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Purpose: Cryptogenic epilepsy, the group of epilepsy syndromes for which an etiology is unknown, comprises approximately 20% of all epilepsy syndromes. We selected patients in this subgroup of epilepsy and tested them for evidence of Toxoplasma gondii IgG antibodies by the enzyme-linked immunosorbent assay. T. gondii is found in up to 20% of the U.S. population forming dormant brain cysts in the latent bradyzoite form. We investigated the hypothesis that dormant T. gondii infection might be associated with cryptogenic epilepsy., Methods: We selected patients with cryptogenic epilepsies and tested them for evidence of T. gondii IgG antibodies by the enzyme-linked immunosorbent assay. A control group was also tested for comparison., Results: We have found a statistically-significant elevation of T. gondii antibodies among cryptogenic epilepsy patients as compared to controls [59% increase in optical density (OD), p = 0.013]. This association persisted after adjustment for subjects' gender and age in a multiple logistic regression model; however, it was no longer as statistically significant., Conclusions: Our results suggest that chronic T. gondii infection with brain cysts may be a cause of cryptogenic epilepsy.

The nucleoside triphosphate hydrolase of Toxoplasma gondii is a potent apyrase that is secreted into the parasitophorous vacuole where it appears to be essentially inactive in an oxidized form. Recent evidence shows that nucleoside triphosphate hydrolase can be activated by dithiothreitol in vivo. On reduction of the enzyme, there is a rapid depletion of host cell ATP. Previous results also demonstrate a dithiothreitol induced egress of parasites from the host cell with a concurrent Ca2+ flux, postulated to be a consequence of the release of ATP-dependent Ca2+ stores within the tubulovesicular network of the parasitophorous vacuole. Reduction of the nucleoside triphosphate hydrolase appears crucial for its activation; however, the exact mechanism of reduction/activation has not been determined. Using a variety of techniques, we show here that glutathione promoters activate a Ca2+ flux and decrease ATP levels in infected human fibroblasts. We further show the in vitro activation of nucleoside triphosphate hydrolase by endogenous reducing agents, one of which we postulate might be secreted into the PV by T. gondii. Our findings suggest that the reduction of the parasite nucleoside triphosphate hydrolase, and ultimately parasite egress, is under the control of the parasites themselves.

Background & Aims: Acute inflammatory ileitis occurs in susceptible (C57BL/6) mice after oral infection with Toxoplasma gondii. Overproduction of interferon (IFN)-gamma and synthesis of nitric oxide mediate the inflammation. We evaluated the role of transforming growth factor (TGF)-beta produced by intraepithelial lymphocytes (IELs) in this process., Methods: We analyzed the histologic and immunologic consequences of adoptive transfer of antigen-primed IELs into susceptible mice treated with anti-TGF-beta before oral challenge with T. gondii cysts. An in vitro coculture of enterocytes and IELs assessed the production of chemokines and cytokines in the presence of anti-TGF-beta., Results: Antigen-primed IELs prevent acute ileitis in susceptible mice that is reversed with anti-TGF-beta. Resistant mice (CBA/J) develop ileitis after treatment with anti-TGF-beta. Antigen-primed IELs can induce systemic immunosuppression as measured by depressed IFN-gamma production. In vitro, primed IELs reduce the production of inflammatory chemokines by infected enterocytes and IFN-gamma by splenocytes., Conclusions: Regulation of the ileal inflammatory process resulting from T. gondii is dependent on TGF-beta-producing IELs. The IELs are an essential component in gut homeostasis after oral infection with this parasite.

In this study, we report that infection with Plasmodium yoelii 17XL, a lethal strain of rodent malaria, does not result in death in the DBA/2 strain of mice. In contrast to BALB/c mice, DBA/2 mice developed significantly less parasitemia and never manifested symptoms of cerebral malaria (CM) on infection with this parasite. Moreover, the histological changes evident in the brain of susceptible BALB/c were absent in DBA/2 mice. Interestingly, the resistant DBA/2 mice when treated with recombinant interleukin (IL)-2, were found to develop CM symptoms and the infection became fatal by 6 to 8 days after infection. This condition was associated with an augmented interferon-gamma and nitric oxide production. Unexpectedly, IL-10 levels were also elevated in IL-2-treated DBA/2 mice during late stage of infection (at day 6 of infection) whereas the inverse relationship between IL-10 and interferon-gamma or nitric oxide was maintained in the early stage of infection (at day 3 after infection). The level of tumor necrosis factor-alpha production was moderately increased in the late phase of infection in these mice. Histology of brain from IL-2-treated mice demonstrated the presence of parasitized erythrocytes and infiltration of lymphocytes in cerebral vessels, and also displayed some signs of endothelial degeneration. Confocal microscopical studies demonstrated preferential accumulation of gammadelta T cells in the cerebral vessels of IL-2-treated and -infected mice but not in mice treated with IL-2 alone. The cells recruited in the brain were activated because they demonstrated expression of CD25 (IL-2R) and CD54 (intercellular adhesion molecule 1) molecules. Administration of anti-gammadelta mAb prevented development of CM in IL-2-treated mice until day 18 after infection whereas mice treated with control antibody showed CM symptoms by day 6 after infection. The information concerning creating pathological sequelae and death in an otherwise resistant mouse strain provides an interesting focus for the burden of pathological attributes on death in an infectious disease.

