Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. II. Transfection efficiency in airway epithelium.

A phase I clinical trial was conducted in which recombinant adenovirus containing the cystic fibrosis trans-membrane regulator (CFTR) (Ad2/CFTR) was administered by bronchoscopic instillation or aerosolization to the lungs of cystic fibrosis (CF) patients. In this paper, we evaluate the efficiency of Ad2/CFTR-mediated transduction of bronchial airway cells. The ability of an Ad2/CFTR vector to transduce airway cells was first evaluated in patients to whom the vector was administered by bronchoscopic instillation. Cells at the administration site were collected 2 days after treatment by bronchoscopic brushing. Ad2-specific CFTR DNA was detected in four of five individuals by PCR, and Ad2-specific CFTR RNA was detected in three of five individuals by RT-PCR. Ad2/CFTR-mediated transduction of airway epithelial cells was then determined in CF individuals receiving this vector by aerosol inhalation. Ad2-specific CFTR DNA was detected in 13 of 13 individuals 2 days after aerosolization, and in 3 of 5 individuals 7 days after aerosolization. Ad2-specific RNA was detected in 4 of 13 individuals on day 2, but was not detected in the 5 individuals tested on day 7. The percentage of airway epithelial cells containing nuclear-localized vector DNA was < or =2.4% as determined by fluorescence in situ hybridization (FISH). However, in some cases, a high percentage of nonepithelial mononuclear cells or squamous metaplastic epithelial cells was infected with the adenoviral vector. In conclusion, aerosol administration is a feasible means to distribute adenoviral vectors throughout the conducting airways, but improvements in adenovirus-mediated transduction of airway epithelial cells are necessary before gene therapy for CF will be effective.

Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. I. Methods, safety, and clinical implications.

Cystic fibrosis (CF), an autosomal recessive disorder resulting from mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene, is the most common lethal genetic illness in the Caucasian population. Gene transfer to airway epithelium, using adenoviruses containing normal CFTR cDNA, leads to transient production of CFTR mRNA and, in some studies, to correction of the airway epithelial ion transport defect caused by dysfunctional CFTR. Inflammatory responses to the adenoviral vector have been reported, particularly at high viral titers. We evaluated the effects of adenovirus-mediated CFTR gene transfer to airway epithelium in 36 subjects with CF (34 individuals, 2 of whom received two separate doses of vector), 20 by lobar instillation and 16 by aerosol administration. Doses ranged from 8 x 10(6) to 2.5 x 10(10) infective units (IU), in 0.5-log increments. After lobar administration of low doses there were occasional reports of cough, low-grade temperature, and myalgias. At the highest lobar dose (2.5 x 10(9) IU) two of three patients had transient myalgias, fever, and increased sputum production with obvious infiltrates on CT scan. After aerosol administration there were no significant systemic symptoms until the 2.5 x 10(10) IU dose, when both patients experienced myalgias and fever that resolved within 24 hr. There were no infiltrates seen on chest CT scans in any of the patients in the aerosol administration group. There were no consistent changes in pulmonary function tests or any significant rise in serum IgG or neutralizing antibodies in patients from either group. Serum, sputum, and nasal cytokines, measured before and after vector administration, showed no correlation with adenoviral dose. Gene transfer to lung cells was inefficient and expression was transient. Cells infected with the vector included mononuclear inflammatory cells as well as cuboidal and columnar epithelial cells. In summary, we found no consistent immune response, no evidence of viral shedding, and no consistent change in pulmonary function in response to adenovirus-mediated CFTR gene transfer. At higher doses there was a mild, nonspecific inflammatory response, as evidenced by fevers and myalgias. Overall, vector administration was tolerated but transfer of CFTR cDNA was inefficient and transgene expression was transient for the doses and method of administration used here.

A clinical inflammatory syndrome attributable to aerosolized lipid-DNA administration in cystic fibrosis.

