Chinh Nguyen

Recent Advances in Our Understanding of the Environmental, Epidemiological, Immunological, and Clinical Dimensions of Coccidioidomycosis

SUMMARY Coccidioidomycosis is the endemic mycosis caused by the fungal pathogens Coccidioides immitis and C. posadasii. This review is a summary of the recent advances that have been made in the understanding of this pathogen, including its mycology, genetics, and niche in the environment. Updates on the epidemiology of the organism emphasize that it is a continuing, significant problem in areas of endemicity. For a variety of reasons, the number of reported coccidioidal infections has increased dramatically over the past decade. While continual improvements in the fields of organ transplantation and management of autoimmune disorders and patients with HIV have led to dilemmas with concurrent infection with coccidioidomycosis, they have also led to advances in the understanding of the human immune response to infection. There have been some advances in therapeutics with the increased use of newer azoles. Lastly, there is an overview of the ongoing search for a preventative vaccine.

A Randomized, Double-Blind Placebo Controlled Trial of Balapiravir, a Polymerase Inhibitor, in Adult Dengue Patients

Background. Dengue is the most common arboviral infection of humans. There are currently no specific treatments for dengue. Balapiravir is a prodrug of a nucleoside analogue (called R1479) and an inhibitor of hepatitis C virus replication in vivo. Methods. We conducted in vitro experiments to determine the potency of balapiravir against dengue viruses and then an exploratory, dose-escalating, randomized placebo-controlled trial in adult male patients with dengue with <48 hours of fever. Results. The clinical and laboratory adverse event profile in patients receiving balapiravir at doses of 1500 mg (n = 10) or 3000 mg (n = 22) orally for 5 days was similar to that of patients receiving placebo (n = 32), indicating balapiravir was well tolerated. However, twice daily assessment of viremia and daily assessment of NS1 antigenemia indicated balapiravir did not measurably alter the kinetics of these virological markers, nor did it reduce the fever clearance time. The kinetics of plasma cytokine concentrations and the whole blood transcriptional profile were also not attenuated by balapiravir treatment. Conclusions. Although this trial, the first of its kind in dengue, does not support balapiravir as a candidate drug, it does establish a framework for antiviral treatment trials in dengue and provides the field with a clinically evaluated benchmark molecule. Clinical Trials Registration. NCT01096576.

Lower Antibody Levels to Staphylococcus aureus Exotoxins Are Associated With Sepsis in Hospitalized Adults With Invasive S. aureus Infections

BACKGROUND Staphylococcus aureus has numerous virulence factors, including exotoxins that may increase the severity of infection. This study was aimed at assessing whether preexisting antibodies to S. aureus toxins are associated with a lower risk of sepsis in adults with S. aureus infection complicated by bacteremia. METHODS We prospectively identified adults with S. aureus infection from 4 hospitals in Baltimore, MD, in 2009–2011. We obtained serum samples from prior to or at presentation of S. aureus bacteremia to measure total immunoglobulin G (IgG) and IgG antibody levels to 11 S. aureus exotoxins. Bacterial isolates were tested for the genes encoding S. aureus exotoxins using polymerase chain reaction (PCR). RESULTS One hundred eligible subjects were included and 27 of them developed sepsis. When adjusted for total IgG levels and stratified for the presence of toxin in the infecting isolate as appropriate, the risk of sepsis was significantly lower in those patients with higher levels of IgG against α-hemolysin (Hla), δ-hemolysin (Hld), Panton Valentine leukocidin (PVL), staphylococcal enterotoxin C-1 (SEC-1), and phenol-soluble modulin α3 (PSM-α3). CONCLUSIONS Our results suggest that higher antibody levels against Hla, Hld, PVL, SEC-1, and PSM-α3 may protect against sepsis in patients with invasive S. aureus infections.

NEU1 Sialidase Expressed in Human Airway Epithelia Regulates Epidermal Growth Factor Receptor (EGFR) and MUC1 Protein Signaling

