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View Full Details and Buy. Complementary Documents and Links:. Preface This work began as a small collaborative project in , a complement to our drug discovery and clinical development activities. We aim to discover novel treatments and prevention methods for major infectious neglected diseases. In developing countries where these diseases are endemic, Novartis will make treatments available to poor patients without profit.
NITD is also a center for teaching and training of postdoctoral fellows and graduate students, especially young scientists from countries where these diseases are endemic. Far from being an ivory tower, NITD is able to operate because of the collaborations we have made with academic, clinical, nongovernment, and commercial institutions at all levels. This publication is the product of one such collaboration and without making any grand claims is intended as a simple, useful resource-a visual reference which will allow an appreciation of the histopathological differences of TB between different animal models.
It is not intended for consultant histopathologists, but for all scientists and students working in the field of TB. This atlas provides a visual comparison of histopathological manifestations of TB disease in different animal species and man. However, one has to keep in mind that disease expression in animal models is dependent upon the TB strain used, the number of bacilli for infection, the route of infection, the timing, and the animal strain.
Standardization is not possible, and many clinical terms do not have parallels in animal models. Nonetheless, we hope the images provided are helpful to those involved in research practice.
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Browse Publishers. Cipemastat concentration in eluate was measured as the area under the curve for mass transition peaks. Rabbits were imaged using a CereTom 8-slice clinical computed tomography CT scanner with a CT reconstructions were viewed using VivoQuant software Invicro. Lung cavities were radiologically identified from CT scan reconstructions as a contiguous set of volume elements voxels whose size is defined by the limit of resolution of the CT scanner, within the lungs, with densities close to air — to Hounsfield units [HU] and encapsulated by consolidation, defined as a continuous region of voxels with densities similar to water — to HU.
This radiological definition was consistent with the consensus definition for cavities advanced by Gadkowski and Stout [ 2 ]. Contiguous airspace and consolidation regions were selected by connected thresholding in the density range for each landmark. Continuity of consolidation around the airspace was confirmed by eye. After 14 weeks, rabbits were sedated and euthanized. Gross images were obtained using a Nikon D digital camera and a Nikon NIKKOR lens and analyzed with ImageJ to identify the areas of visually diseased lung as a fraction of the total area of splayed lung [ 32 ].
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This fraction was used as an estimate of the percentage of diseased lung. Transverse lung slices were collected for paraffin embedding and histologic sectioning.
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Regions of interest ROI included the full thickness of the cavity wall while excluding necrotic debris and air space at the cavity interior. Positive staining was calculated as a percentage of the total ROI. At 24 hours, an equal volume of ethylenediaminetetraacetic acid was added to stop the cleavage reaction, and the products were briefly boiled and subjected to sodium dodecyl sulfate—polyacrylamide gel electrophoresis on a Mini-Protean TGX precast gel Bio-Rad, Hercules, California.
Previous investigations suggested that sensitization with heat-killed mycobacteria prior to infection increased the frequency and severity of cavitation in rabbit models [ 26 , 31 ]. We reasoned that multiple aerosol challenges with virulent M. To test this hypothesis, we conducted a limited-power study by exposing rabbits to M. For our studies, we defined exposure as the product of the bacterial concentration in the aerosol inoculum and the total time spent in the aerosol chamber. One group of rabbits received 5 aerosol challenges with M.
A second group of rabbits received a single aerosol challenge with M. These challenges corresponded to an extrapolated day 1 bacterial implantation of colony-forming units CFUs per exposure in the OD 0. We confirmed that the cumulative exposure was the same for both groups by measuring the CFUs in the aerosol inoculum Figure 1A. Infection parameters and disease patterns for rabbits challenged in the single and repetitive exposure groups.
A , Experimental exposure conditions and timing. Single exposure group rabbits received a single implantation with approximately bacteria on day 8 and sham exposures on days 1, 4, 12, and Repetitive exposure group rabbits received 5 repetitive exposures resulting in implantation of approximately bacteria on each of days 1, 4, 8, 12, and Exposure was calculated based on the colony-forming units CFU in the aerosol inoculum.
B , Frequency of cavitation among rabbits in the single and repetitive exposure groups. C , Number of cavities per animal in the single and repetitive exposure groups. Cavity counts are only plotted for the animals that demonstrated cavitation.
D , Quantification of the fraction of lung identified as diseased by gross observation for rabbits in the single exposure and repetitive exposure groups. To address the possibility that the infection chamber does not achieve aerosol concentrations of bacilli that are linearly correlated with the OD of the aerosol inoculum, we performed a titration assay to compare the OD of the aerosol inoculum with the day 1 implantation of bacteria in rabbit lungs, and showed a linear correlation in the OD range used in these experiments Supplementary Figure 1.
Indeed, our data indicate that it is likely that repetitive exposure rabbits were infected on every occasion of exposure, as bacteria were recovered from the lungs of all rabbits in the lowest OD exposure group of our chamber titration experiment Supplementary Figure 1. We monitored rabbits using CT scans. Of those animals that developed cavities, those in the repetitive exposure group showed a trend toward more cavities per animal than those in the single exposure group Figure 1C. On week 10 of the experiment, the rabbits were killed and the lungs were fixed.
