Biomedical subjects

I M Orme

Publications and source records attributed to I M Orme.

At least 19 recordsLinked to original sources

PubMed2003

Activation marker expression on bovine peripheral blood gammadelta T cells during post-natal development and following vaccination with a commercial polyvalent viral vaccine.

Understanding the immunological function of bovine gammadelta T cells is essential for evaluating their role in the response to infectious agents and for determining the potential of targeting this population with vaccines. This study examined the age dependent changes of circulating CD2(+) and CD2(-) gammadelta T cells as well as differences in the expression of activation markers between these two populations. Changes in activation marker expression following vaccination with Vira Shield 5 are also discussed. CD62L was expressed on all CD2(-) gammadelta T cells but only a subset of CD2(+) gammadelta T cells and following vaccination there was a significant decrease in the percentage of CD2(-)/CD62L(+) gammadelta T cells but not CD2(+)/CD62L(+) gammadelta T cells. Both CD2(-) and CD2(+) gammadelta T cells consistently expressed high levels of CD44. The majority of both CD2(-) and CD2(+) gammadelta T cells also expressed CD45R, however, more of the CD2(-) cells were CD45R(neg/lo). Following vaccination there was a significant decrease in the percentage of CD2(-) and CD2(+) gammadelta T cells that expressed CD44 and CD45R. These data indicate significant differences in activation expression on CD2(-) and CD2(+) gammadelta T cells, which adds to the growing evidence that there may be functional as well as phenotypic differences between these two populations of bovine gammadelta T cells.

Age Factors↗

PubMed2002

Dose of BCG does not influence the efficient generation of protective immunity in mice challenged with Mycobacterium tuberculosis.

It is generally agreed that BCG vaccination is relatively ineffective in adults exposed to tuberculosis infection. The reasons for this may well be multiple, and may include the possibility that higher doses of BCG may induce a mixed TH1 and TH2 response, which may lessen the protective effect of the vaccine. To test this hypothesis, mice were vaccinated with a range of doses of BCG and then challenged by the intravenous or aerogenic routes with virulent Mycobacterium tuberculosis. While the data support the hypothesis that a TH2 response is induced by higher doses of BCG, this was found to have no influence whatsoever on the capacity of the vaccinated mouse to express acquired specific resistance to the challenge infection.

Animals↗

PubMed2002

Characterization of virulence, colony morphotype and the glycopeptidolipid of Mycobacterium avium strain 104.

SETTING: Members of the Mycobacterium avium complex (MAC) are responsible for mycobacterial disease in children, the aged and in immunocompromised individuals. The complex consists of different species, serovars and morphologic forms that vary in virulence. One isolate of the MAC is currently being sequenced (MAC 104) and was chosen based on its derivation from an AIDS patient and the fact that it could be genetically manipulated. OBJECTIVE: MAC 104 was therefore analyzed for virulence, colony morphotype and expression of the glycopeptidolipid (GPL) responsible for serotying differences and the rough to smooth morphological switch. RESULTS: The isolate was found to be virulent in the murine model of low-dose aerosol infection in that it could colonize the lung, proliferate within the tissue and disseminate to other organs. MAC 104 expressed a variety of colony morphotypes, the most prevalent of which were smooth opaque, smooth transparent and rough. All three morphotypes could persist in the lung; however, the transparent and rough morphotypes grew more rapidlyinvivo. The rough morphotype was unusual in that it expressed an atypical form of the GPL usually absent from rough morphotypes. CONCLUSION: This characterization complements the genome data and confirms that MAC 104 behaves similarly to other MAC isolates.

Animals↗

PubMed2001

Inflammation and lymphocyte activation during mycobacterial infection in the interferon-gamma-deficient mouse.

Interferon-gamma is a pivotal cytokine in the protective response to tuberculosis. In its absence rampant bacterial growth results in tissue destruction and death. While macrophage activation is key, this pleiotropic cytokine has other secondary but significant roles. To investigate these roles, both intravenous and aerosol infection of the IFN-gamma gene disrupted (GKO) mouse was performed. For the first time we describe the very similar growth of bacteria, during the initial phase of infection, between control and GKO mice. During this initial phase, however, very different histopathologic consequences between control and GKO mice were observed. Key observations included an early increased accumulation of granulocytes and a much more rapid and pronounced interstitial pneumonia in the GKO mice. As infection developed, GKO mice mounted an antigen-specific response; however, lymphocyte activation was much more rapid in these mice. Of interest is the fact that this increased rapidity occurred prior to significant differences in bacterial number. Taken together these data support a role for IFN-gamma in limiting both initial cellular recruitment and acquired lymphocytic responses to mycobacterial infection. This role may be key in surviving the kind of chronic stimulatory disease caused by Mycobacterium tuberculosis.