Objective: The aim was to determine the ability of macrophage-activated killer cells (MAK cells) obtained from peripheral blood of normal volunteers to kill glioblastoma multiforme (GBM) cell lines. Another goal was to investigate whether a bispecific antibody (bsAb) MDX-447, recognizing the high-affinity Fc receptor for IgG (FcgammaRI) and epidermal growth factor receptor (EGFR), would enhance MAK cell tumoricidal activity., Methods: Monocytes, from leukapheresis product, were isolated by countercurrent elutriation and differentiated into MAK cells by culture with granulocyte/macrophage-colony-stimulating factor, vitamin D3 and interferon gamma. Cells were checked for sterility, endotoxin and phenotypic markers. MAK cell functional activity was measured by a flow-cytometric phagocytosis assay. Target cells, a carcinoma cell line and two glioma cell lines expressing EGFR, were stained with PKH-26. MAK cells were labeled with fluorescein-conjugated anti-CD14. Combined effectors, targets and bsAb were incubated and the percentage of MAK cells with phagocytosed targets was determined by flow cytometry., Conclusion: We demonstrate that a large number of highly purified monocytes, isolated from peripheral blood, can be differentiated into MAK cells for use as an adjuvant for cancer treatment. After culture these cells are sterile, endotoxin-free and comprise more than 95% MAK cells. Increased amounts of CD14, CD64 and HLA-DR, which are characteristics of macrophage activation, were expressed. MAK cells were extremely phagocytic in comparison to monocytes, even in the absence of bsAb. Moreover, bsAb enhanced the tumoricidal activity of elutriated MAK cells targeted against GBM cell lines. Therefore, intracavity MAK cells armed with MDX-447 could be an effective adoptive immunotherapy for EGFR-positive GBM.

To assess the relationship between the changes of cellular components and the production of Th1 cytokine in the immune tissue, inbred C57BL/6 mice were orally infected with 40 cysts of 76K strain of Toxoplasma gondii. The sequential change of cell differentials and IFN-gamma production of splenocytes were analyzed by Diff-Quik stain and RT-PCR. There were no significant proportional changes of cellular components of splenocytes until day 4 postinfection (PI) as compared to those of day 0, and the relative percentage of macrophages and neutrophils/eosinophils increased significantly (p < 0.01) thereafter. The expression of IFN-gamma mRNA of CD3- cells was observed from day 1 PI at a low level. However, IFN-gamma production of CD3+ cells increased significantly from day 4 PI (p < 0.01) which progressively increased thereafter. These findings provide the relative percentages of granulocytes and macrophages were increased in conjunction with increase of total number of splenocytes after oral infection with T. gondii in the susceptible murine hosts, and lymphocytes were the major cellular components and the important source of IFN-gamma.

In this study, we examined whether the PTN strain (isolated from an AIDS patient) of Toxoplasma gondii could induce cross-protection in mice against infection with a lethal dose of the PLK strain. Mice were first infected with tachyzoites (5 x 10(5)) of PTN and 5 days later challenged with PLK (1 x 10(5), LD(90)) parasites. None of these mice succumbed to infection until day 21 after infection, whereas 100% of the mice given the same dose of PLK infection alone died between 5 and 11 days after infection. The protection was accompanied by an increased expansion of NK cells and CD4 + T cells. This condition was associated by increased production of IFN-gamma and an augmented number of IFN-gamma-producing cells in the spleen. Further, PTN + PLK-infected mice showed higher production of TNF-alpha and nitrite compared to PLK-infected mice. Mice infected with the PTN strain had an enhanced capacity to activate the immune system early in infection since they produced higher levels of IFN-gamma, TNF-alpha, and NO than PLK-infected mice. Administration of anti-IFN-gamma mAb or anti-asialo GM1 antibody resulted in 100 and 20% mortality, respectively, in PTN-infected mice but no death in PTN + PLK-infected mice. Together, these results suggest that early production of IFN-gamma and NK-cell activity is important in protection against PTN infection, whereas in PTN + PLK infection components of adaptive immunity rapidly developed following elaboration of an effective early innate immune response., (Copyright 1999 Academic Press.)

