American Society of Transplantation executive summary on pediatric lung transplantation.

Lung transplantation in children poses distinctly different challenges from those seen in the adult population. This consensus statement reviews the experience in the field of pediatric lung transplantation and highlights areas that deserve further investigation.

A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects with mild lung disease.

Cystic fibrosis (CF) is one of the most common autosomal recessive disorders in North America, leading to significant morbidity and early mortality. The defect in the cystic fibrosis transmembrane conductance regulator protein (CFTR) function can be corrected in vitro by gene replacement with a wild-type gene. A Phase I, single administration, dose escalation trial was designed and executed to assess safety and delivery of tgAAVCF, an adeno-associated virus (AAV) vector encoding the human CFTR cDNA, by nebulization to the lungs of CF subjects. Four cohorts of three subjects each were administered increasing doses of the study agent, beginning with 10(10) DNase-resistant particles (DRP) and escalating in log increments up to 10(13) DRP. Sequential bronchoscopies were performed to gather analytical samples throughout the study. All 12 subjects completed the study. There were a total of 242 adverse events (AEs), six of which were defined as serious and three of which were defined as possibly being related to the study drug. A clear dose-response relationship was observed in vector gene transfer. A maximum of 0.6 and 0.1 vector copies per brushed cell were observed 14 days and 30 days, respectively, following nebulization of 10(13) DRP tgAAVCF, and this declined to nearly undetectable levels by day 90. Vector gene transfer was evenly distributed throughout the fourth airway generation following single-dose administration. RNA-specific PCR did not detect vector-derived mRNA. This Phase I trial shows that aerosolized tgAAVCF is safe and widely delivered to the proximal airways of CF subjects by nebulization.

Preimmunization anti-pneumococcal antibody levels are protective in a majority of patients with cystic fibrosis.

OBJECTIVE: Although invasive pneumococcal disease is infrequent in cystic fibrosis (CF), it is recommended that all patients with CF receive pneumococcal immunization. As part of a comprehensive program to immunize our clinic population, we obtained preimmunization anti-pneumococcal antibody levels. We hypothesized that the percentage of CF patients without protective levels of anti-pneumococcal antibody levels would be high, as they are exposed to frequent antibiotic therapy that may eradicate organisms before generation of an antibody response. METHODS: An observational study of 100 patients with CF, aged 1 to 39 years, was conducted in a regional CF center. Preimmunization anti-pneumococcal antibody levels against 6 serotypes were measured by enzyme-linked immunosorbent assay. Protective antibody levels were defined as >200 ng/mL. RESULTS: A majority of CF patients-61% to 100%, depending on age and serotype-had protective levels of pneumococcal antibody. There was a significant positive correlation between antibody level and age for 5 of the 6 serotypes tested. CONCLUSIONS: In contradistinction to our hypothesis, the majority of CF patients have protective preimmunization anti-pneumococcal antibody levels. However, a significant proportion-between 17% and 39%, depending on the serotype-did not exhibit adequate levels. Therefore, we concur with current recommendations for pneumococcal immunization in CF.

Bronchoscopically administered recombinant human DNase for lobar atelectasis in cystic fibrosis.

Lobar atelectasis is a common complication of cystic fibrosis. The majority of cases respond to intravenous antibiotics and chest physiotherapy. In a subgroup of patients, atelectasis is resistant to medical therapy, and its persistence in the pediatric population is associated with a poor prognosis. Bronchoscopic instillation of human recombinant DNase expanded atelectatic lobes in three children resistant to at least 2 weeks of medical therapy. This method of administration of DNase has been successful in resistant cases of lobar atelectasis in patients with chronic obstructive pulmonary disease, quadriplegia, and status asthmaticus. Purulent cystic fibrosis sputum has a very high DNA content, and DNA has been shown to become more pourable in vitro when treated with rhDNase. Bronchoscopic instillation of rhDNase should be considered in cases of persistent lobar atelectasis unresponsive to medical therapy.

FEV(1) as a guide to lung transplant referral in young patients with cystic fibrosis.

