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1.
Infect Immun ; 75(3): 1186-95, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17189432

ABSTRACT

Burkholderia pseudomallei, the causative agent of melioidosis, is an important human pathogen in Southeast Asia and northern Australia for which a vaccine is unavailable. A panel of 892 double signature-tagged mutants was screened for virulence using an intranasal BALB/c mouse model of infection. A novel DNA tag microarray identified 33 mutants as being attenuated in spleens, while 6 were attenuated in both lungs and spleens. The transposon insertion sites in spleen-attenuated mutants revealed genes involved in several stages of capsular polysaccharide biosynthesis and DNA replication and repair, a putative oxidoreductase, ABC transporters, and a lipoprotein that may be important in intercellular spreading. The six mutants identified as missing in both lungs and spleens were found to have insertions in recA involved in the SOS response and DNA repair; putative auxotrophs of leucine, threonine, p-aminobenzoic acid, and a mutant with an insertion in aroB causing auxotrophy for aromatic compounds were also found. Murine challenge studies revealed partial protection in BALB/c mice vaccinated with the aroB mutant. The refined signature-tagged mutagenesis approach developed in this study was used to efficiently identify attenuating mutants from this highly pathogenic species and could be applied to other organisms.


Subject(s)
Burkholderia pseudomallei/genetics , Burkholderia pseudomallei/pathogenicity , Genes, Bacterial , Melioidosis/microbiology , Mutagenesis, Insertional , Animals , Burkholderia pseudomallei/growth & development , Female , Lung/metabolism , Lung/microbiology , Mice , Mice, Inbred BALB C , Spleen/metabolism , Spleen/microbiology
2.
Infect Immun ; 69(9): 5661-70, 2001 Sep.
Article in English | MEDLINE | ID: mdl-11500442

ABSTRACT

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-alpha), and to a lesser extent interleukin 18 (IL-18), substantially reduced MMP production in response to mycobacteria. Exogenous addition of TNF-alpha or IL-18 induced macrophages to express MMPs, even in the absence of bacteria. The immunoregulatory cytokines gamma interferon (IFN-gamma), IL-4, and IL-10 all suppressed BCG-induced MMP production, but through different mechanisms. IFN-gamma treatment increased macrophage secretion of TNF-alpha but still reduced their MMP activity. Conversely, IL-4 and IL-10 seemed to act by reducing the amount of TNF-alpha 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-alpha, IL-18, and IFN-gamma. These findings indicate a possible contribution of MMPs to tissue remodeling processes that occur in mycobacterial infections.


Subject(s)
Macrophages, Peritoneal/enzymology , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 9/metabolism , Mycobacterium bovis/immunology , Mycobacterium tuberculosis/immunology , Tuberculosis/microbiology , Animals , Cells, Cultured , Macrophage Activation , Macrophages, Peritoneal/microbiology , Mice , Mice, Inbred BALB C , Mice, SCID , Tuberculosis/enzymology , Tumor Necrosis Factor-alpha/metabolism
3.
Infect Immun ; 69(2): 1142-50, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11160012

ABSTRACT

Auxotrophic mutants of Mycobacterium tuberculosis have been proposed as new vaccine candidates. We have analyzed the virulence and vaccine potential of M. tuberculosis strains containing defined mutations in genes involved in methionine (metB), proline (proC), or tryptophan (trpD) amino acid biosynthesis. The metB mutant was a prototrophic strain, whereas the proC and trpD mutants were auxotrophic for proline and tryptophan, respectively. Following infection of murine bone marrow-derived macrophages, H37Rv and the metB mutant strain survived intracellularly for over 10 days, whereas over 90% of proC and trpD mutants were killed during this time. In SCID mice, both H37Rv and the metB mutant were highly virulent, with mouse median survival times (MST) of 28.5 and 42 days, respectively. The proC mutant was significantly attenuated (MST, 130 days), whereas the trpD mutant was essentially avirulent in an immunocompromised host. Following infection of immunocompetent DBA mice with H37Rv, mice survived for a median of 83.5 days and the metB mutant now showed a clear reduction in virulence, with two of five infected mice surviving for 360 days. Both proC and trpD mutants were avirulent (MST of >360 days). In vaccination studies, prior infection with either the proC or trpD mutant gave protection equivalent (proC mutant) to or better (trpD mutant) than BCG against challenge with M. tuberculosis H37Rv. In summary, proC and trpD genes are essential for the virulence of M. tuberculosis, and mutants with disruptions in either of these genes show strong potential as vaccine candidates.


