Correlates of survival after autoantibody reduction therapy for acute IPF exacerbations.

BACKGROUND: No medical treatment has proven efficacy for acute exacerbations of idiopathic pulmonary fibrosis (AE-IPF), and this syndrome has a very high mortality. Based on data indicating humoral autoimmune processes are involved in IPF pathogenesis, we treated AE-IPF patients with an autoantibody reduction regimen of therapeutic plasma exchange, rituximab, and intravenous immunoglobulin. This study aimed to identify clinical and autoantibody determinants associated with survival after autoantibody reduction in AE-IPF. METHODS: Twenty-four(24) AE-IPF patients received the autoantibody reduction regimen. Plasma anti-epithelial autoantibody titers were determined by HEp-2 indirect immunofluorescence assays in 22 patients. RESULTS: Mean age of the patients was 70 + 7 years old, and 70% were male. Beneficial clinical responses that occurred early during therapy were a favorable prognostic indicator: supplemental O2 flows needed to maintain resting SaO2>92% significantly decreased and/or walk distances increased among all 10 patients who survived for at least one year. Plasma anti-HEp-2 autoantibody titers were ~-three-fold greater in survivors compared to non-survivors (p<0.02). Anti-HEp-2 titers >1:160 were present in 75% of the evaluable one-year survivors, compared to 29% of non-survivors, and 10 of 12 patients (83%) with anti-HEP-2 titers <1:160 died during the observation period (Hazard Ratio = 3.3, 95% Confidence Interval = 1.02-10.6, p = 0.047). CONCLUSIONS: Autoantibody reduction therapy is associated with rapid reduction of supplemental oxygen requirements and/or improved ability to ambulate in many AE-IPF patients. Facile anti-epithelial autoantibody assays may help identify those most likely to benefit from these treatments.

Decrements of body mass index are associated with poor outcomes of idiopathic pulmonary fibrosis patients.

BACKGROUND: The processes that result in progression of idiopathic pulmonary fibrosis (IPF) remain enigmatic. Moreover, the course of this disease can be highly variable and difficult to accurately predict. We hypothesized analyses of body mass index (BMI), a simple, routine clinical measure, may also have prognostic value in these patients, and might provide mechanistic insights. We investigated the associations of BMI changes with outcome, plasma adipokines, and adaptive immune activation among IPF patients. METHODS: Data were analyzed in an IPF discovery cohort (n = 131) from the University of Pittsburgh, and findings confirmed in patients from the University of Alabama at Birmingham (n = 148). Plasma adipokines were measured by ELISA and T-cell phenotypes determined by flow cytometry. RESULTS: Transplant-free one-year survivals in subjects with the greatest rates of BMI decrements, as percentages of initial BMI (>0.68%/month), were worse than among those with more stable BMI in both discovery (HR = 1.8, 95%CI = 1.1-3.2, p = 0.038) and replication cohorts (HR = 2.5, 95%CI = 1.2-5.2, p = 0.02), when adjusted for age, baseline BMI, and pulmonary function. BMI decrements >0.68%/month were also associated with greater mortality after later lung transplantations (HR = 4.6, 95%CI = 1.7-12.5, p = 0.003). Circulating leptin and adiponectin levels correlated with BMI, but neither adipokine was prognostic per se. BMI decrements were significantly associated with increased proportions of circulating end-differentiated (CD28null) CD4 T-cells (CD28%), a validated marker of repetitive T-cell activation and IPF prognoses. CONCLUSIONS: IPF patients with greatest BMI decrements had worse outcomes, and this effect persisted after lung transplantation. Weight loss in these patients is a harbinger of poor prognoses, and may reflect an underlying systemic process, such as adaptive immune activation.

Fortification of Preservation Solution With Nitroprusside Does Not Alter Lung Allograft Survival in Clinical Human Lung Transplantation.

