Azithromycin Polarizes Macrophages to an M2 Phenotype via Inhibition of the STAT1 and NF-κB Signaling Pathways.

Azithromycin is effective at controlling exaggerated inflammation and slowing the long-term decline of lung function in patients with cystic fibrosis. We previously demonstrated that the drug shifts macrophage polarization toward an alternative, anti-inflammatory phenotype. In this study we investigated the immunomodulatory mechanism of azithromycin through its alteration of signaling via the NF-κB and STAT1 pathways. J774 murine macrophages were plated, polarized (with IFN-γ, IL-4/-13, or with azithromycin plus IFN-γ) and stimulated with LPS. The effect of azithromycin on NF-κB and STAT1 signaling mediators was assessed by Western blot, homogeneous time-resolved fluorescence assay, nuclear translocation assay, and immunofluorescence. The drug's effect on gene and protein expression of arginase was evaluated as a marker of alternative macrophage activation. Azithromycin blocked NF-κB activation by decreasing p65 nuclear translocation, although blunting the degradation of IκBα was due, at least in part, to a decrease in IKKß kinase activity. A direct correlation was observed between increasing azithromycin concentrations and increased IKKß protein expression. Moreover, incubation with the IKKß inhibitor IKK16 decreased arginase expression and activity in azithromycin-treated cells but not in cells treated with IL-4 and IL-13. Importantly, azithromycin treatment also decreased STAT1 phosphorylation in a concentration-dependent manner, an effect that was reversed with IKK16 treatment. We conclude that azithromycin anti-inflammatory mechanisms involve inhibition of the STAT1 and NF-κB signaling pathways through the drug's effect on p65 nuclear translocation and IKKß.

Source control review in clinical trials of anti-infective agents in complicated intra-abdominal infections.

In clinical trials of complicated intra-abdominal infections, assessment of adequacy of the initial surgical approach to the management of the infection is of considerable importance in determining outcome. Antibiotic therapy would not be expected to adequately treat the infection if the surgical procedure was inadequate with respect to source control. Inclusion of such cases in an efficacy analysis of a particular therapeutic antibiotic may confound the results. We analyzed the source control review process used in double-blind clinical trials of antibiotics in complicated intra-abdominal infections identified through systematic review. We searched MEDLINE (PubMed) and ClinicalTrials.gov databases to identify relevant articles reporting results from double-blind clinical trials that used a source control review process. Eight prospective, randomized, double-blind, multicenter, clinical trials of 5 anti-infective agents in complicated intra-abdominal infections used a source control review process. We provide recommendations for an independent, adjudicated source control review process applicable to future clinical trials.

Azithromycin increases in vitro fibronectin production through interactions between macrophages and fibroblasts stimulated with Pseudomonas aeruginosa.

OBJECTIVES: Chronic azithromycin therapy has been associated with improved clinical outcomes in patients with cystic fibrosis (CF) who are chronically infected with Pseudomonas aeruginosa. We have previously demonstrated that azithromycin polarizes macrophages towards an alternatively activated phenotype, thereby blunting inflammation associated with infection. Because this phenotype is pro-fibrotic, it is important to evaluate azithromycin's consequential effects upon fibroblast function and extracellular matrix (ECM) protein production. METHODS: We co-cultured macrophages and fibroblasts together and stimulated them by adding P. aeruginosa or lipopolysaccharide to assess the ability of azithromycin to alter the macrophage phenotype, along with the impact exerted upon the production of fibronectin and other effectors that govern tissue remodelling, including transforming growth factor ß (TGFß), matrix metalloproteinase-9 (MMP-9) and arginase. We supported these studies by evaluating the impact of azithromycin treatment on these proteins in a mouse model of P. aeruginosa infection. RESULTS: Azithromycin increased arginase expression in vitro, as well as the activation of latent TGFß, consistent with polarization to the alternative macrophage phenotype. While the drug increased fibronectin concentrations after stimulation in vitro, secretion of the ECM-degrading enzyme MMP-9 was also increased. Neutralization of active TGFß resulted in the ablation of azithromycin's ability to increase fibronectin concentrations, but did not alter its ability to increase MMP-9 expression. In P. aeruginosa-infected mice, azithromycin significantly decreased MMP-9 and fibronectin concentrations in the alveolar space compared with non-treated, infected controls. CONCLUSIONS: Our results suggest that azithromycin's effect on MMP-9 is regulated independently of TGFß activity. Additionally, the beneficial effects of azithromycin may be partially due to effects on homeostasis in which ECM-degrading mediators like MMP-9 are up-regulated early after infection. This may impact the damaging effects of inflammation that lead to fibrosis in this patient population.

