Exploring antiviral and anti-inflammatory effects of thiol drugs in COVID-19.

The redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) is important for the binding of SARS-2-S to angiotensin-converting enzyme 2 (ACE2), suggesting that drugs with a functional thiol group ("thiol drugs") may cleave cystines to disrupt SARS-CoV-2 cell entry. In addition, neutrophil-induced oxidative stress is a mechanism of COVID-19 lung injury, and the antioxidant and anti-inflammatory properties of thiol drugs, especially cysteamine, may limit this injury. To first explore the antiviral effects of thiol drugs in COVID-19, we used an ACE-2 binding assay and cell entry assays utilizing reporter pseudoviruses and authentic SARS-CoV-2 viruses. We found that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus infection. The most potent drugs were effective in the low millimolar range, and IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. To determine if thiol drugs have antiviral effects in vivo and to explore any anti-inflammatory effects of thiol drugs in COVID-19, we tested the effects of cysteamine delivered intraperitoneally to hamsters infected with SARS-CoV-2. Cysteamine did not decrease lung viral infection, but it significantly decreased lung neutrophilic inflammation and alveolar hemorrhage. We speculate that the concentration of cysteamine achieved in the lungs with intraperitoneal delivery was insufficient for antiviral effects but sufficient for anti-inflammatory effects. We conclude that thiol drugs decrease SARS-CoV-2 lung inflammation and injury, and we provide rationale for future studies to test if direct (aerosol) delivery of thiol drugs to the airways might also result in antiviral effects.

Myocyte repolarization modulates myocardial function in aging dogs.

Studies of myocardial aging are complex and the mechanisms involved in the deterioration of ventricular performance and decreased functional reserve of the old heart remain to be properly defined. We have studied a colony of beagle dogs from 3 to 14 yr of age kept under a highly regulated environment to define the effects of aging on the myocardium. Ventricular, myocardial, and myocyte function, together with anatomical and structural properties of the organ and cardiomyocytes, were evaluated. Ventricular hypertrophy was not observed with aging and the structural composition of the myocardium was modestly affected. Alterations in the myocyte compartment were identified in aged dogs, and these factors negatively interfere with the contractile reserve typical of the young heart. The duration of the action potential is prolonged in old cardiomyocytes contributing to the slower electrical recovery of the myocardium. Also, the remodeled repolarization of cardiomyocytes with aging provides inotropic support to the senescent muscle but compromises its contractile reserve, rendering the old heart ineffective under conditions of high hemodynamic demand. The defects in the electrical and mechanical properties of cardiomyocytes with aging suggest that this cell population is an important determinant of the cardiac senescent phenotype. Collectively, the delayed electrical repolarization of aging cardiomyocytes may be viewed as a critical variable of the aging myopathy and its propensity to evolve into ventricular decompensation under stressful conditions.

Similar to Those Who Are Breastfed, Infants Fed a Formula Containing 2'-Fucosyllactose Have Lower Inflammatory Cytokines in a Randomized Controlled Trial.

BACKGROUND: Evidence suggests that human milk oligosaccharides (HMOs) provide multiple benefits to infants, including prebiotic effects, gut maturation, antimicrobial activities, and immune modulation. Clinical intervention studies with HMOs are required to confirm these benefits in infants. OBJECTIVE: Our objective was to investigate the effects of feeding formulas supplemented with the HMO 2'-fucosyllactose (2'-FL) on biomarkers of immune function in healthy term infants. METHODS: We performed a substudy nested within a randomized, double-blind, controlled growth and tolerance study in healthy singleton infants (birth weight ≥2490 g) who were enrolled by 5 d of life and exclusively formula-fed (n = 317) or breastfed (n = 107) from enrollment to 4 mo of age. Formula-fed infants were randomly assigned to receive 1 of 3 formulas, all containing 2.4 g total oligosaccharides/L [control: galacto-oligosaccharides (GOS) only; experimental formulas: GOS + 0.2 or 1.0 g 2'-FL/L], and compared with a breastfed reference group. For this substudy, blood samples were drawn from infants at 6 wk of age (n = 31-42/group). Peripheral blood mononuclear cells (PBMCs) were isolated for cellular phenotyping and stimulated ex vivo with phytohemagglutinin for proliferation and cell cycle progression or respiratory syncytial virus (RSV). Cytokine concentrations were measured in plasma and in ex vivo-stimulated culture supernatants. RESULTS: Breastfed infants and infants fed either of the experimental formulas with 2'-FL were not different but had 29-83% lower concentrations of plasma inflammatory cytokines than did infants fed the control formula [interleukin (IL) receptor antagonist (IL-1ra), IL-1α, IL-1ß, IL-6, and tumor necrosis factor α (TNF-α)] (P ≤ 0.05). In ex vivo RSV-stimulated PBMC cultures, breastfed infants were not different than either of the groups fed formula with 2'-FL, but they had lower concentrations of TNF-α (31%) and interferon γ (IFN-γ 54%) (P ≤ 0.05) and tended to have lower IL-1ra (25%) and IL-6 (38%) (unadjusted P ≤ 0.05) and IL-1ß (30%) (unadjusted P = 0.06) than did infants fed the control formula. CONCLUSIONS: Our data indicate that infants fed formula supplemented with 2'-FL exhibit lower plasma and ex vivo inflammatory cytokine profiles, similar to those of a breastfed reference group. This trial was registered at clinicaltrials.gov as NCT01808105.

