The case against anergy testing as a routine adjunct to tuberculin skin testing.

Although anergy testing is commonly used to help interpret negative tuberculin skin test results, the validity of this approach has not been demonstrated. Specific issues include lack of a standardized protocol for antigen selection, number needed to reliably evaluate inability to respond, and uniform criteria for defining cutaneous reactivity, as well as regional variation in skin test reactivity. Tuberculin skin testing is used to screen for latent infection and to evaluate the need for isoniazid prophylaxis. The presence or absence of reactivity to control antigens does not affect this decision. The results of anergy testing also do not predict the risk for progression to active disease in either HIV-negative or HIV-positive patients. In HIV-negative patients with active tuberculosis, 10% to 20% have negative tuberculin test results, and 5% to 10% have a negative tuberculin result but have a positive reaction to another antigen. A negative tuberculin skin test result does not exclude either latent infection or active disease, even in the presence of a reaction to other antigens. Neither anergy testing nor tuberculin testing obviates the need for microbiologic evaluation when there is suspicion for active tuberculosis infection. Therefore, anergy testing is not useful in screening for asymptomatic tuberculous infection or for diagnosing active tuberculosis.

Effect of cardiogenic and noncardiogenic pulmonary edema on histamine responsiveness in sheep.

We compared the effects of cardiogenic pulmonary edema, brief pulmonary vascular congestion without frank edema, and noncardiogenic pulmonary edema on responsiveness to inhaled histamine in chronically instrumented awake sheep. Histamine responsiveness was measured before and after 1) cardiogenic pulmonary edema induced by raising left atrial pressure to 35 cmH2O (Pla) for 3.5 h by partial obstruction of flow across the mitral valve, 2) brief cardiogenic congestion via Pla for 0.5 h, 3) noncardiogenic pulmonary edema induced by 25 mg/kg intravenous perilla ketone (PK), and 4) 3.5 h of monitoring without Pla or PK (controls). Treatment for 3.5 h with Pla (n = 9) and PK (n = 11) each significantly lessened the histamine dose required to cause a fall to 65% of baseline dynamic lung compliance (ED65Cdyn), i.e., increased responsiveness. Sheep treated for 0.5 h with Pla (n = 7) and controls (n = 5) showed no significant change in ED65Cdyn. Intravenous atropine (0.1 mg/kg) before the second histamine challenge altered neither the reduction of ED65Cdyn in Pla (n = 8) and PK (n = 9) sheep nor the ED65Cdyn level of controls (n = 9). These data imply that the local effects of edema, rather than bronchial vascular hemodynamics, cholinergic reflexes, and permeability changes, are germane to lung hyperresponsiveness during pulmonary edema in sheep.

Plasmid-liposome transfer of the alpha 1 antitrypsin gene to cystic fibrosis bronchial epithelial cells prevents elastase-induced cell detachment and cytokine release.

Human neutrophil elastase (NE) stimulates release of neutrophil chemotactic activity by a bronchial epithelial cell line and from nasal epithelial cells. In this article, we show that NE stimulates the production of neutrophil chemotactic activity by 2CFSMEo-cells, a transformed cystic fibrosis bronchial epithelial cell line. The production of chemotactic activity is dose- and time-dependent and can be blocked by preincubation of NE with alpha 1 antitrypsin (alpha1AT). Incubation of the NE-stimulated culture supernatant with neutralizing concentrations of rabbit anti-human interleukin 8 antibody completely neutralizes the chemotactic activity. Transfection of 2CFSMEo- cells with the eukaryotic expression vector pCMV4alpha1AT, complexed to cationic liposomes in a 1:3 wt/wt ratio, results in at least a 10-fold increase in measured human alpha1AT protein in culture supernatant. Detection of human alpha1AT mRNA by reverse transcriptase polymerase chain reaction in total RNA from transfected, but not untransfected cells, confirms successful gene transfer. Compared with untransfected cells, transfer of the human alpha1AT gene decreases chemotactic activity in culture supernatant and prevents cell detachment after NE exposure. Our data indicate that alpha1AT gene transfer is capable of blocking at least some of the biological effects of free elastase on cultured epithelial cells.

