Aerosol Infection of Mice with Mycobacteria Using a Nose-Only Exposure Device

Tsenova, Liana; Moreira, André L.; Party, Esmeralda; Freedman, Victoria H.; Kaplan, Gilla

doi:10.1177/109135059700200307

Cited by 12 publications

(9 citation statements)

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“…were infected via the respiratory route as described previously (28). Mice were inoculated by exposure to an aerosolized suspension of the parental or ⌬nrdZ strains of M. tuberculosis by using a nose-only exposure system (In-Tox Products, Albuquerque, N.M.) (42). This procedure resulted in implantation of approximately 100 organisms into the lungs of each mouse, which was confirmed by plating lung homogenates on Middlebrook 7H11 plates 3 h postinfection.…”

Section: Methodsmentioning

confidence: 99%

Ribonucleotide Reduction in Mycobacterium tuberculosis : Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases

Dawes

Warner

Tsenova³

et al. 2003

Infect Immun

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Mycobacterium tuberculosis, the causative agent of tuberculosis, possesses a class Ib ribonucleotide reductase (RNR), encoded by the nrdE and nrdF2 genes, in addition to a putative class II RNR, encoded by nrdZ. In this study we probed the relative contributions of these RNRs to the growth and persistence of M. tuberculosis. We found that targeted knockout of the nrdF2 gene could be achieved only in the presence of a complementing allele, confirming that this gene is essential under normal, in vitro growth conditions. This observation also implied that the alternate class Ib small subunit encoded by the nrdF1 gene is unable to substitute for nrdF2 and that the class II RNR, NrdZ, cannot substitute for the class Ib enzyme, NrdEF2. Conversely, a ⌬nrdZ null mutant of M. tuberculosis was readily obtained by allelic exchange mutagenesis. Quantification of levels of nrdE, nrdF2, nrdF1, and nrdZ gene expression by real-time, quantitative reverse transcription-PCR with molecular beacons by using mRNA from aerobic and O 2 -limited cultures showed that nrdZ was significantly induced under microaerophilic conditions, in contrast to the other genes, whose expression was reduced by O 2 restriction. However, survival of the ⌬nrdZ mutant strain was not impaired under hypoxic conditions in vitro. Moreover, the lungs of B6D2/F 1 mice infected with the ⌬nrdZ mutant had bacterial loads comparable to those of lungs infected with the parental wild-type strain, which argues against the hypothesis that nrdZ plays a significant role in the virulence of M. tuberculosis in this mouse model.Mycobacterium tuberculosis is a formidable human pathogen that is estimated to infect one-third of the world's population (2). The success of this pathogen is attributable to its remarkable ability to persist for prolonged periods in a clinically latent state from which it is able to reactivate and cause disease. During the course of infection in humans, the tubercle bacillus is likely to encounter environments where there is limited O 2 availability, most notably the fibrous granulomas (9). This notion has underpinned efforts to identify the metabolic changes that occur in M. tuberculosis in response to hypoxia as a means of modeling the changes that may be associated with clinical latency (13, 37, 44). The value of this approach was underscored by a recent report (38) suggesting that during stationary infection in mice, the bacilli may be in a physiological state that approximates the nonreplicating persistence achieved in the widely used model of adaptation of M. tuberculosis to hypoxia in vitro developed by Wayne and coworkers (44,45).An important class of enzymes that includes both O 2 -dependent and O 2 -independent forms is the ribonucleotide reductases (RNRs), which catalyze the reduction of ribonucleotides to deoxyribonucleotides. These enzymes perform an essential role in the cycling of nucleotides in the cell and during replication of the chromosome in all organisms and provide attractive targets for antiproliferative drugs (40) and subunit ...

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Section: Methodsmentioning

confidence: 99%

Ribonucleotide Reduction in Mycobacterium tuberculosis : Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases

Dawes

Warner

Tsenova³

et al. 2003

Infect Immun

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“…For each experiment, frozen stocks of bacteria were thawed, followed by 30 s sonication in a water bath sonicator (Laboratory Supplies, Hicksville, NY; Model G112SPIT) to disperse clumps. A Lovelace nebulizer (In-Tox Products, Albuquerque, NM) was used to generate the aerosol from 1.5 to 8 ϫ 10 6 bacilli͞ml in 10 ml of saline containing 0.04% Tween 80 (16). For each experiment, 24 mice were exposed for 30 min to the aerosol, resulting in implantation of 50-600 organisms into the lungs of each mouse.…”

Section: Methodsmentioning

confidence: 99%

Virulence of aMycobacterium tuberculosisclinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α/β

Manca

Tsenova

Bergtold

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-␥ production by spleen and lymph node cells from infected mice. In addition, 2-to 4-fold lower levels of tumor necrosis factor-␣ (TNF-␣), IL-6, IL-12, and IFN-␥ mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-␣ mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-␣͞␤. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

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“…Mice were exposed to the aerosol for 20 min using a Lovelace nebulizer (In-Tox Products, Albuquerque, NM). This implanted between 20 and 50 bacilli in the lungs of the mice, as confirmed by plating lung homogenates 3 h after infection (34). Quantification of CFU in lung, spleen, and liver tissue was carried out as follows.…”

Section: Methodsmentioning

confidence: 99%

The Phenolic Glycolipid ofMycobacterium tuberculosisDifferentially Modulates the Early Host Cytokine Response but Does Not in Itself Confer Hypervirulence

et al. 2008

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Mycobacterium tuberculosis possesses a diversity of potential virulence factors including complex branched lipids such as the phenolic glycolipid PGL-tb. PGL-tb expression by the clinical M. tuberculosis isolate HN878 has been associated with a less efficient Th1 response and increased virulence in mice and rabbits. It has been suggested that the W-Beijing family is the only group of M. tuberculosis strains with an intact pks1-15 gene, required for the synthesis of PGL-tb and capable of producing PGL-tb. We have found that some strains with an intact pks1-15 do not produce PGL-tb while others may produce a variant of PGL-tb. We examined the early host cytokine response to infection with these strains in vitro to better understand the effect of PGL-tb synthesis on immune responses. In addition, we generated a PGL-tb-producing H37Rv in order to determine the effect of PGL-tb production on the host immune response during infection by a strain normally devoid of PGL-tb synthesis. We observed that PGL-tb production by clinical M. tuberculosis isolates affected cytokine production differently depending on the background of the strain. Importantly, while ectopic PGL-tb production by H37Rv suppressed the induction of several pro-and anti-inflammatory cytokines in vitro in human monocytes, it did not lead to increased virulence in infected mice and rabbits. Collectively, our data indicate that, while PGL-tb may play a role in the immunogenicity and/or virulence of M. tuberculosis, it probably acts in concert with other bacterial factors which seem to be dependent on the background of the strain.

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Aerosol Infection of Mice with Mycobacteria Using a Nose-Only Exposure Device

Abstract: The effectiveness of an aerosol producing system (In-Tox Products, Albuquerque, NM)

Cited by 12 publications

References 14 publications

Ribonucleotide Reduction in Mycobacterium tuberculosis : Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases

Ribonucleotide Reduction in Mycobacterium tuberculosis : Function and Expression of Genes Encoding Class Ib and Class II Ribonucleotide Reductases

Virulence of aMycobacterium tuberculosisclinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α/β

The Phenolic Glycolipid ofMycobacterium tuberculosisDifferentially Modulates the Early Host Cytokine Response but Does Not in Itself Confer Hypervirulence

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