In vitro regulation of gene expression of pregnancy-associated proteins and cytokines in bovine endometrial epithelial cells and bovine trophoblastic cells by infection with Neospora caninum.

Abortion caused by the parasite Neospora caninum is an important threat to the livestock industry worldwide. Trophoblasts and caruncular cells play major roles in initiating innate immune responses and controlling parasite infection at the fetal-maternal interface. In the present study, bovine uterine epithelial cells (BUECs) and bovine trophoblastic (BT) cells treated with bovine interferon-gamma (IFN-γ), IFN-alpha (IFN-α) and IFN-tau (IFN-τ) followed by infection with N. caninum were examined by measuring the mRNA expression levels of numerous pregnancy-associated proteins and observing parasite growth to elucidate the host-parasite interaction at the uteroplacental region. N. caninum infection increased the expression of prolactin-related protein 1 (PRP1), pregnancy-associated glycoprotein 1 (PAG1), and cytokines (TNF-α, IL-8 and IL-10) in BUECs and of IL-8 in BT cells. Bovine IFN-γ inhibited IL-8 and TNF-α expression in BUECs and IL-8 in BT cells. In contrast, the expression of the interferon-stimulated gene OAS1 was significantly increased by treatment of the infected BT cells with IFN-γ. However, treatment with bovine IFNs did not inhibit N. caninum growth in either cell line. In conclusion, our results suggest that bovine IFN-γ plays a crucial role in control of pathogenesis in uterus and induction of inflammatory response in the placental region following N. caninum infection, rather than growth inhibition of the parasites.

An Ionizable Lipid Material with a Vitamin E Scaffold as an mRNA Vaccine Platform for Efficient Cytotoxic T Cell Responses.

RNA vaccines based on lipid nanoparticles (LNPs) with in vitro transcribed mRNA (IVT-mRNA) encapsulated are now a currently successful but still evolving modality of vaccines. One of the advantages of RNA vaccines is their ability to induce CD8+ T-cell-mediated cellular immunity that is indispensable for excluding pathogen-infected cells or cancer cells from the body. In this study, we report on the development of LNPs with an enhanced capability for inducing cellular immunity by using an ionizable lipid with a vitamin E scaffold. An RNA vaccine that contained this ionizable lipid and an IVT-mRNA encoding a model antigen ovalbumin (OVA) induced OVA-specific cytotoxic T cell responses and showed an antitumor effect against an E.G7-OVA tumor model. Vaccination with the LNPs conferred protection against lethal infection by Toxoplasma gondii using its antigen TgPF. The vitamin E scaffold-dependent type I interferon response was important for effector CD8+ T cell differentiation induced by the mRNA-LNPs. Our findings also revealed that conventional dendritic cells (cDCs) were essential for achieving CD8+ T cell responses induced by the mRNA-LNPs, while the XCR1-positive subset of cDCs, cDC1 specialized for antigen cross-presentation, was not required. Consistently, the mRNA-LNPs were found to selectively transfect another subset of cDCs, cDC2 that had migrated from the skin to lymph nodes, where they could make vaccine-antigen-dependent contacts with CD8+ T cells. The findings indicate that the activation of innate immune signaling by the adjuvant activity of the vitamin E scaffold and the expression of antigens in cDC2 are important for subsequent antigen presentation and the establishment of antigen-specific immune responses.

Macrophage Stimulation as a Useful Approach for Immunoscreening of Potential Vaccine Candidates Against Toxoplasma gondii and Neospora caninum Infections.

