Immune deficiencies and parasitic diseases.
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Animals↗
Biomedical subjects
C A Hunter
Publications and source records attributed to C A Hunter.
Intestinal pathology during acute toxoplasmosis is IL-4 dependent and unrelated to parasite burden.
The role of interleukin-4 (IL-4) during the course of Toxoplasma gondii infection was studied using IL-4-/- mice and their wild-type (WT) counterparts on a C57BL/6 background. Following oral infection with T. gondii tissue cysts an exacerbative role for IL-4 was demonstrated and IL-4-/- mice were found to be more resistant to infection than WT mice as measured by significantly reduced mortality. Furthermore pathology in the small intestine was less severe in IL-4-/- mice although conversely liver pathology was greater than in wild-type mice. Significantly, plasma IL-12 and IFN-gamma levels, which peaked at days 6 and 8, respectively, were higher in IL-4-/- mice. The exacerbatory role of IL-4 in the intestine was found by competitive RT-PCR not to be associated with increased parasite burdens but was related to comparative expression of IL-10.
Acute Disease↗
IL-10 is not required to prevent immune hyperactivity during memory responses to Toxoplasma gondii.
Primary infection of IL-10 knockout (KO) mice with the protozoan parasite Toxoplasma gondii leads to a CD4(+)-T-cell dependent shock-like reaction with high systemic levels of IL-12 and IFN-gamma, severe liver pathology and death of mice. In the present study, this immune-mediated pathology was prevented by treatment of IL-10 KO mice with the anti-parasitic drug sulfadiazine, allowing these mice to progress to the chronic phase of infection. To address the role of endogenous IL-10 in the regulation of secondary immune responses to T. gondii, IL-10 KO mice were infected with the avirulent Me49 strain of this parasite, treated with sulfadiazine for 2 weeks starting at day 3 p.i., and were rechallenged 6 weeks p.i. with RH, a highly virulent strain of T. gondii. In these studies, chronically infected IL-10 KO mice survived secondary infection with RH and controlled parasite load. Although serum levels of IL-12 and IFN-gamma were higher in IL-10 KO mice than in wild type (WT) mice 8 days after RH rechallenge, these levels were well controlled in the absence of endogenous IL-10, suggesting that IL-10 is not required to down-regulate cytokine production during the memory response. Antigen-specific ex vivo recall responses further revealed that splenocytes from chronically infected WT and IL-10 KO mice responded to parasite antigen with similar production of IL-12 and IFN-gamma, and there was also no significant difference in ex vivo production of these cytokines by splenocytes in response to parasite antigen 7 days after secondary infection with T. gondii. Furthermore, IL-10 KO mice immunized with the Ts-4 vaccine-strain of T. gondii were protected when rechallenged with the virulent RH strain. Together, these studies demonstrate that the inhibitory effect of IL-10, which is required to prevent immune-mediated pathology during primary infection, is not required to prevent immune hyperactivity during a secondary response to T. gondii, and a highly effective memory response is generated in the absence of endogenous IL-10.
Animals↗
Suppression of NF-kappaB activation by infection with Toxoplasma gondii.
The interaction of host cells with microbial products or their invasion by pathogens frequently results in activation of the NF-kappaB family of transcription factors. The studies presented here reveal that in vivo, infection with Toxoplasma gondii results in the activation of NF-kappaB. To determine whether host cells could activate NF-kappaB in response to invasion by T. gondii, Western blots, immunofluorescence, and electrophoretic mobility shift assays were used to assess the response of host cells to infection. In these studies, infection of macrophages or fibroblasts with T. gondii did not result in the activation of NF-kappaB. In addition, the ability of lipopolysaccharide to activate NF-kappaB was impaired in cultures of macrophages infected with T. gondii. Together, these data demonstrate that invasion of cells by T. gondii does not lead to the activation of NF-kappaB and suggest that the parasite may actively interfere with the pathways that lead to NF-kappaB activation.
Active Transport, Cell Nucleus↗
Host-pathogen interactions: subversion and utilization of the NF-kappa B pathway during infection.
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Animals↗
Quantitative measurements of edge-to-face aromatic interactions by using chemical double-mutant cycles.
Synthetic H-bonded zipper complexes have been used to quantify the magnitude of an edge-to-face aromatic interaction between a benzoyl group and an aniline ring. Four chemical double-mutant cycles were constructed by using a matrix of nine closely related complexes in which the aromatic rings were sequentially substituted for alkyl substituents. The stability constants and three-dimensional structures of the complexes were determined by using 1H NMR titrations in deuterochloroform at room temperature. The value of the interaction energy is similar in all cases, the average is -1.4 +/- 0.5 kJ mol(-1). The scope and limitations of the double-mutant approach are explored, and the consequences of conformational equilibria are discussed.
