The structure of Sec12 implicates potassium ion coordination in Sar1 activation.

Coat protein II (COPII)-coated vesicles transport proteins and lipids from the endoplasmic reticulum to the Golgi. Crucial for the initiation of COPII coat assembly is Sec12, a guanine nucleotide exchange factor responsible for activating the small G protein Sar1. Once activated, Sar1/GTP binds to endoplasmic reticulum membranes and recruits COPII coat components (Sec23/24 and Sec13/31). Here, we report the 1.36 Å resolution crystal structure of the catalytically active, 38-kDa cytoplasmic portion of Saccharomyces cerevisiae Sec12. Sec12 adopts a ß propeller fold. Conserved residues cluster around a loop we term the "K loop," which extends from the N-terminal propeller blade. Structure-guided site-directed mutagenesis, in conjunction with in vitro and in vivo functional studies, reveals that this region of Sec12 is catalytically essential, presumably because it makes direct contact with Sar1. Strikingly, the crystal structure also reveals that a single potassium ion stabilizes the K loop; bound potassium is, moreover, essential for optimum guanine nucleotide exchange activity in vitro. Thus, our results reveal a novel role for a potassium-stabilized loop in catalyzing guanine nucleotide exchange.

Palliative care referrals after lung transplantation in major transplant centers in the United States.

OBJECTIVE: Although lung transplantation is a widely used treatment modality for patients with end-stage lung disease, its long-term outcomes are limited. Including palliative approaches in the care of lung transplant recipients may be beneficial; however, systematic information regarding the utilization of palliative care services for lung recipients is lacking. DESIGN AND SETTING: Of the 27 transplant centers meeting the inclusion criteria (an annual lung transplant volume >or=15 for the past 5 years and the availability of palliative care or pain services at the center), 74 clinicians representing either the transplant or palliative care program from 18 centers completed surveys. RESULTS: Both transplant and palliative care clinician respondents strongly favored the idea of integrating palliative care into lung transplant care. However, the number of palliative care referrals made during the last year was low (

CD28 down-regulation on CD4 T cells is a marker for graft dysfunction in lung transplant recipients.

RATIONALE: Repeated antigen-driven proliferations cause CD28 on T cells to down-regulate. We hypothesized that alloantigen-induced proliferations could cause CD28 down-regulation in lung transplant recipients. OBJECTIVES: To ascertain if CD28 down-regulation on CD4 T cells associated with manifestations of allograft dysfunction in lung transplant recipients. METHODS: Peripheral blood CD4 T cells from 65 recipients were analyzed by flow cytometry, cytokine multiplex and proliferative assays, and correlated with clinical events. MEASUREMENTS AND MAIN RESULTS: Findings that CD28 was present on less than 90% of total CD4 T cells were predominantly seen among the recipients with bronchiolitis obliterans syndrome (specificity = 88%). Perforin and granzyme B were produced by >50% of the CD4(+)CD28(null) cells, but less than 6% of autologous CD4(+)CD28(+) cells (P < 0.006). CD4(+)CD28(null) cells also had increased productions of proinflammatory cytokines, but less frequently expressed regulatory T-cell marker FoxP3 (2.1 +/- 1.3%), compared with autologous CD4(+)CD28(+) (9.5 +/- 1.4; P = 0.01). Cyclosporine A (100 ng/ml) inhibited proliferation of CD4(+)CD28(null) cells by 33 +/- 11% versus 68 +/- 12% inhibition of CD4(+)CD28(+) (P = 0.025). FEV(1) fell 6 months later (0.35 +/- 0.04 L) in recipients with CD4(+)CD28(+)/CD4(total) less than 90% (CD28% Low) compared with 0.08 +/- 0.08 L among CD4(+)CD28(+)/CD4(total) (CD28% High) greater than 90% (CD28% High) recipients (P = 0.013). Two-year freedom from death or retransplantation in CD28% Low recipients was 32 +/- 10% versus 78 +/- 6% among the CD28% High subjects (P < 0.0001). CONCLUSIONS: CD28 down-regulation on CD4 cells is associated with bronchiolitis obliterans syndrome and poor outcomes in lung transplantation recipients. CD4(+)CD28(null) cells have unusual, potentially pathogenic characteristics, and could be important in the progression of allograft dysfunction. These findings may illuminate a novel paradigm of transplantation immunopathogenesis, and suggest that CD28 measurements could identify recipients at risk for clinical deteriorations.

