James H. Dauber

MMP1 and MMP7 as Potential Peripheral Blood Biomarkers in Idiopathic Pulmonary Fibrosis

Background Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease associated with substantial morbidity and mortality. The objective of this study was to determine whether there is a peripheral blood protein signature in IPF and whether components of this signature may serve as biomarkers for disease presence and progression. Methods and Findings We analyzed the concentrations of 49 proteins in the plasma of 74 patients with IPF and in the plasma of 53 control individuals. We identified a combinatorial signature of five proteins—MMP7, MMP1, MMP8, IGFBP1, and TNFRSF1A—that was sufficient to distinguish patients from controls with a sensitivity of 98.6% (95% confidence interval [CI] 92.7%–100%) and specificity of 98.1% (95% CI 89.9%–100%). Increases in MMP1 and MMP7 were also observed in lung tissue and bronchoalveolar lavage fluid obtained from IPF patients. MMP7 and MMP1 plasma concentrations were not increased in patients with chronic obstructive pulmonary disease or sarcoidosis and distinguished IPF compared to subacute/chronic hypersensitivity pneumonitis, a disease that may mimic IPF, with a sensitivity of 96.3% (95% CI 81.0%–100%) and specificity of 87.2% (95% CI 72.6%–95.7%). We verified our results in an independent validation cohort composed of patients with IPF, familial pulmonary fibrosis, subclinical interstitial lung disease (ILD), as well as with control individuals. MMP7 and MMP1 concentrations were significantly higher in IPF patients compared to controls in this cohort. Furthermore, MMP7 concentrations were elevated in patients with subclinical ILD and negatively correlated with percent predicted forced vital capacity (FVC%) and percent predicted carbon monoxide diffusing capacity (DLCO%). Conclusions Our experiments provide the first evidence for a peripheral blood protein signature in IPF to our knowledge. The two main components of this signature, MMP7 and MMP1, are overexpressed in the lung microenvironment and distinguish IPF from other chronic lung diseases. Additionally, increased MMP7 concentration may be indicative of asymptomatic ILD and reflect disease progression.

Carbon Monoxide Induces Cytoprotection in Rat Orthotopic Lung Transplantation via Anti-Inflammatory and Anti-Apoptotic Effects

Successful lung transplantation has been limited by the high incidence of acute graft rejection. There is mounting evidence that the stress response gene heme oxygenase-1 (HO-1) and/or its catalytic by-product carbon monoxide (CO) confers cytoprotection against tissue and cellular injury. This led us to hypothesize that CO may protect against lung transplant rejection via its anti-inflammatory and antiapoptotic effects. Orthotopic left lung transplantation was performed in Lewis rat recipients from Brown-Norway rat donors. HO-1 mRNA and protein expression were markedly induced in transplanted rat lungs compared to sham-operated control lungs. Transplanted lungs developed severe intraalveolar hemorrhage, marked infiltration of inflammatory cells, and intravascular coagulation. However, in the presence of CO exposure (500 ppm), the gross anatomy and histology of transplanted lungs showed marked preservation. Furthermore, transplanted lungs displayed increased apoptotic cell death compared with the transplanted lungs of CO-exposed recipients, as assessed by TUNEL and caspase-3 immunostaining. CO exposure inhibited the induction of IL-6 mRNA and protein expression in lung and serum, respectively. Gene array analysis revealed that CO also down-regulated other proinflammatory genes, including MIP-1alpha and MIF, and growth factors such as platelet-derived growth factor, which were up-regulated by transplantation. These data suggest that the anti-inflammatory and antiapoptotic properties of CO confer potent cytoprotection in a rat model of lung transplantation.

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.

Immunologic Abnormalities in Sarcoidosis

Patients with active sarcoidosis (acute and chronic) have a depression in systemic cell-mediated immunity manifested by a reduction in the number of circulating T cells and impaired responses of these cells to polyclonal mitogens and recall antigens. These abnormalities are absent in patients with resolved disease and contrast with heightened B-cell activity. The latter includes elevated serum immunoglobulins and the presence of autoantibodies and circulating immune complexes. Similarly, many humoral abnormalities (for example, immune complexes) are absent in patients with resolved disease. Studies of bronchoalveolar cells have revealed changes that are opposite to what is found in peripheral blood. The number of lymphocytes recovered by bronchoalveolar lavage is increased. This is mainly due to an increase in the number of T cells and a subpopulation of activated T cells. These findings suggest that the lung (when involved) is the site of an immune inflammatory response.

Identification, cellular localization, isolation, and characterization of human Clara cell-specific 10 KD protein.

Human lung lavage proteins were fractionated by centrifugation and molecular sieving. An antiserum to the post-albumin fraction of the soluble proteins reacted with a 10 KD protein and this protein was isolated by conventional chromatography. The protein, which has a pI of 4.8, consists of two 5 KD polypeptides and is rich in glutamic acid, leucine, serine, and aspartic acid amino acids. The protein does not bind to concanavalin A, pancreatic elastase, leukocyte elastase, or trypsin, and lacks anti-protease activity. It constitutes about 0.15% of the soluble proteins in lung lavage. Antibodies to the 10 KD protein specifically and exclusively stain Clara cells in human, dog, and rat. Staining of granules of Clara cells was prominent in the distal bronchioles; however, the non-ciliated cells of respiratory bronchioles did not stain for the 10 KD protein. This 10 KD protein appears in fetal lungs at 21 weeks of gestation, and was present in about 10% of the primary pulmonary adenocarcinomas. As a specific marker for Clara cells, this protein could be useful in the study of development, regulation of secretion, and pathobiology of these cells.