Journal Article↗
A supramolecular system for quantifying aromatic stacking interactions.
A supramolecular complex for investigating the thermodynamic properties of intermolecular aromatic stacking interactions has been developed. The conformation of the complex is locked in a single well-defined conformation by an array of H-bonding interactions that force two aromatic rings on one end of the complex into a stacked geometry. Chemical double-mutant cycles have been used to measure an anthracene-aniline interaction (+0.6 +/- 0.8 kJ mol(-1)) and a pentafluorophenyl-aniline interaction (-0.4 +/- 0.9 kJ mol(-1)) in this system. Although the interactions are very weak, the pentafluorophenyl interaction is attractive, whereas the anthracene interaction is repulsive: this is consistent with the dominance of pi-electron electrostatic interactions. The nitropyrrole subunits used to control the conformation of these complexes lead to problems of aggregation and multiple conformational equilibria. The implications for the thermodynamic analysis are examined in detail, and the double-mutant-cycle approach is found to be remarkably robust with respect to such effects, since systematic errors in individual experiments are removed in a pair-wise fashion when the cycle is constructed.
Journal Article↗
Quantitative determination of intermolecular interactions with fluorinated aromatic rings.
The chemical double mutant cycle approach has been used to investigate substituent effects on intermolecular interactions between aromatic rings and pentafluorophenyl pi-systems. The complexes have been characterised using 1H and 19F NMR titrations, X-ray crystal structures of model compounds and molecular mechanics calculations. In the molecular zipper system used for these experiments, H-bonds and the geometries of the interacting surfaces favour the approach of the edge of the aromatic ring with the face of the pentafluorophenyl pi-system. The interactions are generally repulsive and this repulsion increases with more electron-withdrawing substituents up to a limit of +2.2 kJ mol(-1), when the complex distorts to minimise the unfavourable interaction. Strongly electron-donating groups cause a change in the geometry of the aromatic interaction and attractive stacking interactions are found (-1.6 kJ mol(-1) for NMe2). These results are generally consistent with an electrostatic model: the polarisation of the pentafluorophenyl ring leads to a partial positive charge located at the centre and this leads to repulsive interactions with the positive charges on the protons on the edge of the aromatic ring; when the aromatic ring has a high pi-electron density there is a large electrostatic driving force in favour of the stacked geometry which places this pi-electron density over the centre of the positive charge on the pentafluorophenyl group.
Crystallography, X-Ray↗
Sequence-structure relationships in DNA oligomers: a computational approach.
A collective-variable model for DNA structure is used to predict the conformation of a set of 30 octamer, decamer, and dodecamer oligomers for which high-resolution crystal structures are available. The model combines an all-atom base pair representation with an empirical backbone, emphasizing the role of base stacking in fixing sequence-dependent structure. We are able to reproduce trends in roll and twist to within 5 degrees across a large database of both A- and B-DNA oligomers. A genetic algorithm approach is used to search for global minimum structures and this is augmented by a grid search to identify local minimums. We find that the number of local minimums is highly sequence dependent, with certain sequences having a set of minimums that span the entire range between canonical A- and B-DNA conformations. Although the global minimum does not always agree with the crystal structure, for 24 of the 30 oligomers, we find low-energy local minimums that match the experimental step parameters. Discrepancies throw some light on the role of crystal packing in determining the solid-state conformation of double-helical DNA.
Algorithms↗
Knot tied around an octahedral metal centre.
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Biopolymers↗
Cutting edge: systemic inhibition of angiogenesis underlies resistance to tumors during acute toxoplasmosis.
The ability of various infections to suppress neoplastic growth has been well documented. This phenomenon has been traditionally attributed to infection-induced concomitant, cell-mediated antitumor immunity. We found that infection with Toxoplasma gondii effectively blocked neoplastic growth of a nonimmunogenic B16.F10 melanoma. Moreover, this effect was independent of cytotoxic T or NK cells, production of NO by macrophages, or the function of the cytokines IL-12 and TNF-alpha. These findings suggested that antitumor cytotoxicity was not the primary mechanism of resistance. However, infection was accompanied by strong, systemic suppression of angiogenesis, both in a model system and inside the nascent tumor. This suppression resulted in severe hypoxia and avascular necrosis that are incompatible with progressive neoplastic growth. Our results identify the suppression of tumor neovascularization as a novel mechanism critical for infection-induced resistance to tumors.
