Granuloma encapsulation is a key factor for containing tuberculosis infection in minipigs.
ABSTRACT A transthoracic infection involving a low dose of Mycobacterium tuberculosis has been used to establish a new model of infection in minipigs. The 20-week monitoring period showed a marked Th1 response and poor humoral response for the whole infection. A detailed histopathological analysis was performed after slicing the formalin-fixed whole lungs of each animal. All lesions were recorded and classified according to their microscopic aspect, their relationship with the intralobular connective network and their degree of maturity in order to obtain a dissemination ratio (DR) between recent and old lesions. CFU counts and evolution of the DR with time showed that the proposed model correlated with a contained infection, decreasing from week 9 onwards. These findings suggest that the infection induces an initial Th1 response, which is followed by local fibrosis and encapsulation of the granulomas, thereby decreasing the onset of new lesions. Two therapeutic strategies were applied in order to understand how they could influence the model. Thus, chemotherapy with isoniazid alone helped to decrease the total number of lesions, despite the increase in DR after week 9, with similar kinetics to those of the control group, whereas addition of a therapeutic M. tuberculosis fragment-based vaccine after chemotherapy increased the Th1 and humoral responses, as well as the number of lesions, but decreased the DR. By providing a local pulmonary structure similar to that in humans, the mini-pig model highlights new aspects that could be key to a better understanding tuberculosis infection control in humans.
- SourceAvailable from: Tjip van der Werf[Show abstract] [Hide abstract]
ABSTRACT: For eradication of tuberculosis (TB) by 2050, the declared aim of the Stop TB Partnership, novel treatment strategies are indispensable. The emerging epidemic of multi-drug resistant (MDR) TB has fuelled the debate about TB vaccines, as increasing numbers of patients can no longer be cured by pharmacotherapy. Of several proposed modalities, TB vaccines administered in therapeutic manner represents a promising alternative, despite the controversial history due to the occurrence of exacerbated immune response. A modified concept of immunotherapy is required in order to justify further exploration. In this paper we systematically reviewed the most advanced therapeutic vaccines for TB. We address the rationale of immunotherapeutic vaccination combined with optimized pharmacotherapy in active TB. We summarize preclinical and patient data regarding the five most advanced therapeutic vaccines currently in the pipeline. Of the five products that have been tested in animal models and in humans during active or latent TB, the quality of the published clinical reports of two of these products justify further studies in patients with active TB. This systematic review fuels further clinical evaluation eventually including head-to-head comparative studies.Vaccine 04/2014; · 3.77 Impact Factor
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ABSTRACT: Although contacts between tuberculosis patients may result in multiple consecutive infections (MCI), no experimental animal models consider this fact when used in basic studies. Moreover, the current TB vaccine (BCG) has demonstrated a limited protection in humans. In this study we evaluate the effect of tuberculosis MCI by way of a simple mathematical analysis using data from the low dose aerosol murine experimental model. The results show that a higher number of, or shorter intervals between, multiple consecutive infections reduce the protective effect of BCG. This is due to both the increase in bacillary load at the stationary level of the infection, and the protective immune response induced by the infection itself. This factor must therefore be taken into account when designing new prophylactic strategies as candidate vaccines for the replacement of BCG.PLoS ONE 01/2014; 9(4):e94736. · 3.53 Impact Factor
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ABSTRACT: Confrontation between invading microbial pathogens and host defense systems involves intricate cellular and molecular interactions. Here we discuss the virulence factors as trumps, overriding the contest in favor of the tubercle bacillus (Mycobacterium tuberculosis). It evolved a number of molecular constituents, which can interfere with antigen presentation and Toll receptor function, thus impairing immune defenses. It also evolved stress responses, which can drive its cell cycle into a non-replicating, low metabolic mode. Although the low counts of latent bacilli prevent their direct detection, we contend that they retain a capacity to survive for long periods in foamy macrophages and within the necrotic parts of lung granulomas. We attributed significance to drainage of M. tuberculosis by the alveolar fluid: while out-flow is responsible for the clearance, the reverse-flow has an important capacity to re-infect the lungs and to transmit the infection to new recipients. We consider the cycling between replicating and latent organisms to be a continuous process, which is a departure from the concept of long-lived dormant organisms, with a capacity to resuscitate. These aspects impinge also on the actions of isoniazid (INH) chemotherapy and on the topography of human lung lesions. Eventually, fibrosis of the connective tissue of the lungs is known to encapsulate lung lesions, thus limiting the impact of both outward and reverse drainage. In conclusion, the novelty of our views on M. tuberculosis-host interactions rests in the dynamic perception of M. tuberculosis latency and its evolutionary importance for the pathogenesis of tuberculosis.Enfermedades Infecciosas y Microbiología Clínica 03/2011; 29 Suppl 1:14-9. · 1.48 Impact Factor
Granuloma Encapsulation Is a Key Factor for Containing
Tuberculosis Infection in Minipigs
Olga Gil1,2., Ivan Dı ´az3., Cristina Vilaplana1,2, Gustavo Tapia4, Jorge Dı ´az1,2, Marı ´a Fort3, Neus
Ca ´ceres1,2, Sergio Pinto1,2, Joan Cayla `5, Leigh Corner6, Mariano Domingo3, Pere-Joan Cardona1,2*
1Unitat de Tuberculosi Experimental (UTE), Institut per a la Investigacio ´ en Cie `ncies de la Salut Germans Trias i Pujol, Universitat Auto `noma de Barcelona, Badalona,
Catalonia, Spain, 2CIBER Enfermedades Respiratorias, Instituto Carlos III, Palma de Mallorca, Spain, 3Centre de Recerca en Sanitat Animal (CReSA) (UAB-IRTA), Bellaterra,
Catalonia, Spain, 4Pathology Department, Hospital Universitari Germans Trias i Pujol, Badalona, Catalonia, Spain, 5Tuberculosis Investigation Unit of Barcelona, Servei
d’Epidemiologia, Age `ncia de Salut Pu ´blica, Barcelona, Catalonia, Spain, 6School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Dublin,
A transthoracic infection involving a low dose of Mycobacterium tuberculosis has been used to establish a new model of
infection in minipigs. The 20-week monitoring period showed a marked Th1 response and poor humoral response for the
whole infection. A detailed histopathological analysis was performed after slicing the formalin-fixed whole lungs of each
animal. All lesions were recorded and classified according to their microscopic aspect, their relationship with the intralobular
connective network and their degree of maturity in order to obtain a dissemination ratio (DR) between recent and old
lesions. CFU counts and evolution of the DR with time showed that the proposed model correlated with a contained
infection, decreasing from week 9 onwards. These findings suggest that the infection induces an initial Th1 response, which
is followed by local fibrosis and encapsulation of the granulomas, thereby decreasing the onset of new lesions. Two
therapeutic strategies were applied in order to understand how they could influence the model. Thus, chemotherapy with
isoniazid alone helped to decrease the total number of lesions, despite the increase in DR after week 9, with similar kinetics
to those of the control group, whereas addition of a therapeutic M. tuberculosis fragment-based vaccine after chemotherapy
increased the Th1 and humoral responses, as well as the number of lesions, but decreased the DR. By providing a local
pulmonary structure similar to that in humans, the mini-pig model highlights new aspects that could be key to a better
understanding tuberculosis infection control in humans.