Pulmonary function outcomes in bronchopulmonary dysplasia through childhood and into adulthood: implications for primary care.

Bronchopulmonary dysplasia (BPD) results from prematurity and surfactant deficiency with contributing factors from barotrauma, volutrauma, and oxygen toxicity from supportive mechanical ventilation care and infection. These factors result in chronic inflammation with recurring cycles of lung damage and repair that impair alveolarisation and vascularisation in developing infant lungs. With advancement in the understanding of its pathophysiology and resulting therapy, BPD has evolved into a different disorder which has been coined the 'new' BPD. As these patients age, primary care physicians need to understand the impact on pulmonary function. This discussion reviews the pulmonary function outcomes resulting from BPD through later childhood and young adulthood.

Azithromycin alters macrophage phenotype and pulmonary compartmentalization during lung infection with Pseudomonas.

Infection with mucoid strains of Pseudomonas aeruginosa in chronic inflammatory diseases of the airway is difficult to eradicate and can cause excessive inflammation. The roles of alternatively activated and regulatory subsets of macrophages in this pathophysiological process are not well characterized. We previously demonstrated that azithromycin induces an alternatively activated macrophage-like phenotype in vitro. In the present study, we tested whether azithromycin affects the macrophage activation status and migration in the lungs of P. aeruginosa-infected mice. C57BL/6 mice received daily doses of oral azithromycin and were infected intratracheally with a mucoid strain of P. aeruginosa. The properties of macrophage activation, immune cell infiltration, and markers of pulmonary inflammation in the lung interstitial and alveolar compartments were evaluated postinfection. Markers of alternative macrophage activation were induced by azithromycin treatment, including the surface expression of the mannose receptor, the upregulation of arginase 1, and a decrease in the production of proinflammatory cytokines. Additionally, azithromycin increased the number of CD11b(+) monocytes and CD4(+) T cells that infiltrated the alveolar compartment. A predominant subset of CD11b(+) cells was Gr-1 positive (Gr-1(+)), indicative of a subset of cells that has been shown to be immunoregulatory. These differences corresponded to decreases in neutrophil influx into the lung parenchyma and alteration of the characteristics of peribronchiolar inflammation without any change in the clearance of the organism. These results suggest that the immunomodulatory effects of azithromycin are associated with the induction of alternative and regulatory macrophage activation characteristics and alteration of cellular compartmentalization during infection.

Aerosolized vancomycin for the treatment of MRSA after lung transplantation.

Lung transplantation is the only life-prolonging therapy available for cystic fibrosis (CF) patients with end-stage lung disease. The presence of pathogens in the airways of CF patients prior to transplantation is the major risk factor for infection in the post-transplantation period, with methicillin-resistant Staphylococcus aureus (MRSA) having a growing impact. Aerosolized vancomycin has been used successfully in the treatment of MRSA in the CF population but its use after lung transplantation has not been previously reported. We report the case of a lung transplant recipient who was successfully treated for MRSA infection with aerosolized vancomycin.

Pathogenesis of bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) refers to a heterogeneous group of lung disorders in infants that is commonly associated with prematurity and surfactant deficiency. BPD results from the complex interplay between impairments in the premature lung such as surfactant deficiency, perinatal insults such as infection, and damage resulting from supportive care of the infant due to barotrauma or volutrauma from mechanical ventilation and oxygen toxicity from supplemental oxygen administration. These factors result in chronic inflammation in the infant lung with recurring cycles of lung damage and repair that may impair alveolarization and vascularization in the developing lungs. As our insight in how to treat BPD improves along with the ability to do so with developing technology and therapies, the underlying pathogenesis will also change. The term 'new' BPD is now commonly used, to describe the changes seen in the post-surfactant era. This discussion reviews the pathogenesis of BPD according to the current medical literature.

Airway dehiscence after lung transplantation in a patient with cystic fibrosis.

The presence of resistant pathogens in the lower airways of patients with cystic fibrosis (CF) is not an absolute contraindication for lung transplantation. We describe a case in which a patient with CF died as a result of an anastomotic dehiscence, ischemia, and infection with linezolid-resistant methicillin-resistant Staphylococcus aureus. We review infection issues during the post-lung-transplant period and related anastomotic dehiscence in CF.

