CD8+ T Lymphocytes Are the Major Cell Population Involved in the Early Gamma Interferon Response and Resistance to Acute Primary Toxoplasma gondii Infection in Mice

Gamma interferon (IFN-γ) is known to be a major mediator influencing host defense against Toxoplasma (T.) gondii. To evaluate lymphocyte populations involved in this cytokine-mediated early resistance to T. gondii, the effects of in vivo administration of monoclonal antibodies (MAbs) against T-cell subsets and anti-asialo GM1 antibody on the course of infection and IFN-γ response were investigated in mice infected acutely with this parasitic protozoan. A single injection of anti-CD8 MAb on day ?1 or day 4 severely exacerbated the infection, in accordance with a marked suppression of endogenous IFN-γ production. Moreover, the administration of anti-IFN-γ MAb on day 0 but not later than day 4 resulted in a total abrogation of resistance to T. gondii, suggesting that endogenous IFN-γ produced during the first several days of infection is critical for the generation of antitoxoplasmal resistance in mice. In contrast, no significant increase in mortality was observed when injected with either anti-CD4 MAb or anti-asialo GM1 antibody on day ? 1, while these antibodies reduced significantly the ability of mice to produce IFN-γ. Indeed, simultaneous depletion of CD4⁺ and CD8⁺ cells had no greater suppressive effect on host defense and endogenous IFN-γ production than depletion of CD8⁺ cells alone. Together, these results suggest that CD8⁺ T cells play a central role for resolution of acute toxoplasmosis by participating in endogenous IFN-γ production. The possible role of early produced IFN-γ in the development of protective immune response to T. gondii is also discussed.     

Failure of Mouse Peritoneal Macrophage Activation by the Purified Excreted/Secreted Antigens of Toxoplasma gondii

This study was carried out to evaluate the effect of excreted/secreted antigens on macrophages infected by Toxoplasma gondii. Six proteins 28, 30, 45, 58, 63 and 145 kDa were separated by different chromatographic techniques. Mouse peritoneal macrophages were treated in vitro and in vivo with these purified fractions. Penetration and proliferation assays of T. gondii in the macrophages were performed in vitro. The different antigens used did not change the rate of penetration and proliferation of the parasites. Therefore, the secreted products, which are capable of provoking an immune response, could not directly activate the macrophages. Furthermore, the secreted products were not cytotoxic and neither did they possess a visible phospholipasic activity which would have increased penetration.     

A novel B-2 suppressor cell regulating susceptibility/resistance of mice to Toxoplasma gondii infection

Irradiation treatment enhanced resistance of C57BL/6, but not BALB/c against Toxoplasma gondii infection. Six Gy-irradiated (IR) C57BL/6 recipients of B-2 cells from T. gondii-infected C57BL/6 died after infection. B-2 suppressor cells from infected C57BL/6 enhanced production of IL-4 and IL-10 in peritoneal exudate cells (PECs), and down-regulated NO release in peritoneal macrophages after infection. On the other hand, B-2 suppressor cells were not detected in a strain, BALB/c, resistant against infection. These data indicated that irradiation-sensitive B-2 cells regulated susceptibility/resistance in mice against T. gondii infection.     

Induction of protective immunity by primed B-1 cells in Toxoplasma gondii -infected B cell-deficient mice

We examined the role of B‐1 cells in protection against Toxoplasma gondii infection using B cell‐deficient mice (μMT mice). We found that primed but not naïve B‐1 cells from wild‐type C57BL/6 mice protected B cell‐deficient recipients from challenge infection. All μMT mice transferred with primed B‐1 cells survived more than 5 months after T. gondii infection, whereas 100% of μMT mice transferred with naïve B‐1 cells succumbed by 18 days after infection. Additionally, high expression of both T help (Th) 1‐ and Th2‐type cytokines and a high level of nitric oxide production were observed in T. gondii‐infected μMT mice transferred with primed B‐1 cells. Thus, it was clearly demonstrated that B‐1 cells play an important role in host protection against T. gondii infection in μMT mice.     