Forced expiratory volume in one second (FEV(1)) is widely used to guide referral of patients with cystic fibrosis (CF) for lung transplantation. We reasoned that the best FEV(1) in a 6-month period (bFEV(1)) would be a useful marker of the need for transplant referral. We examined both the rate of decline and different threshold values of bFEV(1) as prognostic indicators in young CF patients. In a case-control analysis, rates of decline in and threshold values of bFEV(1) of 28 patients dying between ages 7-18 years from 1980-1997 were compared to those of 28 age- and gender-matched controls. The threshold analysis of bFEV(1) values was then applied to all patients under age 19 years followed in our clinic from 1993-1997. The rate of decline in bFEV(1) differed for cases and controls over the 4-year period prior to death, but not from years 2-4 prior to death, the time at which transplant referral decisions should be made. A bFEV(1) value of 50% predicted at 2 years prior to the death of the case selected 14 of 28 cases and one control. When applied to all pediatric patients followed from 1993-1997, a bFEV1 threshold of 50% predicted selected 2 of the 3 patients who died and 3 of the 140 patients who survived. Consideration of lung transplantation referral should begin when young CF patients have a best FEV(1) <50% predicted despite aggressive conventional treatment.

Nonproteolytic role for the urokinase receptor in cellular migration in vivo.

The urokinase receptor (uPAR) binds and localizes urokinase activity at cellular surfaces, facilitating fibrinolysis and cellular migration at sites of tissue injury. uPAR also participates in cellular signaling and regulates integrin-dependent adhesion and migration in vitro. We now report evidence that uPAR occupancy regulates cellular migration in vivo in the absence of functional urokinase. Recombinant murine KC (1.5 microg), a potent neutrophil chemoattractant, was delivered to the lungs of wild-type, urokinase-deficient or uPAR-deficient mice 18 h after intraperitoneal injection of 200 microg human immunoglobulin G (IgG) or a fusion protein composed of an amino-terminal receptor-binding fragment of urokinase and a human IgG Fc fragment (GFD-Fc). Whole lung lavage for recovery of leukocytes was performed 4 h later. KC treatment resulted in a 100-fold increase in lavage neutrophils. GFD-Fc injection resulted in >50% reduction in neutrophil influx in both wild-type and urokinase-deficient animals but had no effect on uPAR -/- mice. A concomitant reduction in alveolar protein leakage but no change in numbers of circulating neutrophils accompanied this attenuated inflammatory response. The reduction in neutrophil influx induced by GFD-Fc is thus related to uPAR occupancy and yet not due to disruption of uPAR-mediated proteolysis. These observations verify that protease-independent functions of uPAR operate in vivo and identify uPAR as a potential target for regulation of inflammatory processes characterized by neutrophil-mediated injury.

Role of urokinase receptor and caveolin in regulation of integrin signaling.

Emerging evidence indicates a prominent role for non-integrin membrane adaptors in the dynamic regulation of integrin signaling. Two such integrin-associated proteins are the glycosylphosphatidyl-inositol (GPI)-linked urokinase receptor (u-PAR) and the cholesterol-binding protein, caveolin-1. Recent studies indicate that caveolin is required for the association of Src-family kinases with beta 1 integrins. Loss of caveolin/beta 1 integrin association results in loss of ligand-induced focal adhesion kinase (FAK) phosphorylation and impaired development of focal adhesion sites. Similarly, fibronectin-dependent fyn signaling through alpha 5/beta 1 leading to mitogen-activated protein (MAP) kinase activation requires the presence of caveolin-1. Caveolin binds Src-family kinases and such binding maintains these kinases in an inactive state. Current evidence favors a model in which ligand-induced integrin clustering, a central event in integrin activation, promotes caveolin oligomerization leading to release and/or activation of Src-family kinases and initiation of integrin signaling. The presence of u-PAR promotes these events because the extracellular domain(s) of u-PAR binds to beta 1 and beta 2 integrins and the GPI anchor of u-PAR, like that of other GPI-anchored proteins, interacts with cholesterol-rich membrane domains enriched in caveolin and tyrosine kinases. Integrins, caveolin, and u-PAR form interdependent functional complexes, promoting the association of integrins with caveolin-rich signaling domains. During states of accelerated cellular migration, such as during inflammation and tumorigenesis, expression of u-PAR may be a key facilitator of integrin signaling. Interruption of u-PAR/integrin interactions may be a strategy to regulate cellular migration in these settings.

Pediatric lung transplantation and "lessons from Green Surgery.".

