Sensitivity of Types 1 and 2 Herpesvirus hominis to an Interferon Inducer, Poly I:C

Type 1 strains of Herpesvirus hominis were more resistant than type 2 strains to the antiviral effects of polyinosinic acid-polycytidilic acid in primary cultures of rabbit kidney cells.     

Interferon Induction by Poly(I):Poly(C) Enclosed in Phospholipid Particles

Liposomes were prepared with phospholipids (sphingomyelin, lecithin, and phosphatidylethanolamine) in combination with cholesterol and charged lipids (dicetyl phosphate and stearylamine) and contained either poly(I):poly(C) or poly(I). Neutral and positively charged liposomes attached much better to L-929 cells in tissue culture than did negatively charged particles. Liposomes were toxic to L cells at relatively low concentrations, making the determination of antiviral activity induced by particles containing poly(I):poly(C) difficult to measure by the plaque reduction assay. When injected into mice, all of the liposomes containing poly(I):poly(C), except phosphatidylethanolamine liposomes, greatly potentiated and extended the serum interferon response of poly(I):poly(C). Lecithin and sphingomyelin liposomes given intravenously were ten times more effective than free poly(I):poly(C) in stimulating production of serum interferon. Sphingomyelin liposomes containing [(14)C]poly(I):poly(C) were 88% cleared from the bloodstream of mice by 3 min after intravenous injection. Most of the radioactivity (70%) was captured by the liver and remained there for at least 4 h. By 2 h, 7% of the radioactivity could be found in the spleen. Five percent of the radioactivity was found in the lungs at 30 min, with decreasing amounts thereafter. Small amounts of radioactivity were found in the muscle and kidneys. The spleen was shown to contain appreciable levels of interferon at 4 h, and low levels were found in the liver. Radioactivity accumulated slowly in the liver following an intraperitoneal injection of sphingomyelin liposomes containing [(14)C]poly(I):poly(C). By 4 h, 26% of the dose was recovered from the liver and 4.9% from the spleen, with small amounts in the lung, kidney, and omentum.     

Expression of Indoleamine 2,3-Dioxygenase, Tryptophan Degradation, and Kynurenine Formation during In Vivo Infection with Toxoplasma gondii: Induction by Endogenous Gamma Interferon and Requirement of Interferon Regulatory Factor 1

The induction of indoleamine 2,3-dioxygenase (INDO) expression and the tryptophan (Trp)-kynurenine (Kyn) metabolic pathway during in vivo infection with Toxoplasma gondii was investigated. Decreased levels of Trp and increased formation of Kyn were observed in the lungs, brain, and serum from mice infected with T. gondii. Maximal INDO mRNA expression and enzyme activity were detected in the lungs at 10 to 20 days postinfection. Further, the induction of INDO mRNA expression, Trp degradation and Kyn formation were completely absent in tissues from mice deficient in IFN-gamma (IFN-gamma(-/-)) or IFN regulatory factor -1 (IRF-1(-/-)). These findings indicate the important role of endogenous IFN-gamma and IRF-1 in the in vivo induction of the Trp-Kyn metabolic pathway during acute infection with T. gondii. In contrast, expression of INDO mRNA and its activity was preserved in the tissues of TNF-receptor p55- or inducible nitric oxide synthase-deficient mice infected with T. gondii. Together with the results showing the extreme susceptibility of the IFN-gamma(-/-) and the IRF-1(-/-) mice to infection with T. gondii, our results indicate a possible involvement of INDO and Trp degradation in host resistance to early infection with this parasite.     

Acute induction of joint inflammation in the rat by Poly I · Poly C

Intraarticular injection of interferon inducer, double-stranded polyinosinate-polycytidylate (Poly I · C) caused acute synovitis in rats. This acute inflammatory response was accompanied by an increased concentration of prostaglandin E (PGE) in the synovial tissue. Double-stranded polyadenylate-polyuridylate (Poly A · Poly U) was less potent than Poly I · Poly C in inducing synovitis and increasing PGE concentration, while single-stranded polyinosinate (Poly I) or polycytidylate (Poly C) were inactive in these respects. Intraarticular injection of partially purified mouse fibroblast interferon also induced synovial inflammation. The present results suggest that interferon may be a mediator of viral inflammatory responses.Investigation supported in part by a grant from the Chief Scientist's Office, Ministry of Health, Israel.     

