Concentration of antifungal agents within host cell membranes: a new paradigm governing the efficacy of prophylaxis.

Posaconazole prophylaxis has proven highly effective in preventing invasive fungal infections, despite relatively low serum concentrations. However, high tissue levels of this agent have been reported in treated patients. We therefore hypothesized that the intracellular levels of antifungal agents are an important factor in determining the success of fungal prophylaxis. To examine the effect of host cell-associated antifungals on the growth of medically important molds, we exposed cells to antifungal agents and removed the extracellular drug prior to infection. Epithelial cells loaded with posaconazole and its parent molecule itraconazole, but not other antifungals, were able to inhibit fungal growth for at least 48 h and were protected from damage caused by infection. Cell-associated posaconazole levels were 40- to 50-fold higher than extracellular levels, and the drug was predominantly detected in cellular membranes. Fungistatic levels of posaconazole persisted within epithelial cells for up to 48 h. Therefore, the concentration of posaconazole in mammalian host cell membranes mediates its efficacy in prophylactic regimens and likely explains the observed discrepancy between serum antifungal levels and efficacy.

Attenuation of endothelium-dependent hyperpolarizing factor by bacterial lipopolysaccharides.

Endothelium-dependent hyperpolarizing factor (EDHF) is an important contributor to agonist-induced vascular dilation. Recent studies suggest that bacterial lipopolysaccharides attenuate endothelium-dependent dilation. Whether or not this effect is mediated through inhibition of EDHF is not known. We studied the in vitro influence of Escherichia coli lipopolysaccharides on endothelium-dependent smooth muscle dilation and hyperpolarization in porcine coronary arteries. Endothelium-intact porcine coronary arterial rings were examined after 20 h of incubation with either saline or E. coli lipopolysaccharides (100 microg/ml). Endothelium-dependent dilation elicited by increasing concentrations of bradykinin was significantly attenuated by lipopolysaccharides. Baseline values of smooth muscle membrane potential were not influenced by lipopolysaccharides. However, lipopolysaccharides significantly attenuated bradykinin-induced smooth muscle membrane hyperpolarization. Our results suggest that attenuation of EDHF is an important mechanism through which lipopolysaccharides influence vascular dilation in severe sepsis.

Low systemic vascular resistance state in patients undergoing cardiopulmonary bypass.

OBJECTIVE: To determine the prevalence, hemodynamic characteristics, and risk factors for the low systemic vascular resistance (SVR) state in patients who have undergone cardiopulmonary bypass. DESIGN: Prospective cohort study. SETTING: The intensive care unit of a tertiary care hospital. PATIENTS: Seventy-nine consecutive patients who underwent coronary artery bypass graft, mitral valve, or aortic valve procedures. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Low SVR was defined as an indexed systemic vascular resistance (SVRi) of <1800 dyne x sec/cm5 x m2 at two consecutive times postoperatively. SVRi, cardiac index, mean arterial pressure, temperature, and central venous pressure were recorded before bypass and at 0, 1, 2, 4, 8, and 16 hrs after bypass. We recorded age, gender, urgency of operation, use of angiotensin-converting enzyme inhibitors and calcium channel blockers, ejection fraction, pump time, cross-clamp time, use of antifibrinolytics, type of oxygenator, amrinone use, postoperative biochemical and hematologic values, medication use, fluid balance, intensive care unit admission duration, and hospital admission duration. We assessed the role of diabetes mellitus, current smoking, and systemic hypertension. The incidence of the low-SVR state was 35 of 79 patients during a 3-month period (44%). At 8 hrs postoperatively, the SVRi in low-SVR and non-low-SVR patients was 1594+/-50 (SEM) and 2103+/-56 (SEM) dyne x sec/cm5 x m2, respectively (p < .001). In low-SVR patients, there was an initial and sustained increase in cardiac index and central venous pressure that preceded the decrease in mean arterial pressure. The decrease in mean arterial pressure was maximal at 8 hrs postoperatively. Patients with low SVR were more likely to have longer cross-clamp times, to be male, and to have lower postoperative platelet counts (p < .05 for all). Low-SVR patients were less likely to require dobutamine in the first 4 hrs postoperatively. CONCLUSIONS: Low SVR, a probable manifestation of systemic inflammatory response syndrome, is common in patients after cardiopulmonary bypass. These patients may respond better to a vasopressor to restore vascular tone than to volume loading to further increase cardiac index.