Background: Nitric oxide improves gas exchange following primary lung allograft dysfunction. Nitroprusside, a potent nitric oxide donor, has reduced reperfusion injury and improved oxygenation in experimental lung transplantation. Methods: We sought to study the effect on lung allograft outcomes of fortifying the preservation solution with nitroprusside. We conducted a single-center clinical study of 46 consecutive lung recipients between 1998 and 2000: 24 patients received donor organs preserved in modified Euro-Collins solution with prostaglandin E1 (PGE1) (control group), and 22 patients received organs preserved in modified Euro-Collins with PGE1 and nitroprusside (NP group). The primary endpoint was overall survival. Results: Baseline characteristics were similar between the groups except for a significantly longer graft ischemic time in the NP group vs the control group (253.3 ± 52 vs 225.3 ± 41 minutes, respectively, P=0.04). No significant differences were found in partial pressure arterial oxygen to fraction inspired oxygen ratio at ≤48 hours, primary graft dysfunction, or bronchiolitis obliterans-free days. Overall survival at 1, 3, and 5 years was 89%, 73%, and 63% in the control group and 76%, 38%, and 23% in the NP group. Log-rank survival analysis showed that the NP group had a significantly increased risk of mortality (P=0.034) compared to the control group. Conclusion: The addition of nitroprusside to the lung transplant perfusate in this clinical trial did not improve survival; however, a large randomized trial would likely reduce confounding ischemia times and increase the power of the study.

CFTR targeting during activation of human neutrophils.

Cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel, plays critical roles in phagocytic host defense. However, how activated neutrophils regulate CFTR channel distribution subcellularly is not well defined. To investigate, we tested multiple Abs against different CFTR domains, to examine CFTR expression in human peripheral blood neutrophils by flow cytometry. The data confirmed that resting neutrophils had pronounced CFTR expression. Activation of neutrophils with soluble or particulate agonists did not significantly increase CFTR expression level, but induced CFTR redistribution to cell surface. Such CFTR mobilization correlated with cell-surface recruitment of formyl-peptide receptor during secretory vesicle exocytosis. Intriguingly, neutrophils from patients with ΔF508-CF, despite expression of the mutant CFTR, showed little cell-surface mobilization upon stimulation. Although normal neutrophils effectively targeted CFTR to their phagosomes, ΔF508-CF neutrophils had impairment in that process, resulting in deficient hypochlorous acid production. Taken together, activated neutrophils regulate CFTR distribution by targeting this chloride channel to the subcellular sites of activation, and ΔF508-CF neutrophils fail to achieve such targeting, thus undermining their host defense function.

Autoantibody-Targeted Treatments for Acute Exacerbations of Idiopathic Pulmonary Fibrosis.

BACKGROUND: Severe acute exacerbations (AE) of idiopathic pulmonary fibrosis (IPF) are medically untreatable and often fatal within days. Recent evidence suggests autoantibodies may be involved in IPF progression. Autoantibody-mediated lung diseases are typically refractory to glucocorticoids and nonspecific medications, but frequently respond to focused autoantibody reduction treatments. We conducted a pilot trial to test the hypothesis that autoantibody-targeted therapies may also benefit AE-IPF patients. METHODS: Eleven (11) critically-ill AE-IPF patients with no evidence of conventional autoimmune diseases were treated with therapeutic plasma exchanges (TPE) and rituximab, supplemented in later cases with intravenous immunoglobulin (IVIG). Plasma anti-epithelial (HEp-2) autoantibodies and matrix metalloproteinase-7 (MMP7) were evaluated by indirect immunofluorescence and ELISA, respectively. Outcomes among the trial subjects were compared to those of 20 historical control AE-IPF patients treated with conventional glucocorticoid therapy prior to this experimental trial. RESULTS: Nine (9) trial subjects (82%) had improvements of pulmonary gas exchange after treatment, compared to one (5%) historical control. Two of the three trial subjects who relapsed after only five TPE responded again with additional TPE. The three latest subjects who responded to an augmented regimen of nine TPE plus rituximab plus IVIG have had sustained responses without relapses after 96-to-237 days. Anti-HEp-2 autoantibodies were present in trial subjects prior to therapy, and were reduced by TPE among those who responded to treatment. Conversely, plasma MMP7 levels were not systematically affected by therapy nor correlated with clinical responses. One-year survival of trial subjects was 46+15% vs. 0% among historical controls. No serious adverse events were attributable to the experimental medications. CONCLUSION: This pilot trial indicates specific treatments that reduce autoantibodies might benefit some severely-ill AE-IPF patients. These findings have potential implications regarding mechanisms of IPF progression, and justify considerations for incremental trials of autoantibody-targeted therapies in AE-IPF patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT01266317.