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.

In this study, we describe protection of BALB/c mice by immunization with heat-killed T. gondii tachyzoites against infection with Plasmodium yoelii 17XL which causes cerebral malaria and death in mice by day 7-8 post infection. Immunization resulted significant reduction in parasitemia at the peak period of infection. Protection induced by heat-killed T. gondii was associated with marked increase in NK cell number and IFN-gamma mRNA expression early in the infection. The level of IFN-gamma or TNF-alpha was found to diminish in T. gondii-treated mice as the infection progressed to the late stage. This declined response of IFN-gamma or TNF-alpha was associated with marked increase in the expression of IL-10, a counterregulatory cytokine. Pretreatment of mice with live T. gondii induced poor level of protection as compared with that of heat-killed parasites. Mice that received P. yoelii infection alone, had an elevated IFN-gamma response in the late stage of infection. Development of cerebral malaria in untreated mice was accompanied by an augmented production of TNF-alpha and nitric oxide (NO), the proinflammatory mediators. These findings suggest that nonspecific immunization with T. gondii leads to restoration of an early IFN-gamma response in P. yoelii-infected mice and in the establishment of an immunoregulatory mechanism that effectively antagonizes the disease-promoting effects of proinflammatory cytokines in the late phase of infection.

A potential role for T-cell receptor (TCR)-gamma(delta) cells in response to Toxoplasma gondii has been described. This study was undertaken to evaluate whether exogenous rIL-15 can enhance the production of gamma(delta) T cells in response to infection with T. gondii. For this, C57BL/6 and TCR alpha-gene deficient mice (alpha-/-) were vaccinated with Toxoplasma lysate antigen (TLA) in combination with interleukin (IL)-15. The cellular and humoral immune response was assessed following immunization. Administration of IL-15/TLA to alpha-/-mice lengthened survival time post lethal challenge. Several immunological parameters were increased in the alpha-/-mice following immunization with IL-15/TLA including serum immunoglobulin (Ig)G1 and IgG2a antibody titres and splenocyte lymphoproliferation in response to parasite antigen. Further, the CTL response to parasite infected target cells as well as the production of interferon gamma was enhanced by IL-15/TLA administration in the alpha-/-mice. These observations indicate that the gamma(delta) T cells response to this parasite can be enhanced by the administration of exogenous IL-15 in the presence of parasite lysate antigen.

Encephalitozoon cuniculi is a protozoan parasite that has been implicated recently as a cause of opportunistic infection in immunocompromised individuals. Protective immunity in the normal host is T cell-dependent. In the present study, the role of individual T cell subtypes in immunity against this parasite has been studied using gene knockout mice. Whereas CD4-/- animals resolved the infection, mice lacking CD8+ T cells or perforin gene succumbed to parasite challenge. The data obtained in these studies suggest that E. cuniculi infection induces a strong and early CD8+ T response that is important for host protection. The CD8+ T cell-mediated protection depends upon the CTL activity of this cell subset, as the host is rendered susceptible to infection in the absence of this function. This is the first report in which a strong dependence upon the cytolytic activity of host CD8+ T cells has been shown to be important in a parasite infection.

Toxoplasma gondii Ag-primed intraepithelial lymphocytes (IEL) from the mouse intestine have been shown to be protective against an lethal parasite challenge when adoptively transferred into recipient mice. In the present study, we observed that Ag-primed IEL traffic to the intestine of naive mice following i.v. administration. Primed and CD8beta+ IEL were the most efficient cells at homing to the host organ. In congenic mice, IEL migrated from intestine within several hours posttransfer. On Ag reexposure, the primed IEL return to the intestine where they enhance resistance as determined by reduction in the number of brain cysts. Treatment of recipient mice with anti-alpha4 and anti-alphaE Abs partially inhibited IEL intestinal homing. The Ab treatment dramatically impaired resistance to a subsequent oral infection. These finding indicate that lymphocyte homing is an important parameter in establishing long term immunity to recurrent infection with this parasite.

The importance of IFN-gamma in regulating the host CD8+ T cell response during microbial infection has not been delineated. Mice deficient for the p40 chain of the IL-12 heterodimer have impaired IFN-gamma production and are susceptible to infection with the intracellular parasite Toxoplasma gondii. The administration of exogenous IFN-gamma to parasite-infected p40-/- mice increases survival and up-regulates the depressed CD8+ T cell response following infection. CD8+ T cells isolated from cytokine-treated p40-/- mice exhibit an increase in both precursor CTL frequency and IFN-gamma production compared with untreated controls. The enhancement of the CD8+ T cell response is independent of CD4+ T cell help. These CD8+ T cells induce protective immunity against a lethal challenge when adoptively transferred into naive p40-/- and IFN-gamma-/- mice. These observations indicate that IFN-gamma can regulate the CD8+ T cell response during T. gondii infection.

Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p-Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non-WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98-RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.

Functional and quantitative analysis of splenic T cell immune responses following oral Toxoplasma gondii infection in mice. Experimental Parasitology 91, 212-221. Immunity to Toxoplasma gondii is mediated primarily by the host T cell response. Although there is considerable information regarding host immunity following intraperitoneal infection with tachyzoites, little information is available regarding naturally acquired infection following peroral infection with bradyzoites. In this study, a sequential quantitative analysis of the cell-mediated immune response was performed at the single cell level. To assess the kinetics of this response and parasitic loads, inbred mice were orally infected with the 76K strain bradyzoites of T. gondii. Within 24 h of infection, follicular hyperplasia followed by infiltration with histiocytes, macrophages, and apoptotic bodies was observed in the spleens of infected mice. T. gondii were detected from day 1, and counts increased gradually during the experimental period. Splenocyte DNA synthesis to antigen and mitogen was severely suppressed at days 7 and 10. The percentages of NK1.1(+) or delta gamma T cells were increased from day 1, whereas CD4(+) and CD8alpha+ T cells were signficantly increased after day 7 postinfection. CD25 expression and intracellular IFN-gamma production increased in NK1.1(+) cells on day 1 and by all other T cell subsets after day 4. Intracellular IL-4 did not increase until day 7, and IL-10 production was increased in all T cell subsets after day 4. Together, these findings indicate that oral infection with T. gondii stimulates a strong cellular immune response that appears to polarize toward an early Th1 response. However, within 7 days, a strong immune Th2 regulatory response as well as high parasitic loads can be observed, with a reduction in lymphoproliferation to mitogen stimulation, increased production of IL-4 and IL-10, and evidence of T cell apoptosis in the splenic immune compartment., (Copyright 1999 Academic Press.)

Dendritic cells (DC) are potent antigen-presenting cells that can stimulate T cell responses by secreting cytokines. During Toxoplasma gondii infection, host immunity is mediated by interferon-gamma, which is induced by interleukin-12 (IL-12). Whether T. gondii infection would stimulate human DC to produce IL-12 was determined. DC were generated from human peripheral blood mononuclear cells cultured with recombinant human granulocyte-macrophage colony-stimulating factor and recombinant human IL-4. DC secreted high levels of IL-12 in response to lipopolysaccharide but not to either live T. gondii tachyzoites or soluble antigen. However, IL-12 production in response to T. gondii was observed when DC were cocultured in contact with lymphocytes isolated from seropositive donors. Ligation of CD40:CD154 was partially essential for IL-12 secretion. These data demonstrate that signals obtained from contact with sensitized lymphocytes are critical for human DC to secrete IL-12 in response to T. gondii.

Genes encoding the Phe-Gly (FG) repeat-containing nucleoporins NUP98 and CAN/NUP214 are at the breakpoints of several chromosomal translocations associated with human acute myeloid leukemia (AML), but their role in oncogenesis is unclear. Here we demonstrate that the NUP98-HOXA9 fusion gene encodes two nuclear oncoproteins with either 19 or 37 NUP98 FG repeats fused to the DNA binding and PBX heterodimerization domains of the transcription factor HOXA9. Both NUP98-HOXA9 chimeras transformed NIH 3T3 fibroblasts, and this transformation required the HOXA9 domains for DNA binding and PBX interaction. Surprisingly, the FG repeats acted as very potent transactivators of gene transcription. This NUP98-derived activity is essential for transformation and can be replaced by the bona fide transactivation domain of VP16. Interestingly, FG repeat-containing segments derived from the nucleoporins NUP153 and CAN/NUP214 functioned similarly to those from NUP98. We further demonstrate that transactivation by FG repeat-rich segments of NUP98 correlates with their ability to interact functionally and physically with the transcriptional coactivators CREB binding protein (CBP) and p300. This finding shows, for the first time, that a translocation-generated fusion protein appears to recruit CBP/p300 as an important step of its oncogenic mechanism. Together, our results suggest that NUP98-HOXA9 chimeras are aberrant transcription factors that deregulate HOX-responsive genes through the transcriptional activation properties of nucleoporin-specific FG repeats that recruit CBP/p300. Indeed, FG repeat-mediated transactivation may be a shared pathogenic function of nucleoporins implicated human AML.

