Evidence That Humoral Allograft Rejection in Lung Transplant Patients Is Not Histocompatibility Antigen-Related

We have recently recognized humoral rejection (HR) in lung allograft recipients and its association with acute and chronic graft dysfunction. We have shown that C4d, a stable marker of classic complement activation, is deposited in lung allografts, correlating with clinical rejection and parenchymal injury. The antigenic target may be endothelium in the setting of recurrent acute rejection while varying components of the bronchial wall may be important in chronic graft dysfunction. We sought to establish whether there is a role for antibodies with histocompatibility antigen specificity in the lung humoral allograft phenomenon. Flow cytometric and ELISA assays to assess donor-specific antigens were conducted on sera from 25 lung transplant recipients who had experienced one or more episodes of clinical rejection; in addition, the serum samples were tested for evidence of antiendothelial cell antibody activity. Morphologically, each case had biopsies showing septal capillary injury with significant deposits of immunoreactants with microvascular localization and positive indirect immunofluorescent antiendothelial cell antibody assay. Panel-reactive antibody testing showed absence of MHC Class I/II alloantibodies; ELISA based crossmatch detecting donor-specific MHC Class I/II specific antibodies was negative. HR can occur in the absence of antibodies with HLA specificity; antigenic targets may be of endothelial cell origin.     

Impact of Allograft Injury Time of Onset on the Development of Chronic Lung Allograft Dysfunction After Lung Transplantation

The impact of allograft injury time of onset on the risk of chronic lung allograft dysfunction (CLAD) remains unknown. We hypothesized that episodes of late‐onset (≥6 months) allograft injury would produce an augmented CXCR3/ligand immune response, leading to increased CLAD. In a retrospective single‐center study, 1894 transbronchial biopsy samples from 441 lung transplant recipients were reviewed for the presence of acute rejection (AR), lymphocytic bronchiolitis (LB), diffuse alveolar damage (DAD), and organizing pneumonia (OP). The association between the time of onset of each injury pattern and CLAD was assessed by using multivariable Cox models with time‐dependent covariates. Bronchoalveolar lavage (BAL) CXCR3 ligand concentrations were compared between early‐ and late‐onset injury patterns using linear mixed‐effects models. Late‐onset DAD and OP were strongly associated with CLAD: adjusted hazard ratio 2.8 (95% confidence interval 1.5–5.3) and 2.0 (1.1–3.4), respectively. The early‐onset form of these injury patterns did not increase CLAD risk. Late‐onset LB and acute rejection (AR) predicted CLAD in univariable models but lost significance after multivariable adjustment for late DAD and OP. AR was the only early‐onset injury pattern associated with CLAD development. Elevated BAL CXCR3 ligand concentrations during late‐onset allograft injury parallel the increase in CLAD risk and support our hypothesis that late allograft injuries result in a more profound CXCR3/ligand immune response.     

Survival After Lung Transplantation of Cystic Fibrosis Patients Infected with Burkholderia cepacia Complex

Within the Burkholderia cepacia complex (Bcc), B. cenocepacia portends increased mortality compared with other species. We investigated the impact of Bcc infection on mortality and re-infection following lung transplant (LT). Species designation for isolates from Bcc-infected patients was determined using 16S rDNA and recA gene analyses. Of 75 cystic fibrosis patients undergoing LT from September 1992 to August 2002, 59 had no Bcc and 16 had Bcc (including 7 B. cenocepacia ) isolated in the year before LT. Of the latter, 87.5% had Bcc recovered after transplantation, and all retained their pretransplant strains. Survival was 97%, 92%, 76% and 63% for noninfected patients; 89%, 89%, 67% and 56% for patients infected with Bcc species other than B. cenocepacia; and 71%, 29%, 29% and 29% for patients with B. cenocepacia (p = 0.014) at 1 month, 1 year, 3 years and 5 years, respectively. Patients infected with B. cenocepacia before transplant were six times more likely to die within 1 year of transplant than those infected with other Bcc species (p = 0.04) and eight times than noninfected patients (p < 0.00005). Following LT, infection with Bcc species other than B. cenocepacia does not significantly impact 5-year survival whereas infection with B. cenocepacia pretransplant is associated with decreased survival.     

