Acute respiratory disease is associated with significant morbidity and mortality in influenza. Because antiviral drugs are only effective early in infection, new agents are needed to treat nonvaccinated patients presenting with late-stage disease, particularly those who develop acute respiratory distress syndrome. We found previously that the de novo pyrimidine synthesis inhibitor A77-1726 reversed the influenza-induced impairment of alveolar fluid clearance. Patients with acute respiratory distress syndrome and intact alveolar fluid clearance demonstrate lower mortality than those with compromised fluid clearance. We therefore investigated the effects of treatment with nebulized A77-1726 (67.5 mg/kg) on indices of cardiopulmonary function relevant to the diagnosis of acute respiratory distress syndrome. BALB/cAnNCr mice (8–12 wk old) were inoculated intranasally with 10,000 plaque-forming units/mouse influenza A/WSN/33 (H1N1). Pulse oximetry was performed daily. Alveolar fluid clearance, lung water, and lung mechanics were measured at 2 and 6 days after inoculation in live, ventilated mice by BSA instillation, magnetic resonance imaging, and forced-oscillation techniques, respectively. A77-1726 treatment at 1 day after inoculation delayed mortality. Treatment on Days 1 or 5 reduced viral replication on Day 6, and improved alveolar fluid clearance, peripheral oxygenation, and cardiac function. Nebulized A77-1726 also reversed influenza-induced increases in lung water content and volume, improved pulmonary mechanics, reduced bronchoalveolar lavage fluid ATP and neutrophil content, and increased IL-6 concentrations. The ability of A77-1726 to improve cardiopulmonary function in influenza-infected mice and to reduce the severity of ongoing acute respiratory distress syndrome late in infection suggests that pyrimidine synthesis inhibitors are promising therapeutic candidates for the management of severe influenza.
alveolar fluid clearance; pulmonary edema; antiviral agents; lung function; ARDS
MicroRNA-29b (miR-29b) expression has been shown to be reduced in non-small–cell lung cancer (NSCLC) tissues. Here, we have identified the oncogene cyclin-dependent protein kinase 6 (CDK6) as a direct target of miR-29b in lung cancer. We hypothesized that in vivo restoration of miR-29b and thus targeting of genes important to tumor initiation and progression may represent an option for lung cancer treatment. We developed a cationic lipoplexes (LPs)-based carrier that efficiently delivered miR-29b both in vitro and in vivo. LPs containing miR-29b (LP-miR-29b) efficiently delivered miR-29b to NSCLC A549 cells, reduced the expression of key targets CDK6, DNMT3B, and myeloid cell leukemia sequence 1 (MCL1), as well as cell growth and clonogenicity of A549 cells. In addition, the IC50 for cisplatin in the miR-29b–treated cells was effectively reduced. In a xenograft murine model, LPs efficiently accumulated at tumor sites. Systemic delivery of LP-miR-29b increased the tumor miR-29b expression by approximately fivefold, downregulated the tumor mRNA expression of CDK6, DNMT3B, and MCL1 by ~57.4, ~40.5, and ~52.4%, respectively, and significantly inhibited tumor growth by ~60% compared with LP-miR-NC (negative control). Our results demonstrate that cationic LPs represent an efficient delivery system that holds great potential in the development of miRNA-based therapeutics for lung cancer treatment.
