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									                                                      Duke University
                                                      Medical Center                                                                                                           The Role of Surfactant Protein A & D in Lung Allograft Rejection
                                                                                                                                                                               Matthew Schechter, BSc, C. Ashley Finlen Copeland, MSW, Francine Kelly, BS, Scott M. Palmer, MD, MHS.
                                                                                                                                                                               Division of Pulmonary and Critical Care Medicine - Duke University Medical Center, Durham, NC

                                                                                 ABSTRACT                                                                                                                                                                             HYPOTHESES                                                                                                                     Figure 1: Recipient T11208C genotype (SP-D 3’ UTR) affects                                                                                                                              Figure 4: SP-D levels in transplanted lung influenced by the recipient’s
  Background Lung transplantation is characterized by high rates of rejection, especially compared to other types of solid organ transplants. Our lab has                                                                                                                                                                                                                                            progression to BOS > 5 yrs. post transplant                                                                                                                                                                            genotype
  developed the hypothesis that the lung’s innate immune system, unique among transplanted organs, influences the outcome of lung transplantation. The                                                                               Donor single nucleotide polymorphisms (SNPs) within the SP-A and SP-D
  pulmonary collectins, surfactant protein (SP)-A and SP-D, regulate the innate host defense of the lung and may provide a link between the innate and
  acquired immune systems. Although these proteins have been implicated in numerous lung diseases, their role in lung transplantation remains unstudied.
                                                                                                                                                                                                                                      genes explain the interindividual variation in the development of PGD                                                                                                                                                                                                                                                                                                                                p = .0622                                                                      p = .0322
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           p = .0705                                                                                  p = .0731
  We hypothesized that single nucleotide polymorphisms (SNPs) in the SP-A and SP-D genes influence the development of early and late graft dysfunction.                                                                              SNPs within the recipient SP-A and SP-D genes explain the interindividual
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     900                                                                                  900

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           Mean SP-D Concentration
  Therefore, we determined the effect of SP-A and SP-D polymorphisms in the donor on the development of primary graft dysfunction (PGD) and the effect of

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             Mean SP-D Concentration
                                                                                                                                                                                                                                      variation in the development of BOS                                                                                                                                                                                                                                                                                                                                            800                                ANOVA: p = .0813
  these polymorphisms in the recipient on the development of bronchiolitis obliterans syndrome (BOS), the major cause of late death after lung                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            800                                 ANOVA:
  transplantation. We also theorized that SP-D levels are associated with allograft function, and therefore followed the trends of SP-D levels in a group of                                                                         SP-D levels in the bronchioalveolar lavage fluid are associated with chronic                                                                                                                                                                                                                                                                                                   700                                                                                  700                                 p = .0931
  patients that eventually developed BOS and a matched BOS-free cohort.                                                                                                                                                               allograft function.                                                                                                                                                                                                                                                                                                                                                            600                                                                                  600