Immunologic reactivity to lipid-DNA conjugates has traditionally been viewed as less of an issue than with viral vectors. We performed a dose escalation safety trial of aerosolized cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the lower airways of eight adult cystic fibrosis patients, and monitored expression by RT-PCR. The cDNA was complexed to a cationic lipid amphiphile (GL-67) consisting of a cholesterol anchor linked to a spermine head group. CFTR transgene was detected in three patients at 2-7 days after gene administration. Four of the eight patients developed a pronounced clinical syndrome of fever (maximum of 103.3EF), myalgias, and arthralgia beginning within 6 hr of gene administration. Serum IL-6 but not levels of IL-8, IL-1, TNF-alpha, or IFN-gamma became elevated within 1-3 hr of gene administration. No antibodies to the cationic liposome or plasmid DNA were detected. We found that plasmid DNA by itself elicited minimal proliferation of peripheral blood mononuclear cells taken from study patients, but led to brisk immune cell proliferation when complexed to a cationic lipid. Lipid and DNA were synergistic in causing this response. Cellular proliferation was also seen with eukaryotic DNA, suggesting that at least part of the immunologic response to lipid-DNA conjugates is independent of unmethylated (E. coli-derived) CpG sequences that have previously been associated with innate inflammatory changes in the lung.

Effect of noninvasive positive-pressure ventilation on survival in amyotrophic lateral sclerosis.

BACKGROUND: Noninvasive positive-pressure ventilation may benefit patients with amyotrophic lateral sclerosis and respiratory insufficiency. OBJECTIVE: To determine 1) whether patients tolerant of noninvasive positive-pressure ventilation have better survival than intolerant patients and 2) whether bulbar symptoms account for intolerance of noninvasive positive-pressure ventilation. DESIGN: Observational cohort study. SETTING: Tertiary care referral center. PATIENTS: 39 patients with amyotrophic lateral sclerosis who were treated with noninvasive positive-pressure ventilation. INTERVENTION: Noninvasive positive-pressure ventilation was started for patients with new orthopnea, new hypercapnia, or both. Patients were divided into two groups: those tolerant of and those intolerant of noninvasive positive-pressure ventilation. RESULTS: The risk for death from onset of respiratory insufficiency was higher for intolerant patients than for tolerant patients (relative risk, 3.1 [95% CI, 1.8 to 9.6]). Moderate or severe bulbar symptoms were more prevalent among intolerant patients than among tolerant patients (67% compared with 33%; P = 0.04). CONCLUSIONS: Among patients with amyotrophic lateral sclerosis, those who are tolerant of noninvasive positive-pressure ventilation have better survival than do those who are intolerant. Bulbar symptoms partially account for intolerance of noninvasive positive-pressure ventilation.

Safety of a single aerosol administration of escalating doses of the cationic lipid GL-67/DOPE/DMPE-PEG5000 formulation to the lungs of normal volunteers.

Several groups are assessing the use of cationic lipids for respiratory gene therapy. To date no human data are available regarding the safety of intra-pulmonary cationic lipid delivery. In preparation for a trial of pulmonary delivery of the CFTR gene, we have assessed the safety of nebulised lipid GL-67/DOPE/DMPE-PEG5000 (GL-67A), the cationic lipid formulation to be used in this study. Fifteen healthy volunteers were given incremental doses of GL-67A via a Pari LC Jet nebuliser; three volunteers in each of five dosing cohorts with a week interval between cohorts. Markers of safety included clinical assessment, measurement of lung function, chest CT scan, serological testing and analysis of induced sputum. Measurements were taken before administration and at intervals up to 21 days thereafter. No adverse clinical events were seen or any statistically significant changes in spirometry or gas transfer. There were no clinically significant changes in any of the blood parameters and no CT changes were seen. Comparisons of the cellular subpopulations (neutrophils, eosinophils, lymphocytes and macrophages) in induced sputum showed no significant alterations following administration of the GL-67A. This study suggests that a single application of aerosol formulation of GL-67A does not result in clinically detectable changes when given by nebulisation into the lungs of normal volunteers and provides an indication of a lipid dose tolerated in man.

