Interstitial pneumonitis and the risk of chronic allograft rejection in lung transplant recipients.

BACKGROUND: The presence of interstitial pneumonitis (IP) on surveillance lung biopsy specimens in lung transplant recipients is poorly described, and its impact on posttransplant outcomes is not established. The following study assessed the association of posttransplant IP with the development of bronchiolitis obliterans syndrome (BOS). METHODS: We examined all recipients of primary cadaveric lung transplants at our institution between January 1, 2000, and December 31, 2007 (N = 145). Patients had bronchoscopies with BAL, and transbronchial biopsies performed for surveillance during posttransplant months 1, 3, 6, and 12 as well as when clinically indicated. Patients were given a diagnosis of IP if, in the absence of active infection and organizing pneumonia, they showed evidence of interstitial inflammation and fibrosis on two or more biopsy specimens. RESULTS: IP was a significant predictor of BOS (OR, 7.84; 95% CI, 2.84-21.67; P < .0001) and was significantly associated with time to development of BOS (hazard ratio, 3.8; 95% CI, 1.93-7.39; P = .0001) within the first 6 years posttransplant. The presence of IP did not correlate with a significantly higher risk of mortality or time to death. There was no association between the presence of IP and the development of or time to acute rejection. CONCLUSIONS: The presence of IP on lung transplant biopsy specimens suggests an increased risk for BOS, which is independent of the presence of acute cellular rejection.

[Predictors of osteopenic syndrome in idiopathic pulmonary fibrosis].

AIM: To evaluate the functional state of bone tissue in patients with idiopathic pulmonary fibrosis (IPF), waiting for lung transplantation, and to determine possible predictors of lower bone mineral density (BMD) in this pathology in the pretransplantation period. SUBJECTS AND METHODS: Forty-nine IPF patients waiting for lung transplantation were examined. The patients' mean age was 53.4 +/- 6.4 years. BMD in the lumbar spine (L(II)-L(IV)) and femoral neck (FN) was estimated using dual-energy X-ray absorptiometry. All the patients underwent external respiratory function test, pulmonary diffusing capacity (DL(CO)), gasometry, and 6-minute walk test (6'WT). RESULTS: Osteopenia was recorded in 77% of the examinees, of them 28% had osteoporosis (OP). Normal BMD in both L(II)-L(IV) and FN was found only in 13% of the patients. The T score in L(II)-L(IV) was directly related to body mass index. There was a direct correlation between BMD in L(II)-L(IV) and FN, forced vital capacity (FVC), DL(CO), and arterial blood oxygen saturation (SaO2) and an inverse correlation with arterial carbon dioxide partial pressure (pCO2). No significant correlation was found between the distance covered in 6'WT, FEV1, pO2, and BMD in both L(II)-L(IV) and FN. Six (15%) subjects had atraumatic fractures at different sites. CONCLUSION: Osteopenia is a common systemic manifestation in patients with IPF in the pre-transplantation period. BMI, FVC, exercise desaturation, and DL(CO) may be considered as predictors for the development of OP initiated by IPF.

Surgical lung biopsy in transplant patients with diffuse lung disease: how much worse when the lung is the graft?

BACKGROUND: There are no data that compare the clinical presentation and results of surgical lung biopsy (SLB) for diffuse lung disease (DLD) in lung transplant patients, in contrast to individuals with other type of solid organ grafts. Our objective was to compare the clinical picture, radiologic pattern, pathology results, and outcomes of SLB for DLD in these two subsets of patients. METHODS: We retrospectively reviewed the clinical records of transplant patients undergoing SLB for DLD at our institution between 2004 and 2011. Patients with lung transplants and those with other transplants were compared. RESULTS: During the study period, 1,232 solid organ transplants were done at our institution. Of these, 49 patients (4%) had DLD that needed SLB for diagnosis, and 24 of these patients had a lung transplant. Dyspnea and a radiologic reticular pattern were more frequent in lung transplant patients, 21 of 24 vs 11 of 25 (p = 0.001) and 14 of 24 vs 7 of 25 (p = 0.03), respectively. Although postoperative complications and in-hospital deaths were more common in lung transplant patients, the differences were not statistically significant. Having the SLB performed for diagnosis, as opposed to being conducted for DLD that did not improve on medical treatment, had a protective effect on multivariate analysis (hazard ratio, 0.39; 95% confidence interval, 0.16 to 0.96; p = 0.042). A prior lung transplant was the only independent predictor of survival (hazard ratio, 4.62; 95% confidence interval, 1.53 to 13.92, p = 0.006). CONCLUSIONS: It is relatively uncommon for a solid organ transplant patient with DLD to require a SLB. Clinical and radiologic presentation differ in patients with lung transplants compared with other transplants. Postoperative outcomes are not significantly different between the groups. SLB performed early in the course of the disease might be beneficial. Having a lung transplant is a significant negative predictor of survival.

