Effect of Charge on the Deposition of Electrostatically Charged Inhalable Aerosol in Lung Model

Inhalable drugs are widely used for treating lung diseases such as asthma, emphysema, and cystic fibrosis. The aerosol particles in these inhalable drugs may be charged electrostatically. The deposition of these inhaled therapeutic aerosol particles in the different regions of the lung depends on the particle aerodynamic diameter, electrostatic charge distribution, particulate number density, breathing rate, aerodynamics of the lung, ambient temperature, and relative humidity (RH). The primary mechanisms for lung deposition of inhaled particles are impaction, gravitational settling, diffusion, interception, and electrostatic attraction. To simulate lung deposition, electrostatically charged aerosol particles are introduced through a throat section into a glass bead lung model. The E-SPART analyzer was used to measure aerosol deposition as a function of the particle charge and size. Experiments were carried out to determine the increase in deposition efficiency as a function of the net charge-to-mass ratio (Q/M) of aerosol particles. Using a fairly monodisperse aerosol of 5.0 um count median aerodynamic diameter, it was found that the total deposition efficiency increased from 54% to 91% when Q/M increased from 0.5 to 9.67 |muC/g. The data show that enhanced delivery of the therapeutic aerosol in the lung can be achieved by controlling the electrostatic charge on the inhaled aerosol particles

Infection with Francisella tularensis LVS clpB Leads to an Altered yet Protective Immune Response

ABSTRACT Bacterial attenuation is typically thought of as reduced bacterial growth in the presence of constant immune pressure. Infection with Francisella tularensis elicits innate and adaptive immune responses. Several in vivo screens have identified F. tularensis genes necessary for virulence. Many of these mutations render F. tularensis defective for intracellular growth. However, some mutations have no impact on intracellular growth, leading us to hypothesize that these F. tularensis mutants are attenuated because they induce an altered host immune response. We were particularly interested in the F. tularensis LVS (live vaccine strain) clpB (FTL_0094) mutant because this strain was attenuated in pneumonic tularemia yet induced a protective immune response. The attenuation of LVS clpB was not due to an intracellular growth defect, as LVS clpB grew similarly to LVS in primary bone marrow-derived macrophages and a variety of cell lines. We therefore determined whether LVS clpB induced an altered immune response compared to that induced by LVS in vivo . We found that LVS clpB induced proinflammatory cytokine production in the lung early after infection, a process not observed during LVS infection. LVS clpB provoked a robust adaptive immune response similar in magnitude to that provoked by LVS but with increased gamma interferon (IFN-γ) and interleukin-17A (IL-17A) production, as measured by mean fluorescence intensity. Altogether, our results indicate that LVS clpB is attenuated due to altered host immunity and not an intrinsic growth defect. These results also indicate that disruption of a nonessential gene(s) that is involved in bacterial immune evasion, like F. tularensis clpB , can serve as a model for the rational design of attenuated vaccines

Development of an aerosol model of Cryptococcus reveals humidity as an important factor affecting the viability of Cryptococcus during aerosolization.

Cryptococcus is an emerging global health threat that is annually responsible for over 1,000,000 infections and one third of all AIDS patient deaths. There is an ongoing outbreak of cryptococcosis in the western United States and Canada. Cryptococcosis is a disease resulting from the inhalation of the infectious propagules from the environment. The current and most frequently used animal infection models initiate infection via liquid suspension through intranasal instillation or intravenous injection. These models do not replicate the typically dry nature of aerosol exposure and may hinder our ability to decipher the initial events that lead to clearance or the establishment of infection. We have established a standardized aerosol model of murine infection for the human fungal pathogen Cryptococcus. Aerosolized cells were generated utilizing a Collison nebulizer in a whole-body Madison Chamber at different humidity conditions. The aerosols inside the chamber were sampled using a BioSampler to determine viable aerosol concentration and spray factor (ratio of viable aerosol concentration to total inoculum concentration). We have effectively delivered yeast and yeast-spore mixtures to the lungs of mice and observed the establishment of disease. We observed that growth conditions prior to exposure and humidity within the Madison Chamber during exposure can alter Cryptococcus survival and dose retained in mice

Aerosolization at high humidity increases spray factor and viable aerosols for <i>Cryptococcus</i> during aerosolization in the Madison Chamber.

<p>(A and C ) Log spray factor plotted against relative humidity for broth-grown (A) <i>C. neoformans</i> var. <i>grubii</i> H99 (p = 0.0005, R² = 0.802) and (C) <i>C. gattii</i> EJB18 (p = 0.06, R² = 0.534). Spray factor increases with relative humidity. (B and D) Log aerosol concentration plotted against relative humidity for broth-grown (B) <i>C. neoformans</i> var. <i>grubii</i> H99 (p = 0.011, R² = 0.57) and (D) <i>C. gattii</i> EJB18 (p = 0.12, R² = 0.402). Log aerosol concentration increases with relative humidity. Filled circles and squares represent <i>C. neoformans</i> var. <i>grubii</i> (H99) and open circles and squares represent <i>C. gattii</i> EJB18.</p

Growth on agar increases spray factor and viable aerosols for <i>Cryptococcus</i> during aerosolization in the Madison Chamber.

