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Prevalence of Polyreactive Innate-Like B Cells Among Graft-Infiltrating B Cells in Human Cardiac Allograft Vasculopathy
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The Journal of Heart and Lung Transplantation, Vol 35, No 4S, April 2016
Bilateral LuTX was performed in 95%, and single LuTX or combined HLTX was performed in 2,5 % each. In 60 % whole lungs were transplanted and in 40 % lobar Tx was performed. 5 children were transplanted using living donors. Extracorporeal circulation was used in 78% of the transplantations (55 pats with ECMO, 9 with HLM). 1- and 5-yr patient survival rates were 78% and 64%, respectively. Analyzing different eras of transplantation (1990-2002 vs 2002-2014) suggests an improvement over the years with a 5-yr survival rate of 45.5% vs 71.16% in the second decade (p= 0.002). A high rate of successful re-transplantations (5 years survival after first ReTX 62.3%) prolonged total patient survival. Freedom from CLAD at 5 years post-transplant was 71%. Analysis of 1134 LuTX in adult patients in the same period of time (2002– 2014) in our institution revealed a 1 and 5 year survival rate of 81 % and 70 %, and a freedom from CLAD of 72 % at 5 years. Conclusion: Lung transplantation in children and pediatric patients in the recent decade provides results equal to those achieved in the adult population.
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The Hard Truth: Outcomes of Adolescent Recipients Following Lung Transplantation M. Paraskeva ,1 L.B. Edwards,2 B. Levvey,1 J. Stehlik,2 S. Goldfarb,2 R.D. Yusen,2 G.P. Westall,1 G.I. Snell.1 1Lung Transplant Service, Alfred Hospital, Melbourne, Australia; 2ISHLT Transplant Registry, Dallas, TX.
Increased Angiogenesis and Non-Surgical Bleeding in Patients with Continuous-Flow Left Ventricular Assist Devices Are Mediated by Thrombin-Induced Angiopoietin-2 C.E. Tabit ,1 G.H. Kim,1 S.E. Fedson,2 G. Sayer,1 M.J. Coplan,1 V. Jeevanandam,3 N. Uriel,1 J.K. Liao.1 1Cardiology, The University of Chicago Medical Center, Chicago, IL; 2Baylor Center for Medical Ethics and Health Policy, Baylor University, Houston, TX; 3Cardiac and Thoracic Surgery, The University of Chicago Medical Center, Chicago, IL.
Purpose: Adolescent transplant recipients have worse long-term survival compared to children and adults across different SOT groups. Recently adolescence has been defined as 10-24yrs taking into account the biological and social transitions occurring in this period. As adolescents span both pediatric and adult registries with outcomes reported for 11-17 and 18-34yo, much of adolescence is invisible. Methods: The ISHLT registry (Jan05-Jun13) was used to examine the outcomes of: 1) adolescent (10-24yo) lung transplant recipients (LTR) 2) the tertiles of adolescence:10-14; 15-19 and 20-24yo. Results: 24,730 LTR < 65yo were included. Adolescents made up 9% (n= 2319) of the cohort, 88% were between 15-24yrs. Most had CF (75% vs 13% p< 0.0001) and compared to adults more females (57% vs 43%, p< 0.0001) were transplanted. Adolescents received younger (p< 0.001), non-smoking (p= 0.005) donors but were more often hospitalized (32% vs 16% p< 0.0001), ventilated (11% vs 6% p< 0.0001) or on ECMO (5% vs 2% p< 0.0001) than adults. There was no difference in HLA match but PRA was higher in adults (p= 0.002). Adolescent survival was poor. They exhibited similar survival to 50-65yo (fig 1). Their 3-yr survival was reduced compared to children (65% vs 73% p= 0.006) and adults: including 25-34yo (65% vs 75% p< 0.00001) and 35-49yo (65% vs 71% p< 0.00001). This difference persisted when analysis conditional on 1-year survival was done. 15-19yo had the poorest outcomes. Survival was reduced at 1-year compared to 10-14yo (82% vs 88% p= 0.017) and 3-yrs compared to 10-14yo (59% vs 73% p< 0.00001) and 20-25yo (59% vs 65% p= 0.00003). Conclusion: Adolescent LTR have poorer survival than children and adults. 15-19yo are at highest risk a fact not apparent when the pediatric and adult registries are examined in isolation. This may reflect the biological and social transitions of adolescence which may influence immunological function and adherence and should influence our reporting of outcomes and management strategies if we are to improve adolescent survival.