Subject(s)
BCG Vaccine/immunology , Mycobacterium tuberculosis/pathogenicity , Proline/biosynthesis , Tryptophan/biosynthesis , Animals , Mice , Mice, Inbred BALB C , Mice, Inbred DBA , Mice, SCID , Mutation , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/metabolism , Virulence
4.
J Immunol ; 166(2): 1097-105, 2001 Jan 15.
Article in English | MEDLINE | ID: mdl-11145690

ABSTRACT

The bacterium Burkholderia pseudomallei causes a life-threatening disease called melioidosis. In vivo experiments in mice have identified that a rapid IFN-gamma response is essential for host survival. To identify the cellular sources of IFN-gamma, spleen cells from uninfected mice were stimulated with B. pseudomallei in vitro and assayed by ELISA and flow cytometry. Costaining for intracellular IFN-gamma vs cell surface markers demonstrated that NK cells and, more surprisingly, CD8(+) T cells were the dominant sources of IFN-gamma. IFN-gamma(+) NK cells were detectable after 5 h and IFN-gamma(+) CD8(+) T cells within 15 h after addition of bacteria. IFN-gamma production by both cell populations was inhibited by coincubation with neutralizing mAb to IL-12 or IL-18, while a mAb to TNF had much less effect. Three-color flow cytometry showed that IFN-gamma-producing CD8(+) T cells were of the CD44(high) phenotype. The preferential activation of NK cells and CD8(+) T cells, rather than CD4(+) T cells, was also observed in response to Listeria monocytogenes or a combination of IL-12 and IL-18 both in vitro and in vivo. This rapid mechanism of CD8(+) T cell activation may be an important component of innate immunity to intracellular pathogens.


Subject(s)
Burkholderia pseudomallei/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Interferon-gamma/biosynthesis , Lymphocyte Activation , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/microbiology , Cells, Cultured , Female , Immunologic Memory , Interferon Inducers/pharmacology , Interleukin-12/physiology , Interleukin-18/physiology , Interphase/immunology , Intracellular Fluid/immunology , Intracellular Fluid/microbiology , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Killer Cells, Natural/microbiology , Kinetics , Listeriosis/immunology , Listeriosis/metabolism , Listeriosis/microbiology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Spleen/cytology , Spleen/immunology , Spleen/metabolism , Spleen/microbiology , Tumor Necrosis Factor-alpha/physiology
5.
Immunology ; 104(4): 439-46, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11899430

ABSTRACT

Adrenaline is a catecholamine hormone secreted by the adrenal medulla in response to acute stress. Previous studies have shown that adrenaline suppresses the nitric oxide (NO) response of murine macrophages (M phi s) stimulated in vitro with lipopolysaccharide (LPS). We have now extended these studies to examine the effects of adrenaline on the production of tumour necrosis factor alpha (TNF-alpha) and interleukin-10 (IL-10). Our results showed that NO, TNF-alpha and IL-10 were concurrently produced following in vitro LPS (10 micrograms/ml) stimulation of murine peritoneal M phi s. Adrenaline suppressed both NO and TNF-alpha with concomitant up-regulation of the IL-10 response above that seen with LPS alone. In this in vitro model of LPS stimulation we demonstrated that TNF-alpha was required for NO production, as the TNF-alpha neutralizing monoclonal antibody, TN3.19.12, abolished the response; in contrast, IL-10 suppressed NO. In order to determine any functional consequence of adrenaline-mediated IL-10 augmentation on NO production, M phi s were stimulated with LPS and specific neutralizing anti-IL-10 antibodies were added to the cultures. The LPS NO response was suppressed to 43% of the control value by adrenaline (10(-8) M) and an irrelevant control antibody had no effect on the adrenaline-mediated inhibition of NO, but anti-IL-10 treatment restored the NO response to levels similar to those observed with LPS alone. Furthermore, we demonstrated that exogenous TNF-alpha, at a dose range of 1.9-50 ng per ml, also restored the nitrite response to LPS in the presence of adrenaline. Together, the observations that neutralization of IL-10 and addition of TNF-alpha abrogate adrenaline's inhibition of NO, suggest that this hormone suppresses NO partly through up-regulation of IL-10 which, in turn, may suppress TNF-alpha that is required for NO production. Finally, we also observed that the M phi-activating cytokine, interferon-gamma (IFN-gamma), attenuated the inhibitory effect of adrenaline on the LPS NO response.


