The majority of patients who develop bronchiolitis obliterans, after lung transplantation, die within 2-3 yrs after onset since treatment with conventional immunosuppression is typically ineffective. A case/control study was conducted in lung transplant recipients with biopsy-documented bronchiolitis obliterans to determine whether aerosol cyclosporin use contributed to increased survival.The cases comprised 39 transplant recipients who received open-label aerosol cyclosporin treatment in addition to conventional immunosuppression. The controls were transplant recipients treated with conventional immunosuppression alone. There were 51 controls from the University of Pittsburgh Medical Center and 100 from a large multicentric database (Novartis Lung Transplant Database).Forced expiratory volume in one second expressed as a percentage of the predicted value was an independent predictor of survival in all patients with bronchiolitis obliterans. Cox proportional-hazards analysis revealed a survival advantage for aerosol cyclosporin cases compared to the Pittsburgh control group. A survival advantage was also seen when comparing study cases to multicentric controls.Aerosol cyclosporin, given with conventional immunosuppression to lung transplant recipients with bronchiolitis obliterans, provides a survival advantage over conventional therapy alone.

BACKGROUNDConventional regimens of immunosuppressive drugs often do not prevent chronic rejection after lung transplantation. Topical delivery of cyclosporine in addition to conventional systemic immunosuppression might help prevent acute and chronic rejection events.METHODSWe conducted a single-center, randomized, double-blind, placebo-controlled trial of inhaled cyclosporine initiated within six weeks after transplantation and given in addition to systemic immunosuppression. A total of 58 patients were randomly assigned to inhale either 300 mg of aerosol cyclosporine (28 patients) or aerosol placebo (30 patients) three days a week for the first two years after transplantation. The primary end point was the rate of histologic acute rejection.RESULTSThe rates of acute rejection of grade 2 or higher were similar in the cyclosporine and placebo groups: 0.44 episode (95 percent confidence interval, 0.31 to 0.62) vs. 0.46 episode (95 percent confidence interval, 0.33 to 0.64) per patient per year, respectively (P=0.87 by Poisson regression). Survival was improved with aerosolized cyclosporine, with 3 deaths among patients receiving cyclosporine and 14 deaths among patients receiving placebo (relative risk of death, 0.20; 95 percent confidence interval, 0.06 to 0.70; P=0.01). Chronic rejection-free survival also improved with cyclosporine, as determined by spirometric analysis (10 events in the cyclosporine group and 20 events in the placebo group; relative risk of chronic rejection, 0.38; 95 percent confidence interval, 0.18 to 0.82; P=0.01) and histologic analysis (6 vs. 19 events, respectively; relative risk, 0.27; 95 percent confidence interval, 0.11 to 0.67; P=0.005). The risks of nephrotoxic effects and opportunistic infection were similar for patients in the cyclosporine group and the placebo group.CONCLUSIONSInhaled cyclosporine did not improve the rate of acute rejection, but it did improve survival and extend periods of chronic rejection-free survival. (ClinicalTrials.gov number, NCT00268515.).

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are widely used antilipidemic agents that are also immunomodulatory. We evaluated possible effects of these agents after lung transplantation by comparing outcomes of 39 allograft recipients, who were prescribed statins for hyperlipidemia, with those of 161 contemporaneous control recipients who did not receive these drugs. Acute rejection (>or= Grade II) was less frequently found in the statin group (15.1 versus 25.6% of biopsies, p < 0.01). None of 15 recipients started on statins during postoperative Year 1 developed obliterative bronchiolitis, whereas the cumulative incidence of this complication among control subjects was 37% (p < 0.01). Total cellularity, as well as proportions of inflammatory neutrophils and lymphocytes, were significantly lower in bronchoalveolar lavages of statin recipients. Among double lung recipients, those taking statins had significantly better spirometry: FVC (80 +/- 2 versus 70 +/- 1%) and FEV1 (87 +/- 2 versus 70 +/- 1%), as percentages of predicted values, and absolute FEV1/FVC (83.4 +/- 1.2 versus 78.6 +/- 0.5) (all p < 0.01). The 6-year survival of recipients taking statins (91%) was much greater than that of control subjects (54%) (p < 0.01). These data suggest statin use may have substantial clinical benefits after pulmonary transplantation.