Down-regulation of host immune response to Toxoplasma gondii is associated with the expression of specific cytokines, in particular IL-10, and the induction of CD4+ T cell anergy. In the present study we report that the expression of both CD4 and CD2 antigen is down-regulated during the acute phase of infection. A decrease in the expression of CD2 was apparent during the acute phase of T. gondii infection in three genetically distinct strains of mice, CBA/J, C57BL/6, and BALB/c. The lymphoproliferative response induced by cross-linked anti-CD3 mAb or by Con A was markedly depressed. This suppressed response was associated with a reduction in the influx of Ca2+. We have examined whether lymphocytes from T. gondii mice maintain NF-AT transcription factors in the nucleus where they participate in the Ca2+-dependent induction of genes required for lymphocyte activation and proliferation. Immunofluorescence with confocal microscopy using an Ab to NF-ATc demonstrates a decrease in translocation of NF-ATc in T lymphocytes from acutely infected mice. Together, these results suggest that the defect in T cell expansion that occurs during acute murine toxoplasmosis is related to reduced activity of NF-AT, a calcium-dependent transcription factor required for T cell proliferation.

Intraepithelial lymphocytes (IEL) of the intestine represent an important barrier in the prevention of infection against orally acquired pathogens. Adoptive transfer of Ag-primed IEL into a naive host can protect against challenge. Using a murine model, we demonstrate in two genetically distinct mouse strains (C57BL/6 and CBA/J) that protective IEL can be isolated at specific times after oral infection with cysts containing bradyzoites. Adoptive transfer of IEL obtained from the intestine of infected mice at these specific times can provide long term protection, as determined by mortality and cyst number against challenge. The protective IEL appear to be CD8+, TCR-alpha/beta and are at least partially dependent upon the presence of TCR-gamma/delta T cells in the host. Endogenous production of the pivotal cytokine, IFN-gamma, is essential for host immunity. These findings demonstrate that gut-derived IEL represent a potentially important mechanism to provide long term immunity to the host.

Induction of reactive nitrogen intermediates by IFN-gamma is presumed an important mechanism of host resistance against acute and chronic infection with Toxoplasma gondii. Although nitric oxide (NO) has been shown to be important in the control of parasite replication in vivo, the role of this molecule in vaccine-based immunity against T. gondii is unknown. Mice with a targeted disruption of inducible NO synthase (iNOS) were immunized with an avirulent temperature-sensitive strain of this parasite (ts-4). Both the parental C57BL/6 and the iNOS(-/-) mice survived infection with the ts-4 mutant. Oral challenge of the vaccinated mice with a lethal dose of cysts containing bradyzoites resulted in reduced parasite burden and increased survival compared with nonvaccinated control mice. Host immunity in the iNOS(-/-) mice, similar to that observed in the parental strain, appears dependent upon both IFN-gamma and CD8+ T cells. These findings suggest that although vaccine-based long-term immunity against T. gondii is dependent upon the induction of IFN-gamma, it does not rely upon the anti-microbial effect of NO.

Almost 80% of patients with AIDS die from infections other than human immunodeficiency virus (HIV). These infections usually occur late in the course of disease when CD4(+) T-cell count has fallen below 200 permm(3) cells per milliliter. Most of these infections are caused by organisms that do not normally afflict healthy individuals and are thus considered to be opportunistic. In this article, Lloyd Kasper and Dominique Buzoni-Gatel review the host-parasite interaction for four important pathogens: Candida albicans and Pneumocystis carinii (usually non-invasive pathogens), Cryptosporidium parvum (invades the cells but remains localized in the gut) and Toxoplasma gondii (penetrates through the gut to cause systemic infection). These organisms, which generally cause limited or even insignificant clinical evidence of infection in the normal host, were chosen because of their high prevalence in AIDS patients and because they exhibit different invasive abilities. The reason why individuals with AIDS are susceptible to this particular group of pathogens is uncertain.

Production of nitric oxide by macrophages is believed to be an important microbicidal mechanism for a variety of intracellular pathogens, including Toxoplasma gondii. Mice with a targeted disruption of the inducible nitric oxide synthase gene (iNOS) were infected orally with T. gondii tissue cysts. Time to death was prolonged compared with parental controls. Histologic analysis of tissue from infected mice showed scattered small foci of inflammation with parasites in various tissues of iNOS-/- mice, whereas tissue from the parental C57BL/6 mice had more extensive tissue inflammation with few visible parasites. In particular, extensive ulceration and necrosis of distal small intestine and fatty degeneration of the liver was seen in the parental mice at day 7 postinfection, as compared with the iNOS-/- mice where these tissues appeared normal. Serum interferon gamma and tumor necrosis factor alpha levels postinfection were equally elevated in both mouse strains. Treatment of the parental mice with a NO synthase inhibitor, aminoguanidine, prevented early death in these mice as well as the hepatic degeneration and small bowel necrosis seen in acutely infected control parentals. These findings indicate that NO production during acute infection with T. gondii can kill intracellular parasites but can be detrimental, even lethal, to the host.