Long-Term Immunity to Lethal Acute or Chronic Type II Toxoplasma gondii Infection Is Effectively Induced in Genetically Susceptible C57BL/6 Mice by Immunization with an Attenuated Type I Vaccine Strain

C57BL/6 (B6) mice are genetically highly susceptible to chronic type II Toxoplasma gondii infections that invariably cause lethal toxoplasmic encephalitis. We examined the ability of an attenuated type I vaccine strain to elicit long-term immunity to lethal acute or chronic type II infections in susceptible B6 mice. Mice immunized with the type I cps1-1 vaccine strain were not susceptible to a lethal (100-cyst) challenge with the type II strain ME49. Immunized mice challenged with 10 ME49 cysts exhibited significant reductions in brain cyst and parasite burdens compared to naive mice, regardless of the route of challenge infection. Remarkably, cps1-1 strain-immunized B6 mice chronically infected with ME49 survived for at least 12 months without succumbing to the chronic infection. Potent immunity to type II challenge infections persisted for at least 10 months after vaccination. While the cps1-1 strain-elicited immunity did not prevent the establishment of a chronic infection or clear established brain cysts, cps1-1 strain-elicited CD8⁺ immune T cells significantly inhibited recrudescence of brain cysts during chronic ME49 infection. In addition, we show that uracil starvation of the cps1-1 strain induces early markers of bradyzoite differentiation. Collectively, these results suggest that more effective immune control of chronic type II infection in the genetically susceptible B6 background is established by vaccination with the nonreplicating type I uracil auxotroph cps1-1 strain.Toxoplasma gondii is a common and significant obligate intracellular pathogen of humans and animals. There are three clonal types that exist which are also thought to be derived when T. gondii acquired oral infectivity (50). Virulence in mice is strain specific where type I clones are universally virulent, type II clones are of intermediate virulence, and type III clones are avirulent. Ingestion of contaminated food sources is the most common route of human infection, resulting in systemic disease that can be divided into two stages: the acute disseminating tachyzoite stage and the chronic encysted bradyzoite stage (12). Recrudescent infections arising from reactivation of preexisting chronic latent cyst stages are particularly severe in the context of immune deficiency such as AIDS (38), and improved treatments and the development of vaccines to reduce disease burden are important therapeutic objectives. Strategies with the potential to eradicate the latent cyst stages present in already-infected individuals could be helpful but, unfortunately, the biology of cyst development, as well as the immune control mechanisms of latent stages, are relatively poorly understood at this time. Clearly, CD8⁺ T cells and gamma interferon (IFN-γ) are significant effectors in mediating resistance to acute and chronic T. gondii infection (17, 19).Numerous studies have evaluated responses to vaccines based on protein or DNA components of T. gondii with various degrees of success (1, 2, 4, 5, 9, 11, 22, 24, 25, 28, 36, 37, 40, 44, 46). Virulent parasite strains, as well as attenuated T. gondii strains, have been paramount in dissecting the immunobiology of host response in regard to understanding adaptive immune responses that may be helpful in vaccine design. Dense granule protein 6 (GRA6), GRA4, and rhoptry bulb protein 7 (ROP7) were recently identified as parasite antigens possessing a H-2L^d-restricted major histocompatibility complex class I (MHC-I) epitope that correlates with stage-specific expression and resistance to lethal chronic type II infections in the H-2L^d background (BALB/c). These data further define a potential molecular basis for genetic susceptibility to lethal type II chronic infections in the C57BL/6 H-2^b MHC-I-restricted background (6, 16). Vaccine models using either live attenuated parasites, such as type I strain ts-4, or irradiated tachyzoites, have had the greatest success in providing complete protection against lethal type I challenges. These studies also report more significant reductions in type II cyst burdens than component vaccines or whole-dead parasite vaccines (42, 48, 53, 54). However, live parasite-based vaccines such as strain ts-4 are still slowly replicating and retain a significant potential for virulence in the immunocompromised host. Furthermore, immune protection elicited by strain ts-4 is not long-lasting and significantly decreases within months after immunization (27).From the same parental RH strain that strain ts-4 was developed (45), our laboratory developed a fully attenuated nonreplicating type I cps1-1 strain that exhibits a severe uracil auxotrophy. The cps1-1 strain in a single immunization elicits complete immune protection and is able to clear high lethal dose virulent type I infection (14). Significantly, this highly attenuated strain is completely avirulent at extreme doses in immunocompromised hosts, such as in Tyk2^−/− mice (49), which cannot control inflammation, and also in IFN-γ^−/− mice (14, 20). The cps1-1 strain elicits potent Th1 immunity to lethal type I challenge infection after immunization of BALB/c, C57BL/6, Tyk2^−/− (C57BL/6), or MyD88^−/− (C57BL/6) mice (13, 14, 20, 49, 51, 56). Immunity to lethal type I challenge infection induced by the cps1-1 strain is dependent on CD8⁺ T cells (20), local production of IFN-γ (20), and interleukin-12 (IL-12) p70 (20, 51, 56). Remarkably, the potent immunity elicited by vaccination with the cps1-1 strain does not require systemic IFN-γ (20).We show here that the immunity induced in C57BL/6 mice after vaccination with the cps1-1 strain provides a surprisingly effective and complete protection from a lethal oral or intraperitoneal (i.p.) challenge infection of type II cysts from the ME49 strain. We address the durability of cps1-1 strain-induced immunity to lethal type II cyst challenge infection by different routes and find that immunization with the cps1-1 strain provides long-term protective immunity to lethal type II challenge. Vaccination with the cps1-1 strain also markedly reduces the cyst burden and protects susceptible C57BL/6 mice from succumbing to chronic infection. CD8⁺ immune T cells elicited by vaccination with the cps1-1 strain prevent cyst recrudescence during chronic infection.     