Citation: Gil O, Dı ´az I, Vilaplana C, Tapia G, Dı ´az J, et al. (2010) Granuloma Encapsulation Is a Key Factor for Containing Tuberculosis Infection in Minipigs. PLoS
ONE 5(4): e10030. doi:10.1371/journal.pone.0010030
Editor: T. Mark Doherty, Statens Serum Institute, Denmark
Received January 21, 2010; Accepted March 15, 2010; Published April 6, 2010
Copyright: ? 2010 Gil et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Archivel Farma S.L. supported the study by providing the vaccine RUTIH. The Spanish Ministry of Health (FIS PI080785; National Plan I+D+I FIS CM06/
00123); the Spanish Ministry of Science and Innovation (BIO2005-07949-C02-02; CIBER Enfermedades Respiratorias, programa CRP-TB); the Bill and Melinda Gates
Foundation (Grand Challenges GC12) and the European Community’s 7th Framework Programme (FP7/2007-2013: STOPLATENT-TB project under grant
agreement 200999) collaborated in funding the study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
Competing Interests: PJC is co-inventor of the patent claiming RUTIH as a therapeutic vaccine. Regulatory approval and further clinical development is
currently being undertaken by a spin-off, the biopharmaceutical company Archivel Farma S.L., in collaboration with the Institut Germans Trias i Pujol. PJC is the
Scientific Director of this spin-off. The authors confirm that this does not alter their adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: firstname.lastname@example.org
. These authors contributed equally to this work.
Mycobacterium tuberculosis causes up to 100 million new cases of
latent tuberculosis infection (LTBI) every year. The fact that this
infection can persist in the host for years explains why LTBI is so
prevalent. Indeed, it is estimated that a third of humankind (more
than 2.5 billion people)  already has LTBI. Progression towards
active tuberculosis (TB) (from 5 to 25% of infected people) is
relatively low, although even this low percentage represents the
induction of 9 million new TB cases every year . Standard
treatment for LTBI requires the administration of a potentially
hepatotoxic drug, namely isoniazid (INH), for between 6 and 9
months, which results in important compliance problems. Low
compliance is the main reason why health systems are reluctant to
provide LTBI treatment except in those countries that want to
maintain their current low incidence of TB  or in the case of
HIV co-infection . A shorter non-toxic treatment is therefore
crucial to generalize LTBI control and to promote a global
decrease in TB incidence, although this necessarily implies a better
knowledge of the evolution of LTBI in order to generate new
prophylactic and therapeutic approaches.
The diagnosis of LTBI in humans is based on a normal chest X-
ray, together with a positive reaction to the tuberculin skin test (TST)
and a lack of clinical signs and symptoms suggestive of active
tuberculosis . Recently, new tests based on the detection of effector
T-cells reacting against antigens secreted by active growing bacilli
(ESAT-6/CFP-10) in peripheral blood, the so-called T-cell interferon
gamma assay (TIGRAS), have been introduced . Despite this,
human LTBI is still mainly diagnosed by indirect methods , and
little is known about its histopathological evolution.
Different experimental models have been used to better
understand LTBI evolution, the most common being those
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generated in mice. This host is able to control the bacillary load
once a strong Th1 immune response has been triggered and a
chronic phase generated  [8,9], even with a demonstrated
presence of non-replicating bacilli [10,11]. However, and despite
surviving the infection for a long time, progressive infiltration of
the lung occurs [12,13] and all mice finally die from tuberculosis.
Murine lesions are characterized by the lack of intragranuloma-
tous necrosis, a discrete fibrotic reaction, lack of encapsulation and
a strong lymphocytic presence. The predominance of a strong Th1
response led this response (with very low toxicity for the
parenchyma) to be considered an ‘‘ideal’’ response to be
reproduced and promoted to avoid evolution towards active TB
in humans, as a Th1/Th2 response is seen in humans and human
lesions are characterized by a strong tissue toxicity (i.e. necrosis
and fibrosis) . The lesions in guinea pig infection (with a
relatively low survival time once infected ), on the other hand,
better resemble human ones as they generate a strong inflamma-
tory response with strong fibrosis (although with no external
encapsulation) and strong intragranulomatous necrosis followed by
mineralization, mostly in primary lesions, where non-replicating
bacilli can be detected . Guinea pigs are also characterized by
a strong inflammatory response in the hilar lymph-nodes ,
which appears to be an attempt to avoid the systemic
dissemination that would otherwise lead to rapid death, as in the
case of humans with detectable bacilli in the spleen .
Other animal hosts have been used to better understand human
LTBI. Zebra fish infected with M. marinum, for example, have
provided insights into how mycobacteria exploit the granuloma
during the innate immune phase for local expansion and systemic
dissemination . Cattle have also been used as a model due to
their ‘‘human-like’’ control of the infection and similar immune
response in peripheral blood. Indeed, the cattle model is nowadays
used as a model to test new prophylactic vaccines against human
TB . However, the first evidence for progression from LTBI to
active disease, including cavity formation, came from an
experimental model using the Cynomolgus macaque , which
shows a wide spectrum of human-like lesions.
A histopathological description of LTBI in humans was
obtained from the studies performed by Canetti in the middle of
the last century . This author described the presence of a
‘‘benign evolution’’ of the infection in those necropsy subjects
without active TB, with small granulomas encapsulated by a
fibrotic ring and mineralized necrosis in the center. This is
probably the best histopathological description of LTBI as it shows
an efficacious control of the infection that defines this process.
There is still, however, a need to ascertain the role of the
parenchyma structure in the evolution of LTBI. The parenchyma in
humans and other large animals is subdivided by connective tissue
septa into portions of around one to two cubic centimeters, which
may play a roleincontrolling the dissemination ofpulmonarylesions
. Lungs are lined by a connective tissue band with a mesothelial
surface facing the pleural space. As is the case for the lungs of many
larger species, such as pigs, cows, and horses, the human lung has
extensive interlobular and intralobular connective tissue, which joins
the major vessels and the bronchi to the pleural surface. Such
structures are not, however, present in smaller species, such as mice,
guinea pigs, rabbits or macaques .
In light of the factors discussed above, we therefore considered
that an experimental model in a large animal was urgently
required to gain a better understanding of LTBI. As mentioned
above, there is some previous experience using cattle as a model to
test new prophylactic vaccines against human TB . We
discarded this option, however, as cattle are ruminants and
therefore have a different physiology. We therefore decided to
develop a swine model, as pigs generate lesions that closely
resemble those in human TB patients [24,25] and there is a
precedent regarding the development of an experimental swine
model using M. bovis .
This study presents for the first time the evolution of M. tuberculosis
infection in minipigs and shows how different treatment regimes can
influenceit. The results presented herein demonstrate that this model
might resembles LTBI in humans as it shows a strong ability to
control infection by generating a strong Th1 response together with a
strong local response based on the induction of a fibrotic process,
which encapsulates the lesions and limits the constant induction of
new lesions, and causes intragranulomatous necrosis and calcification
of the lesions that entraps and stresses the remaining non-replicating
bacilli inside the lesions.