A critical role of acute bronchoconstriction in the mortality associated with high-dose sarin inhalation: effects of epinephrine and oxygen therapies.

Sarin is an organophosphate nerve agent that is among the most lethal chemical toxins known to mankind. Because of its vaporization properties and ease and low cost of production, sarin is the nerve agent with a strong potential for use by terrorists and rouge nations. The primary route of sarin exposure is through inhalation and, depending on the dose, sarin leads to acute respiratory failure and death. The mechanism(s) of sarin-induced respiratory failure is poorly understood. Sarin irreversibly inhibits acetylcholine esterase, leading to excessive synaptic levels of acetylcholine and, we have previously shown that sarin causes marked ventilatory changes including weakened response to hypoxia. We now show that LD50 sarin inhalation causes severe bronchoconstriction in rats, leading to airway resistance, increased hypoxia-induced factor-1α, and severe lung epithelium injury. Transferring animals into 60% oxygen chambers after sarin exposure improved the survival from about 50% to 75% at 24h; however, many animals died within hours after removal from the oxygen chambers. On the other hand, if LD50 sarin-exposed animals were administered the bronchodilator epinephrine, >90% of the animals survived. Moreover, while both epinephrine and oxygen treatments moderated cardiorespiratory parameters, the proinflammatory cytokine surge, and elevated expression of hypoxia-induced factor-1α, only epinephrine consistently reduced the sarin-induced bronchoconstriction. These data suggest that severe bronchoconstriction is a critical factor in the mortality induced by LD50 sarin inhalation, and epinephrine may limit the ventilatory, inflammatory, and lethal effects of sarin.

The neonatal susceptibility window for inhalant allergen sensitization in the atopically predisposed canine asthma model.

Allergic asthma often begins in early life and, although many risk factors have been enumerated, the specific factors that initiate disease progression in an individual remain unclear. Using our dog model of early life allergen inhalation, we tested the hypothesis that the atopically biased neonatal immune system would exhibit tolerance to ragweed if allowed to mature normally before exposure or artificially through innate immune stimulation with early life exposure. Dogs were subjected to a series of inhalational ragweed exposures from 1 to 20 weeks old, with or without inhalation of a Toll-like receptor 4 (TLR4) agonist (CRX-527), or from 13 to 31 weeks old. Serum allergen-specific antibody response was assessed at 4, 8 and 20 weeks after the last sensitizing exposure. At 24 or 35 weeks old, airway hyper-responsiveness to methacholine and ragweed challenges and pulmonary inflammation by bronchoalveolar lavage were tested 1 and 4 days after ragweed challenge at 28 or 39 weeks old. Allergen-free immune maturation resulted in no airway hyper-responsiveness and very little ragweed-specific IgE relative to the control group, but eosinophilia developed upon ragweed challenge. TLR4 agonism yielded no airway hyper-responsiveness, but a strong airway neutrophilia developed upon ragweed challenge. Our data indicate that an atopic predisposition creates a critical window in which allergen exposure can lead to an asthmatic phenotype. Allergen-free immune maturation may lead to allergen tolerance. TLR4 agonism before early life allergen exposure may abrogate the development of allergen-specific bronchonconstriction, but allergen-specific pulmonary inflammation remains a strong concern.

