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Malaysian Journal of Medical Sciences
School of Medical Sciences, Universiti Sains Malaysia
ISSN: 1394-195X
Vol. 18, Num. 1, 2011, pp. 12-15
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Malaysian Journal of Medical Sciences, Vol. 18, No. 1, 2011, pp. 12-15
Special Communication
Modelling of Cerebral Tuberculosis: Hope for Continuous
Research in Solving the Enigma of the Bottom Billion's Disease
Rogelio HERNÁNDEZ PANDO
Experimental Pathology Section, Department of Pathology, National Institute
of Medical Sciences and Nutrition “Salvador Zubirán”, Calle
Vasco de Quiroga 15, Tlalpan, CP 14000, México DF, México
Correspondence: Dr Rogelio Hernández Pando,
MD, MSc and PhD in Immunology (National University of Mexico),
Experimental Pathology Section,
Department of Pathology,
National Institute of Medical Sciences and Nutrition “Salvador Zubirán”, Calle
Vasco de Quiroga 15, Tlalpan,
CP 14000, México D.F. México,
Tel: (+52-55) 54 85 34 91,
Fax: (+52-55) 54 85 34 91,
Email: rhpando @quetzal.innsz.mx, rhdezpando@hotmail.com
Submitted: 23 Oct 2010
Accepted: 25 Oct 2010
Code Number: mj11003
Abstract
Cerebral tuberculosis is a severe type of extrapulmonary disease that is highly
predominant in children. It is thought that meningeal tuberculosis, the most
common form of cerebral tuberculosis, begins with respiratory infection followed
by early haematogenous dissemination to extrapulmonary sites involving the
brain. Host genetic susceptibility factors and specific mycobacteria substrains
could be involved in the development of this serious form of tuberculosis.
In this editorial the different animal models of cerebral tuberculosis are
commented, highlighting a recently described murine model in which BALB/c mice
were infected by the intratracheal route with clinical isolates, which exhibited
rapid dissemination and brain infection. These strains were isolated from the
cerebrospinal fluid of patients with meningeal tuberculosis; they showed specific
genotype and induced a peculiar immune response in the infected brain. This
model could be a useful tool to study host and bacilli factors involved in
the pathogenesis of the most severe form of tuberculosis.
Keywords: experimental models, infectious diseases, meningeal tuberculosis,
mice, Mycobacterium, virulence
Tuberculosis and the Central Nervous System
Tuberculosis involvement of the central nervous system (CNS) is a significant
and serious type of extrapulmonary disease. It constitutes approximately 5%–15%
of the extrapulmonary cases, and in developing countries, it has high predominance
in children (1). There are different clinical/pathological manifestations of
cerebral tuberculosis; the most common is tuberculous meningitis, followed
by tuberculoma, tuberculous abscess, cerebral miliary tuberculosis, tuberculous
encephalopathy, tuberculous encephalitis, and tuberculous arteritis (2). Cerebral
tuberculosis is often fatal and mainly caused by Mycobacterium
tuberculosis;
other non-tuberculous mycobacteria such as M.
avium-intracellulare can also
produce CNS tuberculosis, mainly in human immunodeficiency virus (HIV)-infected
persons (2).
It is believed that cerebral tuberculosis, like any other forms of tuberculosis,
begins with respiratory infection followed by early haematogenous dissemination
to extrapulmonary sites, including the CNS. On the basis of their clinical
and experimental observations, Rich and McCordock (3) suggested that cerebral
tuberculosis develops in two stages. Initially, small tuberculous lesions (Rich’s
foci) develop in the brain during the stage of bacteraemia of the primary tuberculosis
infection or shortly afterwards. These early tuberculous lesions can be located
in the meninges, the subpial or subependymal surface of the brain, and may
remain dormant for long time. Later, rupture or growth of one or more of the
small lesions produces development of various types of CNS tuberculosis. Rupture
into the subarachnoideal space or into the ventricular system produce meningitis,
the most common form of cerebral tuberculosis.