The fact that the local pulmonary structure in this animal model
is almost identical to that in humans is of great significance as it
may lead to a better understanding of the mechanisms of LTBI
induction and allow new prophylactic and therapeutic approaches
designed to enhance the strong control of LTBI to be tested.
1. The natural course of the infection in the minipig
model: mimicking human LTBI
At the first time point selected to evaluate the minipig model,
namely week 5 post-infection, the bacillary load was found to be
high at all sites examined: lung, lymph nodes and extrapulmonary
samples (spleen, liver, kidneys, extrapulmonary lymph nodes and
others). Furthermore, control of the bacillary count tended to
stabilize from week 9 to the end of the experiment (Figure 1).
Indeed, bacilli could only be detected in the BAL culture for one
animal at week 5 and in the blood cultures of another animal at
week 9. BAL samples clearly show the presence of foamy
macrophages through time.
The kinetics of the cellular immune response are shown in
Figures 2 and 3. A peak in the evolution was detected 9 weeks after
Figure 1. CFU values in the lungs. Data (mean and standard
deviation) are presented by comparing an inoculated lobe with others
(pictures A and B), lung lymph nodes (C), and extrapulmonary samples
(D). Every sample collected weighted 2 g. approximately. * signifies a
statistically significant difference in the one-way ANOVA test (p,0.005).
CT = control group; INH = chemotherapy with isoniazid; VAC = group
treated with vaccine therapy.
TB Infection in Minipig
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the challenge in non-treated animals, irrespective of the cytokine
evaluated or the techniques or antigens used to stimulate the
samples. Levels tended to decrease from week 9 onwards to a
residual level, which persisted. Statistically significant differences
were only encountered in the ELISPOT assay for this time point
when the 16 kDa and Ag85B stimulations were used (one-way
ANOVA, p,0.05). IL-4 and IL-10 levels remained undetectable
in all animals. As for the humoral response, only one animal in the
control group was found to have serological PPD-specific
antibodies from week 13 to the end of the experiment (Figure 4).
Despite the presence of an active infection with a proven
continuous residual CFU and cellular immune response, the
animals appeared to be in good health, as shown by the welfare
checklist conducted as part of the daily clinical monitoring, and
continued to gain weight until the end of the experiment. All these
data highlight the similarity of our model with the course of
human LTBI in terms of the immunological response in peripheral
blood, the asymptomatic evolution and the hypothetical control of
the CFU concentration in tissue.
2. The pathology permitted a classification of
granulomas according to their characteristics and
showed the role of the intralobular septa network in the
Figure 5 shows the aspect of the lungs once fixed with formalin.
Only confluent lesions related to the site of bacillary inoculation
(the caudal left lung) could be detected before slicing. Small lesions
(0.5–2 mm in diameter) could be seen in the other lobes after
careful examination of every slice. Figure 5 also shows the
reactivity of the intralobular septa in the vicinity of a lesion.
Enlargement of the septa was observed when they were touching a
granuloma, even in the presence of a small lesion.
A careful examination of 235 lesions permitted us to observe
differenttypesof granulomas.Early-phasegranulomas seemed barely
organized and resembled a mouse-type granuloma, with a non-
organized mixture of macrophages, neutrophils and lymphocytes
(Figure 6A and B), whereas later-phase lesions (Figures 6C to F) were
moreorganized and offered a more symmetrical sphere-like aspect. A
44.1% of Phase II and a 92.5% of Phase III lesions were
encapsulated. These lesions showed a fully organized structure as
they mainly contained myofibroblasts along with a very low
proportion of lymphocytes and macrophages surrounding a
macrophage-based layer containing foamy macrophages and
apoptotic cells (mainly neutrophils) around the central necrotic area.
In Phase III lesions, the lymphocytes appeared to be mainly
accumulated as a thin layer outside the capsule and mineralization
was present in all cases, as demonstrated by addition of von Kossa
stain (Figure 7). Evolution towards the later phase was mainly based
on the increase of the mineralization area and reduction of the
surrounding cellularity, together with a reduction in size (Figure 8).
According to these data, and after careful comparison of the
microscopic preparations and observation with a stereoscopic
microscope, we were able to classify the granulomas according to
their major recognizable characteristics (Figure 9 and Table 1) as
follows: Phase I granulomas were characterized by the presence of
Figure 2. Evaluation of the cellular immune response by ELISA assay. ESAT-6 and PPD-specific IFN-c, IL-12 and TNF-a production after
stimulation of PBMCs, as determined by ELISA assay. Means and standard error of the means (SEM) are drawn in gray and connected by dotted lines.
The black boxes show the period of INH treatment and the black arrows the time of therapeutic vaccination.
TB Infection in Minipig
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an irregular infiltration with no intragranulomatous necrosis.
Phase II was considered when structured and the first signs of
intragranulomatous necrosis appeared, as revealed by the presence
of an opaque area inside the infiltration. Phase III occurred when
mineralization dominated the lesion, giving it the appearance of a
shiny opaque area, and it became compact and has a cartilaginous
Figure 3. Evaluation of the cellular immune response by ELISPOT assay. Evaluation of ESAT-6, Ag85B, 16 kDa and PPD-specific IFN-c
production after stimulation of PBMCs, as determined by ELISPOT assay. Means and SEM are drawn in black and connected by dotted lines. The black
boxes show the period of INH treatment and the black arrows the time of therapeutic vaccination.
Figure 4. Evaluation of the humoral response. Humoral response against PPD is presented. The dotted line indicates the background threshold.
Means and SEM are drawn in black and connected by dotted lines. The dark-grey boxes show the period of INH treatment and the black arrows the
time of therapeutic vaccination.
TB Infection in Minipig
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consistency when pressed with forceps. We considered this a sign
that the lesion had become highly fibrotic, which is not seen in
earlier lesions. Finally, phase IV lesions are characterized by the
predominance of mineralization with a thin surrounding infiltra-
tion. When two or more lesions were found together, they are
classified as coalescent.
After evaluating a total of 4981 lesions (Table 2) we attempted
to determine the kinetics of the recent (phase I and II) and older
(phase III and IV) lesions (Figure 10). Recent lesions were initially
predominant but decreased by week 9, when the number of old
ones increased, thus showing their maturation. Both types of
lesions tended to decrease from this point onwards, with the older
ones predominating. Remarkably, one animal showed no lesions
at week 13.
In light of the physiological value of the classification between
recent and old lesions (recent ones can disseminate bacilli as they
are still not well encapsulated), we determined the dissemination
ratio (DR) by dividing the number of recent lesions by the older
ones in each animal at each time point. Figure 11 shows that
evolution of the DR fits a negative exponential regression
(p,0.0001), thus demonstrating that, after an initial dissemination
period, the generation of new lesions decreased at week 9 to
become almost constant (DR of between 0.8 and 0.34) at week 20.
This evolution was well supported by the CFU data, which also
show a strong control after week 9 (Figure 1).
The presence of acid-fast bacilli inside the granulomas was
hardly detected during the whole experiment. The maximum
observed was two bacilli per lesion, inside the necrotic tissue in all
cases, using the auramine technique (data not shown).
