Toxoplasma gondii binds sheep prolactin.

Taking into account the literature reports on the involvement of prolactin (PRL) in the regulation of immunity against Toxoplasma gondii, we decided to check whether this parasite has the ability to bind the lactotrophic hormone. We examined T. gondii binding of sheep fluoresceine- and biotine-labeled prolactin isolated from pituitary (shPRL). In this work we announced for the first time that shPRL was bound to live tachyzoites of RH (type I) and ME49 (type II) strains. Furthermore, by use of competitive inhibition analysis, we confirmed that this binding was specific for both tested T. gondii strains.

Inhibitory effect of prolactin on Toxoplasma proliferation in peripheral blood mononuclear cells from patients with hyperprolactinemia.

Despite many years of studies on the mechanisms of immunological defence responses induced in host organisms by Toxoplasma, no satisfactory immunoprophylaxis or chemotherapy have yet been established for humans. Thus, alternative methods to prevent toxoplasmosis and to enhance the efficacy of currently used antitoxoplasmic drugs are under evaluation. In this work, we strove to determine the influence of human prolactin (endogenous present in serum--sPRL and recombinant--rhPRL) on the course of Toxoplasma infection of peripheral blood mononuclear cells (PBMC) originating from female hyperprolactinemia patients. This study revealed that exogenous rhPRL as well as autologous sPRL from inactivated sera significantly restricted intracellular growth of Toxoplasma in PBMC cultures. Moreover, analysis of IL-10 production by PBMC infected with Toxoplasma and cultured in the presence of sPRL showed a positive correlation between sPRL concentration and the level of IL-10. The obtained results could indicate the possible protective action of PRL in a host organism infected with Toxoplasma and suggest that a significant increase in the serum PRL level, during pregnancy for instance, might significantly limit the risk of Toxoplasma spreading and could play an important role in natural protection against toxoplasmosis. The mechanism of inhibitory effect of PRL needs further detailed study.

Tachyzoite-specific isoform of Toxoplasma gondii lactate dehydrogenase is the target antigen of a murine CD4(+) T-cell clone.

In two-dimensionally separated Toxoplasma gondii lysate, mouse Th1 clone 3Tx15 detects two proteins of apparent molecular weight 40000 and pI of 5.8 and 5.9. Microsequencing of peptide fragments from tryptic digestion of one of these proteins yielded partial sequences of T. gondii lactate dehydrogenase (LDH)1. As shown by Western blot, toxoplasmic LDH co-migrates in two-dimensional gel electrophoresis with both T-cell antigenic proteins. With synthetic peptides spanning the complete primary structure of T. gondii LDH1, the T-cell epitope was mapped to a nine amino acid partial sequence which exhibits a motif for binding to I-E(k), the class II restriction element of antigen recognition by clone 3Tx15. From the two known isoforms of T. gondii LDH, clone 3Tx15 specifically recognises tachyzoite LDH1, but not bradyzoite LDH2, as shown with the corresponding epitope peptides and recombinant proteins. Antigen-presenting cells infected with live bradyzoites stimulate 3Tx15 T cells, while killed bradyzoites provide no antigenic stimulus. This finding implies that a transformation into the tachyzoite stage occurs in cells challenged with bradyzoites. Although LDH1 represents one major constituent of the tachyzoite proteome, the protein does not seem to be immunogenic in T. gondii infection of mice. This is evident from the lack of serum anti-LDH immunoreactivity and the failure of adoptively transferred 3Tx15 T cells to protect against lethal challenge. In conclusion, a T-cell-stimulatory Toxoplasma antigen is identified by means of a novel, high-resolution T-cell blot technique, the clones antigenic fine specificity allowing detection of parasite-stage conversion.

Attenuation of mouse-virulent Toxoplasma gondii parasites is associated with a decrease in interleukin-12-inducing tachyzoite activity and reduced expression of actin, catalase and excretory proteins.