Modelling of Cerebral Tuberculosis
Experimental animal models of cerebral tuberculosis have been established
in rabbits (4,5), mouse (6,7), and pigs (8). Although they reproduce in some
extend the human lesions, these models are artificial because they use the
direct intracerebral or intravenous route of infection, instead of the natural
respiratory route. Thus, it is important to establish an experimental model
which reproduces more closely the human disease, including the initial respiratory
natural route of infection. However, such model is difficult to achieve because
of the highly efficient CNS protection conferred by the blood-brain-barrier
(BBB). BBB is composed of tightly associated brain microvascular endothelial
cells covered by pericytes and outgrowths of astrocytes (cytoplasmic end feet).
This structure efficiently prevents CNS infection by many microorganisms, including
mycobacteria. Thus, to produce CNS infection, some microorganisms have evolved
specific virulence factors that permit, first, endothelial attachment and internalization,
followed by brain parenchyma invasion (9). This is the case of bacterial proteins
IbeA, IbeB, AslA, YijP, and OMPA expressed by neurotropic Escherichia coli,
or meningococcal surface proteins Opa, Opc, and PiIC among others.
Recent in vitro studies have shown that M. tuberculosis can adhere, invade,
and traverse endothelial cells (10), and clinical–epidemiological studies
have shown distinct genotype in strains isolated from tuberculous patients’ cerebrospinal
fluid (CSF) (11), which suggest strain-dependent neurovirulence and neurotropism.
We recently informed the results from an experimental study in which, using
a model of pulmonary tuberculosis in BALB/c mouse infected by the intratracheal
route, three different M. tuberculosis clinical isolates obtained from CSF
of meningeal tuberculous patients were able to rapidly disseminate and infect
the mouse brain (12). These clinical strains were isolated from patients with
meningeal tuberculosis in Colombia, and they showed a distinctive genotype.
They extensively disseminated by haematogenous route after one day of intratracheal
infection and rapidly produced tuberculous lesions in the mice brain. As mentioned
before, it has been established that mycobacteria reach the CNS by the haematogenous
route secondary to pulmonary infection (3). This experimental model is the
first one that reproduced this situation, confirming that the strain type is
directly related with the ability to disseminate by the haematogenous route,
and add M. tuberculosis to the list of microorganism families in which some
members or substrains have certain ability to infect the CNS.
Comprehensive clinical-epidemiological studies have identified several risk
factors for meningeal tuberculosis; these include age less than 40 years, HIV
infection, and certain ethnic populations. The latter factor suggests significant
interplay between host genetic background and specific strains of mycobacteria
(11). In fact, it has been recently shown association between the development
of tuberculous meningitis and single nucleotide polymorphism in the Toll-interleukin-1
receptor domain containing adaptor protein (TIRAP) and Toll-like receptor (TLR-2)
genes (13).
Our strains are from the Euro-American lineage which was recently reported
to be more pulmonary than meningeal. It has been proposed that Euro–American
strains are less capable of extra pulmonary dissemination due the lack of pks
15/1 intact gene. Pks gene participates in the production of phenolic glycolipid
(PGL), which inhibits the innate immune response and may be responsible for
dissemination and CNS infection. Our Euro–American isolates are unable
to express PGL; however they efficiently disseminate and produced brain infection,
suggesting that other mycobacterial molecules could participate in this process.
We consider mycobacterial heparin-binding haemaglutinin adhesin as a good candidate
because this molecule triggers receptor-mediated bacilli adherence and invasion
to epithelial cells, and extrapulmonary dissemination. Another potential participating
molecule is histone-like protein (HLP), which permits to M. leprae interacts
with laminin on the surface of Schwann cells, facilitating its invasion. HLP
is also expressed by M. tuberculosis, and could participate in the infection
of the nervous cells after endothelial barrier traverse. This study is now
in progress in our laboratory.
