We analyzed the gene expression profile under specific conditions during reversible transition of M. tuberculosis cells to the "non-culturable" (NC) state in a prolonged stationary phase. More than 500 genes were differentially regulated, while 238 genes were upregulated over all time points during NC cell formation. Approximately a quarter of these upregulated genes belong to insertion and phage sequences indicating a possible high intensity of genome modification processes taking place under transition to the NC state. Besides the high proportion of hypothetical/conserved hypothetical genes in the cohort of upregulated genes, there was a significant number of genes belonging to intermediary metabolism, respiration, information pathways, cell wall and cell processes, and genes encoding regulatory proteins. We conclude that NC cell formation is an active process involved in the regulation of many genes of different pathways. A more detailed analysis of the experimental data will help to understand the precise molecular mechanisms of dormancy/latency/persistence of M. tuberculosis in the future. The list of upregulated genes obtained in this study includes many genes found to be upregulated in other models of M. tuberculosis persistence. Thirteen upregulated genes, which are common for different models, can be considered as potential targets for the development of new anti-tuberculosis drugs directed mainly against latent tuberculosis.
The role of the alternative sigma factor ςB in Staphylococcus epidermidis was investigated by the construction, complementation, and characterization of a sigB deletion mutant. Electrophoretic analyses confirmed a profound influence of ςB on the expression of exoproteins and cytoplasmic proteins. Detailed investigation revealed reduced lipase and enhanced protease activity in the ςB mutant. Furthermore, no significant influence of ςB on heterologous biofilm formation or on the activity of the global regulator agr was detected.
The intracellular parasite Toxoplasma gondii has the capacity to persist in the brain within neurons. In this study we demonstrated that T. gondii infected murine cerebellar neurons in vitro and replicated within these cells. Stimulation with gamma interferon (IFN-γ) and/or tumor necrosis factor (TNF) did not enable neurons to inhibit parasite invasion and replication. Cultured neurons constitutively produced interleukin 1 (IL-1), IL-6, macrophage inflammatory protein 1α (MIP-1α), and MIP-1β but not transforming growth factor β1 (TGF-β1), IL-10, and granulocyte-macrophage colony-stimulating factor. Neuronal expression of some cytokines (IL-6, TGF-β1) and chemokines (MIP-1β) was regulated by infection and/or by IFN-γ and TNF.
Onchocerca volvulus is a human pathogenic filarial parasite which, like other parasitic nematodes, is capable of surviving in an immunologically competent host by employing a variety of immune evasion strategies and defense mechanisms including the detoxification and repair mechanisms of the glutathione S-transferases (GSTs). In this study we analyzed the glycosylation pattern and the immunological properties of extracellular O. volvulus GST1a and -1b (OvGST1a and -1b). The enzymes differ in only 10 amino acids, and both are glycoproteins that have cleavable signal peptides and unusual N-terminal extensions. These characteristics have not been described for other GSTs so far. Mass spectrometry analyses indicate that both enzymes carry high-mannose type oligosaccharides on at least four glycosylation sites. Glycosylation sites 1 to 3 of OvGST1a (OvGST1b sites 2 to 4) are occupied by truncated N-glycans (Man2GlcNAc2 to Man5GlcNAc2), and N glycosylation site 4 of OvGST1a (OvGST1b site 5) carries Man5GlcNAc2 to Man9GlcNAc2. To analyze the capacity of these secretory GSTs to stimulate host immune responses, we studied the antibody responses of onchocerciasis patients against the native affinity-purified OvGST1a and -1b. By enzyme-linked immunosorbent assay we showed that OvGST1a and -1b are immunodominant antigens, with less than 7% nonresponder patients. A direct comparison of the antibody responses to the glycosylated and deglycosylated forms demonstrates the high immunogenicity of the N-glycans. Analyses of the antibody responses to the unusual N-terminal extension show an enhanced recognition of this portion by patients as opposed to recognition of the recombinant protein without extension.
