using TIMP-1 bone marrow (BM) chimeric mice, we observed that the TIMP-1-deficient phenotype was abolished in wild-type recipients of TIMP-1-deficient BM but not in TIMP-1-deficient recipients of wild-type BM. Acute lung injury in TIMP-1-deficient mice was accompanied by exaggerated gelatinase-B activity in the alveolar compartment. TIMP-1 deficiency did not alter neutrophil chemotactic factor accumulation in the injured lung nor neutrophil migration in response to chemotactic stimuli in vivo or in vitro. Moreover, TIMP-1 deficiency did not modify collagen accumulation after bleomycin injury. Our results provide direct evidence that TIMP-1 contributes significantly to the regulation of acute lung injury, functioning to limit inflammation and lung permeability.
American Journal of Respiratory Cell and Molecular Biology 10/2005; 33(3):271-9. · 5.13 Impact Factor
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ABSTRACT: The role of pulmonary function before stem cell transplant as a potential risk factor for the development of early post-transplant respiratory failure and mortality is controversial.
We conducted a retrospective analysis of the pretransplant pulmonary function of 2,852 patients who received their transplant between 1990 and 2001.
Pretransplant FEV(1), FVC, total lung capacity (TLC), diffusing capacity of carbon monoxide (DL(CO)), and the alveolar-arterial oxygen tension difference P(A-a)O(2) were measured and assessed for association with development of early respiratory failure and mortality in Cox proportional hazard logistic models.
In multivariate analyses, progressive decrease of all lung function parameters was associated with a stepwise increase in risk of developing early respiratory failure and mortality when assessed in independent models. On the basis of a significant correlation between FEV(1) and FVC (r = 0.81), FEV(1) and TLC (r = 0.61), and FVC and TLC (r = 0.80), and a lack of correlation between FEV(1) and DL(CO), we developed a pretransplant lung function score based on pretransplant FEV(1) and DL(CO) to determine the extent of pulmonary compromise before transplant. Multivariate analysis indicated that higher pretransplant lung function scores are associated with a significant increased risk for developing early respiratory failure (category II hazard ratio [HR], 1.4; category III HR, 2.2; category IV HR, 3.1; p < 0.001) and death (category II HR, 1.2; category III HR, 2.2; category IV HR, 2.7; p < 0.005).
These results suggest that not only does compromised pretransplant lung function contribute to the risk for development of early respiratory failure and mortality but this risk may be estimated before transplant by grading the extent of FEV(1) and DL(CO) compromise.
American Journal of Respiratory and Critical Care Medicine 09/2005; 172(3):384-90. · 11.08 Impact Factor
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ABSTRACT: Chronic hypoxic pulmonary hypertension is an important clinical disorder causing significant morbidity. Despite recent discoveries, many molecular mechanisms involved in its pathogenesis remain unexplored. We have undertaken a systematic and unbiased approach to gain global insights into this complex process. By combining transcriptional profiling with rigorous statistical methods and cluster analysis, we identified the dominant temporal patterns of gene expression during progression and regression of hypoxic pulmonary hypertension. We next integrated these results with global gene annotation analysis to identify key biological themes involved in the development and resolution of hypoxic pulmonary hypertension and vascular remodeling. This novel approach assigned biological roles to thousands of candidate genes based on their temporal expression profiles and membership in specific biological modules. Our procedure confirmed several molecular pathways and gene products known to be important in hypoxic pulmonary hypertension. Furthermore, we discovered several novel candidates and molecular mechanisms, including IQ motif containing GTPase-activating protein-1 (IQGAP1), decorin, insulin-like growth factor binding protein-3 (IGFBP3), and lactotransferrin, that may play crucial roles in hypoxic pulmonary hypertension and vascular remodeling. Our methodology of integrating transcriptional profiling, cluster analysis, and global gene annotation provides new insights into the pathophysiology of pulmonary hypertension and is applicable to other models of human disease.
Physiological Genomics 07/2005; 22(1):14-23. · 2.73 Impact Factor
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ABSTRACT: Trafficking of lymphocytes to lung is a critical component of pulmonary immune defense and surveillance. Selectins, expressed on vascular endothelium, regulate T lymphocyte emigration into tissues, such as skin, but the role of the selectins in trafficking of T cells to lung has not been well characterized. Here, we used a model of lung inflammation induced by adoptive transfer of alloreactive Th1 cells to analyze the role of P- and E-selectin in Th1 cell trafficking to lung in vivo. We found that both P- and E-selectin play an important role in Th1 lymphocyte migration to lung. We confirmed that the Th1 cells express P-selectin glycoprotein ligand-1, which was functional in binding to P- and E-selectin in vitro. However, our studies reveal that a ligand distinct from P-selectin glycoprotein-1 also binds these selectins in vitro and appears to play a physiologic role in in vivo emigration of Th1 lymphocytes into the lung.
