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Biomedical subjects

L H Kasper

Publications and source records attributed to L H Kasper.

At least 55 records · Page 3Linked to original sources
PubMed1997

Molecular analysis and characterization of a protein involved in the replication of intracellular Toxoplasma gondii.

Previous studies in our laboratory have identified a cytoplasmic protein (p97) of T. gondii that is involved in the process of intracellular parasite replication. Monoclonal antibody inhibits parasite replication in vitro and recognizes a protein of approximate 97 kDa by Western blot analysis. Using biotinylation, we demonstrate that p97 is not expressed on the surface of the tachyzoite. Polyclonal sera raised against the purified native protein was used to isolate a cDNA of 3.3 kb from a library. The product of this gene expresses a protein of approximate Mr 97 kDa that is reactive to the antibody (1B8) raised against the native antigen. The protein sequence of this product suggests that it is within the cytoplasm as suggested by the lack of a signal sequence or hydrophobic trans-membrane domain. This protein fails to dissociate into a monomer in the presence of non-ionic detergents as shown by gel filtration and density gradient. Southern blot analysis demonstrates a homologous gene sequence in two closely related Apicomplexa, Neospora caninum and Besnoitia jellisoni suggesting this protein is conserved among certain species of the Sarcocystidae.

Amino Acid Sequence↗
PubMed1997

A dichotomous role for nitric oxide during acute Toxoplasma gondii infection in mice.

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.

Animals↗
PubMed1997

Adoptive transfer of gut intraepithelial lymphocytes protects against murine infection with Toxoplasma gondii.

Intraepithelial lymphocytes (IEL) of the gut represent a primary immune barrier against infection by orally acquired pathogens. Naturally acquired infection with Toxoplasma gondii induces the proliferation of CD8+ T cells in both the gut and spleen. Gut-derived CD8alpha/beta+ IEL exhibit MHC-restricted cytotoxicity against parasite-infected enterocytes and macrophages. In a murine model, we demonstrate that the adoptive transfer of IEL obtained from inbred mice at day 11 postinfection is able to protect against a virulent challenge in syngenic recipients. In CBA mice, the parasite cyst load within the brain of the recipients receiving primed IEL was reduced by 90%. In BALB/c and C57BL/6 mice, a 50% decrease in mortality was observed following adoptive transfer of primed IEL. To determine the T cell subset responsible for protective immunity, a purified CD8alpha/beta+ IEL population was isolated from infected mice at day 11 postinfection. These cells were able to protect naive mice by adoptive transfer against a lethal parasite challenge. RNA analysis by reverse-transcriptase PCR revealed that primed CD8alpha/beta+ IEL produce significant message for IFN-gamma, an essential cytokine for host protection against toxoplasmosis. Administration of anti-IFN-gamma at the time of adoptive transfer of primed IEL abrogated protection. The adoptive transfer of these protective IEL was not restricted to the Ld class I locus. These data demonstrate that IFN-gamma-producing IEL may be an important primary barrier against acute and perhaps recurrent infection with T. gondii.

Adoptive Transfer↗
PubMed1997

Neospora caninum: role for immune cytokines in host immunity.

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.

Animals↗
PubMed1997

Toxoplasma gondii: dithiol-induced Ca2+ flux causes egress of parasites from the parasitophorous vacuole.

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.

Animals↗
PubMed1996

Induction of gammadelta T cells during acute murine infection with Toxoplasma gondii.

The importance of the host gammadelta T cell response to microbial pathogens is not yet fully understood. We report that inbred mice infected with T. gondii developed a gammadelta T cell proliferative response to parasite Ag. Mice depleted of either the alphabeta or gammadelta TCR were found to be significantly more susceptible to infection than the parent mouse strain. Proliferation of gammadelta T cells was observed in mice deficient in the TCR-alphabeta in response to UV-irradiated parasites. These T cells lyse parasite-infected syngenic macrophages. Adoptive transfer of these gammadelta T cells into beta2 microglobulin-deficient mice that have been depleted of both CD4+ and NK cells prolongs survival against acute parasite challenge when compared with nontransferred controls. The gammadelta T cells isolated from infected alpha -/- mice express a 10-fold increase in mRNA and produce high titers of IFN-gamma. These data suggest that gammadelta T cells may play an important role in the early host response to infection with T. gondii.