Subject(s)
Adrenergic Agonists/pharmacology , Epinephrine/pharmacology , Interleukin-10/biosynthesis , Macrophages, Peritoneal/drug effects , Nitric Oxide/biosynthesis , Tumor Necrosis Factor-alpha/biosynthesis , Animals , Cells, Cultured , Dose-Response Relationship, Immunologic , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/immunology , Macrophage Activation/drug effects , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/metabolism , Male , Mice , Nitrites/metabolism , Tumor Necrosis Factor-alpha/pharmacology
6.
Infect Immun ; 68(11): 6127-32, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11035715

ABSTRACT

The early role of natural killer cells and gamma delta T cells in the development of protective immunity to the blood stage of nonlethal Plasmodium yoelii infection was studied. Splenic cytokine levels were measured 24 h after infection of natural killer cell-depleted immunodeficient and littermate mice or transiently T-cell-depleted normal mice. Splenic gamma interferon levels were significantly increased above background in immunodeficient and littermate mice 24 h after infection. Depletion of natural killer cells resulted in markedly depressed gamma interferon levels and poor control of parasitemia, particularly in severe combined immunodeficient mice. In the littermates, gamma interferon levels were partially reduced, but parasitemias were resolved normally. However, in athymic mice, natural killer cell depletion had no effect on gamma interferon production. Levels of tumor necrosis factor alpha were increased in all animals 24 h after infection, and responses were not affected by natural killer cell depletion. However, in T-cell-depleted animals, both gamma interferon and tumor necrosis factor alpha levels were decreased 24 h after infection, and depleted mice were unable to control their parasitemia. These results suggest that the early production of both cytokines is important in the early control of parasitemia and that both natural killer and gamma delta T cells contribute equally towards their production. The data also suggest that the subsequent resolution of infection requires early production of gamma interferon, which might act by switching on the appropriate T-helper-cell subsets and other essential parasitotoxic effector mechanisms.


Subject(s)
Malaria/immunology , Plasmodium yoelii/immunology , T-Lymphocytes/immunology , Animals , Immunity, Innate , Interferon-gamma/biosynthesis , Killer Cells, Natural/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Receptors, Antigen, T-Cell, gamma-delta/analysis , Thy-1 Antigens/physiology , Tumor Necrosis Factor-alpha/biosynthesis
7.
Med Mycol ; 37(3): 183-94, 1999 Jun.
Article in English | MEDLINE | ID: mdl-10421850

ABSTRACT

Aspergillus fumigatus causes life-threatening invasive pulmonary aspergillosis in the immunocompromised patient. In this study we have used a murine model of intratracheal challenge with A. fumigatus to investigate the recruitment of inflammatory cells in the lung and the expression of proinflammatory cytokines and chemokines. Our results show that A. fumigatus causes an acute pulmonary inflammatory response which is dominated by neutrophils and to a lesser extent macrophages. During the peak of infection, proinflammatory cytokines (TNF-alpha, GM-CSF and IL-1beta) and chemokines (MIP-1alpha, MCP-1 and MIP-2), are induced within the lung. Furthermore, treatment of mice with neutralizing anti-TNF-alpha and anti-GM-CSF mAbs reduced the influx of neutrophils into the lung and delayed fungal clearance. Our observations show that Aspergillus conidia are effective inducers of host chemokine responses both in vitro and in vivo. Furthermore, TNF-alpha and GM-CSF play a central role in the recruitment of neutrophils into the lung in response to this clinically important pathogen.


