An ultrastructural study of the early development and tissue cyst formation of Toxoplasma gondii in the brains of mice

The ultrastructural features of the early development and tissue cyst formation of Toxoplasma gondii were examined in the brains of mice at various intervals from 7 days to 22 months post inoculation (PI). At 11 days PI toxoplasms, with the ultrastructural features of the proliferative (endozoite) form, were identified undergoing multiplication within both inflammatory and neural cells. Early tissue cyst formation was also observed, predominantly within neurons. By 21 days PI the proliferative forms had disappeared and only developing tissue cysts containing densely packed cystozoites were present. The proportion of dividing cystozoites decreased with increasing size and age of the cysts. The wall of the tissue cyst developed as an adaptation of the lining of the parasitophorous vacuole. In the majority of older cysts, numerous tubular structures were present beneath the cyst wall. All the cysts observed were retained within intact host cells. The only morphological change with increasing age was that a proportion of the older cysts contained loosely packed cystozoites in an electron lucent ground substance. There was no evidence of any degenerative changes within the cystozoites.Abbreviations C conoid - Cy cystozoite - D daughter anlage - En endozoite - HC host cell cytoplasm - HE host cell rough endoplasmic reticulum - HM host cell mitochondrion - HN host cell nucleus - M micronene - Me limiting membrane of parasitophorous vacuole/cyst wall - MP micropore - MT subpellicular microtubule - MV microvilli - N nucleus - NV neurosecretory vesicle - P polysaccharide granule - PL pellicle - PV parasitophorous vacuole - R rhoptry - Ri ribosome - S synapse - T tubular structure - K vesicle - W cyst wall     

Temporal and spatial distribution of Toxoplasma gondii differentiation into Bradyzoites and tissue cyst formation in vivo

During Toxoplasma gondii infection, a fraction of the multiplying parasites, the tachyzoites, converts into bradyzoites, a dormant stage, which form tissue cysts localized mainly in brain, heart, and skeletal muscles that persist for several years after infection. At this stage the parasite is protected from the immune system, and it is believed to be inaccessible to drugs. While the long persistence of tissue cysts does not represent a medical problem for healthy individuals, this condition represents a major risk for patients with a compromised immune system, who can develop recrudescent life-threatening T. gondii infections. We have investigated for the first time the dynamics and the kinetics of tachyzoite-to-bradyzoite interconversion and cyst formation in vivo by using stage-specific bioluminescent parasites in a mouse model. Our findings provide a new framework for understanding the process of bradyzoite differentiation in vivo. We have also demonstrated that complex molecules such as d-luciferin have access to tissue cysts and are metabolically processed, thus providing a rationale for developing drugs that attack the parasite at this developmental stage.     

Molecular cloning, sequencing and expression of a serine proteinase inhibitor gene from Toxoplasma gondii

A cDNA clone from a Toxoplasma gondii tachyzoite cDNA library encoding a serine proteinase inhibitor (serpin) was isolated. The 1376 bp cDNA sequence encodes a 294 amino acid protein with a putative signal peptide of 23 amino acids resulting in a mature protein with a predicted mass of 30,190 Da and a pI of 4.86. This protein has internal sequence similarity of residues 30-66, 114-150, 181-217 and 247-283 indicating a four-domain structure. The four domains exhibit high identity to serine proteinase inhibitors belonging to the non-classical Kazal-type family. The gene is single copy in the tachyzoite haploid genome of RH strain and was amplified by polymerase chain reaction (PCR). Several introns were identified. The sequence encoding the mature protein was amplified by PCR, cloned into the pQE30 vector and expressed in Escherichia coli. Specific antiserum generated against the recombinant protein was used in immunoblot assay and two bands of 38 and 42 kDa were detected in a whole parasite homogenate. The recombinant protein showed trypsin-inhibitory activity, one of the two potential specificities. We discuss the possible roles that T. gondii serpin(s) may play in the survival of the tachyzoites in the host.     