INTRODUCTIONBronchoscopy with transbronchial biopsy (TBBx) and bronchoalveolar lavage is useful and safe for diagnosing acute rejection and infection in lung transplant recipients. However, its role is less well defined in determining the etiology of allograft dysfunction in the setting of respiratory failure necessitating mechanical ventilation.METHODSWe retrospectively identified 41 mechanically ventilated patients with respiratory failure in whom 42 TBBx were followed within a 10 day period by surgical lung biopsy (SLBx) to determine the sensitivity, specificity, and positive and negative predictive values of TBBx compared with SLBx.RESULTSThe sensitivity, specificity, and positive and negative predictive values of TBBx for all episodes of acute rejection and for significant episodes of acute cellular rejection were 53.3% and 36.0%; 91.7% and 94.1%; 94.1% and 90.0%; 44.0% and 50.0%, respectively. A significantly higher histologic grade was noted on SLBx compared with TBBx specimens obtained within a 10-day period (2.39 +/- 1.02 vs 0.97 +/- 0.11, p <or= 0.0001). Performing SLBx in this setting increased histopathologic diagnoses by 33% and resulted in treatment changes in 15 of 41 (37%) patients.CONCLUSIONSTransbronchial biopsy has low sensitivity, high specificity, and high positive predictive value for diagnosing acute rejection. We found a significant tendency for TBBx to underestimate the presence and severity of clinically significant grades of rejection while simultaneously overestimating the presence of clinically insignificant rejection. Adding SLBx is valuable in lung transplant recipients with respiratory failure who require mechanical ventilation.

Actin released from damaged cells after a variety of tissue injuries appears to be involved in multiple organ dysfunction syndrome. Under experimental conditions, when the quantity of actin present in plasma is made to exceed the protective capacity of the actin-scavenging mechanism, microembolism and pulmonary vascular angiopathy have been noted in rats. It remains to be determined whether this injury is a result of a direct toxic effect or occurs indirectly via platelet activation or fibrin interactions. We examined the effect of sera from patients with adult respiratory distress syndrome (ARDS), as well as G-actin added to normal serum, on the viability, morphology, and function of cultured sheep pulmonary artery endothelial cells (SPAEC). Both patient sera and normal sera to which actin was added were toxic in the cell culture model; this toxicity could be abrogated, at least partially, by preincubation with gelsolin, which is known to complex with actin. A significant portion of the toxicity of sera from patients with ARDS was sensitive to heat (56 degrees C), suggesting an important role of complement. Sera from patients with ARDS were shown to contain filaments of F-actin by immunoblot and rhodamine phalloidin staining after ultracentrifugation. Thus, saturation of the actin-scavenging system by addition of exogenous G-actin to plasma produces direct pulmonary endothelial cell injury. Furthermore, plasma from patients with ARDS secondary to bacterial pneumonia is toxic to SPAEC, and a small but significant contributory role of actin is apparent in these studies.

Cytokines and lipopolysaccharide (LPS) are known to be injurious to vascular endothelial cells (ECs), but the influence of adjacent vascular smooth muscle cells (SMCs) on this injury is unknown. Exposure of cultured rat (RPMECs) or human (HPMECs) pulmonary microvascular ECs on tissue culture plastic to a mixture of cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) and LPS (cytomix) resulted in a significant increase in (51)Cr release to 35-40%. When unstimulated RPMECs were cocultured with cytomix-pretreated rat pulmonary microvascular SMCs (RPMSMCs) there was an increase in (51)Cr release to 8.4%, which was nitric oxide dependent. However, when RPMECs or HPMECs were stimulated in direct contact with their respective SMCs, rather than a further increase in cytomix-induced injury (e.g., >35-40%), (51)Cr release decreased to <10%. This cytoprotection was fully reproduced with fixed RPMSMCs, and partially reproduced by plating HPMECs on gelatin. These data show that the direct toxicity of a cytokine and endotoxin mixture on cultured ECs can be reduced by contact with vascular smooth muscle.