Two fibrotic processes, the patterns of which are shown in
Figure 12, were present during granuloma evolution. The central
pattern was initially characterized by the presence of proliferation,
active fibroblasts expressing CD10 and a disorganized net of
reticulin based on type III collagen. Once phase II appeared,
myofibroblasts (recognized by the presence of antibodies against
the actin in smooth muscle cells) organized the reticulin network
and the cellular proliferation and CD10 expression appeared to be
located around the capsule, at the periphery, where both reticulin
and Masson’s trichromic stains showed thick, wavy collagen fibers.
As in the intralobular septa, these were easily recognized by
antibodies against collagen type I.
As the encapsulation process was detected in a high number of
in order to ascertain its origin; the results are presented in Table 3. At
the univariate level, encapsulation was found to be related to the
granuloma’s evolutive phase, its size, and contact with the
intralobular connective network (in particular with the medium-large
influence). Multivariate analysis confirmed the influence of the
evolutive phase and contact between the granuloma and the
intralobular septa that join the major vessels and bronchi to the
pleura. Although this was observed using a 2D method, the
percentage of contact (56.5%) was high enough to predict a much
larger percentage if different cuts of the granulomas were assayed
using a 3D approach. The area of the granulomas appeared to be less
important, although it should be noted that encapsulated lesions
appeared to be significantly larger (0.76 vs 0.23 mm2, p,0.001) than
non-encapsulated ones (Figure 8). Interestingly, a number of Phase I
lesions were also found to be encapsulated, a fact that was linked to
the presence of neighboring larger granulomas stimulating the same
Previous studies have already differentiated between old (initial
or primary) granulomatous lesions and new (secondary) ones, both
in rabbits  or in guinea-pigs, mainly accordingly to the size of
the lesions (larger in the former) and also by the lack of
intragranulomatous necrosis in the case of guinea-pigs .
However, our study provides, for the first time, a clear
histopathological evolution of the lesions in the context of what
could be considered as a ‘‘human-like’’ experimental LTBI model.
The process described was marked by two kind of fibrotic
Figure 5. Macroscopic pathology of the whole lung showing the intralobular septa network. Macroscopic pathology of the whole lung
(A), sliced lobes (B) and the inoculation site (C). White arrows indicate normal septa and the increased thickness of those surrounding lesions (D).
TB Infection in Minipig
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structures: an internal one based on the production of collagen III
organized by myofibroblasts, and an external one directed by the
contact with intralobular septa, based on type I collagen. The
evolution of new and old lesions provides a dissemination ratio
that could be useful for understanding the persistence of LTBI.
3. Modification of the natural course of the infection in
the minipig model by adding two different therapeutic
Animalswere observed daily to ensuretheirgood health according
to a pre-established welfare-status schedule. No local effect was
detected at the inoculation site at any time (data not shown). Systemic
adverse effects were monitored by visual inspection and by
monitoring the weight and body temperature. There were no
apparent differences among the three experimental groups, although
weight tended to increase in all groups and temperature to decrease,
whilst remaining in the physiological range. These changes were
considered related to the age of the animals.
Neither of the treatments applied resulted in a statistically
significant difference in the bacillary counts at any site with respect
to the control group.
As shown in Figures 2 and 3, INH treatment did not affect the
evolution of the cellular immune response with time, although a
Figure 6. Microscopic evolution of recent lesions, showing the relationship between the granulomas and the intralobular septa. A–
D: Phase I lesions; E and F: Phase II lesions. Images A and B show the initial evolution phase where the granuloma touches an intralobular septa but
there is still no fibroblast proliferation. This can be seen in images C and D, where the septa increase in thickness and start to surround the
granuloma, as shown by the white dotted lines. Images E and F show how the granuloma is finally surrounded by a thick collagenic mantle. Pictures
A, C and D were stained with haematoxylin and eosin (H&E), while B, D and F were stained with Masson’s trichromic. The original magnification of
the large images is 640 whereas all insets, except F (x100), are magnified 6400. The green arrows show the septa and the dotted white lines the
trajectory of the capsule.
TB Infection in Minipig
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slight decrease could be seen from week 13 onwards. In contrast,
the ELISPOT assay showed a transient increase in IFN-c SFU
after both the first and second vaccine inoculations (using antigen
85B, PPD, ESAT-6 or 16 kDa as stimuli). This increase was
statistically significant when compared to the other two experi-
mental groups. Vaccine therapy also induced a transient increase
in IFN-c, IL-12 and TNF-a one week after the first vaccine
inoculation in some animals, as detected using the ELISA
technique (Figure 2). This increase was confirmed statistically
upon comparison with the other groups. IL-4 and IL-10 levels
remained undetectable in all animals.
An increase of serological PPD-specific antibodies was observed
after the second vaccine inoculation in all vaccinated animals.
Two animals from the vaccinated group already showed positive
PPD-specific antibodies in their sera at week 9, before the first
inoculation, and these two animals also had the highest amount of
antibodies at the end of the study, thus suggesting a boosting
As regards the kinetics of recent and old lesions, INH treatment
during weeks 5 to 9 resulted in a reduction of all lesions, thereby
reflecting a decrease in the maturation process. Remarkably, only
two lesions could be detected in one animal belonging to the INH
group at week 13 (Figure 12). Interestingly, vaccine therapy
increased both types of lesions, with the number of old lesions
showing a higher increase. This treatment also decreased the CFU
numbers of the inoculated lobe (Figure 1).
Although we have already shown that DR decreased sharply from
week 9 to a residual constant value untilthe end of the experiment for
the non-treated animals, INH treatment stopped this decrease,
apparently by stopping the progression of lesions to phases III and IV
but without changing the overall evolution defined by the control
group. Interestingly, vaccine therapy modified this evolution by
Figure 7. Microscopic evolution of the old lesions. Once the granuloma is structured, the necrotic process starts and calcification appears.
Images A and B (phase III) show a well-structured and encapsulated granuloma with necrotic calcification. Images C and D show lesions of an
advanced evolution (phase IV), with granulomas containing a large amount of calcification and fibrosis. Samples A and C were stained with H&E,
whereas samples B and D were half stained with Masson’s trichromic and von Kossa stain, which shows calcification in black. Pictures E to G were
stained with H&E, Masson’s trichromic and von Kossa stain, respectively. Lesions 1 and 2 are phase II lesions and differ only in the initial mineralization
seen in lesion 2. The other lesions are all phase III lesions that have progressed differently. Original magnification is 610.
Figure 8. Evolution of the area of granulomas according to their evolutive phase. Individual data are presented in relation to the evolutive
state of the lesion (A) or its encapsulation status (B). Mean and quartiles are presented in each case. Inter-group differences were determined by
Dunn’s One Anova on ranks test and are marked with * if statistically significant (p,0.05).
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reducing the DR to 0.195 at week 20, a fact strongly related to the
increased number of old lesions (Figure 10).
In conclusion, treatment of the infection with short-term
chemotherapy reduced the number of lesions but appears to also
reduce their maturation towards old lesions. In contrast, the
addition of a therapeutic vaccination increased the number of
lesions, mainly the old ones, tends to reduce the CFU counts, and
could be linked to an increase in the (cellular and humoral)
immunological responses induced.
