Lung transplant referral practice patterns: a survey of cystic fibrosis physicians and general pulmonologists.

BACKGROUND: Many individuals with cystic fibrosis (CF) die from respiratory failure without referral for lung transplant. Physician practices that may expedite, delay, or preclude referral, are poorly understood. METHODS: Two parallel, web-based surveys focusing on lung transplant referral triggers and barriers, as well as pre-referral evaluation, were emailed to pulmonologists practicing in the New England region. One questionnaire was sent to CF providers (n = 61), and the second to general pulmonary providers practicing at the same institutions (n = 61). RESULTS: There were 43 (70%) responses to the CF provider survey, and 25 (41%) responses to the general pulmonary ('non-CF') provider survey. Primary reasons for CF providers to refer their patients included: rapidly declining lung function (91%) and a forced expiratory volume in 1 s (FEV1) below 30% predicted (74%). The greatest barriers to referral for both CF and non-CF providers included active tobacco use (65 and 96%, respectively, would not refer), and active alcohol or other substance use or dependence (63 and 80%). Furthermore, up to 42% of CF providers would potentially delay their referral if triple-combination therapy or other promising new, disease-specific therapy were anticipated. In general, non-CF providers perform a more robust pre-referral medical work-up, while CF providers complete a psychosocial evaluation in higher numbers. Across both groups, communication with lung transplant programs was reported to be inadequate. CONCLUSIONS: Physician-level barriers to timely lung transplant referral exist and need to be addressed. Enhanced communication between lung transplant programs and pulmonary providers may reduce these barriers.

Bronchial epithelial injury in the context of alloimmunity promotes lymphocytic bronchiolitis through hyaluronan expression.

Epithelial injury is often detected in lung allografts, however, its relation to rejection pathogenesis is unknown. We hypothesized that sterile epithelial injury can lead to alloimmune activation in the lung. We performed adoptive transfer of mismatched splenocytes into recombinant activating gene 1 (Rag1)-deficient mice to induce an alloimmune status and then exposed these mice to naphthalene to induce sterile epithelial injury. We evaluated lungs for presence of alloimmune lung injury, endoplasmic reticulum (ER) stress, and hyaluronan expression, examined the effect of ER stress induction on hyaluronan expression and lymphocyte trapping by bronchial epithelia in vitro, and examined airways from patients with bronchiolitis obliterans syndrome and normal controls histologically. We found that Rag1-deficient mice that received mismatched splenocytes and naphthalene injection displayed bronchial epithelial ER stress, peribronchial hyaluronan expression, and lymphocytic bronchitis. Bronchial epithelial ER stress led to the expression of lymphocyte-trapping hyaluronan cables in vitro. Blockade of hyaluronan binding ameliorated naphthalene-induced lymphocytic bronchitis. ER stress was present histologically in >40% of bronchial epithelia of BOS patients and associated with subepithelial hyaluronan deposition. We conclude that sterile bronchial epithelial injury in the context of alloimmunity can lead to sustained ER stress and promote allograft rejection through hyaluronan expression.

Posttransplant lymphoproliferative disease after lung transplantation.

Posttransplant lymphoproliferative disease (PTLD) after lung transplantation occurs due to immunosuppressant therapy which limits antiviral host immunity and permits Epstein-Barr viral (EBV) replication and transformation of B cells. Mechanistically, EBV survives due to latency, escape from cytotoxic T cell responses, and downregulation of host immunity to EBV. Clinical presentation of EBV may occur within the lung allograft early posttransplantation or later onset which is more likely to be disseminated. Improvements in monitoring through EBV viral load have provided a means of earlier detection; yet, sensitivity and specificity of EBV load monitoring after lung transplantation may require further optimization. Once PTLD develops, staging and tissue diagnosis are essential to appropriate histopathological classification, prognosis, and guidance for therapy. The overall paradigm to treat PTLD has evolved over the past several years and depends upon assessment of risk such as EBV-naïve status, clinical presentation, and stage and sites of disease. In general, clinical practice involves reduction in immunosuppression, anti-CD20 biologic therapy, and/or use of plasma cell inhibition, followed by chemotherapy for refractory PTLD. This paper focuses upon the immunobiology of EBV and PTLD, as well as the clinical presentation, diagnosis, prognosis, and emerging treatments for PTLD after lung transplantation.

