Biomedical subjects

Robert J North

Publications and source records attributed to Robert J North.

13 recordsLinked to original sources

PubMed2006

Expression levels of Mycobacterium tuberculosis antigen-encoding genes versus production levels of antigen-specific T cells during stationary level lung infection in mice.

Mycobacterium tuberculosis lung infection in mice was controlled at an approximately stationary level after 20 days of log linear growth. Onset of stationary level infection was associated with the generation by the host of T helper type 1 (Th1) immunity, as evidenced by the accumulation of CD4 Th1 cells specific for the early secretory antigen (ESAT-6) of M. tuberculosis encoded by esat6, and for a mycolyl transferase (Ag85B) encoded by fbpB. CD4 T cells specific for these antigens were maintained at relatively high numbers throughout the course of infection. The number of CD4 T cells generated against ESAT-6 was larger than the number generated against Ag85B, and this was associated with a higher transcription level of esat6. The total number of transcripts of esat6 increased during the first 15 days of infection, after which it decreased and then approximately stabilized at 10(6.5) per lung. The total number of fbpB transcripts increased for 20 days of infection before decreasing and then approximately stabilizing at 10(4.8) per lung. The number of transcripts of esat6 per colony-forming unit of M. tuberculosis fell from 8.6 to 0.8 after day 15, and of fbpB from 0.3 to less than 0.02 after day 10, suggesting that at any given time during stationary level infection the latter gene was expressed by a very small percentage of bacilli. Expressed at an even lower level was an M. tuberculosis replication gene involved in septum formation (ftsZ), indicating that there was no significant turnover of the M. tuberculosis population during stationary level infection.

Acyltransferases↗

PubMed2005

Superior virulence of Mycobacterium bovis over Mycobacterium tuberculosis (Mtb) for Mtb-resistant and Mtb-susceptible mice is manifest as an ability to cause extrapulmonary disease.

Mice of a Mycobacterium tuberculosis-resistant (BALB/c) and of a M. tuberculosis-susceptible (DBA/2) strain proved considerably more susceptible, and equally so, to infection with Mycobacterium bovis than with M. tuberculosis when infection was initiated via the iv route. Infection with M. tuberculosis was eventually controlled at an approximately stationary level in the lungs, livers, spleens and kidneys of BALB/c mice, and in all of these organs except the lungs in DBA/2 mice. M. tuberculosis-infected DBA/2 mice died with a much shorter median survival time (MST) than M. tuberculosis-infected BALB/c mice. By contrast, infection with M. bovis killed mice of both strains with the same and much shorter MST. Unexpectedly, M. bovis caused progressive infection and pathology in the livers of BALB/c mice, but not in this organ in DBA/2 mice. More importantly, this pathogen caused progressive infection and infection-induced pathology in the kidneys and adrenal glands of both strains of mice. It is proposed that disease of the adrenal glands might serve to explain why M. bovis caused mice of both strains to die with the same much shorter MST.

Adrenal Gland Diseases↗

PubMed2005

Changes in energy metabolism of Mycobacterium tuberculosis in mouse lung and under in vitro conditions affecting aerobic respiration.

Transcription profiling of genes encoding components of the respiratory chain and the ATP synthesizing apparatus of Mycobacterium tuberculosis was conducted in vivo in the infected mouse lung, and in vitro in bacterial cultures subjected to gradual oxygen depletion and to nitric oxide treatment. Transcript levels changed dramatically as infection progressed from bacterial exponential multiplication (acute infection) to cessation of bacterial growth (chronic infection) in response to host immunity. The proton-pumping type-I NADH dehydrogenase and the aa3-type cytochrome c oxidase were strongly down-regulated. Concurrently, the less energy-efficient cytochrome bd oxidase was transiently up-regulated. The nitrate transporter NarK2 was also up-regulated, indicative of increased nitrate respiration. The reduced efficiency of the respiratory chain was accompanied by decreased expression of ATP synthesis genes. Thus, adaptation of M. tuberculosis to host immunity involves three successive respiratory states leading to decreased energy production. Decreased bacterial counts in mice infected with a cydC mutant (defective in the cytochrome bd oxidase-associated transporter) at the transition to chronic infection provided initial evidence that the bd oxidase pathway is required for M. tuberculosis adaptation to host immunity. In vitro, NO treatment and hypoxia caused a switch from transcription of type I to type II NADH dehydrogenase. Moreover, cytochrome bd oxidase expression increased, but cytochrome c oxidase expression decreased slightly (nitric oxide) or not at all (hypoxia). These specific differences in respiratory metabolism during M. tuberculosis growth arrest in vitro and in vivo will guide manipulation of in vitro conditions to model bacterial adaptation to host immunity.

