Azithromycin Fails to Prevent Accelerated Airway Obliteration in T-bet-/- Mouse Lung Allograft Recipients.

BACKGROUND: Cellular and molecular mechanisms of acute and chronic lung allograft rejection have yet to be clearly defined, and obliterative bronchiolitis (OB) remains the primary limitation to survival in lung transplant recipients (LTRs). We have previously shown that T-bet-deficient recipients of full major histocompatibility complex (MHC)-mismatched, orthotopic left lung transplants develop accelerated obliterative airway disease (OAD) in the setting of acute cellular rejection characterized by robust alloimmune CD8+ interleukin (IL)-17 and interferon (IFN)-γ responses that are attenuated with neutralization of IL-17. Azithromycin has been shown to be beneficial in some LTRs with bronchiolitis obliterans syndrome/OB. Here, we evaluated the effects of azithromycin on rejection pathology and T-cell effector responses in T-bet-/- recipients of lung transplants. METHODS: Orthotopic left lung transplantation was performed in BALB/c → B6 wild type or BALB/c → B6 T-bet-/- strain combinations as previously described. Mice treated with azithromycin received 10 mg/kg or 50 mg/kg subcutaneously daily. Lung allograft histopathology was analyzed at day 10 or day 21 post-transplantation, and neutrophil staining for quantification was performed using anti-myeloperoxidase. Allograft mononuclear cells were isolated at day 10 for T-cell effector cytokine response assessment using flow cytometry. RESULTS: We show that while azithromycin significantly decreases lung allograft neutrophilia and CXCL1 levels and attenuates allospecific CD8+ IL-17 responses early post-transplantation, OAD persists in T-bet-deficient mice. CONCLUSIONS: Our results indicate that lung allograft neutrophilia is not essential for the development of OAD in this model and suggest allospecific T-cell responses that remain despite marked attenuation of CD8+ IL-17 are sufficient for obliterative airway inflammation and fibrosis.

CD154 blockade abrogates allospecific responses and enhances CD4(+) regulatory T-cells in mouse orthotopic lung transplant.

Acute cellular rejection (ACR) is a common and important clinical complication following lung transplantation. While there is a clinical need for the development of novel therapies to prevent ACR, the regulation of allospecific effector T-cells in this process remains incompletely understood. Using the MHC-mismatched mouse orthotopic lung transplant model, we investigated the short-term role of anti-CD154 mAb therapy alone on allograft pathology and alloimmune T-cell effector responses. Untreated C57BL/6 recipients of BALB/c left lung allografts had high-grade rejection and diminished CD4(+) : CD8(+) graft ratios, marked by predominantly CD8(+) >CD4(+) IFN-γ(+) allospecific effector responses at day 10, compared to isograft controls. Anti-CD154 mAb therapy strikingly abrogated both CD8(+) and CD4(+) alloeffector responses and significantly increased lung allograft CD4(+) : CD8(+) ratios. Examination of graft CD4(+) T-cells revealed significantly increased frequencies of CD4(+) CD25(+) Foxp3(+) regulatory T-cells in the lung allografts of anti-CD154-treated mice and was associated with significant attenuation of ACR compared to untreated controls. Together, these data show that CD154/CD40 costimulation blockade alone is sufficient to abrogate allospecific effector T-cell responses and significantly shifts the lung allograft toward an environment predominated by CD4(+) T regulatory cells in association with an attenuation of ACR.

Leukocytes can enhance platelet-mediated aggregation and thromboxane release via interaction of P-selectin glycoprotein ligand 1 with P-selectin.

BACKGROUND: Platelet--leukocyte conjugates have been observed in patients with unstable coronary syndromes and after cardiopulmonary bypass. In vitro, the binding of platelet P-selectin to leukocyte P-selectin glycoprotein ligand-1 (PSGL1) mediates conjugate formation; however, the hemostatic implications of these cell--cell interactions are unknown. The aims of this study were to determine the ability of leukocytes to modulate platelet agonist--induced aggregation and secretion in the blood milieu, and to investigate the role of P-selectin and PSGL-1 in mediating these responses. METHODS: Blood was drawn from healthy volunteers for in vitro analysis of platelet agonist--induced aggregation, secretion (adenosine triphosphate, beta-thromboglobulin, and thromboxane), and platelet-leukocyte conjugate formation. Experiments were performed on live cells in whole blood or plasma to simulate physiologic conditions. Whole-blood impedance and optical aggregometry, flow cytometry, and enzyme-linked immunosorbent assays were performed in the presence and absence of blocking antibodies to P-selectin and PSGL1. The platelet-specific agonists, thrombin receptor activating peptide and adenosine diphosphate, were used to elicit platelet activation responses. RESULTS: Inhibition of platelet--leukocyte adherence by P- selectin and PSGL1 antibodies decreased agonist--induced aggregation in whole blood. The presence of leukocytes in platelet-rich plasma increased aggregation, and this increase was attenuated by P-selectin blocking antibodies. Data from flow cytometry confirmed that platelet-leukocyte conjugate formation contributed to aggregation responses. Blocking antibodies reduced platelet agonist--induced thromboxane release but had no impact on adenosine triphosphate and beta-thomboglobulin secretion. CONCLUSIONS: Leukocytes can enhance platelet agonist--induced aggregation and thromboxane release in whole blood and platelet-rich plasma under shear conditions in vitro. Interaction of platelet P-selectin with leukocyte PSGL1 contributes substantially to these effects.