Increased plasma mannose binding lectin levels are associated with bronchiolitis obliterans after lung transplantation.

BACKGROUND: Long-term lung allograft survival is limited by bronchiolitis obliterans syndrome (BOS). Mannose binding lectin (MBL) belongs to the innate immune system, participates in complement activation, and may predispose to graft rejection. We investigated mannose binding (MBL) during cold ischemia and in tissue samples from explanted lungs with BOS, and assessed MBL and complement proteins in plasma post-lung transplantation relative to BOS staging. METHODS: MBL was detected by immunohistochemistry lung tissue at the time of cold ischemia and in samples with BOS. MBL was assayed in the peripheral blood of 66 lung transplant patients transplanted between 1990-2007. RESULTS: MBL localized to vasculature and basement membrane during cold ischemia and BOS. Patients further out post-lung transplant > 5 years (n = 33), had significantly lower levels of MBL in the blood compared to lung transplant patients < 5 years with BOS Op-3 (n = 17), 1738 ± 250 ng/ml vs 3198 ± 370 ng/ml, p = 0.027, and similar levels to lung transplant patients < 5 years with BOS 0 (n = 16), 1738 ± 250 ng/ml vs 1808 ± 345 ng/ml. MBL levels in all BOS 0 (n = 30) vs. all BOS Op-3 (n = 36) were 1378 ± 275 ng/ml vs. 2578 ± 390 ng/ml, p = 0.001, respectively. C3 plasma levels in BOS 0 (n = 30) vs. BOS Op-3 (n = 36) were 101 ± 19.8 mg/ml vs. 114 ± 25.2 mg/ml, p = 0.024, respectively. CONCLUSIONS: MBL localizes within the lung during graft ischemia and BOS, higher levels of plasma MBL are associated with BOS Op-3 and < 5 years post-transplant, and higher level of plasma complement protein C3 was associated with BOS Op-3 clinical status. MBL may serve as a biomarker for poorer outcome post-lung transplantation.

Post-transplantation lymphoproliferative disease: Epstein-Barr virus DNA levels, HLA-A3, and survival.

RATIONALE: Elevation in Epstein-Barr virus (EBV) circulating DNA has been proposed as a marker for development of post-transplant lymphoproliferative disease (PTLD), but few published data exist in the study of lung-transplant recipients. OBJECTIVES: To determine if elevated EBV DNA levels, in combination with other risk factors, were predictive of PTLD. METHODS: We conducted a retrospective, single-center study examining all lung transplant recipients (n = 296) and EBV DNA levels (n = 612) using real-time TaqMan polymerase chain reaction. There were 13 cases of PTLD overall, of which 5 occurred in the era of EBV DNA monitoring. MEASUREMENTS AND MAIN RESULTS: EBV DNA levels were distributed differently among seropositive and seronegative patients, with the latter having higher values (P < 0.0001). Among the cohort of pretransplantation seropositive patients, there was one diagnosed with PTLD. The EBV DNA level in this patient was elevated at the time of PTLD diagnosis (sensitivity = 100%, specificity = 100% for PTLD). Among the cohort of pretransplantation seronegative patients, there were four with a diagnosis of PTLD. In all four patients, the EBV DNA level was detectable (sensitivity = 100%, specificity = 24%), but in only two was it elevated (sensitivity = 50%, specificity = 22%). HLA-A3 expression in the recipient and/or donor conferred additional risk for PTLD among the seronegative patients (P = 0.026 to 0.003). No other PTLD risk factor was found. CONCLUSIONS: EBV DNA levels are a useful but imperfect predictor of PTLD in patients with lung transplants. Pretransplant EBV status affected the results of the assay and should be considered when interpreting test results. HLA-A3 was strongly linked to PTLD and may be a novel marker of PTLD risk.