Malignancy in lung transplantation: biomarkers, gender differences, and consideration of a systems biology approach.

We present here new molecular tools such as gene expression microarrays as promising biomarkers for diagnosis and prognosis in lung cancer as well as for an early diagnosis of malignancy after lung transplantation. Data regarding gender differences in survival, recently collected in a single-institution study, may allow better targeting of immunosuppression goals to maintain effectiveness while minimizing cancer development and progression. Combining these differing types of data using an integrative approach that is the central theme of systems biology may allow us to provide powerful and more individualized risks of cancer after lung transplantation.

Gender differences in survival after lung transplant: implications for cancer etiology.

Predictors of survival after lung transplant were analyzed in a large cohort of 990 lung transplanted patients from a single center. The overall survival was 41.6%, (41.5% in males, and 41.8% in females), the average length of the follow up was 45.84+/-51.98 months (range 0-282.47 months). Females tend to live longer than males: 50.75+/-55.41 months versus 40.64+/-47.60 months, respectively. Males had a risk of dying during the follow up that was 1.18 (95% CI 1.01-1.40) relative to females, after adjusting for ethnicity, age, smoking status, diagnosis and donor characteristics. Females who had at least one full term pregnancy during their life had better survival rates than females who had no full term pregnancies. Our results of a better survival after lung transplant in females, and among them in those who had at least a full term pregnancy support the hypothesis of a hormonal contribution to survival and of the development of immunotolerance after pregnancy. This model could be useful for understanding the role of immunity in cancer development.

Course of illness after the onset of chronic rejection in lung transplant recipients.

BACKGROUND: Despite the overall negative impact of chronic rejection on quality of life and survival after lung transplant, the specific clinical indicators of deterioration have not been identified. OBJECTIVES: To describe the course of illness after the onset of chronic rejection, including demographic and transplant variables, morbidity, mortality, health resource utilization, and end-of-life care, and to identify clinical indicators of deterioration in health and limited survival after the onset of chronic rejection. METHODS: The medical records of 311 recipients of lung transplants between 1998 and 2004 were reviewed retrospectively to identify 60 recipients who experienced chronic rejection. RESULTS: Median survival after chronic rejection was 31.34 months. Time to rejection (mean, 26.05 months; SD, 16.85) was significantly correlated with overall survival without need of a retransplant (r = 0.64; P < .001). The earlier the onset of chronic rejection or the need for oxygen at home, the shorter was the period of survival after chronic rejection and the more frequent were hospital and intensive care unit admissions and prolonged stays. Of the 26 recipients who died, 65% died at the transplant center, and all but 1 died in the intensive care unit; 3 died after multiple attempts of cardiopulmonary resuscitation; life support was ultimately withdrawn in 69%. CONCLUSIONS: Lung transplant recipients who experience chronic graft rejection have high rates of morbidity, mortality, and health resource utilization; however, the course of illness after chronic rejection is highly variable.

Binding of mRNA to the bacterial translation initiation complex.

Translation initiation is a key step for regulating the synthesis of several proteins. In bacteria, translation initiation involves the interaction of the mRNA with the ribosomal small subunit. Additionally, translation initiation factors 1, 2, and 3, and the initiator tRNA, also assemble on the ribosomal small subunit and are essential for efficiently recruiting an mRNA for protein biosynthesis. In the following chapter, we describe fluorescence-based methods for studying the interaction of mRNA with the bacterial initiation complex. Model mRNAs with a covalently attached fluorescent probe showed an increase in fluorescence intensity when bound to the bacterial initiation complex. Utilizing the increase in fluorescence intensity upon mRNA binding to the bacterial initiation complex, we determined the equilibrium binding constants and the association and dissociation rate constants. These methods are important for quantitatively analyzing the effects of mRNA secondary structure and the role of the initiation factors in recruitment of mRNA by the bacterial initiation complex.