Ca2+ is an essential activator of motility in the obligate intracellular parasite Toxoplasma gondii. Ca2+ ionophore A23187 and intracellular microinjection of Ca2+ initiate motility of parasites residing in parasitophorous vacuoles (PV). The source of Ca2+ and the mechanism by which it activates motility in vivo remain uncertain. Exposure of the parasites to dithiothreitol (DTT) can activate egress of previously nonmotile intravacuolar parasites within 60 sec. DTT is also known to activate both isoforms of the highly concentrated nucleoside triphosphate hydrolase (NTPase) produced by T. gondii. Using an adherent cell analysis system (ACAS) for Ca2+ imaging, a brief 15-50% increase in intra-PV fluorescence ratio was observed after exposure of infected fibroblasts to 5 mM DTT. Chelation of intracellular Ca2+ with BAPTA-AM and extracellular Ca2+ with EGTA blocked the DTT effect; however, this chelation did not prevent the activation of parasites nor the Ca2+ response to the Ca2+ ionophore ionomycin, suggesting that the Ca2+ that activates motility may reside near or within the parasite itself. This result demonstrates that an increase in Ca2+ within the vacuole precedes the onset of motility and the correlation of the DTT effect on motility and tachyzoite NTPase suggests that NTPase activation may be involved in the Ca2+ flux.

Neospora caninum is a coccidial protozoan parasite that infects a large range of mammals including dogs, cats, mice, and cattle. Morphologically, N. caninum appears indistinguishable from Toxoplasma gondii, although they are genetically distinct. To date there have been no reported cases of this infection in humans, although nonhuman primates may be susceptible to infection. Inbred A/J mice develop no clinical and little histologic evidence of infection in spite of a high-dose inoculum of N. caninum. Splenocytes obtained from infected mice proliferate in vitro in response to both N. caninum and T. gondii-soluble antigen. A transient state of T cell hyporesponsiveness to parasite antigen and mitogen was observed at Day 7 p.i. This downregulatory response could be partially reversed by the addition of the nitric oxide antagonist LNMMA, but not antibody to IL-10. Mice infected with N. caninum produce significant quantities of IL-12 and IFN gamma, most evident shortly after infection. In vivo, antibody to IL-12 is able to neutralize immune resistance to the parasite. Moreover, in vivo depletion of IFN gamma with antibody renders the mice susceptible to infection. These observations suggest that N. caninum induces a T cell immune response in the infected host that is at least partially mediated by IL-12 and IFN gamma.

In this study, we describe the functional alterations in the host immune system that occur following acute infection with Plasmodium yoelii. Further, we have addressed the question whether the transient condition of altered immune responsiveness can be restored by a cytokine therapy. The lymphoproliferative response towards concanavalin A (Con A) or to cross-linked anti-CD3 mAb was significantly diminished in acutely infected mice compared to immune and normal animals. This condition was associated with poor production of IL-2. In vivo treatment with recombinant IL-2 (rIL-2) resulted in marked diminution of parasitemia (from 24% +/- 6% to 8% +/- 3%) in mice during the acute phase of infection. Despite this diminution in parasitemia, 70% of the IL-2 treated mice died by day 17 post infection. In vivo treatment with rIL-2 led to a partial but significant restoration in lymphoproliferative response to TCR-mediated (cross-linked anti-CD3 mAb) or to Con A-induced stimulation in acutely infected mice. The transcripts for IL-4, IL-5, GM-CSF, and TNF-alpha were expressed in the splenocytes from acutely infected mice not treated with rIL-2. mRNAs for IL-2, IFN-gamma, IL-6, IL-10 which were not detected in acutely infected mice could be reversed by administration of rIL-2 in vivo. We suggest that some of the hyporesponsive T-cells in the acute phase of infection have the potential to be reversed, and this reversal is manifested also at the level of cytokine gene expression.

