Samuel A. Yousem

Obliterative bronchiolitis after lung and heart-lung transplantation: An analysis of risk factors and management

With a prevalence of 34% (55/162 at-risk recipients) and a mortality of 25% (14/55 affected recipients), obliterative bronchiolitis is the most significant long-term complication after pulmonary transplantation. Because of its importance, we examined donor-recipient characteristics and antecedent clinical events to identify factors associated with development of obliterative bronchiolitis, which might be eliminated or modified to decrease its prevalence. We also compared treatment outcome between recipients whose diagnosis was made early by surveillance transbronchial lung biopsy before symptoms or decline in pulmonary function were present versus recipients whose diagnosis was made later when symptoms or declines in pulmonary function were present. Postoperative airway ischemia, an episode of moderate or severe acute rejection (grade III/IV), three or more episodes of histologic grade II (or greater) acute rejection, and cytomegalovirus disease were risk factors for development of obliterative bronchiolitis. Recipients with obliterative bronchiolitis detected in the preclinical stage were significantly more likely to be in remission than recipients who had clinical disease at the time of diagnosis: 81% (13/15) versus 33% (13/40); p < 0.05). These results indicate that acute rejection is the most significant risk factor for development of obliterative bronchiolitis and that obliterative bronchiolitis responds to treatment with augmented immunosuppression when it is detected early by surveillance transbronchial biopsy.

Inhibition and Role of let-7d in Idiopathic Pulmonary Fibrosis

RATIONALE Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and usually lethal fibrotic lung disease characterized by profound changes in epithelial cell phenotype and fibroblast proliferation. OBJECTIVES To determine changes in expression and role of microRNAs in IPF. METHODS RNA from 10 control and 10 IPF tissues was hybridized on Agilent microRNA microarrays and results were confirmed by quantitative real-time polymerase chain reaction and in situ hybridization. SMAD3 binding to the let-7d promoter was confirmed by chromatin immunoprecipitation, electrophoretic mobility shift assay, luciferase assays, and reduced expression of let-7d in response to transforming growth factor-beta. HMGA2, a let-7d target, was localized by immunohistochemistry. In mice, let-7d was inhibited by intratracheal administration of a let-7d antagomir and its effects were determined by immunohistochemistry, immunofluorescence, quantitative real-time polymerase chain reaction, and morphometry. MEASUREMENTS AND MAIN RESULTS Eighteen microRNAs including let-7d were significantly decreased in IPF. Transforming growth factor-beta down-regulated let-7d expression, and SMAD3 binding to the let-7d promoter was demonstrated. Inhibition of let-7d caused increases in mesenchymal markers N-cadherin-2, vimentin, and alpha-smooth muscle actin (ACTA2) as well as HMGA2 in multiple epithelial cell lines. let-7d was significantly reduced in IPF lungs and the number of epithelial cells expressing let-7d correlated with pulmonary functions. HMGA2 was increased in alveolar epithelial cells of IPF lungs. let-7d inhibition in vivo caused alveolar septal thickening and increases in collagen, ACTA2, and S100A4 expression in SFTPC (pulmonary-associated surfactant protein C) expressing alveolar epithelial cells. CONCLUSIONS Our results indicate a role for microRNAs in IPF. The down-regulation of let-7d in IPF and the profibrotic effects of this down-regulation in vitro and in vivo suggest a key regulatory role for this microRNA in preventing lung fibrosis. Clinical trial registered with www.clinicaltrials.gov (NCT 00258544).

Egr-1 Regulates Autophagy in Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease

Background Chronic obstructive pulmonary disease (COPD) is a progressive lung disease characterized by abnormal cellular responses to cigarette smoke, resulting in tissue destruction and airflow limitation. Autophagy is a degradative process involving lysosomal turnover of cellular components, though its role in human diseases remains unclear. Methodology and Principal Findings Increased autophagy was observed in lung tissue from COPD patients, as indicated by electron microscopic analysis, as well as by increased activation of autophagic proteins (microtubule-associated protein-1 light chain-3B, LC3B, Atg4, Atg5/12, Atg7). Cigarette smoke extract (CSE) is an established model for studying the effects of cigarette smoke exposure in vitro. In human pulmonary epithelial cells, exposure to CSE or histone deacetylase (HDAC) inhibitor rapidly induced autophagy. CSE decreased HDAC activity, resulting in increased binding of early growth response-1 (Egr-1) and E2F factors to the autophagy gene LC3B promoter, and increased LC3B expression. Knockdown of E2F-4 or Egr-1 inhibited CSE-induced LC3B expression. Knockdown of Egr-1 also inhibited the expression of Atg4B, a critical factor for LC3B conversion. Inhibition of autophagy by LC3B-knockdown protected epithelial cells from CSE-induced apoptosis. Egr-1 −/− mice, which displayed basal airspace enlargement, resisted cigarette-smoke induced autophagy, apoptosis, and emphysema. Conclusions We demonstrate a critical role for Egr-1 in promoting autophagy and apoptosis in response to cigarette smoke exposure in vitro and in vivo. The induction of autophagy at early stages of COPD progression suggests novel therapeutic targets for the treatment of cigarette smoke induced lung injury.