cationic lipoplexes; lung cancer; microRNA
Each year, approximately 20% of asthmatics in the United States experience acute symptom exacerbations, which commonly result from pulmonary viral infections. The majority of asthma exacerbations in very young children follow infection with respiratory syncytial virus (RSV). However, pathogenic mechanisms underlying induction of asthma exacerbations by RSV are not well understood. We therefore investigated the effect of post-sensitization RSV infection on lung function in ovalbumin (OVA)-sensitized BALB/c mice as a model of RSV asthma exacerbations. OVA sensitization of uninfected female BALB/c mice increased bronchoalveolar lavage fluid (BALF) eosinophil levels and induced airway hyperresponsiveness to the muscarinic agonist methacholine, as measured by the forced-oscillation technique. In contrast, intranasal infection with replication-competent RSV strain A2 for 2–8 days reduced BALF eosinophil counts and reversed airway hyperresponsiveness in a pertussis toxin-sensitive manner. BALF levels of the chemokine keratinocyte cytokine (KC; a murine homolog of interleukin-8) were elevated in OVA-sensitized, RSV-infected mice and reversal of methacholine hyperresponsiveness in these animals was rapidly inhibited by KC neutralization. Hyporesponsiveness could be induced in OVA-sensitized, uninfected mice by recombinant KC or the Gαi agonist melittin. These data suggest that respiratory syncytial virus induces KC-mediated activation of Gαi, resulting in cross-inhibition of Gαq-mediated M3-muscarinic receptor signaling and reversal of airway hyperresponsiveness. As in unsensitized mice, KC therefore appears to play a significant role in induction of airway dysfunction by respiratory syncytial virus. Hence, interleukin-8 may be a promising therapeutic target to normalize lung function in both asthmatics and non-asthmatics with bronchiolitis. However, the OVA-sensitized, RSV-infected mouse may not be an appropriate model for investigating the pathogenesis of viral asthma exacerbations.
Cadmium is a toxic heavy metal ranked seventh on the Priority List of Hazardous Substances. As a byproduct of smelters, cadmium is a prevalent environmental contaminant. It is also a major component of cigarette smoke, and its inhalation is associated with decreased pulmonary function, lung cancer, and chronic obstructive pulmonary disease. Ion channels, including the cystic fibrosis transmembrane conductance regulator (CFTR), play a central role in maintaining fluid homeostasis and lung functions. CFTR is mostly expressed in epithelial cells, and little is known about the effect of cadmium exposure on lung epithelial cell function. We show that exposure to cadmium decreases the expression of the CFTR protein and subsequent chloride transport in human airway epithelial cells in vitro. Impairment of CFTR protein expression was also observed in vivo in the lung of mice after intranasal instillation of cadmium. We established that the inhibitory effect of cadmium was not a nonspecific effect of heavy metals, as nickel had no effect on CFTR protein levels. Finally, we show that selected antioxidants, including alpha-tocopherol (vitamin E), but not N-acetylcysteine, can prevent the cadmium-induced suppression of CFTR. In summary, we have identified cadmium as a regulator of the CFTR chloride channel present in lung epithelial cells. Future strategies to prevent the deleterious effect of cadmium on epithelial cells and lung functions may benefit from the finding that alpha-tocopherol protects CFTR expression and function.
CFTR; cadmium; airway epithelial cells; vitamin E
We have reported that moderate-intensity aerobic exercise training attenuates airway inflammation in mice sensitized/challenged with ovalbumin (OVA). The current study determined the effects of repeated bouts of aerobic exercise at a moderate intensity on airway hyperresponsiveness (AHR) in these mice. Mice were sensitized/challenged with OVA or saline and exercised at a moderate intensity 3 times/week for 4 weeks. At protocol completion, mice were analyzed for changes in AHR via mechanical ventilation. Results show that exercise decreased total lung resistance 60% in OVA-treated mice as compared with controls; exercise also decreased airway smooth muscle (ASM) thickness. In contrast, exercise increased circulating epinephrine levels 3-fold in saline- and OVA-treated mice. Because epinephrine binds β2-adrenergic receptors (AR), which facilitate bronchodilatation, the role of β2-AR in exercise-mediated improvements in AHR was examined. Application of the β2-AR antagonist butoxamine HCl blocked the effects of exercise on lung resistance in OVA-treated mice. In parallel, ASM cells were examined for changes in the protein expression of β2-AR and G-protein receptor kinase-2 (GRK-2); GRK-2 promotes β2-AR desensitization. Exercise had no effect on β2-AR expression in ASM cells of OVA-treated mice; however, exercise decreased GRK-2 expression by 50% as compared with controls. Exercise also decreased prostaglandin E2 (PGE2) production 5-fold, but had no effect on E prostanoid-1 (EP1) receptor expression within the lungs of OVA-treated mice; both PGE2 and the EP1 receptor have been implicated in β2-AR desensitization. Together, these data indicate that moderate-intensity aerobic exercise training attenuates AHR via a mechanism that involves β2-AR.