Subject(s)
Aspergillosis/immunology , Aspergillus fumigatus/immunology , Chemokine CCL2/biosynthesis , Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Interleukin-1/biosynthesis , Macrophage Inflammatory Proteins/biosynthesis , Monokines/biosynthesis , Neutrophils/immunology , Tumor Necrosis Factor-alpha/biosynthesis , Animals , Cells, Cultured , Chemokine CCL3 , Chemokine CCL4 , Chemokine CXCL2 , Cricetinae , Disease Models, Animal , Female , Lung/immunology , Lung/microbiology , Macrophages, Alveolar/immunology , Macrophages, Alveolar/microbiology , Mice , Mice, Inbred BALB C , Neutralization Tests
8.
Infect Immun ; 67(7): 3593-600, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10377144

ABSTRACT

Burkholderia pseudomallei, the causative agent of melioidosis, is a gram-negative bacterium capable of causing either acute lethal sepsis or chronic but eventually fatal disease in infected individuals. However, despite the clinical importance of this infection in areas where it is endemic, there is essentially no information on the mechanisms of protective immunity to the bacterium. We describe here a murine model of either acute or chronic infection with B. pseudomallei in Taylor Outbred (TO) mice which mimics many features of the human pathology. Intraperitoneal infection of TO mice at doses of >10(6) CFU resulted in acute septic shock and death within 2 days. In contrast, at lower doses mice were able to clear the inoculum from the liver and spleen over a 3- to 4-week period, but persistence of the organism at other sites resulted in a chronic infection of between 2 and 16 months duration which was eventually lethal in all of the animals tested. Resistance to acute infection with B. pseudomallei was absolutely dependent upon the production of gamma interferon (IFN-gamma) in vivo. Administration of neutralizing monoclonal antibody against IFN-gamma lowered the 50% lethal dose from >5 x 10(5) to ca. 2 CFU and was associated with 8,500- and 4,400-fold increases in the bacterial burdens in the liver and spleen, respectively, together with extensive destruction of lymphoid architecture in the latter organ within 48 h. Neutralization of either tumor necrosis factor alpha or interleukin-12 but not granulocyte-macrophage colony-stimulating factor, also increased susceptibility to infection in vivo. Together, these results provide the first evidence of a host protective mechanism against B. pseudomallei. The rapid production of IFN-gamma within the first day of infection determines whether the infection proceeds to an acute lethal outcome or becomes chronic.


Subject(s)
Burkholderia pseudomallei , Interferon-gamma/immunology , Melioidosis/immunology , Acute Disease , Animals , Burkholderia pseudomallei/pathogenicity , Chronic Disease , Humans , Immunity, Innate , Interleukin-12/immunology , Melioidosis/physiopathology , Mice , Tumor Necrosis Factor-alpha/immunology
9.
Infect Immun ; 66(8): 3626-34, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9673242

ABSTRACT

The mycobacterial 19-kilodalton antigen (19Ag) is a highly expressed, surface-associated glycolipoprotein which is immunodominant in infected patients and has little homology with other known proteins. To investigate the pathogenic significance of the 19Ag, site-directed mutagenesis of the Mycobacterium intracellulare 19Ag gene was carried out by using a suicide vector-based strategy. Allelic replacement of the 19Ag gene of a mouse-avirulent M. intracellulare strain, 1403, was achieved by double-crossover homologous recombination with a gentamicin resistance gene-mutated allele. Unfortunately, an isogenic 19Ag was not achievable in the mouse-virulent strain, D673. However, a 19Ag mutant was successfully constructed in M. intracellulare FM1, a chemically mutagenized derivative of strain D673. FM1 was more amenable to genetic manipulation and susceptible to site-directed mutagenesis of the 19Ag gene yet retained the virulent phenotype of the parental strain. No deleterious effects of 19Ag gene mutation were observed during in vitro growth of M. intracellulare. Virulence assessment of the isogenic 19Ag mutants in a mouse infection model demonstrated that the antigen plays no essential role in the growth of M. intracellulare in vivo. Site-directed mutagenesis of the 19Ag gene demonstrated that it plays no essential role in growth and pathogenicity of M. intracellulare; however, the exact nature of its biological function remains unknown.