Aspergillus galactomannan antigen in the bronchoalveolar lavage fluid for the diagnosis of invasive aspergillosis in lung transplant recipients.

BACKGROUND: The clinical utility of Platelia Aspergillus enzyme immunoassay (EIA) for galactomannan (GM) antigen detection in bronchoalveolar lavage (BAL) for the diagnosis of invasive aspergillosis (IA) in lung transplant recipients is not known. METHODS: BAL fluid samples from consecutive lung transplant recipients who underwent bronchoscopy were prospectively analyzed for GM. RESULTS: A total of 333 BAL samples from 116 patients were tested. Invasive aspergillosis was documented in 5.2% (6/116) of the patients. Samples analyzed included 9 BALs from two patients with proven IA, 19 BALs from four patients with probable IA, and 305 BALs from 110 patients without IA. At the index cutoff value of > or =0.5, the sensitivity was 60%; specificity was 95%, with positive and negative likelihood ratios of 14 and 0.41, respectively. Increasing the index cutoff value to > or =1.0 yielded a sensitivity of 60%, a specificity of 98%, and the positive and negative likelihood ratios of 28 and 0.40, respectively. Two of six patients with IA receiving antifungal prophylaxis had false-negative results. CONCLUSIONS: A Platelia EIA index cut-off > or =1.0 in the BAL fluid in a lung transplant recipient with a compatible clinical illness may be considered as suggestive of IA.

Evaluation of the electronic version of the questionnaire for lung transplant patients.

UNLABELLED: Context-Recent modifications to the QLTP (Questionnaire for Lung Transplant Patients), including changing items from dichotomous to multiple dimension scaling, adding psychological symptoms, and converting to an electronic format (e-QLTP), made it necessary to reevaluate its reliability, validity, recipient satisfaction, and feasibility of administering the e-QLTP in the clinical setting. Purpose-To report the final modifications, psychometric properties, recipient satisfaction, and feasibility of administering the e-QLTP, a patient report outcome measure of symptoms and activity tolerance. Methods-Sixty lung recipients completed the original QLTP and the e-QLTP and rated their satisfaction with the e-version during a routine posttransplant evaluation; 65% (38 of 60) also completed a retest version. Correlations were computed for retest stability, concurrent validity between versions of the QLTP, and construct validity among the subscales of the e-QLTP and forced expiratory volumes in 1 second. Using the After Scenario Questionnaire, participants rated their satisfaction with the ease, amount of time, and support information when completing the e-QLTP. RESULTS: The e-QLTP and subscales were internally consistent (alpha = .73 - .90) and stable (intraclass correlations = .47 - .93). Significant correlations (P = .001) were found between the e-QLTP and the original QLTP (r = 0.53-0.56) and between the e-QLTP subscales and forced expiratory volumes in 1 second (r = 0.51 - 0.53). The overall mean satisfaction score was 1.27 (+/- 0.47). Conclusions-The e-QLTP is a reliable and valid measure of physical and psychological symptoms after lung transplantation. It is feasible to complete in the clinical setting and recipients are highly satisfied with its use. Its computerized functionality enhances assessment and management of symptoms over time.

Considerations for optimal management of patients with pulmonary arterial hypertension: a multi-stakeholder roundtable discussion.