Azithromycin alters macrophage phenotype.

OBJECTIVES: To investigate the in vitro effects of azithromycin on macrophage phenotype. Utilizing a mouse macrophage cell line (J774), we examined the effect of azithromycin on the properties that define classical macrophage activation (M1) and alternative macrophage activation (M2). METHODS: J774 cells were cultured in the presence of azithromycin and stimulated with classical activation [interferon-gamma (IFNgamma)] and alternative activation [interleukin (IL)-4 and IL-13] cytokines along with lipopolysaccharide (LPS). Macrophages were analysed for inflammatory cytokine production, surface receptor expression, inducible nitric oxide synthase (iNOS) protein expression and arginase activity. RESULTS: Azithromycin altered the overall macrophage phenotype. Azithromycin-treated J774 macrophages demonstrated a significantly reduced production of the pro-inflammatory cytokines IL-12 and IL-6, increased production of the anti-inflammatory cytokine IL-10 and decreased the ratio of IL-12 to IL-10 by 60%. Receptor expression indicative of the M2 phenotype (mannose receptor and CD23) was increased, and receptor expression typically up-regulated in M1 cells (CCR7) was inhibited. The presence of azithromycin increased arginase (M2 effector molecule) activity 10-fold in cells stimulated with IFNgamma and LPS, and iNOS protein (M1 effector molecule) concentrations were attenuated by the drug. CONCLUSIONS: These data provide evidence that azithromycin affects the inflammatory process at the level of the macrophage and shifts macrophage polarization towards the alternatively activated phenotype. This recently defined M2 phenotype has been described in conditions in which pulmonary inflammation and fibrosis are major determinants of clinical outcome, but the concept of antibiotics altering macrophage phenotype has not yet been critically evaluated.

Postexposure prophylaxis: a guide for prevention of human immunodeficiency virus transmission in orthopedic surgery.

Approximately half of orthopedic surgeons reported operating on at least 1 patient with known HIV infection. Knowledge of postexposure prophylaxis guidelines may prevent HIV transmission and avert unnecessary exposure to antiretroviral agents.

Testing practices and knowledge of HIV among prenatal care providers in a low seroprevalence state.

As the prevalence of heterosexually transmitted HIV increases among women of childbearing age in the United States, so too does the potential for vertical transmission from mother to child. Early maternal diagnosis and appropriate management are critical to minimizing the risk of perinatal infection. We designed a study to evaluate current prenatal care provider testing practices and knowledge of HIV as it relates to pregnancy in a low seroprevalence state. A written questionnaire was mailed to 642 prenatal care providers in Kentucky. Responses were compared to a similar survey conducted in 1998 and to current federal guidelines for HIV management. Nearly all respondents reported to offer HIV testing to all prenatal patients, demonstrating a marked improvement since 1998 (p < 0.001). However, clinicians did not report adequate follow-up when testing is refused and appear to have limited knowledge of the disease as it relates to pregnancy. Only 9.3% of respondents demonstrated proficiency on two knowledge assessment questions. Those with previous experience treating prenatal patients with HIV were more likely to respond correctly (odds ratio [OR] 3.03; 95% confidence interval [CI] 1.08-8.50). Providers with little experience treating patients with HIV may not possess the basic knowledge required to manage the disease during pregnancy. Additional educational interventions are needed in low seroprevalence areas to ensure the appropriate treatment of all HIV-positive pregnant patients and to minimize the risk of preventable perinatal transmission.

Yersinia pestis YadC: a novel vaccine candidate against plague.