Use of Dense Granule Antigen GRA6 in an Immunoglobulin G Avidity Test To Exclude Acute Toxoplasma gondii Infection during Pregnancy

The usefulness of a specific immunoglobulin G (IgG) avidity enzyme-linked immunosorbent assay (ELISA) based on recombinant GRA6 antigen for distinguishing between acute and chronic Toxoplasma infection was investigated. Two sets of serum samples obtained from pregnant women with acute, chronic, or no Toxoplasma infection collected in France and Iran were used. Among the French subjects, 19 of 20 (95%) women who experienced seroconversion during the past 4 months before sampling displayed low-avidity IgG antibodies against GRA6, while all 17 (100%) women with chronic infection had high-avidity antibodies. When the Euroimmun IgG avidity ELISA was used, 15 of 19 (78.9%) recently infected women had low-avidity antibodies, and 20 of 22 (90.9%) women with chronic infection displayed high-avidity antibodies. The results suggested better performance of the GRA6 avidity ELISA than the Euroimmun avidity ELISA for exclusion of a recent infection occurring less than 4 months previously. Similarly, all 35 Iranian women with acute Toxoplasma infection had low-avidity antibodies against GRA6, whereas all 34 women with chronic infection displayed IgG antibodies of high avidity, indicating the value of GRA6 avidity testing for ruling out a recent infection. Avidity tests based on lysed whole-cell Toxoplasma gondii antigen are currently used to exclude recently acquired infections; however, the use of recombinant antigen(s) might improve the diagnostic performance of avidity tests and facilitate the development of more standardized assays.Congenital toxoplasmosis may occur when maternal infection is acquired during pregnancy and results in severe fetopathy or miscarriage (28, 38). While the rate of fetal infection with Toxoplasma gondii is extremely low in preconception infections, the transmission rate increases and the severity of fetal infection decreases as gestational age progresses (7, 18). Fortunately, prenatal treatment of the infection is effective for reducing both the incidence of clinical manifestations in infected newborns (8) and the maternal-fetal transmission rate (6, 29, 37). It is, therefore, essential to estimate the gestational age of the primary Toxoplasma infection as precisely as possible for the proper clinical management of pregnant women.Diagnosis of acute toxoplasmosis depends mainly on serological testing, as the infection is asymptomatic in 93% to 97% of pregnant women (10, 27). In countries where a prenatal screening of T. gondii infection is performed, detection of seroconversion or a significant rise in the specific immunoglobulin G (IgG) titer establishes a recently acquired infection. In most countries, however, a single serum sample from a pregnant woman is available to determine whether the infection occurred during gestation. IgM antibodies have been traditionally known as the markers of acute infection; however, persistent specific IgM has been found for up to several years after primary infection (15, 24, 39). Measurement of specific IgG avidity was shown to be an effective confirmatory test to help discriminate between recently