Acute Disease↗
PubMed1996

IL-15 augments CD8+ T cell-mediated immunity against Toxoplasma gondii infection in mice.

Cytokines of the Th1 profile are important mediators of protective host immunity against Toxoplasma gondii infection in mice. In this study we describe the effect of the recently identified cytokine, IL-15, on prevention of murine infection with T. gondii. Administration of exogenous rIL-15 with soluble Toxoplasma lysate Ag (TLA) provides complete protection against a lethal parasite challenge, whereas treatment with either rIL-15 or TLA alone is not protective. Following immunization with TLA/rIL-15, there is a significant proliferation of splenocytes expressing the CD8+ phenotype in response to TLA. A significant rise in the level of serum IFN gamma was observed in vaccinated mice. Adoptive transfer of CD8+ T cells, but not CD4+ T cells, from TLA/rIL-15-vaccinated mice protects naive mice from a lethal parasite challenge. These CD8+ T cells exhibit enhanced CTL activity against target macrophages infected with T. gondii. Mice that have been immunized are protected against lethal parasite challenge for at least 1 mo postvaccination. These observations demonstrate that TLA when administered with exogenous rIL-15 generates toxoplasmacidal Ag-specific CD8+ T cells. These T cells proliferate upon exposure to parasite Ag, exhibit long term memory CTL against infected target cells, and may be involved in host immune memory to this parasite.

Adjuvants, Immunologic↗
PubMed1996

Production of nitric oxide (NO) is not essential for protection against acute Toxoplasma gondii infection in IRF-1-/- mice.

Production of nitric oxide (NO) by macrophages is important for the killing of intracellular pathogens. IFN-gamma and LPS stimulate NO production by transcriptional up-regulation of inducible nitric oxide synthetase (iNOS). In the present study we used mice with a targeted disruption of the IFN regulatory factor-1 gene (IRF-1-/-) to investigate the importance of NO in the host immune response against Toxoplasma gondii, a major cause of infection in newborns and those with AIDS. IRF-1-/- mice were more susceptible to acute Toxoplasma infection, and treatment with either exogenous IFN-gamma or in vivo neutralization of endogenous IFN-gamma had little effect on their susceptibility to infection. However, administration of exogenous IL-12 was able to prolong survival even when IFN-gamma was depleted. An in vivo depletion study suggested that the mechanism of this protective response is mediated in part by CD4+ T cells. The administration of IL-12 could not overcome the inhibition of lymphoproliferative response in T. gondii-infected mice and treatment with N-monomethyl-L-arginine (L-NMMA), a nitric oxide synthase (iNOS) antagonist in vitro was unable to reverse the immunosuppression. In response to Toxoplasma infection, splenocytes from IRF-1-/- mice exhibited increased production of IL-10 as well as a 30-fold increase in its message expression. These studies indicate that NO may not be essential for host immunity to the parasite, and moreover that IL-12 appears to induce an IFN-gamma-independent mechanism of protection against this opportunistic pathogen.

Animals↗
PubMed1996

Activation-mediated CD4+ T cell unresponsiveness during acute Toxoplasma gondii infection in mice.