Purpose: Non-surgical bleeding (NSB) is a common adverse event in patients with Continuous-Flow Left Ventricular Assist Devices (LVADs). NSB is often caused by the development of arteriovenous malformations (AVMs), a process which may reflect dysregulated angiogenesis. Angiopoietin-2 (Ang-2) is a potent stimulator of angiogenesis produced by endothelial cells in response to activation of the thrombin receptor (PAR-1). We hypothesized that activation of the coagulation system leads to expression of Ang-2 in LVAD patients which induces angiogenesis and is associated with NSB. Methods: Ang-2, sP-Selectin, β -Thromboglobulin (β -TG), thrombin, and Factor XIIa were measured by ELISA or Western blot in blood samples from patients with heart failure (HF), LVAD, or orthotopic heart transplant (OHT). Endothelial biopsy was performed and Ang-2 protein expression was measured by quantitative immunofluorescence. Angiogenesis was determined by in vitro tube formation of cultured human umbilical vein endothelial cells (HUVECs) on Matrigel incubated with serum from each patient with or without Ang-2-blocking antibody. Ang-2 gene expression was measured by RT-PCR in HUVECs incubated with plasma from each patient with or without the thrombin receptor blocker Vorapaxar. Results: Compared with patients with HF or OHT, serum levels and endothelial expression of Ang-2 were higher in LVAD patients (p< 0.01 and p< 0.05, respectively). This corresponded with increased angiogenic potential of serum from patients with LVADs (p< 0.001), which was normalized with Ang-2 blockade. Further, plasma from LVAD patients contained higher amounts of thrombin (p= 0.018), Factor XIIa (p< 0.05), sP-Selectin (p< 0.01), and β -TG (p< 0.05), and thrombin receptor blockade normalized the increase in Ang-2 gene expression in endothelial cells (p< 0.001). LVAD patients with serum Ang-2 above the mean (12.32 ng/mL) had a 33-fold increase in 3-month risk of NSB compared with patients below the mean (p= 0.003). Conclusion: Activation of the coagulation system leading to thrombininduced Ang-2 over-expression was observed in LVAD patients. This was associated with increased angiogenesis in vitro and higher incidence of NSB. Ang-2, therefore, may contribute to AVM formation and subsequent bleeding in LVAD patients. 7( 5) Prevalence of Polyreactive Innate-Like B Cells Among Graft-Infiltrating B Cells in Human Cardiac Allograft Vasculopathy D. Chatterjee ,1 C. Moore,2 B. Gao,3 K.J. Clerkin,1 S.B. See,1 D. Shaked,1 K.J. Rogers,1 S. Nunez,1 Y. Veras,1 L. Addonizio,1 M.M. Givertz,4 Y. Naka,1 D. Mancini,1 R. Vasilescu,5 C. Marboe,5 S. Restaino,1 J.C. Madsen,2 E. Zorn.1 1Dept. of Medicine, Columbia University, New York, NY; 2Massachusetts General Hospital Transplant Center, Boston, MA; 3First Hospital of Jilin University, Changchun, China; 4Brigham and Women’s
Abstracts S37 Hospital, Boston, MA; 5Dept. of Pathology & Cell Biology, Columbia University, New York, NY. Purpose: Cardiac allograft vasculopathy (CAV) is a leading cause of death in long-term heart transplant survivors. Dense B cell infiltrates are common in heart transplants with CAV. To understand the role of these cells, we conducted detailed examination of graft-infiltrating B cells with regards to their phenotype, clonality, reactivity and functional interplay with other infiltrating immune cells. Methods: We collected 55 cardiac allograft explants and histologically determined the presence of CAV based on intimal thickening of intramural vessels. We analyzed lymphoid infiltration near epicardial coronary arteries and their association with donor specific antibodies (DSA). To study the clonality of infiltrating B cells, we analyzed the immunoglobulin heavy chain variable region (IGVH) repertoire in 6 CAV explants using next generation sequencing. Lastly, we isolated and immortalized B cell clones directly from three