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,§
*
Departments of Molecular Genetics and Biochemistry, and Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; and Departments of
Pathology,
Medicine, and
§
Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10467
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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, TNF-
, and inducible nitric oxide
synthase were all expressed throughout the latent phase of infection.
Reactivation of latent tuberculous infection by aminoguanidine
treatment was confirmed using a second murine tuberculosis model based
on treatment with antimycobacterial drugs. Results obtained using this
drug-based model also suggested the existence of an RNI-independent
antimycobacterial mechanism(s) operative in the latent phase of
infection. Together, these data suggest that both RNI-dependent and
-independent mechanisms contribute to the prevention of tuberculous
reactivation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The basis for the reactivation theory is largely derived from clinical studies of tuberculosis (reviewed in Refs. 2 and 4). In chronic tuberculosis, the apical lung segments are by far the most common sites of active disease (2). Remarkably, apical scarring could be detected in a significant number of older persons long before the development of active pulmonary tuberculosis (4). These clinical data support the concept that chronic tuberculosis in adults, particularly the older population, is generally caused by reactivation of viable bacilli harbored in dormant foci in the apexes of the lung (2, 4). More importantly, clinical pathologic studies have shown that apical scars in humans without active tuberculosis contain cultivable tubercle bacilli, and such areas of pulmonary fibrosis can progress to active disease (2, 4).
It is well established that cell-mediated immunity is critical in host defense against M. tuberculosis (reviewed in 5 . From clinical evidence, it is generally accepted that suppression of cellular immunity associated with corticosteroid therapy can result in reactivation of latent tuberculous infection (4, 6). The correlation between cell-mediated immunity and tuberculosis is, however, best illustrated by the remarkable susceptibility of individuals with AIDS to M. tuberculosis (7, 8, 9). Since reactivation plays a significant role in the pathogenesis of tuberculosis in HIV-infected persons, whose T cell-mediated immune responses are severely compromised, it is likely that attenuation of cell-mediated antimycobacterial mechanisms can lead to recrudescence of latent tuberculous infection.
A potent cell-mediated antimycobacterial mechanism is effected via
macrophage inducible nitric oxide synthase
(iNOS)3 production of toxic
reactive nitrogen intermediates (RNI). This
L-arginine-dependent antimicrobial mechanism of macrophages
has been established to be effective against M. tuberculosis
both in vitro (10, 11) and in vivo (12, 13, 14). Thus, inhibition of iNOS
function by chemical inhibitors such as
NG-monomethyl L-arginine (NMMA) or
aminoguanidine (AG) during acute M. tuberculosis infection
led to fulminant and rapidly fatal disease progression associated with
heavy bacterial burden in the lung, liver, and spleen (12). In
addition, disruption of the function of TNF- and IFN-, the two
cytokines critical for signaling RNI production by macrophages,
compromised host defense against M. tuberculosis. Mice with
disruptions in the genes for IFN- or the 55-kDa TNF receptor were
unable to produce RNI early in infection and quickly succumbed to
M. tuberculosis infection (13, 14). Although the importance
of these immune responses for control of acute tuberculous infection
has been established, the contribution of macrophage antimicrobial RNI
production to containing latent tuberculous infection has not been
examined. Using two murine experimental tuberculosis models, the
present study provides evidence suggesting that RNI play a significant
role in the prevention of tuberculousreactivation. In addition, the
results suggest the existence of an RNI-independent antimycobacterial
mechanism(s) that participates in the containment of latent
tuberculosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Eight- to ten-week-old C57BL/6 female mice (The Jackson Laboratory, Bar Harbor, ME) were maintained in specific pathogen-free facilities. All experiments were performed in biosafety level 3 animal laboratories.
Mycobacteria and infection
M. tuberculosis strain Erdman was prepared and maintained as previously described (10, 13, 14). Bacterial stocks, harvested from tissues of infected mice and expanded once (first passage), were stored at -80°C until use. Infection of mice was achieved i.v., via tail vein, at a dose of 5 x 103 to 1 x 104 viable CFU. For the low dose model, AG (2.5%, w/v) was given ad libitum in drinking water (12) beginning 6 mo postinfection. For the antimycobacterial drug-based model, mice were treated for 1 mo with isoniazid (0.1 g/l) and pyrazinamide (15 g/l) in drinking water ad libitum beginning 4 wk postinfection. AG treatment (2.5%, w/v, in drinking water) was initiated 1 wk after completion of antibiotic therapy. In both models, M. tuberculosis-infected control mice received plain drinking water. During the course of the study, moribund animals were killed to avoid suffering and were scored as succumbing to M. tuberculosis infection.