Determinants of Toxoplasma gondii virulence are still unknown, although genetic markers associated with T. gondii pathogenicity or host susceptibility to infection have been identified. To define indicator proteins of mouse virulence, type I strain parasites were attenuated by continuous passage in fibroblast culture and compared with the parental strain passaged in mice. The loss of acute virulence, evident by a 1000-fold higher pathogen dose causing 100% lethality in mice correlated with a less efficient infection of inflammatory cells at the site of inoculation, while parasite proliferation and invasiveness in vitro proved unimpaired. Infection with the attenuated parasites elicited earlier local interleukin-12 and strong interferon-gamma responses in vivo, although the activity that triggers interleukin-12 secretion in macrophages is reduced in the attenuated compared to the virulent strain variant. The interleukin-12-inducing T. gondii stimulus was identified as a protein(s) present in tachyzoite excretory products. Comparative proteome analysis combined with immunodetection and quantitation of a variety of T. gondii antigens indicated that the steady-state levels of actin, catalase, microneme protein 5, as well as dense granule proteins 1, 2, 3, 4, 5, 7, 8 and nucleoside triphosphate hydrolase 1 are decreased in the attenuated phenotype, whereas the surface antigen 1 and rhoptry protein 1 are produced at a similar level by virulent and attenuated parasites. In conclusion, these findings reveal a correlation between the efficient establishment of T. gondii infection in vivo and parasite synthesis of actin, catalase and several excretory proteins, and thus postulate a role for these molecules in acute virulence.

Towards the Toxoplasma gondii proteome: position of 13 parasite excretory antigens on a standardized map of two-dimensionally separated tachyzoite proteins.

High resolution two-dimensional separation of Toxoplasma gondii tachyzoite lysate revealed up to 224 distinct protein spots in Coomassie-stained gel. Computional matching of 14 digitized gels yielded a standard two-dimensional proteome map. The excretory T. gondii dense granule proteins GRA1-GRA8, S16/acid phosphatase, nucleoside triphosphate hydrolase, and H4 were identified by Western blotting of both total gel and isolated protein spots. In addition, two excretory antigens defined by parasite-specific monoclonal T cells, p36 and p40, were mapped by a novel T-cell blotting technique based on electroeluting single protein spots and testing the eluates for antigenic activity against the T-cell clones. In summary, these results represent a first step in Toxoplasma proteome analysis.

Serodiagnostics of Toxoplasma gondii invasions.

Diagnosis of T. gondii invasions is based mainly on serological tests, because of their relative simplicity and availability. In the present work we examined efficiency of immunoblot and two inhouse ELISA assays in anti-Toxoplasma antibody detection, comparing our results with those obtained using commercial ELISA kit of Organon Teknika. We found that inhouse ELISAs and immunoblot can be helpful in diagnosing toxoplasmosis.

[Immunity in Toxoplasma gondii infections]. / Odpornosc w zarazeniach wywolywanych przez Toxoplasma gondii.

The article presents selected data concerning pathogenesis, clinical manifestations, natural and specific resistance and vaccines against T. gondii infections. Toxoplasma gondii protozoan has been recognized as one of the most successful parasites infecting any nucleated cell of human individuals and most warm-blooded animals. The infection causes life-threatening disease in individuals with defective immunity such as fetuses, AIDS patients and transplant recipients. In immunocompetent humans toxoplasmosis is usually asymptomatic. Following an acute phase of infection characterized by systemic spread of rapidly dividing tachyzoites, bradyzoites encyst in various host tissues (brain, muscles) and may persist there lifelong. Toxoplasmosis in controlled by vigorous cell-mediated immune response capable of killing infected cells and parasites. As shown in the mouse experimental model natural killer cells (NK) are critical for the resistance at the early stage of primary infections, whereas adaptive immunity depends on T lymphocytes. Both CD4+ Th1 and CD8+ Tc1 lymphocytes are believed to mediate protection by producing of IFN-gamma, a pitoval cytokine that induces anti-Toxoplasma effector mechanisms in macrophages.

[Antigens of Toxoplasma gondii]. / Antygeny Toxoplasma gondii.

The paper presents antigenic structure of T. gondii tachyzoites and bradyzoites in respect of potential use of some chosen antigens for diagnostic and immunoprophylactic purposes.

[Identification of Toxoplasma gondii antigens recognized by specific T lymphocytes]. / Identyfikacja antygenów Toxoplasma gondii rozpoznawanych przez swoiste limfocyty T.

The paper presents chosen experimental strategies for identifying of Toxoplasma gondii antigens recognized by specific T lymphocytes.

[Antigens of Toxoplasma gondii]. / Antygeny Toxoplasma gondii.

The paper presents antigenic structure of T. gondii tachyzoites and bradyzoites in respect of potential use of some chosen antigens for diagnostic and immunoprophylactic purposes.

