Vincent G. Valentine

Extended Valganciclovir Prophylaxis to Prevent Cytomegalovirus After Lung Transplantation: A Randomized, Controlled Trial

BACKGROUND Cytomegalovirus (CMV) is the most prevalent opportunistic infection after lung transplantation. Current strategies do not prevent CMV in most at-risk patients. OBJECTIVE To determine whether extending prophylaxis with oral valganciclovir from the standard 3 months to 12 months after lung transplantation is efficacious. DESIGN Randomized, clinical trial. Patients were randomly assigned by a central automated system to treatment or placebo. Patients and investigators were blinded to treatment status. (ClinicalTrials.gov registration number: NCT00227370) SETTING Multicenter trial involving 11 U.S. lung transplant centers. PATIENTS 136 lung transplant recipients who completed 3 months of valganciclovir prophylaxis. INTERVENTION 9 additional months of oral valganciclovir (n = 70) or placebo (n = 66). MEASUREMENTS The primary end point was freedom from CMV disease (syndrome or tissue-invasive) on an intention-to-treat basis 300 days after randomization. Secondary end points were CMV disease severity, CMV infection, acute rejection, opportunistic infections, ganciclovir resistance, and safety. RESULTS CMV disease occurred in 32% of the short-course group versus 4% of the extended-course group (P < 0.001). Significant reductions were observed with CMV infection (64% vs. 10%; P < 0.001) and disease severity (110 000 vs. 3200 copies/mL, P = 0.009) with extended treatment. Rates of acute rejection, opportunistic infections, adverse events, CMV UL97 ganciclovir-resistance mutations, and laboratory abnormalities were similar between groups. During the 6 months after study completion, a low incidence of CMV disease was observed in both groups. LIMITATION Longer-term effects of extended prophylaxis were not assessed. CONCLUSION In adult lung transplant recipients who have received 3 months of valganciclovir, extending prophylaxis by an additional 9 months significantly reduces CMV infection, disease, and disease severity without increased ganciclovir resistance or toxicity. A beneficial effect with regard to prevention of CMV disease seems to extend at least through 18 months after transplantation.

CD28 Down-Regulation on Circulating CD4 T-Cells Is Associated with Poor Prognoses of Patients with Idiopathic Pulmonary Fibrosis

Background Although the etiology of idiopathic pulmonary fibrosis (IPF) remains perplexing, adaptive immune activation is evident among many afflicted patients. Repeated cycles of antigen-induced proliferation cause T-cells to lose surface expression of CD28, and we hypothesized this process might also occur in IPF. Methodology/Principal Findings Peripheral blood CD4 T-cells from 89 IPF patients were analyzed by flow cytometry and cytokine multiplex assays, and correlated with clinical events. In comparison to autologous CD4+CD28+cells, the unusual CD4+CD28null lymphocytes seen in many IPF patients had discordant expressions of activation markers, more frequently produced cytotoxic mediators perforin (2.4±0.8% vs. 60.0±7.4%, p<0.0001) and granzyme B (4.5±2.8% vs.74.9±6.5%, p<0.0001), produced greater amounts of many pro-inflammatory cytokines, and less frequently expressed the regulatory T-cell marker FoxP3 (12.9±1.1% vs. 3.3±0.6% p<0.0001). Infiltration of CD4+CD28null T-cells in IPF lungs was confirmed by confocal microscopy. Interval changes of CD28 expression among subjects who had replicate studies were correlated with conterminous changes of their forced vital capacities (rs = 0.49, p = 0.012). Most importantly, one-year freedom from major adverse clinical events (either death or lung transplantation) was 56±6% among 78 IPF patients with CD4+CD28+/CD4total≥82%, compared to 9±9% among those with more extensive CD28 down-regulation (CD4+CD28+/CD4total<82%) (p = 0.0004). The odds ratio for major adverse events among those with the most extensive CD28 down-regulation was 13.0, with 95% confidence intervals 1.6-111.1. Conclusions/Significance Marked down-regulation of CD28 on circulating CD4 T-cells, a result of repeated antigen-driven proliferations, is associated with poor outcomes in IPF patients. The CD4+CD28null cells of these patients have potentially enhanced pathogenic characteristics, including increased productions of cytotoxic mediators and pro-inflammatory cytokines. These findings show proliferative T-cell responses to antigen(s) resulting in CD28 down-regulation are associated with progression and manifestations of IPF, and suggest assays of circulating CD4 T-cells may identify patients at greatest risk for clinical deterioration.