Pulmonary Capillaritis as a Manifestation of Acute Humoral Allograft Rejection Following Infant Lung Transplantation

Pulmonary capillaritis has been described in adult lung transplant recipients, but has not been previously reported in pediatric recipients. We report a case of posttransplant pulmonary capillaritis in an 8-month-old infant, and demonstrate evidence of C4d deposition and B-lymphocytes in the allograft, donor anti-HLA antibodies in the serum and a clinical and immunohistochemical response to anti-CD20 monoclonal antibody (rituximab) therapy. These findings strongly support the hypothesis that pulmonary capillaritis may represent a form of acute humoral rejection in the lung allograft that is less common than, and clinically and histologically distinct from, typical acute cellular rejection.     

Ultra-Late Antibody-Mediated Rejection 30 Years After a Living-Related Renal Allograft

Antibody-mediated renal allograft rejection has become increasingly recognized and more clearly defined through the use of flow cytometry cross-matching and the deposition of C4d in renal allograft biopsies. All of the cases reported thus far have developed an antibody within 10 years of transplantation, and many lacked HLA and/or donor specificity. The present patient developed an anti-HLA donor-specific antibody between the 22nd and 30th year after a living-related renal transplant. At the 30th year post-transplantation, she experienced a rise in the serum creatinine from 0.7 to 1.9 mg/dL associated with transplant biopsy C4d deposition in peritubular capillaries and glomeruli. After the replacement of azathioprine with mycophenolate mofetil, and six apheresis treatments followed by two infusions of IVIG, the renal function stabilized at 1.9 mg/dL, 33 years after transplantation. Antibody-mediated rejection must be considered as a possible cause or renal allograft dysfunction at all time periods after transplantation.     

Alemtuzumab in the Treatment of Refractory Acute Rejection and Bronchiolitis Obliterans Syndrome After Human Lung Transplantation

Despite substantial improvements in early survival after lung transplantation, refractory acute rejection (RAR) and bronchiolitis obliterans syndrome (BOS) remain major contributors to transplant-related morbidity and mortality. We have utilized alemtuzumab, a humanized anti-CD52 antibody which results in potent lymphocyte depletion, in consecutive patients with RAR (n = 12) or BOS (n = 10). All patients failed conventional treatment with methylprednisolone and antithymocyte globulin and received strict infection prophylaxis. Alemtuzumab significantly improved histological rejection scores in RAR. Total rejection grade/biopsy was 1.98 +/- 0.25 preceding alemtuzumab versus 0.33 +/- 0.14 posttreatment, p-value <0.0001 (with a similar number of biopsies/patient per respective time interval). Freedom from BOS was observed in 65% of RAR patients 2 years after alemtuzumab treatment. Although there was no statistically significant change in forced expiratory volume in 1 second (FEV1) before and after alemtuzumab treatment in patients with BOS, a stabilization or improvement in BOS grade occurred in 70% of patients. Patient survival 2 years after alemtuzumab for BOS was 69%. Despite a dramatic decline in CD4 counts in alemtuzumab-treated patients, only one patient developed a lethal infection. Thus, we provide the first evidence that alemtuzumab is a potentially useful therapy in lung transplant recipients with RAR or BOS.     