Adenosine Triphosphate↗

PubMed2005

Properties and protective value of the secondary versus primary T helper type 1 response to airborne Mycobacterium tuberculosis infection in mice.

Mice immunized against Mycobacterium tuberculosis (Mtb) infection by curing them of a primary lung infection were compared with naive mice in terms of the ability to generate a Th1 cell immune response and to control growth of an airborne Mtb challenge infection. Immunized mice generated and expressed Th1 cell immunity several days sooner than naive mice, as demonstrated by an earlier increase in the synthesis in the lungs of mRNA for Th1 cytokines and for inducible nitric oxide synthase, an indicator of macrophage activation. This Th1 cytokine/mRNA synthesis was accompanied by an earlier accumulation of Mtb-specific Th1 cells in the lungs and the presence of CD4 T cells in lesions. An earlier generation of immunity was associated with an earlier inhibition of Mtb growth when infection was at a 1-log lower level. However, inhibition of Mtb growth in immunized, as well as in naive, mice was not followed by resolution of the infection, but by stabilization of the infection at a stationary level. The results indicate that there is no reason to believe that the secondary response to an Mtb infection is quantitatively or qualitatively superior to the primary response.

Animals↗

PubMed2004

Immunity to tuberculosis.

Only 5 to 10% of immunocompetent humans are susceptible to tuberculosis, and over 85% of them develop the disease exclusively in the lungs. Human immunodeficiency virus (HIV)-infected humans, in contrast, can develop systemic disease that is more quickly lethal. This is in keeping with other evidence showing that susceptible humans generate some level of Th1 immunity to Mycobacterium tuberculosis (Mtb) infection. Tuberculosis in mice is also exclusively a lung disease that is progressive and lethal, in spite of the generation of Th1-mediated immunity. Thus mouse tuberculosis is a model of tuberculosis in susceptible humans, as is tuberculosis in guinea pigs and rabbits. Inability to resolve infection and prevent disease may not be a consequence of the generation of an inadequate number of Th1 cells but of an intrinsic deficiency in macrophage function that prevents these cells from expressing immunity. If this proves to be true, vaccinating susceptible humans against tuberculosis will be a difficult task.

Animals↗

PubMed2003

Susceptibility to tuberculosis: a locus on mouse chromosome 19 (Trl-4) regulates Mycobacterium tuberculosis replication in the lungs.

The mouse DBA/2 (D2) strain is very susceptible to infection with virulent Mycobacterium tuberculosis, whereas C57BL/6 (B6) is much more resistant. Infection of D2 and B6 mice with M. tuberculosis H37Rv by the respiratory route is biphasic: during the first 3 weeks, there is rapid bacterial growth in the lung of both strains, whereas beyond this point replication stops in B6 but continues in D2, causing rapidly fatal pulmonary disease. To identify the genes regulating growth of M. tuberculosis in the lungs of these two strains, 98 informative (B6 x D2) F2 mice were infected by the respiratory route with M. tuberculosis H37Rv (2 x 102 colony-forming units), and the extent of bacterial replication in the lungs at 90 days was used as a quantitative measure of susceptibility in a whole-genome scan. Quantitative trait locus mapping identified a major locus on chromosome 19 (Tuberculosis resistance locus-4, Trl-4; logarithm of odds 5.6), which regulated pulmonary replication of M. tuberculosis and accounted for 25% of the phenotypic variance. B6 alleles at Trl-4 were inherited in an incompletely dominant fashion and associated with reduced bacterial replication. An additional effect of a locus (Trl-3), previously shown to affect survival to i.v. infection with M. tuberculosis, was also noted. F2 mice homozygous for B6 alleles at both Trl-3 and Trl-4 were as resistant as B6 parents, whereas mice homozygous for D2 alleles were as susceptible as D2 parents. These results suggest a strong genetic interaction between Trl-3 and Trl-4 in regulating pulmonary replication of M. tuberculosis.

Animals↗

PubMed2003

Increased interleukin-10 expression is not responsible for failure of T helper 1 immunity to resolve airborne Mycobacterium tuberculosis infection in mice.