The first step required for certain neurotropic microorganisms to cause CNS
infection is to penetrate the BBB. The basic and first element of BBB is microvascular
endothelial cells that differ from those in other tissues by tight junctions
with high electrical resistance and a relatively low number of pinocytotic
vesicles. Recently, an in vitro model using human brain microvascular endothelial
cells showed that the reference strain M. tuberculosis H37Rv can invade and
traverse these cells, using a process that requires active cytoskeleton rearrangements.
By microarray expression profiling, the authors found 33 genes that were overexpressed
during endothelial cells invasion, suggesting that the products of these genes
might participate in this process (10). Perhaps different gene profile could
be expressed by our strains than the laboratory reference strain H37Rv used
in the in vitro system that our experimental model showed limited ability to
infect the brain.
Intravenous infection of C57Bl mice with M.
avium induces brain infection,
and the number of bacteria increases with the duration and level of bacteraemia,
which depend of the inoculum size (7). One of our strains (code 209) was highly
virulent; it induced more rapid animal death and high bacilli burdens in the
lung, liver, spleen, and particularly in the blood. In order to study if hypervirulence
could be related to dissemination and brain infection, we infected animals
with other highly virulent strains isolated from the sputum of patients with
pulmonary tuberculosis. Interestingly, these pulmonary strains produced minimal
brain infection, suggesting that hypervirulence is not always related with
the ability to CNS infection. Moreover, infection of mice with the other CSF-isolated
strains (codes 136 and 28), which efficiently infected the brain, produced
higher or similar mice survival and bacilli loads than infection of mice with
mild, virulent pulmonary strains.
The most distinctive histological features were small or middle size nodules
constituted by lymphocytes and macrophages located in the cerebral parenchyma
near to piamadre or below the ependimal cell layer (Rich’s nodules).
During late infection these nodules were larger and connected with the subarachnoideal
space producing mild or extensive inflammatory infiltrate in meninges. Another
interesting histological observation was the presence of extracellular positive
acid fast bacilli or intracellular in astrocytes or microglia cells without
inflammatory response. Immunohistochemical detection of mycobacterial antigens
showed strong positivity in activated microglia, astrocytes, ependimal cells,
and meningothelial cells, indicating that all these cell types are able to
phagocytose mycobacteria or their debries. Indeed, under physiological conditions,
basal expression of significant innate immunity receptors involved in mycobacteria
phagocytosis (such as TLR-2 and TLR-4) were detected in the meninges, choroids
plexus, and circumventricular brain area, which lack BBB and are more exposed
to pathogens. Microglia and astrocytes can also express these receptors. Thus,
in brain mycobacterial infection specific bacterial antigens and their host
cell receptors, as well as innate immunity receptors are involved.
In many areas where we found strong mycobacterial-antigen immunostaining
in CNS cells, there were not inflammatory infiltrate. Thus, an efficient modulation
of inflammation is produced in the brain which could avoid or delay tissue
damage and signs of neurological lesion, even in the presence of high amount
of bacilli. Th-2 cells could participate in this process, due to its efficient
activity to suppress Th-1 high activity that induces excessive inflammation
and tissue damage. Indeed, the Th-2 response also has beneficial activity for
CNS, favouring healing and supporting neuronal survival. We found progressive
IL-4 expression in the brain of mice infected with either of the three different
clinical strains isolated from the CSF of meningeal tuberculous patients, while
reference strain H37Rv induced low expression of Th-2 cytokine. Another significant
anti-inflammatory cytokine localized in activated microglia is transforming
growth factor beta (TGFβ). We found high gene expression of TGFβ in
the brain of mice infected with CSF clinical isolates, and our histological
studies showed positive immunostaining in macrophages located in Rich’s
nodules, as well as in activated microglia and capillary endothelial cells
from distant areas of the inflammatory response. Thus, neuroprotection and
suppression by specific cytokines should be a significant factor to avoid excessive
inflammation and tissue destruction in mycobacterial CNS infection. This is
in agreement with the observation that during late infection, when high bacilli
loads in the brain were determined, none of the infected mice developed evident
clinical signs of neurological damage, such as seizures or paralysis. The same
situation has been reported in mice infected with high doses of M.
avium by
the intravenous route (7), or even in mice infected directly in the brain (6).