A roundtable panel of national and regional managed care decision makers and providers met to discuss pulmonary arterial hypertension (PAH) and strategies for management. As a rare, complex disease with high economic costs and potentially devastating outcomes, PAH necessitates that managed care providers balance optimal care with efficient use of healthcare resources. PAH specialists are recognized by health plans as knowledgeable experts and integral partners in managing patients and resources. The diagnosis of PAH must be confirmed by a right heart catheterization. Available therapies are indicated almost exclusively for patients with PAH (riociguat is also indicated in chronic thromboembolic pulmonary hypertension) and target 1 of 3 pathways: endothelin receptor antagonists for the endothelin pathway; phosphodiesterase type-5 inhibitors and soluble guanylate cyclase stimulators for the nitric oxide pathway; and prostanoids as well as a prostacyclin receptor agonist for the prostacyclin pathway, with combination therapy becoming more common. Even in the modern treatment era, as shown in the REVEAL and French registries, PAH is often diagnosed years after symptoms first appear, which leads to a poor prognosis and increased burden on the healthcare system. Facilitating treatment of patients with PAH through centers of excellence, and coordinating care management between health plans and providers with evidence-based approaches can lead to both better results for patients and lower healthcare costs. When PAH experts have access to the right treatments for the right patients at the right time, they can work with insurers to improve the health of patients with PAH while helping to reduce the impact on the healthcare system.

Rapid kinetic analysis of EF-G-dependent mRNA translocation in the ribosome.

Precise and coordinated movement of the tRNA-mRNA complex within the ribosome is a fundamental step during protein biosynthesis. The molecular mechanism for this process is still poorly understood. Here we describe a new sensitive method for monitoring elongation factor G-dependent translocation of the mRNA in the ribosome. In this method, the fluorescent probe pyrene is covalently attached to the 3' end of a short mRNA sequence at position +9. Translocation of the mRNA by one codon results in a significant decrease in the fluorescence emission of pyrene and can be used to directly monitor mRNA movement using rapid kinetic methods. Importantly, this method offers the flexibility of using any tRNA or tRNA analog in order to elucidate the molecular mechanism of translocation. Our results show that the mRNA is translocated at the same rate as the tRNAs, which is consistent with the view that the movement of the tRNAs and the mRNA are coupled in the ribosome. Furthermore, an anticodon stem-loop analog of tRNA is translocated from the ribosomal A site at a rate constant that is 350-fold lower than peptidyl tRNA, indicating that the D stem, T stem and acceptor stem of A site tRNA contribute significantly to the rate of translocation.

Mediastinal abscess due to passage of a broncholith.

We report a case of a 32-year-old woman who, after passage of broncholiths, developed a mediastinal abscess that required surgical drainage for treatment. Previously reported infectious complications resulting from broncholiths include obstructive pneumonitis and recurrent aspiration pneumonitis secondary to bronchoesophageal fistulas. Because radiographic evidence of abnormal calcification in the chest is common, but rarely is associated with broncholithiasis, the patient's history of lithoptysis was crucial to determining the underlying etiology of her abscess.

The course of neurofibromatosis type 1 on immunosuppression after lung transplantation: report of 2 cases.

We describe 2 patients with neurofibromatosis type 1 (NF1) who underwent lung transplantation. Case 1 reports a patient with NF1 with no complications 5 years after bilateral lung transplantation. Case 2 details a patient with NF1 diagnosed with both post-transplant lymphoproliferative disorder (PTLD) and massive intra-abdominal sarcoma consistent with a malignant nerve sheath tumor 9 months after lung transplantation. There was no clinical evidence of sarcoma preceding or immediately following lung transplant as demonstrated by a normal abdominal sonogram 3 months post-transplantation. Although an increased risk for cancer has been documented in NF1, the rapid malignant degeneration of a neurofibroma following transplantation raises concern about immunosuppression and transplantation candidacy among individuals with NF1.

Cadaveric donor selection and management.

The current availability of lung donors is far exceeded by the number of potential transplant recipients who are waiting for an organ. This disparity results in significant morbidity and mortality for those on the waiting list. Although it is desirable to increase overall consent rates for organ donation, doing so requires an intervention to affect societal response. In contrast, increased procurement of organs from marginal donors and improved donor management may be realized through increased study and practice changes within the transplant community. Transplantation of organs from marginal or extended-criteria donors may result in some increase in complications or mortality, but this possibility must be weighed against the morbidity and risk of death risk faced by individuals on the waiting list. The effects of this trade-off are currently being studied in kidney transplantation, and perhaps in the near future lung transplantation may benefit from a similar analysis. Until that time, the limited data regarding criteria for donor acceptability must be incorporated into practice to maximize the overall benefits of lung transplantation.