asthma; airway hyperresponsiveness; exercise; β2-adrenergic receptor
We investigated the mechanisms by which respiratory syncytial virus (RSV) infection decreases vectorial Na+ transport across respiratory epithelial cells. Mouse tracheal epithelial (MTE) cells from either BALB/c or C57BL/6 mice and human airway H441 cells were grown on semipermeable supports under an air–liquid interface. Cells were infected with RSV-A2 and mounted in Ussing chambers for measurements of short-circuit currents (Isc). Infection with RSV for 24 hours (multiplicity of infection = 1) resulted in positive immunofluorescence for RSV antigen in less than 10% of MTE or H441 cells. In spite of the limited number of cells infected, RSV reduced both basal and amiloride-sensitive Isc in both MTE and H441 cells by approximately 50%, without causing a concomitant reduction in transepithelial resistance. Agents that increased intracellular cAMP (forskolin, cpt-CAMP, and IBMX) increased mainly Cl− secretion in MTE cells and Na+ absorption in H441 cells. RSV infection for 24 hours blunted both variables. In contrast, ouabain sensitive Isc, measured across apically permeabilized H441 monolayers, remained unchanged. Western blot analysis of H441 cell lysates demonstrated reductions in α- but not γ-ENaC subunit protein levels at 24 hours after RSV infection. The reduction in amiloride-sensitive Isc in H441 cells was prevented by pretreatment with inhibitors of de novo pyrimidine or purine synthesis (A77-1726 and 6-MP, respectively, 50 μM). Our results suggest that infection of both murine and human respiratory epithelial cells with RSV inhibits vectorial Na+ transport via nucleotide release. These findings are consistent with our previous studies showing reduced alveolar fluid clearance after RSV infection of BALB/c mice.
short circuit current; epithelial Na+ channels; H441 cells; uridine triphosphate; A77-1726
Previous studies have suggested that the asthmatic responses of airway inflammation, remodeling, and hyperresponsiveness (AHR) are interrelated; in this study, we used exercise to examine the nature of this interrelationship. Mice were sensitized and challenged with ovalbumin (OVA); mice were then exercised via running on a motorized treadmill at a moderate intensity. Data indicate that, within the lungs of OVA-treated mice, exercise attenuated the production of inflammatory mediators, including chemokines KC, RANTES, and MCP-1 and IL-12p40/p80. Coordinately, OVA-treated and exercised mice displayed decreases in leukocyte infiltration, including eosinophils, as compared with sedentary controls. Results also show that a single bout of exercise significantly decreased phosphorylation of the NFκB p65 subunit, which regulates the gene expression of a wide variety of inflammatory mediators. In addition, OVA-treated and exercised mice exhibited decreases in the levels of Th2-derived cytokines IL-5 and IL-13 and the prostaglandin PGE2, as compared with sedentary controls. In contrast, results show that a single bout of exercise had no effect on AHR in OVA-treated mice challenged with increasing doses of aerosolized methacholine (0–50 mg/ml) as compared with sedentary mice. Exercise also had no effect on epithelial cell hypertrophy, mucus production, or airway wall thickening in OVA-treated mice as compared with sedentary controls. These findings suggest that a single bout of aerobic exercise at a moderate intensity attenuates airway inflammation but not AHR or airway remodeling in OVA-treated mice. The implication of these findings for the interrelationship between airway inflammation, airway remodeling, and AHR is discussed.
asthma; aerobic exercise; airway inflammation; remodeling; hyperresponsiveness
Rationale: Pulmonary infections can impair alveolar fluid clearance (AFC), contributing to formation of lung edema. Effects of influenza A virus (IAV) on AFC are unknown.
Objectives: To determine effects of IAV infection on AFC, and to identify intercellular signaling mechanisms underlying influenza-mediated inhibition of AFC.
Methods: BALB/c mice were infected intranasally with influenza A/WSN/33 (10,000 or 2,500 focus-forming units per mouse). AFC was measured in anesthetized, ventilated mice by instilling 5% bovine serum albumin into the dependent lung.