American Journal of Respiratory Cell and Molecular Biology 03/2004; 30(2):220-7. · 5.13 Impact Factor
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Yoshiki Akatsuka,
Edus H Warren,
Ted A Gooley,
Anthony G Brickner,
Ming-Tseh Lin,
John A Hansen,
Paul J Martin, David K Madtes,
Victor H Engelhard,
Toshitada Takahashi,
Stanley R Riddell
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ABSTRACT: We recently identified a new minor histocompatibility antigen, termed HA-8, which is presented by human leucocyte antigen (HLA)-A*0201 or HLA-A*0202 and expressed ubiquitously among tissues. A retrospective analysis of 577 Caucasian patients with HLA-A*0201 or A*0202 who had received a haematopoietic stem cell transplant from a human leucocyte antigen (HLA)-identical sibling was conducted to determine whether HA-8 disparity correlated with clinical outcome. HA-8 disparity was detected in 72 recipients, and grades II-IV graft-versus-host disease (GVHD) occurred in 46 (64%), compared with 251 (50%) of the 503 patients without HA-8 disparity. After adjusting for known risk factors for acute GVHD, this difference was statistically significant (odds ratio, 1.8; 95% confidence interval, 1.0-3.1; P = 0.04). However, the hazards of clinical extensive chronic GVHD, overall mortality and recurrent malignancy were not statistically significantly different between the two groups. These data suggest that the increased risk of acute GVHD associated with recipient HA-8 disparity was not sufficient to change other clinical outcomes.
British Journal of Haematology 12/2003; 123(4):671-5. · 4.94 Impact Factor
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ABSTRACT: Bronchiolitis obliterans organizing pneumonia (BOOP) has been reported following hematopoietic stem cell (HSC) transplantation, but the clinical features and risk factors for this disorder have not been well characterized. This case-control study of 49 patients with histologic BOOP and 161 control subjects matched by age and year of transplantation describes the clinical features and analyzes the risk factors for BOOP following HSC transplantation. Data on clinical features and outcome were collected by chart review. Odds ratios, estimating the relative risk of BOOP in allogeneic HSC recipients, were calculated by conditional logistic regression with adjustment for potential confounding factors. Clinical features of BOOP in this population were similar to idiopathic BOOP and BOOP occurring in other disease settings. There was an association between acute and chronic graft-versus-host disease (GVHD) and the subsequent development of BOOP (odds ratios, 3.8 [95% CI, 1.2 to 12.3] and 3.1 [95% CI, 1.1 to 9.2], respectively). Patients with BOOP were more likely to have acute GVHD involving the skin (odds ratio, 4.6; P =.005) and chronic GVHD involving the gut (odds ratio, 6.6; P =.018) and oral cavity (odds ratio, 5.9; P =.026). This study shows that histologic BOOP following HSC transplantation has clinical features that resemble idiopathic BOOP and is strongly associated with prior acute and chronic GVHD. These results have important implications for the care of patients who develop respiratory symptoms after HSC transplantation and may help elucidate the pathogenesis of idiopathic BOOP.
Blood 12/2003; 102(10):3822-8. · 9.90 Impact Factor
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Takahiro Fukuda,
Robert C Hackman,
Katherine A Guthrie,
Brenda M Sandmaier,
Michael Boeckh,
Michael B Maris,
David G Maloney,
H Joachim Deeg,
Paul J Martin,
Rainer F Storb, David K Madtes
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ABSTRACT: Idiopathic pneumonia syndrome (IPS) is a significant noninfectious complication of hematopoietic stem cell transplantation (HSCT). We compared the incidences and outcomes of IPS among patients who underwent allogeneic HSCT after nonmyeloablative (n = 183) compared with conventional (n = 917) conditioning between December 1997 and December 2001. Patients given nonmyeloablative conditioning were older than those given conventional conditioning (median ages, 53 vs 41 years; P =.001). The cumulative incidence of IPS was significantly lower at 120 days after nonmyeloablative conditioning than conventional conditioning (2.2% vs 8.4%; P =.003). In addition, greater patient age (older than 40 years), diagnosis of acute leukemia or myelodys-plastic syndrome, and severe acute graft-versus-host disease were associated with significantly increased risks for IPS. Among older patients (older than 40 years) given conventional conditioning, high-dose total body irradiation (TBI) was associated with an increased risk for IPS than were non-TBI-based regimens (16% vs 5.8%; P =.001). IPS occurred early after transplantation, progressed rapidly, and was associated with a high mortality rate (75%) despite aggressive support. Initiation of mechanical ventilation and the presence of renal insufficiency at IPS onset were associated with increased risks for death after IPS. These findings support the concept that lung damage from the conditioning regimen plays a crucial role in the development of IPS after HSCT.
Blood 11/2003; 102(8):2777-85. · 9.90 Impact Factor
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ABSTRACT: We report two cases of pulmonary venocclusive disease (PVOD) in children with acute lymphoblastic leukemia treated by marrow allograft transplantation following conditioning with high-dose 1-3 bis chloroethyl-1 nitrosourea (BCNU), etoposide (VP-16), and cyclo-phosphamide (Cy). Both patients developed symptomatic pulmonary hypertension documented by right heart catheterization. Open-lung biopsy of one patient demonstrated PVOD evident even on frozen sections stained with hematoxylin and eosin. High-dose methylprednis-olone was associated with significant clinical improvement in both patients. Pulmonary symptoms resolved in one patient who subsequently died in leukemic relapse. PVOD resolved in the other patient, only to recur when steroids were discontinued and then again respond to reinstitution of therapy. More aggressive therapy for malignant diseases may increase the incidence of PVOD. Prompt recognition of its subtle clinical and histological manifestations allows early institution of steroid therapy, which may be beneficial.
(C) Williams & Wilkins 1989. All Rights Reserved.
Transplantation 05/1989; 47(6). · 4.00 Impact Factor