fresh cardiac allografts with CAV and analyzed the reactivity profiles of their secreted antibodies using ELISAs and flow cytometry. Results: All allografts had CAV. 93% of these explants had B cell infiltration near coronary arteries. Antibody-producing plasma cells and macrophages could also be detected in 85% and 95% of CAV explants, respectively. Significant fraction of infiltrating macrophages had an M2 phenotype (CD68+ CD163+). Notably, B cell infiltrates were not associated with circulating DSA, indicating that the function of these cells was unrelated to the production of anti-HLA antibodies. IGVH repertoire analyses revealed that certain B cell clones had undergone robust proliferation at multiple locations in the graft tissue. Lastly, a majority of immortalized B cell clones isolated from 3 different allograft specimens had characteristics of innate-like B cells and secreted natural antibodies reactive to multiple auto-antigens and/or to apoptotic cells. Conclusion: This study revealed a high frequency of B cell infiltrates around coronary arteries in heart transplants with CAV independent of DSA. Moreover, the unexpected enrichment of polyreactive innate-like B cells, among infiltrating cells, strongly supports their role in the immune reaction associated with CAV, especially in association with neighboring macrophages. Supported by NIH AI116814, ROTRF 46510926, S10RR027050-01A1. 7 ( 6) Feasibility of Detecting Fungal DNA in Exhaled Breath Condensate by the Luminex Multiplex xTAG Fungal PCR Assay in Lung transplant Recipients: A Pilot Study A. Bhimji ,1 L.G. Singer,2 D. Kumar,2 A. Humar,2 R. Pavan,2 H. Zhang,3 C. Rotstein,2 S. Keshavjee,2 T. Mazzulli,2 S. Husain.2 1Laboratory Medicine & Pathobiology, University of Toronto, Toronto, ON, Canada; 2Medicine, University Health Network, Toronto, ON, Canada; 3Luminex Molecular Diagnostics, Toronto, ON, Canada. Purpose: Exhaled breath condensate (EBC) collection provides a noninvasive means of sampling the airway lining fluid. We assessed the suitability of EBC to detect fungal DNA. Methods: A prospective study was conducted to investigate the feasibility of detecting clinically relevant fungi (23 species and one internal control) in EBC specimens from lung transplant recipients using the Luminex multiplex xTAG fungal ASR Assay. PCR results in EBC specimens were compared with conventional diagnostic tests, such as culture and galactomannan (GM), in concomitant bronchoalveolar lavage (BAL) samples. Results: We tested 202 EBCs negative (n = 166) or positive (n = 36) for fungi. The xTAG ASR assay in EBC specimens and BAL fungal cultures yielded concurrent negative results in 162 (80%) samples and positive results in 14 (7%) samples. The xTAG ASR assay yielded sensitivity, specificity, positive and negative predictive values of 38.9%, 97.6%, 77.8%, and 88.0%. Identification of species was provided by the xTAG ASR assay for 17 unspeciated BAL fungal cultures [A. flavus (n = 3), A. fumigatus (n = 1), A. terreus (n = 1), C. albicans (n = 1), C. guilliermondii (n = 2), C. parapsilosis (n = 2), H. capsulatum (n = 3), M. indicus (n = 1), C. parapsilosis + A. flavus (n = 1), C. parapsilosis + C. bertholletiae (n = 1), C. parapsilosis + S. apiospermum (n = 1)]. Discordant results were obtained in 4 samples which were culture positive for A. fumigatus, but negative for the xTAG ASR assay. PCR results were also compared to BAL GM ELISA values (n = 190). Concurrent negative
results were seen in 158 (83%) samples and positive results in 5 (3%) samples. The xTAG ASR assay displayed sensitivity, specificity, positive and negative predictive values of 15.6%, 100%, 100%, and 85.4% when compared to GM ELISA. Conclusion: These results provide proof-of-concept that detection of fungal DNA is feasible in EBC. Further evaluation to determine its role in prophylaxis and treatment is warranted.