PX3.102, a novel chinese herb extract, diminishes chronic airway allograft rejection.

More effective immunosuppressants are needed to improve lung-transplantation survival. PX3.102 is a novel immunosuppressant isolated from a mixture of traditional Chinese herbs. We tested its protective role on chronic lung rejection in the heterotopic tracheal transplant model. C57BL/6 mice received BALB/c tracheal grafts and were treated with PX3.102, cyclosporine A, or vehicle. PX3.102 improved tracheal allograft lumen patency (*P<0.01 vs. vehicle and P=0.14 vs. cyclosporine A) but not epithelialization (P>0.2 vs. vehicle). Subsequent in vitro studies demonstrated that PX3.10 was toxic to fully differentiated human tracheal epithelial cells in a dose-dependent manner. PX3.102 markedly suppressed antigen-specific lymphocyte proliferation in vitro at a concentration 10 times lower than cyclosporine A. In conclusion, PX3.102, a promising and potent immunosuppressant, although exhibiting toxicity to airway epithelial cells at high doses, is effective in inhibiting chronic airway allograft rejection.

Human leukocyte antigen mismatches predispose to the severity of bronchiolitis obliterans syndrome after lung transplantation.

BACKGROUND: Obliterative bronchiolitis (OB) is the most important cause of long-term morbidity and mortality in lung transplant recipients, and probably results from alloimmune airway injury. Bronchiolitis obliterans syndrome (BOS), defined as a staged decline in pulmonary function, is the clinical correlate of OB. OBJECTIVE: Evaluation of the risk and severity of BOS on the basis of the incompatibility of donor and recipient human leukocyte antigen (HLA) molecules. DESIGN: Retrospective cohort study. SETTING: Large university hospital. PARTICIPANTS: Lung transplant recipients between January 1990 and January 2000. MEASUREMENTS: We determined the BOS stage using internationally promulgated guidelines with a minor modification on all recipients at their 4-year transplant anniversary. Recipients whose graft function had deteriorated or who died due to causes other than BOS were excluded from the study. HLA loci mismatches and other covariables, including recipient age, donor age, cytomegalovirus (CMV) mismatch, cold ischemic time, use of cardiopulmonary bypass, ventilatory days, episodes of acute rejection and CMV pneumonitis, mean trough cyclosporin A (CsA) level, episodes of subtherapeutic CsA levels, and histopathology of OB and diffuse alveolar damage were entered into the analysis of BOS predictors. RESULTS: Sixty-four patients met the inclusion and exclusion criteria of the study at the 4-year posttransplant time point. In univariate analyses, the number of combined HLA-A and HLA-B mismatches was strongly associated with the stage of BOS at 4 years (p = 0.002). This association remained significant after the inclusion of other potential risk factors for BOS in multiple linear regression models. Pretransplant and posttransplant proportional odds models confirmed that the increasing number of combined HLA-A and HLA-B mismatches increased the overall severity of BOS (adjusted odds ratio, 1.84 [p = 0.035] vs 1.69 [p = 0.067], respectively). A trend toward significance was seen with HLA-DR mismatching (p = 0.17). CONCLUSIONS: The degree of HLA class I mismatching between donors and recipients predisposes lung transplant recipients to the development and severity of BOS.

Stem cells and repair of lung injuries.

Fueled by the promise of regenerative medicine, currently there is unprecedented interest in stem cells. Furthermore, there have been revolutionary, but somewhat controversial, advances in our understanding of stem cell biology. Stem cells likely play key roles in the repair of diverse lung injuries. However, due to very low rates of cellular proliferation in vivo in the normal steady state, cellular and architectural complexity of the respiratory tract, and the lack of an intensive research effort, lung stem cells remain poorly understood compared to those in other major organ systems. In the present review, we concisely explore the conceptual framework of stem cell biology and recent advances pertinent to the lungs. We illustrate lung diseases in which manipulation of stem cells may be physiologically significant and highlight the challenges facing stem cell-related therapy in the lung.