Pathogenicity of Toxoplasma gondii through B-2 cell-mediated downregulation of host defense responses

IFN-gamma is the primary mediator of anti-parasite effector mechanisms against Toxoplasma gondii. After intraperitoneal infection with the Fukaya strain of T. gondii, unirradiated IFN-gamma knock-out (GKO) mice transferred with wild type (WT) CD8+ effector T cells from infected mice failed to induce the production of IFN-gamma and died, whereas irradiated (IR) GKO mice transferred with WT CD8+ T cells induced IFN-y production and survived more than 6 months. IR GKO mice transferred with WT CD8+ T cells together with GKO B-2 cells died 8 days after infection, whereas those transferred with WT CD8+ T cells together with B-la or T cells survived. B-2 cells of infected GKO mice activated CD11b+ cells for IL-4 production, and down-regulated NO release, STAT1 phosphorylation, and interferon regulatory factor-1 expression in the peritoneal exudates cells of IR GKO mice transferred with WT CD8+ T cells together with GKO B-2 cells after infection. Thus, B-2 cells in T. gondii-infected mice act as suppressor cells in the host defense of infected mice.     

Protective effect of vaccination with Toxoplasma lysate antigen and CpG as an adjuvant against Toxoplasma gondii in susceptible C57BL/6 mice

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems to both humans and livestock and of great economic impact worldwide. Oligodeoxynucleotides (ODN) which contain immunostimulatory CG motifs (CpG ODN) can promote Th1 responses, an adjuvant activity that is desirable for vaccination against intracellular pathogens. We investigated the feasibility of using CpG as an adjuvant combined with Toxoplasma lysate antigen (TLA) as a vaccine against toxoplasmosis. Genetically susceptible C57BL/6 mice were vaccinated with TLA with or without CpG ODN as an adjuvant and then challenged with 85 cysts of the moderately virulent RRA (Beverley) strain of T. gondii. Prior to challenge infection, immunization with TLA plus CpG ODN directed cellular and humoral immunity toward a Th1 pattern, characterized by enhanced INF gamma production by splenic cells in response to TLA, and enhanced production of toxoplasma-specific IgG and IgG (2a) antibodies. Consequently, CpG/TLA-treated mice showed prolonged survival and 64% reduction in brain parasite burden compared to non-CpG/TLA treated group. Our results suggest that CpG ODN would provide a stable and effective adjuvant for use in vaccination against toxoplasmosis.     

Peroral infectivity of Toxoplasma gondii in bile and feces of interferon-gamma knockout mice

Toxoplasama gondii appeared in the bile and feces of interferon-gamma knockout (GKO) but not wild type mice on days 7-8 after peroral infection with T. gondii cysts of Fukaya strain. Both tachyzoite-specific SAG1 and bradyzoite-specific T.g. HSP30 mRNAs were detected in the bile and feces of GKO mice. Tachyzoites converted to bradyzoites by culturing in the bile. By feeding uninfected mice with the bile and washed feces of T. gondii-infected GKO mice, T. gondii-specific antibody formation in the serum and cyst formation in the brain were observed. The novel migration route of T. gondii from liver to bile and feces in GKO mice was confirmed.     

The role of IFN-gamma and Toll-like receptors in nephropathy induced by Toxoplasma gondii infection

The pathologic links between Toxoplasma gondii infections and renal diseases have not yet been established. Gamma interferon (IFN-gamma) and Toll-like receptors (TLRs) are involved in the host defense mechanism against T. gondii infection. The role of IFN-gamma and TLRs in renal function of T. gondii -infected mice was studied using wild type (WT), TLR2-deficient and TLR4-deficient mice perorally infected with cysts of an avirulent cyst-forming Fukaya strain of T. gondii. T. gondii was abundant in kidneys in IFN-gamma KO (GKO) mice as determined by a quantitative competitive-polymerase chain reaction (QC-PCR). But, T. gondii was not detected in kidneys in WT, TLR2-deficient and TLR4-deficient mice. Interestingly, renal function of TLR2-deficient and TLR4-deficient mice was damaged as evaluated by serum creatinine, serum blood urea nitrogen (BUN), and urine albumin/creatinine ratio (ACR), whereas renal function of GKO and WT mice was not damaged. Histopathology of TLR2-deficient mice exhibited glomerular and extracellular matrix swelling with advancing glomerular tissue proliferation, thickened Bowman's capsules and vacuolization of tubules. Renal immunofluorescence study of T. gondii -infected TLR2-deficient mice displayed positive staining of the glomerular basement membrane, mesangial areas and peritubular capillaries. The damage of kidney from TLR4-deficient mice was less severe compared to TLR2-deficient mice, and histopathological damage of kidney was not observed in WT and GKO mice. These results indicate that TLR2, but not IFN-gamma, plays a role in the protection of the renal function against T. gondii infection.     

