G.A. Lombard

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.

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.

Infections in Lung Allograft Recipients: Ganciclovir Era

BACKGROUND Infections are common after lung transplantation. This report analyzes infections and associated pathogens identified in 202 lung transplant recipients. METHODS Infections were tallied according to sites of infection and associated pathogen(s). Infection events were also categorized by post-operative Days 0 to 100, 101 to 365, and after 365, and normalized to 100 patient-days before and after bronchiolitis obliterans syndrome (BOS). RESULTS From November 1990 to November 2005, 202 patients received 208 lung transplants. The follow-up was 702.4 patient-years. A total of 178 lung transplant patients developed 859 infections, with 944 pathogens identified. Infections were in the lung in 559 (65.1%), mucocutaneous (skin, wound, catheter-related, and oral) in 88 (10.2%), in the blood in 85 (9.8%), and in other sites (urine, bowel, eye, and peritoneum) in 127 (14.8%). Most lung pathogens were bacterial (83.6%), and 57.9% were Pseudomonas aeruginosa. Fungi comprised 10.6%, with Aspergillus spp the most common (67.1%) isolate. Cytomegalovirus pneumonitis was seen in 4.3% of respiratory infections. BOS was diagnosed in 87 patients (43.1% of the total). Of all infections seen in the BOS population, there were 0.42 episodes/100 patient-days and 0.70 episodes/100 patient-days before and after BOS, respectively (p = 0.5). CONCLUSIONS These data provide an updated infection profile in the ganciclovir era after lung transplantation. When compared with pre-ganciclovir times, post-transplant cytomegalovirus infection incidence has notably declined, with filamentous fungi emerging as prevalent pathogens in its place. Such findings are important for refining management of infections in order to offer more stringent treatment against aggressive pathogens.

Clostridium difficile colitis in lung transplantation

Purpose. Clostridium difficile colitis (CDC) is the most common nosocomial infection of the gastrointestinal tract in patients with recent antibiotic use or hospitalization. Lung transplant recipients receive aggressive antimicrobial therapy postoperatively for treatment and prophylaxis of respiratory infections. This report describes the epidemiology of CDC in lung recipients from a single center and explores possible associations with bronchiolitis obliterans syndrome (BOS), a surrogate marker of chronic rejection.

Single-institution study evaluating the utility of surveillance bronchoscopy after lung transplantation.

BACKGROUND Many lung transplant physicians advocate surveillance bronchoscopy with transbronchial lung biopsy and bronchoalveolar lavage (TBB/BAL) to monitor lung recipients despite limited evidence this strategy improves outcomes. This report compares rates of infection (INF), acute rejection (AR), bronchiolitis obliterans syndrome (BOS) and survival in lung allograft recipients managed with surveillance TBB/BAL (SB) versus those with clinically indicated TBB/BAL (CIB). METHODS We reviewed 47 consecutive recipients transplanted between March 2002 and August 2005. Of these recipients, 24 consented to a multi-center trial requiring SB and 23 were managed by our usual practice of CIB. Rates of freedom from INF, AR, BOS and survival were compared. BOS and AR were diagnosed according to published guidelines from the International Society for Heart and Lung Transplantation. RESULTS A total of 240 TBB/BALs were performed. CIB and SB groups underwent 84 (3.7 +/- 3.4/patient) and 156 (6.5 +/- 2.0/patient) TBB/BALs, respectively. In the SB group, 54 (2.2 +/- 1.6/patient) TBB/BALs were true surveillance procedures, whereas 102 (4.2 +/- 2.3/patient) were clinically indicated. No AR episode requiring treatment was detected by true surveillance. Freedom from respiratory INF, AR, BOS and survival in the SB and CIB groups showed no significant differences. Five patients in the CIB group remained stable without requiring TBB/BAL. In the SB group, 4 previously asymptomatic patients developed pneumonia within 2 weeks of surveillance TBB/BAL. CONCLUSIONS With no obvious advantage identified, surveillance bronchoscopy may pose a risk to stable lung transplant recipients. A multi-center, controlled trial is required to validate the utility and safety of surveillance bronchoscopy in lung transplantation.

Clinical spectrum of gram‐positive infections in lung transplantation

Purpose. Gram‐positive (GP) organisms are among the most common cause of infections in early postsurgical and immunocompromised populations. Patients recovering from lung transplantation (LT) are particularly susceptible owing to the physiologic stress imposed by surgery and induction with intense immunosuppression. Sites, types, and timing of GP infections following LT  are not well documented. This report describes the clinical spectrum of GP infections and their effects on surgical airway complications (SAC) and bronchiolitis obliterans syndrome (BOS) following LT.

Analysis of pulmonary non-tuberculous mycobacterial infections after lung transplantation

Non‐tuberculous mycobacteria (NTM) are important pathogens in lung transplant recipients. This study describes the spectrum of NTM respiratory tract infections and examines the association of NTM infections with lung transplant complications.

Probable Phaeoacremonium parasiticum as a cause of cavitary native lung nodules after single lung transplantation

Lung nodules after lung transplantation most often represent infection or post‐transplant lymphoproliferative disorder in the allograft. Conversely, native lung nodules in single lung transplant recipients are more likely to be bronchogenic carcinoma. We present a patient who developed native lung cavitary nodules. Although malignancy was anticipated, evaluation revealed probable Phaeoacremonium parasiticum infection. Phaeoacremonium parasiticum is a dematiaceous fungus first described as a cause of soft tissue infection in a renal transplant patient. Lung nodules have not been previously described and this is the first case, to our knowledge, of P. parasiticum identified after lung transplantation.