With a view to determining whether failure of mice to resolve Mycobacterium tuberculosis (Mtb) infection is a consequence of downregulation of T helper 1 (Th1) immunity by interleukin (IL)-10, mice deleted of the gene for IL-10 were compared with wild-type (WT) mice in terms of their ability to make IL-10 mRNA, generate Th1-mediated immunity [as measured by synthesis of mRNA for interferon-gamma (IFN-gamma)], IL-12p40 and inducible nitric oxide synthase (iNOS), and to control lung infection. It was found that the response of WT mice to infection included a substantial and sustained increase in IL-10 mRNA synthesis in the lungs. A Th1 response in the lungs of WT and IL-10-/- mice was evidenced by a large and sustained increase in the synthesis of mRNA for IFN-gamma, IL-12p40 and iNOS, with somewhat higher levels of these mRNA species being made in the lungs of IL-10-/- mice, particularly at an early stage of infection. However, IL-10-/- mice were no more capable than WT mice at combating infection.

Animals↗

PubMed2003

DNA alkylation damage as a sensor of nitrosative stress in Mycobacterium tuberculosis.

One of the cellular consequences of nitrosative stress is alkylation damage to DNA. To assess whether nitrosative stress is registered on the genome of Mycobacterium tuberculosis, mutants lacking an alkylation damage repair and reversal operon were constructed. Although hypersensitive to the genotoxic effects of N-methyl-N'-nitro-N-nitrosoguanidine in vitro, the mutants displayed no phenotype in vivo, suggesting that permeation of nitrosative stress to the level of cytotoxic DNA damage is restricted.

Alkylation↗

PubMed2002

Expression of Th1-mediated immunity in mouse lungs induces a Mycobacterium tuberculosis transcription pattern characteristic of nonreplicating persistence.

The lung is the primary target of infection with Mycobacterium tuberculosis. It is well established that, in mouse lung, expression of adaptive, Th1-mediated host immunity inhibits further multiplication of M. tuberculosis. Here, real-time RT-PCR was used to define the pattern of expression against time of lung infection of key genes involved in Th1-mediated immunity and of selected genes of M. tuberculosis. Inhibition of bacterial multiplication was preceded by increased mRNA synthesis for IFN-gamma and inducible NO synthase (NOS2) and by NOS2 protein synthesis in infected macrophages. Concurrently, the pattern of transcription of bacterial genes underwent dramatic changes. mRNA synthesis increased for alpha-crystallin (acr), rv2626c, and rv2623 and decreased for superoxide dismutase C (sodC), sodA, and fibronectin-binding protein B (fbpB). This pattern of M. tuberculosis transcription is characteristic of the nonreplicating persistence [Wayne, L. G. & Sohaskey, C. D. (2001) Annu. Rev. Microbiol. 55, 139-163] associated with adaptation of tubercle bacilli to hypoxia in vitro. Based on this similarity, we infer that host immunity induces bacterial growth arrest. In IFN-gamma gene-deleted mice, bacterial growth was not controlled; NOS2 protein was not detected in macrophages; sodC, sodA, and fbpB transcription showed no decrease; and acr, rv2626c, and rv2623 transcription increased only at the terminal stages of lung pathology. These findings define the transcription signature of M. tuberculosis as it transitions from growth to persistence in the mouse lung. The bacterial transcription changes measured at onset of Th1-mediated immunity are likely induced, directly or indirectly, by nitric oxide generated by infected macrophages.

Animals↗

PubMed2002

Virulent but not avirulent Mycobacterium tuberculosis can evade the growth inhibitory action of a T helper 1-dependent, nitric oxide Synthase 2-independent defense in mice.