Little is known about the histopathological evolution of human
LTBI, although some findings have helped to determine the
evolution of the lesions [21,29].
In general, a strong inflammatory response is induced, with the
presence of giant multinuclear cells, a strong fibrotic response
encapsulating the lesion, and a large intragranulomatous necrosis,
which finally calcifies over time. The presence of bacilli in these
lesions is seldom observed. As a chest X-ray is able to detect old
calcified lesions, it is traditionally considered that, upon infection
with M. tuberculosis, an initial dissemination of lesions, which might
evolve towards calcification, takes place before the immune
response is triggered . It was demonstrated a long time ago
that bacilli can persist in a latent stage in a small percentage
(around 1%) of these evolved lesions . This is the origin of the
traditional theory, which postulates that these bacilli can
resuscitate after some type of local immunodepression and
produce active TB . This theory has, however, been
superseded to some extent by other less disseminated and/or less
accepted theories developed to explain LTBI: external reinfection
as the most probable cause of TB in adults ; M. tuberculosis
infection undergoing a constant process of autoinoculation, with
bacilli constantly being drained out of the lesions and inducing
febrile episodes [31,32]; and the more recent hypothesis that
considers LTBI to be a consequence of a constant endogenous
reinfection process .
As the ‘‘perfect’’ animal model that mimics LTBI has still not
been found, we present a new animal model using minipigs. In this
study we describe for the first time a comprehensive evolution of
the lesions caused by M. tuberculosis in a large animal that might fit
the traditional view of control of this infection in humans, which
leads to an LTBI. This is seen in a context characterized by a
maintained Th1 response, control of the bacillary load, the
constant induction of recent lesions, and their resolution (towards
old lesions) as a result of a strong local fibrotic response in which
the local intralobular septa seem to play a crucial role in
granuloma encapsulation. This response seems to play a major
role in the evolution of granulomas by curtailing the dissemination
of bacilli towards the alveolar space, thereby explaining the sudden
drop in the induction of new lesions 9 weeks after the challenge. As
this dissemination still exists, albeit at a low level, these findings
also support the Dynamic Hypothesis, which postulates that
maintenance of an LTBI requires a constant endogenous
reinfection , and might explain the success of humans in
controlling TB infections and the low progression towards active
Interestingly, the initial granulomatous dissemination observed
also fits with the work developed in the zebrafish model infected
with M. marinum, where infected macrophages from primary
granulomas could disseminate to initiate new secondary granulo-
mas during the innate immune phase of the infection .
The initial lesions in this model are similar to the initial murine
lesions, in other words a mixture of neutrophils, macrophages and
lymphocytes without much organization, with the periphery open
to the alveolar spaces. However, very few acid-fast bacilli are seen
in minipigs, whereas they are highly abundant in mice, thus
reflecting a similar degree of ‘‘human-like’’ low tolerance to the
bacilli as such a low concentration is enough to trigger a strong
granulomatous reaction. As in mice, a close relation to the alveolar
spaces still exists in this phase, thus allowing the constant drainage
of infected foamy macrophages towards this space [13,34]. As in
guinea pigs or larger mammals, this drainage is very effective as
the alveolar spaces are large enough not to retain them locally, as
Figure 9. Macroscopic evolution of the lesions. Classification of the
fixed pulmonary lesions as they appear under the stereoscopic
microscope. Considering the histological evolution of the lesions, and
taking into account the sequential appearance of encapsulation and
calcification, we have divided the lesions into four phases. Phase I is
characterized by the presence of cellular infiltration (A and B). The
intragranulomatous necrosis, which is characterized by the presence of an
opaque zone inside the granuloma (C and D), appears during Phase II and
structuration of the lesion starts. Phase III (E and F) involves the onset of
in size. These lesions are characterized by the cartilaginous texture of the
lesion when touched with the forceps. Phase IV lesions (G and H) are
characterized by predominance of the calcification and a thin surrounding
infiltration. Original magnification is 610. Scale bar: 1 mm.
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is the case in mice. This phase is, however, rather limited in time,
as also demonstrated by the scarcity of foamy macrophages
detected in the BAL. This phenomenon could be a consequence of
the entrapment of foamy macrophages in the granuloma during
encapsulation and might therefore play a role in the generation of
intragranulomatous necrosis [35,36].
Our data permit the lesions to be classified and show that the
evolution of the granulomas is marked by fibroblast proliferation
and transformation into myofibroblasts, which contract the lesions
by structuring the reticulin fibers, thereby allowing the original
structure to be maintained against external mechanical forces. The
granuloma itself develops with a spherical form due to an energy-
minimization process [37,38]. This follows the usual healing
process in the lung once a lesion has been induced. Upon onset of
infection, intracellular bacillary growth mainly causes necrosis of
the infected macrophages, thus maintaining the inflammatory
response. In this scenario, secretion of TNF by macrophages is
crucial for both fibrin and collagen deposition, which results in a
provisional extracellular matrix . This inflammatory response
also generates apoptotic neutrophils and macrophages which, once
phagocytosed, stimulate the production of TGF-b and thus stop
the inflammatory response and provoke an anti-inflammatory
process, thereby promoting fibrosis [40,41,42]. TGF-b also
promotes fibroblast accumulation and, together with mechanical
stress, induces the transformation into myofibroblasts  ,
whose presence has already been linked to mycobacterial
The animal model studied herein also shows two sclerotic
processes that have classically been described in the human
‘‘benign’’ evolution of the infection , namely an increase in
fibroblast proliferation, which leads to the internal accumulation of
myofibroblasts, and the formation of a capsule around the
granuloma, as a continuum of the interlobular septum, once the
granuloma has reached it. The similarities with human granulo-
mas might then be a consequence of the presence of intralobular
septa in minipigs. These structures are highly sensitive to
mechanical stress and react to it with fibroblast proliferation and
thickening after collagen production . This capsule appears to
be vital to prevent the constant drainage of latent bacilli towards
the alveolar spaces, as animals that are not able to structure it,
such as mice and guinea pigs, constantly have infected foamy
macrophages in the BAL [13,34,45]. This process of healing and
avoiding constant bacillary dissemination has been known for
some time and was the rationale behind the most successful pre-
chemotherapy TB therapy, namely collapse therapy, which
effectively reduced the appearance of new lesions, avoided
liquefaction and also increased local fibrosis and healing by
increasing the stasis of the blood circulation [21,32].
It has been speculated that both fibrosis and the anti-inflammatory
response restrict the arrival of new macrophages that are able to
phagocytose the apoptotic bodies , thus allowing them to
accumulate and favoring the mineralization process. Calcification
could also be promoted by the destruction of foamy macrophages,
which could be both a source of intragranulomatous necrosis [35,36]
accumulation of calcium and phosphate ions along with an increase
in the local pH [47,48] [49,50]. Both these factors could induce a
multi-stress scenario against the bacilli, thereby contributing to
control of the bacillary load, as has already been demonstrated in the
guinea-pig model .