Effect of the NADPH oxidase inhibitor apocynin on ischemia-reperfusion lung injury.

Apocynin (4-hydroxy-3-methoxy-acetophenone) inhibits NADPH oxidase in activated polymorphonuclear (PMN) leukocytes, preventing the generation of reactive oxygen species. To determine if apocynin attenuates ischemia-reperfusion lung injury, we examined the effects of apocynin (0.03, 0.3, and 3 mM) in isolated in situ sheep lungs. In diluent-treated lungs, reperfusion with blood (180 min) after 30 min of ischemia (ventilation 28% O(2), 5% CO(2)) caused leukocyte sequestration in the lung and increased vascular permeability [reflection coefficient for albumin (sigma(alb)) 0.47 +/- 0.10, filtration coefficient (K(f)) 0.14 +/- 0.03 g. min(-1). mmHg(-1). 100 g(-1)] compared with nonreperfused lungs (sigma(alb) 0.77 +/- 0. 03, K(f) 0.03 +/- 0.01 g. min(-1). mmHg(-1). 100 g(-1); P < 0.05). Apocynin attenuated the increased protein permeability at 0.3 and 3 mM (sigma(alb) 0.69 +/- 0.05 and 0.91 +/- 0.03, respectively, P < 0. 05); K(f) was decreased by 3 mM apocynin (0.05 +/- 0.01 g. min(-1). mmHg(-1). 100 g(-1), P < 0.05). Diphenyleneiodonium (DPI, 5 microM), a structurally unrelated inhibitor of NADPH oxidase, worsened injury (K(f) 0.32 +/- 0.07 g. min(-1). mmHg(-1). 100 g(-1), P < 0.05). Neither apocynin nor DPI affected leukocyte sequestration. Apocynin and DPI inhibited whole blood chemiluminescence and isolated PMN leukocyte-induced resazurin reduction, confirming NADPH oxidase inhibition. Apocynin inhibited pulmonary artery hypertension and perfusate concentrations of cyclooxygenase metabolites, including thromboxane B(2). The cyclooxygenase inhibitor indomethacin had no effect on the increased vascular permeability, suggesting that cyclooxygenase inhibition was not the explanation for the apocynin results. Apocynin prevented ischemia-reperfusion lung injury, but the mechanism of protection remains unclear.

Prospective, randomized, controlled clinical trial comparing traditional versus reduced tidal volume ventilation in acute respiratory distress syndrome patients.

OBJECTIVE: To assess the safety and potential efficacy of a mechanical ventilation strategy designed to reduce stretch-induced lung injury in acute respiratory distress syndrome. DESIGN: Prospective, randomized, controlled clinical trial. SETTING: Eight intensive care units in four teaching hospitals. PATIENTS: Fifty-two patients with acute respiratory distress syndrome. INTERVENTIONS: Traditional tidal volume patients: tidal volume 10-12 mL/kg ideal body weight, reduced if inspiratory plateau pressure was > 55 cm H2O (7.3 kPa). Small tidal volume patients: tidal volume 5-8 mL/kg ideal body weight, to keep plateau pressure < 30 cm H2O (4.0 kPa). MEASUREMENTS AND MAIN RESULTS: Mean tidal volumes during the first 5 days in traditional and small tidal volume patients were 10.2 and 7.3 mL/kg, respectively (p < .001), with mean plateau pressure = 30.6 and 24.9 cm H2O (3.3 kPa), respectively (p < .001). There were no significant differences in requirements for positive end-expiratory pressure or FIO2, fluid intakes/outputs, requirements for vasopressors, sedatives, or neuromuscular blocking agents, percentage of patients that achieved unassisted breathing, ventilator days, or mortality. CONCLUSIONS: The reduced tidal volume strategy used in this study was safe. Failure to observe beneficial effects of small tidal volume ventilation treatment in important clinical outcome variables may have occurred because a) the sample size was too small to discern small treatment effects; b) the differences in tidal volumes and plateau pressures were modest; or c) reduced tidal volume ventilation is not beneficial.