Chemicals and reagents
All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). 7H9 medium and 7H10 agar substrates were obtained from Difco (Detroit, MI). Reagents for histopathologic studies and the RT-PCR were purchased from Vector Laboratories, Inc. (Burlingame, CA), and Life Technologies (Gaithersburg, MD), respectively.
Quantitation of viable bacilli in infected tissues
Portions (25%) of the lung, liver, and spleen of killed mice were homogenized in PBS containing 0.05% Tween-80 (12, 13, 14). Dilutions of organ homogenates were plated on 7H10 agar plates, and CFU were counted following 21-day incubation at 37°C. There were three to five mice per group.
Histopathologic studies
Tissue samples for histopathologic studies were prepared as previously described (12, 13, 14). Briefly, tissues were fixed in 10% buffered formalin before paraffin embedment. Hematoxylin and eosin and Ziehl-Neelsen acid fast staining of serial sections from paraffin blocks was examined to assess pathologic changes and bacillary load, respectively. The number and size of granulomas in five to seven random 10x fields was determined for each liver sample studied. The extent of granulomatous infiltration in infected liver tissues was assessed by the Cell Analysis Systems Micrometer version 1.0 program (Elmhurst, IL) according to the manufacturer’s instructions and was expressed as the granulomatous index, which indicates the ratio of area occupied by granulomas to the total area examined.
Immunohistochemical staining
Formalin-fixed, paraffin-embedded tissues were used for immunohistochemical studies by the method of microwave Ag retrieval (15, 16). Preliminary studies have shown that this method yields the best results for staining by an affinity-purified rabbit polyclonal Ab against murine macrophage iNOS (provided by Dr. Charles Lowenstein, Johns Hopkins School of Medicine, Baltimore, MD) (14). Paraffin-embedded tissues were sectioned at 5 to 6 µm, deparaffinized, and subjected to microwave treatment in citrate buffer (pH 6.0) before immunostaining (15, 16). Anti-iNOS Abs were applied at a dilution of 1/100 for 4 h and detected using the avidin-biotin complex-based method according to the supplier’s instructions (Vector Laboratories); peroxidase-diaminobenzidine was used for development. The sections were counterstained with hematoxylin.
RT-PCR for IFN-, TNF-, and iNOS gene expression in tissues
Expression of IFN-, TNF-, and iNOS in lung tissue of mice
was examined by quantitative competitive RT-PCR as described previously
(14, 17). Fresh lung tissue (three mice were analyzed for each time
point) was flash-frozen in liquid nitrogen and stored at -80°C until
use. RNA was prepared by homogenizing frozen tissue in Trizol according
to the manufacturer’s protocol and treated with DNase to remove any
contaminating DNA. RNA (5 µg) was reverse transcribed using
SuperScript II (Life Technologies, Grand Island, NY). Quantitative
competitive RT-PCR was performed as described previously (14, 17).
Briefly, cDNA was standardized with primers for hypoxanthine-guanine
phosphoribosyl transferase using a competitor plasmid that contains
sequences for a number of different genes (18). Following
standardization, equivalent amounts of cDNA were used in competitive
PCR, using primers for each gene of interest (IFN-, TNF-, and
iNOS) (18). PCR products were resolved on agarose gels and visualized
with ethidium bromide.