Nitric oxide (NO) regulates prostaglandin H synthase (PGHS) activity in various cell types, but reports conflict in regard to its stimulatory versus inhibitory role. Murine lung endothelial cells infected with a retroviral vector expressing the human inducible NO synthase gene were used to prevent ambiguous effects of NO from either exogenous chemical donors or cytokine-stimulated cells. Low concentrations of endogenous NO led to a dose-dependent increase in 6-keto PGF1α production (p < 0.05), whereas the highest production of NO resulted in lower 6-keto PGF1α production. These data demonstrate a complex regulation of PGHS activity by NO that needs to be considered when proposing a physiological or pathophysiological role for NO.

The activity of small GTP-binding proteins is regulated by a critical step in posttranslational processing, namely, the addition of isoprenoid lipids farnesyl and geranylgeranyl, mediated by the enzymes farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I), respectively. We have developed compounds that inhibit these enzymes specifically and in this study sought to determine their effects on smooth muscle cells (SMC) from the pulmonary microvasculature. We found that the GGTase I inhibitor GGTI-298 suppressed protein geranylgeranylation and blocked serum-dependent growth as measured by thymidine uptake and cell counts. In the absence of serum, however, GGTI-298 induced apoptosis in these cells as measured by both DNA staining and flow cytometry. The FTase inhibitor FTI-277 selectively inhibited protein farnesylation but had a minor effect on growth and no effect on apoptosis. To further investigate the role of geranylgeranylated proteins in apoptosis, we added the cholesterol synthesis inhibitor lovastatin, which inhibits the biosynthesis of farnesyl and geranylgeranyl pyrophosphates. This also induced apoptosis, but when geranylgeraniol was added to replenish cellular pools of geranylgeranyl pyrophosphate, apoptosis was reduced to baseline. In contrast, farnesol achieved only partial rescue of the cells. These results imply that geranylgeranylated proteins are required for growth and protect SMC against apoptosis. GGTase I inhibitors may be useful in preventing hyperplastic remodeling and may have the potential to induce the apoptotic regression of established vascular lesions.

Pulmonary arterial microvascular smooth muscle function governs many aspects of lung physiology and pathophysiology. Acutely, microvascular smooth muscle cells (SMC) modulate pulmonary vascular resistance; chronically, they contribute to vascular remodeling. Recent work has also suggested a possible immune function for pulmonary smooth muscle through cytokine-stimulated nitric oxide production. To facilitate study of the mechanisms underlying these functions, we have developed methods for isolating pulmonary arterial microvessels from the rat and culturing SMC from these vessels. The pulmonary arterial circulation was filled with a suspension of iron oxide in agar, and a subpleural tissue sample was obtained. The vessels were cleared of surrounding lung parenchyma by partial collagenase digestion, and the iron-containing arteries were separated magnetically. The diameter of the harvested arteries confirmed an intraacinar origin, and the cultured cells expressed smooth muscle isoforms of alpha-actin and myosin but did not take up acetylated low density lipoprotein. To assess a possible immune effector role for these cells, confluent monolayers were stimulated with cytokines and endotoxin. At 24 h, immunofluorescent staining for inducible nitric oxide synthase was prominent within these cells. Nitric oxide production, as measured by nitrite levels in the cell-conditioned medium, was also markedly elevated but reduced by adding N(G)-monomethyl-L-arginine. We conclude that rat pulmonary arterial microvascular SMC can be obtained by the iron oxide infusion method and that these cells express an inducible nitric oxide synthase after cytokine stimulation.