Potential paracrine role of the pericardium in the regulation of cardiac function.

OBJECTIVE: Both coronary and endocardial endothelium regulate cardiac contractile function via paracrine pathways. We investigated whether pericardial fluid (PF) and pericardial mesothelial cells (PMC) could exert a similar paracrine action. METHODS: Both PF and PMC were extracted from sheep pericardial space. Endothelin-1, prostaglandins and atrial natriuretic factor were measured in PF in vivo. In the other hand, PMC were grown on T-75 flasks and microcarrier beads to investigate endothelin-1, nitric oxide and prostaglandin pathways in vitro. In addition, effects of PF and PMC effluent were tested on adult rat cardiac myocyte contraction in vitro. RESULTS: In vitro, cultured PMC expressed endothelin-1 mRNA but not the endothelial nitric oxide synthase III, and released endothelin-1 and prostaglandins. Both PF and cultured PMC superfusate induced a potent, rapidly reversible decrease in the shortening of isolated rat cardiac myocytes. This effect was not associated with changes in intracellular calcium. In vivo, prostaglandins, atrial natriuretic factor and endothelin were present in PF. A greater concentration of atrial natriuretic factor was present in PF than in serum, suggesting molecular diffusion from the myocardium to PF. Preliminary results show that the instillation of vasoactive agents into the pericardial space of dogs rapidly alter coronary and systemic vascular tone, consistent with a molecular diffusion of these substances from PF into the myocardium and circulation. CONCLUSIONS: In addition to its mechanical role, the pericardium may contribute to the integration and the regulation of cardiovascular function via a paracrine mechanism.

Endothelium-independent relaxation of aortic rings by the nitric oxide synthase inhibitor diphenyleneiodonium.

1. The flavoprotein binder diphenyleneiodonium (DPI) is a potent, irreversible inhibitor of nitric oxide synthase (NOS), but produces only a transient pressor response following systemic administration to animals, despite evidence of persistent NOS inhibition. To characterize further the effects of DPI on vascular tone, isometric tension was recorded from rat isolated aortic rings mounted between steel wires in an organ bath. 2. The NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 1 mM) initiated an additional contraction of prostaglandin F2 alpha-preconstricted rings with endothelium which was sustained throughout the period of L-NAME exposure (+234 +/- 39% at 15 min). In contrast, addition of DPI (5 microM) to rings with endothelium produced a transient initial contraction (+111 +/- 27% at 2 min) followed by a more sustained relaxation (-27 +/- 19% at 15 min, P < 0.001 vs L-NAME). 3. The contraction to DPI was also observed in rings without endothelium, was abolished by L-NAME pretreatment, and was unaffected by the alpha-adrenoreceptor inhibitor prazosin. Relaxation in response to DPI was not inhibited by endothelium removal or by pretreatment with either L-NAME or with the ATP-sensitive potassium channel blocker glibenclamide. 4. The endothelium-independent relaxation to DPI was inhibited at 23 degrees C and its time course was delayed by pretreatment with the guanylate cyclase inhibitor methylene blue. 5. Thus, in addition to a transient initial contraction due to NOS inhibition, DPI produces an endothelium-independent, temperature-dependent relaxation which appears in part due to activation of guanylate cyclase. This relaxant effect of DPI may explain the transient nature of its pressor effect in vivo despite sustained NOS inhibition.

Coronary alpha 1-adrenergic constrictor tone varies with intensity of exercise.

This study tested the hypothesis that an alpha-adrenergic coronary constrictor tone increases with the intensity of exercise and imposes a limitation on transmural myocardial blood flow and contractile function during strenuous levels of exercise. Nine (9) dogs were chronically instrumented to measure left circumflex blood flow (CBF), global myocardial contractile function (dP/dtmax), and regional myocardial contractile function (maximal rate of segmental shortening, dL/dtmax). The dogs were subjected to a graded sub-maximal exercise test with increasing workloads encompassing 4.8 kph and 6.4 kph, 0, 4, 8, 12, and 16% incline. On separate days, either vehicle (sterile water) or the specific alpha 1-adrenergic receptor antagonist prazosin (1 microgram.kg-1.min-1) was infused into the circumflex artery during exercise. Removal of an alpha 1-receptor mediated coronary constrictor tone resulted in a 15 +/- 7%, 24 +/- 9%, and 35 +/- 10% greater increase in CBF compared with vehicle at the three most strenuous levels of exercise, respectively. Regional left ventricular blood flow was measured using labeled microspheres in four (4) additional dogs. Endocardial and epicardial blood flow increased equally by 16% during exercise after prazosin, such that the endocardial/epicardial flow ratio did not change. The augmentation in CBF after alpha 1-blockade was associated with significant increases in both regional and global left ventricular contractile function. These studies indicate that a uniformly distributed transmural coronary alpha 1-constrictor tone increases in magnitude with increasing levels of exercise intensity and, as a result, imposes a significant limitation on myocardial function.