Statistical analysis
To ensure the normality assumption of the analysis, spleen, liver, and lung CFU were transformed using log base 10. These datasets were subjected to analysis by SAS program version 6.12 (SAS Institute, Cary, NC), using the general linear model.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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There are basically two murine models that have been described for
the study of latent tuberculosis. The first involves a relatively low
dose M. tuberculosis infection (the low dose model) that is
solely controlled by the host’s immune response and remains quiescent
for 15 to 18 mo, at which time reactivation occurs, presumably due to
the immunocompromised state associated with aging (19). The second
model uses a short course of antituberculous drugs (the drug model) to
reduce the bacterial burden to a very low or undetectable level in the
infected host (20, 21, 22, 23, 24); the infection in these mice can reactivate upon
cessation of antibiotics (20, 21, 22, 23, 24) or upon treatment with
immunosuppressive drugs such as glucocorticoid (22, 23, 24). Although
neither is an exact replica of latent tuberculosis in man, each model
possesses attributes that mimic human latency. In the low dose model,
the host immune response is solely responsible for controlling the
initial infection and maintaining the bacterial load at a stable level
for many months, a situation similar to that in human latent
tuberculosis. The weakness of this model is the relatively high
bacillary burden in the spleen and lung of infected mice (Fig. 1
). By contrast, the very low bacterial
load in the organs of infected animals in the drug model is more akin
to that in human latency; however, the effect of antibiotics on the
initial host-bacterial interaction may be a confounding factor. Given
that no true animal latency model exists, both the low dose and the
drug-based models were used in this study to elucidate the mechanisms
involved in the containment of latent tuberculosis.
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A) indicated that the liver is the most resistant
organ to M. tuberculosis, and the lung is the most
susceptible, as previously noted (25). Most importantly, there is an
extended period, beginning 1 mo postinfection and extending for at
least 10 mo, during which the infected host appears clinically healthy,
and the bacillary burden is stably maintained. We reasoned that this is
a suitable phase of infection in which to study latent tuberculosis.
Indeed, in a related model, studies performed to examine the host-
bacterium equilibrium 3 to 6 mo after infection with virulent M.
tuberculosis provided evidence suggesting that during this period,
bacilli were viable but not multiplying in the tissues (26). For the
drug model, C57BL/6 mice were infected as described above. Four weeks
following infection, mice received a 1-mo course of antimycobacterial
therapy with isoniazid and pyrazinamide. This treatment resulted in the
reduction of bacterial burden in all tissues examined to 
200
bacilli/organ (see below). This low level of bacillary load was
maintained for at least 6 mo after completion of antimycobacterial
treatment. The NOS inhibitor AG reactivates latent tuberculosis
The NOS inhibitor AG was used as a pharmacologic probe (12) to assess the role of toxic nitrogen oxides in localizing M. tuberculosis during the quiescent phase of infection. AG was used in this study because it is the most cost-effective of the various commonly used iNOS inhibitors. More importantly, by comparing the effects of AG on murine tuberculosis with those of the chemically distinct NMMA, we have previously established the validity of using either one of these NOS inhibitors in investigating the role of RNI in defense against M. tuberculosis infection in mice (12). One advantage of NMMA is that it allows correlation of in vivo RNI production to disease progression, because this NOS inhibitor has the capacity to abrogate virtually all RNI production in acute murine tuberculosis (12). By contrast, we and others (12, 27, 28) have demonstrated that reduction of nitrogen oxide production in vivo by AG can be incomplete, probably because of the relatively selective effect of AG against the inducible form of NOS. However, the cost of NMMA prohibited its use in these lengthy experiments.
M. tuberculosis-infected mice treated with AG at 6 mo
postinoculation developed reactivated tuberculosis in the low dose
model. This AG-induced reactivation was associated with progressively
fatal disease (mean survival time of 62 ± 2 days post-AG
treatment) as well as increased bacillary burden compared with those in
control mice (Fig. 1B), all of which survived the
experiment (12 mo).
Interestingly, as is well described for acute murine tuberculosis, the
kinetics of bacillary proliferation in the lung, liver, and spleen of
the AG-reactivated mice followed distinct patterns (Fig. 1B). The effect of AG-induced reactivation on
bacterial burden is most prominent in the lung. The kinetics of
pulmonic mycobacterial multiplication were progressive, with CFU
reaching as high as 109 at later time points (1000-fold
more than that in the lung of control mice). In contrast, the number of
hepatic CFU in AG-treated mice did not increase until 8 wk post-AG
treatment, suggesting that the liver is relatively resistant to
reactivated M. tuberculosis infection. The kinetics of
reactivation observed in the spleen followed yet another pattern.
Unlike the rapidly progressive expansion of CFU seen in the lung,
splenic bacterial multiplication followed a much slower course, even
though the bacillary loads of these two organs at the time of
initiation of AG treatment were comparable.