Subject(s)
Antigens, Bacterial/genetics , Bacterial Proteins/genetics , Lipoproteins/genetics , Mutagenesis, Site-Directed , Mycobacterium avium Complex/genetics , Mycobacterium avium Complex/pathogenicity , Animals , Antigens, Bacterial/physiology , Bacterial Proteins/physiology , Base Sequence , Blotting, Southern , DNA, Bacterial , Disease Models, Animal , Female , Genetic Complementation Test , Genetic Vectors , Humans , Lipoproteins/physiology , Mice , Mice, Inbred BALB C , Mice, SCID , Molecular Sequence Data , Mycobacterium avium Complex/growth & development , Mycobacterium avium Complex/isolation & purification , Mycobacterium avium-intracellulare Infection/microbiology , Rabbits , Virulence
11.
Infect Immun ; 65(8): 3074-9, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9234756

ABSTRACT

Immunological control of infection with cryptosporidia in mice is dependent on CD4+ T cells and the production of gamma interferon (IFN-gamma), but to date, the mucosal T cells which produce IFN-gamma local to the infection have not been characterized. We previously showed that immunity against the gastric parasite Cryptosporidium muris could be adoptively transferred to adult SCID (severe combined immunodeficiency) mice with small intestinal intraepithelial lymphocytes (IEL) from previously infected immunocompetent mice, but only if the donor CD4+ T cells were intact. The present investigation examined whether IFN-gamma was important in the effector mechanisms mediated by immune IEL in SCID mice. The development of resistance against C. muris infection in SCID mice given immune IEL was prevented by treatment with a hamster anti-mouse IFN-gamma-neutralizing monoclonal antibody, but following cessation of antibody treatment, the mice recovered from infection. In further experiments, an enzyme-linked immunospot (ELISPOT) technique was used to compare frequencies of IFN-gamma-producing cells in activated T-cell populations from C. muris-immune and naive donor mice. Stimulation with concanavalin A or a rat anti-mouse CD3 monoclonal antibody resulted in detection of greater numbers of cells producing IFN-gamma from immune than naive IEL populations. Small numbers of IEL from C. muris-immune mice, but not from naive mice, also produced IFN-gamma when cultured with soluble oocyst antigen, but this occurred only if gamma-irradiated spleen cells were cocultured with the immune IEL. These results suggested that IEL were important in the generation of immunity to Cryptosporidium and that one of their crucial functions was to produce IFN-gamma at the site of infection.


Subject(s)
Cryptosporidiosis/immunology , Interferon-gamma/biosynthesis , Intestines/immunology , T-Lymphocytes/immunology , Animals , Female , Immunity, Mucosal , Lymphocyte Activation , Mice , Mice, SCID , Rats
12.
Immunology ; 91(2): 289-96, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9227330

ABSTRACT

Phagocytosis is a fundamental process in innate resistance to infection. We have used the pathogenic yeast Cryptococcus neoformans to study the interaction of this encapsulated organism with murine macrophages in vitro. In the absence of exogenous opsonins the encapsulated yeast is almost totally resistant to ingestion by murine macrophages. Owing to its ability to activate the alternative complement pathway, the anti-phagocytic properties of the polysaccharide capsule can be partially overcome following opsonization in vitro with non-immune mouse serum and subsequent phagocytosis via complement receptors. Here, we demonstrate the importance of the complement receptor type 3 (CR3) in in vitro phagocytosis of the yeast and in in vivo resistance to infection. In vitro, 70% of a population of resident murine macrophages are able to ingest C. neoformans and then only inefficiently (1-2 organisms per cell). Previously we have shown that tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) efficiently enhance ingestion of serum-opsonized encapsulated C. neoformans, and we now show that the cytokines convert a population of resident macrophages to a state where all the cells are competent for ingestion of large numbers of yeasts (6-8 per cell). We also show that these cytokines have a direct effect on CR3, as enhanced levels of complement-opsonized sheep red blood cells (EIgMC) bind to macrophages activated in this way. However, cytokines that have previously been shown to enhance phagocytosis of EIgMC have no effect on ingestion of encapsulated C. neoformans. These results demonstrate that the cytokines regulating CR3-dependent ingestion of C. neoformans are different to those regulating ingestion of EIgMC and reinforce the importance of studying pathogens rather than inert ligands in understanding the regulation of phagocytosis.