The impact of primary immunization route on the outcome of infection with SARS-CoV-2 in a hamster model of COVID-19.

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has resulted in over 6.7 million deaths worldwide. COVID-19 vaccines administered parenterally via intramuscular or subcutaneous (SC) routes have reduced the severity of respiratory infections, hospitalization rates, and overall mortality. However, there is a growing interest in developing mucosally delivered vaccines to further enhance the ease and durability of vaccination. This study compared the immune response in hamsters immunized with live SARS-CoV-2 virus via SC or intranasal (IN) routes and assessed the outcome of a subsequent IN SARS-CoV-2 challenge. Results showed that SC-immunized hamsters elicited a dose-dependent neutralizing antibody response but of a significantly lower magnitude than that observed in IN-immunized hamsters. The IN challenge with SARS-CoV-2 in SC-immunized hamsters resulted in body weight loss, increased viral load, and lung pathology than that observed in IN-immunized and IN-challenged counterparts. These results demonstrate that while SC immunization renders some degree of protection, IN immunization induces a stronger immune response and better protection against respiratory SARS-CoV-2 infection. Overall, this study provides evidence that the route of primary immunization plays a critical role in determining the severity of a subsequent respiratory infection caused by SARS-CoV-2. Furthermore, the findings suggest that IN route of immunization may be a more effective option for COVID-19 vaccines than the currently used parenteral routes. Understanding the immune response to SARS-CoV-2 elicited via different immunization routes may help guide more effective and long-lasting vaccination strategies.

Increased lethality of respiratory infection by Streptococcus pneumoniae in the Dp16 mouse model of Down syndrome.

Objectives: We sought to investigate whether the Dp16 mouse model of Down syndrome (DS) is more susceptible to severe and lethal respiratory tract infection by Streptococcus pneumoniae. Study Design: We infected controls and Dp16 mice with Streptococcus pneumoniae and measured survival rates. We compared cytokine production by primary lung cell cultures exposed to Streptococcus pneumoniae. We examined lung protein expression for interferon signaling related pathways. We characterized the histopathology and quantified the extent of bronchus-associated lymphoid tissue. Finally, we examined mouse tissues for the presence of oligomeric tau protein. Results: We found that the Dp16 mouse model of DS displayed significantly higher susceptibility to lethal respiratory infection with Streptococcus pneumoniae compared to control mice. Lung cells cultured from Dp16 mice displayed unique secreted cytokine profiles compared to control mice. The Dp16 mouse lungs were characterized by profound lobar pneumonia with massive diffuse consolidation involving nearly the entire lobe. Marked red hepatization was noted, and Dp16 mice lungs contained numerous bronchus-associated lymphoid tissues that were highly follicularized. Compared to uninfected mice, both control mice and Dp16 mice infected with Streptococcus pneumoniae showed evidence of oligomeric tau aggregates. Conclusions: Increased susceptibility to severe respiratory tract infection with Streptococcus pneumoniae in Dp16 mice closely phenocopies infection in individuals with DS. The increase does not appear to be linked to overexpression of mouse interferon genes syntenic to human chromosome 21.

Design and validation of an exposure system for efficient inter-animal SARS-CoV-2 airborne transmission in Syrian hamsters.

IMPORTANCE: The main route of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission is airborne. However, there are few experimental systems that can assess the airborne transmission dynamics of SARS-CoV-2 in vivo. Here, we designed, built, and characterized a hamster transmission caging and exposure system that allows for efficient SARS-CoV-2 airborne transmission in Syrian hamsters without contributions from fomite or direct contact transmission. We successfully measured SARS-CoV-2 viral RNA in aerosols and demonstrated that SARS-CoV-2 is transmitted efficiently at either a 1:1 or 1:4 infected index to naïve recipient hamster ratio. This is meaningful as a 1:4 infected index to naïve hamster ratio would allow for simultaneous comparisons of various interventions in naïve animals to determine their susceptibility to infection by aerosol transmission of SARS-CoV-2. Our SARS-CoV-2 exposure system allows for testing viral airborne transmission dynamics and transmission-blocking therapeutic strategies against SARS-CoV-2 in Syrian hamsters.