AG treatment also evoked pathologic responses closely resembling those
of an acute infection. Significantly, as early as 2 wk after initiation
of AG treatment, mice in the latent phase of infection developed
hepatosplenomegaly, a characteristic finding in acute tuberculosis. The
most prominent component of these host responses was the granulomatous
reaction, the hallmark of tuberculosis. Upon AG treatment, the
granulomas enlarged and became more structured and cellular (Fig. 2, A–D). In tissues
obtained from infected mice treated with AG, the area occupied by
granulomas far exceeded that in untreated controls (Table I). This AG-induced granulomatous
reaction was most apparent in the liver and least apparent in the lung.
The discrepancy is probably related to the numbers of bacilli in the
tissues studied during the latent phase of infection. To contain the
infection foci in the lung, which carried a higher bacillary load
compared with that of the liver (Fig. 1A), a
relatively vigorous tissue response was maintained, even at the
quiescent state of infection. As a result, augmentation of this degree
of tissue reaction secondary to AG-induced reactivation may not be
readily appreciated. Nevertheless, the effect of AG on the
granulomatous reaction in the pulmonic tissues of mice with quiescent
disease became obvious at later time points: by 10 wk post-AG
treatment, lung granulomas of AG-reactivated mice had progressed
remarkably(Fig. 2F), and on examining multiple tissue
sections, there was evidence of necrosis. In contrast, the vigorous
AG-induced granulomatous reaction in the liver, which suggests hepatic
recrudescence of tuberculosis, began to subside at 6 wk post-treatment
(Fig. 2, C and D). This latter observation
suggests that the liver is uniquely resistant to M.
tuberculosis during the reactivated phase of infection. Together,
these results provide evidence that in the low dose model, RNI may play
a role in the containment of latent tuberculosis.
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, as measured by ELISA, following in vitro
stimulation with Con A and PPD (data not shown). These results suggest
that the AG-induced reactivation of latent tuberculosis is not due to
suppression of the T cell response. Rather, AG induces recrudescence of
quiescent disease via direct suppression of RNI production by the
macrophage L-arginine-dependent antimicrobial
mechanism. Expression of iNOS in latent and reactivated tuberculosis
To further characterize the role of RNI in the containment of
M. tuberculosis in the low dose model, tissue expression of
iNOS was assessed by immunohistochemical studies using an
affinity-purified polyclonal Ab against murine macrophage iNOS (14).
The results of these studies revealed that iNOS is expressed in all
latently infected tissues examined, suggesting that RNI are necessary
for the control of latent M. tuberculosis infection (Fig. 3).
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). This AG-induced, reactivation-associated increase in
iNOS expression was most apparent in the liver (Fig. 3, A–D). While enhancement of iNOS expression in the
liver was remarkable after 2 wk of AG treatment, that in the lung was
apparent only at later times (Fig. 3). This discrepancy may be due to
the higher baseline level of iNOS expression in the lung compared with
that in the liver during the latent phase of infection, thus rendering
a further increase in pulmonic expression not readily apparent.
Interestingly, at 6 wk after initiation of AG treatment, when the
regression of hepatic granulomatous reaction began (Fig. 2, C and D), iNOS expression in the liver was
also noted to start decreasing (Fig. 3). These results further
suggest that the liver is unique in its ability to adequately control
AG-reactivated tuberculosis in the low dose model. Since AG is
effective in attenuating RNI production in vivo in various animal
models of infectious diseases, including tuberculosis (12, 28), the
relative resistance of the liver to M. tuberculosis in the
reactivation phase of the infection may be due to macrophage
iNOS-independent antimycobacterial mechanisms.
Finally, the expression of iNOS during the quiescent phase of infection
in the low dose model of latent tuberculosis is further reinforced by
the detection of iNOS mRNA in latently infected tissue for up to at
least 8 mo postinfection by competitive RT-PCR analysis (Fig. 4).
