Successful treatment of toxoplasmic encephalitis diagnosed early by polymerase chain reaction after allogeneic hematopoietic stem cell transplantation: two case reports and review of the literature.

Toxoplasmic encephalitis represents a rare, but often fatal infection after allogeneic hematopoietic stem cell transplantation. Polymerase chain reaction (PCR)-based preemptive therapy is considered promising for this disease, but is not routinely applied, especially in low seroprevalence countries including Japan. We encountered 2 cases of toxoplasmic encephalitis after transplantation that were successfully treated. The diagnosis of toxoplasmic encephalitis in these cases was confirmed by PCR testing when neurological symptoms were observed. Both patients received pyrimethamine and sulfadiazine treatments within 2 weeks of the development of neurological symptoms, and remained free of recurrence for 32 and 12 months. These results emphasized the importance of the PCR test and immediate treatment after diagnosis for the management of toxoplasmic encephalitis.

Heat stress-induced modulation of host defense against Toxoplasma gondii infection in mice.

This study examined the effects of burn injury on murine immune response against Toxoplasma gondii infection. Male C57BL/6 mice were divided into 3 groups: T. gondii infection (group T), burn injury (group B), and burn injury followed by T. gondii infection (group BT). The survival of group BT was significantly lower than those of group B and group T. Parasite abundance in the tissues was determined by quantitative competitive-polymerase chain reaction. Group BT exhibited significantly higher numbers of T. gondii than group T. Antibody production against T.g.HSP30 in group BT was significantly lower than that in group T, whereas no significant difference was observed in SAG1-specific antibody production. Delayed-type hypersensitivity (DTH) specific for 2,4-dinitrofluorobenzene (DNFB) of both group B and group BT was significantly lower than that of group T. One week after infection, serum interferon-gamma (IFN-gamma) and interleukin (IL)-10 levels in group BT were significantly lower, whereas serum IL-6 levels were significantly higher than in group T Serum TNF-alpha levels in both group T and group BT were elevated at 1 wk after infection, although there was no significant difference between them. Serum IFN-gamma, IL-10, and TNF-alpha levels in group B were not elevated during the experimental term. In conclusion, the impaired antigen-specific antibody production and DTH response, together with the modulated patterns of cytokine responses, seemed to be strongly involved in the development of burn-induced immunosuppression and the consequent increased susceptibility to T. gondii infection in mice.

Involvement of MyD88 in host defense and the down-regulation of anti-heat shock protein 70 autoantibody formation by MyD88 in Toxoplasma gondii-infected mice.

This study investigated the influence of TLR (toll-like receptor)4, TLR2, and MyD88 in Toxoplasma gondii-infected wild-type (WT) mice and TLR4-, TLR2-, and MyD88-deficient mice. Ninety-five percent of MyD88-deficient mice died 10-16 days after intraperitoneal infection with 100 cysts of T. gondii Fukaya strain, whereas 95-100% of TLR4- and TLR2-deficient mice and WT C57BL/6 (B6) mice survived for more than 7 wk after T. gondii infection. The distribution of T. gondii in various organs of TLR4-, TLR2-, and MyD88-deficient mice and WT B6 mice was assessed 2 wk after T. gondii intraperitoneal infection using quantitative competitive polymerase chain reaction. In MyD88-deficient mice, high levels of T. gondii load were observed in the brain, tongue, heart, lungs, spleen, liver, mesenteric lymph node, and kidneys after infection. The T. gondii load was significantly increased in the lungs in both TLR4- and TLR2-deficient mice compared with WT B6 mice. High levels of anti-mouse heat shock protein (mHSP)70 autoantibody and anti-T. gondii HSP70 antibody production were detected in the sera from MyD88-deficient mice.

Organ infectivity of Toxoplasma gondii in interferon-gamma knockout mice.