Mitogen-activated protein kinases mediate activator protein-1-dependent human inducible nitric-oxide synthase promoter activation.

Inducible nitric-oxide synthase (iNOS) is an important signaling protein involved in the regulation of biological processes (e.g. vasodilation, inflammation) and is subject to transcriptional regulation by cytokines and lipopolysaccharide (LPS). Full activation of the human iNOS (hiNOS) promoter by cytokines (i.e., tumor necrosis factor-alpha, interleukin-1beta, interferon-gamma (IFN-gamma)) required downstream and upstream nuclear factor-kappaB (-115, -8283) and activator protein-1 (AP-1) (-5115, -5301) transcription factor binding sites. Human lung epithelial (A549) cells were transiently transfected with luciferase reporter plasmids containing an 8.3-kilobase human iNOS promoter to examine the molecular signaling events necessary for hiNOS transcriptional activation. The combination of LPS and IFN-gamma, but neither alone, increased hiNOS promoter activity 28-fold, in a reaction requiring two critical AP-1 (JunD-Fra-2) promoter binding sites. Mitogen-activated protein kinases (MAPKs) were assessed as potential activators of AP-1 and the hiNOS promoter. Both pharmacological and molecular inhibitors of the extracellular signal-related kinase (ERK) and p38 pathways reduced cytokine mixture (CM)- and LPS/IFN-gamma-induced promoter activation. By gel retardation analysis, the addition of MAP/ERK kinase-1 and p38 inhibitors significantly diminished AP-1 binding in both CM- and LPS/IFN-gamma-stimulated cells. Thus, p38- and ERK-dependent pathways, through effects on the AP-1 complex, activate the hiNOS promoter in cells stimulated with CM or LPS/IFN-gamma.

Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury.

The role of nitric oxide (NO) in lung injury remains unclear. Both beneficial and detrimental roles have been proposed. In this study, we used mutant mice lacking the inducible nitric oxide synthase (iNOS) to assess the role of this isoform in sepsis-associated lung injury. Wild-type and iNOS knockout mice were injected with either saline or Escherichia coli endotoxin (LPS) 25 mg/kg and killed 6, 12, and 24 h later. Lung injury was evaluated by measuring lactate dehydrogenase activity in the bronchoalveolar lavage, pulmonary wet/dry ratio, and immunostaining for nitrotyrosine formation. In the wild-type mice, LPS injection elicited more than a 3-fold rise in lactate dehydrogenase activity, a significant rise in lung wet/dry ratio and extensive nitrotyrosine staining in large airway and alveolar epithelium, macrophages, and pulmonary vascular cells. This was accompanied by induction of iNOS protein and increased lung nitric oxide synthase activity. By comparison, LPS injection in iNOS knockout mice elicited no iNOS induction and no significant changes in lung NOS activity, lactate dehydrogenase activity, lung wet/dry ratio, or pulmonary nitrotyrosine staining. These results indicate that mice deficient in iNOS gene are more resistant to LPS-induced acute lung injury than are wild-type mice.

Prevalence and clinical characteristics of lymphangioleiomyomatosis (LAM) in patients with tuberous sclerosis complex.

The true prevalence of pulmonary lymphangioleiomyomatosis (LAM) in patients with tuberous sclerosis complex (TSC) is unknown. The prevalence of LAM, radiological features, and lung function in patients with TSC was measured. The presence of LAM, as defined by the presence of cysts by high-resolution chest computed tomography (HRCT) scan, was determined in patients with TSC without prior pulmonary disease (Group 1). To determine the significance of early detection, severity of disease in screened patients (Group 1) was compared with that in patients with TSC with a prior diagnosis of LAM (Group 2). Forty-eight patients with TSC and no prior history of LAM were screened. Of the 38 females, 13 (34%) had LAM; LAM was absent in males. Lung function was preserved in patients with TSC who were found to have LAM by screening. In patients previously known to have LAM, FEV(1) and DL(CO) correlated inversely with severity of disease as assessed by CT scan. The prevalence of LAM in women with TSC was 34%, approximately 10-fold that previously reported, consistent with a large hitherto unrecognized subclinical population of patients at risk for pulmonary complications.