  Methods A cohort of lung transplant recipients was established using recipients and donors from Duke University Medical Center. Patients were screened                                                                                                                                                                                                                                                                                                                                                                                                                                                             500                                                                                  500
  for four SP-D polymorphisms and three polymorphisms from each of the SP-A genes (SP-A1 and SP-A2). For the SP-D polymorphisms, commercially                                                                                                                                                                                                                                                                                                                                                                                                                                                                        400                                                                                  400
  available TaqMan assays were used. Given the homology between the two SP-A genes, gene-specific primers were first used to amplify the region                                                                                                                                                                                                                                                                                                                                                                                                                                                                      300                                                                                  300
  containing the polymorphism of interest, followed by allelic determination using custom-designed TaqMan assays. We examined the association between
  donor SP-A & SP-D genotypes and the presence or absence of severe (Grade 3) PGD using a chi-square test and compared the PaO2/FiO2 ratio 24 hours
  post-transplant, based on donor genotype, using an ANOVA. A Kaplan-Meier log rank test was used to determine the effect of recipient SP-A and SP-D                                                                                                                       RESULTS                                                                                                                                                                                                                                                                                                                                   200
  polymorphisms on the onset of BOS and post-transplant graft survival. A commercially-available ELISA for SP-D was performed on bronchoalveolar lavage                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                 TT                TC           CC                                              TT             TC                 CC
  fluid (BALF) collected from transplant recipients during surveillance bronchoscopies. We examined the relationship between recipient SP-D genotypes and                                                                           TABLE 1: SP-A1, SP-A2 & SP-D POLYMORPHISMS ANALYZED IN THE LUNG
  mean SP-D levels over the course of the transplant using ANOVA & Student’s t-tests.                                                                                                                                               TRANSPLANATION POPULATION                                                                                                                                                                                                                                                                                                                                                                          rs1923537 (3' UTR)                                                            rs721917 (Met11Thr)
  Results A total of 407 lung transplant recipients and 212 donors were genotyped for the 10 surfactant SNPs. All SNPs had a no-call rate <5%. Relevant
                                                                                                                                                                                                                                                                                                                                                                      Minor                                                                                                                                                                                                                                                                                                                               900
  data was entered into database in order to establish well-defined clinical phenotypes of the early and late graft function in all transplant patients.                                                                                                                                                                                                                                                                                                                                                                                                                                                             900

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                Mean SP-D Concentration
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          Mean SP-D Concentration
  Homozygosity for the T allele within the 3’ UTR of SP-D was associated with decreased rates of both severe PGD and late (> 5yrs) BOS. The genotype was
                                                                                                                                                                                                                                               Absolute                           Genetic      Amino Acid           Nucleotide                                        allele                                                      1.00                                                                                                                                                                               800                                     p = .0357                                    800                         p = .0369

                                                                                                                                                                                                                                                                                                                               Survival Distribution Function
  also associated with increased SP-D levels in the BALF. Lung transplant recipients with a Thr/Thr genotype at αα11 had significantly decreased SP-D levels                                                                        Gene       position           RS number       region       substitution        substitution                                    frequency
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     700                                                                                  700
  as well as increased rates of severe PGD In a cross-sectional analysis of BALF, we found that BAL SP-D levels were significantly decreased in BOS                                                                                 SP-A1        chr10:           rs1059047        Exon 1          T/C             αα 19: Val →                                       6.5%                                                                                                                                                                                                                                           600                                                                                  600

  patients, with higher BOS grade corresponding to lower of SP-D levels.                                                                                                                                                                       74656728                                                                Ala                                                                                                        0.75
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     500                                                                                  500
  Conclusions These studies indicate that polymorphic forms of SP-D might modulate the immune system’s response to lung transplantation and support our
                                                                                                                                                                                                                                    SP-A1        chr10:           rs1136450        Exon 1          G/C             αα 50: Val →                                      22.7%                                                                                                                                                                                                                                           400                                                                                  400
  overall hypothesis for a role of pulmonary innate immunity in modulating lung allograft outcomes. A decrease in SP-D, due to either genetic or environmental
                                                                                                                                                                                                                                               74656820                                                                Leu                                                                                                                                                                                                                                                                                           300                                                                                  300
  factors, is associated with chronic graft dysfunction. Furthermore, the 3’ UTR variant associated with increased SP-D levels seems to be protective against                                                                                                                                                                                                                                                                     0.50
  severe PGD and late progression to BOS. The pulmonary collectins provide a potentially novel target for therapies designed to help prevent rejection and                                                                          SP-A1        chr10:           rs4253527        Exon 4          C/T            αα 219: Arg →                                       2.2%                                                                                                                                                                                                                                           200                                                                                  200
  improve outcomes following lung transplantation.                                                                                                                                                                                             74658868                                                                Trp                                                                                                                                                                                                                                                                                           100                                                                                  100
                                                                                                                                                                                                                                    SP-A2        chr10:           rs1059046        Exon 1          A/C             αα 9: Asn →                                       17.4%                                                        0.25                                                                                                                                                                                 0                                                                                    0
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      TT                   C*                                                        T*                  CC
                                                                                                                                                                                                                                               75314238                                                                Thr
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      rs1923537 (3' UTR)                                                         rs721917 (Met11Thr)
                                                                   INTRODUCTION                                                                                                                                                     SP-A2        chr10:
                                                                                                                                                                                                                                                                  rs17886395       Exon 2          G/C             αα 91: Ala →
                                                                                                                                                                                                                                                                                                                                                                     10.9%                                                        0.00