Toxoplasmosis and neosporosis are protozoan diseases that adversely affect the medical and additionally veterinary sectors, respectively. Toxoplasmosis is caused by Toxoplasma gondii which infects almost all warm-blooded animals including humans. While, neosporosis is caused by Neospora caninum, which induces infection in many animal species particularly in cattle. Currently, control measures for both infections are defective because of no effective vaccine or treatment. Macrophages constitute the first line of innate immunity, which contributes to the effective elimination of T. gondii or N. caninum. This action is mediated by IL-12, which is critical for the secretion of interferon gamma (IFN-γ). Successful vaccine candidates against both protozoan parasites should possess the ability to induce the cellular immune response and IFN-γ production. In this chapter, we will focus on an efficient immunological approach for discovery of potential vaccine candidates against above-mentioned parasites. Our previous studies revealed a strong correlation between vaccine antigens that enhanced the macrophage secretion of IL-12 and their efficacy as potential vaccine candidates in murine model. In case of T. gondii, peroxiredoxin 1 (TgPrx1) and peroxiredoxin 3 stimulated the production of IL-12 from murine peritoneal macrophages and conferred strong to moderate protection in C57BL/6 mice, respectively. At the same context, Neospora antigens of dense granule protein 6 (NcGRA6) and cyclophilin entrapped with oligo-mannose coated-liposomes stimulated macrophage IL-12 secretion and substantially protected immunized BALB/c mice. Therefore, we can deduce that macrophage stimulation evidenced in IL-12 production can be used as a useful approach for judgment of vaccine efficacy before further evaluation using in vivo experiments. Methods of vaccine preparation and macrophage stimulation will be fully described for TgPrx1 and NcGRA6 as potential vaccine candidates against toxoplasmosis and neosporosis, respectively.

Toll-Like Receptor 2 is Involved in Abnormal Pregnancy in Mice Infected with Toxoplasma gondii During Late Pregnancy.

Infection with Toxoplasma gondii during pregnancy causes failure of pregnancy maintenance, resulting in fetal death, abortion, stillbirth, or premature birth, but the mechanism of disease onset remains unclear. Although Toll-like receptor 2 (TLR2) is expressed on antigen-presenting cells and trophoblasts, the role of TLR2 in T. gondii infection during pregnancy is unknown. In this study, we investigated the role of TLR2 in congenital toxoplasmosis using TLR2-deficient (TLR2-/-) mice. T. gondii infection on gestational day 12.5 (Gd12.5) induced more abnormal pregnancy, including premature birth and stillbirth, in wild-type mice than in TLR2-/- mice. Multiple calcifications were observed in the placentas of the infected wild-type mice. At Gd18.5 (6days postinfection), the parasite numbers in the placenta and uterus and the histological changes did not differ significantly between the wild-type and TLR2-/- mice. However, T. gondii infection reduced the mRNA expression of interleukin-12p40 (IL-12p40) and increased IL-4 and IL-10 mRNAs in the placentas of the wild-type mice. In contrast, the placentas of the TLR2-/- mice showed no changes in the expression of these cytokines, including IL-6 and tumor necrosis factor α, in response to T. gondii infection. Serum interferon-γ levels were significantly lower in the infected TLR2-/- mice than in the infected wild-type mice on Gd18.5. Thus, the TLR2-/- mice were less susceptible to the induction of immune responses by T. gondii infection during late pregnancy. Therefore, TLR2 signaling may play a role in the development of disease states during pregnancy, specifically placental hypofunction.

Disseminated Toxoplasmosis in a Narrow-Ridged Finless Porpoise (Neophocaena asiaeorientalis) with Transplacental Embryonal Transmission.

We describe a case of systemic toxoplasmosis in a female adult narrow-ridged finless porpoise (Neophocaena asiaeorientalis) found in May 2018 inside a gillnet set in the Ariake Sound, southern Japan. The main lesions observed were lymphoplasmacytic and focally necrotizing encephalitis, necrotizing to granulomatous adrenalitis, myocarditis, and inflammation in the intestinal wall, associated with protozoal tissue cysts and tachyzoites. Additionally, the individual had a 5.6 mm (crown-rump length) early-stage embryo in the left uterine horn, which had multifocal necrotizing lesions with intralesional tissue cysts and tachyzoites in the parenchyma. Immunohistochemistry and PCR and sequencing of the internal transcribed spacer 1 region confirmed a Toxoplasma gondii infection. Further genotyping revealed an atypical type II genotype with a type I pattern for the Apico locus. Narrow-ridged finless porpoises are an endangered coastal species already facing various anthropogenic threats. Toxoplasmosis, especially with its ability to transmit to an early-stage embryo, should be considered an emerging threat to this vulnerable species.

Babesia microti Confers Macrophage-Based Cross-Protective Immunity Against Murine Malaria.