Expression of SAG-1 of Toxoplasma gondii in transgenic mice

We describe the expression of SAG-1 cDNA in B6C3F1 mice by microinjecting a 3.3 kbp DNA fragment, consisting of the cytomegalovirus enhancer-chicken β-actin hybrid promoter and SAG-1 into the pronucleus of a fertilized egg at the one-cell stage. Offspring derived from this microinjection were analyzed for the integration and functional expression of the SAG-1 transgene. Steady-state expressions of both the mRNA for SAG-1 and SAG-1 protein product were detected in the brain, thymus, spleen and liver. Approximately 50% of F1 and F2 progeny inherited the SAG-1 transgene from SAG-1 transgenic mice in Mendelian fashion. These results indicated that SAG-1 transgenic lines were established. Transgenic mice harboring the SAG-1 gene will contribute a critical tool of defining the molecular mechanisms of SAG-1 in pathogenesis and host immune response. Received: 28 September 1999 / Accepted: 14 October 1999     

Mucosal and systemic cellular immune responses induced by Toxoplasma gondii antigens in cyst orally infected mice.

This study was performed to determine the T-cellular immune responses following Toxoplasma gondii oral infection and to assess further toxoplasma antigens on their ability to stimulate in vitro mucosal and systemic T-cell immunity. Parasite-specific cellular immune responses in Peyer's patches (PP), in mesenteric lymph nodes (MLN) and in spleen (SPL) were investigated using a lymphoblastic transformation test following oral infection of mice with strain 76K cysts of T. gondii. An early toxoplasma sonicate-induced mucosal T-cell proliferation occurred in MLN and PP with a peak responsiveness on day 6 post-infection (PI) and rapidly reached background levels on day 7 PI in PP and on day 8 PI in mesenteric lymph nodes. A later splenic cellular blastogenesis was observed from day 28 PI and persisted throughout the experiment (day 91). At the time of T-cell proliferation, FACS analyses revealed a decrease in the relative percentages of CD4+ and CD8+ T cells with a predominance of CD8+ lymphocytes which leads to an inversion of the CD4/CD8 ratios. We found that CBA/J is a high responder mouse strain in the induction of mesenteric and splenic T-lymphocyte blastogenesis compared to the intermediate responder BALB/c and low responder C57BL/6. Toxoplasma gondii antigens SAG1 (30,000 MW) and GRA4 (40,000-41,000 MW), which are known to induce locally IgA antibodies, are shown to stimulate primed mucosal T lymphocytes from CBA/J and BALB/c mice whereas no proliferation was demonstrated with C57BL/6 T cells. 229-242 peptide, derived from the deduced amino acid sequence of GRA4, only induces detectable proliferation of primed-CBA/J T lymphocytes. Following oral experimental infection, the in vitro mesenteric response to a toxoplasma sonicate is dominated by a Th2-type cytokine pattern whereas a predominant Th1 cytokine response is observed in the spleen. Finally, in vitro stimulation of mesenteric T cells with the three defined toxoplasma antigens resulted in secretion of interleukin-5 (IL-5) and IL-6 (except for SAG1) and interferon-gamma (IFN-gamma) whereas no detectable IL-2 or IL-4 was observed.     

Toxoplasma gondii: appearance of specific markers during the development of tissue cysts in vitro

 Cultures were initiated in Madin-Darby bovine kidney (MDBK) cells from ME49 strain bradyzoites. Specific antibody staining showed that two populations of parasites exist, one being a predominant population of tachyzoites that were positive for the tachyzoite-specific marker SAG1 and negative for the bradyzoite-specific marker P36. All of these parasites expressed the dense granule molecule GRA5, which in larger clusters was seen faintly in the membrane of the parasitophorous vacuole. No rosette formation or monolayer destruction was observed. Also seen was a sub-population of bradyzoites that were positive for P36 and negative for SAG1. Approximately 90% of these parasites expressed the matrix molecule P29. These parasites were also positive for the dense granule molecule GRA5, which was highly in the wall of the cyst. These bradyzoite clusters contained fewer parasites and were smaller in diameter than those expressing tachyzoite markers. Received: 18 July 1995 / Accepted: 3 November 1995     

Characterization and in vitro translation of Toxoplasma gondii ribonucleic acid

RNA was extracted from purified tachyzoites of Toxoplasma gondii (RH strain) by sequential centrifugation in guanidine hydrochloride, urea, and lithium chloride. The subunits of the RNA were characterized by denaturing and non-denaturing electrophoresis in agarose gels. Poly(A)⁺-RNA, purified by oligo(dT)-cellulose affinity chromatography, was translated in a rabbit reticulocyte lysate assay and the products were immunoprecipitated with an experimentally infected mouse serum and a naturally infected human serum. After sodium dodecyl sulphate-polyacrylamide gel electrophoresis, fluorography of the polypeptides confirmed that the mRNA translated specific parasite antigens.     