  Lung transplant is a viable option for patients with end-stage lung disease. Although immediate post-transplant survival has
   improved to over 80% at 1 year, the 5-year survival rate remains poor at 50%, well behind other solid organ transplants. 1
                                                                                                                                                                                                                                    SP-A2        chr10:
                                                                                                                                                                                                                                                                  rs1965708        Exon 4          C/A            αα 223: Gln →
                                                                                                                                                                                                                                                                                                                                                                     11.4%                                                                    0            500        1000      1500           2000

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         2500                    3000       3500       4000        4500
                                                                                                                                                                                                                                     SP-D        chr10:           rs1923537        3’ UTR          T/C                    n/a                                        35.4%                                                                STRATA:                        DUTR=CC                                                                     Censored DUTR=CC
  Primary Graft Dysfunction (PGD) is an important early complication of lung allograft transplantation. Severe (Grade 2 & 3)                                                                                                                  75537958                                                                (T11208C)                                                                                                                                         DUTR=TC
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     Censored DUTR=TC
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                     Censored DUTR=TT
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           Of         all the polymorphisms studied, only two SP-D SNPs (3’UTR & Met11Thr)
   PGD occurs in ~12% of transplants, and as many as half of all lung transplant recipients may experience a mild form of this                                                                                                       SP-D        chr10:            rs721917        Exon 2          T/C            αα 11: Met →                                       41.7%                                                                                                                                                                                                                          seemed to influence either early or late lung allograft function
   disease. PGD has a 30-day mortality rate of over 60%.2 PGD is believed to be due to ischemia/reperfusion injury, whereby                                                                                                                    75549076                                                                Thr
   activation of neutrophils leads to an accumulation of reactive oxygen species that causes acute lung injury (similar to ARDS).3                                                                                                   SP-D        chr10:           rs2243639        Exon 5          C/T            αα 160: Ala →                                      43.8%                                                                                                                                                                                                                 The genotype associated with increased SP-D levels in the BAL (TT 3’UTR) was
                                                                                                                                                                                                                                               75692660                                                                Thr
  The long-term survival of lung transplants is limited by bronchiolitis obliterans (BO), characterized by fibrotic scarring and
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    also associated with decreased rates of severe PGD & progression to BOS > 5
   destruction of the bronchioles by a dense, lymphocytic infiltration. BO manifests clinically as progressive airflow obstruction
                                                                                                                                                                                                                                     SP-D        chr10:           rs3088308        Exon 8          A/T            αα 270: Thr →                                       6.2%     Figure 2: Recipient SP-D genotypes are associated with the                                                                                                                                                           years post-transplant
                                                                                                                                                                                                                                               75688804                                                                Ser
   termed bronchiolitis obliterans syndrome (BOS). Nearly half of all lung transplant patients develop BOS within 5 years of                                                                                                                                                                                                                                                   development of severe (Grade 3) PGD 24 hours post-transplant
   transplantation. BO is thought to the result of chronic allograft injury, whether caused by alloimmune responses (acute                                                                                                              TABLE 2: BOS GENETIC ASSOCIATION COHORT                                           NO (%)                                                                                                                                                                                                                                               p = .212
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           The         genotype associated with decreased SP-D levels (Thr/Thr at αα 11) is
   rejection, lymphocytic bronchiolitis) or alloimmune-independent processes (infection, gastroesophageal reflux, PGD).4                                                                                                                (N= 356)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        PGD 0-2
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           300                                                                      associated with increased rates of severe PGD