Isolation of fibroblasts and epithelial cells in bronchoalveolar lavage (BAL).

The long-term outcome of lung transplants is poor with 60%-70% of patients developing chronic rejection. Chronic rejection is manifested histologically by obliterative bronchiolitis with bronchiolitis obliterans syndrome (BOS), the clinical surrogate. Recent studies suggest that fibroblasts and epithelial cells present in bronchoalveolar lavage (BAL) may be a clinically relevant biomarker for BOS. The goal of this investigation was to develop a fast, repeatable method to individually isolate these low-frequency cell types. Fibroblasts and epithelial cells were isolated from BAL using attachment methods and the phenotype of the cells confirmed using immunostaining for vimentin (fibroblasts) and epithelial cell adhesion molecule (EpCAM, epithelial cells). Both fibroblasts and epithelial cells were isolated in every sample of BAL processed with the frequency of fibroblasts ranging from 0.03% to 0.48% and epithelial cells ranging from 0.05% to 1.5% of the total sample. Additional studies were performed using cytospins of cells after macrophages were depleted; cells exhibiting characteristics of both fibroblasts and epithelial cells were observed. The frequency of the cells of interest suggests that conventional methods of immunomagnetic isolation will not be effective in isolating these subpopulations. Finally, some of the low-frequency cells isolated via cytospin exhibit characteristics of epithelial to mesenchymal transition (which was not observed in plating incubations), indicating that the epithelial to mesenchymal cell transition fibroblasts may be nonadherent. In future studies, this technique and dataset may be of use to statistically correlate low-frequency cell type abundance to the onset and development of BOS.

Effect of positive end-expiratory pressure after porcine unilateral left lung transplant.

OBJECTIVES: To evaluate the effects of 2 different levels of positive end-expiratory pressure on pigs who had unilateral lung transplants. MATERIALS AND METHODS: A left lung transplant was performed in 12 pigs. The animals were randomized into 2 groups based on positive end-expiratory pressure: group 1 (5 cm H(2)O) and group 2 (10 cm H(2)O). Hemodynamics, gas exchange, and respiratory mechanics were measured before and after surgery. Cytokines, oxidative stress, and histologic scores were assessed in the lung tissue of each pig. RESULTS: Pigs in group 2 exhibited a significantly higher mean heart rate (P = .006), static compliance (P = .001), lower mean arterial pressure (P = .003), and airway resistance (P = .001) than did pigs in group 1. There were no postoperative differences between the groups in concentrations of thiobarbituric acid reactive substances, superoxide dismutase, and interleukin 8. At the end of the observation period, pigs in group 2 had higher levels of thiobarbituric acid reactive substances (P = .001) and interleukin 8 (P = .05), and pigs in group 1 had higher levels of superoxide dismutase (P = .05) than they did at baseline. CONCLUSIONS: After unilateral lung transplant, higher positive end-expiratory pressure was associated with improved respiratory mechanics, a negative effect on hemodynamics, a stronger inflammatory response, and increased production of reactive oxygen species, but no effect on gas exchange.

Interobserver variability in grading transbronchial lung biopsy specimens after lung transplantation.

BACKGROUND: Acute rejection remains a major source of morbidity after lung transplantation. Given the importance of this diagnosis, an international grading system was developed to standardize the diagnosis of acute lung-allograft rejection. The reliability of this grading system has not been adequately assessed by previous studies. METHODS: We examined the level of agreement in grading transbronchial biopsy specimens obtained from a large multicenter study (AIRSAC [Comparison of a Tacrolimus/Sirolimus/Prednisone Regimen vs Tacrolimus/Azathioprine/Prednisone Immunosuppressive Regimen in Lung Transplantation] trial). Biopsy specimens were initially graded for acute rejection and lymphocytic bronchiolitis by the site pathologist and subsequently graded by a central pathologist. Reliability of interobserver grading was evaluated using Cohen κ coefficients. RESULTS: A total of 481 transbronchial biopsy specimens were graded by both the site and central pathologists. The overall concordance rates were 74% and 89% for grade A and grade B biopsy specimens, respectively. When samples from biopsies performed at different time points after transplantation were assessed, there was a higher level of agreement early (≤ 6 weeks) after transplant compared with later time points for acute rejection. However, there was still only moderate agreement for both grade A (κ score 0.479; 95% CI, 0.29-0.67) and grade B (κ score 0.465; 95% CI, 0.08-0.85) rejection. CONCLUSIONS: These results expand upon previous reports of interobserver variability in grading transbronchial biopsy specimens after lung transplantation. Given the variability in grading these specimens, we advocate further education of the histopathologic findings in lung transplant biopsy specimens, as well as revisiting the current criteria for grading transbronchial biopsy specimens to improve concordance among lung transplant pathologists. TRIAL REGISTRY: ClinicalTrials.gov; No. NCT00321906; URL: www.clinicaltrials.gov.

