7 research outputs found
Location of Intra- and Extracellular M. tuberculosis Populations in Lungs of Mice and Guinea Pigs during Disease Progression and after Drug Treatment
The lengthy treatment regimen for tuberculosis is necessary to eradicate a small
sub-population of M. tuberculosis that persists in certain host
locations under drug pressure. Limited information is available on persisting
bacilli and their location within the lung during disease progression and after
drug treatment. Here we provide a comprehensive histopathological and
microscopic evaluation to elucidate the location of bacterial populations in
animal models for TB drug development
Location of <i>M. tuberculosis</i> bacilli in lungs from <i>M. tuberculosis</i> infected guinea pigs, throughout infection and after 6 weeks of INH or TMC207 drug treatment.
<p>(AR) auramine-rhodamine, hematoxylin QS and DAPI; (H&E) hematoxylin
and eosin. (A, B) Low (A) and high (B) magnifications of an early
primary granuloma in the lungs of <i>M. tuberculosis</i>
infected guinea pigs 4 weeks post-aerosol infection. Primary granulomas
are distinguished from secondary lesions by the presence of necrosis (N)
(H&E staining, 100× and 1000× magnifications). (C) A
secondary lesion in the lungs of an <i>M. tuberculosis</i>
infected guinea pig 4 weeks post-aerosol infection. Secondary lesions
are distinguished from primary granulomas by the lack of central
necrosis in the former (H&E staining, 100× magnification). (D,
E) Low (D) and high (E) magnifications of a primary granuloma from the
lungs of an <i>M. tuberculosis</i> infected guinea pig 4 weeks
after aerosol infection. The majority of AR+ bacilli are
extracellular within the necrotic core (c) (AR, 100× and
200× magnifications). (F) High magnification of area demarcated in
figure 5E (square). Extracellular AR+ bacilli in the necrotic core
of primary granulomas exist as single cells or are situated in clusters
(AR, 1000× magnification). (G, H) Confocal micrographs of a
necrotic granuloma showing an acellular necrotic core (c) with
extracellular AR+ bacilli (red) (AR and DAPI, 200× and
630× magnifications). (I) A primary granuloma from the lungs of an
<i>M. tuberculosis</i> infected guinea pig 10 weeks after
aerosol infection showing advanced calcification and calcification of
the necrotic core (c) and the acellular rim (R) surrounding the core
(H&E staining, 100× magnification). (J) Fluorescent image of a
primary lung granuloma from an <i>M. tuberculosis</i> infected
guinea pig 10 weeks after aerosol infection. Extracellular AR+
bacilli are present within the necrotic core (c) and the acellular rim
(R) surrounding the necrotic regions (AR, 200× magnification). (K)
Cropped image from figure 5I (square) showing extracellular AR+
stained bacilli in the acellular rim. (L) Fluorescent image of a
necrotic primary lung granuloma from an <i>M. tuberculosis</i>
infected guinea pig treated for 6 weeks with INH. Extra-cellular
AR+ bacilli are primarily within the core (c) of the partially
calcified lytic necrosis, and to a lesser extent within the acellular,
uncalcified rim (R) (AR, 200× and 400× magnifications). (M)
A low magnification of the remnant of a primary lung granuloma in an
<i>M. tuberculosis</i> infected guinea pig treated for 6
weeks with TMC207. The primary granuloma shows a caseous necrotic core
(c) surrounded by inflammatory cells (H&E staining, 100×
magnification). (N) Fluorescent image of the caseous necrotic core (c)
in the primary lung granuloma shown in figure 5M taken from a serial
tissue section. The few extra-cellular AR+ bacilli remaining after
TMC207 treatment are primarily located within the central core of
caseous necrosis (AR, 400× magnification).</p
The progression of cellular necrosis that occurs within pulmonary inflammatory lesions during <i>M. tuberculosis</i> infection in GKO mice and the development of hypoxia as revealed by pimonidazole.