Mycobacterium tuberculosis and Mycobacterium avium are facultative intracellular pathogens that are able to survive and replicate in mononuclear phagocytes. Human complement component C3 has previously been shown to mediate attachment and phagocytosis of these bacteria by mononuclear phagocytes. In this study, a C3 ligand affinity blot protocol was used to identify a 30-kDa C3-binding protein in M. tuberculosis and Mycobacterium smegmatis and a 31-kDa C3-binding protein in M. avium. The C3-binding proteins in M. tuberculosis and M. avium localized to the cell membrane fraction and partitioned to the detergent fraction during Triton X-114 phase partitioning. The C3-binding protein from M. tuberculosis was partially purified using a cation exchange column and was shown to bind concanavalin A. The N terminus and an internal fragment of the partially purified C3-binding protein were subjected to amino acid sequence analysis. The resulting amino acid sequences matched the M. tuberculosis heparin-binding hemagglutinin (HbhA) protein. Recombinant full-length HbhA and the C terminus of HbhA fused to maltose-binding protein, but not recombinant HbhA lacking the C-terminal region, bound human C3. Recombinant full-length HbhA coated on polystyrene beads, was found to enhance the adherence and/or phagocytosis of the coated beads to J774.A1 cells in both the presence and absence of human serum. The presence of complement-sufficient serum increased the adherence of the HbhA-coated beads to the J774.A1 cells in a C3-dependent manner. If HbhA within the bacterial cell membrane functions similarly to isolated HbhA, this protein may enhance the adherence and phagocytosis of M. tuberculosis and M. avium to mononuclear phagocytes through the binding of C3 and interaction with C3 receptors on mononuclear phagocytes.
The elucidation of the genomic sequence of Mycobacterium tuberculosis revealed the presence of a novel multigene family designated PE/PE_PGRS that encodes numerous, highly related proteins of unknown function. In this study, we demonstrate that a transposon insertion in a PE_PGRS gene (1818PE_PGRS) found in Mycobacterium bovis BCG Pasteur, which is the BCG homologue of the M. tuberculosis H37Rv gene Rv1818c, introduces new phenotypic properties to this BCG strain. These properties include dispersed growth in liquid medium and reduced infection of macrophages. Complementation of the 1818PE_PGRS::Tn5367 mutant with the wild-type gene restores both aggregative growth (clumping) in liquid medium and reestablishes infectivity of macrophages to levels equivalent to those for the parent BCG strain. Western blot analysis using antisera raised against the 1818PE_PGRS protein shows that PE_PGRS proteins are found in cell lysates of BCG and M. tuberculosis H37Ra and in the cell wall fraction of M. tuberculosis H37Rv. Moreover, immunofluorescent labeling of mycobacteria indicates that certain PE_PGRS proteins are localized at the cell surface of BCG and M. tuberculosis. Together these results suggest that certain PE_PGRS proteins may be found at the surface of mycobacteria and influence both cell surface interactions among mycobacteria as well as the interactions of mycobacteria with macrophages.
Infection of severe combined immunodeficient mice with Babesia sp. strain WA1 was studied to assess the contributions of innate and adaptive immunity in resistance to acute babesiosis. The scid mutation showed little effect in genetically susceptible C3H mice and did not decrease the inherent resistance of C57BL/6 mice to the infection, suggesting that innate immunity plays a central role in determining the course of Babesia infection in these strains. In contrast, the scid mutation dramatically impaired resistance in moderately susceptible BALB/c mice, suggesting that acquired immunity may play an important secondary role. In comparison to their female counterparts, male mice of different genetic backgrounds showed increased resistance to the infection, indicating that the gender of the host may influence protection against babesiosis.