Immune responses and resistance to Toxoplasma gondii in mice immunized with antigens of the parasite incorporated into immunostimulating complexes.

Immunostimulating complexes were prepared with antigens extracted from tachyzoites of Toxoplasma gondii and were used to immunize mice. The major antigens incorporated into the immunostimulating complexes were the P30 and P22 antigens and an antigen with an approximate molecular weight of 6,000. Other antigens of molecular weights above 30,000 were also present. High antibody titers to T. gondii antigens and a delayed-type hypersensitivity reaction were noted for the immunized mice. Challenge of these mice with tachyzoites injected interperitoneally or with oocysts administered orally resulted in a statistically significant (P < 0.001) conditional probability of survival compared with that of controls. In contrast, the differences between immunized mice and controls challenged with tissue cysts did not attain statistical significance.     

HtrA1 is a novel mast cell serine protease of mice and men

Mast cells are rich sources of proteases, such as tryptases and chymases that control many physiological and pathological processes, for example vascular permeability, smooth muscle cell proliferation or extracellular matrix remodeling. Murine mucosal mast cells mature under the influence of TGF-beta and play a role in asthmatic and anti-helminthic immune responses. In an attempt to identify novel genes that are highly upregulated during mucosal mast cell differentiation, we detected HtrA1 protease as a novel protein in mast cells by microarray experiments. HtrA1 level was much higher in murine mucosal than in connective tissue-type mast cells. Furthermore, HtrA1 is not localized in the secretory granules and is constitutively secreted by human mast cells. Although HtrA1 has been attributed a TGF-beta-inhibitory activity, it did not show any influence on TGF-beta-induced mucosal mast cell differentiation. As many extracellular target proteins have been suggested for HtrA1, this protease may participate in the mast cell-induced extracellular remodeling.     

Multicomponent DNA vaccine-encoding Toxoplasma gondii GRA1 and SAG1 primes: anti-Toxoplasma immune response in mice

A multicomponent DNA vaccine, encoding Toxoplasma gondii GRA1 and SAG1, was constructed and tested for its ability to confer protection. BALB/c mice were challenged with tachyzoites of the virulent T. gondii RH strain at 4?weeks following the last immunization, and immune responses and survival times were observed. The results show that vaccination by the multicomponent vaccine prolonged survival of mice challenged with the T. gondii RH strain (from average 4.50?±?0.22 to 7.60?±?0.74?days); induced high levels of IgG antibody (from 0.252?±?0.080 to 0.790?±?0.083), IFN-gamma (from 598.74?±?67.50 to 853.77?±?66.74?pg/ml), and IL-2 (from 89.44?±?10.66 to 192.24?±?19.90?pg/ml); changed the CD4⁺/CD8⁺ lymphocyte ratio (from 1.81?±?0.14 to 1.09?±?0.19); and stimulated NK cell-killing activity (from 46.81?±?3.96 to 64.15?±?7.71?%). These findings demonstrate that a multicomponent DNA vaccine, encoding GRA1 and SAG1, primes a strong humoral and cellular immune response and enhances protection against T. gondii challenge. The new, combined DNA vaccine provides another means to combat T. gondii infection.     

匹多莫德增强弓形虫GRA1蛋白免疫效果的动物实验评价

目的:探讨匹多莫德增强弓形虫GRA1蛋白刺激机体产生免疫应答和保护免疫的效果。方法:匹多莫德和毕赤酵母菌表达的弓形虫GRA1蛋白混合皮下注射免疫小鼠,以PBS作对照,检测其细胞免疫和体液免疫,并观察其受到弓形虫攻击感染后的生存情况。结果:匹多莫德能辅助GRA1蛋白刺激宿主产生较高的细胞免疫和体液免疫,并能提供较好的免疫保护力。GRA1蛋白加匹多莫德组、GRA1蛋白加弗氏完全佐剂组小鼠IFNγ-、特异性IgG显著高于对照组(P<0.01)和GRA1蛋白组(P<0.01);各免疫组CD8+T细胞数量显著高于对照组(P<0.01);GRA1蛋白加匹多莫德组CD4+T细胞数量(P<0.01)、CD4+/CD8+比值显著高于其它免疫组(P<0.05)。各免疫组T细胞增殖活性与PBS对照组比较明显增强(P<0.01),GRA1蛋白加匹多莫德组T细胞增殖活性最明显。小鼠攻击试验表明,GRA1蛋白加匹多莫德组和GRA1蛋白加弗氏完全佐剂组小鼠存活时间明显长于对照组和GRA1蛋白组。结论:匹多莫德与弗氏佐剂具有相似增强免疫应答和提高GRA1蛋白抗原的免疫原性作用,可望用于弓形虫亚单位疫苗的研制。     