Donor-specific antibodies are associated with antibody-mediated rejection, acute cellular rejection, bronchiolitis obliterans syndrome, and cystic fibrosis after lung transplantation.

BACKGROUND: Lung transplantation is limited by chronic lung allograft dysfunction. Acute cellular rejection (ACR) is a risk factor for allograft dysfunction; however, the role of antibody-mediated rejection (AMR) is not well characterized. METHODS: This was a retrospective review from 2007 to 2011 of lung transplant recipients with human leukocyte antigen (HLA) antibody testing using Luminex (Luminex Corp, Austin, TX) single-antigen beads. Statistics included Fisher's exact test for significance. RESULTS: Donor-specific antibodies (DSA) developed in 13 of 44 patients. Of the 13 with DSA, 12 had cystic fibrosis compared with 18 of 31 in the non-DSA group (p = 0.035). Of those with DSAs, 23.1% occurred within the first year, and 69.2% occurred between 1 and 3 years. Twelve of 13 DSA patients had anti-HLA DQ specificity compared with 2 of 31 non-DSA patients (p = 0.0007). AMR developed in 10 of the 13 DSA patients compared with 1 of 31 non-DSA patients (p = 0.0001). The DSA group experienced 2.6 episodes/patient of cellular rejection vs 1.7 episodes/patient in the non-DSA group (p = 0.059). Bronchiolitis obliterans syndrome developed in 11 of 13 in the DSA group vs 10 of 31 in the non-DSA group (p = 0.0024). In the DSA group, 11.5% HLAs matched compared with 20.4% in the non-DSA group (p = 0.093). AMR developed in 11 of 22 patients in the non-DSA HLA group compared with 0 of 22 in the group without non-DSA HLA antibodies (p = 0.002). Survival at 1 and 3 years was 92% and 36% in the DSA group, respectively, and 97% and 65% in the non-DSA group. CONCLUSIONS: DSAs and non-DSAs occur frequently after lung transplantation. DSAs are prevalent in the cystic fibrosis population and are associated with AMR, bronchiolitis obliterans syndrome, and possibly, ACR.

MyD88 provides a protective role in long-term radiation-induced lung injury.

PURPOSE: The role of innate immune regulators is investigated in injury sustained from irradiation as in the clinic for cancer treatment or from a nuclear incident. The protective benefits of flagellin signaling through Toll-like receptors (TLR) in an irradiation setting warrant study of a key intracellular adaptor of TLR signaling, namely Myeloid differentiation primary response factor 88 (MyD88). The role of MyD88 in regulating innate immunity and Nuclear factor kappa-B (NF-κB)-activated responses targets this critical factor for influencing injury and recovery as well as maintaining immune homeostasis. MATERIALS AND METHODS: To examine the role of MyD88, we examined immune cells and factors during acute pneumonitic and fibrotic phases in Myd88-deficient animals receiving thoracic gamma (γ)-irradiation. RESULTS: We found that MyD88 supports survival from radiation-induced injury through the regulation of inflammatory factors that aid in recovery from irradiation. The absence of MyD88 resulted in unresolved pulmonary infiltrate and enhanced collagen deposition plus elevated type 2 helper T cell (Th2) cytokines in long-term survivors of irradiation. CONCLUSIONS: These results based only on a gene deletion model suggest that alterations of MyD88-dependent inflammatory processes impact chronic lung injury. Therefore, MyD88 may contribute to attenuating long-term radiation-induced lung injury and protecting against fibrosis.

'Old' bones in young bodies: the tale of cystic fibrosis.