Animals↗

PubMed2001

The application of proteomics in defining the T cell antigens of Mycobacterium tuberculosis.

The complete sequencing of the Mycobacterium tuberculosis genome offers a unique opportunity to fully elucidate the biology of this human pathogen. One aspect of significant importance is the definition of T cell antigens. This report describes the development and implementation of a proteomic approach to defining such antigens. Large quantities of subcellular protein fractions of M. tuberculosis were resolved by two-dimensional liquid phase electrophoresis (2-D LPE), resulting in 355 and 299 fractions of culture filtrate and cytosolic proteins, respectively. Analysis of these fractions against splenocytes of C57Bl/6 mice infected with M. tuberculosis resulted in the identification of 37 fractions that stimulated a dominant T cell response, as measured by the production of interferon-gamma. Additionally, when the 2-D LPE fractions were assayed against splenocytes harvested at 10 and 40 days post infection significant changes in the T cell response were observed. Molecular characterization of the proteins contained in each of the 38 immunodominant fractions by liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry resulted in the identification of 30 individual proteins. Many of these represented previously defined antigens. However 17 of these proteins were novel T cell antigens. The data presented demonstrate that proteomics offers a rapid and facile approach for elucidation of immunodominant T cell antigens of pathogenic bacteria.

Animals↗

PubMed2001

Tuberculosis in aged gammadelta T cell gene disrupted mice.

In young mice exposed to aerosol infection with Mycobacterium tuberculosis removal of the gammadelta T cell population by targeted gene disruption does not affect the expression of host resistance, but does influence the integrity of the early granulomatous response. The current study demonstrates that in aged gammadelta T cell gene disrupted mice similar immunopathologic changes ensued in both gene knockout and wild type control mice. Changes in cell surface marker expression, evident in other gene knockout models, was not observed in the aged gammadelta T cell knockout mice. These data imply that gammadelta T cell functions previously observed in young mice become much less important as the animal ages.

Aging↗

PubMed2001

The progression of chronic tuberculosis in the mouse does not require the participation of B lymphocytes or interleukin-4.

The aging process is associated with alterations in the immune system. Some of the changes reported are an increase in the proportion of B lymphocytes, and a shift to a TH2-like cytokine environment. It has been hypothesized that the development of immunopathology within the lung during tuberculosis is linked to increased interleukin-4 (IL-4) production. In addition, a role for B cells in maintaining granuloma integrity has been recently proposed. This study investigated the role of B cells and IL-4 during the long-term course of chronic tuberculosis in mice and showed that the course of Mycobacterium tuberculosis infection in the lungs was not influenced by the absence of B lymphocytes or the TH2 cytokine IL-4.

Animals↗

PubMed2001

Tuberculosis vaccine development: recent progress.

Recent years have seen a renewed effort to develop new vaccines against tuberculosis. As a result, several promising avenues of research have developed, including the production of recombinant vaccines, auxotrophic vaccines, DNA vaccines and subunit vaccines. In this article we briefly review this work, as well as consider the pros and cons of the animal models needed to test these new vaccines. Screening to date has been carried out in mouse and guinea pig models, which have been used to obtain basic information such as the effect of the vaccine on bacterial load, and whether the vaccine can prevent or reduce lung pathology. The results to date lead us to be optimistic that new candidate vaccines could soon be considered for evaluation in clinical trials.

Animals↗

PubMed2001

Pentoxifylline treatment of mice with chronic pulmonary tuberculosis accelerates the development of destructive pathology.

It is well established in animal models that production of the cytokine tumour necrosis factor-alpha (TNF-alpha) is essential to the proper expression of acquired specific resistance following infection with Mycobacterium tuberculosis. This gives rise to an apparent state of chronic disease which over the next 100-200 days is characterized by slowly worsening pathological changes in the lung. To determine whether continued TNF-alpha production was harmful during this phase mice were treated with a TNF-alpha inhibitor, pentoxifylline. It was observed that although this therapy did not alter the numbers of bacteria recovered from the lungs of the infected mice, tissue damage within the lung was accelerated. These data thus demonstrate that production of TNF-alpha, already known to be important during the early expression of resistance to tuberculosis, remains important and beneficial during the chronic stage of the disease.

Animals↗

PubMed2001

Immunology and vaccinology of tuberculosis: can lessons from the mouse be applied to the cow?