As regards the influence of different treatment regimes on the
course of the infection, the effect of INH treatment on the
evolution of the lesions is worthy of comment. Thus, although this
treatment prevents the induction of new lesions and reduces their
concentration, it also diminishes the concentration of old lesions,
thus indicating a reduction in the fibrosis process that is probably
related to a reduction in the inflammatory process, as discussed
previously . This could mean that some type of inflammatory
process is needed to induce the fibrotic process, possibly by causing
enough apoptotic bodies to enhance TGF-b production, although
this would need to be demonstrated experimentally. Interestingly,
the addition of a therapeutic vaccine has the opposite effect: it
increases the number of lesions, particularly old ones. This could
reflect a proinflammatory process that might involve the quicker
detection and control of new lesions, due to the increased cellular
and humoral response, and which would finally increase the
fibrotic effect. Previous studies with this particular vaccine have
already revealed its ability to elicit a combined Th1-Th2-Th3
response , a cellular immunity characterized by an increase in
the level of Th1 cells that are able to recognize replicating and
non-replicating bacilli  and a protective humoral response
. This increase in the number of lesions might result from a
protective role, as supported by the reduction in both the number
of CFUs in the inoculated lobe and the dissemination rate.
Overall, the data presented herein suggest a scenario very
similar to human LTBI, and could support the idea of a constant
reinfection process maintaining the LTBI . It is well known
that the chance of developing active TB after infection decreases
exponentially with time . Concurrently, our data also show
how the induction of new lesions decreases suddenly after an initial
dissemination process and remains at a constant residual level,
thus reducing the chance of progression towards active TB .
Finally, these results are also in agreement with the constant
presence of ESAT-6-specific IFN-c spot-forming units in the
peripheral blood of LTBI human subjects, thereby revealing the
presence of short-lived specific effector T-cells . As ESAT-6 is
produced by growing bacilli , the explanation for the constant
presence of these cells might be related to the constant
development of new lesions, as demonstrated in the experimental
model proposed here. These new lesions would be so small
Table 1. Characteristics of the evolution Phase of the lesions.
Phase Irregular margin NecrosisCalcification Cartilaginous consistency Halus size in relation to necrosis
*Phase I lesions are subdivided in a or b regarding its contact with interlobular septae (in the b case).
**N.E. means ‘‘not evaluable’’.
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Table 2. Characteristics of the lesions.
Total lesionsGrouped lesions DR
Phasenmedian 25–75 per.median25–75 per. median 25–75 per.
I 702247 (136.8–328)387 (174–411)22.77 (10.76–44.32)
III8117 (5.75–50.75)17 (5.75–50.75)
Total 990 Median lesions per animal and percentiles (25–75)=404 (180–462)
I62 16 (10–32.5)45 (18–63.75)0.86 (0.78–0.97)
III13848 (18.75–72.75) 60(21.75–75.75)
Total 274Median lesions per animal and percentiles (25–75)=105 (39.8–140)
I 2613 (4–22)17 (7–27)4.85 (2.7–7)
III 105(1–9) 5.5(1–10)
Total45Median lesions per animal and percentiles (25–75)=22.5 (8–37)
I 845 (1.25–60.5)13(4–153.3)0.464 (0.43–0.87)
III487 20(5.75–354.5) 28(7.75–361.8)
Total716 Median lesions per animal and percentiles (25–75)=41 (11.8–515)
I 1102(2–81.5)6 (1.5–93.75)0.35 (0.09–1.9)
III6114 (3.5–38.75) 17 (4.25–42.5)
Total 197 Median lesions per animal and percentiles (25–75)=23 (5.75–136)
Coalescent 45 3.5(1–16)
IV 1093 (1–31)
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(around 0.5–1 mm) that they would not be observable in the chest
X-ray, therefore their presence would be absolutely compatible
with a diagnosis of LTBI .
The ‘‘secret’’ to controlling M. tuberculosis infection might
therefore lie in both avoiding the drainage of non-replicating
bacilli (by encapsulating the lesion) and stopping the inflammatory
response. This would prevent any phagocytosis of the remaining
bacilli, thus stopping their growth. This is a crucial point as
extracellular multiplication of M. tuberculosis in vivo has only been
seen in liquefacted lesions , which are not present in this
scenario. In addition, this anti-inflammatory effect could favour
mineralization of the necrotic tissue, which might be deleterious
for the healing process but would further stress any remaining
The high number of parallelisms found between this infection
model and the process thought to occur in humans makes it
especially useful for further studies devoted to understanding the
evolution of LTBI. Achieving a course of the infection that might
be similar to the human one, including the monitoring of the
immune responses, also provides an opportunity to not only help
new preventive and therapeutic approaches against it to be
designed, but to be tested with some reliability, thereby offering a
completely new field for future studies.
Materials and Methods
1. Experimental Design
pathogen-free (spf) Go ¨ttingen minipigH were obtained from
Ellegaard (Dalmose, Denmark) and fed with maintenance diet
127 (SAFE, Augy, France). All animals were provided with a
highly detailed certificate and were routinely checked for infection
A total of 36 1.5-month-old female specific
Total1559Median lesions per animal and percentiles (25–75)=76 (34–330)
I1565.5 (1–24) 8.5(4–55)0.412 (0.32–0.83)
III 121 11(3–26) 26(6–37)
Total417Median lesions per animal and percentiles (25–75)=38 (11–155)
I9220(3–21) 35(7–41) 0.195(0.18–0.32)
Coalescent 26 4.5 (1–7)
IV198 27 (7–47)
783 Median lesions per animal and percentiles (25–75)=142.5 (44–214)
Table 2. Cont.
Figure 10. Evolution of recent and old lesions. The results show the median of the values represented in Table 2. The gray bar shows the INH
treatment period and vaccine inoculation is represented by the black arrows. CT = control group; INH = chemotherapy with isoniazid; VAC = group
treated with vaccine therapy.
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with more than 40 infectious agents, including 24 bacteria, 12
viruses, 4 parasites and 3 fungi.
Infection and experimental groups.
anesthetized by intramuscular injection of 10 mg/kg of ketamine,
2 mg/kg of xylacine and 2 mg/kg of azaperone before infection
with 26103CFUs of M. tuberculosis diluted in 2 mL of saline. The
Mycobacterium tuberculosis strain used was the H37Rv Pasteur strain,
grown in Proskauer Beck medium containing 0.01% Tween 80 to
mid-log phase and stored at 270uC in 2 mL aliquots until being
used to infect the animals. The transthoracic method was
preferred to an aerosol as it is safer for the professionals
responsible for infecting the animals. The transthoracic infection
was performed with an 18-gauge Tuohy epidural needle (length:
90 mm) (Viggon S.A, Paterna, Spain) between the 8th and 9th rib
of the left lung. Three experimental groups were established
according to the treatment received: infected non-treated
(control; n=15); treated with INH alone (INH; n=12); and
treated with INH plus two doses of M. tuberculosis fragment-based
vaccine (vaccinated; n=6). INH (300 mg; Cemidon, Alcala
Farma, Madrid, Spain) was administered intramuscularly into
the cervical region twice a week for four weeks (weeks 5–9), with
three days between injections. This schedule was chosen to
determine the effects of INH during its most efficacious period,
on the basis of previous bactericidal activity studies ,
assuming that the CFU control would start at week 5 after
challenge, as previously demonstrated in guinea pigs .