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IFN- and TNF- are the key factors that activate the
macrophage RNI-generating pathway (29) and are essential in the control
of acute tuberculous infection (13, 14, 30). Therefore, we evaluated
the expression of these cytokines in the quiescent phase of tuberculous
infection. Examining the lung tissue of infected mice, results obtained
by RT-PCR indicated that both IFN- and TNF- are expressed
throughout the quiescent phase of tuberculous infection (Fig. 4),
suggesting that these cytokines participate in the control of latent
tuberculosis at least in part by maintaining the RNI-generating pathway
in an activated state. Indeed, it was shown recently that
neutralization of TNF- by administration of an adenovirus vector
expressing the extracellular domain of the human p55 TNF receptor
exacerbated acute and chronic M. tuberculosis infection in
mice (31). In summary, the results demonstrating the expression of
iNOS, IFN-, and TNF- in latently infected tissues together with
the ability of AG to trigger clinical reactivation, as manifested by
rapid development of hepatosplenomegaly, a marked granulomatous
response, and increased bacillary burden, strongly suggest that
continuous production of RNI at latent foci of infection plays an
important role in preventing tuberculous reactivation.
AG treatment also reactivates infection in the antimycobacterial drug
model of latent tuberculosis. To more stringently test the role of RNI
in preventing tuberculous reactivation, we examined the effect of AG on
mice latently infected with M. tuberculosis using the drug
model. As in the case of the low dose model, AG treatment resulted in
increased bacterial growth in the lung, liver, and spleen (Fig. 5). Again, the largest increase in
bacillary burden was seen in the lung, where AG treated mice had a
1000-fold more viable CFU than controls by 85 days post-treatment (Fig. 5). The bacterial burden in control mice was maintained at
102 to 103 CFU/organ during the study period
(24 wk). Interestingly, after an initial rise in pulmonary CFU to
105/organ, AG-treated mice in the drug model were able
to stably maintain this bacillary burden for >80 days (Fig. 5). By
contrast, this ability of the lung to apparently stabilize bacterial
multiplication was not seen in the low dose model. In the low dose
model, AG-treated mice could not control disease progression, resulting
in an increase in pulmonary CFU from 6 x 105 to
109 within 80 days after AG treatment was initiated (Fig. 1). These data suggest the existence of an iNOS-independent
antimycobacterial mechanism(s) that may contribute to the control of
latent murine tuberculosis. The recent in vitro demonstration that
various strains of M. tuberculosis may vary widely in their
susceptibility to the toxic effects of RNI provides indirect evidence
that these latter mechanisms exist (32, 33). Given the wide array of
redundant host defense strategies, the existence of an RNI-independent
antimycobacterial mechanism(s) operative in the control of latent
tuberculosis should not be difficult to envision. Although not yet
defined, these iNOS-independent mechanisms may be related to IFN-
and TNF-, given the fact that these two cytokines, which are known
to play critical roles in host defense against M.
tuberculosis, are expressed throughout the quiescent phase of
tuberculous infection.
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The macrophage L-arginine-dependent cytotoxic
pathway mediates a major antimycobacterial mechanism in acute murine
tuberculosis via the generation of toxic RNI (10, 11, 12, 13, 14). Importantly,
iNOS has been shown to be highly expressed in bronchial alveolar
macrophages obtained from pulmonary lavage fluids of patients infected
with M. tuberculosis (34), suggesting a role for RNI in host
defense against the tubercle bacillus in humans. Directly relevant to
this study, the NOS inhibitor
N6-(1-iminoethyl)-L-lysine has been
shown to induce tubercular progression when administered to mice 1 mo
after infection with 105 CFU of the virulent Erdman strain
of M. tuberculosis (35). Although not confirmed by the drug
model, these latter results support the evidence provided by the
present study suggesting that RNI contribute to the control of latent
tuberculosis. In addition, macrophage iNOS has been demonstrated to
participate in controlling latent murine leishmaniasis (36). Using the
two models of latent murine tuberculosis currently available, results
from the present study suggest that both RNI-dependent and -independent
antimycobacterial mechanisms contribute to the prevention of disease
reactivation. Detailed analyses of the low dose model indicated that
the three critical factors whose concerted actions culminate in the
generation of NO and related nitrogen oxides, IFN-, TNF-, and
iNOS, are all expressed during the latent phase of tuberculous
infection. Attenuation of the production of mycobacteriocidal RNI leads
to reactivation of latent infection, evidenced by increases in
bacillary burden and mortality. This reactivation phase is associated
with pathologic findings reminiscent of acute disease,
hepatosplenomegaly and a vigorous granulomatous reaction, with
concomitant enhanced expression of the RNI-generating enzyme iNOS.