7( 7) Graft-Infiltrating Neutrophils Cannibalize Dying Neutrophils to Produce IL-10 and to Protect Lung Transplants from IschemiaReperfusion Injury M. Zhu , X. Wang, D. Scozzi, K.A. Toth, J. Zhu, X. Lin, A.S. Krupnick, D. Kreisel, A.E. Gelman. Department of Surgery, Washington University in Saint Louis, Saint Louis, MO. Purpose: Phagocytosis of apoptotic cells by mononuclear phagocytes (i.e. macrophages) promotes the resolution of inflammation predominantly through immunosuppressive mediator expression. In particular, IL-10 expression has been observed in reperfused lung grafts but precisely how it is generated and whether such de novo expression contributes to protection from lung transplant-mediated ischemia-reperfusion injury (IRI) remains unknown. Methods: As physiological relevant amounts of IL-10 are difficult to measure in vivo, we utilized a mouse orthotopic lung transplant model where donor/ recipients encode a bicistronic transgene that expresses IL-10 and enhanced green fluorescent protein or are deficient in either IL-10 or the transcription factor C/EBP-β , which is required for IL-10 transcript production. IL-10 level and related intracellular signaling mechanisms were investigated by flow cytometry, luminex immunoassay, western blot, fluorescence and transmission electronic microscopy. Results: Surprisingly, protection from IRI was predominantly dependent on IL-10 and C/EBP-β -recipient-derived expression where graft-infiltrating neutrophils are the major source of this cytokine. Furthermore, the adoptive transfer of apoptotic neutrophil enhances graft-infiltrating neutrophil IL-10 expression and promotes IRI attenuation. Transmission electron microscopy and flow cytometry analysis revealed that most IL-10+ neutrophils have cannibalized apoptotic neutrophils. Finally, we established that neutrophil IL-10 expression was dependent on both the engagement of the G2A receptor, which recognizes lysophosphatidylserine, a phospholipid found on dying neutrophils, and the LPS receptor toll-like receptor 4. Conclusion: Taken together, IL-10+ neutrophils protect lung grafts from IRI suggesting that therapies that principally target neutrophil infiltration may have the unintended effect of worsening acute lung injury. 7( 8) Clara Cell Secretory Protein Limits Small Airway Inflammation and Fibrosis Induced by De Novo Donor Specific Antibodies D. Nayak ,1 F. Zhou,2 T. Mohanakumar.3 1Washington University School of Medicine, St. Louis, MO; 2Surgery, Washington University School of Medicine, St. Louis, MO; 3Surgery, Pathology & Immunology, Washington University School of Medicine, St. Louis, MO. Purpose: De novo development of donor specific antibody (Ab) is a major risk factor for chronic rejection following human lung transplantation (LTx). Sensitization to mismatched donor HLA can also induce immune responses to lung-associated antigens (K alpha 1 Tubulin (Kα 1T) and Collagen V (Col V)). It is known that LTx recipients that develop chronic rejection, bronchiolitis obliterans syndrome, have significantly reduced Clara Cell Secretory Protein (CCSP), a component of the bronchiolar surfactants, in lungs. Our
The Journal of Heart and Lung Transplantation, Vol 35, No 4S, April 2016