PURPOSE OF REVIEW: Cystic fibrosis (CF) is the most common genetic disease within the white population and leads to premature respiratory failure. Approximately, 60 000 individuals are currently living with CF in North America and Europe, almost half of whom are adults. RECENT FINDINGS: Dozens of studies across the globe indicate that CF adults have low bone density and increased rates of fractures. This genesis of the problem appears to be in late childhood to adolescence. SUMMARY: Prevention and treatment of CF-related bone disease must address the myriad risk factors (decreased absorption of fat-soluble vitamins due to pancreatic insufficiency, altered sex hormone production, chronic lung infection with increased serum levels of proinflammatory, bone-active cytokines, malnutrition and low body weight, physical inactivity and glucocorticoid therapy) for poor bone health. This review will address the pathogenesis, diagnosis and treatment of bone disease in CF. It will also discuss best practice guidelines for optimizing bone health in patients with CF.

Managing complications following lung transplantation.

Lung transplantation has become a proven therapeutic option for patients with end-stage lung disease, extending life and providing improved quality of life to those who otherwise would continue to be breathless and oxygen-dependent. Over the past 20 years, considerable experience has been gained in understanding the multitude of medical and surgical issues that impact upon patient survival. Today, clinicians have an armamentarium of tools to manage diverse problems such as primary graft dysfunction, acute and chronic allograft rejection, airway anastomotic issues, infectious complications, renal dysfunction, diabetes and osteoporosis, hematological and gastrointestinal problems, malignancy, and other unique issues that confront immunosuppressed solid organ transplant recipients.

Pulmonary immune responses to Propionibacterium acnes in C57BL/6 and BALB/c mice.

Propionibacterium acnes (PA) is a gram-positive anaerobic bacterium implicated as a putative etiologic agent of sarcoidosis. To characterize the pulmonary immune response to PA, C57BL/6 and BALB/c mice were intraperitoneally sensitized and intratracheally challenged with heat-killed bacteria. C57BL/6 mice challenged with PA developed a cellular immune response characterized by elevations in Th1 cytokines/chemokines, increased numbers of lymphocytes and macrophages in lung lavage fluid, and peribronchovascular granulomatous inflammation composed of T- and B-lymphocytes and epithelioid histiocytes. T-lymphocytes in the lung lavage fluid showed a marked CD4+ cell predominance. In contrast, C57BL/6 mice challenged with Staphylococcus epidermidis (SE), another gram-positive commensal of human skin, and BALB/c mice challenged with PA, showed only a modest induction of Th1 cytokines, less pulmonary inflammation, and no granulomatous changes in the lung. Enhancement of Toll-like receptor expression was seen in PA-exposed C57BL/6 mice within 24 h after exposure, suggesting that induction of innate immunity by PA contributes to the robust, polarized Th1 immune response elicited by this bacterium. These findings suggest that PA-induced pulmonary inflammation may be a useful model for testing the contributions of both bacterial and host factors in the development, maintenance, and resolution of granulomatous inflammation in the lung.

Calcineurin inhibitor effects on growth and phenotype of human airway epithelial cells in vitro.

Calcineurin inhibitors (CIs) cyclosporin and tacrolimus form the basis for immunosuppression in lung transplantation, yet also exert biological effects on nonlymphoid tissue. With the advent of inhaled cyclosporin, we hypothesize that the airway epithelium is also subject to CI effects at high doses. The aim of this study was to identify human tracheobronchial epithelial cell (hTBEC) calcineurin gene expression and quantify effects of CIs on hTBEC growth, interleukin-1-beta stimulated IL-8 production and hTBEC phenotype. Cyclophillin B and FK-associated binding protein, calcineurin A (alpha and beta), and NFATC3 and NFAT5 were detected in hTBEC cultures by RT-PCR. Acute and chronic cyclosporine treatment 1000 ng/mL significantly inhibited hTBEC proliferation, while tacrolimus did not (range of 10 ng/mL to 1000 ng/mL for acute treatment, 50 ng/mL for chronic treatment). Cyclosporin at 10,000 ng/mL significantly increased LDH release by well-differentiated hTBEC cultures (n = 6) and trended towards significance at 1000 ng/mL. IL1-beta stimulated IL-8 production was significantly increased in rapidly growing hTBEC cultures (n = 8) treated with cyclosporin (p = 0.049). Prolonged treatment of well-differentiated hTBECs at air-liquid-interface (ALI) with cyclosporin 1000 ng/mL significantly reduced intact multilayered mucociliary epithelium (p = 0.009). Inhibition of hTBEC growth, stimulation of IL-8 production and long-term effects on mucociliary phenotype and intact multi-layered epithelium suggest that cyclosporin may have a direct toxic effect on airway epithelium after transplantation.