Lymphocytic Bronchiolitis After Lung Transplantation Is Associated With Daily Changes in Air Pollution

Acute rejection represents a major problem after organ transplantation, being a recognized risk for chronic rejection and mortality. Recently, it became clear that lymphocytic bronchiolitis (LB, B‐grade acute rejection) is more important than previously thought, as it predisposes to chronic rejection. We aimed to verify whether daily fluctuations of air pollution, measured as particulate matter (PM) are related to histologically proven A‐grade rejection and/or LB and bronchoalveolar lavage (BAL) fluid cellularity after lung transplantation. We fitted a mixed model to examine the association between daily variations in PM₁₀ and A‐grade rejection/LB on 1276 bronchoscopic biopsies (397 patients, 416 transplantations) taken between 2001 and 2011. A difference of 10 μg/m³ in PM₁₀ 3 days before diagnosis of LB was associated with an OR of 1.15 (95% CI 1.04–1.27; p = 0.0044) but not with A‐grade rejection (OR = 1.05; 95% CI 0.95–1.15; p = 0.32). Variations in PM₁₀ at lag day 3 correlated with neutrophils (p = 0.013), lymphocytes (p = 0.0031) and total cell count (p = 0.024) in BAL. Importantly, we only found an effect of PM10 on LB in patients not taking azithromycin. LB predisposed to chronic rejection (p < 0.0001). The risk for LB after lung transplantation increased with temporal changes in particulate air pollution, and this was associated with BAL neutrophilia and lymphocytosis. Azithromycin was protective against this PM effect.     

Bilateral Hematogenous Pseudomonas aeruginosa Endophthalmitis After Lung Transplantation

Pseudomonas aeruginosa commonly colonizes the airways of patients with cystic fibrosis (CF). However, the occurrence of bacteremia with metastatic infection to the eye causing endogenous endophthalmitis is very rare. In the setting of lung transplantation, the significance of P. aeruginosa bacteremia in patients with CF whose airways are colonized before transplantation is unknown. We report a case of bilateral P. aeruginosa endogenous endophthalmitis in a patient with CF after lung transplant without documented bacteremia. The patient presented with acute eye symptoms in the presence of a left atrial thrombus and the disease followed a rapidly progressive course requiring aggressive medical-surgical treatment. Typically P. aeruginosa endophthalmitis has been associated with a poor visual prognosis. However, with combined medical-surgical management this patient retained useful vision in one eye without having retinal detachment or requiring enucleation. Endogenous endophthalmitis should be considered in the differential diagnosis of ocular complaints in patients with CF after lung transplant.     

Allograft Rejection of Small Bowel Transplantation in Pigs

n = 9): nonimmunosuppressed recipients; (2) group 2 (n = 8): FK506-immunosuppressed recipients; (3) group 3 (n = 2): autotransplant controls; and (4) donors (n = 17). Orthotopic small bowel transplantations were performed with Thiry-Vella loops for daily biopsies. The survival rate of group 2 was significantly longer than that of group 1 (P < 0.05). One best survivor in group 2 was killed at postoperative day (POD) 365. Treatment by FK506 prevented rejection, but most of the pigs died of pneumonia. In group 1, rejection began on POD 3 and progressed to severe rejection rapidly within 7 days. In group 2, rejection began from POD 6 to POD 8, but either remained mild or spontaneously improved. The differences in the routine laboratory data and the tumor necrosis factor-α level were not evident between the groups. Histological studies of repeated graft biopsies are thus considered to be essential for detecting signs of graft rejection. (Received for publication on Mar. 26, 1997; accepted on Jan. 6, 1998)     

Prevention of Acute Lung Allograft Rejection in Rat by CTLA4Ig

CTLA4 immunoglobulin (CTLA4Ig), which binds with a high affinity to B7-1 and B7-2, interrupts T-cell activation by inhibiting costimulatory signal. CTLA4Ig has been used in hopes of achieving antigen-specific tolerance induction in several solid organ transplants. In lung allograft rejection, however, its use has been controversial in terms of its effect on prevention of rejection. In the present study, the effect of murine CTLA4Ig on rat-lung allograft rejection was investigated. Rat left-lung transplantation was performed in an RT1 incompatible donor (Brown Norway; BN)-recipient (F344) combination. All allografts (n = 12) without any treatment were rejected within 7 days after transplantation. A single injection of murine form CTLA41g at a dose of 100 microg intraperitoneally (ip) or intravenously (iv) on day 1 post-transplantation achieved long-term graft survival (>90days) in 2/5 (40%) and 3/8 (38%), respectively. Moreover, 6/7 (86%) allografts in rats that received iv injection of 500 microg CTLA4Ig survived more than 90days. Allograft survival in the CTLA4Ig 500 microg iv recipient group was significantly longer than that in the no-treatment control or control immunoglobulin group (p <0.01). Four out of seven recipients bearing functional allografts for more than 90 days with the CTLA4Ig treatment accepted donor-specific skin grafts, whereas all third-party skin grafts (n=3) were rejected. Prevention of rat-lung allograft rejection could be achieved by intravenous administration of CTLA4Ig, resulting in long-term allograft survival with acceptance of donor-specific skin grafts.     