<p>(A) Log spray factor of <i>C. neoformans</i> var. <i>grubii</i> (H99) and <i>C. gattii</i> (EJB18) plotted in respect to growth condition and relative humidity. High humidity increases spray factor for broth-grown cells. (B) Log aerosol concentration of <i>C. neoformans</i> var. <i>grubii</i> (H99) and <i>C. gattii</i> (EJB18) plotted in respect to growth condition and relative humidity. Growth on agar increased log aerosol concentrations. Triangle, square, or circles represent growth conditions denoted in the x-axis for each individual exposure Mean values (n = 3–7, +/− SEM) are plotted. * indicates p<0.01.</p

Aerosol delivery of <i>C. neoformans</i> var. <i>grubii</i> yeast and yeast-spore mixtures.

<p>(A) <i>C. n.</i>var. <i>grubii</i>, H99 (MATα), KN99<b>a</b>, and a mated mixture containing spores was effectively delivered to mice as assayed by dose retained in lungs (95% RH and 1 hour exposure). (A) Increased tissue burden, dissemination to the brain, and (B) decreased weight was observed at 24 days post-exposure and demonstrates the developmental sequelae of cryptococcosis. No <i>Cryptococcus</i> colonization was obtained from brain or spleen tissues 1 hour post exposure. No significant differences in CFUs were observed between H99 (MATα), KN99<b>a</b>, or mated mixtures (with spores) at any 1 or 25 days post exposure. Mean value (n = 3–4, +/− SEM) are plotted.</p

Aerosol delivery of <i>C. neoformans</i> var. <i>grubii</i> (YSB119α and KN99a NEO1) yeast and yeast-spore mixtures.

<p>Animal exposure was performed utilizing <i>Cryptococcus</i> strains containing different drug resistance markers. NAT^R (Red), NEO^R (Green), and NAT^R+NEO^R (Purple) resistant colonies were obtained pre- and post- nebulization in similar proportions indicating both yeast and spores equally survive nebulization. NAT^R+NEO^R resistant colonies were not observed from the BioSampler or lung tissues and we observed a net loss of total cell density (Blue) from post-nebuliztion to BioSampler, to dose presented, and to dose retained.</p

Reduced Virulence of an Extensively Drug-Resistant Outbreak Strain of <i>Mycobacterium tuberculosis</i> in a Murine Model

<div><p>Bacterial drug resistance is often associated with a fitness cost. Large outbreaks of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB have been described that predominately affect persons with HIV infection. We obtained four closely-related <i>Mycobacterium tuberculosis</i> strains (genotype F15/LAM4/KZN) from an outbreak in KwaZulu-Natal (KZN), South Africa, including drug-sensitive, MDR, and XDR clinical isolates. We compared the virulence of these strains in a murine model of aerosol <i>M. tuberculosis</i> infection for four phenotypes: (1) competitive <i>in vivo</i> growth in lung and spleen, (2) non-competitive <i>in vivo</i> growth in lung and spleen, (3) murine survival time, and (4) lung pathology. When mixtures of sensitive, MDR, and XDR KZN strains were aerosolized (competitive model), lung CFUs were similar at 60 days after infection, and spleen CFUs were ordered as follows: sensitive > MDR > XDR. When individual strains were aerosolized (non-competitive model), modest differences in lung and spleen CFUs were observed with the same ordering. C57BL/6, C3H/FeJ, and SCID mice all survived longer after infection with MDR as compared to sensitive strains. SCID mice infected with an XDR strain survived longer than those infected with MDR or sensitive strains. Lung pathology was reduced after XDR TB infection compared to sensitive or MDR TB infection. In summary, increasing degrees of drug resistance were associated with decreasing murine virulence in this collection of KZN strains as measured by all four virulence phenotypes. The predominance of HIV-infected patients in MDR and XDR TB outbreaks may be explained by decreased virulence of these strains in humans.</p></div

Necrosis and apoptosis induction by KZN <i>M. tuberculosis</i> strains <i>in vitro</i>.

<p>Erdman and H37Rv reference strains were compared to sensitive (S), multidrug-resistant (M), and extensively drug-resistant (X) KZN strains. (A) For necrosis measurement, alveolar epithelial cells (A549) were infected with the KZN strains at MOI of 10 for 96 h and the supernatant was assayed for lactate dehydrogenase (LDH). Percentage cytotoxicity was calculated by the following formula: [release of LDH from infected cells (OD490)-release of LDH from uninfected control/maximum LDH release (OD490)] X 100. Data from four independent experiments. (B) Infected A549 cells were lysed and plated to monitor bacterial survival. (C) The percentage of apoptosis of THP1 macrophages infected with the KZN strains at an MOI of 10 was determined by TUNEL staining of DNA fragmentation. Values are means with error bars indicating standard error. Statistically significant differences in necrosis relative to that of Erdman are shown: *, P<0.05; **, P<0.001.</p