Measurements and Main Results: Infection with high-dose IAV resulted in a steady decline in arterial oxygen saturation and increased lung water content. AFC was significantly inhibited starting 1 hour after infection, and remained suppressed through Day 6. AFC inhibition at early time points (1–4 h after infection) did not require viral replication, whereas AFC inhibition later in infection was replication-dependent. Low-dose IAV infection impaired AFC for 10 days, but induced only mild hypoxemia. High-dose IAV infection increased bronchoalveolar lavage fluid ATP and UTP levels. Impaired AFC at Day 2 resulted primarily from reduced amiloride-sensitive AFC, mediated by increased activation of the pyrimidine-P2Y purinergic receptor axis. However, an additional component of AFC impairment was due to activation of A1 adenosine receptors and stimulation of increased cystic fibrosis transmembrane regulator–mediated anion secretion. Finally, IAV-mediated inhibition of AFC at Day 2 could be reversed by addition of β-adrenergic agonists to the AFC instillate.
Conclusions: AFC inhibition may be an important feature of early IAV infection. Its blockade may reduce the severity of pulmonary edema and hypoxemia associated with influenza pneumonia.
orthomyxovirus infections; pneumonia, viral; pulmonary edema; ion transport; adenosine
Despite respiratory syncytial virus (RSV) bronchiolitis remaining the most common cause of lower respiratory tract disease in infants worldwide, treatment has progressed little in the past 30 years. The aim of our study was to determine whether post-infection administration of de novo pyrimidine synthesis inhibitors could prevent the reduction in alveolar fluid clearance (AFC) and hypoxemia that occurs at Day 2 after intranasal infection of BALB/c mice with RSV. BALB/c mice were infected intranasally with RSV strain A2. AFC was measured in anesthetized, ventilated mice after instillation of 5% bovine serum albumin into the dependent lung. Post-infection systemic treatment with leflunomide has no effect on AFC. However, when added to the AFC instillate, leflunomide's active metabolite, A77-1726, blocks RSV-mediated inhibition of AFC at Day 2. This block is reversed by uridine (which allows pyrimidine synthesis via the scavenger pathway) and not recapitulated by genistein (which mimics the tyrosine kinase inhibitor effects of A77-1726), indicating that the effect is specific for the de novo pyrimidine synthesis pathway. More importantly, when administered intranasally at Day 1, A77-1726, but not its vehicle dimethyl sulfoxide, maintains its beneficial effect on AFC and lung water content until Day 2. Intranasal instillation of A77-1726 at Day 1 also reduces bronchoalveolar lavage nucleotide levels, lung inflammation, and hypoxemia at Day 2 without impairing viral replication at Day 2 or viral clearance at Day 8. Post-infection intranasal or aerosolized treatment with pyrimidine synthesis inhibitors may provide symptomatic relief from the pathophysiologic sequelae of impaired AFC in children with RSV bronchiolitis.
paramyxovirus; leflunomide; dihydroorotate dehydrogenase; pulmonary edema
Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants and children worldwide. We wished to determine whether intracheal administration of β-agonists improved alveolar fluid clearance (AFC) across the distal respiratory epithelium of RSV infected mice. Following intranasal infection with RSV strain A2, AFC was measured in anesthetized, ventilated BALB/c mice by instillation of 5% BSA into the dependent lung. We found that direct activation of protein kinase A by forskolin or 8-bromo-cAMP increased AFC at day 2 after infection with RSV. In contrast, short- and long-acting β-agonists had no effect at either day 2 or day 4. Insensitivity to β-agonists was not a result of elevated plasma catecholamines, or lung epithelial cell β-adrenergic receptor degradation. Instead, RSV infected mice had significantly higher levels of phosphorylated PKCζ in the membrane fractions of their lung epithelial cells. In addition, insensitivity to β-agonists was mediated in a paracrine fashion by KC (the murine homolog of CXCL8) and reversed by inhibition of either PKCζ or G protein-coupled receptor kinase 2 (GRK2). These results indicate that insufficient response to β-agonists in RSV may be caused, at least in part, by impaired β-adrenergic receptor signaling, as a consequence of GRK2-mediated uncoupling of β-adrenergic receptors from adenylyl cyclase.