Adenovirus type 5 SARS-CoV-2 vaccines delivered orally or intranasally reduced disease severity and transmission in a hamster model.

Transmission-blocking strategies that slow the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and protect against coronavirus disease 2019 (COVID-19) are needed. We have developed an orally delivered adenovirus type 5-vectored SARS-CoV-2 vaccine candidate that expresses the spike protein. Here, we demonstrated that hamsters vaccinated by the oral or intranasal route had robust and cross-reactive antibody responses. We then induced a postvaccination infection by inoculating vaccinated hamsters with SARS-CoV-2. Orally or intranasally vaccinated hamsters had decreased viral RNA and infectious virus in the nose and lungs and experienced less lung pathology compared to mock-vaccinated hamsters after SARS-CoV-2 challenge. Naïve hamsters exposed in a unidirectional air flow chamber to mucosally vaccinated, SARS-CoV-2-infected hamsters also had lower nasal swab viral RNA and exhibited fewer clinical symptoms than control animals, suggesting that the mucosal route reduced viral transmission. The same platform encoding the SARS-CoV-2 spike and nucleocapsid proteins elicited mucosal cross-reactive SARS-CoV-2-specific IgA responses in a phase 1 clinical trial (NCT04563702). Our data demonstrate that mucosal immunization is a viable strategy to decrease SARS-CoV-2 disease and airborne transmission.

Effects of simulated downwind coal combustion emissions on pre-existing allergic airway responses in mice.

CONTEXT: Coal-fired power plant emissions can contribute a significant portion of the ambient air pollution in many parts of the world. OBJECTIVE: We hypothesized that exposure to simulated downwind coal combustion emissions (SDCCE) may exacerbate pre-existing allergic airway responses. METHODS: Mice were sensitized and challenged with ovalbumin (OVA). Parallel groups were sham-sensitized with saline. Mice were exposed 6 h/day for 3 days to air (control, C) or SDCCE containing particulate matter (PM) at low (L; 100 µg/m³), medium (M; 300 µg/m³), or high (H; 1000 µg/m³) concentrations, or to the H level with PM removed by filtration (high-filtered, HF). Immediately after SDCCE exposure, mice received another OVA challenge (pre-OVA protocol). In a second (post-OVA) protocol, mice were similarly sensitized but only challenged to OVA before air/SDCCE. Measurement of airway hyperresponsiveness (AHR), bronchoalveolar lavage (BAL), and blood collection were performed ~24 h after the last exposure. RESULTS: SDCCE significantly increased BAL macrophages and eosinophils in OVA-sensitized mice from the post-OVA protocol. However, there was no effect of SDCCE on BAL macrophages or eosinophils in OVA-sensitized mice from the pre-OVA protocol. BAL neutrophils were elevated following SDCCE in both protocols in nonsensitized mice. These changes were not altered by filtering out the PM. In the post-OVA protocol, SDCCE decreased OVA-specific IgG1 in OVA-sensitized mice but increased levels of total IgE, OVA-specific IgE and OVA-specific IgG1 and IgG(2a) in non-sensitized animals. In the pre-OVA protocol, SDCCE increased OVA-specific IgE in both sensitized and non-sensitized animals. Additionally, BAL IL-4, IL-13, and IFN-γ levels were elevated in sensitized mice. CONCLUSION: These results suggest that acute exposure to either the particulate or gaseous phase of SDCCE can exacerbate various features of allergic airway responses depending on the timing of exposure in relation to allergen challenge.

Health effects of subchronic inhalation exposure to simulated downwind coal combustion emissions.

CONTEXT: There have been no animal studies of the health effects of repeated inhalation of mixtures representing downwind pollution from coal combustion. Environmental exposures typically follow atmospheric processing and mixing with pollutants from other sources. OBJECTIVE: This was the fourth study by the National Environmental Respiratory Center to create a database for responses of animal models to combustion-derived pollutant mixtures, to identify causal pollutants-regardless of source. METHODS: F344 and SHR rats and A/J, C57BL/6, and BALB/c mice were exposed 6 h/day 7 days/week for 1 week to 6 months to three concentrations of a mixture simulating key components of "downwind" coal combustion emissions, to the highest concentration filtered to remove particulate matter (PM), or to clean air. Emissions from low-sulfur subbituminous coal were modified to create a mixture recommended by an expert workshop. Sulfur dioxide, nitrogen oxides, and PM were the dominant components. Nonanimal-derived PM mass concentrations of nominally 0, 100, 300, and 1000 µg/m(3) were mostly partially neutralized sulfate. RESULTS: Only 17 of 270 species-gender-time-outcome comparisons were significantly affected by exposure; some models showed no effects. There was strong evidence that PM participated meaningfully in only three responses. CONCLUSION: On a total mass or PM mass basis, this mixture was less toxic overall than diesel and gasoline exhausts or wood smoke. The largely sulfate PM contributed to few effects and was the sole cause of none. The study did not allow identification of causal pollutants, but the potential role of NOx in some effects is suggested by the literature.