Reliability of diagnostic criteria for bronchiolitis obliterans syndrome after lung transplantation: a survey.

BACKGROUND: Long-term outcomes after lung transplantation are limited due to chronic lung allograft dysfunction (CLAD). Bronchiolitis obliterans syndrome (BOS) is the most common form of obstructive CLAD and its definition derives from spirometric measurements. Given the importance of this diagnosis, both the accuracy and reliability of the definition of CLAD are crucial in understanding the pathophysiology of this disease to develop therapeutic options and influence outcome after lung transplantation. METHODS: A web-based survey was designed and distributed to members of the Pulmonary Council of the International Society for Heart and Lung Transplantation (ISHLT) to better understand the accuracy and reliability of pulmonary function criteria in diagnosing BOS. Spirometric data from five patient scenarios that were discordant among reviewers regarding BOS determination from the Assessment of Immunosuppressive Regimen in Suppressing Acute and Chronic Rejection (AIRSAC) trial were randomly selected and summarized in this survey. Survey questions included the respondent's general understanding of the BOS definition, the determination of BOS, and difficulties with the current BOS definition. RESULTS: Eighty-seven respondents from the Pulmonary Council of the ISHLT responded to this survey. There was an overall 70% interobserver agreement regarding the presence or absence of BOS. Among those who agreed upon the presence of BOS, there was a 41% interobserver agreement regarding its time of onset. Despite this variability, the majority of respondents were not only familiar and agreed with the BOS criteria, they also felt confident in applying these criteria. CONCLUSIONS: Our survey identified potential limitations with the current criteria for diagnosing BOS. With recognition of the various CLAD phenotypes, further refinements of these diagnostic criteria will allow for an improved ability to identify and characterize patients who develop or are at risk for BOS, prognosticate outcomes, and, most importantly, marshal in future strategies directed at treating and preventing chronic lung dysfunction after lung transplantation.

Decreased incidence of cytomegalovirus infection with sirolimus in a post hoc randomized, multicenter study in lung transplantation.

BACKGROUND: Cytomegalovirus (CMV) is the most common opportunistic infection in lung transplantation. A recent multicenter, randomized trial (the AIRSAC study) comparing sirolimus to azathioprine in lung transplant recipients showed a decreased incidence of CMV events in the sirolimus cohort. To better characterize this relationship of decreased incidence of CMV events with sirolimus, we examined known risk factors and characteristics of CMV events from the AIRSAC database. METHODS: The AIRSAC database included 181 lung transplant patients from 8 U.S.-based lung transplant centers that were randomized to sirolimus or azathioprine at 3 months post-transplantation. CMV incidence, prophylaxis, diagnosis and treatment data were all prospectively collected. Prophylaxis and treatment of CMV were at the discretion of each institution. RESULTS: The overall incidence of any CMV event was decreased in the sirolimus arm when compared with the azathioprine arm at 1 year after lung transplantation (relative risk [RR] = 0.67, confidence interval [CI] 0.55 to 0.82, p < 0.01). This decreased incidence of CMV events with sirolimus remained significant after adjusting for confounding factors of CMV serostatus and CMV prophylaxis. CONCLUSIONS: These data support results from other solid-organ transplantation studies and suggest further investigation of this agent in the treatment of lung transplant recipients at high risk for CMV events.

Interobserver variability in grading transbronchial lung biopsy specimens after lung transplantation.