Growth factor upregulation during obliterative bronchiolitis in the mouse model.

Obliterative bronchiolitis (OB), or chronic allograft rejection, is a major cause of morbidity and mortality after lung transplantation. The goal of these experiments was to determine whether several important growth factors were upregulated during OB in the mouse heterotopic trachea model. Isografts (BALB/c into BALB/c) and allografts (BALB/c into C57BL/6) were implanted in three sets of cyclosporine-treated animals and were harvested from 2 to 10 weeks. Ribonucleic acid was isolated using the cesium chloride-guanidine method and was reverse transcribed and semiquantitated with the polymerase chain reaction using specific primers for platelet-derived growth factor (PDGF)-A and PDGF-B chains, fibroblast growth factor (FGF) isoforms 1 and 2, transforming growth factor-beta, tumor necrosis factor-alpha (TNF-alpha), edothelin-1, (prepro) epidermal growth factor, insulin-like growth factor-1, and beta-actin as a control. Transforming growth factor-beta, TNF-alpha, endothelin-1, and insulin-like growth factor-1 expression were increased 1.5-fold to 5.0-fold (p < or = 0.04 for each) in the allografts compared with the isografts at Weeks 2 through 6. Significantly increased expression of FGF-1, FGF-2, and PDGF-B was noted in the allografts at 4 weeks (p < 0.05 for each), which reversed at 6 and 10 weeks. No differences were found with the PDGF-A chain. The isografts expressed more epidermal growth factor than allografts (p < 0.001). Treatment with a TNF-alpha-soluble receptor (human TNFR:Fc) significantly reduced epithelial injury (p = 0.01) and lumenal obstruction (p = 0.037) in this model. We conclude that increased expression of a large number of growth factors occurs during OB in this model. Growth factor blockade (in particular with regard to TNF-alpha) may be useful in ameliorating OB in this model.

Epithelial kinetics in mouse heterotopic tracheal allografts.

Obliterative bronchiolitis (OB) is the most important cause of graft dysfunction post-lung transplantation. It is likely that the small airway epithelium is a target of the alloimmune response, and that epithelial integrity is a crucial determinant of airway patency. Our goals are to elucidate epithelial cell kinetics in the heterotopic mouse trachea model and to determine potential mechanisms of cell death in allografts. Allografts and isografts were obtained by transplanting BALB/c tracheas into C57BL/6 and BALB/c immunosuppressed and non-immunosuppressed hosts, respectively and harvested from day 3-20. Morphometry, BrdU and TUNEL labeling, and EM studies were performed. Columnar epithelium in isografts and allografts sloughs during day 0-3, but regenerates in both sets of grafts by day 10. Subsequently, allografts become inflamed and denuded, while isografts retain an intact epithelium. Prior to airway denudation, allografts exhibited significantly increased epithelial cell density, BrdU labeling index (LI), and TUNEL positive cells. Epithelial apoptosis was confirmed by electron microscopy. Allograft percent ciliated columnar epithelium and lumenal circumference were significantly decreased. Cyclosporin delayed airway fibrosis but did not alter the progression of the allograft through the phases of early ischemic injury, airway epithelial cell regeneration, and eventual cell death. These studies quantitatively demonstrate that the allograft epithelium actively regenerates in the alloimmune environment, but succumbs to increased apoptotic cell death, underscoring the importance of the airway epithelium as a self-renewing source of alloantigen.