Respiratory bronchiolitis-associated interstitial lung disease and its relationship to desquamative interstitial pneumonia

Respiratory bronchiolitis is a mild inflammatory reaction commonly noted in asymptomatic cigarette smokers. We reviewed 18 cases of respiratory bronchiolitis-associated interstitial lung disease (RB/ILD), which had been diagnosed on the basis of clinical evaluation and open-lung biopsy. All patients were cigarette smokers. The sex distribution of the patients was approximately equal, and their mean age was 36 years. Chest roentgenograms showed reticular or reticulonodular infiltrates in 72% of the patients. Histologically, inflammation of the respiratory bronchioles, filling of the bronchiolar lumens and surrounding alveoli with finely pigmented macrophages, associated interstitial inflammation, and mild fibrosis were noted. In most patients, respiratory improvement ensued when they stopped smoking. Because of histologic similarities to desquamative interstitial pneumonia (DIP), the 18 cases of RB/ILD were compared with 36 cases of DIP. DIP tended to occur in older persons, caused more severe symptoms, displayed ground glass infiltrates on chest roentgenograms, was characterized by more severe interstitial disease on pulmonary function tests, and was often associated with progressive respiratory disease.

Gene Expression Profiles of Acute Exacerbations of Idiopathic Pulmonary Fibrosis

RATIONALE The molecular mechanisms underlying acute exacerbations of idiopathic pulmonary fibrosis (IPF) are poorly understood. We studied the global gene expression signature of acute exacerbations of IPF. OBJECTIVES To understand the gene expression patterns of acute exacerbations of IPF. METHODS RNA was extracted from 23 stable IPF lungs, 8 IPF lungs with acute exacerbation (IPF-AEx), and 15 control lungs and used for hybridization on Agilent gene expression microarrays. Functional analysis of genes was performed with Spotfire and Genomica. Gene validations for MMP1, MMP7, AGER, DEFA1-3, COL1A2, and CCNA2 were performed by real-time quantitative reverse transcription-polymerase chain reaction. Immunohistochemistry and in situ terminal deoxynucleotidyltransferase dUTP nick end-labeling assays were performed on the same tissues used for the microarray. ELISA for alpha-defensins was performed on plasma from control subjects, patients with stable IPF, and patients with IPF-AEx. MEASUREMENTS AND MAIN RESULTS Gene expression patterns in IPF-AEx and IPF samples were similar for the genes that distinguish IPF from control lungs. Five hundred and seventy-nine genes were differentially expressed (false discovery rate < 5%) between stable IPF and IPF-AEx. Functional analysis of these genes did not indicate any evidence of an infectious or overwhelming inflammatory etiology. CCNA2 and alpha-defensins were among the most up-regulated genes. CCNA2 and alpha-defensin protein levels were also higher and localized to the epithelium of IPF-AEx, where widespread apoptosis was also detected. alpha-Defensin protein levels were increased in the peripheral blood of patients with IPF-AEx. CONCLUSIONS Our results indicate that IPF-AEx is characterized by enhanced epithelial injury and proliferation, as reflected by increases in CCNA2 and alpha-defensins and apoptosis of epithelium. The concomitant increase in alpha-defensins in the peripheral blood and lungs may suggest their use as biomarkers for this disorder.

Cytomegalovirus serologic status and postoperative infection correlated with risk of developing chronic rejection after pulmonary transplantation.

Twenty-seven patients received pulmonary transplants during the period since we began routine use of cytomegalovirus-seronegative blood products for CMV-seronegative recipients. Preoperative serologic status of the recipient and the occurrence of cytomegalovirus infection in the postoperative period were correlated with development of obliterative bronchiolitis (OB) as diagnosed by transbronchial biopsy (TBB). Patients included 20 heart-lung and 7 double-lung recipients. OB occurred in 18 of 27 patients. All 3 CMV seronegative recipients receiving lungs from a seropositive donor and 9 of 10 CMV recipients seropositive at the time of transplantation developed OB compared with only 6 of 14 CMV seronegative patients receiving seronegative grafts (P = 0.018). CMV infection occurred in 10/27 patients, of whom 5 were asymptomatic; 90% of these patients developed OB. Donor-specific alloreactivity, based on primed lymphocyte testing (PLT) of bronchoalveolar lavage cells was found at the time of diagnosis of OB in 23 of 27 patients. A positive PLT was significantly associated with the presence of OB (P = 0.017). We conclude that preoperative seropositive status for CMV, grafting of organs from seropositive donors, and postoperative CMV infection are significant risk factors for developing OB. That OB is, in part, an immunologically mediated form of injury and represents chronic rejection is supported by the presence of donor-specific alloreactivity in BAL lymphocytes from all recipients with OB.