Analysis of the safety of bronchoscopy after recent acute myocardial infarction.

Acute myocardial infarction (AMI) is generally considered to increase the risk of flexible fiberoptic bronchoscopy (FFB). Currently, to our knowledge, no data in the literature support or challenge this concept. We conducted a retrospective chart review for the years 1986 to 1994 of 20 patients (14 men) who underwent 21 FFBs while hospitalized for an AMI. The mean age was 63.8 years (range, 38 to 83 years). Ten patients underwent revascularization procedures (eight coronary artery bypass grafting and two percutaneous transluminal coronary angioplasty) before FFB. The mean period between the AMI and FFB was 11.7 days (range, 1 to 30 days). Indications for FFB were pulmonary infiltrate (n = 10), hemoptysis (n = 6), atelectasis (n = 4), and to localize a suspected bronchopleural fistula (n = 1). Procedures performed included airway examination (21), BAL (12), transbronchial biopsy (2), endobronchial biopsy (3), and endobronchial brushing (4). No procedure was interrupted as a result of an adverse event, and five patients died during the same hospitalization. Four of the deaths occurred 6 to 15 days postprocedure; 1 patient (who had active ischemia at the time of FFB) died 4 h postprocedure. We conclude that FFB is safe in the immediate post-AMI period as long as the patient does not have active ischemia at the time of the procedure.

Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane conductance regulator to the nasal epithelium of patients with cystic fibrosis.

Cystic fibrosis (CF) is a common autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator gene. Recombinant adenoviruses have shown promise as vectors for transfer of CF transmembrane conductance regulator cDNA to airway epithelia and correction of the Cl- transport defect. However, because adenovirus-mediated gene transfer is transient, use of adenovirus as a vector for treatment of CF would require repeated administration. Therefore, we evaluated repeat administration of an adenovirus vector to the nasal epithelium of patients with CF with five escalating doses of up to 10(10) infectious units. There were no detectable adverse affects. All subjects were initially seropositive but developed additional humoral immune responses. The vector partially corrected the defect in airway epithelial Cl- transport in some subjects, although there was variability between subjects and there was less correction with subsequent administration, perhaps because the immune response limited gene transfer. Future work must focus on vectors with increased efficiency and with the ability to evade host defenses.

Surfactant downregulates synthesis of DNA and inflammatory mediators in normal human lung fibroblasts.

The initial inflammatory event in the adult respiratory distress syndrome (ARDS) is followed by fibroproliferation and a cascade of fibroblast-derived mediators. Because lung fibroblasts may be exposed to surfactant as well as inflammatory cytokines during ARDS, we hypothesized that surfactant might modulate fibroblast activity. We previously demonstrated that surfactant inhibited production of inflammatory cytokines from endotoxin-stimulated human alveolar macrophages. In the current study the effects of surfactant on normal human lung fibroblast proliferative capacity and mediator production were examined. Both synthetic (Exosurf) and natural (Survanta) surfactant inhibited fibroblast [3H]thymidine incorporation. Examination of pre-S-phase events indicated stimulation of the immediate response gene, c-fos, and no effect on the G1/S cyclin, cyclin D1, suggesting that the surfactant block occurred elsewhere before S phase. The antioxidant N-acetyl-L-cysteine (NAC), like surfactant, inhibited [3H]thymidine incorporation. Furthermore, menadione, a generator of intracellular H2O2, stimulated fibroblast [3H]thymidine incorporation, and this was inhibited by surfactant. Interleukin-1 (IL-1)-stimulated secretion of the inflammatory mediators, IL-6 and prostaglandin E2, was also inhibited by surfactant. These data suggest that surfactant may modify lung fibroblast participation in ARDS sequelae by downregulating DNA synthesis and secondary inflammatory mediator production.

Community-acquired pneumonia: an update.