Expression, characterization and serological reactivity of a 41 kDa excreted-secreted antigen (ESA) from Toxoplasma gondii.

A Toxoplasma gondii tachyzoite expression library was screened with immune sera from T. gondii infected patients. Among others, one gene product reacted strongly with human sera and was further investigated. The gene called B10 was shown to encode a 41 kDa antigen. The complete genomic nucleotide sequence of the B10 protein has been analysed and was shown to contain one intron with conserved splice junctions. Southern blot analysis indicated that B10 is a single-copy gene. The corresponding 1.5 kb cDNA encodes a 318 amino acid sequence of mainly hydrophilic character with a putative signal sequence of 19 amino acids and no further trans-membrane domain. Immunofluorescence assays and immunoblots with a preparation of excreted-secreted antigens (ESA) suggested that the native protein is secreted into the parasitophorous vacuole and its delimiting membrane, indicating that B10 is a member of the ESA family of T. gondii. Recombinant B10 protein exhibited a strong reactivity with human serum samples both in ELISA and in immunoblots.

T cell receptor specificities of Toxoplasma gondii-reactive mouse CD4+ T lymphocytes and Th1 clones.

The Toxoplasma gondii-directed CD4+ T cell response in chronically infected mice was studied with respect to both T cell receptor diversity and antigen specificities. T cell receptor chains V beta 4, 6, 8, 10, and 14 were predominantly found on toxoplasma-reactive CD4+ splenocytes. This repertoire was also detected among T. gondii-specific CD4+ T cell clones. Analysis of clonotypic cytokine profiles revealed typical Th1 clones secreting interleukin-2, interferon-gamma and tumour necrosis factor activity and Th2 clones producing interleukin-4 and interleukin-10. Five distinct toxoplasma antigens (p26, p40, p55, p58 and p60) were detected in electrophoretically separated toxoplasma lysate by five individual Th1 clones. Parallel testing of CD4+ T lymphocytes from infected mice confirmed that these specificities constitute the peak immunogenic fractions of toxoplasma lysate. The expression patterns of two clonotypic, T cell-stimulatory parasite antigens were studied in detail. While p55 was expressed by mouse-virulent and avirulent T. gondii isolates and in both the tachyzoite and bradyzoite stages, p58 was detected only in virulent strains from intraspecies subgroup I. Thus, we describe the heterogeneity of toxoplasmic immunodominant T cell antigens including a 58-kDa group I-restricted molecule which may provide a marker for virulent isolates.

Murine Th1 clone defines a 60 kD tachyzoite antigen of Toxoplasma gondii.

Toxoplasma gondii-specific murine CD4+ T cell clone 3Tx9 belongs to the Th1 subtype by virtue of secreting high levels of interleukin(IL)-2, interferon-gamma and tumor necrosis factor without producing IL4 and IL10. To characterize the clonal antigen, Toxoplasma lysate-was separated by SDS-PAGE and probed in T cell blot analysis with 3Tx9 T cells, revealing a fraction of about 60 kD molecular weight. This fraction proved highly stimulatory also for CD4+ splenocytes isolated from infected mice. The expression pattern of the relevant 60 kD antigen was determined by challenge of clone 3Tx9 with T. gondii strains from all three intraspecies subgroups and tachyzoites versus bradyzoites isolated from two strains as a source of antigen. While the T cell clone reacted with tachyzoites of all strains tested, bradyzoites lacked antigenic activity. Parallel T cell blot and ELISA confirmed co-migration of the T cell-stimulatory antigen p60 and rhoptry proteins ROP1, ROP2,3,4, and ROP5 among which ROP1 is a molecule of similar size and has only been shown on tachyzoites. However, a ROP1 knock-out Toxoplasma mutant still had antigen activity for 3Tx9 T cells. Since the two known tachyzoite-specific proteins, surface antigens SAG1/p30 and SAG2/p22, have a much lower molecular weight, we suggest that p60 represents a new T. gondii tachyzoite marker which is defined by clone 3Tx9.

Detection of a novel 40,000 MW excretory Toxoplasma gondii antigen by murine Th1 clone which induces toxoplasmacidal activity when exposed to infected macrophages.