Current subunit vaccines provide partial protection against pneumonic plague if the infecting Y. pestis strain is encapsulated (F1+). Here we describe YadC, a novel Y. pestis outer membrane protein that provides partial protection against a F1(-) Y. pestis strain. Swiss-Webster mice were immunized subcutaneously with glutathione S-transferase (GST) or His6-tagged (HT) purified fusion proteins (GST-YadC137-409 or HT-LcrV) or buffer emulsified with Alhydrogel. Intravenous challenge with 1 x 10(4) F1(-) Deltapgm Y. pestis CO99-3015 revealed no protection for those mice immunized with GST-Alhydrogel alone, full protection for HT-LcrV-immunized mice, and partial protection for GST-YadC137-409-immunized mice. Similarly, C57BL/6 mice were immunized with GST-YadC137-409, HT-LcrV, or GST all with Alhydrogel adjuvant. After intranasal challenge with 3 x 10(3) F1(-) Y. pestis CO99-3015, 87% of GST-YadC137-409-immunized mice survived pneumonic plague. This is compared to the GST control group (0 surviving mice) and the LcrV-immunized group where 50% survived the challenge. This protection was correlated with a predominantly IgG1 response in LcrV-immunized mice and an IgG1/IgG3 antibody response in YadC-immunized mice. Additionally, we report the cytokine response from HT-LcrV- and GST-YadC137-409-stimulated peripherally derived macrophages. YadC-stimulated cells demonstrated a predominant pro-inflammatory cytokine production. This mixed Thl/Th2 response suggests that YadC's protection may involve a different adaptive immune response than the LcrV protein that currently is part of plague vaccines.

Pneumocystis infection alters the activation state of pulmonary macrophages.

Recent studies show a substantial incidence of Pneumocystis jirovecii colonization and infection in patients with chronic inflammatory lung conditions. However, little is known about the impact of Pneumocystis upon the regulation of pulmonary immunity. We demonstrate here that Pneumocystis polarizes macrophages towards an alternatively activated macrophage-like phenotype. Genetically engineered mice that lack the ability to signal through IL-4 and IL-13 were used to show that Pneumocystis alternative macrophage activation is dependent upon signaling through these cytokines. To determine whether Pneumocystis-induced macrophage polarization would impact subsequent immune responses, we infected mice with Pneumocystis and then challenged them with Pseudomonas aeruginosa 14 days later. In co-infected animals, a higher proportion of macrophages in the alveolar and interstitial spaces expressed both classical and alternatively activated markers and produced the regulatory cytokines TGFß and IL-10, as well as higher arginase levels than in mice infected with P. aeruginosa alone. Our results suggest that Pneumocystis reprograms the overall macrophage repertoire in the lung to that of a more alternatively-activated setpoint, thereby altering subsequent immune responses. These data may help to explain the association between Pneumocystis infection and decline in pulmonary function.

Rhabdomyolysis during therapy with daptomycin.

The use of daptomycin has been associated with an elevation in creatine phosphokinase level, with a reported incidence of 2.8% in phase III clinical trials. Published case reports have documented the presence of myopathy in patients who received daptomycin; however, there have been no previously reported cases of rhabdomyolysis in animals or humans to date. We describe a case of rhabdomyolysis during therapy with daptomycin.

Temporal trends, clinical characteristics, and outcomes of histoplasmosis in a tertiary care center in Kentucky, 2000 to 2009.

The impact of highly active antiretroviral therapy (HAART) on the epidemiology of AIDS-associated histoplasmosis in the past decade is poorly defined. Among 100 patients with histoplasmosis in an endemic region between 2000 and 2009, 42 patients were immunocompetent, 32 were infected with HIV, and 26 were non-HIV-immunocompromised patients. The percentage with HIV decreased 67% in 2000-2001 to 18% in 2008-2009 (P = .004), while the proportion of non-HIV immunocompromised patients increased, 8% to 41% (P = .14). Histoplasma antigen was the most sensitive test (73%), whereas potassium hydroxide examination of clinical specimens was the least sensitive test (33%) in all 3 groups. Bronchoalveloar fluid culture (74%) had the highest yield among the cultures. The relapse rate was higher in HIV-infected patients compared to the other 2 groups (P = .04). The epidemiology of histoplasmosis in our endemic area has changed during the era of HAART. Organ transplantation and increasing use of immunosuppressive agents for chronic inflammatory conditions in non-HIV patients now account for most of the cases of histoplasmosis.

Impact of azithromycin treatment on macrophage gene expression in subjects with cystic fibrosis.

BACKGROUND: Azithromycin treatment improves clinical parameters in patients with CF, and alters macrophage activation from a pro-inflammatory (M1) phenotype to a pro-fibrotic, alternatively activated (M2) phenotype. The transcriptional profile of cells from patients receiving azithromycin is unknown. METHODS: Gene expression in association with macrophage polarization, inflammation, and tissue remodeling was assessed from sputum samples collected from patients with CF. Transcriptional profiles and clinical characteristics, including azithromycin therapy, were compared. RESULTS: Expression of NOS2 and TNFα was decreased in subjects receiving azithromycin, whereas expression of M2-associated genes was unaffected. Principal component analysis revealed gene expression profiles consistent with M1- (MMP9, NOS2, and TLR4) or M2-polarization (CCL18, fibronectin, and MR1) in select subject groups. These expression signatures did not significantly correlate with clinical characteristics. CONCLUSIONS: Pro-inflammatory gene expression was low in subjects receiving AZM. Genes were stratified into groupings characteristic of M1- or M2-polarization, suggesting that overall polarization status is distinct among patient groups.