Malaria and babesiosis, the two primary intraerythrocytic protozoan diseases of humans, have been reported in multiple cases of co-infection in endemic regions. As the geographic range and incidence of arthropod-borne infectious diseases is being affected by climate change, co-infection cases with Plasmodium and Babesia are likely to increase. The two parasites have been used in experimental settings, where prior infection with Babesia microti has been shown to protect against fatal malarial infections in mice and primates. However, the immunological mechanisms behind such phenomena of cross-protection remain unknown. Here, we investigated the effect of a primary B. microti infection on the outcome of a lethal P. chabaudi challenge infection using a murine model. Simultaneous infection with both pathogens led to high mortality rates in immunocompetent BALB/c mice, similar to control mice infected with P. chabaudi alone. On the other hand, mice with various stages of B. microti primary infection were thoroughly immune to a subsequent P. chabaudi challenge. Protected mice exhibited decreased levels of serum antibodies and pro-inflammatory cytokines during early stages of challenge infection. Mice repeatedly immunized with dead B. microti quickly succumbed to P. chabaudi infection, despite induction of high antibody responses. Notably, cross-protection was observed in mice lacking functional B and T lymphocytes. When the role of other innate immune effector cells was examined, NK cell-depleted mice with chronic B. microti infection were also found to be protected against P. chabaudi. Conversely, in vivo macrophage depletion rendered the mice vulnerable to P. chabaudi. The above results show that the mechanism of cross-protection conferred by B. microti against P. chabaudi is innate immunity-based, and suggest that it relies predominantly upon the function of macrophages. Further research is needed for elucidating the malaria-suppressing effects of babesiosis, with a vision toward development of novel tools to control malaria.

Vitamin E Scaffolds of pH-Responsive Lipid Nanoparticles as DNA Vaccines in Cancer and Protozoan Infection.

DNA vaccinations are promising strategies for treating diseases that require cellular immunity (i.e., cancer and protozoan infection). Here, we report on the use of a liposomal nanocarrier (lipid nanoparticles (LNPs)) composed of an SS-cleavable and pH-activated lipidlike material (ssPalm) as an in vivo DNA vaccine. After subcutaneous administration, the LNPs containing an ssPalmE, an ssPalm with vitamin E scaffolds, elicited a higher gene expression activity in comparison with the other LNPs composed of the ssPalms with different hydrophobic scaffolds. Immunization with the ssPalmE-LNPs encapsulating plasmid DNA that encodes ovalbumin (OVA, a model tumor antigen) or profilin (TgPF, a potent antigen of Toxoplasma gondii) induced substantial antitumor or antiprotozoan effects, respectively. Flow cytometry analysis of the cells that had taken up the LNPs in draining lymph nodes (dLNs) showed that the ssPalmE-LNPs were largely taken up by macrophages and a small number of dendritic cells. We found that the transient deletion of CD169+ macrophages, a subpopulation of macrophages that play a key role in cancer immunity, unexpectedly enhanced the activity of the DNA vaccine. These data suggest that the ssPalmE-LNPs are effective DNA vaccine carriers, and a strategy for avoiding their being trapped by CD169+ macrophages will be a promising approach for developing next-generation DNA vaccines.

Detection of antibodies against Toxoplasma gondii in cats using an immunochromatographic test based on GRA7 antigen.

Toxoplasmosis, caused by Toxoplasma gondii, is one of the most common parasitic diseases worldwide. The GRA7 of T. gondii (TgGRA7) is an essential component of the parasitophorous vacuole (PV) and PV membrane surrounding the tachyzoites and the cyst wall of the bradyzoites. While it has been widely employed as antigen for ELISA, there is only one study that has reported its potential as antigen for immunochromatographic test (ICT) in pigs. There is no study yet documenting its potential for ICT serodiagnosis of T. gondii infection in cats. In this study, we assessed the efficacy of an ICT using TgGRA7 in the detection of Toxoplasma infections in 100 cats and compared the results with iELISAs using TgGRA7 and lysate antigens of T. gondii strains, RH, PLK, and VEG. Our results revealed that TgGRA7-ICT is a reliable test for the diagnosis of anti-T. gondii antibodies in cats, producing comparable results as conventional serological methods. This study is the first report on the use of TgGRA7 as ICT antigen for the serodiagnosis of T. gondii infection in cats.