Semiquantitative gross pathologic analysis showed that rabbits in the repetitive aerosol group experienced worse lung disease than those in the single exposure group Figure 1D and Supplementary Figure 2. In light of the limited number of rabbits involved in this study, these data suggest that repetitive exposure caused an increase in the severity of disease as well as the frequency and severity of cavities. Focal matrix depletion precedes TB cavitation, so we sought to define the optimal treatment window for the prevention of cavitation as the weeks before the greatest frequency of cavitation.
To study the dynamics of cavity formation generated by repetitive exposure to M. Timing of cavitation and cavity growth dynamics in the repetitive aerosol method. A , Frequency of cavitation mapped to time after start of infection. Solid line indicates the cumulative frequency of cavitation among the cohort of 9 rabbits; dashed line indicates the frequency of cavitation among the cohort at the specific time point and is distinguished from the solid line by the occurrence of cavity resolution. B , Cavity volume mapped to time after the start of infection for 4 representative cavities demonstrating continuous growth 1 , growth and shrinking behavior 2 and 3 , and growth and resolution 4.
Cavities were identified as lung volumes not connected to the normal bronchial structure with densities between — and — Hounsfield units, and points on the x-axis indicating a noncavitary focus are plotted at the limit of resolution for the computed tomographic CT scanner. The y-axis is plotted using a logarithmic base 3 scale as measured volume varies closely as the cube of the radius of a spheroid object so that relationship among cavity volumes are more comparable to the 2-dimensional reconstructions in C. C , Transverse CT scan reconstructions showing each of the foci identified in B.
Cavity morphology over time was observed by CT scan reconstructions. We used density segmentation analysis to identify cavities as air-filled spaces that were not connected to the bronchial tree.
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From this analysis, we identified 3 patterns of change in cavity morphology: 1 cavity growth Figure 2B and 2C, examples 1, 2, and 3 ; 2 cavity shrinkage Figure 2B and 2C, examples 2 and 3 ; and 3 cavity resolution Figure 2B and 2C, example 4. Together, these data demonstrate that cavities most often formed between 6 and 8 weeks after the initial aerosol exposure and were persistent though dynamic structures between weeks 8 and 16 of the study.
A large body of historic literature, in addition to our own observations, suggests that TB cavities arise from necrotic granulomas. As we had not previously worked with a model that generates cavities by repetitive exposure to M. Histology samples collected from rabbits infected by repetitive exposure displayed many of the microscopic findings described in TB pathology reports Figure 3A [ 10 , 21 , 33 , 34 ]. These hallmarks included granulomas, necrotic granulomas, and cavities. Histologic observations from the repetitive exposure model show that the cytoarchitecture between necrotic granulomas and cavities was similar, further supporting a close relationship between the 2 lesions Figures 3A , 3B , and 4C.
Histologic patterns of tuberculous lesions in rabbits infected by repetitive aerosol exposure. C , Overview of the model for collagenase-mediated destruction of extracellular matrix in proximity to a cavity. Collagen enrichment in lung lesions from rabbits infected with Mycobacterium tuberculosis. A , Examples of hematoxylin and eosin—stained tissue fields used for quantification of collagen enrichment analysis. Black rectangles represent example high-resolution fields used for quantification in B and traces in C.
Arrowheads indicate examples of the histologic regions surveyed during the relative collagen quantification reported in B. B , Relative enrichment in collagen identified by blue hues in the Masson trichrome stain. Multiple surveys were taken from nonoverlapping areas within each region, and the number above each category indicates the number of unique lesions surveyed. C , Relative tissue density gray line, right y-axis and collagen density black line, left y-axis along linear traces crossing 2 granulomas, 2 necrotic granulomas, and 2 cavities.
All traces are set to the same x- and y-scale, and the minor hash marks in the lowest plot show the regular pattern of surveys continued along each lesion. Dotted lines indicate cavity space on histology. The cavities generated by repetitive exposure were marked by large proliferations of acid-fast bacteria along their inner surface and a wall enriched with fibrosis Figure 3B. Because our investigations are predicated on the pathologic observation that matrix depletion predisposes to cavitation, we also confirmed that collagen matrix depletion was a hallmark of cavitary lesions from repetitive exposure Figure 4.
These observations show that repetitive aerosol exposure in rabbits generates a spectrum of histologic lesions commonly observed in TB pathology studies and validates the model for our studies by showing that pathologic matrix depletion is modeled by rabbits following repetitive aerosol infection. We previously showed that MMP-1 transcripts accumulated in the areas near M.
Cipemastat is a potent inhibitor of MMP-1 and was originally developed by the Roche Corporation as an antiarthritis agent [ 28 , 29 ]. We first confirmed that cipemastat did not have intrinsic antimycobacterial properties which our stock of cipemastat was able to inhibit MMP-1 in vitro Supplementary Table 1 and Supplementary Figure 4. Next, we conducted a 3-rabbit pharmacokinetic study to confirm that cipemastat was orally bioavailable in rabbits and had suitable kinetics for a daily dosing regimen Table 1. These results suggest that cipemastat shows good oral bioavailability in rabbits and confirms that a daily dosing regimen is sufficient to maintain plasma concentration levels above the IC 50 during most of a hour period.