Thiol drugs decrease SARS-CoV-2 lung injury in vivo and disrupt SARS-CoV-2 spike complex binding to ACE2 in vitro.

Neutrophil-induced oxidative stress is a mechanism of lung injury in COVID-19, and drugs with a functional thiol group ("thiol drugs"), especially cysteamine, have anti-oxidant and anti-inflammatory properties that could limit this injury. Thiol drugs may also alter the redox status of the cysteine-rich SARS-CoV-2 spike glycoprotein (SARS-2-S) and thereby disrupt ACE2 binding. Using ACE2 binding assay, reporter virus pseudotyped with SARS-CoV-2 spikes (ancestral and variants) and authentic SARS-CoV-2 (Wuhan-1), we find that multiple thiol drugs inhibit SARS-2-S binding to ACE2 and virus entry into cells. Pseudoviruses carrying variant spikes were less efficiently inhibited as compared to pseudotypes bearing an ancestral spike, but the most potent drugs still inhibited the Delta variant in the low millimolar range. IC50 values followed the order of their cystine cleavage rates and lower thiol pKa values. In hamsters infected with SARS-CoV-2, intraperitoneal (IP) cysteamine decreased neutrophilic inflammation and alveolar hemorrhage in the lungs but did not decrease viral infection, most likely because IP delivery could not achieve millimolar concentrations in the airways. These data show that thiol drugs inhibit SARS-CoV-2 infection in vitro and reduce SARS-CoV-2-related lung injury in vivo and provide strong rationale for trials of systemically delivered thiol drugs as COVID-19 treatments. We propose that antiviral effects of thiol drugs in vivo will require delivery directly to the airways to ensure millimolar drug concentrations and that thiol drugs with lower thiol pKa values are most likely to be effective.

Linear and conformation specific antibodies in aged beagles after prolonged vaccination with aggregated Abeta.

Previously we showed that anti-Abeta peptide immunotherapy significantly attenuated Alzheimer's-like amyloid deposition in the central nervous system of aged canines. In this report we have characterized the changes that occurred in the humoral immune response over 2.4years in canines immunized repeatedly with aggregated Abeta(1-42) (AN1792) formulated in alum adjuvant. We observed a rapid and robust induction of anti-Abeta antibody titers, which were associated with an anti-inflammatory T helper type 2 (Th2) response. The initial antibody response was against dominant linear epitope at the N-terminus region of the Abeta(1-42) peptide, which is identical to the one in humans and vervet monkeys. After multiple immunizations the antibody response drifted toward the elevation of antibodies that recognized conformational epitopes of assembled forms of Abeta and other types of amyloid. Our findings indicate that prolonged immunization results in distinctive temporal changes in antibody profiles, which may be important for other experimental and clinical settings.

Formulation development and in vivo evaluation of a new dry powder formulation of albuterol sulphate in beagle dogs.

PURPOSE: Study objectives were to develop, characterize, and evaluate a novel excipient for dry powder inhalation formulations in a canine model with a model compound. METHODS: Dry powder inhalation formulations of albuterol sulphate were developed and compared to a commercially available nebulizer albuterol solution formulation. In vitro analysis indicated a high fine-particle fraction (FPF, >70%) and a respirable particle size ( approximately 2.5 microm MMAD). Each inhalation formulation, including controls, was delivered targeting a deposited lung dose of 10 microg/kg albuterol. Active formulations were evaluated for pharmacokinetic (PK) profile and bronchodilatory effects in a ragweed-sensitized dog model of allergic airway responses. RESULTS: In vitro, the dextran spray-dried formulated materials showed that aerosol performance, including FPF, MMAD, glass transition temperature, and amorphous characteristics, were all largely unaffected by amount of drug loaded. Both the commercial and the dry powder formulations attenuated the ragweed-induced bronchoconstriction by 91.59 +/- 3.60 and 81.28 +/- 9.29%, respectively. The PK profiles for both albuterol formulations were similar, as were the corresponding T(max), C(max) and T(1/2). CONCLUSIONS: Results indicate that dextran 10 has promise as a novel excipient for dry powder inhalation drug delivery, in a preclinical setting, over a wide range of drug loadings.