Despite the discovery of new pathogens and the evolving problem of antibiotic resistance, the basic trends in community-acquired pneumonia remain remarkably constant. This article reviews the common pathogens, new pathogens, their clinical presentations, the diagnostic workup, the decision to hospitalize, antibiotic resistance, and antibiotic choices.

Expression of blood group antigen A by stage I non-small cell lung carcinomas.

To clarify the significance of blood group antigen A (BAA) expression by neoplastic cells, we studied patients who had curative resections of stage I non-small cell lung carcinomas. Immunohistochemical staining using monoclonal antibodies was used to detect BAA expression by paraffin-embedded carcinoma cells. One hundred three patients were studied; mean age was 62.6 years, and 70 (68%) were male. Histologic types were as follows: adenocarcinoma, 52 (50.5%); squamous cell, 25 (24.3%); large cell, 24 (23.3%); and adenosquamous, 2 (1.9%). Histologic grades were as follows: I, 13 (12.6%); II, 26 (25.3%); and III, 64 (62.1%). All patients had American Joint Committee on Cancer stage I tumors: 65 patients (63.1%) had T1 tumors, and 38 (36.9%) had T2 tumors. Recurrences developed in 25 (24.3%) and metachronous malignancies in 4 (3.9%). Survival was 75% +/- 4.8% at 3 years and 66.6% +/- 7.5% at 5 years. Eighty-nine patients (86.4%) were blood group A and 14 (13.6%) were AB. Ninety-five (92.2%) were secretors of BAA and 8 (7.8%) were not. The expression of BAA by neoplastic cells was not detectable in 34 (33%), trace (1% to 5% of neoplastic cells) in 10 (9.7%), 1+ (6% to 25%) in 8 (7.8%), 2+ (26% to 50%) in 12 (11.7%), 3+ (51% to 75%) in 12 (11.7%), and 4+ (76% to 100%) in 27 (26.2%). The pattern of neoplastic cell staining was homogeneous in 14 patients (20.3%) and heterogeneous in 55 (79.7%). Carcinoma recurrence, overall survival, and event-free survival were not related to secretor status, BAA expression, or pattern of staining.(ABSTRACT TRUNCATED AT 250 WORDS)

Presentation and management of bronchogenic cysts in the adult.

Bronchogenic cysts are congenital anomalies of the bronchial tree that are often asymptomatic at presentation in adults. Management of asymptomatic bronchogenic cyst in this population remains controversial. Eighteen patients with bronchogenic cysts were treated at our institution since 1975. At initial presentation, 10 patients (56 percent) were asymptomatic and 8 (44 percent) were symptomatic. Cough and pain were the most frequent symptoms. Two patients presented with potentially serious complications, one with respiratory distress from airway compression and the other with infection and airway fistulae. Chest radiographs were abnormal but nondiagnostic in 17 out of 18 (94 percent) patients. Chest computerized tomography (CT) scans were abnormal in eight of eight (100 percent) patients, but they confirmed the benign cystic nature in only five of eight (62.5 percent). Overall, considering the use of all imaging modalities and clinical suspicion, bronchogenic cyst was considered in the preoperative differential diagnosis in only 11 of 18 (61 percent) patients. Fifteen of 18 cysts were resected initially. Three of the asymptomatic patients who were followed up initially ultimately required resection because of the development of symptoms. A trend toward increased postoperative complications was noted in patients who were symptomatic at the time of surgery (27 percent vs 14 percent). In conclusion, adult patients with asymptomatic bronchogenic cyst may develop symptoms over time. Symptoms in adults can sometimes be potentially serious. Since a confident preoperative diagnosis is not always possible and because surgical complications may be more common in the symptomatic patient, we recommend surgical resection of all suspected bronchogenic cysts in operable candidates.

Characterization of exosurf (surfactant)-mediated suppression of stimulated human alveolar macrophage cytokine responses.