Pathologic findings in lung allografts with anti-HLA antibodies.

BACKGROUND: Despite data indicating a positive correlation between donor-specific anti-HLA antibodies (DSAs) and early development of bronchiolitis obliterans syndrome (BOS) in lung allografts, the role of an antibody-mediated process in acute and chronic lung allograft rejection has not been elucidated. In this study we evaluated pathologic features of transplant lung biopsies in patients with and without DSAs. METHODS: Forty-one lung transplant biopsies from 41 patients at our institution were included in our study. The biopsy H&E slides were reviewed in a blinded fashion, and scored for presence of microvascular inflammation, acute rejection, bronchiolar inflammation and acute lung injury, as well as diffuse alveolar damage (DAD). Microvascular inflammation was graded by the presence of capillary neutrophils on a scale of 0 to 4(+). For immunohistochemical analysis, the pattern and intensity of staining for C4d and C3d deposition were evaluated in airways and alveolar capillaries. RESULTS: Histopathology suspicious for antibody-mediated rejection (AMR)-defined as≥2(+) neutrophilic infiltration and/or DAD-were more common in DSA-positive cases than controls (11 of 16 vs 6 of 25, p<0.01). Evidence of allograft dysfunction was significantly more common among patients with both DSA and suspicious histopathology compared with controls (5 of 10 vs 3 of 25, p = 0.03). The combination of DSAs and histopathology suspicious for AMR was associated with both BOS (p = 0.002) and mortality (p = 0.03). Immunohistochemistry for C3d and C4d showed no correlation with each other, DSAs or histopathology. CONCLUSIONS: Grade 2(+) neutrophilic infiltration is the histopathologic finding most closely related to DSAs with graft dysfunction and development of BOS in lung transplant recipients and may be a marker for AMR.

Association of large-airway lymphocytic bronchitis with bronchiolitis obliterans syndrome.

RATIONALE: Lung transplantation offers great promise for otherwise terminal lung diseases, but the development of bronchiolitis obliterans syndrome (BOS) continues to limit survival. Although acute rejection and lymphocytic bronchiolitis have been identified as risk factors for the development of BOS, it is unclear whether large-airway lymphocytic inflammation conveys the same risk. OBJECTIVES: We evaluated lymphocytic bronchitis on endobronchial biopsies as a risk factor for BOS and mortality. METHODS: Endobronchial biopsies were collected and graded during surveillance after lung transplantation. We assessed samples with negative cultures collected in the first 90 days from 298 subjects and compared large-airway lymphocytic bronchitis assessed by a 0-2 "E-score" and with standard A and BR pathology scores for acute rejection and small-airway lymphocytic bronchiolitis, respectively. MEASUREMENTS AND MAIN RESULTS: We found surprisingly little association between large- and small-airway lymphocytic inflammation scores from a given bronchoscopy. Endobronchial lymphocytic bronchitis was more prevalent in subjects in BOS stage 0p and BOS stages 1-3 at the time of biopsy. Within 90 days after transplantation, increasing maximum E-score was associated with greater risk of BOS (adjusted hazard ratio, 1.76; 95% confidence interval, 1.11-2.78; P = 0.02) and in this analysis 90-day maximum E-scores were the only score type predictive of BOS (P < 0.01). CONCLUSIONS: These results support a multicenter study to evaluate endoscopic biopsies for the identification of patients at increased risk for BOS. The association of endobronchial lymphocytic inflammation and BOS may have mechanistic implications.

Leaky lysosomes in lung transplant macrophages: azithromycin prevents oxidative damage.