Toxoplasma Effector TgIST Targets Host IDO1 to Antagonize the IFN-γ-Induced Anti-parasitic Response in Human Cells.

Toxoplasma gondii is an important human and animal pathogen that causes life-threatening toxoplasmosis. Interferon-γ (IFN-γ) is critical for anti-T. gondii cell-autonomous immunity in both humans and mice. To proliferate efficiently within the hosts, virulent strains of T. gondii can suppress IFN-γ-dependent immunity. During parasite infection, it is well-characterized that various virulence effectors are secreted to transcriptionally or post-translationally target IFN-γ-inducible GTPases, which are essential for anti-parasite responses in mice. However, the role of IFN-γ-inducible GTPases in anti-T. gondii responses in human cells is controversial since they are non-functional or absent in humans. Instead, IFN-γ-induced tryptophan degradation by indole-2,3-dioxygenase (IDO) is important for the anti-T. gondii human response. To date, the T. gondii virulent mechanism targeting IDO in human cells remains elusive. Here we show that although humans possess two IDO isozymes, IDO1 and IDO2, human cells of various origins require IDO1 but not IDO2 for IFN-γ-induced cell-autonomous immunity to T. gondii. T. gondii secretes an effector TgIST to inhibit IDO1 mRNA expression. Taken together, the data suggests that T. gondii possesses virulence programs operated by TgIST to antagonize IFN-γ-induced IDO1-mediated anti-parasite cell-autonomous immunity in human cells.

A clinical case of neosporosis in a 4-week-old holstein friesian calf which developed hindlimb paresis postnatally.

A 4-week-old female Holstein Friesian calf presented with hindlimb paresis. Neurologic examination of spinal reflexes revealed depressed or absent reflexes of the hindlimbs. Menace responses on both sides disappeared on examination of cranial nerves. The calf was finally diagnosed with Neospora caninum infection by pathological findings including nonsuppurative inflammation associated with cysts in the cerebrum and spinal cord. High levels of antibody against recombinant surface antigen 1 of N. caninum (NcSAG1) were detected by ELISA from both serum and cerebrospinal fluid (CSF) samples. This result suggests that detection of antibodies against N. caninum by NcSAG1-ELISA in serum and CSF could be useful for the clinical diagnosis of neosporosis in calves with acquired neurological signs.

CCR5 Is Involved in Interruption of Pregnancy in Mice Infected with Toxoplasma gondii during Early Pregnancy.

Toxoplasmosis can cause abortion in pregnant humans and other animals; however, the mechanism of abortion remains unknown. C-C chemokine receptor type 5 (CCR5) is essential for host defense against Toxoplasma gondii infection. To investigate the relationship between CCR5 and abortion in toxoplasmosis, we inoculated wild-type and CCR5-deficient (CCR5-/-) mice with T. gondii tachyzoites intraperitoneally on day 3 of pregnancy (embryonic day 3 [E3]). The pregnancy rate decreased as pregnancy progressed in infected wild-type mice. Histopathologically, no inflammatory lesions were observed in the fetoplacental tissues. Although wild-type mice showed a higher parasite burden at the implantation sites than did CCR5-/- mice at E6 (3 days postinfection [dpi]), T. gondii antigen was detected only in the uterine tissue and not in the fetoplacental tissues. At E8 (5 dpi), the embryos in infected wild-type mice showed poor development compared with those of infected CCR5-/- mice, and apoptosis was observed in poorly developed embryos. Compared to uninfected mice, infected wild-type mice showed increased CCR5 expression at the implantation site at E6 and E8. Furthermore, analyses of mRNA expression in the uterus of nonpregnant and pregnant mice suggested that a lack of the CCR5 gene and the downregulation of tumor necrosis factor alpha (TNF-α) and CCL3 expression at E6 (3 dpi) are important factors for the maintenance of pregnancy following T. gondii infection. These results suggested that CCR5 signaling is involved in embryo loss in T. gondii infection during early pregnancy and that apoptosis is associated with embryo loss rather than direct damage to the fetoplacental tissues.