Previous studies in our laboratory demonstrated that the synthetic surfactant Exosurf (Burroughs Wellcome Co.) inhibited endotoxin-stimulated cytokine secretion from human alveolar macrophages in vitro. The purpose of the present study was to further characterize the suppressive effects of Exosurf, which consists of dipalmitoylphosphatidylcholine (DPPC), cetyl alcohol (spreading agent), and tyloxapol (nonionic dispersing agent). Suppression was not stimulus specific in that Exosurf also significantly reduced cytokine production elicited by either Staphylococcus aureus or recombinant interleukin-1. Suppression was also mediated by a modified bovine surfactant (Survanta), which, in contrast to Exosurf, contains the surfactant-associated proteins B and C, and several different phospholipids, but no cetyl alcohol or tyloxapol. This suggests that suppression of macrophage cytokines is not specific to Exosurf. Both cell associated and secreted tumor necrosis factor and interleukin-1 were reduced by Exosurf, indicating that Exosurf is not simply blocking cytokine release. At 3 h, cytokine mRNA levels were not different between Exosurf-treated and untreated cells. However, at 8 and 24 h, cytokine mRNA levels were lower in Exosurf-treated cells. The observations that mRNA levels were decreased at 8 and 24 h and that cellular cytokine release was not blocked suggest that Exosurf's effect may in part be pretranslationally mediated. Collectively, these data add to previous work indicating that pulmonary surfactant may play a critical role in reducing inflammatory cytokine production associated with the adult respiratory distress syndrome and similar disorders.

Right pleural effusion due to a migrating ventriculoperitoneal shunt.

BACKGROUND: Pseudotumor cerebri or benign intracranial hypertension is a collection of disorders characterized by papilledema and increased intracranial pressure without associated neurologic abnormalities and wherein the cerebrospinal fluid composition is normal. Therapy may consist of diuretics, acetazolamide, and, in more severe cases, ventricular shunts. SUMMARY: A patient with pseudotumor cerebri and a ventriculoperitoneal shunt presented with dyspnea early in her pregnancy. Chest roentgenography revealed a right pleural effusion and a shunt catheter in the right pleural space. The patient underwent four thoracenteses, and a new shunt was placed after the baby was born. CONCLUSIONS: Physicians should recognize the potential for a ventriculoperitoneal shunt to migrate from the peritoneum to the pleural space. In a pregnant patient, it is reasonable to perform serial thoracenteses as a temporizing measure, and the shunt can be definitively revised in an elective surgical procedure postpartum.

Diagnostic utility of lactate dehydrogenase levels in patients receiving aerosolized pentamidine.

Serum lactate dehydrogenase (LDH) levels are elevated in patients with Pneumocystis carinii pneumonia (PCP). In patients receiving aerosolized pentamidine (AP), PCP may be less severe and present with more localized radiographic infiltrates. We evaluated the diagnostic utility of the serum LDH level in 11 patients developing PCP while receiving AP. Serum LDH levels were increased 72.7 +/- 19.0 percent over mean baseline levels and 76.4 +/- 26 percent over the upper limit of normal value for our laboratory with the development of PCP. An elevated serum LDH value remains a useful diagnostic adjunct in the patient developing PCP while receiving AP. Isolated LDH values must be interpreted with caution given the potential for marked variability in the baseline LDH value.

Lung transplantation: the Cleveland Clinic experience.

BACKGROUND: Lung transplantation has been steadily developing as a therapeutic option for end-stage lung disease. METHODS: Retrospective analysis of all 26 patients who underwent lung transplantation at the Cleveland Clinic Foundation between February 1990 and February 1992. RESULTS: Nineteen single-lung transplantations and seven bilateral lung transplantations were performed. The 1-year actuarial survival for all recipients was 65%. A trend was noted towards better survival in recipients with emphysema (100%) and poorer survival in those with pulmonary hypertension (37.5%). Fungal sepsis and reimplantation lung injury were the most common causes of death, and most deaths (8 of 9) occurred within the first 4 weeks. Of 119 pulmonary complications, 82% occurred in the first 3 months, with infection (39%) and acute rejection (29%) being the most common. Bacterial and fungal infections occurred mainly in the first month, and cytomegalovirus infections occurred mainly in the second and third months. The majority of survivors have shown improvement in functional status. CONCLUSIONS: The early perioperative and 1-month post-transplantation period appears critical to long-term survival. Even though the complications are numerous, they are usually manageable, and, in general, do not result in long-term morbidity.