To analyse target molecules of the CD4+ T-cell response to toxoplasma infection, a panel of Toxoplasma gondii-specific murine CD4+ T-cell clones has been established. Clone 3Tx15, belonging to the T helper 1 (Th1) subtype, abolished intracellular parasite growth when co-cultured with macrophages and live toxoplasma at a ratio of 2:2:1. This effect results from macrophage toxoplasmicidal activity induced upon parasite-dependent cellular interaction, an irrelevant Th1 clone failed in this three-party system. Clone 3Tx15 detects its corresponding antigen in the supernatant of infected cells and also reacts with a host cell-free preparation of T. gondii-excreted/secreted antigens. T-cell blot analysis of two-dimensionally separated toxoplasma lysate revealed a molecular weight of about 40,000 for the fractions stimulating clone 3Tx15. As checked in parallel enzyme-linked immunosorbent assay, the 40,000 MW T-cell antigen co-migrates with the excretory protein GRA4, the sole 40,000 MW T. gondii antigen hitherto known to be recognized by T lymphocytes. Nevertheless, neither recombinant GRA4 nor immunoaffinity-purified natural GRA4 was stimulatory for clone 3Tx15. Our findings thus demonstrate that Th1 clone 3Tx15 which induces toxoplasmicidal activity during antigenic interaction with infected macrophages defines a new 40,000 MW excretory T. gondii antigen.

The cells of monocyte-macrophage lineage in mice with the 2-month polyethylene implantation.

End-point-attached heparinized polyethylene (H-PE) was implanted for 2 months into the peritoneum of C57B1/6 mice. The proliferation of bone marrow cells (BMCs) from implanted and non-implanted mice was investigated in M-CSF supplemented medium, in the presence or absence of macrophage-specific monoclonal antibodies (mAbs). The mAb HC 7.67.B, recognizing a surface determinant on immature monocytoid cells, inhibited the proliferation of BMCs from H-PE implanted mice without any influence on the proliferation of BMCs from non-implanted animals. The peritoneal macrophages from H-PE implanted mice demonstrated enhanced production of fibronectin (Fn) in comparison to the macrophages from non-implanted animals. Our results suggest changes in the differentiation of murine monocyte-macrophage lineage in the mice bearing H-PE implants for 2 months.

Administration of IL-12 during ongoing immune responses fails to permanently suppress and can even enhance the synthesis of antigen-specific IgE.

The synthesis of antibodies of the IgE isotype in mice largely depends on IL-4, a cytokine that is released by T lymphocytes of the Th2 subtype. IL-12 is a cytokine considered to direct Th cell development into a Th1 direction and to suppress Th2 responses including the synthesis of IgE. Here we report about the influence of IL-12 on the IgE responses of mice immunized with protein antigens adsorbed to aluminum hydroxide. To avoid problems with the detection of IgE caused by an excess of competitive IgG antibodies produced in IL-12-treated mice, serum IgE was first extracted from the serum by plate-bound anti-IgE mAb and then determined either as total IgE or as antigen-specific IgE by using biotinylated anti-IgE or biotinylated antigen. Depending on the strain of mice and the dose of IL-12 injected together with the antigen, IL-12 can either temporarily suppress or augment the synthesis of (antigen-specific) IgE antibodies. This applies for CBA/J mice immunized six times in biweekly intervals with minute (0.1 micrograms/injection) or three-times with large (5 micrograms/injection) amounts of the bee venom allergen phospholipase A2 (PLA2). Under both conditions the antibody response is characterized by the production of predominantly IgG1 as well as IgE but very little IgG2a, IgG2b and IgG3 antibodies. Simultaneous application of low doses of IL-12 (1 or 10 ng/day) led to a 2- to 4-fold enhancement of IgE production (PLA2-specific IgE or total IgE). Only a high dose of 1 micrograms IL-12/day resulted in a 3- to 10-fold reduction of the IgE response. This suppression was not stable, however, because the synthesis of IgE antibodies was stimulated to a high level when these mice subsequently received a second course of immunizations in the absence of IL-12. Likewise, the synthesis of IgE was only temporarily suppressed by IL-12 treatment in CBA/J mice immunized with keyhole limpet hemocyanin (KLH) as antigen. However, application of low (10 ng/day) or high (1 microgram/day) doses of IL-12 during the primary course of immunizations of CBA/J mice with KLH suppressed the IgE response slightly or strongly respectively. In striking contrast, the KLH-specific IgE response of BALB/c mice was upregulated even when high doses of IL-12 (1 microgram/day) were injected simultaneously with the immunizations. Thus, these results demonstrate a great variability regarding the influence of IL-12 treatment on ongoing IgE responses in vivo.