Effects of concentrated ambient particles and diesel engine exhaust on allergic airway disease in Brown Norway rats.

Increased concentrations of airborne fine particulate matter (PM2.5; particulate matter with an aerodynamic diameter < or = 2.5 microm) are associated with increases in emergency room visits and hospitalizations of asthmatic patients. Emissions from local stationary combustion sources (e.g., coal-burning power plants) or mobile motor vehicles (e.g., diesel-powered trucks) have been identified as potential contributors to the development or exacerbation of allergic airway disease. In the present study, a rodent model of allergic airway disease was used to study the effects of concentrated ambient particles (CAPs) or diesel engine exhaust (DEE) on the development of allergic airway disease in rats sensitized to the allergen ovalbumin (OVA). The overall objective of our project was to understand the effects of PM2.5 on the development of OVA-induced allergic airway disease. Our specific aims were to test the following hypotheses: (1) exposure to CAPs during OVA challenge enhances epithelial remodeling of the airway and inflammation in rats previously sensitized to the allergen; and (2) exposure to DEE during OVA sensitization, or during OVA challenge, exacerbates epithelial remodeling of the airway and inflammation in rats. In the DEE studies, Brown Norway (BN) rats were sensitized with three daily intranasal (IN) instillations of 0.5% OVA, and then two weeks later were challenged with IN OVA or saline for 3 consecutive days. Rats were exposed to DEE diluted to mass concentrations of 30 or 300 microg/m3 diesel exhaust particles (DEPs) or to filtered air during either the sensitization or challenge periods. For the CAPs studies, the same OVA sensitization and challenge rat model was used but exposures to Detroit, Michigan, CAPs were limited to the OVA challenge period. Two separate 3-day CAPs exposures were conducted (week 1, high mean mass concentration = 595 microg/m3; week 2, low mean mass concentration = 356 microg/m3) during OVA challenge. In both the DEE and CAPs studies, rats were killed 24 hours after the last OVA challenge, bronchoalveolar lavage fluid (BALF) was collected and analyzed for cellularity and secreted mediators, and lungs and nose were processed for histopathologic examination and morphometric analysis of intraepithelial mucosubstances (IM). The results of our animal inhalation studies in the southwest (SW) Detroit community, an area with elevated ambient PM2.5 concentrations, suggested that, during allergen challenge, exposure to CAPs that were predominantly associated with emissions from combustion sources markedly enhanced the OVA-induced allergic airway disease, which was characterized by an increased infiltration in the lungs of eosinophilic and lymphocytic inflammation, increased IM in conducting airways, and increased concentrations in BALF of mucin-specific proteins and inflammatory cytokines. These findings suggest that urban airborne PM2.5 derived from stationary combustion sources (e.g., refineries, coal-burning power plants, waste-treatment plants) may enhance the development of human allergic airway diseases like childhood asthma. Previous animal inhalation studies in this community have also suggested that these fine, ambient combustion-derived particles may also exacerbate preexisting allergic airway disease. In contrast to our CAPs studies in Detroit, the controlled DEE exposures of allergen-sensitized BN rats, during either allergen sensitization or challenge periods, caused only a few mild modifications in the character of the allergen-induced disease. This finding contrasts with other reported studies that indicate that DEPs at relatively higher exposure doses do enhance allergic airway disease in some rodent models. The reasons for these disparities between studies likely reflect differences in exposure dose, animal models, the timing of exposures to the allergens and DEP exposures, the methods of allergen sensitization and challenge, or physicochemical differences among DEEs.

Effects of gasoline engine emissions on preexisting allergic airway responses in mice.