While γ/δ T cells are involved in host defense and immunopathology in a variety of infectious diseases, their precise role is not yet clearly defined. In the absence of γ/δ T cells, mice die after infection with a dose of Listeria monocytogenes that is not lethal in immunologically intact animals. Morbidity might result from insufficient levels of cytokines normally produced by γ/δ T cells or conversely from an excess of cytokines due to a lack of down-regulation of the inflammatory response in the absence of γ/δ T cells. Consistent with a regulatory role, we found that systemic levels of proinflammatory cytokines (interleukin-6 [IL-6], IL-12, and gamma interferon [IFN-γ]) were significantly higher in the absence of γ/δ T cells during the innate phase of the response. Using combinations of genetically altered and immunodepleted mice, we found evidence for γ/δ T-cell-mediated regulation of IFN-γ production by multiple cell types of both lymphoid and myeloid lineages. The antigen-specific α/β T-cell response that followed the exaggerated innate response was also increased in γ/δ T-cell-deficient mice. These findings are consistent with an emerging picture from a variety of immune response models of a critical role for γ/δ T cells in down-modulation of the immune response.
Mycobacterium tuberculosis alone induces small, donor-variable amounts of tumor necrosis factor alpha (TNF-α) from primary human monocytes in vitro. However, TNF-α release is increased 5- to 500-fold when fixed activated T cells (FAT) or their isolated, unfixed membranes are added to this system. This FAT-induced synergy was at least as potent as that induced by gamma interferon (IFN-γ) at 100 U/ml. FAT-enhanced TNF-α production is at least in part transcriptionally mediated, as reflected by quantitative changes in TNF-α mRNA between 2 and 6 h poststimulation. Unlike IFN-γ-cocultured cells, FAT-treated monocytes appeared not to have enhanced TNF-α message stability, suggesting that de novo transcription may be involved in this effect. Furthermore, M. tuberculosis alone induced only minimal DNA binding of monocyte NF-κB, but cells treated with M. tuberculosis and FAT potentiated NF-κB activity more effectively. It is therefore possible that one mechanism by which FAT synergize with M. tuberculosis to stimulate TNF-α production is via NF-κB-enhanced transcription. These data strongly suggest that in the interaction of cells involved in the immune response to M. tuberculosis, T-cell stimulation of monocyte TNF-α production involves a surface membrane interaction(s) as well as soluble mediators.
Echinococcus multilocularis causes alveolar echinococcosis, one of the most lethal helminthic (accidental) infections in humans, as the life cycle predominantly includes wildlife rodents as intermediate hosts. The physical barrier between the proliferating parasitic metacestode and the host tissue is the acellular laminated layer (LL), which is characterized by its rich high-molecular-weight polysaccharide composition. Conversely to a crude protein-rich vesicular fluid antigen, a major carbohydrate antigen of the LL—the Em2(G11) antigen—did not stimulate murine T-cell proliferation in vitro. In fact, the persistent metacestode growth and antigenic stimulation induced a Th2 shift in vivo following conventional infection by intraperitoneal inoculation of 100 metacestode vesicles into C57/BL6 mice. Concurrently, the expression of Th1 cytokines (interleukin-2 and gamma interferon) remained persistently low until the late stage of chronic infection. In comparison to a recombinant proteinic II/3 antigen, the specific immunoglobulin G (IgG) response against the Em2(G11) antigen (including all IgG isotypes) maintained persistently low avidity. Furthermore, the Em2(G11) antigen induced a specific IgM and IgG response in T-cell-deficient athymic nude, TCRβ−/−, major histocompatibility complex class II (MHCII)−/−(CD4-deficient), and CD40−/− mice. The Em2(G11)-specific IgG synthesized in nude TCRβ−/− and MHCII−/− mice was predominantly of the IgG3 and IgG2a isotypes and of the IgG3 and IgG2b isotypes in CD40−/− mice. This finding suggested that in vivo, the IgG response to major carbohydrate antigen Em2(G11) of E. multilocularis could take place independently of αβ+ CD4+ T cells and in the absence of CD40-CD40 ligand interactions; thus, the Em2(G11) antigen of the acellular LL represents a T-cell-independent antigen. Functionally, the encapsulating LL, and especially its major carbohydrate antigen, Em2(G11), seems to be one of the key factors in the parasite's survival strategy and acts by modulating the host immune response by virtue of its T-cell-independent nature.