Bilateral LuTX was performed in 95%, and single LuTX or combined HLTX was performed in 2,5 % each. In 60 % whole lungs were transplanted and in 40 % lobar Tx was performed. 5 children were transplanted using living donors. Extracorporeal circulation was used in 78% of the transplantations (55 pats with ECMO, 9 with HLM). 1- and 5-yr patient survival rates were 78% and 64%, respectively. Analyzing different eras of transplantation (1990-2002 vs 2002-2014) suggests an improvement over the years with a 5-yr survival rate of 45.5% vs 71.16% in the second decade (p= 0.002). A high rate of successful re-transplantations (5 years survival after first ReTX 62.3%) prolonged total patient survival. Freedom from CLAD at 5 years post-transplant was 71%. Analysis of 1134 LuTX in adult patients in the same period of time (2002– 2014) in our institution revealed a 1 and 5 year survival rate of 81 % and 70 %, and a freedom from CLAD of 72 % at 5 years. Conclusion: Lung transplantation in children and pediatric patients in the recent decade provides results equal to those achieved in the adult population.
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The Hard Truth: Outcomes of Adolescent Recipients Following Lung Transplantation M. Paraskeva ,1 L.B. Edwards,2 B. Levvey,1 J. Stehlik,2 S. Goldfarb,2 R.D. Yusen,2 G.P. Westall,1 G.I. Snell.1 1Lung Transplant Service, Alfred Hospital, Melbourne, Australia; 2ISHLT Transplant Registry, Dallas, TX.
Increased Angiogenesis and Non-Surgical Bleeding in Patients with Continuous-Flow Left Ventricular Assist Devices Are Mediated by Thrombin-Induced Angiopoietin-2 C.E. Tabit ,1 G.H. Kim,1 S.E. Fedson,2 G. Sayer,1 M.J. Coplan,1 V. Jeevanandam,3 N. Uriel,1 J.K. Liao.1 1Cardiology, The University of Chicago Medical Center, Chicago, IL; 2Baylor Center for Medical Ethics and Health Policy, Baylor University, Houston, TX; 3Cardiac and Thoracic Surgery, The University of Chicago Medical Center, Chicago, IL.
Purpose: Adolescent transplant recipients have worse long-term survival compared to children and adults across different SOT groups. Recently adolescence has been defined as 10-24yrs taking into account the biological and social transitions occurring in this period. As adolescents span both pediatric and adult registries with outcomes reported for 11-17 and 18-34yo, much of adolescence is invisible. Methods: The ISHLT registry (Jan05-Jun13) was used to examine the outcomes of: 1) adolescent (10-24yo) lung transplant recipients (LTR) 2) the tertiles of adolescence:10-14; 15-19 and 20-24yo. Results: 24,730 LTR < 65yo were included. Adolescents made up 9% (n= 2319) of the cohort, 88% were between 15-24yrs. Most had CF (75% vs 13% p< 0.0001) and compared to adults more females (57% vs 43%, p< 0.0001) were transplanted. Adolescents received younger (p< 0.001), non-smoking (p= 0.005) donors but were more often hospitalized (32% vs 16% p< 0.0001), ventilated (11% vs 6% p< 0.0001) or on ECMO (5% vs 2% p< 0.0001) than adults. There was no difference in HLA match but PRA was higher in adults (p= 0.002). Adolescent survival was poor. They exhibited similar survival to 50-65yo (fig 1). Their 3-yr survival was reduced compared to children (65% vs 73% p= 0.006) and adults: including 25-34yo (65% vs 75% p< 0.00001) and 35-49yo (65% vs 71% p< 0.00001). This difference persisted when analysis conditional on 1-year survival was done. 15-19yo had the poorest outcomes. Survival was reduced at 1-year compared to 10-14yo (82% vs 88% p= 0.017) and 3-yrs compared to 10-14yo (59% vs 73% p< 0.00001) and 20-25yo (59% vs 65% p= 0.00003). Conclusion: Adolescent LTR have poorer survival than children and adults. 15-19yo are at highest risk a fact not apparent when the pediatric and adult registries are examined in isolation. This may reflect the biological and social transitions of adolescence which may influence immunological function and adherence and should influence our reporting of outcomes and management strategies if we are to improve adolescent survival.