Obliterative bronchiolitis after lung and heart-lung transplantation

Obliterative bronchiolitis (OB) has emerged as the main cause of morbidity and mortality in the long-term follow-up after lung and heart-lung transplantation. The pathogenesis of OB is multifactorial, with acute rejection and cytomegalovirus infection being the main risk factors for the development of OB. The final common pathway of all inciting events seems to be an alloimmune injury, with subsequent release of immunologic mediators and production of growth factors leading to luminal obliteration and fibrous scarring of the small airways. Analyzing the 14 years of experience in 163 patients at Stanford University, we found a current incidence of bronchiolitis obliterans syndrome or histologically proven OB within the first 3 years after lung and heart-lung transplantation of 36.3%, with an overall prevalence of 58.1% after heart-lung and 51.4% after lung transplantation. Both pulmonary function indices (forced expiratory flow between 25% and 75% of forced vital capacity and forced expiratory volume in 1 second) and transbronchial biopsies have proven helpful in diagnosing bronchiolitis obliterans syndrome or OB at an early stage. Early diagnosis of OB and improved management have achieved survival rates in patients with OB after 1, 3, 5, and 10 years of 83%, 66%, 46%, and 22%, compared with 86%, 83%, 67%, and 67% in patients without OB. Recently, different experimental models have been developed to investigate the cellular and molecular events leading to OB and to evaluate new treatment strategies for this complication, which currently limits the long-term success of heart-lung and lung transplantation.

Everolimus versus azathioprine in maintenance lung transplant recipients: An international, randomized, double-blind clinical trial

Everolimus is a proliferation signal inhibitor with immunosuppressive activity that may reduce the rate of progression of chronic rejection, bronchiolitis obliterans syndrome (BOS), after lung transplantation. In a randomized, double‐blind clinical trial, 213 BOS‐free maintenance patients received everolimus (3 mg/day) or azathioprine (AZA, 1–3 mg/kg/day) in combination with cyclosporine and corticosteroids. The prospectively defined primary endpoint was the incidence of efficacy failure (decline in FEV1 >15%[ΔFEV1 >15%], graft loss, death or loss to follow‐up) at 12 months. Incidence of efficacy failure at 12 months was significantly lower in the everolimus group than AZA (21.8% vs. 33.9%; p = 0.046); at 24 months, rates of efficacy failure became similar between the groups. At 12 months, the everolimus group had significantly reduced incidences of ΔFEV1 >15%, ΔFEV1 >15% with BOS, and acute rejection. At 24 months, only incidence of acute rejection remained significantly less in the everolimus group. Treatment discontinuations (particularly due to adverse events), serious adverse events and high serum creatinine values were more common with everolimus. For the first time, a drug has demonstrated significant slowing of loss in lung function, suggesting that patients kept on prolonged maintenance treatment with everolimus may benefit from replacing AZA with everolimus 3 months after lung transplantation.

Cellular and Humoral Autoreactivity in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a morbid, refractory lung disorder with an unknown pathogenesis. To investigate potential adaptive immune mechanisms in IPF, we compared phenotypes and effector functions of peripheral CD4 T cells, autoantibody production, and proliferative responses of pulmonary hilar lymph node CD4 T cells to autologous lung extracts from afflicted patients and normals. Our results show that greater proportions of peripheral CD4 T lymphocytes in IPF subjects expressed MHC class II and CD154 (CD40L), and they more frequently elaborated TGF-β1, IL-10, and TNF-α. Abnormal CD4 T cell clonal expansions were found in all IPF patients, and 82% of these subjects also had IgG autoantibodies against cellular Ags. IPF lung extracts stimulated proliferations of autologous CD4 T cells, unlike preparations from normals or those with other lung diseases, and the IPF proliferative responses were enhanced by repeated cycles of stimulation. Thus, CD4 T cells from IPF patients have characteristics typical of cell-mediated pathologic responses, including augmented effector functions, provision of facultative help for autoantibody production, oligoclonal expansions, and proliferations driven by an Ag present in diseased tissues. Recognition that an autoreactive immune process is present in IPF can productively focus efforts toward identifying the responsible Ag, and implementing more effective therapies.