Glutamine synthetase (GS) and superoxide dismutase (SOD), large multimeric enzymes that are thought to play important roles in the pathogenicity of Mycobacterium tuberculosis, are among the bacterium's major culture filtrate proteins in actively growing cultures. Although these proteins lack a leader peptide, their presence in the extracellular medium during early stages of growth suggested that they might be actively secreted. To understand their mechanism of export, we cloned the homologous genes (glnA1 and sodA) from the rapid-growing, nonpathogenic Mycobacterium smegmatis, generated glnA1 and sodA mutants of M. smegmatis by allelic exchange, and quantitated expression and export of both mycobacterial and nonmycobacterial GSs and SODs in these mutants. We also quantitated expression and export of homologous and heterologous SODs from M. tuberculosis. When each of the genes was expressed from a multicopy plasmid, M. smegmatis exported comparable proportions of both the M. tuberculosis and M. smegmatis GSs (in the glnA1 strain) or SODs (in the sodA strain), in contrast to previous observations in wild-type strains. Surprisingly, recombinant M. smegmatis and M. tuberculosis strains even exported nonmycobacterial SODs. To determine the extent to which export of these large, leaderless proteins is expression dependent, we constructed a recombinant M. tuberculosis strain expressing green fluorescent protein (GFP) at high levels and a recombinant M. smegmatis strain coexpressing the M. smegmatis GS, M. smegmatis SOD, and M. tuberculosis BfrB (bacterioferritin) at high levels. The recombinant M. tuberculosis strain exported GFP even in early stages of growth and at proportions very similar to those of the endogenous M. tuberculosis GS and SOD. Similarly, the recombinant M. smegmatis strain exported bacterioferritin, a large (∼500-kDa), leaderless, multimeric protein, in proportions comparable to GS and SOD. In contrast, high-level expression of the large, leaderless, multimeric protein malate dehydrogenase did not lead to extracellular accumulation because the protein was highly unstable extracellularly. These findings indicate that, contrary to expectations, export of M. tuberculosis GS and SOD in actively growing cultures is not due to a protein-specific export mechanism, but rather to bacterial leakage or autolysis, and that the extracellular abundance of these enzymes is simply due to their high level of expression and extracellular stability. The same determinants likely explain the presence of other leaderless proteins in the extracellular medium of actively growing M. tuberculosis cultures.
The present study was initiated to gain insight into the interaction between splenic dendritic cells (DC) and Salmonella enterica serovar Typhimurium in vivo. Splenic phagocytic cell populations associated with green fluorescent protein (GFP)-expressing bacteria and the bacterium-specific T-cell response were evaluated in mice given S. enterica serovar Typhimurium expressing GFP and ovalbumin. Flow cytometry analysis revealed that GFP-positive splenic DC (CD11c+ major histocompatibility complex class II-positive [MHC-II+] cells) were present following bacterial administration, and confocal microscopy showed that GFP-expressing bacteria were contained within CD11c+ MHC-II+ splenocytes. Furthermore, splenic DC and T cells were activated following Salmonella infection. This was shown by increased surface expression of CD86 and CD40 on CD11c+ MHC-II+ cells and increased CD44 and CD69 expression on CD4+ and CD8+ T cells. Salmonella-specific gamma interferon (IFN-γ)-producing cells in both of these T-cell subsets, as well as cytolytic effector cells, were also generated in mice given live bacteria. The frequency of Salmonella-specific CD4+ T cells producing IFN-γ was greater than that of specific CD8+ T cells producing IFN-γ in the same infected animal. This supports the argument that the predominant source of IFN-γ production by cells of the specific immune response is CD4+ T cells. Finally, DC that phagocytosed live or heat-killed Salmonella in vitro primed bacterium-specific IFN-γ-producing CD4+ and CD8+ T cells as well as cytolytic effector cells following administration into naïve mice. Together these data suggest that DC are involved in priming naïve T cells to Salmonella in vivo.