Subject(s)
Cryptococcosis/immunology , Cryptococcus neoformans/immunology , Cytokines/immunology , Macrophage-1 Antigen/immunology , Phagocytosis/immunology , Animals , Cell Culture Techniques , Erythrocytes/immunology , Female , Granulocyte-Macrophage Colony-Stimulating Factor/immunology , Macrophage Activation , Macrophages, Peritoneal/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Tumor Necrosis Factor-alpha/immunology , Zymosan/pharmacology
15.
Parasitol Res ; 83(3): 257-63, 1997.
Article in English | MEDLINE | ID: mdl-9089722

ABSTRACT

Studies with murine infection models have shown that immunity to the protozoan parasite Cryptosporidium involves T-cells and interferon-gamma (IFN-gamma) activity. The present study was performed to compare the course of infection of Cryptosporidium muris in major histocompatibility complex (MHC) congeneic strains of mice and examine the relationship between susceptibility to infection and production of T-cell cytokines. In experiments with BALB mice, the BALB/c strain (H-2d) produced significantly fewer oocysts and recovered from infection sooner than the BALB/B (H-2b) or BALB/K (H-2k) strains. BALB/B X BALB/c F1 hybrid mice were found to express the more susceptible phenotype of the BALB/B parent strain, indicating that the gene(s) in the H-2 locus conferring increased susceptibility to C. muris infection was dominant. At different times during infection of the resistant BALB/c strain and the susceptible BALB/B strain, splenocytes were cultured with soluble parasite antigen and measurements were made of production of a number of T-cell cytokines. Similar patterns of increasing levels of IFN-gamma and interleukin 2 (IL-2) were observed in both the resistant and susceptible strains during the patent stage of infection, indicating that production of these type 1 T-helper-cell (TH1) cytokines (i.e. involved in cell-mediated responses) correlated with the development of immunity. This also suggested that the increased susceptibility of BALB/B mice was not associated with a defective TH1 cytokine response. In the study of TH2 cytokines (involved in induction of an antibody response), low levels of IL-10 were detected during infection of BALB/c and BALB/B mice. In contrast, although IL-4 was released by splenocytes of both strains, significantly larger amounts were obtained from cells of the susceptible BALB/B mice in the early stages of infection. Thus, the H-2-dependent variation in susceptibility to infection between these BALB strains correlated with a difference in the pattern of IL-4 secretion.


Subject(s)
Cryptosporidiosis/immunology , Cytokines/biosynthesis , H-2 Antigens/immunology , T-Lymphocytes/immunology , Animals , Cells, Cultured , Cryptosporidium/immunology , Disease Susceptibility , Female , Immunity, Innate , Lymphocyte Subsets , Lymphoid Tissue/cytology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Rats
16.
Int Immunol ; 8(8): 1299-310, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8918699

ABSTRACT

Infection with the virulent Mycobacterium avium strain TMC 724 caused progressive infection in C57BL/6 and BALB/c mice, while infection with a less virulent strain (M. avium SE 01) resulted in chronically persistent bacterial loads. Livers of mice infected with TMC 724 were characterized by progressively expanding tumor-like infiltrations of epithelioid macrophages, while SE 01 induced well-developed, compact epithelioid granulomas that remained constant in size and number for at least 4 months. When C57BL/6 mice were depleted of CD4+ T cells by i.p. administration of specific mAb at the time of infection, their capacity to initiate granuloma formation was completely abrogated during the first 4 weeks of infection. Semi-quantitative competitive RT-PCR of liver homogenates obtained 3 weeks after infection revealed that depletion of CD4+ T cells was accompanied by a 25-fold reduced expression of IFN-gamma mRNA and a 5-fold reduced expression of tumor necrosis factor (TNF)-alpha mRNA when compared to control infected mice. Granuloma morphology in response to either TMC 724 or SE 01 was similar in immunodeficient SCID mice to that observed in syngeneic BALB/c mice. However, SCID mice developed granulomas in a delayed fashion and were less efficient in surrounding infected Kupffer cells with an inflammatory infiltration. The delayed kinetics of granuloma initiation in infected SCID mice was paralleled by a lower mRNA expression for IFN-gamma and TNF-alpha compared to that observed in infected BALB/c mice. mAb-mediated neutralization of IFN-gamma in BALB/c mice significantly reduced inflammatory infiltrations and granuloma formation. These data support the conclusion that CD4+ T cells accelerate granuloma formation by enhancing the production of TNF-alpha and IFN-gamma at the site of infection.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Granuloma/etiology , Liver Diseases/etiology , Mycobacterium avium , Tuberculosis/etiology , Animals , Base Sequence , DNA Primers/genetics , Granuloma/immunology , Granuloma/pathology , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Liver Diseases/immunology , Liver Diseases/pathology , Lymphocyte Depletion , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Neutralization Tests , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tuberculosis/immunology , Tuberculosis/pathology , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics
17.
Infect Immun ; 64(7): 2556-62, 1996 Jul.
Article in English | MEDLINE | ID: mdl-8698479