Control of infection with virulent Mycobacterium tuberculosis (Mtb) in mice is dependent on the generation of T helper (Th)1-mediated immunity that serves, via secretion of interferon (IFN)-gamma and other cytokines, to upregulate the antimycobacterial function of macrophages of which the synthesis of inducible nitric oxide synthase (NOS)2 is an essential event. As a means to understanding the basis of Mtb virulence, the ability of gene-deleted mice incapable of making NOS2 (NOS2(-/-)), gp91(Phox) subunit of the respiratory burst NADPH-oxidase complex (Phox(-/-)), or either enzyme (NOS2/Phox(-/-)), to control airborne infection with the avirulent R1Rv and H37Ra strains of Mtb was compared with their ability control infection with the virulent H37Rv strain. NOS2(-/-), Phox(-/-), and NOS2/Phox(-/-) mice showed no deficiency in ability to control infection with either strain of avirulent Mtb. By contrast, NOS2(-/-) mice, but not Phox(-/-) mice, were incapable of controlling H37Rv infection and died early from neutrophil-dominated lung pathology. Control of infection with avirulent, as well as virulent Mtb, depended on the synthesis of IFN-gamma, and was associated with a substantial increase in the synthesis in the lungs of mRNA for IFN-gamma and NOS2, and with production of NOS2 by macrophages at sites of infection. The results indicate that virulent, but not avirulent, Mtb can overcome the growth inhibitory action of a Th1-dependent, NOS2-independent mechanism of defense.

Animals↗

PubMed2002

Evidence inconsistent with a negative influence of T helper 2 cells on protection afforded by a dominant T helper 1 response against Mycobacterium tuberculosis lung infection in mice.

Mice incapable of generating an efficient Th2 response because of functional deletion of the genes for signal transducer and activation of transcription 6 (Stat6), interleukin-4 receptor alpha chain (IL-4Ralpha), or IL-4 plus IL-13 (IL-4/IL-13) were no more resistant than wild-type (WT) mice to airborne infection with virulent Mycobacterium tuberculosis. WT mice were able to control infection and hold it at a stationary level following 20 days of log linear M. tuberculosis growth. Likewise, infection was kept under control and was held at the same stationary level in IL-4/IL-13(-/-) mice but progressed to a slightly higher level in Stat6(-/-) and IL-4Ralpha(-/-) mice. The onset of stationary-level infection in WT mice was associated with the expression of Th1-mediated immunity, as evidenced by an approximately 100- to 1,000-fold increase in the lungs in the synthesis of mRNA for IL-12, gamma interferon (IFN-gamma), and inducible nitric oxide synthase (NOS2) that was sustained for at least 100 days. IL-12 is essential for the induction of Th1 immunity, IFN-gamma is a key Th1 cytokine involved in mediation of immunity, and NOS2 is an inducible enzyme of macrophages and is needed by these cells to express immunity. In response to infection, the lungs of Stat6(-/-) mice showed increases in synthesis of mRNA for IL-12, IFN-gamma, and NOS2 similar to that seen in WT mice. In IL-4/IL-13(-/-) mice, however, synthesis of mRNA for IFN-gamma and NOS2 reached higher levels than in WT mice. These results argue against the notion that a Th2 response is partly or wholly responsible for the inability of Th1-mediated immunity to resolve infection with a virulent strain of M. tuberculosis.

Animals↗

PubMed2002

Expression of NADPH oxidase-dependent resistance to listeriosis in mice occurs during the first 6 to 12 hours of liver infection.

Wild-type mice inoculated with Listeria monocytogenes intravenously were capable of reducing the bacterial load in their livers by 90% within 6 h. In contrast, mice with deletions of the gene for NADPH oxidase were incapable of expressing this early oxygen-dependent anti-Listeria defense and consequently showed higher levels of liver infection at later times.

Animals↗

PubMed1996

The requirement of tumour necrosis factor-alpha and interferon-gamma for the expression of protective immunity to secondary murine tularaemia depends on the size of the challenge inoculum.

The present study was conducted to determine the extent to which the cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) are required to protect against primary or secondary murine tularaemia caused by the live vaccine strain of the facultative intracellular bacterium Francisella tularensis. It is shown that non-immune mice treated with neutralizing monoclonal antibodies (mAbs) against TNF-alpha and IFN-gamma are rendered defenceless against otherwise sublethal intravenous inocula of the bacterium. Treatment with either of the anti-cytokine mAbs resulted in even a very small inoculum of 500 c.f.u. of the pathogen multiplying unrestrictedly in the livers, spleens and lungs of non-immune mice to rapidly reach lethal numbers. By contrast, Francisella-immune mice treated with either of the mAbs remained capable of resolving secondary infection with 50-fold larger inocula. However, the need for TNF-alpha and IFN-gamma for controlling secondary tularaemia became critical when challenge inocula exceeded 10(6) c.f.u. Overall, the results imply that different defence mechanisms operate to control primary versus secondary murine tularaemia. Additionally, they show that the need for TNF-alpha and IFN-gamma to combat secondary infection depends on the size of the challenge inoculum.

Animals↗