Acute Disease↗
A role for CD44 in the production of IFN-gamma and immunopathology during infection with Toxoplasma gondii.
The interaction of activated CD44 with its ligand, low m.w. hyaluronan, is involved in inflammation, but no role has been identified for this interaction in the regulation of an immune response to infection. In these studies, infection of C57BL/6 mice with Toxoplasma gondii resulted in increased expression of CD44 on T cells, B cells, NK cells, and macrophages, and a small percentage of CD4(+) T cells express an activated form of CD44. Administration of anti-CD44 to infected mice prevented the development of a CD4(+) T cell-dependent, infection-induced inflammatory response in the small intestine characterized by the overproduction of IFN-gamma. The protective effect of anti-CD44 treatment was associated with reduced production of IFN-gamma, but not IL-12, in vivo and in vitro. Furthermore, the addition of low m.w. hyaluronan to cultures of splenocytes or purified CD4(+) T cells from infected mice resulted in the production of high levels of IFN-gamma, which was dependent on IL-12 and TCR stimulation. Together, these results identify a novel role for CD44 in the regulation of IFN-gamma production by CD4(+) T cells during infection and demonstrate a role for CD44 in the regulation of infection-induced immune pathology.
Adjuvants, Immunologic↗
Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens.
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.
Animals↗
IL-10 mediates susceptibility to Leishmania donovani infection.
Human visceral leishmaniasis (VL) results in a severe and potentially fatal systemic disease, accompanied by cellular immune depression. The production of IL-10 correlates with ongoing disease and it has been suggested that the cellular immune depression that accompanies active disease may be due to a predominance of IL-10 production rather than a lack of IFN-gamma production, which is essential for optimal macrophage activation and parasite elimination. To examine the role of IL-10 in resistance during L. donovani infection (a causative agent of VL), the course of infection was examined in mice lacking the gene for IL-10. BALB/c IL-10-/-, as well as C57BL/6 IL-10-/- mice, were highly resistant to L. donovani infection, as evidenced by liver parasite burdens which were tenfold lower than those in control mice after 14 days of infection. Enhanced resistance was accompanied by increased production of IFN-gamma and nitric oxide in BALB/c IL-10-/- mice. Susceptibility to infection in BALB/c IL-10-/- mice was enhanced following in vivo treatment with a neutralizing antibody to IFN-gamma or IL-12. Together these studies demonstrate for the first time that IL-10 is a critical component of the immune response that inhibits resistance to L. donovani.
Animals↗
Interleukin-10 does not contribute to the pathogenesis of a virulent strain of Toxoplasma gondii.
Interleukin (IL)-10 is an inhibitor of cell mediated immunity and an antagonist of the development of protective immune responses associated with resistance to T. gondii. These observations led to the hypothesis that the production of IL-10 could contribute to the ability of T. gondii to replicate and survive in an immune competent host. To determine whether the production of IL-10 affects the ability of the RH strain of T. gondii to cause a lethal infection in mice, we compared the immune response to RH in IL-10+/+ and IL-10-/- BALB/c mice. Both groups of mice produced comparable amounts of IL-12 and interferon (IFN)-gamma and had similar mortality curves and parasite burdens. The use of green fluorescent protein-labelled parasites allowed us to infect IL-10+/+ and IL-10-/- mice and use a fluorescence-activated cell sorter to distinguish infected and uninfected populations of macrophages and compare their expression of CD80, CD86 and major histocompatibility complex (MHC) class II. Although infected cells expressed higher overall levels of these molecules than uninfected cells, there were no differences between cells isolated from IL-10+/+ and IL-10-/- mice. Taken together, these results indicate that IL-10 is not required for the virulence of the RH strain of T. gondii, nor is it involved in the regulation of the CD80, CD86 and MHC class II molecules during RH-infection.
Animals↗
Dichloroacetate improves cardiac efficiency after ischemia independent of changes in mitochondrial proton leak.