ABSTRACT

The role of gut intraepithelial lymphocytes (IEL) in immunity to cryptosporidial infection was investigated with a murine infection model involving Cryptosporidium muris. Oocyst shedding was monitored in severe combined immunodeficiency (SCID) mice infected with C. muris following intravenous injection of mesenteric lymph node (MLN) cells or intestinal IEL from BALB/c donor mice which were naive or previously infected with C. muris. SCID mice receiving no lymphoid cells developed chronic infections and excreted large numbers of oocysts until the end of the experiment. SCID mice injected with IEL from immune animals, however, were able to overcome the infection, and furthermore, these animals produced fewer oocysts and recovered sooner than ones which received IEL or MLN cells from naive BALB/c donors. Similar levels of protection were obtained in SCID mice injected with either 2 X 10(6) IEL or MLN cells from immune donor mice. Depletion of CD4+ cells from immune IEL, however, abrogated the ability to transfer immunity to SCID mice, while depletion of CD8+ cells only marginally reduced the protective capacity of immune IEL. Finally, control SCID mice which received no lymphocytes had < or = 1% CD4+ cells in the IEL from the small intestine, whereas the IEL from SCID mice recovered from infection, as a result of injection with immune IEL, contained 15% CD4+ cells. Thus, the ability to control C. muris infection correlated with the presence of the protective CD4+ cells in the gut epithelium.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , Cryptosporidiosis/immunology , Intestinal Mucosa/immunology , Animals , CD4-Positive T-Lymphocytes/pathology , CD4-Positive T-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Cell Movement , Cryptosporidiosis/pathology , Cryptosporidium/immunology , Epithelium/immunology , Epithelium/pathology , Female , Immunocompetence , Immunotherapy, Adoptive , Intestinal Mucosa/pathology , Male , Mice , Mice, Inbred BALB C , Mice, SCID
18.
Eur J Immunol ; 26(2): 356-64, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8617304

ABSTRACT

We have used severe-combined immunodeficient (SCID) mice to examine the immunoregulatory effects of interleukin (IL)-10 on innate resistance to infection with Listeria monocytogenes. Addition of heat killed Listeria to spleen cells from naive SCID mice resulted in secretion of interferon (IFN)-gamma from natural killer cells in vitro. This response was enhanced up to 15-fold in the presence of exogenous IL-2, but was completely ablated by addition of IL-10 with IC50 of less than 0.5 U/ml. Infection of SCID mice with viable Listeria in vivo resulted in a prolonged course of infection eventually causing death by 12-14 days, whereas daily administration of IL-10 increased bacterial replication in the liver and spleen by up to 1000-fold resulting in death by day 4 post-infection. The immunosuppressive actions of IL-10 in vivo were also observed in immunocompetent BALB/c mice, whereas doses as low as 100 U/day converted a sublethal infection to 100% mortality. To study the events controlling expression of endogenous IL-10, peritoneal macrophage monolayers were challenged with Listeria after preincubation with a panel of recombinant cytokines. IFN-gamma primed macrophages for enhanced tumor necrosis factor (TNF) secretion, but inhibited IL-10 production, whereas granulocyte/macrophage colony-stimulating factor (CSF), macrophage CSF and also IL-4 enhanced macrophage IL-10 responses after ingestion of Listeria in vitro. Finally, monoclonal antibody neutralization of IFN-gamma during infection of SCID mice with Listeria inhibited TNF-alpha mRNA, but augmented expression of IL-10 mRNA in infected tissues. These results demonstrate that exogenous Il-10 is a potent immunosuppressive cytokine in the context of infection with an intracellular bacterium and that expression of endogenous IL-10 versus TNF is differentially regulated by the cytokine environment of the macrophage.