Sequencing of the entire genome of Mycobacterium tuberculosis identified a novel multigene family composed of two closely related subfamilies designated PE and PE_PGRS. The major difference between these two families is the presence of a domain containing numerous Gly-Ala repeats extending to the C terminus of the PE_PGRS genes. We have used a representative PE_PGRS gene from M. tuberculosis, Rv1818c (1818PE_PGRS), and its amino-terminal PE region (1818PE), to investigate the immunological response to these proteins during experimental tuberculosis and following immunization with DNA constructs. During infection of mice with M. tuberculosis, a significant humoral immune response was observed against recombinant 1818PE_PGRS but not toward the 1818PE protein. Similarly, immunization with a 1818PE_PGRS DNA construct induced antibodies directed against 1818PE_PGRS but not against 1818PE proteins, and no humoral response was induced by 1818PE DNA. These results suggest that certain PE_PGRS genes are expressed during infection of the host with M. tuberculosis and that an antibody response is directed solely against the Gly-Ala-rich PGRS domain. Conversely, splenocytes from 1818PE-vaccinated mice but not mice immunized with 1818PE_PGRS secreted gamma interferon following in vitro restimulation and demonstrated protection in the mouse tuberculosis challenge model. These results suggest that the PE vaccine can elicit an effective cellular immune response and that immune recognition of the PE antigen is influenced by the Gly-Ala-rich PGRS domain.
The generation of nitric oxide (NO) by activated macrophages is believed to control mycobacterial infection in the murine system. In this study we examined the effect of Mycobacterium bovis BCG infection on the l-arginine-dependent NO pathway in J774.1 murine macrophages. We have confirmed previous results by demonstrating that stimulation of J774.1 with lipopolysaccharide (LPS) and gamma interferon (IFN-γ) results in an increase in the uptake of 3H-labeled l-arginine and a concomitant increase in the production of NO. We have also shown that BCG can mimic LPS treatment, leading to enhanced l-[3H]arginine uptake by IFN-γ-stimulated macrophages. Lipoarabinomannan, a component of the BCG cell wall that is structurally similar to LPS, is not responsible for the uptake stimulation in IFN-γ stimulated macrophages. Although we demonstrated that there was a 2.5-fold increase in NO production by macrophages 4 h after LPS–IFN-γ stimulation, BCG infection (with or without IFN-γ stimulation) did not lead to the production of NO by the macrophages by 4 h postinfection. At 24 h postinfection, the infected macrophages that were stimulated with IFN-γ produced amounts of NO similar to those of macrophages stimulated with LPS–IFN-γ. This suggests that there are multiple regulatory pathways involved in the production of NO. Finally, our data suggest that increased expression of the arginine permease, MCAT2B, after 4 h of LPS–IFN-γ treatment or BCG infection–IFN-γ treatment is not sufficient to account for the increases in l-[3H]arginine uptake detected. This suggests that the activity of the l-arginine transporter(s) is also altered in response to macrophage activation.
Buruli ulcer, caused by Mycobacterium ulcerans, is characterized by deep and necrotizing skin lesions, mostly on the arms and legs. Together with tuberculosis and leprosy, this mycobacterial disease has become a major health problem in tropical and subtropical regions, particularly in central and western Africa. No specific vaccine is available for Buruli ulcer. There is, however, evidence in the literature that suggests a cross-reactive protective role of the tuberculosis vaccine M. bovis BCG. To identify potential mechanisms for this cross-protection, we identified and characterized the M. ulcerans homologue of the important protective mycobacterial antigen 85 (Ag85A) from BCG. The homologue is well conserved in M. ulcerans, showing 84.1% amino acid sequence identity and 91% conserved residues compared to the sequence from BCG. This antigen was sufficiently conserved to allow cross-reactive protection, as demonstrated by the ability of M. ulcerans- infected mice to exhibit strong cellular immune responses to both BCG and its purified Ag85 complex. To further address the mechanism of cross-reactive protection, we demonstrate here that prior vaccination with either BCG or plasmid DNA encoding BCG Ag85A is capable of significantly reducing the bacterial load in the footpads of M. ulcerans- infected mice, as determined by Ziehl-Neelsen staining and by actual counting of CFU on 7H11 Middlebrook agar. Together, the results reported here support the potential of a cross-protective Ag85-based future vaccine against tuberculosis, Buruli ulcer, and leprosy.