To determine the influence of interferon (IFN)-gamma on the organ infectivity and on the genetic susceptibility of susceptible (C57BL/6) and resistant (BALB/c) strains after peroral infection with cysts of Toxoplasma gondii. IFN-gamma knockout (KO) mice in C57BL/6 and BALB/c backgrounds were utilized. The kinetics of the changes in T. gondii abundance were evaluated with a quantitative competitive polymerase chain reaction assay in various organs at different times after peroral infection. In IFN-gamma KO mice, a T. gondii-specific gene, SAG1, was detected in all organs examined, and the protozoan proliferated much more actively than in wild-type mice. The abundance of T. gondii was much higher in mesenteric lymph nodes and the heart than in other organs. In contrast, in the nervous system organs and kidneys, only a weakly detectable reaction was observed. Toxoplasma gondii grew at a more rapid rate in the organs of IFN-gamma KO C57BL/6 mice than in the organs of IFN-gamma KO BALB/c mice during the course of infection. Destruction of the IFN-gamma gene showed remarkable effects on the infectivity in both susceptible and resistant mice.

Establishment of gene-vaccinated skin grafting against Toxoplasma gondii infection in mice.

Vaccine effects of in vivo gene-vaccinated skin graft were evaluated against Toxoplasma gondii (T. gondii) infection. By using a gene gun, cDNA coding T. gondii SAG1 molecule was intracutaneously vaccinated into C57BL/6 (B6; a susceptible strain), BALB/c (a resistant strain) and (C57BL/6 x BALB/c) F1 (CBF1) mice, and the gene-vaccinated skin of these strains was transplanted to CBF1 mice. Regarding the antibody production against SAG1, CBF1-recipient mice transplanted with the SAG1 gene-vaccinated B6 skin were high responders, whereas CBF1 mice skin grafted with vaccinated skin of both BALB/c and CBF1 mice were low responders. The donor-derived LC/DC migrated to the draining lymph nodes of the recipients from the skin graft within 3 days. The vaccine effect against T. gondii challenge infection was obtained in CBF1 mice which received the skin graft of the SAG1 gene-vaccinated BALB/c mice.

Toxoplasma gondii Hsp70 as a danger signal in toxoplasma gondii-infected mice.

Toxoplasma gondii Hsp70, T gondii Hsp30/bag1, and surface antigen 1 messenger RNAs were shown to be useful in analyzing stage conversion of T gondii between bradyzoites and tachyzoites. The high-level expression of T gondii Hsp70 was correlated with mortality in interferon-gamma knockout mice infected with T gondii. Tgondii Hsp70 inhibited the induction of nitric oxide release by peritoneal macrophages of T gondii-infected mice. These findings identify T gondii Hsp70 as a danger signal during lethal, acute T gondii infection.

Anti-HSP70 autoantibody formation by B-1 cells in Toxoplasma gondii-infected mice.

Formation of anti-Toxoplasma gondii HSP70 (TgHSP70) antibody cross-reactive to mouse HSP70 (mHSP70) was observed in the sera of BALB/c (a resistant strain) and C57BL/6 (B6; a susceptible strain) mice after peroral infection with T. gondii cysts of the Fukaya strain. The levels of anti-mHSP70 immunoglobulin G (IgG) autoantibody production in B6 mice were higher than those in BALB/c mice. The isotype and subclass of IgG of anti-TgHSP70 monoclonal antibodies cross-reactive to mHSP70 were mu and gamma3. Anti-mHSP70 autoantibody in T. gondii-infected BALB/c and B6 mice was shown to be produced by the CD5(+) subset of B cells (B-1a cells) but not by conventional B cells (B-2 cells). The epitopes recognized by anti-mHSP70 autoantibody were located primarily in the C-terminal fragment of mHSP70.

A role of carboxy-terminal region of Toxoplasma gondii-heat shock protein 70 in enhancement of T. gondii infection in mice.