Interleukin-12 profoundly up-regulates the synthesis of antigen-specific complement-fixing IgG2a, IgG2b and IgG3 antibody subclasses in vivo.

The influence of the cytokine interleukin-12 (IL-12) on humoral immune responses was studied in vivo. CBA/J mice immunized with protein antigens (keyhole limpet hemocyanin, phospholipase A2) adsorbed to aluminum hydroxide (Alum) develop a Th2-like immune response characterized by the production of large amounts of IgG1 as well as some IgE but little IgG2a, IgG2b and IgG3 antibodies. IL-12 is a cytokine that promotes the development and the activation of Th1 cells. Th1 cells are involved in the induction of cellular immunity, which is characterized by low or absent antibody production. On the other hand, some Th1-like immune responses are associated with a strong antibody production of the IgG2a, IgG2b and IgG3 subclasses. Thus, we investigated whether treatment with IL-12 would down-regulate the humoral immune response or stimulate antibody production of the IgG2a, IgG2b and IgG3 subclasses. We observed that: 1) administration of IL-12 to mice together with protein antigens adsorbed to Alum strongly enhanced the humoral immune response by increasing the synthesis of antigen-specific antibodies of the IgG2a, IgG2b and IgG3 subclasses 10- to 1000-fold. The synthesis of IgG1 was not or only slightly (2-5-fold) enhanced, whereas that of the IgE isotype was suppressed. 2) These effects of IL-12 were observed when high (10 micrograms, 100 micrograms) or low doses (0.1 microgram) of antigen were used for immunization. 3) Titration of IL-12 in vitro revealed that IgG2a is strongly up-regulated over a wide dose range of IL-12 (10 to 1000 ng/day). 4) The effects of IL-12 in vivo are at least partially interferon (IFN)-gamma-dependent because an anti-IFN-gamma mAb in combination with IL-12 prevented most of the enhanced IgG2a production. 5) Mice receiving IL-12 showed a strong up-regulation of IFN-gamma but no inhibition of IL-5 synthesis by spleen cells activated ex vivo with antigen. These results suggest that IL-12 is a potent adjuvant for enhancing humoral immunity to protein antigens adsorbed to Alum, primarily by inducing the synthesis of the complement-fixing IgG subclasses 2a, 2b and 3.

Cellular immune response to Listeria in genetically resistant and susceptible mouse strains.

Relatively Listeria-resistant C57B1/6 mice and more susceptible to the infection DBA/2 mice were immunized with Listeria-antigen (LA). Immunized DBA/2 mice developed weaker delayed hypersensitivity to LA and still eliminated listeriae less effectively than identically immunized C57B1/6 mice. An accessory function of LA-pulsed macrophages of normal C57B1/6 mice was only slightly enhanced as compared with LA-pulsed macrophages of DBA/2 mice. It is suggested that some suppressor lymphocytes, IgM+ and/or FcR+, could be responsible for the enhanced susceptibility of DBA/2 mice to listeriosis.

Innate anti-listerial resistance of mice differing in their susceptibility to listeriosis.

The work was designated to compare the influence of an active immunization on the expression of anti-listerial resistance of relatively resistant to listeriosis C57B1/6 mice as compared with more susceptible to the infection DBA/2 mice. Although, specific immunization of DBA/2 mice enhanced their anti-listerial resistance but immunized DBA/2 mice still eliminated Listeria rods less effectively than immunized C57B1/6 mice. It means that innate difference in anti-listerial resistance between C57B1/6 and DBA/2 mice was maintained after immunizing them with the same number of alive bacteria. Greater anti-listerial resistance of C57B1/6 versus DBA/2 mice is associated with an increased accumulation of inflammatory Ms and PMNs in their peritonea and an increased capacity of their PMNs to restrict Listeria growth.

Phagocytosis and killing of Listeria by guinea pig macrophages and neutrophils.

The rate of the phagocytosis and intracellular killing of Listeria monocytogenes by guinea pig macrophages and neutrophils in vitro was determined. The anti-bacterial activity of the phagocytes against virulent Listeria monocytogenes was compared with their activity against avirulent strain of Listeria and Proteus mirabilis. It is suggested that the contribution of the macrophages and the neutrophils to anti-bacterial protection can depend on physiological state of bacteria.