Cardiac response to acute coronary artery occlusion in exercise-trained dogs.

Exercise training is thought to exert a beneficial effect on cardiovascular function, but its effect in the normal heart following acute coronary artery occlusion is still uncertain. Studies were performed in 12 untrained (UT) and 14 endurance-trained (ET) pentobarbital anesthetized dogs. Left ventricular pressure (LVP), heart rate (HR), percent regional myocardial segmental shortening (%SL), and peripheral coronary pressure (PCP) distal to the occlusion were measured during control conditions and during a 2-min circumflex artery occlusion (CAO). During CAO, LVP, dP/dtmax, and %SL in the ischemic region were significantly reduced in both UT and ET dogs. There was no significant difference between the two groups. In addition, PCP decreased to 27 +/- 5 mm Hg and 26 +/- 9 mm Hg in the UT and ET groups, respectively, during CAO indicating no difference in coronary collateral perfusion between the groups. Regional myocardial blood flow was measured using tracer microspheres in eight of the UT and six of the ET dogs, and the decrease in blood flow to the ischemic zone during CAO was similar in both groups. These results indicate that 12-wk of endurance training does not exert a protective effect on myocardial contractile function or on myocardial perfusion in the central ischemic region during CAO in the anesthetized dog.

Effects of a coronary alpha 1-constriction on transmural left ventricular flow and contractile function.

Modulation of myocardial contractile function and perfusion by alpha 1-adrenergic receptors were examined in anesthetized dogs during left stellate ganglion stimulation. In 11 dogs, stellate stimulation significantly increased heart rate, mean arterial pressure, left ventricular systolic pressure, maximal rate of left ventricular pressure generation, segmental shortening and rate of shortening in anterior and posterior ventricular regions, and myocardial oxygen extraction. Myocardial lactate extraction decreased. The selective alpha 1-adrenergic antagonist prazosin (0.5 mg) injected into the circumflex artery during stellate stimulation caused significant additional increases in maximal rate of left ventricular pressure generation by 19 +/- 5% and in rate of shortening in posterior subendocardium by 20 +/- 6%. No changes were observed in posterior subepicardial or anterior subendocardial segmental contractile function. Myocardial oxygen and lactate extractions returned to their control values following prazosin injection. Regional left ventricular perfusion was measured using tracer microspheres in five additional dogs. Stellate stimulation increased subepicardial and subendocardial perfusion by 30%. Prazosin increased both subepicardial and subendocardial perfusion by an additional 36%. Stellate stimulation increased norepinephrine concentration in the coronary sinus, but no further increase was noted after blockage of alpha 1-receptors by prazosin. Thus, during sympathetic stimulation, an alpha 1-vasoconstriction existed uniformly across the left ventricular wall. However, blockade of this vasoconstriction was associated with an increase in contractile function only in the deeper muscle layers.

Augmentation of coronary flow improves myocardial function in exercise.

We compared the effects of increasing coronary blood flow (CBF) by intracoronary alpha 1-adrenergic blockade or by a direct vasodilator in six chronically instrumented dogs undergoing submaximal exercise. On different days, CBF, left ventricular pressure (LVP), regional myocardial segment length (SL), and arterial blood pressure (ABP) were measured at rest, during strenuous exercise before administration of vasodilating agents, and during exercise after administration of the vasodilating agents prazosin (0.5 mg) or adenosine (10-100 micrograms/min continuous infusion) through an indwelling circumflex artery catheter. Exercise resulted in a significant increase in CBF, which was accompanied by significant increases in maximum rate of LVP generation (dP/dtmax), percentage of SL shortening (%SL) and maximum rate of SL shortening (dL/dtmax) in both the anterior and posterior regions of the left ventricle. As compared with the exercise level alone, prazosin administration or adenosine infusion during exercise significantly increased CBF by 22 and 26%, respectively. Furthermore, these increases in CBF were followed by significant increases in both dP/dtmax and dL/dtmax over exercise levels without prazosin administration or adenosine infusion. These results suggest that myocardial contractile function may be flow-limited under conditions of submaximal exercise and that coronary vasodilation, either by alpha 1-adrenergic blockade or by a direct vasodilator, improves contractile function.

Laryngeal rhabdomyosarcoma. Case report and literature review.

A case report is presented, believed to be the first documented example of pleomorphic rhabdomyosarcoma developing in the larynx of a child. A review of the literature then follows, with evaluation of the epidemiology, treatment options, and survival of patients with this disease. Cure of this malignancy is more likely with early diagnosis and aggressive treatment.