Persistent macrophage/microglial activation and myelin disruption after experimental autoimmune encephalomyelitis in tissue inhibitor of metalloproteinase-1-deficient mice

Increased leukocyte trafficking into the parenchyma during inflammatory responses in the central nervous system (CNS) is facilitated by the extracellular proteolytic activities of matrix metalloproteinases that are regulated, in part, by the endogenous tissue inhibitors of metalloproteinases (TIMPs). In experimental autoimmune encephalomyelitis (EAE), TIMP-1 gene expression is induced in astrocytes surrounding inflammatory lesions in the CNS. The physiological importance of this temporal and spatial relationship is not clear. Herein, we have addressed the functional role of TIMP-1 in a myelin oligodendrocyte glycoprotein (MOG35-55)-induced model of EAE using TIMP-1-deficient (TIMP-1-/-) C57BL/6 mice. Although CD4+ T-cell immune responses to myelin in wild-type (WT) and TIMP-1-/- mice were similar, analysis of CNS tissues from TIMP-1-/- mice after EAE revealed more severe myelin pathology than that of WT mice. This disruption of myelin was associated with both increased lymphocyte infiltration and microglial/macrophage accumulation in the brain parenchyma. These findings suggest that induction of TIMP-1 by astrocytes during EAE in WT mice represents an inherent cytoprotective response that mitigates CNS myelin injury through the regulation of both immune cell infiltration and microglial activation.     

Mycophenolic acid induces differentiation of Toxoplasma gondii RH strain tachyzoites into bradyzoites and formation of cyst-like structure in vitro

Parasitology Research - The biochemical and structural changes that occur during the conversion of Toxoplasma gondii tachyzoites to bradyzoites and the formation of tissue cyst are not well...     

Pinocytic rates of macrophages from mice immunized against Toxoplasma gondii and macrophages stimulated to inhibit toxoplasma in vitro.

The rate of pinocytosis by macrophages when measured by uptake of horseradish peroxidase was significantly reduced during toxoplasma infection of the cells in vitro when the macrophages were from toxoplasma-immune mice and when control cells were stimulated in vitro to inhibit toxoplasma multiplication. There was, however, no direct correlation between reduced pinocytosis in this model and inhibition or enhancement of toxoplasma multiplication. We conclude that a reduced pinocytic rate is a feature of the unstimulated toxoplasma-immune macrophage, but this change in rate alone does not correlate with the cell's ability to inhibit toxoplasma. In addition, we observed that enhanced pinocytosis as seen in the elicited macrophage was not a requirement for inhibition of toxoplasma multiplication.     

Fusidic acid is an effective treatment against Toxoplasma gondii and Listeria monocytogenes in vitro, but not in mice

Fusidic acid is a bacteriostatic antibiotic that inhibits the growth of bacteria by preventing the release of translation elongation factor G (EF-G) from the ribosome. The apicomplexan parasite Toxoplasma gondii has an orthologue of bacterial EF-G that can complement bacteria and is necessary for parasite virulence. Fusidic acid has been shown to be effective in tissue culture against the related pathogen Plasmodium falciparum, and current drug treatments against T. gondii are limited. We therefore investigated the therapeutic value of fusidic acid for T. gondii and found that the drug was effective in tissue culture, but not in a mouse model of infection. To determine whether this trend would occur in another intracellular pathogen that elicits a T helper 1-type immune response, we tested the efficacy of fusidic acid for the bacterium Listeria monocytogenes. Similar to its effects on T. gondii, fusidic acid inhibits the growth of L. monocytogenes in vitro, but not in mice. These findings highlight the necessity of in vivo follow-up studies to validate in vitro drug investigations.     

Defect of hepatocyte growth factor activator inhibitor type 1/serine protease inhibitor, Kunitz type 1 (Hai-1/Spint1) leads to ichthyosis-like condition and abnormal hair development in mice

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1^+/+ blastocysts with Hai-1/Spint1^−/− embryonic stem cells successfully generated high-chimeric Hai-1/Spint1^−/− embryos (B6Hai-1^−/−High) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1^−/− mice was caused by placental dysfunction. However, newborn B6Hai-1^−/−High mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development.