The role of chloride anion and CFTR in killing of Pseudomonas aeruginosa by normal and CF neutrophils

Chloride anion is essential for myeloperoxidase (MPO) to produce hypochlorous acid (HOCl) in polymorphonuclear neutrophils (PMNs). To define whether chloride availability to PMNs affects their HOCl production and microbicidal capacity, we examined how extracellular chloride concentration affects killing of Pseudomonas aeruginosa (PsA) by normal neutrophils. PMN‐mediated bacterial killing was strongly dependent on extracellular chloride concentration. Neutrophils in a chloride‐deficient medium killed PsA poorly. However, as the chloride level was raised, the killing efficiency increased in a dose‐dependent manner. By using specific inhibitors to selectively block NADPH oxidase, MPO, and cystic fibrosis transmembrane conductance regulator (CFTR) functions, neutrophil‐mediated killing of PsA could be attributed to three distinct mechanisms: CFTR‐dependent and oxidant‐dependent; chloride‐dependent but not CFTR‐ and oxidant‐dependent; and independent of any of the tested factors. Therefore, chloride anion is involved in oxidant‐ and nonoxidant‐mediated bacterial killing. We previously reported that neutrophils from CF patients are defective in chlorination of ingested bacteria [ 1 ], suggesting that the chloride channel defect might impair the MPO‐hydrogen peroxide‐chloride microbicidal function. Here, we compared the competence of killing PsA by neutrophils from normal donors and CF patients. The data demonstrate that the killing rate by CF neutrophils was significantly lower than that by normal neutrophils. CF neutrophils in a chloride‐deficient environment had only one‐third of the bactericidal capacity of normal neutrophils in a physiological chloride environment. These results suggest that CFTR‐dependent chloride anion transport contributes significantly to killing PsA by normal neutrophils and when defective as in CF, may compromise the ability to clear PsA.

Effect of Etiology and Timing of Respiratory Tract Infections on Development of Bronchiolitis Obliterans Syndrome

BACKGROUND Among the many potential risk factors influencing the development of bronchiolitis obliterans syndrome (BOS), acute cellular rejection is the most frequently identified. Despite the unique susceptibility of the lung allograft to pathogens, the association with respiratory tract infections remains unclear. In this study we analyze the role respiratory tract infections have on the development of BOS after lung transplantation. METHODS Data from a single center were analyzed from 161 lung recipients transplanted from November 1990 to November 2005, and who survived >180 days. Univariate and multivariate Cox regression analyses were performed using BOS development and the time-scale was reported with hazard ratios (HRs) and confidence intervals (CIs). RESULTS Significant findings by univariate analysis per 100 patient-days prior to BOS onset included acute rejection, cytomegalovirus (CMV) pneumonitis, Gram-negative respiratory tract infections, Gram-positive respiratory tract infections and fungal pneumonias. Multivariate analysis indicated acute rejection, Gram-negative, Gram-positive and fungal pneumonias with HRs (CI) of 84 (23 to 309), 6.6 (1.2 to 37), 6,371 (84 to 485,000) and 314 (53 to 1,856) to be associated with BOS, respectively. Acute rejection, CMV pneumonitis, Gram-positive pneumonia and fungal pneumonitis in the first 100 days had HRs (CI) of 1.8 (1.1 to 3.2), 3.1 (1.3 to 6.9), 3.8 (1.5 to 9.4) and 2.1 (1.1 to 4.0), respectively, and acute rejection and fungal pneumonitis in the late post-operative period with HRs (CI) of 2.3 (1.2 to 4.4) and 1.5 (1.1 to 1.9), respectively. CONCLUSIONS In addition to acute rejection, pneumonias with GP, GN and fungal pathogens occurring prior to BOS are independent determinants of chronic allograft dysfunction. Early recognition and treatment of these pathogens in lung transplant recipients may improve long-term outcomes after transplantation.