                                                                                                                                                                                                                                                                                                                                                                               PGD Grade @ 24 hrs
                                                                                                                                                                                                                                        Male                                                                     206 (57.9%)                                                                                                                                                            PGD 3
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           These results suggest that SP-D levels in the BALF of lung transplant patients

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                       PaO2/FiO2 ratio
  Any incidence of PGD is associated with poorer overall transplant function and increased long-term complications, including                                                                                                          Female                                                                   150 (42.1%)
   development of BOS.2,3                                                                                                                                                                                                               Median Age at Transplant                                                 56 (42, 62)                                                                                        50                                                              p = .028                                                                                                        may be influenced by the recipient’s genotype. Whether the differences in SP-D
  Although the exact mechanism of neither PGD nor BOS/BO is completely understood, increasing evidence indicates that the                                                                                                              Native Disease                                                                                                                                                                                                                                                                                                                                              levels directly contribute to the development of either PGD or late BOS requires
   pulmonary innate immune system contributes to the development and progression of both conditions.                                                                                                                                         Obstructive                                                         157 (44.1%)                                                                                        25                                                                                                     100
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    further investigation.
                                                                                                                                                                                                                                             Pulmonary Vascular                                                  5 (1.4%)

  Surfactant protein (SP)-A and SP-D, members of the collectin family of soluble pattern recognition receptors, regulate the
                                                                                                                                                                                                                                                                                                                 69 (19.4%)
                                                                                                                                                                                                                                                                                                                 125 (35.1%)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           This study represents the largest analysis of donor and recipient genotypes upon
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    lung transplantation outcomes; however further validation of these results in


   pulmonary innate immune response & are known to affect both acute and chronic inflammation. 5                                                                                                                                                                                                                                                                                                                                                                                                                                                 TT            TC             CC
                                                                                                                                                                                                                                        Bilateral Transplant                                                     330 (92.7%)                                                                                                                      SP-D 3' UTR
  SP-A & SP-D are synthesized and secreted by type II pneumocytes. These cells are damaged by chronic inflammation, and                                                                                                                Single Transplant                                                        26 (7.3%)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                          SP-D 3' UTR
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    independent multicenter cohorts is needed.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               p = .151
   evidence suggests that over time, recipient-derived cells repopulate the damaged lung epithelium, meaning that both donor                                                                                                            Patients with Acute Rejection (AR)                                       282 (79.2%)                                                                              100                                                                                                                                               p = .052
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                           Given
                                                                                                                                                                                                                                          Median Number of Episodes/Patient                                        2 (1, 3)
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                        PGD 0-2                                                                                                             the diverse functions of SP-A & SP-D in pulmonary immunity, future
                                                                                                                                                                                                                                                                                                                                                                               PGD Grade @ 24 hrs.
   and recipient genotypes may influence protein function. 6,7                                                                                                                                                                                                                                                                                                                                                                                                                          PGD 3                             300
                                                                                                                                                                                                                                          Median Cumulative Rejection Grade/Patient                                3 (2, 6)                                                                                                                                                                                                                                                                         studies should examine manipulation of these innate molecules in the prevention