Subject(s)
Interleukin-10/administration & dosage , Interleukin-10/pharmacology , Listeriosis/immunology , Animals , Cells, Cultured , Female , Immunity, Innate/drug effects , Interferon-gamma/biosynthesis , Interferon-gamma/drug effects , Interferon-gamma/pharmacology , Interleukin-10/biosynthesis , Interleukin-2/administration & dosage , Interleukin-2/pharmacology , Listeriosis/metabolism , Macrophage Activation/drug effects , Macrophages/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, SCID , Species Specificity , Spleen/cytology , Tumor Necrosis Factor-alpha/pharmacology
19.
Infect Immun ; 63(9): 3360-6, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7642263

ABSTRACT

Cryptococcus neoformans is an opportunistic pathogen in AIDS patients causing disseminated disease and lethal meningitis after inhalation of acapsular or sparsely encapsulated yeast cells. In this study we have investigated whether a recently described family of primitive opsonins, termed collectins, contribute to innate resistance against C. neoformans. The pulmonary surfactant proteins SP-A and SP-D as well as the serum collectins mannose-binding protein and CL-43 bound in a calcium-dependent manner to acapsular C. neoformans in vitro. Binding was concentration dependent and abolished by competition with defined mono- and oligosaccharides. In contrast, no binding of the collectins was observed with the encapsulated form of the yeast. Furthermore, binding of purified collectin SP-D, but not SP-A, mannose-binding protein, or CL-43, led to a concentration-dependent agglutination of acapsular C. neoformans. These data indicate that collectins recognize carbohydrate structures in the cell wall of an initial infectious form of C. neoformans and may play a role in early antifungal defenses in the lung.


Subject(s)
Agglutinins/physiology , Cryptococcus neoformans/immunology , Glycoproteins/physiology , Opsonin Proteins/metabolism , Pulmonary Surfactants/physiology , Humans , Proteolipids/physiology , Pulmonary Surfactant-Associated Protein A , Pulmonary Surfactant-Associated Protein D , Pulmonary Surfactant-Associated Proteins
20.
Parasite Immunol ; 17(9): 459-64, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8552414

ABSTRACT

This study was designed to investigate and characterize T-cell responses which lead to elimination of a primary infection of Cryptosporidium muris in BALB/c mice. The proliferative response of spleen cells to parasite antigen was measured by uptake of 3H-thymidine and, in parallel, supernatants were removed from cells to measure levels of IFN-gamma, TNF, IL-2 and IL-4 by ELISA. Oocyst excretion in faeces was first detected on day 10 post infection (p.i.); the level of shedding subsequently increased until day 14 and then declined until no oocysts were detected by day 25. The proliferative response of spleen cells from infected animals was similar to control levels up to day 14 p.i. but increased significantly on day 21 and was even greater on day 26. IFN-gamma and IL-2 were detected initially on day 14 p.i. and significantly higher concentrations were found on days 21 and 26. IL-4 secretion was also detected, but not until day 21 p.i., and production of TNF was not found at any time. Depletion of T-cells or CD4+ cells from spleen cells cultured with antigen resulted in a significant decrease in the levels of cytokine detected. These results indicated, therefore, that in BALB/c mice there was a correlation between the development of immunity to C. muris infection and both a parasite antigen-specific proliferative response and Th1 and Th2 cytokine production by spleen cells.


Subject(s)
Cryptosporidiosis/immunology , Cytokines/metabolism , Lymphocyte Activation , Th1 Cells/immunology , Th2 Cells/immunology , Animals , Cryptosporidium/immunology , Interferon-gamma/metabolism , Interleukin-2/metabolism , Interleukin-4/metabolism , Mice , Mice, Inbred BALB C , Tumor Necrosis Factor-alpha/metabolism
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