Moreover, this schedule had already been found to be effective
in large animals . The therapeutic vaccine used was RUTIH,
a vaccine manufactured by Archivel Farma, s.l. (Badalona,
Catalonia, Spain), under GMP standards. RUTIH vaccine is
based on M. tuberculosis fragments, detoxified and liposomed as
published elsewhere . The vaccine is still in clinical
development and not commercially available, thus was kindly
provided by the manufacturer. RUTIH was administered twice,
at weeks 9 and 12, 21 days apart, according to previous protocols
The animals were
supervised by the Animal Care Committee of the Universitat
Auto `noma de Barcelona in accordance with current EU legislation
regarding the protection of experimental animals. All experiments
were performed in a BL3 facility inside the CReSA building at the
Universitat Auto `noma de Barcelona. Animals were observed daily
according to a welfare schedule and check list (approved by the
CRESA Animal Welfare Committee) to monitor the clinical
aspects after the infection and to ensure the safety of the
All experimental procedures were approved and
Figure 12. Characterization of the dual fibrotic responses in
granuloma evolution. Figures A, B and H show reticulin stain of initial
a green arrow in B and H). C and D present immunostaining with anti
and III granulomas, respectively. E and F also show the differences
between these evolutive phases with the same proliferation pattern
stained with Ki 67. G shows recognition of the capsule by anti collagen
type 1 antibodies, whereas I and J show the identification of
myofibroblasts using anti smooth muscle and anti HHF35 antibodies
respectively. Original magnification is 6200. Scale bar: 100 mm.
Figure 11. Evolution of the dissemination rate (DR). Individual
data points are represented by full or open circles in control (CT) and
isoniazid-treated (INH) groups respectively, and are adjusted to an
exponential regression. The continuous and dotted lines represents the
adjustment for the CT (y=5620.4521.071 ? x+ 0.5460) and INH
(y=295.6220.4848?x+0.8793) groups. Both adjustments were statistically
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treatments tested. They were also weighed every week in order to
monitor their health status.
2. Post-mortem analysis
with an intravenous overdose of pentobarbital sodium at week 20
post-innoculation (Vetoquinol, Madrid, Spain). The minipigs were
subjected to a detailed post-mortem examination . All obvious
lymph nodes present at each site were collected, including the
axilar (right and left), inguinal (right and left), mesenteric, hepatic,
middle retropharyngeal (right and left), tonsil (right and left),
tracheobronchial and caudal mediastinal lymph nodes. A small
portion of each lymph node was removed for histology and the
remainder retained for bacteriology. Bacteriology tissue samples
were collected using an aseptic technique. Gross lesions from the
lungs were detected by observation or palpation, recorded and
collected for histopathology and culture. If no lung lesions were
identified, a sample (approx. 2 g) was collected from the dorsal
margin of each lobe for culture. The remainder of the lungs was
fixed in formalin in all cases for subsequent slicing at 2-mm
intervals. The cut surfaces were further examined for gross lesions
and collected for histopathology. Each slice was also carefully
examined for microscopic lesions under a binocular stereoscopic
microscope SMZ800 (Nikon Instruments Inc., Tokyo, Japan).
Bronchoalveolar lavage (BAL).
removal of the lungs, a total of 200 mL of physiological serum was
The animals were euthanised
After sacrifice and aseptic
introduced through the trachea and recovered by aspiration with a
100 mL syringe. The volume recovered was then divided into two
tubes: one to determine the CFU and the other for cytology. The
latter tube was centrifuged at 300 g for 15 minutes and the sample
stained with Oil Red and examined by electron microscopy to
identify foamy macrophages.
Samples of lung lobes and pulmonary and
extrapulmonary lymph nodes from each animal were extracted to
determine the bacterial burden. Lung tissue and BAL samples
were homogenized and decontaminated according to a previously
reported procedure , whereas lymph node samples were not.
The number of viable bacteria was determined by plating serial
dilutions of whole organ homogenates and BAL on nutrient
Middlebrook 7H11 agar (BD Diagnostics, Spark, USA) and
Lowenstein-Jensen medium (Biomedics, Madrid, Spain). Bacterial
colonies were counted after incubation for 28 days at 37uC. Blood
cultures were performed until week 13 by inoculating 5 mL of
whole blood to BacT/ALERT MB flasks (Biomerieux, Marcy
L’Etolile, France). These samples were incubated for 30 days at
37uC before being considered negative, as recommended by the
Histopathology and histometry.
were fixed in 10% buffered formalin, sectioned at 4 mm and
stained with haematoxylin-eosin and Masson trichromic, and for
acid-fast bacteria following the Ziehl–Neelsen and auramine
methods. Von Kossa stain was also used to evaluate the
Table 3. Predictive factors in granuloma encapsulation.
VARIABLEUNIVARIATE LEVELMUTIVARIATE LEVEL
r-valueOR (95% CI)
r-valueAdjusted OR (95% CI)
Total (% encapsulated)
II 34 (44,1)2,5 (1,0–6,4)2,5 (1,0–5,9)
III93 (92,5)38,8 (14,2–110,5)41,6 (16,2–107,3)
IV29 (93,1)42,6 (8,6–268,8) 39,2 (8,5–181,7)
Contact with Pleura:
Yes18 (61,1)0,94 (0,3–2,8)
Contact with vessel/bronchi
Yes156 (71,2)3,00 (1,62–5,4)
Contact with Septae:
No 117 (55,6)0,031 0,0451
Yes 118 (69,5)1,8 (1,0–3,2)2,1 (1.0–4,3)
Contact with the connective
No29 (34,5) 0,021-
Yes206 (66,5) 3,8 (1,6–9,3)
Area size in mm2:
0.24–0.4958 (63,8) 5,8 (2,4–14,1)
0.50–0.9759 (74,6) 9,6 (3,9–24,5)
0.98–3.2758 (89,7) 28,5 (9,2–93,2)
OR: Odds ratio. CI: Confidence Interval.
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presence of calcified material in the necrotic tissue . A reticulin
silver stain was run to determine the organisation of collagen in the
granuloma. Reticulin mainly stains the thin fibrils of type III
collagen, although type I collagen can also be stained when
organized into thick fibrils assembled in parallel bundles . The
small and large axis of each granuloma were recorded from a
random sample of Masson trichromic cuts using a DS-Fi1 camera
attached to an Eclipse 50i microscope using the NIS-Elements D
version 3.0x software package (Nikon Instruments Inc., Tokyo,
Japan). The area of 235 granulomas was obtained using the
formula of the area of an oval; Pi*a*b, where a=half length of
major axis (horizontal) and b=half length of minor axis (vertical).