Eradication of Pseudomonas aeruginosa in an adult patient with cystic fibrosis.

PURPOSE: The use of a continuous infusion of a ß-lactam antibiotic in combination with high-dose, extended-interval amino-glycoside therapy for eradication of Pseudomonas aeruginosa in an adult patient with cystic fibrosis (CF) is reported. SUMMARY: Testing of an expectorated sputum sample taken during routine evaluation of a 32-year-old woman with CF isolated P. aeruginosa; the patient's medical record indicated no prior episodes of P. aeruginosa colonization. In an initial attempt to eradicate the organism, the woman received outpatient therapy with oral ciprofloxacin twice daily combined with an aminoglycoside (tobramycin solution) by nebulization twice daily. After a culture four weeks later again isolated P. aeruginosa, the patient was hospitalized, and i.v. antimicrobial therapy was initiated. The inpatient treatment regimen consisted of continuous-infusion cefepime 6 g (100 mg/kg/24 hr) and i.v. tobramycin 700 mg (12 mg/kg/24 hr), with both drugs administered via a peripherally inserted central catheter, for two weeks. A bronchoalveolar lavage fluid culture performed two months after completion of the i.v. antimicrobial regimen, as well as several sputum cultures obtained during the subsequent three years, tested negative for P. aeruginosa. CONCLUSION: The administration of continuous-infusion cefepime and high-dose, extended-interval tobramycin led to the successful eradication of P. aeruginosa in an adult patient with CF.

Aerosolized amphotericin for the treatment of allergic bronchopulmonary aspergillosis.

Allergic bronchopulmonary aspergillosis (ABPA) is a complex hypersensitivity reaction to Aspergillus fumigatus that occur frequently in patients with cystic fibrosis (CF). Recurrent episodes of bronchial obstruction, inflammation, and mucoid impaction occur in ABPA and results in bronchiectasis, fibrosis, and respiratory failure. The treatment of ABPA includes corticosteroids to reduce the acute inflammation and intraconazole to reduce the fungal colonization load in order to reduce lung injury. This case discusses the successful use of aerosolized amphotericin B for the treatment of ABPA in a 14-year-old patient with CF listed for lung transplant. The patient required fewer hospitalizations, and both oral corticosteroids and anti-fungal therapy were eventually stopped.

Characterization of macrophage activation states in patients with cystic fibrosis.

BACKGROUND: Chronic airway inflammation characterizes patients with cystic fibrosis (CF). The role of alternative macrophage activation in this disease course is unknown. OBJECTIVE: We evaluated markers of alternative and classical macrophage activation in the lungs of patients with CF and evaluated these characteristics in the context of Pseudomonas aeruginosa (PA) infection, immunomodulatory drug therapy and pulmonary function. METHODS: Bronchoalveolar lavage or spontaneously expectorated sputum samples were collected from 48 CF patients. Clinical data were related to macrophage surface expression of mannose receptor (MR) (up-regulated in alternatively activated macrophages) and TLR4 (up-regulated in classically activated macrophages). Also, the activity of the alternatively activated macrophage effector molecule arginase was compared among patient groups, and pro- and anti-inflammatory cytokines produced by alternatively and classically activated macrophages were measured. RESULTS: There were significant differences between PA-infected and -uninfected patients in several clinical measurements. PA-infected patients exhibited increased use of azithromycin, up-regulation of MR on CD11b+ cells and increased arginase activity in their lung samples, and had a strong inverse relationship between MR and arginase activity to FEV(1). Upon further analysis, PA-infected patients who were treated with azithromycin had the highest arginase activity and the highest number of macrophages that were MR+TLR4-, and both of these markers were inversely related to the FEV(1). CONCLUSIONS: Our findings suggest an increase in both MR and arginase expression as pulmonary function declines in PA-infected patients with CF. These markers of an alternatively activated macrophage phenotype give cause for future study to define the function of macrophage activation states in the CF lung.