Dichloroacetate (DCA) is a pyruvate dehydrogenase activator that increases cardiac efficiency during reperfusion of ischemic hearts. We determined whether DCA increases efficiency of mitochondrial ATP production by measuring proton leak in mitochondria from isolated working rat hearts subjected to 30 min of ischemia and 60 min of reperfusion. In untreated hearts, cardiac work and efficiency decreased during reperfusion to 26% and 40% of preischemic values, respectively. Membrane potential was significantly lower in mitochondria from reperfused (175.6 +/- 2.2 mV) versus aerobic (185.8 +/- 3.1 mV) hearts. DCA (1 mM added at reperfusion) improved recovery of cardiac work (1.9-fold) and efficiency (1.5-fold) but had no effect on mitochondrial membrane potential (170.6 +/- 2.9 mV). At the maximal attainable membrane potential, O(2) consumption (nmol O(2) x mg(-1) x min(-1)) did not differ between untreated or DCA-treated hearts (128.3 +/- 7.5 and 120.6 +/- 7.6, respectively) but was significantly greater than aerobic hearts (76.6 +/- 7.6). During reperfusion, DCA increased glucose oxidation 2.5-fold and decreased H(+) production from glucose metabolism to 53% of untreated hearts. Because H(+) production decreases cardiac efficiency, we suggest that DCA increases cardiac efficiency during reperfusion of ischemic hearts by increasing the efficiency of ATP use and not by increasing the efficiency of ATP production.
Adenosine Triphosphate↗
Identification of a role for NF-kappa B2 in the regulation of apoptosis and in maintenance of T cell-mediated immunity to Toxoplasma gondii.
The NF-kappaB family of transcription factors are involved in the regulation of innate and adaptive immune functions associated with resistance to infection. To assess the role of NF-kappaB(2) in the regulation of cell-mediated immunity, mice deficient in the NF-kappaB(2) gene (NF-kappaB(2)(-/-)) were challenged with the intracellular parasite Toxoplasma gondii. Resistance to this opportunistic pathogen is dependent on the production of IL-12, which is required for the development of innate NK cell and adaptive T cell responses dominated by the production of IFN-gamma necessary to control replication of this parasite. Although wild-type controls were resistant to T. gondii, NF-kappaB(2)(-/-) mice developed severe toxoplasmic encephalitis and succumbed to disease between 3 and 10 wk following infection. However, NF-kappaB(2) was not required for the ability of macrophages to produce IL-12 or to inhibit parasite replication and during the acute stage of infection, NF-kappaB(2)(-/-) mice had no defect in their ability to produce IL-12 or IFN-gamma and infection-induced NK cell responses appeared normal. In contrast, during the chronic phase of the infection, susceptibility of NF-kappaB(2)(-/-) mice to toxoplasmic encephalitis was associated with a reduced capacity of their splenocytes to produce IFN-gamma associated with a loss of CD4(+) and CD8(+) T cells. This loss of T cells correlated with increased levels of apoptosis and with elevated expression of the pro-apoptotic molecule Fas by T cells from infected NF-kappaB(2)(-/-) mice. Together, these results suggest a role for NF-kappaB(2) in the regulation of lymphocyte apoptosis and a unique role for this transcription factor in maintenance of T cell responses required for long-term resistance to T. gondii.
Animals↗
Identification of STAT4-dependent and independent mechanisms of resistance to Toxoplasma gondii.
The capacity of IL-12 to stimulate T and NK cell production of IFN-gamma is required for resistance to Toxoplasma gondii. To identify the transcription factors involved in this mechanism of resistance, mice deficient in STAT4, a protein involved in IL-12 signaling, were infected with T. gondii and their immune responses were analyzed. STAT4-/- mice were unable to control parasite replication and died during the acute phase of infection, whereas wild-type mice controlled parasite replication and survived this challenge. The susceptibility of STAT4-/- mice to toxoplasmosis correlated with a defect in their ability to produce IFN-gamma in response to infection, whereas administration of IFN-gamma to these mice inhibited parasite replication and delayed time to death. Interestingly, analysis of infected STAT4-/- mice revealed that these mice did produce low levels of IFN-gamma during infection, and the ability of splenocytes from infected or uninfected STAT4-/- mice to produce IFN-gamma was enhanced by the addition of IL-2 plus IL-18. Moreover, administration of IL-2 plus IL-18 to STAT4-/- mice resulted in elevated serum levels of IFN-gamma associated with a decreased parasite burden and delayed time to death. In vivo depletion studies demonstrated that the ability of IL-2 plus IL-18 to mediate STAT4-independent resistance to T. gondii is dependent on NK cell production of IFN-gamma. Together, these studies identify STAT4 as an important transcription factor required for development of the innate NK and adaptive T cell responses necessary for resistance to T. gondii. However, other signaling pathways can be used to bypass STAT4-dependent production of IFN-gamma and enhance innate resistance to T. gondii.
Adjuvants, Immunologic↗