acquired and distant infections (4, 16, 20, 22, 30, 31). In fact, it was shown that the combination of a sensitive test for T. gondii IgM and a specific IgG avidity assay is the best tool for determining the time of infection (34). The functional affinity (avidity) of specific IgG antibodies is low in the beginning of infection and usually increases with time by antigen-driven B-cell selection. However, low-avidity antibodies may persist for several months after acute infection, indicating that the avidity test is best used to rule out a recent infection in individuals with high-avidity results (5, 20, 30). Some studies reported the presence of very rare borderline- or high-avidity antibodies in acute infection with T. gondii (11, 20-22). The discordant results of avidity assays are attributed to the presence of antiparasitic treatment and immunodeficiency status; however, the complex nature of lysed whole-cell T. gondii antigen might cause such results (4, 35). Recombinant antigens might improve the performance of avidity assays for distinguishing between acute and chronic infection (1, 32, 33). Furthermore, preparation of recombinant antigens with more constant quality, compared to T. gondii antigen prepared from parasites grown either in cell culture or in mice peritoneal cavities, would facilitate development of better standardized assays (2).In this study, we developed an IgG avidity test based on recombinant GRA6 antigen and evaluated its value for differentiation between recently acquired and distant Toxoplasma infections in pregnant women. The potential usefulness of the GRA6 avidity test was compared with that of the Euroimmun IgG avidity enzyme-linked immunosorbent assay (ELISA; Euroimmun, Lübeck, Germany) for exclusion of a recent Toxoplasma infection that occurred less than 4 months before.     

2005年湛江市、韶关市、汕头市人兽弓形虫感染状况调查

目的了解广东省湛江市、韶关市、汕头市的人兽弓形虫感染情况。方法收集3个地区人、猪、鼠血清，采用酶联免疫吸附试验（ELISA）间接法检测弓形虫IgG抗体。结果湛江市检测猪血清279份，阳性13份，阳性率为4．66％，检测鼠血清93份，阳性7份，阳性率7．53％；韶关市检测人血清42份，阳性0份，检测鼠血清95份，阳性3份，阳性率3．16％；汕头市检测人血清50份，阳性6份，阳性率为12．00％，检测鼠血清100份，阳性3份，阳性率为4．00％。结论汕头市人群存在弓形虫感染，湛江市、韶关市、汕头市兽类存在弓形虫感染，3个地区的鼠类弓形虫感染率无统计学意义，家鼠、野鼠的弓形虫感染率也无统计学意义。     

Toxoplasma gondii : an ultrastructural study of host-cell invasion by the bradyzoite stage

The invasion of Toxoplasma gondii tachyzoites and bradyzoites was followed in bovine kidney cells via electron microscopy. The process of invasion differed between bradyzoites and tachyzoites. In the early stages of entry there was evidence of localised formation of membrane projections in the host cell adjacent to the parasite. Parasite reorientation and rhoptry release appeared to be necessary for invasion; however, the tight junction could not be clearly discerned and there was no evidence of constriction or of any membrane shedding from the parasite. The resulting parasitophorous vacuole was smaller than the tachyzoite vacuole and parasites were frequently found to lie immediately under the host cell membrane. The vacuole was rapidly adapted by the release and formation of an intra-phagosomal membrane network, while the parasitophorous vacuole formed a relationship with host-cell endoplasmic reticulum. Received: 28 March 1998 / Accepted: 28 April 1998     