The nature of both innate and acquired immunity in the lungs are both still poorly understood, and how these two sets of mechanisms intersect with each other may have considerable bearing on the overall expression of host resistance. While the central role of CD4 T cells in the acquired phase is well established, cells bearing this marker may also contribute to innate immunity in the guise of both NK-positive and negative innate populations capable of secreting gamma interferon. In contrast, expansion of an antigen-specific memory T cell population is the purpose of current vaccine strategies, and several interesting and promising types of new candidates are briefly discussed.

Animals↗

PubMed2001

Characterization of murine lung dendritic cells infected with Mycobacterium tuberculosis.

Lung dendritic cells were identified by immunohistochemistry in lung tissue sections from C57BL/6 mice. Following isolation from the lungs using CD11c magnetic beads, the flow cytometric analysis of I-A(b+) and CD11c(+) cells indicated a mixed population of dendritic cells at different stages of maturation, with most expressing an immature phenotype. When cultured for 7 days with recombinant murine granulocyte-macrophage colony-stimulating factor, 99% of cells were CD11c(+) and had a morphology typical of immature dendritic cells. These cells were negative for CD34, CD14, and CD8 alpha antigens but expressed low levels of the myeloid marker F4/80 and moderate levels of MAC3. All expressed high levels of CD11a (LFA-1), CD11b (Mac1), and CD54 antigens, with low levels of class II major histocompatibility complex. Most cells expressed CD80 but only a small percentage of cells were positive for CD40 and CD86. Both overnight and 7-day cultures of lung dendritic cells were able to phagocytose Mycobacterium tuberculosis, and this was associated with the production of interleukin-12 and stimulation of both naïve and immune T cells to produce gamma interferon.

Animals↗

PubMed2001

Temporal and spatial arrangement of lymphocytes within lung granulomas induced by aerosol infection with Mycobacterium tuberculosis.

The progression of the immune response in the lungs after aerosol infection with Mycobacterium tuberculosis is a complex cellular event dominated by macrophages and lymphocytes. Although the phenotype of lymphocytes participating in this response is becoming increasingly well characterized, the dynamic influx of these cells during the infection and their spatial arrangements within the lung tissue are still poorly understood. This study shows that in the first month after aerosol infection with M. tuberculosis there was a steady increase in the percentages of total CD3+, CD3+ CD4+ and CD3+ CD8+ cells, with consistently larger numbers of CD3+ CD4+ cells than of CD3+ CD8+ cells. As granuloma formation continued, the granuloma was found to consist of macrophages, CD4, and CD8 T cells, as well as a smaller number of B cells. Whereas CD4 T cells formed organized aggregates, CD8 T cells were fewer and more scattered and tended to be more prominent toward the periphery of the granulomas. The possible ramifications of the juxtapositions of these two major T-cell subsets are discussed.

Animals↗

PubMed2001

Boosting vaccine for tuberculosis.

An effective new vaccine for the control of tuberculosis is badly needed. While current research attempts to improve vaccination are concentrating on new prophylactic or immunotherapeutic vaccines, virtually no emphasis has been placed on boosting individuals already inoculated with Mycobacterium bovis BCG. It is shown here that mice vaccinated with BCG gradually lose their capacity to resist an aerosol challenge infection as they age. However, if these mice are inoculated with the 30-kDa mycolyl transferase A protein in midlife, after challenge when aged they express levels of protection in the lungs comparable to those of young mice, associated with minimal pathological damage.

Acyltransferases↗

PubMed2001

Immunological basis for reactivation of tuberculosis in mice.

In this study different inbred strains of mice appeared to control and contain a low dose aerosol infection with Mycobacterium tuberculosis in a similar manner, giving rise to a chronic state of disease. Thereafter, however, certain strains gradually began to show evidence of regrowth of the infection, whereas others consistently did not. Using C57BL/6 mice as an example of a resistant strain and CBA/J mice as an example of a strain susceptible to bacterial growth, we found that these animals revealed distinct differences in the cellular makeup of lung granulomas. The CBA/J mice exhibited a generally poor lymphocyte response within the lungs and vastly increased degenerative pathology at a time associated with regrowth of the infection. As a possible explanation for these events, it was then observed that the CBA/J mouse strain was also less able to upregulate adhesion molecules, including CD11a and CD54, on circulating lymphocytes. These results therefore suggest that a failure to control a chronic infection with M. tuberculosis may reflect an inability to localize antigen-specific lymphocytes within the lung.

Animals↗

PubMed2001

Cu,Zn superoxide dismutase of Mycobacterium tuberculosis contributes to survival in activated macrophages that are generating an oxidative burst.