BACKGROUND: Acute rejection remains a major source of morbidity after lung transplantation. Given the importance of this diagnosis, an international grading system was developed to standardize the diagnosis of acute lung-allograft rejection. The reliability of this grading system has not been adequately assessed by previous studies. METHODS: We examined the level of agreement in grading transbronchial biopsy specimens obtained from a large multicenter study (AIRSAC [Comparison of a Tacrolimus/Sirolimus/Prednisone Regimen vs Tacrolimus/Azathioprine/Prednisone Immunosuppressive Regimen in Lung Transplantation] trial). Biopsy specimens were initially graded for acute rejection and lymphocytic bronchiolitis by the site pathologist and subsequently graded by a central pathologist. Reliability of interobserver grading was evaluated using Cohen κ coefficients. RESULTS: A total of 481 transbronchial biopsy specimens were graded by both the site and central pathologists. The overall concordance rates were 74% and 89% for grade A and grade B biopsy specimens, respectively. When samples from biopsies performed at different time points after transplantation were assessed, there was a higher level of agreement early (≤ 6 weeks) after transplant compared with later time points for acute rejection. However, there was still only moderate agreement for both grade A (κ score 0.479; 95% CI, 0.29-0.67) and grade B (κ score 0.465; 95% CI, 0.08-0.85) rejection. CONCLUSIONS: These results expand upon previous reports of interobserver variability in grading transbronchial biopsy specimens after lung transplantation. Given the variability in grading these specimens, we advocate further education of the histopathologic findings in lung transplant biopsy specimens, as well as revisiting the current criteria for grading transbronchial biopsy specimens to improve concordance among lung transplant pathologists. TRIAL REGISTRY: ClinicalTrials.gov; No. NCT00321906; URL: www.clinicaltrials.gov.

Clarithromycin for prevention of bronchiolitis obliterans syndrome in lung allograft recipients.

BACKGROUND: Bronchiolitis obliterans syndrome (BOS) is the major limitation to long-term survival following lung transplantation and strategies to reduce its incidence have remained elusive. Macrolides may stabilize lung function in patients with established BOS. Their role, however, in prevention of BOS remains unexamined. METHODS: Survival and BOS-free survival of 102 lung allograft recipients (LARs), transplanted at a single center between July 1995 and December 2001 who routinely received clarithromycin, were compared with two different control groups. The first control group consisted of 44 LARs from the same center who were transplanted from January 2002 onwards and did not receive clarithromycin. The second control group consisted of a contemporaneous cohort of 5089 recipients, transplanted between 1995 and 2001, reported to the United Network for Organ Sharing database. RESULTS: When compared with the first control group, BOS-free survival was reduced in LARs receiving clarithromycin. Univariate (hazard ratio [HR] 3.13, p-value = 0.004) and multivariate (HR 3.49, p-value = 0.04) analyses showed that routine use of clarithromycin was associated with an increased risk of developing BOS. When compared with the second control group, the five-yr survival of clarithromycin group was similar (p-value = 0.24). CONCLUSIONS: Routine use of clarithromycin does not delay development of BOS or improve survival.

Mucous solids and liquid secretion by airways: studies with normal pig, cystic fibrosis human, and non-cystic fibrosis human bronchi.

To better understand how airways produce thick airway mucus, nonvolatile solids were measured in liquid secreted by bronchi from normal pig, cystic fibrosis (CF) human, and non-CF human lungs. Bronchi were exposed to various secretagogues and anion secretion inhibitors to induce a range of liquid volume secretion rates. In all three groups, the relationship of solids concentration (percent nonvolatile solids) to liquid volume secretion rate was curvilinear, with higher solids concentration associated with lower rates of liquid volume secretion. In contrast, the secretion rates of solids mass and water mass as functions of liquid volume secretion rates exhibited positive linear correlations. The y-intercepts of the solids mass-liquid volume secretion relationships for all three groups were positive, thus accounting for the higher solids concentrations in airway liquid at low rates of secretion. Predictive models derived from the solids mass and water mass linear equations fit the experimental percent solids data for the three groups. The ratio of solids mass secretion to liquid volume secretion was 5.2 and 2.4 times higher for CF bronchi than for pig and non-CF bronchi, respectively. These results indicate that normal pig, non-CF human, and CF human bronchi produce a high-percent-solids mucus (>8%) at low rates of liquid volume secretion (≤1.0 µl·cm(-2)·h(-1)). However, CF bronchi produce mucus with twice the percent solids (~8%) of pig or non-CF human bronchi at liquid volume secretion rates ≥4.0 µl·cm(-2)·h(-1).