Paramyxovirus; Protein kinase C; G protein-coupled receptor kinase 2; CXCL8
Despite respiratory syncytial virus (RSV) bronchiolitis remaining the most common cause of lower respiratory tract disease in infants worldwide, treatment has progressed little in the past 30 years.
To determine whether postinfection administration of de novo pyrimidine synthesis inhibitors could prevent the reduction in alveolar fluid clearance (AFC) and hypoxemia that occurs at day 2 following intranasal infection of BALB/c mice with RSV.
BALB/c mice were infected intranasally with RSV strain A2. AFC was measured in anesthetized, ventilated mice following instillation of 5% BSA into the dependent lung.
Post-infection systemic treatment with leflunomide has no effect on AFC. However, when added to the AFC instillate, leflunomide’s active metabolite, A77-1726, blocks RSV-mediated inhibition of AFC at day 2. This block is reversed by uridine (which allows pyrimidine synthesis via the scavenger pathway) and not recapitulated by genistein (which mimics the tyrosine kinase inhibitor effects of A77-1726), indicating that the effect is specific for the de novo pyrimidine synthesis pathway. More importantly, when administered intranasally at day 1, A77-1726, but not its vehicle DMSO, maintains its beneficial effect on AFC and lung water content until day 2. Intranasal instillation of A77-1726 at day 1 also reduces BAL nucleotide levels, lung inflammation, and hypoxemia at day 2 without impairing viral replication at day 2 or viral clearance at day 8.
Post-infection intranasal or aerosolized treatment with pyrimidine synthesis inhibitors may provide symptomatic relief from the pathophysiologic sequelae of impaired AFC in children with RSV bronchiolitis.
Paramyxovirus; leflunomide; dihydroorotate dehydrogenase; pulmonary edema
Rationale: Previously, we demonstrated that intranasal infection of BALB/c mice with respiratory syncytial virus (RSV) resulted in an early 40% reduction in alveolar fluid clearance (AFC), an effect mediated via P2Y purinergic receptors.
Objectives: To confirm that RSV-induced inhibition of AFC is mediated by uridine triphosphate (UTP), and to demonstrate that inhibition of de novo pyrimidine synthesis with leflunomide prevents increased UTP release after RSV infection, and thereby also prevents inhibition of AFC by RSV.
Methods: BALB/c mice were infected intranasally with RSV strain A2. AFC was measured in anesthetized, ventilated mice by instillation of 5% bovine serum albumin into the dependent lung. Some mice were pretreated with leflunomide or 6-mercaptopurine.
Measurements and Main Results: RSV-mediated inhibition of AFC is associated temporally with a 20-nM increase in UTP and ATP content of bronchoalveolar lavage fluid, hypoxemia, and altered nasal potential difference. RSV-mediated nucleotide release, AFC inhibition, and physiologic sequelae thereof can be prevented by pretreatment of mice with the de novo pyrimidine synthesis inhibitor leflunomide, which is not toxic to the mice, and which does not affect RSV replication in the lungs. In contrast, pretreatment of mice with 6-mercaptopurine, an inhibitor of de novo purine synthesis, has no beneficial effect on AFC or other indicators of disease progression. Finally, RSV-mediated inhibition of AFC is prevented by volume-regulated anion channel inhibitors.
Conclusion: Pyrimidine synthesis or release pathways may provide novel therapeutic targets to counter the pathophysiologic sequelae of impaired AFC in RSV disease.
ion transport; paramyxovirus infections; pneumonia, viral; pulmonary edema
Rationale: Mycoplasma pneumoniae is a significant cause of pneumonia in humans.
Objectives: To determine the impact of mycoplasma infection and the host inflammatory response on alveolar type II (ATII) cell ion transport in vivo and in vitro.
Methods: Mice were infected with M. pulmonis for measurements of alveolar fluid clearance (AFC) in vivo and isolation of ATII cells. ATII cells were infected in vivo for determination of epithelial Na+ channel (ENaC) total and cell surface protein levels by biotinylation and Western blot and in vitro for whole cell patch clamp recording and measurement of nitric oxide (NO) production by chemiluminescence.