Macrophages produce reactive oxygen species and reactive nitrogen species that have potent antimicrobial activity. Resistance to killing by macrophages is critical to the virulence of Mycobacterium tuberculosis. M. tuberculosis has two genes encoding superoxide dismutase proteins, sodA and sodC. SodC is a Cu,Zn superoxide dismutase responsible for only a minor portion of the superoxide dismutase activity of M. tuberculosis. However, SodC has a lipoprotein binding motif, which suggests that it may be anchored in the membrane to protect M. tuberculosis from reactive oxygen intermediates at the bacterial surface. To examine the role of the Cu,Zn superoxide dismutase in protecting M. tuberculosis from the toxic effects of exogenously generated reactive oxygen species, we constructed a null mutation in the sodC gene. In this report, we show that the M. tuberculosis sodC mutant is readily killed by superoxide generated externally, while the isogenic parental M. tuberculosis is unaffected under these conditions. Furthermore, the sodC mutant has enhanced susceptibility to killing by gamma interferon (IFN-gamma)-activated murine peritoneal macrophages producing oxidative burst products but is unaffected by macrophages not activated by IFN-gamma or by macrophages from respiratory burst-deficient mice. These observations establish that the Cu,Zn superoxide dismutase contributes to the resistance of M. tuberculosis against oxidative burst products generated by activated macrophages.

Animals↗

PubMed2001

CD8- and CD95/95L-dependent mechanisms of resistance in mice with chronic pulmonary tuberculosis.

The role of CD8 T lymphocytes in the immune response to Mycobacterium tuberculosis infection remains enigmatic, with persuasive reports of both cytolytic and noncytolytic (cytokine-mediated) responses to infection. To address the importance of the cytolytic mechanisms, mice with targeted disruptions for CD8 and perforin or with gene mutations in the CD95/ CD95L signaling pathway were exposed to pulmonary infection. All mice tested showed no differences in their ability to contain the growth of infection during the early phase of disease. As the chronic phase of the disease ensued, however, both CD8- and CD95/CD95L-deficient mice gradually lost their ability to limit bacterial growth. This was associated with a tendency toward pyogenic inflammation in the lung. This tendency was not seen in the perforin gene-disrupted mice. In CD8 gene-disrupted mice, the ability to generate interferon-gamma secreting T cells was unimpaired. Although these cells were capable of entering the lung they were unable to influence the increasing bacterial load in this organ.

Animals↗

PubMed2001

The search for new vaccines against tuberculosis.

The failure of the BCG vaccine for tuberculosis in large, controlled clinical trials, coupled with the gradual consensus that it is mostly ineffective in preventing adult pulmonary disease in endemic areas, has led to a concerted effort to develop a new generation of vaccines. This work is ongoing in a variety of areas, including DNA vaccines, subunit vaccines, recombinant vaccines, and auxotrophic vaccines. Several such candidates are giving promising results in mouse and guinea pig, aerosol-challenge infection models and should move to clinical trials in the near future.

Animals↗

PubMed2000

T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective antigen associated with the early control of infection.

Infection of C57BL/6 mice with Mycobacterium tuberculosis results in the development of a progressive disease during the first 2 wk after challenge. Thereafter, the disease is controlled by the emergence of protective T cells. We have used this infection model in conjunction with direct T cell expression cloning to identify Ags involved with the early control of the disease. A protective M. tuberculosis-specific CD4 T cell line derived from mice at 3 wk postchallenge was used to directly screen an M. tuberculosis genomic expression library. This screen resulted in the identification of a genomic clone comprising two putative adjacent genes with predicted open reading frames of 10 and 41 kDa, MTB10 and MTB41, respectively (the products of Rv0916c and Rv0915c, respectively, in the TubercuList H37Rv database). MTB10 and MTB41 belong to the PE and PPE family of proteins recently identified to comprise 10% of the M. tuberculosis genome. Evaluation of the recombinant proteins revealed that MTB41, but not MTB10, is the Ag recognized by the cell line and by M. tuberculosis-sensitized human PBMC. Moreover, C57BL/6 mice immunized with MTB41 DNA developed both CD4- (predominantly Th1) and CD8-specific T cell responses to rMTB41 protein. More importantly, immunization of C57BL/6 mice with MTB41 DNA induced protection against infection with M. tuberculosis comparable to that induced by bacillus Calmette-Guérin. Thus, the use of a proven protective T cell line in conjunction with the T cell expression cloning approach resulted in the identification of a candidate Ag for a subunit vaccine against tuberculosis.

Animals↗