Gasoline-powered vehicle emissions contribute significantly to ambient air pollution. We hypothesized that exposure to gasoline engine emissions (GEE) may exacerbate preexisting allergic airway responses. Male BALB/c mice were sensitized by injection with ovalbumin (OVA) and then received a 10-min aerosolized OVA challenge. Parallel groups were sham-sensitized with saline. Mice were exposed 6 h/day to air (control, C) or GEE containing particulate matter (PM) at low (L), medium (M), or high (H) concentrations, or to the H level with PM removed by filtration (high-filtered, HF). Immediately after GEE exposure mice received another 10-min aerosol OVA challenge (pre-OVA protocol). In a second (post-OVA) protocol, mice were similarly sensitized but only challenged to OVA before air or GEE exposure. Measurements of airway hyperresponsiveness (AHR), bronchoalveolar lavage (BAL), and blood collection were performed approximately 24 h after the last exposure. In both protocols, M, H, and HF GEE exposure significantly decreased BAL neutrophils from nonsensitized mice but had no significant effect on BAL cells from OVA-sensitized mice. In the pre-OVA protocol, GEE exposure increased OVA-specific IgG(1) but had no effect on BAL interleukin (IL)-2, IL-4, IL-13, or interferon (IFN)-gamma in OVA-sensitized mice. Nonsensitized GEE-exposed mice had increased OVA-specific IgG(2a), IgE, and IL-2, but decreased total IgE. In the post-OVA protocol, GEE exposure reduced BAL IL-4, IL-5, and IFN-gamma in nonsensitized mice but had no effect on sensitized mice. These results suggest acute exposure to the gas-vapor phase of GEE suppressed inflammatory cells and cytokines from nonsensitized mice but did not substantially exacerbate allergic responses.

Health effects of inhaled gasoline engine emissions.

Despite their prevalence in the environment, and the myriad studies that have shown associations between morbidity or mortality with proximity to roadways (proxy for motor vehicle exposures), relatively little is known about the toxicity of gasoline engine emissions (GEE). We review the studies conducted on GEE to date, and summarize the findings from each of these studies. While there have been several studies, most of the studies were conducted prior to 1980 and thus were not conducted with contemporary engines, fuels, and driving cycles. In addition, many of the biological assays conducted during those studies did not include many of the assays that are conducted on contemporary inhalation exposures to air pollutants, including cardiovascular responses and others. None of the exposures from these earlier studies were characterized at the level of detail that would be considered adequate today. A recent GEE study was conducted as part of the National Environmental Respiratory Center (www.nercenter.org). In this study several in-use mid-mileage General Motors (Chevrolet S-10) vehicles were purchased and utilized for inhalation exposures. An exposure protocol was developed where engines were operated with a repeating California Unified Driving Cycle with one cold start per day. Two separate engines were used to provide two cold starts over a 6-h inhalation period. The exposure atmospheres were characterized in detail, including detailed chemical and physical analysis of the gas, vapor, and particle phase. Multiple rodent biological models were studied, including general toxicity and inflammation (e.g., serum chemistry, lung lavage cell counts/differentials, cytokine/chemokine analysis, histopathology), asthma (adult and in utero exposures with pulmonary function and biochemical analysis), cardiovascular effects (biochemical and electrocardiograph changes in susceptible rodent models), and susceptibility to infection (Pseudomonas bacteria challenge). GEE resulted in significant biological effects for upregulation of MIP-2, clearance of Pseudomonas bacteria, development of allergic response after in utero exposure, and cardiovascular indicators of vasoconstriction, oxidant stress, and damage.

Targeting Host Cell Surface Nucleolin for RSV Therapy: Challenges and Opportunities.

Nucleolin (NCL) has been reported as a cellular receptor for the human respiratory syncytial virus (RSV). We studied the effects of re-purposing AS1411, an anti-cancer compound that binds cell surface NCL, as a possible novel strategy for RSV therapy in vitro and in vivo. AS1411 was administered to RSV-infected cultures of non-polarized (HEp-2) and polarized (MDCK) epithelial cells and to virus-infected mice and cotton rats. Results of in vitro experiments showed that AS1411, used in micromolar concentrations, was associated with decreases in the number of virus-positive cells. Intranasal administration of AS1411 (50 mg/kg) to RSV-infected mice and cotton rats was associated with partial reductions in lung viral titers, decreased virus-associated airway inflammation, and decreased IL-4/IFN-γ ratios when compared to untreated, infected animals. In conclusion, our findings indicate that therapeutic use of AS1411 has modest effects on RSV replication and host response. While the results underscore the challenges of targeting cell surface NCL as a potential novel strategy for RSV therapy, they also highlight the potential of cell surface NCL as a therapeutic target.