Immunization with Toxoplasma gondii peroxiredoxin 1 induces protective immunity against toxoplasmosis in mice.

To develop a vaccine against Toxoplasma gondii, a vaccine antigen with immune-stimulating activity is required. In the present study, we investigated the immunogenicity and prophylactic potential of T. gondii peroxiredoxin 1 (TgPrx1). The TgPrx1 was detected in the ascitic fluid of mice 6 days postinfection, while specific antibody levels were low in the sera of chronically infected mice. Treatment of murine peritoneal macrophages with recombinant TgPrx1 triggered IL-12p40 and IL-6 production, but not IL-10 production. In response to TgPrx1, activation of NF-kB and IL-6 production were confirmed in mouse macrophage cell line (RAW 264.7). These results suggest the immune-stimulating potentials of TgPrx1. Immunization of mice with recombinant TgPrx1 stimulated specific antibody production (IgG1 and IgG2c). Moreover, spleen cell proliferation and interferon-gamma production significantly increased in the TgPrx1- sensitized cells from mice immunized with the same antigen. Immunization with TgPrx1 also increased mouse survival and decreased cerebral parasite burden against lethal T. gondii infection. Thus, our results suggest that TgPrx1 efficiently induces humoral and cellular immune responses and is useful as a new vaccine antigen against toxoplasmosis.

Changes in neurotransmitter levels and expression of immediate early genes in brain of mice infected with Neospora caninum.

Neospora caninum is an obligate intracellular parasite that causes neurological disorders in dogs and cattle. The majority of host animals are asymptomatic at the chronic stage of infection. However, it remains unclear whether cerebral function is normal in asymptomatic animals. In this study, mice were infected with N. caninum (strain Nc-1) and their brains were examined to understand changes in cerebral function at the chronic stage of infection. Mice infected with N. caninum showed impaired locomotor activity, but no differences in clinical symptoms were observed. In the brains of infected mice, parasites were distributed throughout the brain and histological lesions were observed everywhere except for the cerebellum. Expression levels of proinflammatory cytokines, interferon-gamma and tumour necrosis factor-alpha, were highly upregulated in several brain regions of infected mice. Additionally, the level of neurotransmitters glutamate, glycine, gamma-aminobutyric acid, dopamine and 5-hydroxytryptamine, were altered in infected mice compared with those of uninfected mice. Interestingly, the expression levels of immediately early genes, c-Fos and Arc, in the brain of infected mice were lower than those of in uninfected mice. Our findings may provide insight into neurological disorders associated with N. caninum infection.

Characterization of Toxoplasma gondii glyoxalase 1 and evaluation of inhibitory effects of curcumin on the enzyme and parasite cultures.

BACKGROUND: The glyoxalase pathway, which includes two enzymes, glyoxalase 1 and 2 (Glo1 and Glo2), is a ubiquitous cellular system responsible for the removal of cytotoxic methylglyoxal produced during glycolysis. Protozoan parasites, including Toxoplasma gondii (T. gondii) tachyzoites, produce methylglyoxal because of increased glycolytic fluxes. A Glo1 inhibitor such as curcumin could be considered a drug candidate for anti-protozoan, anti-inflammatory, and anti-cancer therapy. METHODS: The T. gondii Glo1 gene (TgGlo1) was cloned and the recombinant protein was produced. Enzyme kinetics of TgGlo1 and five mutants were evaluated by adding methylglyoxal and glutathione to a reaction mixture. Finally, the inhibitory effects of various concentrations of curcumin on recombinant TgGlo1 were evaluated using in vitro cultures of T. gondii. RESULTS: Active recombinant TgGlo1 was successfully produced and the active sites (E166 and E251) of TgGlo1 were verified by point mutagenesis. Curcumin at the tested doses inhibited the enzymatic activity of recombinant TgGlo1 as well as the parasitic propagation of in vitro-cultured T. gondii. The Ki and IC50 were 12.9 ± 0.5 µM and 38.3 ± 0.9 µM, respectively. CONCLUSION: The inhibitory effect of curcumin on the enzymatic activity of TgGlo1 and parasitic propagation of T. gondii could be explored in the potential development of a potent drug for the treatment of toxoplasmosis. However, considering the fact that curcumin is known to have many effects on other molecules in the micromolar range, further elucidation of curcumin's direct inhibition of the glyoxalase system of T. gondii will be needed.