Suppression of the host immune response by Toxoplasma gondii has been observed in both human and experimental murine infection. In this study, inbred mice were infected with T. gondii. At day 7 post-infection, the lymphoproliferative response to both mitogen and superantigen as well as parasite antigen were found to be significantly depressed. Using a transwell system, it was determined that the reduced proliferative response was due to soluble factor(s) being expressed by splenocytes from the infected mice. Isolation of the splenocytes into an adherent and nonadherent population suggested that both macrophages and T cells were able to produce at least one soluble factor. Tissue culture supernatant derived from the splenocytes of the infected mice contain increased levels of IL-10, whereas measurable IL-2 levels could not be quantitated. At day 7 post-infection, both a biologic assay for IFN-gamma in culture supernatant and the expression of IFN-gamma mRNA in the splenocytes were reduced. Antibody to IL-10 was able to partially neutralize (almost 50%) the in vitro immune downregulation of the tissue culture supernatant. Anti-IL-10 in combination with a nitric oxide (NO) antagonist was able to reverse the inhibitory activity of the culture supernatant by 85%. Since IL-10 is a potent antagonist of IFN-gamma, it may represent a critical cytokine involved in mediating T. gondii induced immunosuppression in the infected host.

During its intracellular life cycle within the infected host cell, Toxoplasma gondii is able to undergo rapid asexual replication. Neither the mechanism by which the parasite initiates this process nor the requirements for maintaining it are understood. We produced a monoclonal antibody, 1B8, that identifies a parasite antigen of approximate M(r) 97 kDa as determined by SDS-PAGE. The epitope recognized by mAb 1B8 appears as a collection of vesicular structures scattered throughout the cell cytoplasm. When RH strain parasites are incubated with mAb 1B8 in the absence of serum complement, parasite growth is inhibited by > 90% as determined by radioisotope incorporation. Both attachment and invasion assays show that neither of these parasite-host cell interactions are inhibited by the mAb. However, a marked reduction in the number of intracellular rosettes was observed following mAb treatment of the parasites. Viable extracellular parasites are able to endocytose mAb 1B8. Once within the parasite cytosol the antibody recognizes the vesicular structures similar to those observed with fixed parasites. Immunofluorescence assays with Besnoitia jellisoni and Plasmodium falciparum show that the epitope recognized by mAb 1B8 is conserved among Coccidiae but not the kinetoplastid Leishmania.

Previous observations have demonstrated that monoclonal and polyclonal antibodies directed at SAG-1, the major surface protein of Toxoplasma gondii, decreased the number of T. gondii that infected fibroblast monolayers. Direct evaluation of parasite-host cell attachment using glutaraldehyde-fixed human fibroblasts and live tachyzoites was performed to determine whether SAG-1 was a ligand for the host cell receptor. The interaction between the fixed cells and T. gondii was specific and saturable as determined by a radioisotope competitive binding assay. Moreover, the specificity of this interaction was confirmed by comparison to another member of the Apicomplexa, Besnoitia jellisoni. Treatment of fresh extracellular T. gondii with rabbit polyclonal anti-SAG-1 serum inhibited parasite attachment to host cells by 71%. A monoclonal antibody (6A8) directed at SAG-1 was able to inhibit parasite binding to fixed host cells by 65%. Other mAb's directed at SAG-1 failed to inhibit parasite attachment in this assay. Fab derived from 6A8 mAb showed dose-dependent inhibition of parasite attachment. At an Fab concentration of 25 micrograms/ml, 47% inhibition was observed. Attachment assays using mutant parasites with defective SAG-1 (PTgA and PTgC) showed significantly reduced binding (26 and 39%) when compared to wild-type (SAG-1+) parentals. Taken together, these observations suggest that SAG-1 is an important parasite ligand that binds to the host cell in the process of T. gondii invasion.

Recent studies indicate that Toxoplasma gondii attachment is mediated via a parasite ligand-host cell receptor interaction. Lloyd Kosper and Jose Mineo here survey factors involved in the attachment to and penetration and invasion of host cells by T. gondii.

Mutants resistant to sinefungin (SF) and hydroxyurea (HU) were isolated from an oocyst-producing strain of Toxoplasma gondii with the aid of mutagenesis with ethylnitrosourea. These mutants were used with previously described mutants resistant to adenine arabinoside (araA) and fluorodeoxyuridine (FUDR) in genetic crosses in cats. In order to interpret the data from crosses in which all four mutants were used to infect the same cat, it was necessary to devise a mathematical expression to predict the recombination frequency for unlinked markers. This frequency was shown in theory to be half of the product of the two parental phenotype frequencies. A series of crosses in which the parental frequencies were systematically varied yielded frequencies of recombination that were in accord with this calculation. The four-way crosses in the same cat showed unlinked recombination between all markers except HU and FUDR. This pair of markers yielded no doubly resistant recombinants, suggesting complete linkage. However, linkage was excluded when a binary cross between the HU- and FUDR-resistant mutants resulted in the normal number of doubly sensitive recombinants. The lack of doubly resistant recombinants was shown to be a consequence of phenotypic suppression of HU resistance by FUDR resistance. This suppression was first demonstrated by showing that an FUDR-resistant mutant selected from an HU-resistant parasite lost the HU resistance. The phenotypically suppressed HU-resistant gene was revealed by genetic crosses with wild type T. gondii. Although both parental stains were sensitive to HU, some of the progeny parasites were resistant.