                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                      PaO2/FiO2 Ratio
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    p = .15
                                                                                                                                                                                                                                        Patients with CMV Pneumonia                                              82 (23.0%)                                                                                                                                                                                               200                                                                       or treatment of PGD and BOS
                                                                                                                                                                                                                                        Patients with Nissen within 6 months of transplant                       94 (26.4%)
                                                                                   METHODS                                                                                                                                              Median Number of PFT’s per Patient                                       28 (19, 36)                                                                                         25
BOS Genetic Association (GA) Cohort:                                                                                                                                                                                                    Median Number of Bronchoscopies/Patient Prior to BOS                     8 (5,12)                                                                                                                                                                                                     50
      Genomic DNA was isolated from 356 patients from a total of 424 lung transplant recipients who met the study inclusion criteria: adult Caucasian patients undergoing first lung transplant at Duke University Medical
      Center between January 1, 1998 and June 25, 2008 who were BOS eligible (e.g. survived longer than 180 days and had pulmonary function data from which to assess BOS). Patients undergoing heart/lung                              Time from Transplant to BOS (Q1, 66th percentile)                        5.82 years (3.15, 8.79)                                                                                      0                                                                                                   0
                                                                                                                                                                                                                                                                                                                                                                                                                                          TT          TC           CC                                                                            TT            TC             CC                                           FUNDING:
      transplantation were excluded from the study. BOS was defined according to standard ISHLT criteria based on serial Forced Expiratory Volume (FEV1) measurements.8 Censor date was July 1, 2009 to ensure every
      patient had at least one year of follow-up. Demographic and post-transplant characteristics of the BOS genetic association cohort are summarized in Table 2.                                                                      Overall Survival (Q1, 60th percentile)                                   8.47 years (4.20, 9.02)                                                                                                       SP-D Met11Thr                                                                                             SP-D Met11Thr                                                     SCCOR grant: NIH / NHLBI 1P50-HL084917-011, Project 3 (to Palmer)
PGD GA Cohort:                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                             K24 HL091140 (Palmer)
      Genomic DNA was isolated from 263 cadaveric donors transplanted at Duke University Medical Center between November 1, 1998 and January 1, 2005. We only included the 188 Caucasian donors in our study.                                                                                                                                                                                                                                                                                                                                                                                                              Stead Scholarship, 2007-2008 (to Schechter)
      PGD-T24 was defined according to the ISHLT guidelines9 using the PaO2 and FiO2 values from the arterial blood gas closest to 24 hrs. post-transplant. PGD grades 0 and 1 were combined into a single grade (0/1) for
      analysis given the lack of chest X-rays 24 hours post-transplant. Demographic and post-transplant characteristics of the PGD genetic association cohort are summarized in table 3.                                             TABLE 3: PGD GENETIC                        NO. (%)        TABLE 4: SP-D CROSS-                                            NO. (%)
Determination of SP-A & SP-D Polymorphisms:
      The surfactant protein SNPs studied are outlined in Table 1. SNPs were chosen for inclusion based upon the following criteria: common (generally >5% MAF) non-synonomous SNPs with a probable functional effect.
                                                                                                                                                                                                                                     ASSOCIATION COHORT
                                                                                                                                                                                                                                                                                                SECTIONAL COHORT
                                                                                                                                                                                                                                                                                                                                                                                                                                          Figure 3: SP-D levels decrease with increasing BOS grade                                                                                                                         REFERENCES:
      Probability of functional effect was determined by a review of the literature to identify SNPs that had been previously associated with other pulmonary diseases and/or were located within a functionally-important region                                                                                                                                                                                                                                                                                                                                                                                          (1) Zhang P, Summer WR, Bagby GJ et al. Innate immunity and pulmonary host defense. Immunol Rev 2000;
      of the protein. For the SP-D SNPs, commercially available TaqMan assays (Applied Biosystems, Foster City, CA) were used for genotyping. Given the homology between the two SP-A genes (SFTPA1 & SFTPA2),                       Male                                     91 (48.4%)        Male                                                            25 (66%)                                                                                                                                                                                                                                                       183:310-51
      gene-specific primers were designed to amplify a 600-800bp region containing the SNP of interest, followed by a custom-designed TaqMan assay (Applied Biosystems, Foster City, CA) in order to determine the                                                                                                                                                                                                                                     *
      genotype of each SNP. The TaqMan assay results were performed at least in duplicate, with 100% reproducibility of results and a no-call rate of <5% for each SP-S SNP.                                                         Female                                   97 (51.6%)        Female                                                          13 (34%)
                                                                                                                                                                                                                                                                                                                                                                                                                                                      **                                                                                                                                                                   (2) Christie JD, Sager JS, Kimmel SE et al. Impact of primary graft failure on outcomes following lung
                                                                                                                                                                                                                                                                                                                                                                                                 SP-D Concentration (ng/mL)