The HLA class II Allele DRB1*1501 is over-represented in patients with idiopathic pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a progressive and medically refractory lung disease with a grim prognosis. Although the etiology of IPF remains perplexing, abnormal adaptive immune responses are evident in many afflicted patients. We hypothesized that perturbations of human leukocyte antigen (HLA) allele frequencies, which are often seen among patients with immunologic diseases, may also be present in IPF patients. METHODS/PRINCIPAL FINDINGS: HLA alleles were determined in subpopulations of IPF and normal subjects using molecular typing methods. HLA-DRB1*15 was over-represented in a discovery cohort of 79 Caucasian IPF subjects who had lung transplantations at the University of Pittsburgh (36.7%) compared to normal reference populations. These findings were prospectively replicated in a validation cohort of 196 additional IPF subjects from four other U.S. medical centers that included both ambulatory patients and lung transplantation recipients. High-resolution typing was used to further define specific HLA-DRB1*15 alleles. DRB1*1501 prevalence in IPF subjects was similar among the 143 ambulatory patients and 132 transplant recipients (31.5% and 34.8%, respectively, p = 0.55). The aggregate prevalence of DRB1*1501 in IPF patients was significantly greater than among 285 healthy controls (33.1% vs. 20.0%, respectively, OR 2.0; 95%CI 1.3-2.9, p = 0.0004). IPF patients with DRB1*1501 (n = 91) tended to have decreased diffusing capacities for carbon monoxide (DL(CO)) compared to the 184 disease subjects who lacked this allele (37.8±1.7% vs. 42.8±1.4%, p = 0.036). CONCLUSIONS/SIGNIFICANCE: DRB1*1501 is more prevalent among IPF patients than normal subjects, and may be associated with greater impairment of gas exchange. These data are novel evidence that immunogenetic processes can play a role in the susceptibility to and/or manifestations of IPF. Findings here of a disease association at the HLA-DR locus have broad pathogenic implications, illustrate a specific chromosomal area for incremental, targeted genomic study, and may identify a distinct clinical phenotype among patients with this enigmatic, morbid lung disease.

Increased risk of venous thromboembolism with a sirolimus-based immunosuppression regimen in lung transplantation.

BACKGROUND: Sirolimus (rapamycin) is a potent anti-proliferative agent with immunosuppressive properties that is increasingly being used in solid-organ and hematopoietic stem cell transplantation. In addition, this drug is being investigated for treatment of a broad range of disorders, including cardiovascular disease, malignancies, tuberous sclerosis, and lymphangeioleiomyomatosis. In this study, we found an increased risk of venous thromboembolism (VTE) in lung transplant recipients treated with a sirolimus (SIR)-based immunosuppressive regimen. METHODS: One hundred eighty-one lung transplant recipients were enrolled in a prospective, multicenter, randomized, open-label trial comparing a tacrolimus (TAC)/SIR/prednisone immunosuppression regimen with a TAC/azathioprine (AZA)/prednisone immunosuppressive regimen. The differences in rates of VTE were examined. RESULTS: There was a significantly higher occurrence of VTE in the SIR cohort [15 of 87 (17.2%)] compared with the AZA cohort [3 of 94 (3.2%)] (stratified log-rank statistic = 7.44, p < 0.01). When adjusted for pre-transplant diagnosis and stratified by transplant center, this difference remained essentially unchanged (hazard ratio for SIR vs AZA = 5.2, 95% confidence interval 1.4 to 19.5, p = 0.01). CONCLUSION: Clinicians prescribing SIR should maintain a high level of vigilance for VTE, particularly among patients with other risk factors for this complication.

Comparison of sirolimus with azathioprine in a tacrolimus-based immunosuppressive regimen in lung transplantation.