Results: Mycoplasma infection significantly inhibited AFC at 24 h and total and amiloride-sensitive AFC by 48 h postinfection (pi). In contrast, infected myeloperoxidase-deficient mice had similar basal and amiloride-sensitive AFC values to uninfected control mice at 48 h pi. Addition of forskolin restored total and amiloride-sensitive AFC to control values at 48 h pi. ATII cells isolated from infected mice demonstrated normal α, β, and γ ENaC total protein levels; however, infected whole-lung cell-surface levels of γ ENaC were significantly decreased. Patch-clamp recordings demonstrated a significant decrease in total and amiloride-sensitive Na+ currents at 24 h pi. ATII cells demonstrated a significant increase in the production of NO at 24 h pi and inhibition of NO by ATII cells before infection reversed the decrease in total Na+ currents.
Conclusions: These data indicate that mycoplasma infection results in decreased AFC and functional ENaC via the production of reactive oxygen nitrogen intermediates.
alveolar fluid clearance; amiloride; chemiluminescence; epithelial sodium channels; nitric oxide synthase; patch clamp
Hantavirus cardiopulmonary syndrome (HCPS) is a life-threatening respiratory disease characterized by profound pulmonary edema and myocardial depression. Most cases of HCPS in North America are caused by Sin Nombre virus (SNV), which is carried asymptomatically by deer mice (Peromyscus maniculatus). The underlying pathophysiology of HCPS is poorly understood. We hypothesized that pathogenic SNV infection results in increased generation of reactive oxygen/nitrogen species (RONS), which contribute to the morbidity and mortality of HCPS. Human disease following infection with SNV or Andes virus was associated with increased nitrotyrosine (NT) adduct formation in the lungs, heart, and plasma and increased expression of inducible nitric oxide synthase (iNOS) in the lungs compared to the results obtained for normal human volunteers. In contrast, NT formation was not increased in the lungs or cardiac tissue from SNV-infected deer mice, even at the time of peak viral antigen expression. In a murine (Mus musculus) model of HCPS (infection of NZB/BLNJ mice with lymphocytic choriomeningitis virus clone 13), HCPS-like disease was associated with elevated expression of iNOS in the lungs and NT formation in plasma, cardiac tissue, and the lungs. In this model, intraperitoneal injection of 1400W, a specific iNOS inhibitor, every 12 h during infection significantly improved survival without affecting intrapulmonary fluid accumulation or viral replication, suggesting that cardiac damage may instead be the cause of mortality. These data indicate that elevated production of RONS is a feature of pathogenic New World hantavirus infection and that pharmacologic blockade of iNOS activity may be of therapeutic benefit in HCPS cases, possibly by ameliorating the myocardial suppressant effects of RONS.
Attenuated simian immunodeficiency viruses (SIVs) have been described that produce low levels of plasma virion RNA and exhibit a reduced capacity to cause disease. These viruses are particularly useful in identifying viral determinants of pathogenesis. In the present study, we show that mutation of a highly conserved tyrosine (Tyr)-containing motif (Yxxφ) in the envelope glycoprotein (Env) cytoplasmic tail (amino acids YRPV at positions 721 to 724) can profoundly reduce the in vivo pathogenicity of SIVmac239. This domain constitutes both a potent endocytosis signal that reduces Env expression on infected cells and a sorting signal that directs Env expression to the basolateral surface of polarized cells. Rhesus macaques were inoculated with SIVmac239 control or SIVmac239 containing either a Tyr-721-to-Ile mutation (SIVmac239Y/I) or a deletion of Tyr-721 and the preceding glycine (ΔGY). To assess the in vivo replication competence, all viruses contained a stop codon in nef that has been shown to revert during in vivo but not in vitro replication. All three control animals developed high viral loads and disease. One of two animals that received SIVmac239Y/I and two of three animals that received SIVmac239ΔGY remained healthy for up to 140 weeks with low to undetectable plasma viral RNA levels and normal CD4+ T-cell percentages. These animals exhibited ongoing viral replication as determined by detection of viral sequences and culturing of mutant viruses from peripheral blood mononuclear cells and persistent anti-SIV antibody titers. In one animal that received SIVmac239Y/I, the Ile reverted to a Tyr and was associated with a high plasma RNA level and disease, while one animal that received SIVmac239ΔGY also developed a high viral load that was associated with novel and possibly compensatory mutations in the TM cytoplasmic domain. In all control and experimental animals, the nef stop codon reverted to an open reading frame within the first 2 months of inoculation, indicating that the mutant viruses had replicated well enough to repair this mutation. These findings indicate that the Yxxφ signal plays an important role in SIV pathogenesis. Moreover, because mutations in this motif may attenuate SIV through mechanisms that are distinct from those caused by mutations in nef, this Tyr-based sorting signal represents a novel target for future models of SIV and human immunodeficiency virus attenuation that could be useful in new vaccine strategies.