These results indicate that attenuation of RNI production in latent
tuberculosis results in disease recrudescence. The role of RNI in
controlling latent tuberculous infection is confirmed using the
drug-based model. Interestingly, data derived from the drug model
suggest that an iNOS-independent antimycobacterial mechanism(s) also
contributes to the prevention of reactivation (Figs. 1 and 5). Thus,
the two models of murine latent tuberculosis employed in this study may
afford a useful system for comparative analysis of antimycobacterial
functions operative during latent tuberculous infection. Because of the
lack of true latent animal tuberculosis, these two models, which, in
the strictest sense, may only represent a chronic persistent form of
tuberculous infection, are an approximation of human latency at best.
Therefore, the significance of the observations of this study to human
latent tuberculosis remains to be rigorously tested.
Although we have demonstrated that AG is a suitable iNOS inhibitor for examining the role of RNI in murine tuberculosis (12), its known biologic targets other than iNOS should not be dismissed lightly. These include the potential of AG to interfere with the metabolism of immunoregulatory polyamines (37) and to chemically modulate certain ligands for the macrophage scavenger receptor (38). However, the significance of these attributes of AG in vivo is unclear. Indeed, AG probably does not significantly affect the intracellular balance of polyamines in vivo, since there is evidence that a compensatory increase in excretion of unmodified polyamines occurs during AG administration, thereby preventing intracellular accumulation (39). The relevance of the ability of AG to modify the ligands for macrophage scavenger receptors is no clearer. In hyperglycemic states, such as in a diabetes model, AG acts as an inhibitor of the formation of advanced glycosylation end products (AGEs) (40). The AGEs are modified biologic proteins, including certain ligands for macrophage scavenger receptors, whose formation is accelerated in the presence of high glucose levels. The ability of AG to inhibit the formation of AGEs in a nonhyperglycemic system, such as our murine latent tuberculosis models, is entirely unknown. Regardless of the in vivo significance of these side effects of AG and other NOS inhibitors, the caveats of using NOS inhibitors as a means to attenuate NO production in any biologic system are noteworthy.
Latent tuberculous infection plays a significant role in the pathogenesis of M. tuberculosis (2, 4), a pathogen that causes 3 million deaths annually worldwide (41, 42). Given that one-third of the world’s population is currently infected with M. tuberculosis, hosts chronically harboring tubercle bacilli in the latent phase constitute a significant reservoir of tuberculosis. These individuals can serve as a source of disease dissemination when reactivation of latent infection occurs. Understanding the mechanisms involved in the development of latent and reactivation tuberculosis is, therefore, of paramount importance to the prevention, treatment, and control of tuberculosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. John Chan, Departments of Medicine and Microbiology and Immunology, Montefiore Medical Center, Albert Einstein College of Medicine, 111 East 210th St., Bronx, NY 10467. E-mail address: ; or Dr. JoAnne L. Flynne, Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Biomedical Science Tower, Room E1240, Pittsburgh, PA 15261. E-mail address:
3 Abbreviations used in this paper: iNOS, inducible nitric oxide synthase; RNI, reactive nitrogen intermediates; NMMA, NG-monomethyl L-arginine; AG, aminoguanidine; PPD, purified protein derivative; AGE, advanced glycosylation end products; H&E, hematoxylin and eosin.
Received for publication July 9, 1997. Accepted for publication October 10, 1997.
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C. G. Feng, D. Jankovic, M. Kullberg, A. Cheever, C. A. Scanga, S. Hieny, P. Caspar, G. S. Yap, and A. Sher Maintenance of Pulmonary Th1 Effector Function in Chronic Tuberculosis Requires Persistent IL-12 Production J. Immunol., April 1, 2005; 174(7): 4185 - 4192. [Abstract] [Full Text] [PDF]
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V. Venketaraman, Y. K. Dayaram, M. T. Talaue, and N. D. Connell Glutathione and Nitrosoglutathione in Macrophage Defense against Mycobacterium tuberculosis Infect. Immun., March 1, 2005; 73(3): 1886 - 1889. [Abstract] [Full Text] [PDF]
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J. M. Tufariello, W. R. Jacobs Jr., and J. Chan Individual Mycobacterium tuberculosis Resuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo Infect. Immun., January 1, 2004; 72(1): 515 - 526. [Abstract] [Full Text] [PDF]
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T. Botha and B. Ryffel Reactivation of Latent Tub |