During the past decade, lung transplantation has emerged as the definitive treatment for children with end-stage lung disease. Pediatric transplantation presents unique challenges with respect to diagnostic indications, donor-recipient size disparities, perioperative management, and growth. Lessons from the early development of cardiac surgery at the University of Minnesota (Green Surgical Service) provide a useful model for novel surgical challenges. Since 1990, 25 lung transplantations have been performed at our institution, including 4 heart-lung, 3 single-lung, 17 bilateral-lung, and 1 living-related lobar allograft. Age at transplantation ranged from 7 months to 27 years. The most common indication was cystic fibrosis. Given the limited donor pool, size disparities between donor and recipient were frequent. Excessive donor size was addressed by parenchymal reduction. Accommodation of small donor allografts was facilitated by elective cardiopulmonary bypass and pulmonary vasodilation using inhaled nitric oxide. Epidural anesthesia was routinely used for postoperative pain management and to enhance good pulmonary hygiene. Immunosuppression is presently achieved using cyclosporine, mycophenolate mofetil, and corticosteroids. Monitoring for rejection is accomplished with spirometry and transbronchial biopsies. Bronchial complications in 2 patients required placement of Palmaz stents. The living-related allograft was performed in a previous bone marrow transplant recipient obviating the need for long-term immunosuppression. The potential for growth of mature lung parenchyma postoperatively was studied and verified in a sheep model. Our experience parallels that of other frontiers such as early cardiac surgery in which medical and technologic innovations can be applied in a supportive environment to permit surgical progress.

Plasmin and plasminogen activator inhibitor type 1 promote cellular motility by regulating the interaction between the urokinase receptor and vitronectin.

The urokinase receptor (uPAR) coordinates plasmin-mediated cell-surface proteolysis and promotes cellular adhesion via a binding site for vitronectin on uPAR. Because vitronectin also binds plasminogen activator inhibitor type 1 (PAI-1), and plasmin cleavage of vitronectin reduces PAI-1 binding, we explored the effects of plasmin and PAI-1 on the interaction between uPAR and vitronectin. PAI-1 blocked cellular binding of and adhesion to vitronectin by over 80% (IC50 approximately 5 nM), promoted detachment of uPAR-bearing cells from vitronectin, and increased cellular migration on vitronectin. Limited cleavage of vitronectin by plasmin also abolished cellular binding and adhesion and induced cellular detachment. A series of peptides surrounding a plasmin cleavage site (arginine 361) near the carboxy-terminal end of vitronectin were synthesized. Two peptides spanning res 364-380 blocked binding of uPAR to vitronectin (IC50 approximately 8-25 microM) identifying this region as an important site of uPAR-vitronectin interaction. These data illuminate a complex regulatory scheme for uPAR-dependent cellular adhesion to vitronectin: Active urokinase promotes adhesion and also subsequent detachment through activation of plasmin or complex formation with PAI-1. Excess PAI-1 may also promote migration by blocking cellular adhesion and/or promoting detachment, possibly accounting in part for the strong correlation between PAI-1 expression and tumor cell metastasis.

Lack of association of secretory component with IgA in J chain-deficient mice.

J chain has been proposed to play a role in the mucosal transport of polymeric Igs (pIg) by the polymeric Ig receptor (pIgR). We have previously reported the generation of J chain-deficient mice. These mice exhibited elevated serum IgA and depleted biliary and fecal IgA levels compared with wild-type mice. We report here that, unlike the IgA levels in bile and feces, IgA levels in local mucosal and glandular secretions were not depressed in J chain-deficient mice. Breast milk, intestinal mucosal surface, and nasal wash IgA levels in the mutant mice were similar to wild-type mice while bronchoalveolar lavage IgA levels were higher in the J chain-deficient animals. Western blot analysis with an Ab to secretory component (SC), the portion of the pIgR that remains bound to pig in secretions, and immunoprecipitation with Abs directed against IgA showed that secreted IgA was associated with SC in wild-type but not J chain-deficient mice. The IgA in wild-type secretions was polymeric while the secretions of J chain-deficient mice contained IgA monomers and other nonpolymeric IgA forms. Thus, J chain is not essential for IgA transport by intestinal, mammary, or respiratory epithelia but is necessary for the stable association of pIgA with SC. Further, we suggest that J chain-deficient IgA is transported into secretions by a different mechanism than wild-type IgA.

Identification of the urokinase receptor as an adhesion receptor for vitronectin.

