Kerry M. Empey

Pharmacologic Advances in the Treatment and Prevention of Respiratory Syncytial Virus

Currently, only 2 drugs have been approved for the treatment of respiratory syncytial virus (RSV). Palivizumab is a monoclonal antibody for the prevention of RSV in high-risk children. Ribavirin is approved for treatment of severe RSV disease; however, its effectiveness in improving outcomes is questionable. During the past 40 years, many obstacles have delayed the development of safe and effective vaccines and treatment regimens. This article reviews these obstacles and presents the novel development strategies used to overcome many of them. Also discussed are promising new antiviral treatment candidates and their associated mechanism of action, the significant advances made in vaccine development, and exciting, new studies directed at improving outcomes through pharmacologic manipulation of the host response to RSV disease.

Macrocytosis as an Indicator of Medication (Zidovudine) Adherence in Patients with HIV Infection

This retrospective chart review was conducted in an outpatient human immunodeficiency virus (HIV) clinic to determine if macrocytosis can be used as a clinical indicator of zidovudine adherence in HIV-infected outpatients. This study included 71 HIV-positive patients who were taking zidovudine and 93 HIV-positive controls who were not taking zidovudine, for 8 weeks or longer. One control and 16 subjects were excluded secondary to insufficient laboratory data or preexistence of other macrocytosis-inducing etiologies. The incidence of macrocytosis (mean corpuscular volume [MCV] >/= 100 fL) was significantly different among subjects and controls: 78% versus 32.6% (p < 0.001), respectively. Adherence (determined by documentation from a physician/pharmacist) was assessed among subjects for whom zidovudine was prescribed, comparing those with and without macrocytosis. Adherence was observed in 77% and 18% of the macrocytosis-positive and macrocytosis-negative subjects, respectively (p < 0.001). Macrocytosis was also observed in patients receiving stavudine (another thymidine analogue, in the class of nucleoside reverse transcriptase inhibitors). Twenty-seven of the 41 patients for whom stavudine was prescribed developed macrocytosis (65.8%). These results indicate that macrocytosis may be useful in assessing adherence to zidovudine-containing antiretroviral regimens and may also have a role in assessing stavudine adherence.

The Effect of an Antimicrobial Formulary Change on Hospital Resistance Patterns

A university hospital formulary change that was designed to reduce the use of the third‐generation cephalosporins ceftazidime and cefotaxime and replace them with the so‐called “fourth‐generation” cephalosporin cefepime was evaluated. A retrospective review of antibiotic use and antimicrobial resistance during two 6‐month periods before and after the formulary change was performed. All hospital patients with vancomycin‐resistant Enterococcus (VRE), ceftazidime‐resistant Klebsiella pneumoniae (CRKP), methicillin‐resistant Staphylococcus aureus (MRSA), piperacillin‐resistant Pseudomonas aeruginosa (PRPA), and ceftazidime‐resistant P. aeruginosa (CRPA) infections were included in the study. Ceftazidime use decreased from 9600 g to 99 g, and cefotaxime use decreased from 6314 g to 732 g, which represented a combined decrease of 89%. Use of cefepime increased from 0 g to 5396 g. Infections from CRKP decreased from 13% to 3%, PRPA infections decreased from 22% to 14%, and CRPA infections decreased from 25% to 15% (p<0.05 for all). Infections from MRSA dropped insignificantly, and VRE infections increased significantly. Substituting cefepime for ceftazidime and cefotaxime while reducing the overall use of cephalosporins appears to decrease rates of CRKP, PRPA, and CRPA.

Stimulation of Immature Lung Macrophages with Intranasal Interferon Gamma in a Novel Neonatal Mouse Model of Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and viral death in infants. Reduced CD8 T-cells and negligible interferon gamma (IFNγ) in the airway are associated with severe infant RSV disease, yet there is an abundance of alveolar macrophages (AM) and neutrophils. However, it is unclear, based on our current understanding of macrophage functional heterogeneity, if immature AM improve viral clearance or contribute to inflammation and airway obstruction in the IFNγ-deficient neonatal lung environment. The aim of the current study was to define the age-dependent AM phenotype during neonatal RSV infection and investigate their differentiation to classically activated macrophages (CAM) using i.n. IFNγ in the context of improving viral clearance. Neonatal and adult BALB/cJ mice were infected with 1×106 plaque forming units (PFU)/gram (g) RSV line 19 and their AM responses compared. Adult mice showed a rapid and robust CAM response, indicated by increases in major histocompatibility complex class II (MHC II), CD86, CCR7, and a reduction in mannose receptor (MR). Neonatal mice showed a delayed and reduced CAM response, likely due to undetectable IFNγ production. Intranasal (i.n.) treatment with recombinant mouse IFNγ (rIFNγ) increased the expression of CAM markers on neonatal AM, reduced viral lung titers, and improved weight gain compared to untreated controls with no detectable increase in CD4 or CD8 T-cell infiltration. In vitro infection of J774A.1 macrophages with RSV induced an alternatively activated macrophage (AAM) phenotype however, when macrophages were first primed with IFNγ, a CAM phenotype was induced and RSV spread to adjacent Hep-2 cells was reduced. These studies demonstrate that the neonatal AM response to RSV infection is abundant and immature, but can be exogenously stimulated to express the antimicrobial phenotype, CAM, with i.n. rIFNγ.