Efficacy of Total Lymphoid Irradiation in Azithromycin Nonresponsive Chronic Allograft Rejection After Lung Transplantation

Bronchiolitis obliterans syndrome (BOS) remains a major problem after lung transplantation. Azithromycin seems to be beneficial in some patients with established BOS. We investigated the efficacy of total lymphoid irradiation (TLI) in 6 BOS patients with a continuous decline in FEV₁, despite treatment with azithromycin for a mean of 12 ± 13 (range, 1-35) months. A historical control group consisted of 5 patients with declining FEV₁, also nonresponders to azithromycin and those not treated with TLI. All 6 TLI patients received the total dose of 8 Gy in 10 sessions. There was a significant change in the decline of the FEV₁ after TLI treatment (from 221 ± 107 to 94 ± 79 mL/mo; P = .041). Three patients died, due to BOS progression, overwhelming pneumonia, and sudden cardiac arrest, respectively, 3.5, 11, and 26 months after TLI; two patients underwent retransplantation at 6 and 19 months after TLI, respectively. The sixth patient remains stable in BOS stage 3 after a follow-up period of 24 months. In the control group, there was no significant change in FEV₁ decline (209 ± 97 mL/mo before versus 193 ± 81 mL/mo after starting azithromycin; P = not significant). Two patients remain stable in BOS stage 3, 1 died of BOS progression, and the 5th patient is scheduled for retransplantation. We conclude that patients who do not or no longer respond to azithromycin may benefit from TLI, as suggested by a decreased rate in decline of the FEV₁.     

Pulmonary Hypertension Associated with Lung Transplantation Obliterative Bronchiolitis and Vascular Remodeling of the Allograft

Pathologic obliterative bronchiolitis (OB)/Bronchiolitis obliterans syndrome (pathologic OB/BOS) is the major obstacle to long-term survival post-lung transplantation (LT). Our group has demonstrated that pulmonary hypertension (PH) complicates the course of chronic inflammatory lung diseases that have similarities to pathologic OB/BOS and that vascular remodeling of the bronchial circulation occurs during BOS. Consequently, we hypothesized that PH is associated with pathologic OB/BOS and may result from a vasculopathy of the allograft pulmonary circulation.
We conducted a single-center, retrospective study and examined the presence of PH and vasculopathy in patients with pathologic OB/BOS. Fifty-two pathologic specimens post-LT were recovered from January 10, 1997 to January 5, 2007 and divided into two groups, those with and without pathologic OB/BOS.PH was defined as a mean pulmonary artery pressure (mPAP) > 25 mmHg by right heart catheterization (RHC) or right ventricular systolic pressure (RVSP) ≥45 mmHg by transthoracic echocardiogram (TTE).
PH was more prevalent in those LT recipients with pathologic OB/BOS (72% vs. 0%, p = 0.003). Furthermore, pulmonary arteriopathy and venopathy were more prevalent in patients with pathologic OB/BOS (84% vs. 4%, p < 0.0001, and 77% vs. 35%, p = 0.004, respectively).
PH is common in LT recipients with pathologic OB/BOS and is associated with a vasculopathy of the allograft pulmonary circulation.     