Clinical trial designs in PAH: shifting from functional measurements to long-term clinical outcomes.

Pulmonary arterial hypertension (PAH) is a rare disease of the pulmonary vasculature that leads to right ventricular dysfunction, right ventricular failure, and premature death. There are a number of medications already on the market, representing different therapeutic classes and possessing multiple mechanisms of action. Three new agents were approved by the US Food and Drug Administration in 2013, and others are currently in development. Recent advancements in PAH have resulted in increased survival and improved quality of life; however, no therapy provides a cure. Experts in the field are now utilizing clinical trial designs and end points that better reflect the disease progression among patients with this chronic disease. Although randomized placebo-controlled monotherapy trials are considered the strongest design, ethical and practical considerations have led to an increasing number of randomized trials designed to compare a PAH-specific treatment with placebo as an add-on to standard therapy. As many patients who enroll in clinical trials are already being treated for their condition, it may be unethical to withdraw or delay lifesaving therapies. The most widely used primary end point for PAH trials, change in 6-minute walk distance (6MWD) from baseline, has substantial limitations. Although it is generally reproducible, inexpensive, and relatively easy to conduct, the 6MWD is not designed to assess disease progression. Recent data have shown that 6MWD has inconsistent correlation with key indicators of disease progression such as hospitalization due to PAH, worsening right-sided heart failure, and death. The Task Force on End Points and Clinical Trial Design that met at the 4th World Symposium on Pulmonary Hypertension (WSPH) in 2008 in Dana Point, California, questioned the clinical relevance of the 6MWD as a primary end point and recommended the use of a composite end point--time to clinical worsening (TTCW)--in phase 3 or pivotal trials. TTCW may include time from randomization to PAH-related hospitalization, need for interventional procedures (ie, lung transplantation or balloon atrial septostomy), and mortality. More recently, at the 5th WSPH, held in 2013 in Nice, France, experts reiterated these recommendations. They further noted that, as clinical trials increasingly allow background therapies and are longer in duration, it may be more meaningful to use primary end points that measure "clinical worsening" rather than 6MWD. This paradigm shift will not only lead to a clearer demonstration of efficacy and safety as new agents come on the market, but will provide important information on long-term benefits (ie, the effects of drugs on clinical deterioration) that can assist payers as they strive to make value-based formulary decisions and provide cost-effective high-quality care.

S1P signaling: new therapies and opportunities.

Development of sphingosine-1-phosphate receptor 1 (S1P1) modulators to dampen inflammation and its sequelae is becoming increasingly promising for treating medical conditions characterized by significant immunopathology. As shown by the non-selective S1P receptor modulator FTY720 (fingolimod [Gilenya(®)]) in the treatment of relapsing-remitting multiple sclerosis (MS), the ability to use S1P1 modulation to precisely block immune cell traffic-immunomodulation-while maintaining immunosurveillance, has opened therapeutic opportunities in various other immune-derived chronic pathologies, including inflammatory bowel disease (IBD), lupus, psoriasis, as well as, potentially, in early acute viral respiratory infection. Proof-of-concept studies across validated animal models with S1P receptor modulators highly selective for S1P1, such as BAF-312 (Siponimod), KRP-203, ONO-4641 (Ceralifimod), ponesimod and RPC-1063, and emerging clinical trials for safety and efficacy in humans, particularly in MS, ulcerative colitis (UC) and psoriasis, have set the stage for us to consider additional testing in various other autoimmune diseases.

World Health Organization Group III pulmonary hypertension.