BACKGROUND: Lung allografts contain large amounts of iron (Fe), which inside lung macrophages may promote oxidative lysosomal membrane permeabilization (LMP), cell death and inflammation. The macrolide antibiotic azithromycin (AZM) accumulates 1000-fold inside the acidic lysosomes and may interfere with the lysosomal pool of Fe. OBJECTIVE: Oxidative lysosomal leakage was assessed in lung macrophages from lung transplant recipients without or with AZM treatment and from healthy subjects. The efficiency of AZM to protect lysosomes and cells against oxidants was further assessed employing murine J774 macrophages. METHODS: Macrophages harvested from 8 transplant recipients (5 without and 3 with ongoing AZM treatment) and 7 healthy subjects, and J774 cells pre-treated with AZM, a high-molecular-weight derivative of the Fe chelator desferrioxamine or ammonium chloride were oxidatively stressed. LMP, cell death, Fe, reduced glutathione (GSH) and H-ferritin were assessed. RESULTS: Oxidant challenged macrophages from transplants recipients without AZM exhibited significantly more LMP and cell death than macrophages from healthy subjects. Those macrophages contained significantly more Fe, while GSH and H-ferritin did not differ significantly. Although macrophages from transplant recipients treated with AZM contained both significantly more Fe and less GSH, which would sensitize cells to oxidants, these macrophages resisted oxidant challenge well. The preventive effect of AZM on oxidative LMP and J774 cell death was 60 to 300 times greater than the other drugs tested. CONCLUSIONS: AZM makes lung transplant macrophages and their lysososomes more resistant to oxidant challenge. Possibly, prevention of obliterative bronchiolitis in lung transplants by AZM is partly due to this action.

Antibody-mediated rejection in a lung transplant recipient after acute stroke.

Antibody-mediated rejection (AMR) is becoming a more recognized problem in lung transplantation. We present a case of late onset AMR in a lung transplant recipient after an acute embolic stroke requiring thrombolytic therapy, who previously had a completely unremarkable course for over 3 years.

The bone marrow functions as the central site of proliferation for long-lived NK cells.

NK cells play an important role in the early defense against invading pathogens. Although it is well established that infection leads to a substantial, local increase in NK cell numbers, little is known about the mechanisms that trigger their proliferation and migration. In this study, we investigated the dynamics of NK cell responses after intranasal respiratory virus infection. We show that NK cell numbers increased in the airways after influenza virus infection but find no evidence of proliferation either at the site of infection or in the draining lymph nodes. Instead, we find that the bone marrow (BM) is the primary site of proliferation of both immature and mature NK cells during infection. Using an adoptive transfer model, we demonstrate that peripheral, long-lived and phenotypically mature NK cells migrate back to the BM and proliferate there, both homeostatically and in response to infection. Thus, the BM is not only a site of NK cell development but also an important site for proliferation of long-lived mature NK cells.

Increased expression of graft intraepithelial T-cell pro-inflammatory cytokines compared with native lung during episodes of acute rejection.

BACKGROUND: We have previously reported that T-cell pro-inflammatory cytokines in the airways are associated with acute lung transplant rejection. However, during acute rejection episodes, we found no significant differences in airway intraepithelial T cell pro-inflammatory cytokines from stable and rejecting patients due to broad cytokine variability between patient groups. To overcome this limitation, we hypothesized that there would be an increase in pro-inflammatory intraepithelial T-cells in the graft compared with native airway during acute rejection. METHODS: Bronchial brushings from patients with stable graft function, evidence of acute rejection, bronchiolitis obliterans syndrome, infection, and healthy controls were stimulated and pro-inflammatory cytokines in intraepithelial T cells from graft and native airway were determined using multiparameter flow cytometry. RESULTS: There was a significant increase in intraepithelial T-cell interferon-γ and tumor necrosis factor (TNF)-α in the graft of patients with acute rejection compared with intraepithelial T cells obtained from the native airway, but no changes were noted among other patient groups. The increase in intraepithelial T-cell TNF-α was more pronounced the higher the acute rejection grade. CONCLUSIONS: The graft airway epithelium is enriched with T cells producing interferon-γ and TNF-α during acute graft rejection. Therapeutic targeting of these pro-inflammatory cytokines and improved monitoring using this assay may reduce acute lung transplant rejection.

Donor lung derived myeloid and plasmacytoid dendritic cells differentially regulate T cell proliferation and cytokine production.