<p>(AR) = auramine-rhodamine, hematoxylin QS and DAPI;
(H&E) = hematoxylin and eosin. (A) An
inflammatory lesion from the lungs of <i>M. tuberculosis</i>
infected GKO mice 22 days after aerosol infection. The lesion shows a
number of alveolar spaces beginning to accumulate granulocytes prior to
the presence of cellular necrosis (H&E staining, 200×
magnification). (B) High magnification of the area demarcated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017550#pone-0017550-g003" target="_blank">figure 3A</a> (circle)
showing an alveolus completely occluded with inflammatory infiltrate
(H&E staining, 1000× magnification). (C) Fluorescent image of
an inflammatory lesion from the lungs of <i>M.
tuberculosis</i> infected GKO mice 18 days post-aerosol
infection showing a highly organized tubulin network with intact nuclei
(Immunofluorescence for Tubulin (green) and MTB (red) and DAPI (blue).
1000× magnification). (D) Immunofluorescent image of an
inflammatory lesion from the lungs of <i>M. tuberculosis</i>
infected GKO mice 28 days post-aerosol TB (red) infection showing loss
of tubulin (green) architecture and degenerated nuclei (blue) indicating
loss of cell viability (1000× magnification). (E) Confocal
microscopy of an inflammatory lesion from the lungs of <i>M.
tuberculosis</i> infected GKO mice 18 days post-aerosol
infection. The nuclei (blue) appear healthy with few numbers of AR+
bacilli (red). (AR staining combined with DAPI; 630× magnification
with additional 2.5× digital zoom). (F) Confocal microscopy of an
inflammatory lesion from the lungs of <i>M. tuberculosis</i>
infected GKO mice 28 days post-aerosol infection. Degenerated fragments
of inflammatory cell nuclei are within alveoli with a high number of
extracellular AR+ bacilli (AR staining combined with DAPI;
630× magnification with additional 2.5× digital zoom). (G)
Immunohistochemistry detecting hypoxia (brown) in a necrotic lung lesion
from an <i>M. tuberculosis</i> infected GKO mouse 29 days
after aerosol infection. The area of central necrosis (N) is surrounded
by epithelioid macrophages near a major airway. (Hematoxylin
counterstain, 40× magnification). (H) High magnification of the
area demarcated within the inflammatory lesion shown in figure 3G
(square). The center of alveoli, which is likely hypoxic, is filled with
cellular debris and fails to stain due to the lack of viable cells
(Hematoxylin counterstain, 400× magnification).</p
Intra-and extracellular <i>M. tuberculosis</i> bacilli in lungs throughout infection from <i>M. tuberculosis</i> infected GKO mice and after 9 days of INH drug treatment.
<p>(AR) auramine-rhodamine, hematoxylin QS and DAPI; (H&E) hematoxylin
and eosin. (A) An inflammatory lesion from lungs of an <i>M.
tuberculosis</i> infected GKO mouse 18 days after aerosol
infection. The inflammatory lesion shows a mix of macrophages and
granulocytes arranged in an unorganized manner (H&E, 100×
magnification). (B) Fluorescent image of an inflammatory lesion from
lungs of an <i>M. tuberculosis</i> infected GKO mouse 18 days
after aerosol infection. Intracellular red fluorescent AR+ stained
bacilli were predominantly found uniformly distributed throughout
inflammatory lesions at this time (AR, 200× magnification). (C)
High magnification fluorescent image of the inflammatory lesion in
figure 2B. The lesion shows intracellular AR+ bacilli located
within various macrophage cells (AR, 400× magnification). (D)
Cropped image taken from figure 2C (square) showing multiple AR+
stained bacilli within a single macrophage cell (AR, digital
magnification). (E) A necrotic inflammatory lesion from lungs of an
<i>M. tuberculosis</i> infected GKO mouse 28 days after
aerosol infection. Multiple foci of intense basophilic staining are seen
in alveolar spaces as they accumulate cellular necrotic debris (H&E
staining, 40× magnification). (F) High magnification of alveolar
spaces filled with cellular necrotic debris located within the
inflammatory lesion depicted in figure 2E (square) (H&E staining,
400× magnification). (G) High magnification of a non-necrotic area
within the inflammatory lesion seen in figure 2E (star) taken from a
serial tissue section. Fluorescent image shows a number of intracellular
AR+ stained bacilli residing within macrophages (AR, 1000×
magnification). (H) High magnification of an alveolar space filled with
necrotic debris located within the inflammatory lesion shown in figure
2E (circle) taken from a serial tissue section. Image shows a high
number of extracellular AR+ stained bacilli residing among cellular
necrotic debris situated in alveolar spaces (AR, 1000×
magnification). (I) Fluorescent image of an inflammatory lesion from
lungs of an <i>M. tuberculosis</i> infected GKO mouse 28 days
after aerosol infection showing intracellular AR+ stained bacilli
within multiple macrophages dispersed in non-granulomatous tissue (AR,
200× magnification). (J) AR+ bacilli within a remaining
inflammatory lesion from lungs of an <i>M. tuberculosis</i>
infected GKO mouse after 10 days of INH treatment (AR, 100×
magnification). (K) Higher magnification of the inflammatory lesion seen
in figure 2J. The lesion shows that the majority of remaining AR+
stained bacilli are intracellular within macrophages (AR, 200×
magnification). (L) High magnification of the inflammatory lesion seen
in figure 2K (square). This fluorescent image shows AR+ bacilli
were predominantly found within macrophage cells comprising the few
remaining inflammatory lesions (AR, 1000× magnification).</p
Numbers of viable <i>M. tuberculosis</i> organisms in lungs of infected IFN-γ knock-out and C57BL/6 mice after drug treatment.