Matrix metalloproteinases (MMPs) constitute a large family of enzymes with specificity for the various proteins of the extracellular matrix which are implicated in tissue remodeling processes and chronic inflammatory conditions. To investigate the role of MMPs in immunity to mycobacterial infections, we incubated murine peritoneal macrophages with viable Mycobacterium bovis BCG or Mycobacterium tuberculosis H37Rv and assayed MMP activity in the supernatants by zymography. Resting macrophages secreted only small amounts of MMP-9 (gelatinase B), but secretion increased dramatically in a dose-dependent manner in response to either BCG or M. tuberculosis in vitro. Incubation with mycobacteria also induced increased MMP-2 (gelatinase A) activity. Neutralization of tumor necrosis alpha (TNF-α), and to a lesser extent interleukin 18 (IL-18), substantially reduced MMP production in response to mycobacteria. Exogenous addition of TNF-α or IL-18 induced macrophages to express MMPs, even in the absence of bacteria. The immunoregulatory cytokines gamma interferon (IFN-γ), IL-4, and IL-10 all suppressed BCG-induced MMP production, but through different mechanisms. IFN-γ treatment increased macrophage secretion of TNF-α but still reduced their MMP activity. Conversely, IL-4 and IL-10 seemed to act by reducing the amount of TNF-α available to the macrophages. Finally, infection of BALB/c or severe combined immunodeficiency (SCID) mice with either BCG or M. tuberculosis induced substantial increases in MMP-9 activity in infected tissues. In conclusion, we show that mycobacterial infection induces MMP-9 activity both in vitro and in vivo and that this is regulated by TNF-α, IL-18, and IFN-γ. These findings indicate a possible contribution of MMPs to tissue remodeling processes that occur in mycobacterial infections.
Virulence variability was investigated by analyzing the experimental pathogenicity of 19 Leishmania major strains in susceptible BALB/c mice. Twelve strains were isolated from Tunisian patients with zoonotic cutaneous leishmaniasis; seven strains were isolated in Syria (n = 1), Saudi Arabia (n = 2), Jordan (n = 2), or Israel (n = 2). BALB/c mice were injected in the hind footpad with 2 × 106 amastigotes of the various isolates, and lesion progression was recorded weekly for 9 weeks. Interleukin-4 (IL-4) and gamma interferon (IFN-γ) production of lymph node mononuclear cells activated in vitro with parasite antigens were evaluated 5 weeks after infection. We show that disease progression induced by different L. major isolates was largely heterogeneous although reproducible results were obtained when using the same isolate. Interestingly, isolates from the Middle East induced a more severe disease than did the majority of Tunisian isolates. Strains with the highest virulence tend to generate more IL-4 and less IFN-γ in vitro at week 5 postinfection as well as higher levels of early IL-4 mRNA in the lymph node draining the inoculation site at 16 h postinfection. These results suggest that L. major isolates from the field may differ in virulence, which influences the course of the disease induced in mice and the type of immune response elicited by the infected host.