Infection of Mouse Neural Progenitor Cells by Toxoplasma gondii Reduces Proliferation,Migration, and Neuronal Differentiation in Vitro

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Roles of IFN-gamma on stage conversion of an obligate intracellular protozoan parasite,Toxoplasma gondii

IFN-gamma downregulates the stage conversion of Toxoplasma gondii (T. gondii), from bradyzoites to tachyzoites, and the expression of heat shock protein 70 (HSP70) of T. gondii (T.g.HSP70) by tachyzoites. T.g.HSP70 has been shown to downregulate NO release from macrophages and also to induce auto-HSP70 antibody formation and IL-10 secretion by VH11-JH1 B-1 cells, resulting in the suppression of host defense responses to T. gondii infection. A novel category of virulent tachyzoite stage of T. gondii expressing T.g.HSP70 (virulent tachyzoite), which indirectly manifests its pathogenicity by downregulating host defense responses, has been proposed.     

Reassessment of the Role of Aromatic Amino Acid Hydroxylases and the Effect of Infection by Toxoplasma gondii on Host Dopamine

Toxoplasma gondii infection has been described previously to cause infected mice to lose their fear of cat urine. This behavioral manipulation has been proposed to involve alterations of host dopamine pathways due to parasite-encoded aromatic amino acid hydroxylases. Here, we report successful knockout and complementation of the aromatic amino acid hydroxylase AAH2 gene, with no observable phenotype in parasite growth or differentiation in vitro and in vivo. Additionally, expression levels of the two aromatic amino acid hydroxylases were negligible both in tachyzoites and in bradyzoites. Finally, we were unable to confirm previously described effects of parasite infection on host dopamine either in vitro or in vivo, even when AAH2 was overexpressed using the BAG1 promoter. Together, these data indicate that AAH enzymes in the parasite do not cause global or regional alterations of dopamine in the host brain, although they may affect this pathway locally. Additionally, our findings suggest alternative roles for the AHH enzymes in T. gondii, since AAH1 is essential for growth in nondopaminergic cells.     

Effects of polyinosinic-polycytidylic acid (Poly I:C) on naloxone-precipitated withdrawal in morphine-dependent mice

Viral infections are frequently found in opioid addicts, subjecting them to immune challenge. However, the effects of immune challenge on opioid withdrawal are not fully understood. In the present study, mice were intraperitoneally injected with 2mg/kg polyinosinic-polycytidylic acid (Poly I:C, a viral mimetic) for 3 days to induce an immune challenge, followed by subcutaneous injection of morphine 3 times per day for 3 days to induce morphine dependence. Withdrawal was induced by an intraperitoneal injection of 5mg/kg naloxone, an opioid receptor antagonist. The results showed that Poly I:C pretreatment did not alter body weight loss, jumping behavior, or locomotion during naloxone-precipitated withdrawal. In contrast, Poly I:C pretreatment significantly increased immobility time in the tail suspension test. Our findings suggest that Poly I:C-induced immune challenge has no effects on acute physical opioid withdrawal symptoms but facilitates depression-like behavior.     

Effect of cyclophosphamide on Toxoplasma gondii infection: reversal of the effect by passive immunization.

Treatment of mice with cyclophosphamide (CY) at a dose of 250 mg/kg body weight 24 hr prior to infection with an avirulent strain of Toxoplasma gondii delays the appearance of antibody by about one week and results in 70% mortality. To discount other effects of CY besides inhibition of antibody synthesis, CY-treated infected mice were passively immunized with a pooled specific serum collected from chronically infected syngeneic animals. Passive immunization reversed the effect of CY treatment if the titre of antibody in recipients reached 1 : 512 or more, as measured by the indirect immunofluorescence technique (IFT). It is therefore suggested that antibody plays an important role in establishing an infection-immunity (premunition) in this system.     