Purpose: Non-surgical bleeding (NSB) is a common adverse event in patients with Continuous-Flow Left Ventricular Assist Devices (LVADs). NSB is often caused by the development of arteriovenous malformations (AVMs), a process which may reflect dysregulated angiogenesis. Angiopoietin-2 (Ang-2) is a potent stimulator of angiogenesis produced by endothelial cells in response to activation of the thrombin receptor (PAR-1). We hypothesized that activation of the coagulation system leads to expression of Ang-2 in LVAD patients which induces angiogenesis and is associated with NSB. Methods: Ang-2, sP-Selectin, β -Thromboglobulin (β -TG), thrombin, and Factor XIIa were measured by ELISA or Western blot in blood samples from patients with heart failure (HF), LVAD, or orthotopic heart transplant (OHT). Endothelial biopsy was performed and Ang-2 protein expression was measured by quantitative immunofluorescence. Angiogenesis was determined by in vitro tube formation of cultured human umbilical vein endothelial cells (HUVECs) on Matrigel incubated with serum from each patient with or without Ang-2-blocking antibody. Ang-2 gene expression was measured by RT-PCR in HUVECs incubated with plasma from each patient with or without the thrombin receptor blocker Vorapaxar. Results: Compared with patients with HF or OHT, serum levels and endothelial expression of Ang-2 were higher in LVAD patients (p< 0.01 and p< 0.05, respectively). This corresponded with increased angiogenic potential of serum from patients with LVADs (p< 0.001), which was normalized with Ang-2 blockade. Further, plasma from LVAD patients contained higher amounts of thrombin (p= 0.018), Factor XIIa (p< 0.05), sP-Selectin (p< 0.01), and β -TG (p< 0.05), and thrombin receptor blockade normalized the increase in Ang-2 gene expression in endothelial cells (p< 0.001). LVAD patients with serum Ang-2 above the mean (12.32 ng/mL) had a 33-fold increase in 3-month risk of NSB compared with patients below the mean (p= 0.003). Conclusion: Activation of the coagulation system leading to thrombininduced Ang-2 over-expression was observed in LVAD patients. This was associated with increased angiogenesis in vitro and higher incidence of NSB. Ang-2, therefore, may contribute to AVM formation and subsequent bleeding in LVAD patients. 7( 5) Prevalence of Polyreactive Innate-Like B Cells Among Graft-Infiltrating B Cells in Human Cardiac Allograft Vasculopathy D. Chatterjee ,1 C. Moore,2 B. Gao,3 K.J. Clerkin,1 S.B. See,1 D. Shaked,1 K.J. Rogers,1 S. Nunez,1 Y. Veras,1 L. Addonizio,1 M.M. Givertz,4 Y. Naka,1 D. Mancini,1 R. Vasilescu,5 C. Marboe,5 S. Restaino,1 J.C. Madsen,2 E. Zorn.1 1Dept. of Medicine, Columbia University, New York, NY; 2Massachusetts General Hospital Transplant Center, Boston, MA; 3First Hospital of Jilin University, Changchun, China; 4Brigham and Women’s
Abstracts S37 Hospital, Boston, MA; 5Dept. of Pathology & Cell Biology, Columbia University, New York, NY. Purpose: Cardiac allograft vasculopathy (CAV) is a leading cause of death in long-term heart transplant survivors. Dense B cell infiltrates are common in heart transplants with CAV. To understand the role of these cells, we conducted detailed examination of graft-infiltrating B cells with regards to their phenotype, clonality, reactivity and functional interplay with other infiltrating immune cells. Methods: We collected 55 cardiac allograft explants and histologically determined the presence of CAV based on intimal thickening of intramural vessels. We analyzed lymphoid infiltration near epicardial coronary arteries and their association with donor specific antibodies (DSA). To study the clonality of infiltrating B cells, we analyzed the immunoglobulin heavy chain variable region (IGVH) repertoire in 6 CAV explants using next generation sequencing. Lastly, we isolated and immortalized B cell clones directly from three