Hydroxyurea inhibited the growth and DNA synthesis of Toxoplasma gondii growing in human fibroblast cells. A concentration of 18 micrograms/ml totally suppressed plaque formation. The synthesis of T. gondii RNA was not acutely inhibited. The parasite was equally sensitive to hydroxyurea when grown in wild type or hydroxyurea resistant host cells. With the aid of chemical mutagenesis, we isolated a stable hydroxyurea resistant mutant of T. gondii. This mutant showed no increased ability to incorporate [3H]uracil into its pyrimidine deoxynucleotide pool. Hydroxyurea depressed the [3H]uracil labeling of the pyrimidine deoxynucleotide pool in the wild type parasite but not in the mutant, suggesting that the mutant ribonucleotide reductase was resistant to the inhibitory effect of the drug.

Purified P30, the principal iodinatable membrane protein of Toxoplasma gondii, induced proliferation of peripheral blood mononuclear cells from seropositive individuals but not from seronegative individuals. Culture supernatants from stimulated cells of seropositive individuals blocked the growth of T. gondii in human fibroblasts, whereas those from antibody-negative individuals failed to do so. The anti-toxoplasmic effect of culture supernatants correlated with the induction of indoleamine 2,3-dioxygenase and the destruction of tryptophan, as previously described for fibroblasts treated with recombinant gamma interferon (IFN-gamma). The anti-toxoplasmic effect was blocked by monoclonal antibody to IFN-gamma. The protective effect correlated with the amount of IFN-gamma in the culture supernatant, as measured by inhibition of viral CPE. Thus, the level of IFN-gamma appears to be an important immune factor in protection against toxoplasmosis in humans.

A 51-year-old diabetic woman developed bilateral rhinoorbitocerebral phycomycosis. Successful treatment was accomplished by correction of the ketoacidosis, surgical debridement, and amphotericin B therapy. Rhinoorbitocerebral phycomycosis is a fulminant and frequently fatal disease most often seen in debilitated hosts. This survival suggests that aggressive therapy is indicated in patients with extensive fungal involvement.

Of the possible iodine-labelled Toxoplasma gondii surface proteins, P30 (apparent Mr 30,000) is the principal one recognized by acute and convalescent anti-toxoplasma sera. This protein which comprises from 3 to 5% of the total parasite protein was used to raise a panel of parasiticidal monoclonal anti-P30 antibodies. One of these monoclonal antibodies was able to select a resistant mutant from a large population of chemically mutagenized wild-type P strain parasites. This mutant retained the wild type sensitivity to other non-P30 parasiticidal monoclonal antibodies as well as polyclonal anti-P30 rabbit sera. Analysis of surface radioiodinated wild type and mutant parasites showed that the mutant had a quantitative reduction in the amount of P30. A comparison of surface biotin labelled wild type and resistant parasites by two dimensional electrophoresis showed that the mutant lacked one and possibly two of several proteins that make up wild type P30. Western blot analysis indicated that the mutant was devoid of antigenically reactive P30. These findings further support the hypothesis that antigenic variants of T. gondii can be induced and may involve the major surface membrane antigens of the parasite.

Toxoplasma gondii, an obligate intracellular parasite, is readily grown in nearly all cultured cells. The host-parasite relationship in these cultures can often be explored by using mutant host cells or mutant parasites. Host cells incapable of incorporating uracil or hypoxanthine, which were excellent precursors for T. gondii, allowed the demonstration that the host cell had no access to the purine or pyrimidine nucleotide pools of the parasite. Conversely, a T. gondii mutant that was defective in the principal pyrimidine salvage pathway allowed the demonstration that the parasite had no access to pyrimidine nucleotide or deoxynucleotide pools of the host cell. One metabolite that must pass from the host cytoplasm to T. gondii is a purine. An absolute defect in purine biosynthesis by the parasite was disclosed by growing T. gondii in a mutant host cell that was, itself, incapable of purine synthesis. T. gondii grew normally at 40 degrees C in a mutant host cell that was incapable of protein synthesis at that temperature. Thus, the parasite did not depend on concomitant protein synthesis in the host cell. An antigenic mutant of T. gondii was isolated with the aid of parasiticidal monoclonal antibody. This mutant lacked a major parasite surface protein, of relative molecular mass (Mr) 22 000. The antibody used to select this mutant immunoprecipitated a protein of this Mr from the wild-type parasite.