Hypericumperforatum (H. perforatum) ethanol extract has been found to inhibit lipopolysaccharide-induced production of inflammatory mediators and cytokines in cultured macrophages. Therefore, it may be able to protect the host from excessive inflammation during viral infection. In the current study, the immune-regulatory effect of H. perforatum extract was evaluated in A549 lung epithelial cells and BALB/c mice exposed to Influenza A/PR/8/34 H1N1 virus. In A549 cells, the extract (30 µg/mL) significantly inhibited influenza virus induced monocyte chemotactic protein (MCP)-1 and interferon-γ induced protein 10 kD (IP-10), but dramatically increased interleukin-6 (IL-6). In mice inoculated intranasally with 107.9 EID50 of Influenza A/PR/8/34 H1N1 (high dose), daily oral treatment of H. perforatum extract at a rate of 110 mg/kg of body weight increased lung viral titer, bronchoalveolar lavage (BAL) pro-inflammatory cytokine and chemokine levels, and the infiltration of pro-inflammatory cells in the lung 5 days post-inoculation, as compared to ethanol vehicle treated mice. Transcription of suppressor of cytokine signaling 3 (SOCS3) was increased by H. perforatum extract both in A549 cells and BALB/c mice, which could have interrupted anti-viral immune response and thus led to the inefficient viral clearance and increased lung inflammation. H. perforatum treatment resulted in minor reduction in viral titer without affecting body weight when mice were inoculated with a lower dose (~105.0 EID50) and H. perforatum was applied in the later phase of infection. Mice challenged intranasally with high dose of influenza virus (107.9 EID50) suffered from a higher mortality rate when dosed with H. perforatum extract. In conclusion, the current study showed that SOCS3 elevation by H. perforatum may cause impaired immune defense against influenza virus infection and lead to higher mortality.
Supportive evidence that apoptosis contributes to loss of CD4+ lymphocytes in human immunodeficiency virus type 1 (HIV-1)-infected humans comes from an apparent lack of abnormal apoptosis in apathogenic lentivirus infections of nonhuman primates, including HIV-1 infection of chimpanzees. Two female chimpanzees were inoculated, one cervically and the other intravenously, with HIV-1 derived from the LAI/LAV-1b strain, which was isolated from a chimpanzee infected with the virus for 8 years. Within 6 weeks of infection, both recipient chimpanzees developed a progressive loss of CD4+ T cells which correlated with persistently high viral burdens and increased levels of CD4+ T-cell apoptosis both in vitro and in vivo. Lymph nodes from both animals also revealed evidence of immune hyperactivation. Intermediate levels of T-cell apoptosis in both peripheral blood and lymph nodes were seen in a third chimpanzee that had been infected with the LAI/LAV-1b strain for 9 years; this animal has maintained depressed CD4/CD8 T-cell ratios for the last 3 years. Similar analyses of cells from 4 uninfected animals and 10 other HIV-1-infected chimpanzees without loss of CD4+ cells revealed no difference in levels of apoptosis in these two control groups. These results demonstrate a correlation between immune hyperactivation, T-cell apoptosis, and chronic loss of CD4+ T cells in HIV-1-infected chimpanzees, providing additional evidence that apoptosis is an important factor in T-cell loss in AIDS. Furthermore, the results show that some HIV-1 strains are pathogenic for chimpanzees and that this species is not inherently resistant to HIV-1-induced disease.