<p>Mice were infected with <i>M. tuberculosis</i> strain Erdman
and treated with drugs starting 18 to 21 days after aerosol infection;
with isoniazid (INH), rifampin (RIF), ethambutol (EMB), gatifloxacin
(Gati) or moxifloxacin (Moxi). Sacrifice times were at 2, 5 and 7 days
of drug treatment. Data points represent mean log<sub>10</sub> viable
bacilli +/− standard error present in whole lung
homogenates.</p
Intracellular <i>M. tuberculosis</i> bacilli in lungs from <i>M. tuberculosis</i> infected C57BL/6 mice, throughout infection and after 6 weeks of MXF drug treatment.
<p>(AR) auramine-rhodamine, hematoxylin QS and DAPI; (H&E) hematoxylin
and eosin. (A) An inflammatory lesion from the lungs of an <i>M.
tuberculosis</i> infected C57BL/6 mouse 4 weeks after aerosol
infection. The cellular architecture of lesions shows a field of
lymphocytes (L) surrounding multiple macrophage aggregates (Φ)
(H&E staining, 200× magnification). (B) An inflammatory lesion
from lungs of an <i>M. tuberculosis</i> infected C57BL/6 mouse
4 weeks after aerosol infection. Intracellular AR+ bacilli were
predominantly found within macrophage rich regions surrounded by
lymphocytes (AR, 400× magnification). (C) High magnification of
the inflammatory lesion shown in figure 4B (circle) showing
intracellular AR+ stained bacilli within macrophages (AR,
1000× magnification). (D) An inflammatory lesion from the lungs of
an <i>M. tuberculosis</i> infected C57BL/6 mouse 9 weeks after
aerosol infection. The lesion shows a distinct rim of lymphocytes (L)
surrounding a core of epithelioid macrophages (Φ). A layer of
foamy macrophages (f) can be seen surrounding the lymphocyte rim
(H&E staining, 200× magnification). (E & F) Fluorescent
images of low (E) and high (F) magnifications of the inflammatory lesion
depicted in figure 4D taken from a serial tissue section. The majority
of AR+ bacilli at this time are found in epitheloid mΦs
(Φ) located within the lymphocyte cuff (L), whereas a lower number
of AR+ bacilli are located in foamy mΦs (f) located at the
peripheral edges of inflammation (AR, 100× and 200×
magnifications). (G) Inflammatory lesion from the lungs of a C57Bl/6
mouse infected with <i>M. tuberculosis</i> and treated with
moxifloxacin for 6 weeks. Cellular architecture of lesions after 6 weeks
of drug treatment is similar to untreated controls and consist of a
mΦ core (Φ) surrounded by lymphocytes (L) and foamy
mΦs (f) (AR, 200× magnification). (H) High magnification of
the peripheral edges of the inflammatory lesion shown in figure 4G. A
few remaining AR+ bacilli are found within foamy macrophages
located outside of the lymphocyte cuff (AR, 1000×
magnification).</p