Role of Toxoplasma gondii HSP70 and Toxoplasma gondii HSP30/bag1 in antibody formation and prophylactic immunity in mice experimentally infected with Toxoplasma gondii.

Production of antibodies against Toxoplasma gondii (T. gondii)-derived stress proteins, T. gondii HSP70 (T.g.HSP70) and T.g.HSP30/bagl, in C57BL/6 and BALB/c mice perorally infected with cysts of the avirulent Fukaya strain of T. gondii was analyzed. Production of anti-T.g.HSP70 IgG antibodies was transient, whereas production of anti-T.g.HSP30/bag1 IgG antibodies persisted after infection in both C57BL/6 and BALB/c mice. C57BL/6 mice, a susceptible strain, predominantly produced IgG antibodies specific for T.g.HSP70, whereas BALB/c mice, a resistant strain, predominantly produced IgG antibodies specific for T.g.HSP30/bag1, after T. gondii infection. Immunization with rT.g.HSP30/bag1 enhanced, whereas immunization with rT.g.HSP70 reduced host protective immunity against T. gondii infection with a cyst-forming avirulent strain, Fukaya, and a virulent strain, RH.     

Seroprevalence of Bartonella henselae,Toxoplasma gondii,FIV and FeLV infections in domestic cats in Japan

Seroprevalence of Bartonella henselae, Toxoplasma gondii, feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections was investigated in 1,447 domestic cats derived from the north (Hokkaido) to the south (Okinawa) prefectures in Japan. Of the cats investigated, 8.8% (128/1,447) were seropositive to B. henselae, 5.4% (78/1,447) to T. gondii, 9.8% (107/1,088) to FIV, and 2.9% (32/1,088) to FeLV, respectively. For B. henselae infection, the positive rate varied from 11.5% in cats of 1 to <2 years old to 7.2% in those over 3 years old. Outdoor cats showed higher positive rate (14.5%) than that (7.0%) in indoor ones. The rate (13.5%) in flea-infested cats was significantly higher than that (7.4%) in flea-negative cats. The positive rates in southern and urban sites were more likely to be higher than those in northern and suburban sites, suggesting that warm and humid environments, density of cat population, and raising status, including hygienic condition and flea infestation in cats may correlate to higher seroprevalence of B. henselae infection. For T. gondii, FIV and FeLV infections, the seroprevalence also tended to be higher in outdoor, flea-infested cats and advanced age groups. For FIV infection, the positive rates in male (14.3%) and outdoor cats (15.0%) were significantly higher than those in female (5.0%) and indoor cats (4.6%). On the other hand, no significant difference in seropositivities was observed in FeLV and T. gondii infections concerning to both genders and raising status.     