RATIONALE: Lung transplantation has evolved into a life-saving therapy for select patients with end-stage lung diseases. However, long-term survival remains limited because of chronic rejection. Sirolimus is beneficial in preventing cardiac rejection and may decrease rejection after lung transplantation. OBJECTIVES: To determine the potential benefit versus risk of sirolimus in lung transplantation. METHODS: We conducted a multicenter randomized, open label controlled trial comparing sirolimus (SIR) with azathioprine (AZA) in a tacrolimus-based immunosuppressive regimen in lung transplantation. The primary end point was the incidence of acute rejection at 1 year after transplantation between the two study groups. MEASUREMENTS AND MAIN RESULTS: One hundred eighty-one patients were randomized to be included in this study. At 1 year after transplantation, there was no significant difference in the incidence of grade A acute rejection between the two study groups. Similarly, the incidence of chronic rejection and graft survival was no different between the two study groups. Cytomegalovirus infection was decreased in the SIR arm compared with the AZA arm (relative risk, 0.67 [95% confidence interval, 0.55, 0.82]; P < 0.01). There was a higher rate of adverse events leading to early discontinuation of SIR (64%) compared with AZA (49%) during the course of this study. CONCLUSIONS: Sirolimus, an mTOR inhibitor, did not decrease the incidence of acute rejection at 1 year compared with azathioprine in lung transplantation. These results differ from previous results in cardiac and renal transplantation and emphasize the need for multicenter randomized controlled trials in lung transplantation. Clinical trial registered with www.clinicaltrials.gov (NCT 00321906).

Emerging bacterial, fungal, and viral respiratory infections in transplantation.

Kidney, liver, heart, pancreas, lung, and small intestine transplantations are viable therapeutic options for patients with end-stage organ failure. Ongoing advancements of surgical techniques, immunosuppressive regimens, and perioperative management have resulted in improved survival of allograft recipients. Despite these refinements, infections still contribute to substantial morbidity and mortality, limiting long-term success rates of these procedures. This article discusses the emerging bacterial, fungal, and viral respiratory infections in transplantation.

Concomitant pulmonary infection with Nocardia transvalensis and Aspergillus ustus in lung transplantation.

Lung infections with Nocardia and Aspergillus spp in lung transplant recipients (LTRs) create diagnostic and therapeutic challenges. The present case illustrates the difficulties in identifying these pathogens in LTRs. A high degree of clinical suspicion and aggressive early management are required to ensure good outcomes. Although prospective data on treating these conditions are scarce, the empiric use of combination broad-spectrum anti-microbials initially seems prudent.

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(+)CD28(null) 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(+)CD28(null) 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 (r(s) = 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(+)/CD4(total)>or=82%, compared to 9+/-9% among those with more extensive CD28 down-regulation (CD4(+)CD28(+)/CD4(total)<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(+)CD28(null) 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.

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 CFTR(inh), 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 approximately 26 mM, significantly lower than that of the control ( approximately 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. CFTR(inh)172 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.

Utility of the 6-minute walk test following lung transplantation.

BACKGROUND: The 6-minute walk test (6-MWT) has replaced standard cardiopulmonary exercises for the evaluation of lung disease. However, data on the utility and characteristics of the 6-MWT following lung transplant are lacking. This study aimed to determine if 6-MWT distance has a normal distribution at 6 months post-transplant and if lower 6-MWT distance was predictive of all-cause mortality. METHODS: We performed a retrospective chart review of 6-MWT data on all patients who were lung transplant recipients at Ochsner Medical Center between 2000 and 2005. Forty-nine lung transplant recipients completed a 6-MWT at 6 months following transplant. Of these 49 patients, 34 had completed both the 6-month and 12-month 6-MWT, and data from these were used to evaluate change in distance walked over time. RESULTS: The mean age was 46 ± 16 years, 57% were female, and 69% received a bilateral lung transplant. Normal distribution by Kolmogorov-Smirnov was demonstrated for 6-MWT distance at 6 months (P  =  0.873). Mean distance walked improved from 348 ± 15 m to 478 ±14 m at 12 months (P  =  0.0001). The 6-MWT distance at 6 months was not a predictor of survival (OR  =  1.002). CONCLUSIONS: Distance for the 6-MWT followed a normal distribution following lung transplant, and distances walked continued to improve for a year following transplant. Although 6-MWT distances are not a predictor of survival, other components of the test may strengthen the predictive value for morbidity and mortality post-transplant.