Translational Research: Animal Models of Obliterative Bronchiolitis after Lung Transplantation

Obliterative bronchiolitis (OB) or chronic graft dysfunction remains the major limitation to long-term success of lung transplantation. Investigation using animal models is a critical component of research to understand the underlying pathological mechanisms and to develop novel preventive and therapeutic strategies for OB. Multiple animal models of OB exist, including orthotopic lung transplantation in rodents and large animals, orthotopic tracheal transplantation and heterotopic transplantation of a trachea in variable sites such as subcutaneous, intraomental and intrapulmonary sites. The most important issue for researchers is not specifically which model is the best but which is the most appropriate model to test their scientific hypothesis. For example, while orthotopic lung transplantation best mimics the overall surgical procedure, a question regarding fibrotic processes of OB may be better answered using heterotopic tracheal transplant models because of their reliable reproducibility of allograft obliterative airway fibrosis. Animal models should be continuously refined, modified and sometimes combined to fit the particular research purpose. We review the available animal models, their modifications and possible applications to assist researchers in choosing the appropriate model for their intended research.     

Caspase Inhibition Improves Ischemia-Reperfusion Injury After Lung Transplantation

Ischemia-reperfusion injury is associated with cell death in many organ systems. The role of programmed cell death (PCD) pathways and the ultimate clinical relevance of PCD in the context of lung transplantation (LTx) are unknown. In randomized and blinded studies, rat single LTx was performed in the presence of caspase inhibitors after 'short' (6 h) and 'long' (18 h) periods of cold ischemic storage. Lung function, electron microscopic morphology, caspase 3, 8 and 9 activities and TUNEL assays were evaluated. Endothelial cells and lymphocytes were observed undergoing apoptotic cell death with electron microscopy. Caspase activities were significantly up-regulated immediately after the initial flush and increased further during short periods of cold ischemic storage. A significant amount of apoptotic cell death was observed after LTx and reperfusion. Caspase inhibition virtually eliminated apoptotic cell death and led to improved lung function after LTx and reperfusion. Activation of caspases during cold ischemia contributes significantly to cell death in LTx. Suppression of caspase activity appears to decrease apoptosis and improve lung function. Clearly, this needs to be investigated further with more experiments to validate the potential role of caspase inhibition as a therapeutic modality in ischemia-reperfusion-induced lung injury.     

Alloantibody Levels and Acute Humoral Rejection Early After Positive Crossmatch Kidney Transplantation

We examined the course of donor ‐ specific alloantibody (DSA) levels early after transplant and their relationship with acute humoral rejection (AHR) in two groups of positive crossmatch (+XM) kidney transplant recipients: High DSA group—41 recipients with a baseline T‐ or B‐cell flow crossmatch (TFXM, BFXM) channel shift ≥300 (molecules of equivalent soluble fluorochrome units (MESF) of approximately 19 300) who underwent pretransplant plasmapheresis (PP), and Low DSA group—29 recipients with a baseline channel shift <300 who did not undergo PP. The incidence of AHR was 39% (16/41) in the High DSA group and 31% (9/29) in the Low DSA group. Overall, mean DSA levels decreased by day 4 posttransplant and remained low in patients who did not develop AHR. By day 10, DSA levels increased in patients developing AHR with 92% (23/25) of patients with a BFXM >359 (MESF of approximately 34 000) developing AHR. The BFXM and the total DSA measured by single antigen beads correlated well across a wide spectrum suggesting that either could be used for monitoring. We conclude that AHR is associated with the development of High DSA levels posttransplant and protocols aimed at maintaining DSA at lower levels may decrease the incidence of AHR.     