SP-D Cross-sectional Analysis Cohort:
                                                                                                                                                                                                                                     Median Age at Transplant                 54 (40, 61)       Median Age at Transplant                                        56 (38, 61)                                                       2000                                          ‡                                                             2000                                                                             transplantation. Chest. 2005; 127:161-5.
      From October 1, 2007 to October 29, 2008, we obtained bronchoalveolar lavage fluid (BALF) from 112 patients who had received a lung transplant at Duke University Medical Center (underwent transplant from 1998-
      2008). Of those patients, 58 patients met the following inclusion criteria: patient received a cadaveric transplant, survived l> 6 months post-transplant and had no concurrent rejection or infection at the time of BAL      Native Disease                                             Native Disease                                                                                                                                                                                                                                                                                                             (3) Carter YM, Gelman AE, Kreisel D. Pathogenesis, management, and consequences of primary graft
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                              p = .0067
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                         SP-D Concentration
      sample. BOS grades (0-p to 3) were assigned based upon the 2001 updated classification for BOS. 8 112 Duke lung transplant recipients (underwent transplant from 1998-2008) were screened for possible inclusion.
                                                                                                                                                                                                                                          Obstructive                         98 (52.1%)             Obstructive                                                15 (39.5%)                                                                                                                                                                                                                                                     dysfunction. Sem Thorac Cardiovas Surg 2008; 20:165-72.
      21 patients had BOS 0-p at the time of BAL collection, and were therefore excluded from the study, leaving 38 patients who compromise the analysis cohort (Table 3). Bronchoalveolar lavage fluid (BALF) was                                                                                                                                                                                                                                                                  * p = .0102
      collected at the time of bronchoscopy, processed and stored at -80ºC until analysis. SP-D levels in the BAL fluid using a commercially-available ELISA (Biovendor, Candler, NC) according to the manufacturers                      Pulmonary Vascular                  13 (6.9%)              Pulmonary Vascular                                         4 (10.5%)                                                         1500                                                                                                        1500                                                                         (4) Sato M, Keshavjee S. Bronchiolitis obliterans syndrome: alloimmune-dependent and -independent injury
      instructions.                                                                                                                                                                                                                                                                                                                                                                                                                                                                 ** p = .0469
                                                                                                                                                                                                                                          Cystic                              32 (17.0%)             Cystic                                                     9 (23.7%)                                                                                                                                                                                                                                                      with aberrant tissue remodeling. Sem Thorac Cardiovas Surg 2008; 20:173-82.
SP-D Longitudinal Analysis Cohort:
      In order to follow SP-D levels during the post-transplant period, we identified 23 patients who eventually developed BOS for whom we had at least four BALF samples. For 14 patients, we had at least two samples                   Restrictive                         45 (23.9%)             Restrictive                                                10 (26.3%)                                                                                                          ‡ p = .0568                                                                                                                            (5) Pastva AM, Wright JR, Williams KL. Immunomodulary roles of surfactant proteins A and D: Implications in
      from before the development of BOS (pre-BOS) and two samples from after the development of BOS (post-BOS). Only one pre-BOS sample was available for three of these patients, while two patients had no pre-                                                                                                                                                                                                                1000                                                                                                        1000                                                                             lung disease. Proc Am Thorac Soc 2008; 4:252-8.
      BOS samples. The remaining three patients had multiple pre-BOS samples, but only one post-BOS BALF sample. The average number of samples per patients was 5.39. We then identified a group 20 patients who                     Bilateral Transplant                     163 (86.7%)       Bilateral Transplant                                            37 (97%)
      remained free of BOS that matched the BOS patients in terms of average number of BALF samples available (5.10 samples per patients; p = .55 vs BOS patient population) and average length of follow-up (1292.8                 Single Transplant                        25 (13.3%)        Single Transplant                                                1 (3%)                                                                                                                                                                                                                                                    (6) Kleeberger W, Versmold A, Rothamel T et al. Increased chimerism of bronchial & alveolar epithelium in