反相高压液相色谱折叠重组人白细胞介素-2

重组人白细胞介素２（ｒｈＩＬ２）基因表达产物在大肠杆菌中以不溶性包涵体形式存在,经过菌体发酵和收集、超声破菌、包涵体抽提、稀释透析初步复性、反相高压液相色谱纯化,终产物纯度大于９９％。反相高压液相色谱不仅可以纯化ｒｈＩＬ２,且使产物的总活性提高７９～９倍,比活性提高１４～１８倍,达到１５×１０７ｕ／ｍｇ蛋白质,蛋白得率为５０％～５６％。结果表明,反相高压液相色谱具有纯化和显著提高ｒｈＩＬ２折叠效率的双重功效,为非ＳＤＳ变复性法生产ｒｈＩＬ２提供了简便有效的方法     

Roles of Toxoplasma gondii-derived heat shock protein 70 in host defense against T. gondii infection

C57BL/6 mice receiving intraperitoneal injection of Toxoplasma gondii -derived heat shock protein 70 (T.g. HSP70) on day 3 post T. gondii infection succumbed by day 9 post infection, while vector protein-injected control mice survived more than 6 months. The deteriorating effect of T.g. HSP70 on host immune responses was dose-dependent. By T.g. HSP70 injection, T. gondii loads increased in various organs of T. gondii-infected mice. Th2 cytokines such as IL-4 and IL-10 were continuously produced from spleen and peritoneal exudate cells of T. gondii -infected mice by injection of T.g. HSP70. Furthermore, nitric oxide production from peritoneal macrophages in T. gondii-infected mice was reduced by T.g. HSP70.     

Induction of Interleukin 2-Responsiveness in Thymocytes of the Transgenic Mice Carrying lck-Transgene

The role of lck gene in T cell proliferation and differentiation was investigated with transgenic mice carrying human lck cDNA whose expression was regulated by the promoter of mouse H-2K^b and the enhancer element of mouse IgH. RNase protection assay revealed that the lck transgene was expressed in the thymus and spleen, whereas immunoblot analysis demonstrated that amounts of p56^lck in freshly isolated lymphoid organs were almost equal between transgenic mice and negative littermates. Cell-surface marker analyses of the thymocytes and peripheral lymphocytes revealed no remarkable difference between both groups. Notable finding is that the thymocytes from transgenic mice showed a significant proliferative response to the stimulation with IL-2, but not the thymocytes from negative littermates. Further analysis revealed that CD4⁺8^– single positive thymocytes proliferated in response to IL-2. While surface expression levels of IL-2Rα and IL-2Rβ of these CD4⁺8^– thymocytes from transgenic and control mice were almost equal before stimulation with IL-2, the expression of IL-2Rβ was induced only in transgenic thymocytes after stimulation with IL-2. Immunoblot analysis demonstrated that the expression of p56^lck of transgenic thymocytes was not down-reguated at 4 hr after stimulaion with IL-2, whereas p56^lck of control ones were not detectable any more at 4 hr after stimulation with IL-2. Moreover, in vitro kinase assay substantiated such unchanged expression of p56^lck in the thymocytes from transgenic mice: the kinase activities of p56^lck did not decrease in thymocytes from transgenic mice after stimulation with IL-2, while kinase activities of control ones were significantly down-regulated by stimulation of IL-2. These results suggested that a significant proliferative response found in the thymocytes from lck-transgenic mice after the stimulation with IL-2 was caused by a constitutive expression of p56^lck in these thymocytes even after the stimulation. Our findings, therefore, support a possibility that p56^lck may play a role in the IL-2R-mediated signaling system in CD4⁺8^– thymocytes.     

Immune response induced by recombinant Mycobacterium bovis BCG expressing ROP2 gene of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite, capable of infecting a variety of mammals and birds. Development of vaccine against T. gondii would be of great medical and veterinary value. In this study, the DNA sequence encoding ROP2 from T. gondii was cloned into the muticopy mycobacterial expression vector, pMV262, under the control of the Bacillus Calmette–Guerin (BCG) hsp60 promoter, and electroporated into BCG. Following selection of kanamycin, the recombinant BCG/pMV262-ROP2 was constructed and the expression of ROP2 was confirmed by Western blotting. The BALB/c mice inoculated with the BCG/pMV262-ROP2 developed specific immune responses against ROP2 protein, and there was an obvious delay in the mortality curve than the control (P < 0.05). These results indicated that M. bovis BCG is an adequate vector to express and present antigens of T. gondii, and it may be used to further study the induction of protective immunity in other animals.     