Respiratory syncytial virus infection enhances Pseudomonas aeruginosa biofilm growth through dysregulation of nutritional immunity

Significance Pseudomonas aeruginosa is the major respiratory pathogen that promotes disease progression in chronic lung diseases such as cystic fibrosis (CF) and resides in antibiotic-resistant biofilm communities in the lungs of patients. Little is known about host factors that contribute to the development of bacterial biofilms in the lung. We have observed that respiratory virus coinfection and the antiviral immune response aid in the transition of P. aeruginosa to a biofilm mode of growth through inappropriate release of the nutrient iron. Defining molecular mechanisms by which P. aeruginosa biofilms develop in the lung affords a better opportunity to target therapies to eliminate life-threatening infections in CF and in other chronic lung diseases. Clinical observations link respiratory virus infection and Pseudomonas aeruginosa colonization in chronic lung disease, including cystic fibrosis (CF) and chronic obstructive pulmonary disease. The development of P. aeruginosa into highly antibiotic-resistant biofilm communities promotes airway colonization and accounts for disease progression in patients. Although clinical studies show a strong correlation between CF patients’ acquisition of chronic P. aeruginosa infections and respiratory virus infection, little is known about the mechanism by which chronic P. aeruginosa infections are initiated in the host. Using a coculture model to study the formation of bacterial biofilm formation associated with the airway epithelium, we show that respiratory viral infections and the induction of antiviral interferons promote robust secondary P. aeruginosa biofilm formation. We report that the induction of antiviral IFN signaling in response to respiratory syncytial virus (RSV) infection induces bacterial biofilm formation through a mechanism of dysregulated iron homeostasis of the airway epithelium. Moreover, increased apical release of the host iron-binding protein transferrin during RSV infection promotes P. aeruginosa biofilm development in vitro and in vivo. Thus, nutritional immunity pathways that are disrupted during respiratory viral infection create an environment that favors secondary bacterial infection and may provide previously unidentified targets to combat bacterial biofilm formation.

Passive Immunization of Neonatal Mice against Pneumocystis carinii f. sp. muris Enhances Control of Infection without Stimulating Inflammation

ABSTRACT Pneumocystis carinii is an opportunistic fungal pathogen that causes life-threatening pneumonia in immunocompromised individuals. Infants appear to be particularly susceptible to infection with Pneumocystis. We have previously shown that there is a significant delay in clearance of the organisms from the lungs of neonatal mice compared to adults. Since alveolar macrophages are the effector cells responsible for killing and clearance of Pneumocystis, we have examined alveolar macrophage activity in neonatal mice. We found that alveolar macrophage activation is delayed about 1 week in Pneumocystis-infected neonates compared to adults. Opsonization of the organism by Pneumocystis-specific antibody resulted in increased clearance of the organism in neonatal mice; however, there was decreased expression of activation markers on neonatal alveolar macrophages and reduced levels of cytokines associated with macrophage activation. Mice born to immunized dams had significant amounts of Pneumocystis-specific immunoglobulin G in their lungs and serum at day 7 postinfection, whereas mice born to naïve dams had merely detectable levels. This difference correlated with enhanced Pneumocystis clearance in mice born to immunized dams. The increase in specific antibody, however, did not result in significant inflammation in the lungs, as no differences in numbers of activated CD4+ cells were observed. Furthermore, there was no difference in cytokine or chemokine concentrations in the lungs of pups born to immune compared to naïve dams. These findings indicate that specific antibody plays an important role in Pneumocystis clearance from lungs of infected neonates; moreover, this process occurs without inducing inflammation in the lungs.

Exogenous Heat-Killed Escherichia coli Improves Alveolar Macrophage Activity and Reduces Pneumocystis carinii Lung Burden in Infant Mice

ABSTRACT Pneumocystis carinii is an opportunistic fungal pathogen that causes life-threatening pneumonia in immunocompromised individuals. Infants appear to be particularly susceptible to Pneumocystis pulmonary infections. We have previously demonstrated that there is approximately a 3-week delay in the clearance of Pneumocystis organisms from pup mouse lungs compared to that in adults. We have further shown that there is approximately a 1-week delay in alveolar macrophage activation in pups versus adult mice. Alveolar macrophages are the primary effector cells responsible for the killing and clearance of Pneumocystis, suggesting that pup alveolar macrophages may be involved in the delayed clearance of this organism. Alveolar macrophages cultured in vitro with Pneumocystis alone demonstrate little to no activation, as indicated by a lack of cytokine production. However, when cultured with lipopolysaccharide (LPS) or zymosan, cytokine production was markedly increased, suggesting that pup alveolar macrophages are specifically unresponsive to Pneumocystis organisms rather than being intrinsically unable to become activated. Furthermore, pup mice treated with aerosolized, heat-killed Escherichia coli in vivo were able to clear Pneumocystis more efficiently than were control mice. Together, these data suggest that while pup alveolar macrophages are unresponsive to P. carinii f. sp. muris organisms, they are capable of activation by heat-killed E. coli in vivo, as well as LPS and zymosan in vitro. The lack of response of pup mice to P. carinii f. sp. muris may reflect protective mechanisms specific to the developing pup lung, but ultimately it results in insufficient clearance of Pneumocystis organisms.