      days vs. 1463.0 days for BOS patients; p = .33). BOS grades (0-p to 3) were assigned based upon the 2001 updated classification for BOS.6 Bronchoalveolar lavage fluid (BALF) was collected at the time of
      bronchoscopy, processed and stored at -80ºC until analysis. SP-D levels in the BAL fluid using a commercially-available ELISA (Biovendor, Candler, NC) according to the manufacturers instructions.                                                                                                                                                                                                                                                                                                                                                                                                                      human lung allografts undergoing chronic injury. Amer J Path 2003; 162:1487-94.
                                                                                                                                                                                                                                     PGD-T24 grade                                              Average time to BOS Onset                                       1494 days                                                         500                                                                                                         500
                                                                                                                                                                                                                                        PGD 0/1                               58 (30.9%)                                                                                                                                                                                                                                                                                                                                   (7) Spencer H, Rampling D, Aurora P et al. Transbronchial biopsies provide longitudinal evidence for epithelial
      In the BOS genetic association cohort, the association of each SNP with survival, time to BOS1 and time to BOS 2/3 using a Kaplan-Meier log rank test. For the PGD cohort, the PaO2/FiO2 ratio at 24h was considered
      as a continuous variable, and the relationship between both donor and recipient genotype was assessed using an ANOVA. Results are expressed as mean ± standard error (SEM). Comparisons between two genotype                      PGD 2                                 52 (27.6%)        BOS Grade at Bronch Date                                                                                                                                                                                                                                                                                                       chimerism in children following sex mismatched lung transplantation. Thorax 2005; 60:60-62.
      groups utilized a Student’s t-test. The presence or absence of severe (Grade 3) PGD at 24h was also considered as an ordinal value, whereby a likelihood ratio was used. In both cohorts, each SNP was considered in              PGD 3                                 46 (24.5%)           No BOS                                                       18 (47%)                                                            0                                                                                                           0                                                                          (8) Christie JD, Carby M, Bag R et al. Report of the ISHLT Working Group on Primary Lung Graft Dysfunction
      a dose-response and recessive inheritance model. A p-value < .05 was considered statistically significant.
                                                                                                                                                                                                                                        Unknown                               32 (17.0%)           BOS 1                                                        10 (26%)                                                                 No BOS            BOS 1        BOS 2       BOS 3                                                              No BOS/BOS 1            BOS 2/3                                         part II: definition. J Heart Lung Transplant 2005; 24:1454-9.
      For the cross-sectional analysis of SP-D levels, the relationship between BOS grade and SP-D concentration was assessed using an ANOVA. Comparisons between two groups, as well as the comparison between
                                                                                                                                                                                                                                                                                                   BOS 2                                                        6 (16%)
      severe (Grade 2/3) BOS and No BOS/BOS 1, were completed using a Student’s t-test. All graphs show average SP-D levels by BOS Grade ± SEM. Association between recipient genotype and average SP-D levels
                                                                                                                                                                                                                                     Median PaO2/FiO2 ratio                   270.0                                                                                                                                                                                                                                                                                                                                        (9) Estenne M, Maurer JR, Boehler A et al. Bronchiolitis obliterans syndrome 2001: an update of the diagnostic
      over the post-transplant period were assessed by ANOVA. The means of two genotypes were compared using a Student’s t-test. All graphs show average SP-D levels by genotype ± SEM. Each SNP was considered                                                                                    BOS 3                                                        4 (11%)                                                                                     BOS Status                                                                                            BOS Status
      in a dose-response and recessive inheritance model. A p-value < .05 was considered statistically significant.                                                                                                                  (24 hours post-transplant)               (187.5, 345.2)                                                                                                                                                                                                                                                                                                                                   criteria. J Heart Lung Transplant 2002; 21:297-310.

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