For immunohistochemical analysis,
4-mm consecutive sections were deparaffinized in xylene and
rehydrated with graded alcohol. Heat-induced antigen retrieval was
carried out in an autoclave with citrate buffer at pH 6.0. Endogenous
peroxidase was blocked with 5% hydrogen peroxide and detection
was performed using an UltraVIEW DAB Detection universal Kit in
a BenchmarkXT device(Roche
Commercially available anti-human antibodies against CD10
(clone 56C6, 1/20, Novocastra-UK), Ki67 (Clone MIB-1, 1/800,
Dako-Denmark), Collagen I (ab6308, 1/500, Abcam-UK), Actin
(Clone HHF35, 1/300, Dako-USA), and Alpha Smooth Muscle
Actin (Clone asm, 1/200, Novocastra-UK) were obtained from the
Semi-thin slices from lung granuloma and BAL.
granulomas were placed in Eppendorf tubes containing the
paraformaldehyde in phosphate buffer 0.1 M) and kept at 4–
8uC for at least 24 h. BAL tubes were centrifuged at 300 g for 10
minutes, then resuspended in 0.5 mL of PBS and 0.5 mL of the
fixation solution 2x and kept at 4–8uC for between 2 and 24 h.
The BAL samples were again centrifuged at 300 g for 10 minutes,
then the supernatant was removed carefully, the fixation solution
1x added slowly and the samples kept at 4–8uC. Granuloma and
BAL samples were then post-fixed in osmium tetroxide,
dehydrated and embedded in epoxy resin. Semi-thin sections
were stained with toluidine blue as described elsewhere .
3. Evaluation of the immune response
The evolution of the cellular immunological response was
monitored by stimulation of PBMCs using different specific
stimuli, as well as detection of several cytokines in plasma. The
humoral response was also evaluated.
Blood was collected using heparinized blood tubes at
the time points described above. Peripheral blood mononuclear
cells (PBMCs) were separated from whole blood by density-
gradient centrifugation with a Histopaque 1077 (Sigma) and
washed. Trypan blue was added to assess their viability.
The abundance of antigen-specific IFN-c secreting cells in the
PBMCs was analyzed by an ELISPOT assay using commercial
mABs (Porcine IFN-c P2G10 and biotin P2C11, BD Biosciences
Pharmingen) according to a previously reported method .
Briefly, Costar 3590 ELISA plates (Corning, New York, USA)
were coated overnight with 5 mg/ml P2G10 capture antibody
diluted in carbonate/bicarbonate buffer. The plates were then
washed and blocked for 1 h at 37uC with 150 ml PBS containing
1% BSA. After removal of the blocking solution, 56105viable
PBMCs were dispensed per well and stimulated with PPD, ESAT-
6, Ag 85B, Ag 16 kDa (all at a final concentration of 10 mg/ml) or
PPD RT-49 and BCG were obtained from the Statens Serum
Institute (SSI, Copenhagen, Denmark). The recombinant M.
tuberculosis antigens ESAT-6 (Rv3875), Ag 85B (Rv1886c) and Ag
16 kDa (Rv2031c) were purchased from Lionex Diagnostics and
Therapeutics GmbH (Braunschweig, Germany).
Unstimulated cells and phytohaemagglutinin (PHA)-stimulated
controls (10 mg/ml) were also included in both experiments. After
20 h incubation at 37uC in a humidified atmosphere of 5% CO2,
the cells were removed and the biotinylated P2C11 detection
antibody was added at 0.5 mg/ml and incubated for 1 h at 37uC.
The reaction was revealed by sequential incubation of plates with
streptavidin-peroxidase and insoluble 3,395,59-tetramethylbenzi-
dine (TMB; Calbiochem, Nottingham, UK). To calculate the
antigen-specific frequencies of IFN-c secreting cells, also known as
spot-forming units (SFUs), the number of spots in unstimulated
wells was subtracted from those in antigen-stimulated wells. The
abundance of antigen-specific IFN-c secreting cells was expressed
as number of responding cells from a total of 106PBMC.
To analyze cell-culture supernatants after stimulation of mini-
pig PBMCs with different specific antigens, cells were plated
(56106per well) in 48-well plates and mock-stimulated or
stimulated with PPD, ESAT-6 (10 ml/ml) or PHA. After
incubation for 20 h at 37uC in a humidified atmosphere of 5%
CO2, cell culture supernatants were collected and frozen at
280uC until needed. Capture ELISAs for IFN-c, TNF-a, IL-4,
IL-10 and IL-12 were performed as mentioned above.
Cytokine concentration in sera was
determined at weeks 0, 2, 5, 9, 10, 12, 13 and 20 using
commercial pairs of antibodies (Porcine IFN-c, BD Biosciences
Pharmingen; TNF-a, IL-4, IL-10 and IL-12; R&D Systems)
according to previously reported procedures  and the
manufacturer’s instructions. The cut-off point for each ELISA
was calculated as the mean+3SD OD of negative controls. The
production of each cytokine was calculated using the linear-
regression formula based on the ODs of the cytokine standards
provided by the manufacturer and expressed as pg/ml.
Costar 3590 ELISA plates (Corning,
New York, USA) were coated overnight with 4 mg/ml of PPD RT-
49 diluted in carbonate/bicarbonate buffer. They were then
washed five times with PBS plus 1% Tween 20 and blocked for 45
minutes at 37uC with 150 ml PBS plus 0.5% casein. After washing,
1/200 diluted sera in PBS plus 0.5% casein was added and
incubated for 1 h at 37uC. The plates were further washed and the
conjugate (diluted in PBS with 1% Tween 20) added. The
conjugate was prepared with 1/10000 protein A-HRP (Sigma,
Laboratories, El Prat del Llobregat, Spain). The reaction was
revealed with soluble TMB (Calbiochem, Nottingham, UK) and
stopped 20 minutes later with 0.5 M H2SO4. The plates were read
at 450 nmusinga conventional
determined for each sample using a control to which PPD RT-
49 had not been added. Results were obtained by analyzing the
sera in duplicate and subtracting the unspecific response. The cut-
off point was calculated as the mean+3SD OD of unspecific
4. Statistical analysis
A descriptive study of the qualitative and quantitative variables
collected was carried out in order to characterise the study
population. Frequency distributions and medians for quantitative
variables were calculated. Proportions were compared between
groups using the chi-squared and, when pertinent, the two-sided
Fisher test. Measures of association were calculated using odds ratios
(OR) along with their 95% confidence intervals (CI). The factors
associated with encapsulation were analysed using logistic
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PLoS ONE | www.plosone.org14April 2010 | Volume 5 | Issue 4 | e10030
regression (stepwise method) including the variables associated
with a p-value of less than 0.15 at the univariate level in the model.
A p-value of less than 0.05 was considered significant. The test of
Hosmer and Lemeshow was used to check the goodness-of-fit of
the models. Analyses were conducted using the SPSS statistical
package, version 13.0 (SPSS Inc, Chicago, IL, USA).
Dr. Evelyn Guirado contributed in obtaining the samples. The technicians
of the Pathology Department stained the cuts and performed the
Conceived and designed the experiments: OG ID CV LC MD PJC.
Performed the experiments: OG ID CV GT JD MF NC SP LC MD PJC.
Analyzed the data: OG ID CV GT NC JC LC MD PJC. Contributed
reagents/materials/analysis tools: ID GT JD MF SP JC MD PJC. Wrote
the paper: OG ID CV NC PJC. All authors listed contributed to
conception and design, or acquisition of data or analysis and interpretation
of data; to drafting the article or revising it critically for important
intellectual content; and gave the final approval of the version to be
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PLoS ONE | www.plosone.org15April 2010 | Volume 5 | Issue 4 | e10030