BACKGROUND: Direct allorecognition, i.e., donor lung-derived dendritic cells (DCs) stimulating recipient-derived T lymphocytes, is believed to be the key mechanism of lung allograft rejection. Myeloid (cDCs) and plasmacytoid (pDCs) are believed to have differential effects on T cell activation. However, the roles of each DC type on T cell activation and rejection pathology post lung transplantation are unknown. METHODS: Using transgenic mice and antibody depletion techniques, either or both cell types were depleted in lungs of donor BALB/c mice (H-2(d)) prior to transplanting into C57BL/6 mice (H-2(b)), followed by an assessment of rejection pathology, and pDC or cDC-induced proliferation and cytokine production in C57BL/6-derived mediastinal lymph node T cells (CD3+). RESULTS: Depleting either DC type had modest effect on rejection pathology and T cell proliferation. In contrast, T cells from mice that received grafts depleted of both DCs did not proliferate and this was associated with significantly reduced acute rejection scores compared to all other groups. cDCs were potent inducers of IFNγ, whereas both cDCs and pDCs induced IL-10. Both cell types had variable effects on IL-17A production. CONCLUSION: Collectively, the data show that direct allorecognition by donor lung pDCs and cDCs have differential effects on T cell proliferation and cytokine production. Depletion of both donor lung cDC and pDC could prevent the severity of acute rejection episodes.

Lung transplantation in the fischer 344-wistar kyoto strain combination is a relevant experimental model to study the development of bronchiolitis obliterans in the rat.

Chronic allograft rejection and bronchiolitis obliterans (BO) limited successful long-term outcome after lung transplantation (LTX). Reliable animal models are needed to study the pathogenesis of BO and to develop effective therapeutic strategies. The relevance of an available experimental LTX model without immunosuppression-the Fischer (F344)-Wistar Kyoto (WKY) rat strain combination-was analyzed. The kinetics of acute and chronic rejection and respective graft histopathology were described in the F344-to-WKY rat strain combination after allogeneic LTX. A modified classification of chronic lung allograft rejection was introduced to describe obliterative changes in the airways after rat LTX. Animals from Harlan Winkelmann (HW) and Charles River (CR) were examined. Within 14 days after LTX, allografts showed moderate to severe acute vascular and bronchiolar inflammation. In contrast to rats from CR, animals from HW showed a delayed acute rejection process. Mid-term phase after LTX (days 20-40) presented a "borderline form" consisting of both acute and first signs of chronic airway rejection. On postoperative day (POD) 60, first signs of airway remodelling and distinct BO were diagnosed in 80% of animals from HW. At the same time, the allografts with BO-like lesions increased up to 100% in rats from CR. The F344-to-WKY rat LTX model allows detailed assessment of all features of acute and chronic pulmonary rejection representing a clinically relevant model. However, due to breeding differences resulting in various sublines of the same rat strain, the source and husbandary history of the animals is important for analysis of immuno-mediated processes.

Revisiting the pathologic finding of diffuse alveolar damage after lung transplantation.

BACKGROUND: Diffuse alveolar damage (DAD) is a non-specific pathologic diagnosis frequently encountered after lung transplantation. We examined the relationship between DAD and different forms of chronic lung allograft dysfunction (CLAD). METHODS: We reviewed the results of 4,085 transbronchial biopsies obtained from 720 lung transplant recipients. DAD detected in biopsies within 3 months and newly detected DAD after 3 months were defined as early DAD and late new-onset DAD, respectively. Among patients with CLAD (FEV(1) <80% baseline), restrictive allograft syndrome (RAS) was defined by a decline in total lung capacity to <90% baseline and bronchiolitis obliterans syndrome (BOS) as CLAD without restrictive allograft syndrome (RAS). Kaplan-Meier analyses and multivariate proportional hazard models were used. RESULTS: DAD was observed in 320 of 720 (44.4%) patients at least once; early and late new-onset DAD were observed in 264 of 707 (37.3%) and 87 of 655 (13.3%) patients, respectively. Early DAD was associated with significantly higher 90-day mortality (20 of 264 [7.6%] vs 11 of 443 [2.5%]; p = 0.001). Moreover, among 502 bilateral lung transplant recipients who had sufficient pulmonary function tests to distinguish BOS and RAS, early DAD was associated with earlier BOS onset (hazard ratio [HR] 1.24; confidence interval [CI] 1.04 to 1.47; p = 0.017; median time of BOS onset: 2,902 vs 4,005 days). Conversely, treated as a time-varying covariate, late new-onset DAD was a significant risk factor for RAS in a Cox model (HR 36.8; CI 18.3 to 74.1; p < 0.0001). CONCLUSIONS: Early DAD is associated with early mortality and BOS, and late new-onset DAD increases the risk of RAS.