Pulmonary hypertension in the setting of parenchymal lung disease and conditions associated with chronic hypoxemia is commonly encountered in clinical practice and may adversely affect patients' function and mortality. Diagnosis of this subgroup of pulmonary hypertension has evolved but still requires right heart catheterization for confirmation. The primary treatment goal is optimization of the underlying parenchymal lung or hypoxemia-associated condition prior to consideration of pharmacologic therapy. Limited published experience with pulmonary hypertension-specific medications for treatment of WHO Group 3 pulmonary hypertension suggests symptomatic and functional benefit in selected individuals. The potential for worsening ventilation-perfusion matching must be considered in these cases, however, since there is a paucity of data regarding the optimal approach to treatment selection. Ongoing medication trials and further investigation of mechanisms of hypoxic pulmonary vasoconstriction provide hope for these patients who in the past often had only lung transplantation as a potential treatment option.

Five-year outcomes with alemtuzumab induction after lung transplantation.

BACKGROUND: Induction therapy with alemtuzumab, followed by lower than conventional intensity post-transplant immunosuppression (eg, tacrolimus monotherapy), has been associated with reduced morbidity and mortality in abdominal and heart transplantation. We examined 5-year outcomes in lung recipients receiving alemtuzumab in conjunction with reduced-intensity post-transplant immunosuppression (early lower-dose tacrolimus; lower-dose steroids, with or without mycophenolate mofetil), compared with lung recipients receiving other induction agents or no induction in association with post-transplant immunosuppression. METHODS: A retrospective analysis was performed using prospectively collected data from a single-site clinical database of 336 lung recipients (aged ≥ 18) who received allografts between 1998 and 2005, classified by induction type: alemtuzumab, 127; Thymoglobulin, 43; daclizumab, 73; and none, 93. Survival analyses examined patient and graft survival, and freedom from acute cellular rejection (ACR), lymphocytic bronchiolitis, obliterative bronchiolitis (OB), bronchiolitis obliterans syndrome (BOS), and post-transplant lymphoproliferative disorder (PTLD). RESULTS: Five-year patient and graft survival differed by group (p = 0.046, p = 0.038, respectively). Alemtuzumab patient/graft survival rates were 59%/59%. Survival rates were 60%/44% for Thymoglobulin, 47%/46% for no induction, and 44%/41% for daclizumab. Freedom from ACR, lymphocytic bronchiolitis, OB, and BOS differed by group (all values, p < 0.008); alemtuzumab recipients showed greater 5-year freedom from each outcome (30%/82%/86%/54%) than Thymoglobulin (20%/54%/62%/27%), daclizumab (19%/55%/70%/43%), and no-induction groups (18%/70%/69%/46%). The groups did not differ in PTLD rates (≥ 94% free of PTLD at 5 years; p = 0.864). Effects were unchanged after controlling for potential covariates. CONCLUSIONS: Alemtuzumab induction may be associated with improved outcomes in lung transplantation. Randomized controlled trials are needed to establish any effects of this agent.

Exploring the meaning of chronic rejection after lung transplantation and its impact on clinical management and caregiving.

Although the literature continues to portray chronic rejection after lung transplantation as ominous with no known treatment, no studies have examined family and clinician caregivers' perceptions of the diagnosis of chronic rejection and its impact on the course of clinical care. We explored the meaning and impact of chronic rejection from the perspective of family (n=10) and clinician (n=3) caregivers. We found that family caregivers considered the onset of chronic rejection to be inevitable, irreversible, unpredictable, and going back to pretransplant. Clinicians considered chronic rejection as a harbinger of deterioration and peril and expressed trepidation about informing recipients and their family caregivers about the diagnosis. Despite the heightened caregiving duties and challenges of treating chronic rejection, its unpredictable course and the prospect of retransplant instilled hope for stabilization or cure among most clinicians and caregivers, leading them to support recipients' wishes to pursue potentially futile treatments. Until recipients were no longer competent, caregivers believed all treatment options (including retransplant) had been exhausted, or suffering was prolonged, caregivers were reluctant to halt extraordinary treatment measures. Caregivers perceived that certainty regarding poor prognosis was required for palliative care and that palliative care was end-of-life care. Consequently, trials of aggressive treatment typically precluded palliative care.