Minimal Acute Rejection after Lung Transplantation: A Risk for Bronchiolitis Obliterans Syndrome

Bronchiolitis obliterans syndrome (BOS) is a major cause of lung allograft dysfunction. Although previous studies have identified mild to severe rejection (grade>or=A2) as a risk factor for BOS, the role of minimal rejection (grade A1) remains unclear. To determine if A1 rejection by itself is a risk factor for BOS, we performed a retrospective cohort study on 228 adult lung transplant recipients over a 7-year period. Cohorts were defined by their most severe rejection episode (none, A1 only, and >or=A2) and analyzed for the subsequent development and progression of BOS using univariate and multivariate time-dependent Cox regression analysis. In the univariate model, the occurrence of isolated minimal rejection was a risk factor for all stages of BOS. Similarly, multivariate models that included HLA mismatch, cytomegalovirus pneumonitis, community acquired viral infection, underlying disease and type of transplant demonstrated that A1 rejection was a distinct risk factor for BOS. Furthermore, the associated risk with A1 rejection was slightly greater than the risk from >or=A2 and treatment of A1 rejection decreased the risk for subsequent BOS stage 1. We conclude that minimal rejection is associated with an increased risk for BOS development and progression that is comparable to A2 rejection.     

Effect of Continuous Positive Airway Pressure and Upper Airway Surgery on Exhaled Breath Condensate and Serum Biomarkers in Patients With Sleep Apnea

IntroductionStudies on inflammation biomarkers in serum and in exhaled breath condensate (EBC) in obstructive sleep apnea (OSA) have shown conflicting results. The objective of this study is to assess EBC and serum biomarkers in OSA patients at baseline and after continuous positive airway pressure (CPAP) or upper airway surgery (UAS).Patients and methodsNine OSA patients referred for UAS were matched for anthropometric characteristics and apnea–hypopnea index with 20 patients receiving CPAP. pH, nitrite (NO₂⁻), nitrate, and interleukin 6 in EBC and NO₂⁻, nitrate, leukotriene B₄, and interleukin 6 in serum were determined. EBC and serum samples were collected at baseline and 3 months after CPAP or UAS.ResultsPatients’ mean body mass index was 30 (range 24.9–40) kg/m². EBC biomarker levels at baseline were within normal range and did not differ significantly after CPAP or UAS. No significant changes were observed in the serum concentration of the biomarkers determined after CPAP but the serum concentration of NO₂⁻ increased significantly at 3 months after UAS (P=.0078).ConclusionIn mildly obese OSA patients, EBC biomarkers of inflammation or oxidative stress were normal at baseline and remained unchanged 3 months after UAS or CPAP. Although UAS was not effective in terms of reducing OSA severity, it was associated with an increase in serum NO₂⁻.     

Usefulness of Exhaled Nitric Oxide to Guide Risk Stratification for Bronchiolitis Obliterans Syndrome After Lung Transplantation

The aim of this study was to assess fractional exhaled nitric oxide (FeNO) for the early diagnosis of bronchiolitis obliterans syndrome (BOS) after lung transplantation (LTX). 611 FeNO measurements in 166 consecutive patients were classified depending on BOS stage at the time of assessment and course during minimum follow‐up of 3 months: (1) stable non‐BOS, (2) unstable non‐BOS, (3) stable BOS and (4) unstable BOS. Unstable course was defined as new onset of BOS≥1 or progression of BOS. FeNO before unstable course was significantly increased in comparison to their stable counterparts (non‐BOS: 28.9 ± 1.2 ppb, n = 40 vs. 16.4 ± 0.8 ppb, n = 131 and BOS: 32.5 ± 1.3 ppb, n = 35 vs. 15.3 ± 0.8 ppb, n = 26; p = 0.01 each). Average time from FeNO reading to onset of deterioration was 117 ± 9 days in non‐BOS and 136 ± 9 days in BOS patients. The positive and negative predictive value of FeNO >20 ppb for BOS was 69.0% and 96.9%, respectively. Serial measurements demonstrated significantly lower mean individual variation in stable recipients as compared to stable patients switching to unstable course (3.2 ± 0.3 ppb vs. 12.7 ± 1.4 ppb, p = 0.02). In particular, the excellent negative predictive value of persistently low FeNO readings for future BOS make FeNO assessments a useful tool for continuous risk stratification after LTX.