Follicular bronchitis/bronchiolitis

Nineteen open lung biopsies demonstrating follicular bronchitis/bronchiolitis were reviewed with special attention to clinical manifestations. Morphologically, follicular bronchitis/bronchiolitis was represented by coalescent reactive germinal centers adjacent to airways in the absence of clinical or pathologic evidence of chronic obstructive pulmonary disease or bronchiectasis. Three clinicopathologic groups were identified: 1) patients with collagen vascular diseases, especially rheumatoid arthritis and Sjögrens syndrome; 2) patients with a familial form of the disease or with immunodeficiency syndromes; and 3) a heterogeneous group of patients with frequent peripheral blood eosinophilia, suggesting a hypersensitivity reaction. Prognosis was related to age at the time of biopsy and, to some extent, to the clinical group. Steroid therapy had inconsistent effects in all groups identified. The differential diagnosis of lymphoid lesions in the lung is also discussed.

The pulmonary pathologic manifestations of the CREST syndrome

This report evaluates the histopathologic alterations of a series of 17 patients with the CREST syndrome and relates these alterations to clinical and functional abnormalities. Histologic abnormalities were classified into the following four distinct patterns: pulmonary vascular changes, primarily intimal fibroelastosis, associated with and without pulmonary hypertension; a pattern of fibrosis indistinguishable from usual interstitital pneumonia (UIP); small airways disease; and mixtures of these three patterns. Five patients (29%) had clinical and morphologic pulmonary hypertension, while five others showed mild reductions in diffusing capacity, presumably due to vascular compromise. Five patients had UIP-like interstitial fibrosis, with vascular alterations and restrictive lung disease. Only one patient had small airways disease exclusively. Concentric fibrointimal proliferation and occlusion of arterioles was worse in patients with clinical pulmonary hypertension and interstitial fibrosis of the UIP type, and was not always associated with pulmonary fibrosis. Twenty-one percent of patients developed primary lung carcinomas. The CREST syndrome is unique in the spectrum of pulmonary alterations seen in progressive systemic sclerosis for its high incidence of clinical pulmonary hypertension and propensity for the development of pulmonary carcinomas.

Pathologic pulmonary alterations in long-term human heart-lung transplantation

Twenty-one patients with end-stage pulmonary hypertension underwent combined allograft heart-lung transplantation after 1980. Almost 80 per cent of these patients survived beyond the immediate postoperative period, with the longest survival period more than 3 1/2 years at the time of this report. Five patients died in the perioperative or immediate postoperative period, and 11 returned to normal lives with essentially normal pulmonary function. In the remaining five allograft recipients recurrent respiratory infections and progressive obstructive airway disease developed, with superimposed restrictive deficits in three of them. Two open lung biopsies, two autopsies, and one retransplantation were performed in these recipients. Morphologically, these allograft recipients showed extensive bronchiolitis obliterans, interstitial and pleural fibrosis, and accelerated arterial and venous arteriosclerosis. Bronchiolitis obliterans may prove to be a significant complication of heart-lung transplantation.

HLA-specific antibodies are risk factors for lymphocytic bronchiolitis and chronic lung allograft dysfunction

Bronchiolitis obliterans syndrome (BOS) represents a major limitation in lung transplantation. While acute rejection is widely considered the most important risk factor for BOS, the impact of HLA‐specific antibodies is less understood. Of 51 lung recipients who were prospectively tested during a 4.2 ± 1.6‐year period, 14 patients developed HLA‐specific antibodies. A multi‐factorial analysis was performed to correlate the prevalence of BOS with HLA antibodies, persistent‐recurrent acute rejection (ACR‐PR), lymphocytic bronchiolitis, and HLA‐A, ‐B, and ‐DR mismatches. HLA‐specific antibodies were associated with ACR‐PR (10/14 vs. 11/37 with no antibodies, p < 0.05), lymphocytic bronchiolitis (8/14 vs. 10/37, p < 0.05), and BOS (10/14, vs. 9/37, p < 0.005). Other risk factors for BOS were: lymphocytic bronchiolitis (13/18 vs. 6/33 with no lymphocytic bronchiolitis, p < 0.0001), ACR‐PR (12/21 vs. 7/30 with no ACR‐PR, p < 0.05), and the number of HLA‐DR mismatches (1.7 ± 0.48 in BOS vs. 1.2 ± 0.63 without BOS, p < 0.05). The presence of antibodies exhibited a cumulative effect on BOS when it was associated with either lymphocytic bronchiolitis or ACR‐PR. The complex relationship between the development of HLA antibodies and acute and chronic lung allograft rejection determines the importance of post‐transplant screening for HLA‐specific antibodies as a prognostic element for lung allograft outcome.