Success of lung transplantation without surveillance bronchoscopy

Abstract Background: No current evidence demonstrates improved survival or decreased rate of bronchiolitis obliterans syndrome (BOS) despite regularly scheduled fiberoptic bronchoscopy (FOB) with transbronchial biopsy and bronchoalveolar lavage (TBB/BAL) after lung transplantation. Reduced lung function detected with spirometry or oximetry in symptomatic and asymptomatic lung allograft recipients (LARs) may be a more appropriate indication for bronchoscopic sampling. Hypothesis: Clinically indicated TBB/BAL without routine invasive surveillance sampling of the transplanted lung does not decrease survival or increase the rate of BOS in LARs. Methods: We reviewed 91 consecutive LARs transplanted at Ochsner Clinic between January 1995 and December 1999. Clinical indications for FOB with TBB/BAL include 10% decline in forced expiratory volume in 1 second below baseline; 20% decrease in forced expiratory flow rate between 25% and 75% of the forced vital capacity; or unexplained respiratory symptoms, signs, or fever. Along with demographic and clinical data, 1-year and 3-year survival rates for these 91 LARs were compared with 5,430 LARs from the International Society for Heart and Lung Transplantation (ISHLT) Registry transplanted during the same 60-month period. Ten of the 91 patients did not survive to hospital discharge after transplantation. We divided the remaining 81 LARs into 2 subsets: Group A patients ( n = 43) underwent zero to 1 TBB/BAL and Group B patients ( n = 38) required more than 1 procedure. Demographic data, rejection, infection, and incidence of BOS were compared between groups. Results: The 1-year and 3-year survival rates in the Ochsner LAR cohort were 85% and 73%, respectively, vs 72% and 57% in the ISHLT cohort p p p = not significant. We observed twice as many patients with cystic fibrosis and twice as many pneumonia episodes in Group B. The rate of acute rejection in each group was not statistically different. The cumulative incidence of BOS was increased in Group B at 1 year and at 3 years (5% and 56%) when compared with Group A (3% and 13%), p Conclusion: Based on the findings from this observational, single-institution study, clinically indicated TBB/BAL without routine surveillance sampling of the lung allograft is unlikely to pose greater risk than does regularly scheduled bronchoscopy after lung transplantation.

CFTR‐mediated halide transport in phagosomes of human neutrophils

Chloride serves as a critical component of innate host defense against infection, providing the substrate for MPO‐catalyzed production of HOCl in the phagosome of human neutrophils. Here, we used halide‐specific fluorescent sensors covalently coupled to zymosan particles to investigate the kinetics of chloride and iodide transport in phagosomes of human neutrophils. Using the self‐ratioable fluorescent probe specific for chloride anion, we measured chloride dynamics within phagosomes in response to extracellular chloride changes by quantitative fluorescence microscopy. Under the experimental conditions used, normal neutrophils showed rapid phagosomal chloride uptake with an initial influx rate of 0.31 ± 0.04 mM/s (n=5). GlyH‐101, a CFTRinh, decreased the rate of uptake in a dose‐dependent manner. Neutrophils isolated from CF patients showed a significantly slower rate of chloride uptake by phagosomes, having an initial influx rate of 0.043 ± 0.012 mM/s (n=5). Interestingly, the steady‐state level of chloride in CF phagosomes was ∼26 mM, significantly lower than that of the control (∼68 mM). As CFTR transports chloride as well as other halides, we conjugated an iodide‐sensitive probe as an independent approach to confirm the results. The dynamics of iodide uptake by neutrophil phagosomes were monitored by flow cytometry. CFTRinh172 blocked 40–50% of the overall iodide uptake by phagosomes in normal neutrophils. In a parallel manner, the level of iodide uptake by CF phagosomes was only 20–30% of that of the control. Taken together, these results implicate CFTR in transporting halides into the phagosomal lumen.

Ganciclovir for cytomegalovirus: a call for indefinite prophylaxis in lung transplantation.

BACKGROUND Universal ganciclovir (GCV) prophylaxis is a strategy aimed at reducing cytomegalovirus (CMV) infection and delaying the development of bronchiolitis obliterans syndrome (BOS). However, the optimal duration of GCV prophylaxis remains unclear. We report our experience with GCV prophylaxis administered indefinitely and its effect on CMV pneumonitis, BOS and survival after lung transplantation (LT). METHODS One hundred fifty-one patients surviving >100 days after LT were analyzed. GCV was given to 130 CMV donor- or recipient-seropositive patients. Data from 90 patients who received indefinite GCV prophylaxis (IND) and 40 patients who discontinued their GCV prophylaxis (STOP) were compared. RESULTS CMV pneumonitis occurred in 16%, 8%, 17% and 19% of patients in the D+R+, D-R+, D+R- and D-R- groups, respectively. In the STOP cohort, 15 of 40 patients developed CMV pneumonitis (median time 79 days) after GCV was stopped. Ten of these 15 patients developed BOS (median time 116 days) after discontinuing GCV. The risk of CMV pneumonitis in the STOP cohort was significantly higher when GCV prophylaxis was discontinued within the first year. Cumulative incidence of CMV pneumonitis in the IND and STOP groups at 5 years was 2% and 57%, respectively (p < 0.001). BOS-free survival and survival were similar across both groups. CONCLUSIONS Indefinite GCV prophylaxis prevents CMV pneumonitis in 98% of LT recipients. Thirty-eight percent of patients discontinuing prophylaxis developed CMV pneumonitis, 50% of whom progressed to BOS within 1 year. Continuing ganciclovir prophylaxis indefinitely after lung transplantation should be considered.