Urokinase receptors, expressed on surfaces of many cell types, focus to the pericellular space plasminogen-dependent proteolysis important in matrix remodeling and cell movement. We now report that the urokinase receptor (uPAR) is also a high affinity (Kd < 30 nM) receptor for vitronectin. Recombinant uPAR binds vitronectin in the absence of urokinase, but vitronectin binding is promoted by concurrent receptor binding of either urokinase or fragments thereof containing its uPAR binding domain. Stable epithelial cell transfectants expressing membrane-anchored uPAR, but not cells expressing soluble uPAR, become strongly adhesive with altered morphology in the absence of urokinase. These observations identify a new class of vitronectin receptor and imply a duality in function for the receptor that intrinsically links matrix adhesion to regulation of protease activity. Increases in urokinase receptor expression known to be associated with cellular activation and malignant transformation could modulate cellular trafficking and function by promoting attachment to vitronectin.

Reversible cellular adhesion to vitronectin linked to urokinase receptor occupancy.

Urokinase receptors are distributed on surfaces of many cell types where they are thought to focus plasminogen-dependent proteolysis important to migration and tissue remodeling to the immediate pericellular space. In addition to its well characterized role in proteolysis, urokinase receptor binding per se promotes the adhesiveness of leukemic cell lines exposed to differentiating cytokines in vitro. We sought to determine if a serum or matrix component is involved in urokinase-dependent adhesion. We now report that cytokine-stimulated human myelomonocytic cells express a divalent cation- and Arg-Gly-Asp-independent high affinity receptor for urea-purified vitronectin (Kd < 10 nM). Soluble native vitronectin does not effectively bind to the receptor, while cellular adhesion was noted to both urea-purified and native vitronectin when adsorbed to plastic. The activity of this receptor is tightly coupled to urokinase receptor occupancy. Urokinase receptor binding thus induces selective and reversible cellular adhesion to the matrix form of vitronectin. Because transfer of vitronectin-bound plasminogen activator inhibitor type 1 to urokinase promotes rapid turnover of receptor-bound enzyme, these results illuminate a novel binding cycle by which urokinase receptor occupancy coordinately regulates cellular adhesiveness and pericellular proteolysis.

Cytokines induce urokinase-dependent adhesion of human myeloid cells. A regulatory role for plasminogen activator inhibitors.

Differentiation of monocytic precursors often results in adhesive properties thought to be important in migration. In this study, the influence of cytokines, known to induce macrophage differentiation, on the adhesiveness of the monocytic cell line U937 was examined in vitro. Despite development of a macrophage morphology, < 5% of cytokine-stimulated U937 cells were adherent at 24 h. Addition of 1-10 nM urokinase-type plasminogen activator (uPA) induced adherence in the presence of transforming growth factor type beta-1, 1,25-(OH)2 vitamin D3, granulocyte macrophage colony-stimulating factor, or tumor necrosis factor alpha. uPA-dependent adhesiveness was reversible after 24 h of stimulation with cytokines and uPA as adherence was prevented by the subsequent addition of anti-uPA antibodies. Adherence induced by diisopropylfluorophosphate-inactivated uPA was severalfold greater than that seen with active uPA. This difference was largely due to cell-surface turnover of active uPA complexed with plasminogen activator inhibitor (PAI). These data indicate that cytokines prime monocyte progenitors for uPA receptor-mediated signals leading to adherence, continued uPA receptor occupancy is required for adherence, and PAI decreases adherence by promoting clearance of uPA/PAI complexes. Thus the interaction of uPA and PAI at the cell surface, known to affect extracellular matrix proteolysis and hence myeloid cell migration, also regulates adhesion. The coordinated regulation of these two uPA functions by PAI may enhance the migratory potential of monocytic cells.

Task-dependent hemisphere asymmetries of the visual evoked potential.

Stimulus-related and event-related components of the visual evoked potential were recorded in three separate tasks that required language comprehension or spatial discrimination. Task-dependent asymmetries in the evoked potential were found for both the language task and one of the tasks involving spatial discrimination. Moreover, an identical stimulus presented in two different contexts elicited potentials with a significantly different distribution over the two sides of the head. These asymmetries were largely confined to the event-related components and presumably reflect task-dependent differences in neural processing. Reaction-time studies for these tasks, however, indicated that these asymmetries occurred too late to arise from neural structures responsible for language comprehension or spatial discrimination per se, and must therefore reflect subsequent neural events.