Effects of progesterone and estradiol sex hormones on the release of microparticles by RAW 264.7 macrophages stimulated by Poly(I:C)

Microparticles (MPs) are small membrane-bound vesicles that display proinflammatory and prothrombotic properties. These particles can be released by macrophages stimulated by ligands of the Toll-like receptors (TLRs) in a process that depends on nitric oxide (NO) production. Since sex hormones can modulate macrophage responses, we investigated the effects of progesterone and estradiol on macrophage particle release in vitro, comparing the responses with those induced by the glucocorticoid dexamethasone. As a model system for particle release, RAW 264.7 cells were stimulated in vitro with poly(I:C), a ligand of TLR3. Microparticles were measured by flow cytometry, while NO was measured by the Griess reaction. As the results of these studies showed, progesterone but not estradiol can block particle release by RAW264.7 cells treated with poly(I:C); dexamethasone was also active. Furthermore, while progesterone and dexamethasone inhibited NO production under the same culture conditions, neither agent blocked the production of particles stimulated by the NO donors dipropylenetriamine NONOate {(z)-1-[N-(3-aminopropyl)-N-(3-ammoniopropyl)amino] diazen-1-ium-1,2-diolate} and (z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino] diazen-1-ium-1,2-diolate. Studies using RU486 to assess the role of hormone receptors indicated that while this agent blocked the inhibition of particle and NO production by dexamethasone, it did not affect the inhibition by progesterone. Together, these results indicate that progesterone but not estradiol can inhibit particle release by stimulated macrophages and suggest a mechanism that may contribute to the immunomodulatory effects of this sex hormone.     

Changes in body temperature and vasopressin content of brain neurons,in pregnant and non-pregnant guinea pigs,during fevers produced by Poly I : Poly C

The synthetic polyribonucleotide pyrogen Poly I : Poly C (800 g · kg^–1) was injected intramuscularly on alternate days into pregnant and non-pregnant female guinea pigs. Pregnant animals, close to term, had smaller fevers in response to the pyrogen than did non-pregnant animals. Repeated injections of the pyrogen caused sequentially smaller fevers for the first 3–4 injections, particularly in non-pregnant animals, and this appeared to be like the tolerance usually developed to repeated injections of endotoxin. Continued pyrogen injections then caused, in non-pregnant animals, fevers of increasing magnitude until the original fever levels were reached, whereas in pregnant guinea pigs the fever responses remained reduced until parturition. The development of tolerance was associated with an increase in immunoreactivity for arginine vasopressin (AVP) in some neurons in the medial part of the paraventricular nucleus, and in terminals in the lateral septum and amygdala similar to changes found in these areas at term of pregnancy. These observations raise the possibility that AVP in these regions may have a role in the development of tolerance to pyrogens, and further quantitative studies of the AVP content of, and release from, nerve terminals projecting to the limbic system seem warranted.     

Expression and secretion of antiviral factors by trophoblast cells following stimulation by the TLR-3 agonist, Poly(I : C)

BACKGROUND: During pregnancy, the placenta may become exposed to micro-organisms, such as viruses, which may pose a substantial threat to the embryo/fetus well-being. Recent insight into the immunological capabilities of the trophoblast suggests that the placenta may function as an active barrier by recognizing and responding to pathogens through Toll-like receptors (TLRs). METHODS: The objective of this study was to determine whether the engagement of TLR-3 with viral dsRNA by first-trimester trophoblast could induce the production of factors necessary to generate an antiviral response. Therefore, trophoblast cells were exposed to the TLR-3 agonist, Poly(I : C). RESULTS: We report that following stimulation with Poly(I : C), first-trimester trophoblast cells produce interferon beta (IFNbeta) and secretory leukocyte protease inhibitor (SLPI), as well as the intracellular factors 2',5'-oligoadenylate synthetase (OAS), Myxovirus-resistance A (MxA) and apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G). This response is TLR-3 specific because the TLR-4 ligand, lipopolysaccharide (LPS), had no effect on the production of these antimicrobial factors. Furthermore, we describe a positive feedback mechanism in which IFNbeta enhances the antiviral response by promoting the production of OAS, MxA and APOBEC3G. CONCLUSIONS: These findings suggest that trophoblast cells are able to recognize and specifically respond to viral products in a highly regulated fashion and that the placenta may be pivotal in the control of viral infections at the maternal-fetal interface.