Immunomodulatory effects of recombinant interleukin-3 treatment on human alveolar macrophages and monocytes.

The purpose of these studies was to examine the effects of in vivo and in vitro recombinant IL-3 treatment on alveolar macrophage and monocyte activities associated with antitumor and antimicrobial properties. Alveolar macrophages and blood monocytes from 6 patients receiving IL-3 (125-500 micrograms/m2/day) subcutaneously were isolated before therapy and at various times during the 15 days of therapy. Results indicated that tumor necrosis factor-alpha (TNF), interleukin-1 beta (IL-1), and interleukin-6 (IL-6) secretion were enhanced from monocytes of all patients and from alveolar macrophages of patients receiving 500 micrograms/m2/day IL-3. Constitutive cytokine gene expression was present before therapy, but further enhancement was not detectable during therapy, suggesting a rapid time course of cytokine gene transcription and translation. Serum neopterin levels were elevated 2-5 fold in all patient compatible with the presence of augmented monocyte/macrophage activity. Peak levels of neopterin did not coincide with peak levels of cytokine secretion. In vitro studies of IL-3-treated normal alveolar macrophage and monocyte population demonstrated that IL-3 significantly augmented TNF and IL-6 secretion in monocytes, but not in alveolar macrophages. These differences in alveolar macrophage cytokine secretion observed after in vivo and in vitro IL-3 treatment may reflect the involvement of other cell populations in IL-3 modulation of alveolar macrophages in vivo. Monocytes, in contrast were comparably activated by IL-3 whether presented in vitro or in vivo.

Activation of human monocytes and alveolar macrophages by a synthetic peptide of C-reactive protein.

We have previously shown that native human C-reactive protein (CRP) produces antitumor effects in experimental animals, and that these effects are mediated primarily through macrophages. More recently, we have observed that RS-83277, a synthetic peptide derived from CRP, appears to mimic the antitumor effects of native CRP. The purpose of this study was to determine the effects of RS-83277 on normal human monocyte and alveolar macrophage tumoricidal activity, and cytokine secretion. At optimal doses of 250-500 micrograms/ml, RS-83277 significantly enhanced tumoricidal activity of both monocytes and macrophages. RS-83287, a CRP peptide derived from a different site, had no effect at these doses. Specificity of RS-83277 for monocyte/macrophage-mediated cytotoxic activity was demonstrated by the failure of RS-83277 to enhance either natural killer (NK) or lymphokine-activated killer (LAK) cell-mediated activity. RS-83277 also augmented secretion of interleukin-1-beta (IL-1 beta) and interleukin-6 (IL-6) by monocytes. These data suggest a role for synthetic CRP peptide, RS-83277, as a novel biological response modifier in cancer therapy.

Exercise testing, 6-min walk, and stair climb in the evaluation of patients at high risk for pulmonary resection.

To evaluate three types of exercise testing in prediction of death or prolonged mechanical ventilation after lung resection in high-risk patients, 16 patients underwent evaluation prior to resection. Eleven patients (group 1) had minor or no complications (arrhythmia, atelectasis, pneumonia) and five patients (group 2) died within 90 days of surgery. Exercise testing showed that group 1 had a longer 6-min walk distance and a higher stair climb than group 2. The maximum oxygen uptake on a cycle ergometer was not significantly different between groups, although only ten patients completed this test. Group 1 had a significantly greater calculated oxygen uptake with stair climbing than group 2. A 6-min walk distance of greater than 1,000 feet and a stair climb of greater than 44 steps were predictive of successful surgical outcome. Preoperative exercise testing is a useful adjunct to traditional spirometric testing in evaluation of the high-risk surgical patients.