fresh cardiac allografts with CAV and analyzed the reactivity profiles of their secreted antibodies using ELISAs and flow cytometry. Results: All allografts had CAV. 93% of these explants had B cell infiltration near coronary arteries. Antibody-producing plasma cells and macrophages could also be detected in 85% and 95% of CAV explants, respectively. Significant fraction of infiltrating macrophages had an M2 phenotype (CD68+ CD163+). Notably, B cell infiltrates were not associated with circulating DSA, indicating that the function of these cells was unrelated to the production of anti-HLA antibodies. IGVH repertoire analyses revealed that certain B cell clones had undergone robust proliferation at multiple locations in the graft tissue. Lastly, a majority of immortalized B cell clones isolated from 3 different allograft specimens had characteristics of innate-like B cells and secreted natural antibodies reactive to multiple auto-antigens and/or to apoptotic cells. Conclusion: This study revealed a high frequency of B cell infiltrates around coronary arteries in heart transplants with CAV independent of DSA. Moreover, the unexpected enrichment of polyreactive innate-like B cells, among infiltrating cells, strongly supports their role in the immune reaction associated with CAV, especially in association with neighboring macrophages. Supported by NIH AI116814, ROTRF 46510926, S10RR027050-01A1. 7 ( 6) Feasibility of Detecting Fungal DNA in Exhaled Breath Condensate by the Luminex Multiplex xTAG Fungal PCR Assay in Lung transplant Recipients: A Pilot Study A. Bhimji ,1 L.G. Singer,2 D. Kumar,2 A. Humar,2 R. Pavan,2 H. Zhang,3 C. Rotstein,2 S. Keshavjee,2 T. Mazzulli,2 S. Husain.2 1Laboratory Medicine & Pathobiology, University of Toronto, Toronto, ON, Canada; 2Medicine, University Health Network, Toronto, ON, Canada; 3Luminex Molecular Diagnostics, Toronto, ON, Canada. Purpose: Exhaled breath condensate (EBC) collection provides a noninvasive means of sampling the airway lining fluid. We assessed the suitability of EBC to detect fungal DNA. Methods: A prospective study was conducted to investigate the feasibility of detecting clinically relevant fungi (23 species and one internal control) in EBC specimens from lung transplant recipients using the Luminex multiplex xTAG fungal ASR Assay. PCR results in EBC specimens were compared with conventional diagnostic tests, such as culture and galactomannan (GM), in concomitant bronchoalveolar lavage (BAL) samples. Results: We tested 202 EBCs negative (n = 166) or positive (n = 36) for fungi. The xTAG ASR assay in EBC specimens and BAL fungal cultures yielded concurrent negative results in 162 (80%) samples and positive results in 14 (7%) samples. The xTAG ASR assay yielded sensitivity, specificity, positive and negative predictive values of 38.9%, 97.6%, 77.8%, and 88.0%. Identification of species was provided by the xTAG ASR assay for 17 unspeciated BAL fungal cultures [A. flavus (n = 3), A. fumigatus (n = 1), A. terreus (n = 1), C. albicans (n = 1), C. guilliermondii (n = 2), C. parapsilosis (n = 2), H. capsulatum (n = 3), M. indicus (n = 1), C. parapsilosis + A. flavus (n = 1), C. parapsilosis + C. bertholletiae (n = 1), C. parapsilosis + S. apiospermum (n = 1)]. Discordant results were obtained in 4 samples which were culture positive for A. fumigatus, but negative for the xTAG ASR assay. PCR results were also compared to BAL GM ELISA values (n = 190). Concurrent negative
results were seen in 158 (83%) samples and positive results in 5 (3%) samples. The xTAG ASR assay displayed sensitivity, specificity, positive and negative predictive values of 15.6%, 100%, 100%, and 85.4% when compared to GM ELISA. Conclusion: These results provide proof-of-concept that detection of fungal DNA is feasible in EBC. Further evaluation to determine its role in prophylaxis and treatment is warranted.