Cytoprotective Effects of Lysophospholipids from Sea Cucumber Holothuria atra.

Lysophospholipids are important signaling molecules in animals and metazoan cells. They are widely distributed among marine invertebrates, where their physiological roles are unknown. Sea cucumbers produce unique lysophospholipids. In this study, two lysophospholipids were detected in Holothuria atra for the first time, lyso-platelet activating factor and lysophosphatidylcholine, with nuclear magnetic resonance and liquid chromatography-time-of-flight mass spectrometric analyses. The lipid fraction of H. atra contained lyso-platelet activating factor and lysophosphatidylcholine, and inhibited H2O2-induced apoptosis in the macrophage cell line J774A.1. The antioxidant activity of the lysophospholipid-containing lipid fraction of H. atra was confirmed with the oxygen radical absorbance capacity method. Our results suggest that the lysophospholipids from H. atra are potential therapeutic agents for the inflammation induced by oxidative stress.

Macrophages are the determinant of resistance to and outcome of nonlethal Babesia microti infection in mice.

In the present study, we examined the contributions of macrophages to the outcome of infection with Babesia microti, the etiological agent of human and rodent babesiosis, in BALB/c mice. Mice were treated with clodronate liposome at different times during the course of B. microti infection in order to deplete the macrophages. Notably, a depletion of host macrophages at the early and acute phases of infection caused a significant elevation of parasitemia associated with remarkable mortality in the mice. The depletion of macrophages at the resolving and latent phases of infection resulted in an immediate and temporal exacerbation of parasitemia coupled with mortality in mice. Reconstituting clodronate liposome-treated mice at the acute phase of infection with macrophages from naive mice resulted in a slight reduction in parasitemia with improved survival compared to that of mice that received the drug alone. These results indicate that macrophages play a crucial role in the control of and resistance to B. microti infection in mice. Moreover, analyses of host immune responses revealed that macrophage-depleted mice diminished their production of Th1 cell cytokines, including gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). Furthermore, depletion of macrophages at different times exaggerated the pathogenesis of the infection in deficient IFN-γ(-/-) and severe combined immunodeficiency (SCID) mice. Collectively, our data provide important clues about the role of macrophages in the resistance and control of B. microti and imply that the severity of the infection in immunocompromised patients might be due to impairment of macrophage function.

Starvation of low-density lipoprotein-derived cholesterol induces bradyzoite conversion in Toxoplasma gondii.

BACKGROUND: Lacking enzymes for sterol synthesis, the intracellular protozoan Toxoplasma gondii scavenges cholesterol from host cells to multiply. T. gondii has a complex life cycle consisting of two asexual stages; the proliferative stage (tachyzoite), and the latent stage characterized by tissue cysts (bradyzoite). In vitro, bradyzoite development can be induced by mimicking host immune response stressors through treatment with IFN-γ, heat shock, nitric oxide, and high pH. However, the extent to which host nutrients contribute to stage conversion in T. gondii is unknown. In this study, we examined the impact of host cholesterol levels on stage conversion in this parasite. METHODS: Growth of T. gondii tachyzoites (ME49 strain) was investigated in Chinese hamster ovary (CHO) cells using various concentrations of low-density lipoprotein (LDL), oleic acid, or glucose. Squalestatin, which is an inhibitor of squalene synthase and is, therefore, an inhibitor of sterol synthesis, was used to treat the CHO cells. Tachyzoite to bradyzoite conversion rates were analyzed by indirect fluorescent antibody tests. RESULTS: Parasite growth was significantly enhanced by addition of exogenous LDL, whereas no such enhancement occurred with oleic acids or glucose. In ME49, growth inhibition from squalestatin treatment was not obvious. Although growth of the RH strain was unaffected by squalestatin in the presence of lipoprotein, in its absence growth of this strain was suppressed. The frequency of BAG1-positive vacuoles in ME49 increased under lipoprotein-free conditions. However, addition of exogenous LDL did not increase tachyzoite to bradyzoite conversion in this strain. Furthermore, treatment with squalestatin did not enhance stage conversion. CONCLUSION: Our results suggest that LDL-derived cholesterol levels play a crucial role in bradyzoite conversion in T. gondii.