The role of genetic factors in clinical tuberculosis is increasingly recognized; how such factors regulate the immune response to Mycobacterium tuberculosis in healthy individuals is unclear. In this study of 255 adult twin pairs residing in The Gambia, West Africa, it is apparent that memory T-cell responses to secreted mycobacterial antigens (85-kDa antigen complex, “short-term culture filtrate,” and peptides from the ESAT-6 protein), as well as to the 65-kDa heat shock protein, are subject to effective genetic regulation. The delayed hypersensitivity response to intradermal tuberculin also demonstrates significant genetic variance, while quantitative T-cell and antibody responses to the 38-kDa cell membrane protein appear to be determined largely by environmental factors. Such findings have implications for vaccine development.
T-cell-mediated immunity is known to play a central role in the host response to Candida albicans. T-cell clones are useful tools for the exact identification of fungal T-cell epitopes and the processing requirements of C. albicans antigens. We isolated human T-cell clones from an HLA-DRB1*1101 healthy donor by using an antigenic extract (MP-F2) of the fungus. Specific clones were T-cell receptor α/β and CD4+/CD8− and showed a T-helper type 1 cytokine profile (production of gamma interferon and not interleukin-4). The large majority of these clones recognized both the natural (highly glycosylated) and the recombinant (nonglycosylated) 65-kDa mannoprotein (MP65), an MP-F2 minor constituent that was confirmed to be an immunodominant antigen of the human T-cell response. Surprisingly, most of the clones recognized two synthetic peptides of different MP65 regions. However, the peptides shared the amino acid motif IXSXIXXL, which may be envisaged as a motif sequence representing the minimal epitope recognized by these clones. Three clones recognized natural and pronase-treated MP65 but did not detect nonglycosylated, recombinant MP65 or the peptides, suggesting a possible role for polysaccharides in T-cell recognition of C. albicans. Finally, lymphoblastoid B-cell lines were efficient antigen-presenting cells (APC) for recombinant MP65 and peptides but failed to present natural, glycosylated antigens, suggesting that nonprofessional APC might be defective in processing highly glycosylated yeast proteins. In conclusion, this study provides the first characterization of C. albicans-specific human T-cell clones and provides new clues for the definition of the cellular immune response against C. albicans.
Gamma interferon (IFN-γ), a pleiotropic cytokine, is now known to be produced by macrophages as well as by NK cells, γδ cells, and activated T cells. The autocrine biological functions of IFN-γ on the macrophage include the upregulation of major histocompatibility complex MHC class II and the activation to an antiviral state. In this study, the production of IFN-γ by macrophages was demonstrated to correspond to antibacterial activity. Legionella pneumophila replicates intracellularly in thioglycolate (TG)-elicited macrophages (TG-macrophages) from A/J mice, while TG-macrophages from BALB/c mice restrict bacterial growth after an initial period of growth. BALB/c TG-macrophages were shown to express IFN-γ mRNA at 24 and 28 h, which corresponded to the initiation of anti-L. pneumophila activity. Moreover, IFN-γneutralization by antibody treatment of the cultures resulted in increased L. pneumophila growth in the macrophages. In contrast, no IFN-γ mRNA was expressed in TG-macrophages from A/J mice, where L. pneumophila grew unrestricted. As would be expected, IFN-γ treatment decreased bacterial growth. An IFN-γ-mediated antibacterial activity was, however, inducible in A/J macrophages by the addition of interleukin-12 following L. pneumophila infection. Thus, autocrine IFN-γ is involved in anti-L. pneumophila activity associated with different growth patterns and appears to be important during intracellular infection.