Background: Airway epithelia express sialoglycoproteins that respond to danger signals and initiate repair programs. Results: NEU1 sialidase desialylates EGFR and MUC1 in airway epithelia to regulate their responsiveness to ligands and adhesiveness to P. aeruginosa. Conclusion: NEU1 provides an additional level of regulation over airway epithelial responsiveness to ligands and pathogens. Significance: The downstream effects of EGFR desialylation require further investigation. Epithelial cells (ECs) lining the airways provide a protective barrier between the external environment and the internal host milieu. These same airway epithelia express receptors that respond to danger signals and initiate repair programs. Because the sialylation state of a receptor can influence its function and is dictated in part by sialidase activity, we asked whether airway epithelia express catalytically active sialidase(s). Human primary small airway and A549 ECs expressed NEU1 sialidase at the mRNA and protein levels, and NEU1 accounted for >70% of EC sialidase activity. Blotting with Maackia amurensis and peanut agglutinin lectins established epidermal growth factor receptor (EGFR) and MUC1 as in vivo substrates for NEU1. NEU1 associated with EGFR and MUC1, and NEU1-EGFR association was regulated by EGF stimulation. NEU1 overexpression diminished EGF-stimulated EGFR Tyr-1068 autophosphorylation by up to 44% but enhanced MUC1-dependent Pseudomonas aeruginosa adhesion by 1.6–1.7-fold and flagellin-stimulated ERK1/2 activation by 1.7–1.9-fold. In contrast, NEU1 depletion increased EGFR activation (1.5-fold) and diminished MUC1-mediated bacterial adhesion (38–56%) and signaling (73%). These data indicate for the first time that human airway epithelia express catalytically active NEU1 sialidase that regulates EGFR- and MUC1-dependent signaling and bacterial adhesion. NEU1 catalytic activity may offer an additional level of regulation over the airway epithelial response to ligands, pathogens, and injurious stimuli.

NEU1 and NEU3 Sialidase Activity Expressed in Human Lung Microvascular Endothelia: NEU1 RESTRAINS ENDOTHELIAL CELL MIGRATION, WHEREAS NEU3 DOES NOT*

Background: The vascular endothelial surface is highly sialylated. Results: Vascular endothelia express catalytically active NEU1 and NEU3 sialidases, and NEU1 restrains the endothelial migratory response to wounding. Conclusion: NEU1 regulates endothelial remodeling in response to injury. Significance: Learning how NEU1 and NEU3 regulate sialylated molecules on the endothelial surface is key to understanding endothelial receptor-ligand, cell-cell, and host-pathogen interactions. The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.

Neuraminidase Reprograms Lung Tissue and Potentiates Lipopolysaccharide-Induced Acute Lung Injury in Mice

We previously reported that removal of sialyl residues primed PBMCs to respond to bacterial LPS stimulation in vitro. Therefore, we speculated that prior desialylation can sensitize the host to generate an enhanced inflammatory response upon exposure to a TLR ligand, such as LPS, in a murine model of acute lung injury. Intratracheal instillation of neuraminidase (NA) 30 min prior to intratracheal administration of LPS increased polymorphonuclear leukocytes (PMNs) in the bronchoalveolar lavage fluid and the wet-to-dry lung weight ratio, a measure of pulmonary edema, compared with mice that received LPS alone. Administration of NA alone resulted in desialylation of bronchiolar and alveolar surfaces and induction of TNF-α, IL-1β, and chemokines in lung homogenates and bronchoalveolar lavage fluid; however, PMN recruitment in mice treated with NA alone did not differ from that of PBS-administered controls. NA pretreatment alone induced apoptosis and markedly enhanced LPS-induced endothelial apoptosis. Administration of recombinant Bcl-2, an antiapoptotic molecule, abolished the effect of NA treatment on LPS-induced PMN recruitment and pulmonary edema formation. We conclude that NA pretreatment potentiates LPS-induced lung injury through enhanced PMN recruitment, pulmonary edema formation, and endothelial and myeloid cell apoptosis. A similar “reprogramming” of immune responses with desialylation may occur during respiratory infection with NA-expressing microbes and contribute to severe lung injury.

Bacillus anthracis-derived edema toxin (ET) counter-regulates movement of neutrophils and macromolecules through the endothelial paracellular pathway

BackgroundA common finding amongst patients with inhalational anthrax is a paucity of polymorphonuclear leukocytes (PMNs) in infected tissues in the face of abundant circulating PMNs. A major virulence determinant of anthrax is edema toxin (ET), which is formed by the combination of two proteins produced by the organism, edema factor (EF), which is an adenyl cyclase, and protective antigen (PA). Since cAMP, a product of adenyl cyclase, is known to enhance endothelial barrier integrity, we asked whether ET might decrease extravasation of PMNs into tissues through closure of the paracellular pathway through which PMNs traverse.ResultsPretreatment of human microvascular endothelial cell(EC)s of the lung (HMVEC-L) with ET decreased interleukin (IL)-8-driven transendothelial migration (TEM) of PMNs with a maximal reduction of nearly 60%. This effect required the presence of both EF and PA. Conversely, ET did not diminish PMN chemotaxis in an EC-free system. Pretreatment of subconfluent HMVEC-Ls decreased transendothelial 14 C-albumin flux by ~ 50% compared to medium controls. Coadministration of ET with either tumor necrosis factor-α or bacterial lipopolysaccharide, each at 100 ng/mL, attenuated the increase of transendothelial 14 C-albumin flux caused by either agent alone. The inhibitory effect of ET on TEM paralleled increases in protein kinase A (PKA) activity, but could not be blocked by inhibition of PKA with either H-89 or KT-5720. Finally, we were unable to replicate the ET effect with either forskolin or 3-isobutyl-1-methylxanthine, two agents known to increase cAMP.ConclusionsWe conclude that ET decreases IL-8-driven TEM of PMNs across HMVEC-L monolayers independent of cAMP/PKA activity.