Alveolar macrophages support interferon gamma-mediated viral clearance in RSV-infected neonatal mice.

BackgroundPoor interferon gamma (IFNγ) production during respiratory syncytial virus (RSV) is associated with prolonged viral clearance and increased disease severity in neonatal mice and humans. We previously showed that intra-nasal delivery of IFNγ significantly enhances RSV clearance from neonatal lungs prior to observed T-lymphocyte recruitment or activation, suggesting an innate immune mechanism of viral clearance. We further showed that alveolar macrophages dominate the RSV-infected neonatal airways relative to adults, consistent with human neonatal autopsy data. Therefore, the goal of this work was to determine the role of neonatal alveolar macrophages in IFNγ-mediated RSV clearance.MethodsClodronate liposomes, flow cytometry, viral plaque assays, and histology were used to examine the role of alveolar macrophages (AMs) and the effects of intra-nasal IFNγ in RSV infected neonatal Balb/c mice. The functional outcomes of AM depletion were determined quantitatively by viral titers using plaque assay. Illness was assessed by measuring reduced weight gain.ResultsAM activation during RSV infection was age-dependent and correlated tightly with IFNγ exposure. Higher doses of IFNγ more efficiently stimulated AM activation and expedited RSV clearance without significantly affecting weight gain. The presence of AMs were independently associated with improved RSV clearance, whereas AM depletion but not IFNγ exposure, significantly impaired weight gain in RSV-infected neonates.ConclusionWe show here for the first time, that IFNγ is critical for neonatal RSV clearance and that it depends, in part, on alveolar macrophages (AMs) for efficient viral clearing effects. Early reductions in viral burden are likely to have profound short- and long-term immune effects in the vulnerable post-natally developing lung environment. Studies are ongoing to elucidate the pathologic effects associated with early versus delayed RSV clearance in developing neonatal airways.

Alveolar Macrophages in Neonatal Mice Are Inherently Unresponsive to Pneumocystis murina Infection

ABSTRACT Pneumocystis pneumonia was first diagnosed in malnourished children and has more recently been found in children with upper respiratory symptoms. We previously reported that there is a significant delay in the immune response in newborn mice infected with Pneumocystis compared to adults (Garvy BA, Harmsen AG, Infect. Immun. 64:3987–3992, 1996, and Garvy BA, Qureshi M, J. Immunol. 165:6480–6486, 2000). This delay is characterized by the failure of neonatal lungs to upregulate proinflammatory cytokines and attract T cells into the alveoli. Here, we report that regardless of the age at which we infected the mice, they failed to mount an inflammatory response in the alveolar spaces until they were 21 days of age or older. Anti-inflammatory cytokines had some role in dampening inflammation, since interleukin-10 (IL-10)-deficient pups cleared Pneumocystis faster than wild-type pups and the neutralization of transforming growth factor beta (TGF-β) with specific antibody enhanced T cell migration into the lungs at later time points. However, the clearance kinetics were similar to those of control pups, suggesting that there is an intrinsic deficiency in the ability of innate immunity to control Pneumocystis. We found, using an adoptive transfer strategy, that the lung environment contributes to association of Pneumocystis organisms with alveolar macrophages, implying no intrinsic deficiency in the binding of Pneumocystis by neonatal macrophages. Using both in vivo and in vitro assays, we found that Pneumocystis organisms were less able to stimulate translocation of NF-κB to the nucleus of alveolar macrophages from neonatal mice. These data indicate that there is an early unresponsiveness of neonatal alveolar macrophages to Pneumocystis infection that is both intrinsic and related to the immunosuppressive environment found in neonatal lungs.

Core Competencies for Research Training in the Clinical Pharmaceutical Sciences

Objective. To identify and apply core competencies for training students enrolled in the clinical pharmaceutical scientist PhD training program at the University of Pittsburgh School of Pharmacy. Design. Faculty members reached consensus on the required core competencies for the program and mapped them to curricular and experiential requirements. Assessment. A rubric was created based on core competencies spanning 8 major categories of proficiency, and competencies of clinical versus traditional PhD training were delineated. A retrospective evaluation of the written comprehensive examinations of 12 former students was conducted using the rubric. Students scored above satisfactory in 11 out of 14 comprehensive examination metrics, with a mean score greater than 3.8 on a 5-point scale. Conclusions. The core competencies identified will provide an essential foundation for informed decision-making and assessment of PhD training in the clinical pharmaceutical sciences.