Effects on pulmonary physiology of reamed femoral intramedullary nailing in an open-chest sheep model.

We have recently developed an open-chest sheep model to monitor and study the effects of major orthopedic procedures on pulmonary physiology. In this pilot study, we focused on reamed intramedullary femoral nailing in animals without pulmonary injury. Details of the model are described herein. The control group consisted of sheep that underwent thoracotomy and invasive monitoring only, while the study group also underwent femoral osteotomy, reaming, and intramedullary nailing. Baseline, postthoracotomy, and post-reaming/nailing values were recorded for mean pulmonary arterial pressure, central venous pressure, left arterial pressure, dynamic compliance, arterial blood gas, mixed venous O2, cardiac index, and mean arterial pressure so that hemodynamic and oxygen transport data could be calculated. Postprocedure values were recorded at hourly intervals for 4 h. A physiologically stable, reproducible model was created. No statistically significant differences were found between the control and experimental groups, indicating no adverse effect of femoral reaming/nailing. In one animal, using echocardiography, pulmonary embolization was documented while reaming and inserting the intramedullary nail. Reamed femoral intramedullary nailing is not detrimental to sheep with otherwise normal lungs. This finding suggests that femoral reaming and nailing in trauma patients without associated pulmonary injuries and otherwise normal lungs may be carried out without risk of inducing significant respiratory complications.

The rabbit pulmonary cytochrome P450 arachidonic acid metabolic pathway: characterization and significance.

Cytochrome P450 metabolizes arachidonic acid to several unique and biologically active compounds in rabbit liver and kidney. Microsomal fractions prepared from rabbit lung homogenates metabolized arachidonic acid through cytochrome P450 pathways, yielding cis-epoxyeicosatrienoic acids (EETs) and their hydration products, vic-dihydroxyeicosatrienoic acids, mid-chain cis-trans conjugated dienols, and 19- and 20-hydroxyeicosatetraenoic acids. Inhibition studies using polyclonal antibodies prepared against purified CYP2B4 demonstrated 100% inhibition of arachidonic acid epoxide formation. Purified CYP2B4, reconstituted in the presence of NADPH-cytochrome P450 reductase and cytochrome b5, metabolized arachidonic acid, producing primarily EETs. EETs were detected in lung homogenate using gas chromatography/mass spectroscopy, providing evidence for the in vivo pulmonary cytochrome P450 epoxidation of arachidonic acid. Chiral analysis of these lung EETs demonstrated a preference for the 14(R),15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET enantiomers. Both EETs and vic-dihydroxyeicosatrienoic acids were detected in bronchoalveolar lavage fluid. At micromolar concentrations, methylated 5,6-EET and 8,9-EET significantly relaxed histamine-contracted guinea pig hilar bronchi in vitro. In contrast, 20-hydroxyeicosatetraenoic acid caused contraction to near maximal tension. We conclude that CYP2B4, an abundant rabbit lung cytochrome P450 enzyme, is the primary constitutive pulmonary arachidonic acid epoxygenase and that these locally produced, biologically active eicosanoids may be involved in maintaining homeostasis within the lung.

No lung toxicity after repeated aerosol or intravenous delivery of plasmid-cationic liposome complexes.

The safety aspects of human gene therapy are of paramount importance in developing an ideal system for gene transfer. Lipofection using DNA in the form of a plasmid has been shown to successfully transfect the lungs when administered either intravenously or by aerosol. We have shown that repeated intravenous or aerosol administration of a plasmid containing the recombinant human alpha 1-antitrypsin gene and a cytomegalovirus promoter complexed to cationic liposomes results in no adverse effects on pulmonary histology, lung compliance, lung resistance, or alveolar-arterial oxygen gradient. Immunohistochemistry and Western blot analysis confirm successful gene transfer using this delivery system. We conclude that plasmids complexed to cationic liposomes may be a safe and efficacious delivery system for in vivo gene transfer to the lungs. Using this delivery system, in vivo gene therapy to the lungs can be achieved by either intravenous or aerosol administration of the transgene.