However, we observed significant histological damage in the hippocampus area,
where many neurons showed acidophilic necrosis and extensive gliosis in completely
absence of inflammation. These histological abnormalities should be related
with memory and cognocitive disturbances.
We consider that this experimental model, which demonstrates for the first
time the existence of apparently neurotropic mycobacterial strains, could
be a useful tool to study host and bacilli factors involved in the pathogenesis
of the most severe form of tuberculosis.
References
- Garg RK. Tuberculosis of the central nervous system. Postgrad Med
J. 1999;75(881):133–140.
- Katti MK. Pathogenesis, diagnosis, treatment, and outcome aspects of
cerebral tuberculosis. Med Sci Monit. 2004;10(9):RA215–229.
- Rich AR, McCordock HA. The pathogenesis of tubercular meningitis. Bull
John Hopkins Hosp. 1933;52:5–13.
- Tsenova L, Sokol K, Freedman VH, Kaplan G. A combination of thalidomide
plus antibiotics protect rabbits from mycobacterial meningitis-associated
death. J Infect Dis. 1998;177(6):1563–1572.
- Tsenova L, Bergtold A, Freedman VH, Young RA, Kaplan G. Tumor necrosis
factor alpha is a determinant of pathogenesis and disease progression in
mycobacterial infection in the central nervous system. Proc Natl Acad Sci
USA. 1999;96(10):5657–5662.
- van Well GT, Wieland CW, Florquin S, Roord JJ, van der Poll T, van Furth
AM. A new murine model to study the pathogenesis of tuberculous meningitis.
J Infect Dis. 2007;195(5):694–697.
- Wu HS, Kolonoski P, Chang YY, Bermudez LE. Invasion of the brain and
chronic central nervous system infection after systemic Mycobacterium avium
complex
infection in mice. Infect Immun. 2000;68(5):2979–2984.
- Bolin CA, Whipple DL, Khanna KV, Risdhal JM, Peterson PK, Molitor TW.
Infection of swine with Mycobacterium bovis as a model of human tuberculosis.
J Infect
Dis. 1997;176(6):1559–1566.
- Huang SH, Jong AY. Cellular mechanisms of microbial proteins contributing
to invasion of the blood-brain barrier. Cell Microbiol. 2001;3(5):277–287.
- Jain SK, Paul-Satyassela M, Lamichhane G, Kim KS, Bishai WR. Mycobacterium
tuberculosis invasion and traversal across an in vitro human blood-brain
barrier as a pathogenic mechanism for central nervous system tuberculosis.
J Infect
Dis. 2006;193(9):1287–1295.
- Arvanitakis Z, Long RL, Herschfield ES, Manfreda J, Kabani A, Kunimoto
D, et al. M. tuberculosis molecular variation in CNS infection: Evidence
for strain-dependent neurovirulence. Neurology. 1998;50(6):1827–1832.
- Hernandez Pando R, Aguilar D, Cohen I, Guerrero M, Ribon W, Acosta
P, et al. Specific bacterial genotype of Mycobacterium
tuberculosis cause
extensive
dissemination and brain infection in an experimental model. Tuberculosis
(Edinb). 2010;90(4):268–277.
- Hawn TR, Dustan SJ, Thwaites GE, Simmons CP, Thuong NT, Lan NT, et
al. A polymorphism in Toll-interleukin 1 receptor domain containing adaptor
protein
is associated with suceptibility to meningeal tuberculosis. J Infect Dis.
2006;194(8):1127–1134.
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