Interspecies transmission of influenza A is an important factor in the evolution and ecology of influenza viruses. Marine mammals are in contact with a number of influenza reservoirs, including aquatic birds and humans, and this may facilitate transmission among avian and mammalian hosts. Virus isolation, whole genome sequencing, and hemagluttination inhibition assay confirmed that exposure to pandemic H1N1 influenza virus occurred among free-ranging Northern Elephant Seals (Mirounga angustirostris) in 2010. Nasal swabs were collected from 42 adult female seals in April 2010, just after the animals had returned to the central California coast from their short post-breeding migration in the northeast Pacific. Swabs from two seals tested positive by RT-PCR for the matrix gene, and virus was isolated from each by inoculation into embryonic chicken eggs. Whole genome sequencing revealed greater than 99% homology with A/California/04/2009 (H1N1) that emerged in humans from swine in 2009. Analysis of more than 300 serum samples showed that samples collected early in 2010 (n = 100) were negative and by April animals began to test positive for antibodies against the pH1N1 virus (HI titer of ≥1∶40), supporting the molecular findings. In vitro characterizations studies revealed that viral replication was indistinguishable from that of reference strains of pH1N1 in canine kidney cells, but replication was inefficient in human epithelial respiratory cells, indicating these isolates may be elephant seal adapted viruses. Thus findings confirmed that exposure to pandemic H1N1 that was circulating in people in 2009 occurred among free-ranging Northern Elephant Seals in 2010 off the central California coast. This is the first report of pH1N1 (A/Elephant seal/California/1/2010) in any marine mammal and provides evidence for cross species transmission of influenza viruses in free-ranging wildlife and movement of influenza viruses between humans and wildlife.
Influenza A viruses continue to pose a threat to human health; thus, various vaccines and prophylaxis continue to be developed. Testing of these products requires various animal models including mice, guinea pigs, and ferrets. However, because ferrets are naturally susceptible to infection with human influenza viruses and because the disease state resembles that of human influenza, these animals have been widely used as a model to study influenza virus pathogenesis. In this report, a statistical analysis was performed to evaluate data involving 269 ferrets infected with seasonal influenza, swine influenza, and highly pathogenic avian influenza (HPAI) from 16 different studies over a five year period. The aim of the analyses was to better qualify the ferret model by identifying relationships among important animal model parameters (endpoints) and variables of interest, which include survival, time-to-death, changes in body temperature and weight, and nasal wash samples containing virus, in addition to significant changes from baseline in selected hematology and clinical chemistry parameters. The results demonstrate that a disease clinical profile, consisting of various changes in the biological parameters tested, is associated with various influenza A infections in ferrets. Additionally, the analysis yielded correlates of protection associated with HPAI disease in ferrets. In all, the results from this study further validate the use of the ferret as a model to study influenza A pathology and to evaluate product efficacy.
Critically ill patients frequently develop acute lung injury (ALI). Disturbed alveolar fibrin turnover, a characteristic feature of ALI, is the result of both activation of coagulation and inhibition of fibrinolysis. Nebulized fibrinolytic agents could exert lung–protective effects, via promotion of fibrinolysis as well as anti–inflammation.
Rats were challenged intratracheally with Pseudomonas aeruginosa, resulting in pneumonia as a model for direct ALI, or received an intravenous bolus infusion of lipopolysaccharide, as a model for indirect ALI. Rats were randomized to nebulization of normal saline (placebo), recombinant tissue plasminogen activator (rtPA), or monoclonal antibodies against plasminogen activator inhibitor–type 1 (anti–PAI–1).
Nebulized rtPA or anti–PA1–1 enhanced the bronchoalveolar fibrinolytic system, as reflected by a significant reduction of PAI–1 activity levels in bronchoalveolar lavage fluid, and a consequent increase in plasminogen activator activity (PAA) levels to supranormal values. Both treatments also significantly affected systemic fibrinolysis as reflected by a significant increase in PAA levels in plasma to supranormal levels. Neither nebulized rtPA nor anti–PA1–1 affected pulmonary inflammation. Neither treatment affected bacterial clearance of P. aeruginosa from the lungs in case of pneumonia.
Local treatment with rtPA or anti–PA1–1 affects pulmonary fibrinolysis but not inflammation in models of direct or indirect ALI in rats.