Heart-Lung Transplantation: Lessons Learned and Future Hopes

Since March, 1982, 33 patients have undergone cardiopulmonary transplantation. Nineteen were discharged from the hospital following the operation, and 16 continue to do well. Eight patients have survived 1 year, 5 patients 2 years, and 1 patient 3 years. Often survival has been influenced most by the selection of candidates, as no patient who had undergone a previous sternotomy survived (3 of 3). All 7 early (between 30 and 72 days) and 3 late (145 to 466 days) deaths were related to infection. Methods for ex vivo preservation of the heart-lung bloc have included storage at 4 degrees C, cardiopulmonary bypass and profound hypothermia, and autoperfusion of the heart-lung bloc. The last technique is original and currently is preferred for distant procurement. Because dehiscence of the tracheal anastomosis has occurred in 3 patients, a sutured line is now encircled with a wrap of omentum. Isolated rejection of the lung is frequent in the first three weeks following operation and has been controlled with methylprednisolone. Late survivors have shown a mild restrictive lung disorder that has not progressed between 6 and 24 months. Bronchoalveolar lavage has been useful for diagnosing infection and providing insight into the immunobiology of the transplanted lung. Although mortality and morbidity have been high, the experiences gained through this series will likely result in an improved outlook for future recipients.

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.

Immunologically mediated disease of the airways after pulmonary transplantation

Obliterative bronchiolitis has occurred in eleven of 30 recipients of cardiopulmonary allografts who survived at least 4 months after transplantation, has caused significant morbidity, and has been associated with four of eleven late deaths in this series. Although some improvement, or at least stability, of pulmonary function has followed augmented immune suppression, it appears that once the process is recognized clinically, much of the damage to the airways is irreversible. The histopathology, response to therapy, and, most important, the response of donor specific alloreactivity in the lymphocytes from the lung (bronchoalveolar lavage and peripheral blood) suggest immune- mediated basis for bronchiolitis obliterans. The presence of donor specific alloreactivity detected by primed lymphocyte testing predicted obliterative bronchiolitis in five of six recipients (83% sensitivity, 91% specificity) was absent in ten of eleven recipients who have not as yet developed the process (negative predicted value of 91%). Currently, the presence of a positive primed lymphocyte test in the bronchoalveolar lavage of the cardiopulmonary recipient is an indication for early treatment by augmented immune suppression.

Bronchoalveolar lavage in a patient with chronic berylliosis: evidence for hypersensitivity pneumonitis.

Because immunologic mechanisms are thought to have a role in chronic berylliosis, we studied the immunologic properties of peripheral blood and bronchoalveolar lymphocytes in a patient with this disease. Although the percent of peripheral blood T cells was slightly reduced compared with that in controls (59% versus 70%), there was a large increase in the number and proportion (94% versus 62%) of bronchoalveolar T cells. The percent of activated bronchoalveolar T cells was nearly five times the control value (64.5% versus 14%). Both peripheral blood and bronchoalveolar lymphocytes proliferated in response to BeSO4 and BeF2 exposure but the response of bronchoalveolar lymphocytes was greater than comparable numbers of peripheral blood lymphocytes. These findings are consistent with the concept that chronic berylliosis is a form of hypersensitivity pneumonitis and that bronchoalveolar lavage may be helpful in establishing the diagnosis of this disease.

Treatment of refractory acute allograft rejection with aerosolized cyclosporine in lung transplant recipients

Lung transplant recipients who have persistent acute cellular rejection are at increased risk for the development of chronic rejection, the leading cause of reduced long-term survival. This study evaluated the use of aerosolized cyclosporine as rescue therapy for unremitting acute rejection. Between June 1993 and March 1996, 18 patients with rejection that failed to resolve after therapy with pulse steroids and antilymphocyte globulin were enrolled in the study. Aerosolized cyclosporine A (300 mg) treatment was initiated for 10 consecutive days followed by a maintenance regimen of 3 days per week. Efficacy was assessed by graft histologic and pulmonary function testing. With the use of linear regression, results in these patients were compared with those in 23 control patients, matched for histologic acute rejection, who had continued to receive conventional rescue therapy. Two patients were unable to tolerate the treatments and were withdrawn from the study. Significant improvement in histologic rejection occurred in 14 of the remaining 16 patients after a mean of 37 days of aerosolized cyclosporine therapy. Measures of forced vital capacity and forced expiratory volume in 1 second (change in percent predicted/100 days plus or minus the standard error) increased over time in the treated patients whereas the condition of control patients declined despite repeated attempts at conventional rescue (forced vital capacity, aerosolized cyclosporine group, 4.6 +/- 2.9 vs control group -8.1 +/- 1.9, p = 0.001; forced expiratory volume in 1 second, aerosolized cyclosporine group, 2.1 +/- 4.4 vs control group -9.8 +/- 2.6, p = 0.043). Renal and hepatic toxicity during cyclosporine therapy was not observed. The incidence of acute histologic rejection (> or = A2) decreased from 2.49 +/- 0.68 episodes/100 days before aerosolized cyclosporine therapy to 0.72 +/- 0.3 episodes/100 days (p < 0.05). In summary, aerosolized cyclosporine is a safe and effective therapy for acute rejection that has failed to improve with conventional treatment.