Macrophage depletion prior to Neospora caninum infection results in severe neosporosis in mice.

We observed that murine macrophages showed greater activation and increased interleukin 6 (IL-6), IL-12p40, and interferon gamma (IFN-γ) production during Neospora caninum infection. Many macrophages migrated to the site of infection. Furthermore, macrophage-depleted mice exhibited increased sensitivity to N. caninum infection. This study indicates that macrophages are required for achieving protective immunity against N. caninum.

Overproduction of Toxoplasma gondii cyclophilin-18 regulates host cell migration and enhances parasite dissemination in a CCR5-independent manner.

BACKGROUND: Toxoplasma gondii hijacks host cells to allow it to disseminate throughout a host animal; however, the migratory machinery involved in this process has not been well characterized. We examined the functional role of T. gondii cyclophilin 18 (TgCyp18) in host cell recruitment using recombinant parasites transfected with TgCyp18. RESULTS: High levels of TgCyp18 enhanced IL-12 production in cysteine-cysteine chemokine receptor 5 (CCR5) knockout mice (CCR5-/-) that had been infected peritoneally with T. gondii. Recruitment of CD11b+ cells to the infection site was enhanced in a CCR5-independent manner. T. gondii spread to several organs, particularly the liver, in a TgCyp18-dependent and CCR5-independent manner. Additionally, CCL5 levels were upregulated in macrophages treated with recombinant protein TgCyp18 and in the peritoneal fluids of the infected CCR5-/- mice. Furthermore, the chemokines involved in macrophage migration, CCL2 and CXCL10, were upregulated in the livers of CCR5-/- mice infected with recombinant parasites that had been transfected with TgCyp18. CONCLUSION: TgCyp18 may play a crucial role in macrophage migration, and in assisting with transport of T. gondii via CCR5-independent mechanisms. TgCyp18 may also play a role with CCL5 in the migration of macrophages to the site of infection, and with CCL2 and CXCL10 in the transport of T. gondii-infected cells to the liver.

Molecular characterization and antigenic properties of a novel Babesia gibsoni glutamic acid-rich protein (BgGARP).

Identification and molecular characterization of Babesia gibsoni proteins with potential antigenic properties are crucial for the development and validation of the serodiagnostic method. In this study, we isolated a cDNA clone encoding a novel B. gibsoni 76-kDa protein by immunoscreening of the parasite cDNA library. Computer analysis revealed that the protein presents a glutamic acid-rich region in the C-terminal. Therefore, the protein was designated as B. gibsoni glutamic acid-rich protein (BgGARP). A BLASTp analysis of a translated BgGARP polypeptide demonstrated that the peptide shared a significant homology with a 200-kDa protein of Babesia bigemina and Babesia bovis. A truncated BgGARP cDNA (BgGARPt) encoding a predicted 13-kDa peptide was expressed in Escherichia coli (E. coli), and mouse antisera against the recombinant protein were used to characterize a corresponding native protein. The antiserum against recombinant BgGARPt (rBgGARPt) recognized a 140-kDa protein in the lysate of infected erythrocytes, which was detectable in the cytoplasm of the parasites by confocal microscopic observation. In addition, the specificity and sensitivity of enzyme-linked immunosorbent assay (ELISA) with rBgGARPt were evaluated using B. gibsoni-infected dog sera and specific pathogen-free (SPF) dog sera. Moreover, 107 serum samples from dogs clinically diagnosed with babesiosis were examined using ELISA with rBgGARPt. The results showed that 86 (80.4%) samples were positive by rBgGARPt-ELISA, which was comparable to IFAT and PCR as reference test. Taken together, these results demonstrate that BgGARP is a suitable serodiagnostic antigen for detecting antibodies against B. gibsoni in dogs.