Differential Regulation of HLA-DR Expression and Antigen Presentation in Toxoplasma gondii-Infected Melanoma Cells by Interleukin 6 and Interferon γ

CD4⁺ cytotoxic T lymphocytes (CTL) clones, YT-4 and YT-9, specific for Toxoplasma gondii (T. gondii)-infected melanoma SK-MEL 28 (P36), were generated from the peripheral blood lymphocytes (PBL) of a patient with chronic toxoplasmosis. These CTL clones were shown to secrete significant amounts of interleukin 6 (IL-6) and interferon γ (IFN-γ) upon antigen (Ag)-specific stimulation. Downregulation of human leukocyte antigen (HLA)-DR surface expression and HLA-DR mRNA levels in P36 cells were observed when P36 cells were infected with T. gondii. Such downregulated HLA-DR expressions of 71 gondii-infected P36 cells were upregulated by treatment with both recombinant IL-6 (rIL-6) and recombinant IFN-γ (rIFN-γ). The antigen-presenting ability of T. gondii-infected P36 cells to T. gondii-infected cell-specific CTL was enhanced by rIFN-γ but not by rIL-6. The present study reveals the existence of differential regulation of HLA-DR expression and Ag presentation in T. gondii-infected melanoma cells by IL-6 and IFN-γ.     

Characterization of a new gene WX2 in Toxoplasma gondii

Using hybridization techniques, we prepared the monoclonal antibody （Mab） 7C3-C3 against Toxoplasma gondii. The protection tests showed that the protein （Mab7C3-C3） inhibited the invasion and proliferation of T. gondii RH strain in HeLa cells. The passive transfer test indicated that the antibody significantly prolonged the survival time of the challenged mice. It was also shown that the antibody could be used for the detection of the circulating antigen of T. gondii. After immunoscreening the T. gondii tachyzoite cDNA library with Mab7C3-C3, a new gene wx2 of T. gondii was obtained. Immunofluorescence analysis showed that the WX2 protein was located on the membrane of the parasite. Nucleotide sequence comparison showed 28% identity to the calcium channel α-IE unit and shared with the surface antigen related sequence in some conservative residues. However, no match was found in protein databases. Therefore, it was an unknown gene in T. gondii encoding a functional protein on the membrane of T. gondii. Because it has been shown to have a partial protective effect against T. gondii infection and is released as a circulating antigen, it could be a candidate molecule for vaccine or a novel target for new drugs.     

人重组白细胞介素12在CHO细胞中的表达

将人白细胞介素12（interleukin 12, IL-12）两条链p35及p40全长cDNA分别亚克隆至真核表达载体pcDNA3中,构建了pcDNA3/p35a,pcDNA3/p40a,pcDNA3/p35b,pcDNA3/p40b四种真核细胞重组表达质粒,利用磷酸钙共沉淀法转染中国苍鼠卵巢(CHO)细胞. 通过对阳性克隆的筛选鉴定,获得了稳定表达人IL-12的CHO细胞株,活性最高的一株表达量为105 U/ml.此细胞株经半年的传代培养,能够稳定地分泌IL-12.结果显示：IL-12在CHO细胞中的稳定表达受到重组质粒结构、DNA整合、mRNA转录、蛋白质翻译等多因素的影响,IL-12两个亚基在CHO细胞中的共同表达是产生有生物活性IL-12的基础.     

弓形虫上海株的棒状体蛋白ROP2的克隆表达及纯化

根据已发表基因序列(GenBank登录号为Z36906)设计引物,以弓形虫(Toxoplasma gondii)上海本地株的基因组DNA为模板,扩增编码ROP2(rhpotry protein2)蛋白的基因片段,定向克隆至表达质粒pET32a(+),重组质粒经限制性酶切鉴定后测序,结果表明插入片段长度为1044bp,与GenBank上登录的序列相比,同源性为96%-100%,其中与弓形虫RH株的rop2基因同源性为100%。重组原核表达质粒pET32a-rop2转化至大肠杆菌BL21(DE3),经诱导可表达分子量约60.9kD的融合蛋白,能被感染弓形虫RH株的绵羊阳性血清识别。