Immunological Characterization of Bronchoalveolar Lavage Fluid in Patients With Acute Pulmonary Coccidioidomycosis

BACKGROUND The specific cellular immunological characteristics of bronchoalveolar lavage (BAL) fluid in acute pulmonary coccidioidomycosis have not been defined. METHODS BAL fluid from patients living in a coccidioidomycosis-endemic region of Arizona who were undergoing bronchoscopy because of pulmonary infiltrates was analyzed. Mononuclear cells from BAL fluid and peripheral blood mononuclear cells (PBMCs) were incubated with the coccidioidal antigen T27K in vitro, and cellular immunological assays were performed. RESULTS Forty-six patients were studied. Twelve received a diagnosis of acute pulmonary coccidioidomycosis, 17 received other diagnoses, and 17 had no diagnosis established. There was an increased proportion of polyfunctional CD8(+) T cells after antigen stimulation from subjects with coccidioidomycosis as compared to those with another diagnosis (P = .025). In cells collected from BAL fluid and in PBMCs, the concentrations of interferon γ, tumor necrosis factor α, and interleukin 17 (IL-17) were all significantly increased in samples from those with acute pulmonary coccidioidomycosis, compared with the other 2 groups (for all, P<.05). CONCLUSIONS When incubated in vitro with a coccidioidal antigen preparation, cells from both BAL fluid and peripheral blood obtained from patients with pulmonary coccidioidomycosis demonstrated specific cellular immune responses, including expression of IL-17.

Human airway epithelia express catalytically active NEU3 sialidase

Sialic acids on glycoconjugates play a pivotal role in many biological processes. In the airways, sialylated glycoproteins and glycolipids are strategically positioned on the plasma membranes of epithelia to regulate receptor-ligand, cell-cell, and host-pathogen interactions at the molecular level. We now demonstrate, for the first time, sialidase activity for ganglioside substrates in human airway epithelia. Of the four known mammalian sialidases, NEU3 has a substrate preference for gangliosides and is expressed at mRNA and protein levels at comparable abundance in epithelia derived from human trachea, bronchi, small airways, and alveoli. In small airway and alveolar epithelia, NEU3 protein was immunolocalized to the plasma membrane, cytosolic, and nuclear subcellular fractions. Small interfering RNA-induced silencing of NEU3 expression diminished sialidase activity for a ganglioside substrate by >70%. NEU3 immunostaining of intact human lung tissue could be localized to the superficial epithelia, including the ciliated brush border, as well as to nuclei. However, NEU3 was reduced in subepithelial tissues. These results indicate that human airway epithelia express catalytically active NEU3 sialidase.

Cytokine Profiles from Antigen-Stimulated Whole-Blood Samples among Patients with Pulmonary or Nonmeningeal Disseminated Coccidioidomycosis.

ABSTRACT The outcome of coccidioidomycosis depends on a robust specific cellular immune response. A T-helper type 1 (Th1) cellular immune response has been previously associated with resolution of clinical illness. However, the precise elements of this response and whether cytokines not involved with the Th1 response play a role in coccidioidomycosis are not known. Whole-blood samples were obtained from subjects with active coccidioidomycosis and controls and incubated for 18 h with T27K, a coccidioidal antigen preparation. The supernatant was then assayed for gamma interferon (IFN-γ), interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α), IL-4, IL-6, IL-10, and IL-17A. A total of 43 subjects, 16 with acute pneumonia, 9 with pulmonary sequelae of nodules and cavities, and 18 with nonmeningeal disseminated coccidioidomycosis, were studied. Compared to concentrations in healthy immune and nonimmune donors, the median concentration of IL-17A was significantly higher in those with active coccidioidomycosis (for both, P < 0.01). In addition, IL-6 concentrations were higher while IL-2 and IFN-γ concentrations were significantly lower in those with nonmeningeal disseminated disease diagnosed within 12 months than in those with acute pneumonia (for all, P < 0.05). The cytokine profile among patients with active coccidioidomycosis is distinct in that IL-17A is persistently present. In addition, those with nonmeningeal disseminated disease have an increased inflammatory cytokine response and diminished Th1 responses that modulate over time.