Development of Optimized, Inhalable, Gemcitabine-Loaded Gelatin Nanocarriers for Lung Cancer.

BACKGROUND: Aerosol delivery of chemotherapeutic nanocarriers represents a promising alternative for lung cancer therapy. This study optimized gemcitabine (Gem)-loaded gelatin nanocarriers (GNCs) cross-linked with genipin (Gem-GNCs) to evaluate their potential for nebulized lung cancer treatment. METHODS: Gem-GNCs were prepared by two-step desolvation and optimized through Taguchi design and characterized for physicochemical properties. Particle size and morphology were confirmed by scanning and transmission electron microscopy. In vitro release of Gem from Gem-GNCs performed in Dulbecco's phosphate-buffered saline and simulated lung fluid was evaluated to determine release mechanisms. Particle size stability was assessed under varying pH. Differential scanning calorimetry and powder X-ray diffraction were used to determine the presence and stability of Gem-GNC components and amorphization of Gem, respectively. Gem-GNC efficacy within A549 and H460 cells was evaluated using MTT assays. Mucus rheology upon treatment with Gem-GNCs, lactose, and normal saline control was measured. Andersen cascade impaction identified the aerodynamic particle size distribution of the nebulized formulation. RESULTS: Gem-GNCs had particle size, zeta potential, entrapment efficiency, and loading efficiency of 178 ± 7.1 nm, -18.9 mV, 92.5%, and 9.1%, respectively. The Gem and formulation excipients where molecularly dispersed and configured amorphously. Gem-GNCs were stable at pH 5.4-7.4 for 72 hours. Gem release from Gem-GNCs was governed by non-Fickian controlled release due to diffusion/erosion from a matrix-based nanocarrier. Gem-GNCs elicited a 40% reduction of the complex viscosity η*(1 Hz) of human bronchial epithelial cell mucus containing 3 wt% solids to mimic mild airway disease. The nebulized Gem-GNCs had a mass median aerodynamic diameter (MMAD) of 2.0 ± 0.16 µm, geometric standard deviation (GSD) of 2.7 ± 0.16, and fine particle fraction (FPF) of 75.2% ± 2.4%. The Gem-GNC formulation did not outperform the Gem solution in A549 cells. However, in H460, Gem-GNCs outperformed the Gem IC50 reduction by ∼5-fold at 48 and 10-fold 72 hours. CONCLUSION: Stable, effective, and sustained-release Gem-GNCs were developed. The nebulized Gem-GNCs had satisfactory MMAD, GSD, and FPF and the formulation reduced the dynamic complex viscosity of mucus consistent with increased mobility of nanoparticles.

Aß vaccination in combination with behavioral enrichment in aged beagles: effects on cognition, Aß, and microhemorrhages.

Beta-amyloid (Aß) immunotherapy is a promising intervention to slow Alzheimer's disease. Aging dogs naturally accumulate Aß and show cognitive decline. An active vaccine against fibrillar Aß 1-42 (VAC) in aged beagles resulted in maintenance but not improvement of cognition along with reduced brain Aß. Behavioral enrichment (ENR) led to cognitive benefits but no reduction in Aß. We hypothesized cognitive outcomes could be improved by combining VAC with ENR in aged dogs. Aged dogs (11-12 years) were placed into 4 groups: (1) control/control (C/C); (2) control/VAC (C/V); (3) ENR/control (E/C); and (4) ENR/VAC (E/V) and treated for 20 months. VAC decreased brain Aß, pyroglutamate Aß, increased cerebrospinal fluid Aß 42 and brain-derived neurotrophic factor RNA levels but also increased microhemorrhages. ENR reduced brain Aß and prevented microhemorrhages. The combination treatment resulted in a significant maintenance of learning over time, reduced Aß, and increased brain-derived neurotrophic factor mRNA despite increased microhemorrhages; however, there were no benefits to memory. These results suggest that the combination of immunotherapy with behavioral enrichment leads to cognitive maintenance associated with reduced neuropathology that may benefit people with Alzheimer's disease.