Infection of mice with Toxoplasma gondii has been shown to induce a transient state of immune down-regulation. Earlier reports have demonstrated the role of cytokines, in particular IL-10, in this host response. Here evidence is presented that T. gondii, a major opportunistic pathogen of the newborn and those with AIDS, is able to induce CD4(+) T cell apoptosis in the infected murine host. We have examined the changes in the CD4(+) T cell population that occur during acute infection in an experimental mouse model. Seventy-six percent of the CD4(+) T cell population increased in volume by day 7 post-infection and expressed T cell maturation markers (CD44(hi), IL-2Rhi, Mel-14(lo)). Further noted was a clonal activation of several CD4(+) T cell to mitogen or parasite antigen stimulation was observed, in particular Vbeta5 T cells. Addition of rIL-2 partially restored the CD4(+) T cell proliferative response in vitro. The T cell activation marker CTLA-4 could not be detected and the co-stimulatory molecule, CD28, was down-regulated. Electrophoretic and morphologic analysis of these cells post-culture demonstrated a DNA fragmentation pattern and cell death consistent with apoptosis. These studies demonstrate for the first time in a protozoan parasite that activation induced CD4(+) T cell unresponsiveness occurs during acute T. gondii infection in mice, and may be important in immune down-regulation and parasite persistence in the infected host.

Abatacept↗
PubMed1996

Attachment of Toxoplasma gondii to host cells is host cell cycle dependent.

The initial attachment of Toxoplasma tachyzoites to target host cells is an important event in the life cycle of the parasite and hence critical in the pathogenesis of this infection. The efficiency of Toxoplasma attachment to synchronized populations of Chinese hamster ovary cells and bovine kidney cells was investigated by using a glutaraldehyde-fixed host cell assay system. For both cell lines, parasite attachment increased as the synchronized host cells proceeded from the G1 phase to the mid-S phase and then decreased as the cells entered the G2-M boundary. Postulating that these differences in attachment reflect the upregulation of a specific receptor, polyclonal antibodies were generated against whole MDBK antigen at 0 and 4 h into the S phase. Both antisera were shown to inhibit parasite attachment to both synchronous and asynchronous host cell populations. However, the attachment blockade observed with the 4-h antiserum was significantly greater than that with the 0-h antiserum, completely abolishing the cell cycle-dependent increase in attachment found in control samples. These findings suggest that Toxoplasma tachyzoites bind specifically to a host cell receptor which is upregulated in the mid-S phase of the cell cycle.

Adhesiveness↗
PubMed1996

Toxoplasma gondii-induced immune suppression by human peripheral blood monocytes: role of gamma interferon.

The ability of Toxoplasma gondii to evade the host immune response during primary infection in humans is poorly understood. In murine toxoplasmosis, infected spleen macrophages release soluble factors that mediate a transient immunosuppression, which may allow the parasite to become established. When an enriched population of human monocytes from seronegative individuals was incubated with toxoplasmas in vitro, soluble factors that mediated market suppression of mitogen-induced lymphocyte DNA synthesis were released. Irradiated tachyzoites that do not undergo replication were sufficient stimuli for near-maximal soluble factor release. Up to 50% of the soluble factor-mediated suppression is attributable to a gamma interferon (IFN-gamma)-dependent pathway, and the mediator of the remaining inhibition is neither interleukin-10, transforming growth factor beta, prostaglandin E2, lipoxygenase products, nitric oxide, nor tumor necrosis factor alpha-induced mitochondrial cell-derived reactive oxygen intermediates. IFN-gamma also mediates the up-regulation of an antigen-presenting cell phenotype by both infected and uninfected macrophages. However, IFN-gamma does not activate macrophages to become toxoplasmacidal; instead, intracellular toxoplasmas replicate and reinfect, eventually lysing the macrophage population. These results suggest that T. gondii is able to evade the naive host immune response by induction of soluble immunosuppressive factors that allow the parasite to become established during an acute infection.

Animals↗
PubMed1996

In vivo treatment with interleukin 2 reduces parasitemia and restores IFN-gamma gene expression and T-cell proliferation during acute murine malaria.

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.

Animals↗
PubMed1995

IL-7 stimulates protective immunity in mice against the intracellular pathogen, Toxoplasma gondii.