We randomized 18 rabbits into an 8-rabbit vehicle group and a rabbit cipemastat group. All rabbits received 1 mL of PediaSure per kilogram of body weight. Cipemastat was given orally from study weeks 5 through This treatment window was consistent with the 4 weeks preceding the maximum frequency of cavitation and the time during which we predict that pathologic lesions will undergo matrix depletion Figure 5. Our results are based on 6 control-group rabbits and 7 cipemastat-treated rabbits.
Experimental overview to investigate the pharmacologic inhibition of tissue destruction and cavitation using cipemastat in rabbits infected with Mycobacterium tuberculosis. The predicated temporal window for cavitation was designed based on data presented in Figure 2. During weeks 7, 9, 12, and 14, we performed breath-hold CT scans on all study rabbits.
These CT scans revealed no difference in the number of cavities or severity of cavitation between the control and treatment groups throughout the study Figure 6A and 6B and Supplementary Figure 4. We did notice a repeated trend toward worse cavitary disease among rabbits in the cipemastat-treated group. The animals were sacrificed during week 14 and the lungs were fixed and scored for disease severity by 2 independent blinded observers Figure 6C. We also quantified the extent of disease within the lungs by cutting the lungs in the transverse plane and reporting the overall percentage of all lung slices with grossly visible disease Figure 6C.
Neither severity scoring nor disease quantification showed a difference between experimental groups. The extent of disease severity and cavitation in cipemastat-treated rabbits. A , Average number of cavities per animal for weeks 7, 9, 12, and B , Average volume of the lung identified as cavity volume by computed tomographic scan and segmentation analysis for weeks 7, 9, 12, and C , Disease severity scores of lungs assigned subjectively by 2 independent blinded observers and quantified as the fraction of lung identified as disease in transversely splayed lungs.
D , Quantification of collagen accumulation at the walls of cavities in Mycobacterium tuberculosis —infected rabbits.
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The example of a cavity wall shows regions identified as collagen arrowheads. Tuberculosis lung lesions are often encircled by a fibrotic wall [ 21 ]. This pathologic matrix deposition is also a feature of rabbits modeling cavitary TB Figures 3B and 6D. Because cipemastat inhibits collagenase activity, we predicted that cipemastat administration should increase the collagen content around TB lesions. Using this method, we were unable to identify any difference in the collagenous content of cavity walls, suggesting that cipemastat treatment did not change the phenotype of pathologic collagen accumulation around cavities in the rabbit model Figure 6D.
We have developed a novel model for cavitary TB based on repetitive aerosol exposure to virulent M. Our data show that repetitive exposure over a 2-week period produced more advanced disease and more cavities than a single exposure, even when we carefully adjusted the concentrations bacteria in the aerosol inoculum to provide the same total exposure between groups.
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This finding suggests a link between repetitive exposure and TB exacerbation and is further supported by recent epidemiological evidence that multiple exposures to infected contacts increases the risk of TB progression [ 27 ]. An association between repetitive exposure and severe TB may have important implications for epidemiology and infection control in high-incidence regions of the world [ 37 ]. Our experiments did not evaluate the mechanism of repetitive exposure—related disease exacerbation; however, it is likely that the driver of more severe disease in repeatedly exposed animals is repeated priming of cell-mediated immunity.
Although untested, repetitive exposure may cause a cascading set of T-cell priming and expansion events that disproportionately exacerbate the immune response against M. Our data show that modeling cavitary TB by repetitive aerosol exposure also models a spectrum of human lesions and pathologic matrix depletion associated with caseous and cavitary pulmonary TB [ 39 ].
We took advantage of this model to screen cipemastat, a potent and specific MMP-1 inhibitor, as an inhibitor of cavitation [ 28 ]. Our study was supported by a molecular phenotype in which MMP-1 expression increased around tuberculous lesions with matrix destruction [ 19 ]. In these experiments, we administered cipemastat for 4 weeks preceding the development of caseous and cavitary lesions in the repetitive aerosol model.
However, our results did not show a reduction in cavitation or disease severity. As part of our investigations, we confirmed that the plasma concentrations of cipemastat were well above the IC 50 during the hour dosing cycle. We did not sample the concentration of cipemastat in TB lesions, so it is possible that cipemastat did not reach inhibitory concentrations within granulomas undergoing matrix destruction. Furthermore, MMP activity may be highly localized in pericellular niches [ 40 ]. Alternatively, MMP-1 may act in conjunction with other extracellular collagenases to drive matrix depletion, and the inactivation of MMP-1 did not appreciably change the dynamics of cavity formation, reflecting redundancy in the proteolytic cascade.
Finally, it is possible that MMP-1 is not a mediator of matrix depletion and cavitation.
Our results demonstrate an entirely new system to study TB cavities. We show that repetitive exposure to aerosolized M. Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.