We used Listeria monocytogenes, a gram-positive, facultative intracellular bacterium, to study the gut mucosal immune responses following oral infection. We employed a germfree (GF) mouse model to try to accentuate the development of a humoral mucosal immune response in the gut, and we used oral colonization with one of the mutants, actA-negative (ΔactA) L. monocytogenes, to restrict infection largely to the gut. The ΔactA mutant was able to colonize the intestinal mucosa of formerly GF mice for long periods of time without causing disease while eliciting secretory immunoglobulin A (IgA) responses, as evidenced by gut tissue fragment culture assays. Flow cytometric analyses and immunohistochemical methods showed the development of only minimal germinal center reactions (GCR) in Peyer's patches and more robust GCR in mesenteric lymph nodes. Pronounced increases in total (natural) IgA production occurred in gut tissues by day 7 and were maintained for up to 90 days. Levels of specific IgA were modest in gut tissues on day 14, increased until day 76, and stabilized at day 90. We also observed a significant rise in serum IgA and IgG1 levels following oral infection by listeriae. Upon colonization, the organisms mainly infected the intestines and intestinal lumen, and we only sporadically observed few colony-forming bacteria in the liver and spleen. We observed a marked rise in IgA-secreting cells, including listeria-specific IgA antibody-secreting cells, in the lamina propria of the small intestine by enzyme-linked immunospot assays. To ascertain whether some of the IgA was specific for listeriae, we performed Western blot analysis to test the reactivity of IgA from fragment cultures to antigens in sonicates of L. monocytogenes. We detected IgA binding to antigenic proteins with molecular masses of 96, 60, 40, and 14 kDa in the Listeria sonicates.
Taking advantage of the fact that plasmid DNA encoding a single cytotoxic T lymphocyte (CTL) epitope can induce CTLs, we examined the influence of T-cell responses to dominant epitopes on those to a subdominant epitope derived from Listeria monocytogenes. Our data suggest that interaction between T cells against dominant and subdominant epitopes does not operate in the generation of the hierarchy. Furthermore, we found that a single dominant epitope is sufficient for the induction of protective immunity.
Litomosoides sigmodontis is the only filaria which develops from infective larvae into microfilaria-producing adults in immunocompetent laboratory mice. In this study we report that interleukin-4 knockout (IL-4 KO) mice have an up to 100-fold-higher and a significantly prolonged microfilaremia compared to wild-type BALB/c mice, as well as 20 times more microfilariae in the thoracic cavity, the site of infection. While worm development and adult worm persistence were equivalent in IL-4 KO and wild-type mice, the fertility and length of adult female worms in IL-4 KO mice was clearly enhanced. The high susceptibility to microfilariae in IL-4 KO mice required the presence of adult worms in a full infection cycle since microfilariae loads did not differ much between IL-4 KO and wild-type mice when purified microfilariae were injected into mice. In addition, we found that eosinophilia was diminished and immunoglobulin E (IgE) was absent in IL-4 KO mice. IgE, however, does not seem to be the essential factor for microfilarial containment since microfilaremia was not elevated in B-cell KO mice. In conclusion, IL-4 is shown for the first time to be essential for the control of microfilarial loads but not of adult worm loads in a fully permissive murine filarial infection. IL-4 dependent effector pathways seem to operate on adult worms rather than directly on microfilariae.
In this study, we investigated the potential of a tuberculosis subunit vaccine based on fusion proteins of the immunodominant antigens ESAT-6 and antigen 85B. When the fusion proteins were administered to mice in the adjuvant combination dimethyl dioctadecylammonium bromide-monophosphoryl lipid A, a strong dose-dependent immune response was induced to both single components as well as to the fusion proteins. The immune response induced was accompanied by high levels of protective immunity and reached the level of Mycobacterium bovis BCG-induced protection over a broad dose range. The vaccine induced efficient immunological memory, which remained stable 30 weeks postvaccination.
The proportions of peripheral blood mononuclear cells (PBMC), CD4+ T cells, and CD8+ T cells that produce gamma interferon (IFN-γ) in response to Mycobacterium tuberculosis were markedly reduced in tuberculosis patients, particularly in those with severe disease. Depletion of CD4+ but not CD8+ cells prior to stimulation of PBMC with M. tuberculosis abolished IFN-γ production. These results show that (i) IFN-γ production by CD8+ and CD4+ cells correlates with the clinical manifestations of M. tuberculosis infection and (ii) IFN-γ production by CD8+ cells depends on CD4+ cells.