7( 7) Graft-Infiltrating Neutrophils Cannibalize Dying Neutrophils to Produce IL-10 and to Protect Lung Transplants from IschemiaReperfusion Injury M. Zhu , X. Wang, D. Scozzi, K.A. Toth, J. Zhu, X. Lin, A.S. Krupnick, D. Kreisel, A.E. Gelman. Department of Surgery, Washington University in Saint Louis, Saint Louis, MO. Purpose: Phagocytosis of apoptotic cells by mononuclear phagocytes (i.e. macrophages) promotes the resolution of inflammation predominantly through immunosuppressive mediator expression. In particular, IL-10 expression has been observed in reperfused lung grafts but precisely how it is generated and whether such de novo expression contributes to protection from lung transplant-mediated ischemia-reperfusion injury (IRI) remains unknown. Methods: As physiological relevant amounts of IL-10 are difficult to measure in vivo, we utilized a mouse orthotopic lung transplant model where donor/ recipients encode a bicistronic transgene that expresses IL-10 and enhanced green fluorescent protein or are deficient in either IL-10 or the transcription factor C/EBP-β , which is required for IL-10 transcript production. IL-10 level and related intracellular signaling mechanisms were investigated by flow cytometry, luminex immunoassay, western blot, fluorescence and transmission electronic microscopy. Results: Surprisingly, protection from IRI was predominantly dependent on IL-10 and C/EBP-β -recipient-derived expression where graft-infiltrating neutrophils are the major source of this cytokine. Furthermore, the adoptive transfer of apoptotic neutrophil enhances graft-infiltrating neutrophil IL-10 expression and promotes IRI attenuation. Transmission electron microscopy and flow cytometry analysis revealed that most IL-10+ neutrophils have cannibalized apoptotic neutrophils. Finally, we established that neutrophil IL-10 expression was dependent on both the engagement of the G2A receptor, which recognizes lysophosphatidylserine, a phospholipid found on dying neutrophils, and the LPS receptor toll-like receptor 4. Conclusion: Taken together, IL-10+ neutrophils protect lung grafts from IRI suggesting that therapies that principally target neutrophil infiltration may have the unintended effect of worsening acute lung injury. 7( 8) Clara Cell Secretory Protein Limits Small Airway Inflammation and Fibrosis Induced by De Novo Donor Specific Antibodies D. Nayak ,1 F. Zhou,2 T. Mohanakumar.3 1Washington University School of Medicine, St. Louis, MO; 2Surgery, Washington University School of Medicine, St. Louis, MO; 3Surgery, Pathology & Immunology, Washington University School of Medicine, St. Louis, MO. Purpose: De novo development of donor specific antibody (Ab) is a major risk factor for chronic rejection following human lung transplantation (LTx). Sensitization to mismatched donor HLA can also induce immune responses to lung-associated antigens (K alpha 1 Tubulin (Kα 1T) and Collagen V (Col V)). It is known that LTx recipients that develop chronic rejection, bronchiolitis obliterans syndrome, have significantly reduced Clara Cell Secretory Protein (CCSP), a component of the bronchiolar surfactants, in lungs. Our