Cytokines, in particular IFN-gamma and IL-12, are important in host protection against infection with Toxoplasma gondii. This parasite is a major cause of congenital infection and morbidity in immunosuppressed persons, especially those with AIDS. IL-7, a monomeric protein produced by bone marrow stromal cells and fetal thymus, is able to induce the proliferation of pro-B cells and CD4+ and CD8+ T cells, and to enhance cytotoxicity of CTL and NK cells. Inbred mice were infected with a lethal dose of T. gondii and given IL-7 twice daily. Mice treated with IL-7 beginning at the time of infection survived, whereas mice either treated after infection or not treated died. Phenotypic analysis of splenocytes identified an expansion of NK (asialo GM1+) cells and CD8+ T cell populations. In vivo depletion of NK (asialo GM1+) and CD8+ T cells showed that cells expressing these phenotypes were important for maintaining protection against the parasite. IFN-gamma depletion resulted in complete reversal of the protective effect of IL-7 administration. In vivo depletion of endogenous IL-7 enhanced susceptibility to infection. Cytokine analysis by semiquantitative reverse-transcriptase PCR showed that IL-7 enhances the IFN-gamma response and furthermore reverses the parasite-mediated down-regulatory response on IL-2. These observations indicate that exogenous administration of human rIL-7 is able to protect mice against acute parasite challenge by stimulating IFN-gamma production and augmenting the CD8+ T cell-mediated CTL response.

Animals↗
PubMed1995

IL-10 mediates immunosuppression following primary infection with Toxoplasma gondii in mice.

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.

Animals↗
PubMed1995

Survival of immunoglobulin G-opsonized Toxoplasma gondii in nonadherent human monocytes.

Toxoplasma gondii is a protozoan parasite that is able to penetrate human monocytes by either passive uptake during phagocytosis or active penetration. It is expected that immunoglobulin G (IgG) opsonization will target the parasite to macrophage Fc gamma receptors for phagocytic processing and subsequent degradation. Antibody-opsonized T. gondii tachyzoites were used to infect nonadherent and adherent human monocytes obtained from the peripheral blood of seronegative individuals. The infected monocytes were evaluated for the presence of intracellular parasites and the degree of parasiticidal activity. A marked difference in both the numbers of infected macrophages and numbers of parasites per 100 macrophages was observed in the nonadherent cells when compared with those of the adherent cell population. When macrophage Fc gamma receptors were down-modulated, opsonized tachyzoites retained their ability to penetrate the host cell at a rate similar to that observed for unopsonized parasites. These results suggest that antibody opsonization of T. gondii does not prevent active penetration of human monocytes by the parasite and, furthermore, has little effect on intracellular replication of the parasite.

Animals↗
PubMed1995

A Toxoplasma gondii-derived factor(s) stimulates immune downregulation: an in vitro model.

Suppression of the T-cell lymphoproliferative response and downregulation of interleukin 2 (IL-2) production by Toxoplasma gondii has been observed following in vivo infection. In this study, an experimental in vitro murine system was developed to evaluate the kinetics of these responses. Normal splenocytes from uninfected mice were stimulated with either concanavalin A or an anti-CD3 monoclonal antibody and cocultured with Toxoplasma tachyzoites either directly or separated by a transwell. A progressive decline in the lymphoproliferative response was observed as the concentration of parasites in culture increased. Neither heat-killed nor formaldehyde-fixed parasites stimulated this downregulatory response by the splenocytes. A decline in IL-2 production was associated with the decrease in lymphocyte proliferation. The addition of an antibody to IL-10 or heat-inactivated anti-Toxoplasma sera to the culture supernatant partially neutralized the inhibitory effect on lymphocyte proliferation. Cytokine analysis of the responder splenocytes demonstrated a decrease in the message for IL-2 and IL-2 receptor and an increase in IL-10. Together, these observations suggest that during in vitro culture in a murine system, parasite antigens that stimulate the release of a soluble factor(s), such as IL-10, that inhibits proliferation of mitogen-stimulated T cells are expressed.

Animals↗