We investigated the role of recombinant Toxoplasma gondii heat shock protein (rT.g.HSP) 70-full length, rT.g.HSP70-NH2-terminal region, or rT.g.HSP70-carboxy-terminal region in prophylactic immunity in C57BL/6 mice perorally infected with Fukaya cysts of T. gondii. At 3, 4, 5, and 6 weeks after infection, the number of T. gondii in the brain tissue of each mouse was measured by quantitative competitive-polymerase chain reaction (QC-PCR) targeting the surface antigen (SAG) 1 gene. Immunization with rT.g.HSP70-full length or r.T.g.HSP70-carboxy-terminal region increased the number of T. gondii in the brain tissue after T. gondii infection, whereas immunization with rT.g.HSP70-NH2-terminal region did not. These results suggest that T.g.HSP70-carboxy-terminal region as well as T.g.HSP70-full length may induce deleterious effects on the protective immunity of mice infected with a cyst-forming T. gondii strain, Fukaya.

Toxoplasma gondii: difference of invasion into tissue of digestive organs between susceptible and resistant strain and influence of IFN-gamma in mice inoculated with the cysts perorally.

Because it is widely accepted that there is a significant difference in susceptibility to chronic infection by Toxoplasma gondii among inbred mouse strains with different genetic backgrounds, we compared the distribution of the protozoa in digestive organs at early stages of infection between resistant (BALB/c) and susceptible (C57BL/ 6) mice after peroral infection with Fukaya strain cysts. Furthermore, to determine the influence of interferon gamma (IFN-gamma) on the infectivity of the cysts to the digestive tract, homozygous IFN-gamma knockout mice were utilized. Quantitative competitive polymerase chain reaction (QC-PCR) was employed to assess the distribution of T. gondii in different organs at various times after ingestion of cysts. SAG1, a T. gondii-specific gene, was detected in the small intestine and the caecum in wild-type C57BL/6 mice and in the whole digestive tract in IFN-gamma knockout C57BL/6 at 24 hr after infection. No detectable reaction in QC-PCR was observed in BALB/c mice at 24 hr after ingestion of the cysts. Destruction of the IFN-gamma gene showed less effect on the resistance to infection in BALB/c mice, but remarkable augmentation of infectivity of T. gondii to the rectum and peripheral blood was observed in C57BL/6 mice.

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.

Protective immunity induced by vaccination with SAG1 gene-transfected cells against Toxoplasma gondii-infection in mice.

To develop a vaccine by augmenting the protective cellular immunity against Toxoplasma gondii (T. gondii)-infection, T gondii SAG1 gene-transfectants were established by using RMA.S (H-2b), a murine transporter associated with the antigen processing (TAP) molecule-deficient lymphoma line, as a host antigen-presenting cell (APC). Immunization of C57BL/6 mice with the SAG1-transfected RMA.S induced CD8+ cytotoxic T lymphocytes (CTL) specific for not only SAG1-transfected RMA.S but also T gondii-infected RMA.S, and elicited protective responses to infection with a virulent T. gondii strain, RH.

Human pulmonary dirofilariasis: report of six cases.

We report six cases of pulmonary dirofilariasis diagnosed at our laboratory with clinical and pathological features. The nodules of dirofilariasis were round in three cases as previously reported, however dumbbell-shaped in two cases. The nodule did not attach to the pleura in four cases. Microscopically, the nodules were granulomas composed of central coagulation necrosis and peripheral fibrosis with round cell infiltration, histiocytes, and multinucleated giant cells. Necrotic pulmonary artery with single or multiple sections of degenerated nematode was observed in the center of the nodule. Dilated bronchioles with inflammation were observed in the nodule in four cases. Collapse of the alveoli, organizing pneumonia, hemosiderin-laden macrophages were observed around the nodule. We suppose that the nodule is not an infarction but a granuloma caused by antigen released from the nematode. Because the pulmonary dirofilariasis is difficult to be differentiated from primary or metastatic lung carcinoma, and the inflammation exists around the nodule, the nodule should be removed surgically.

Kinetics in parasite abundance in susceptible and resistant mice infected with an avirulent strain of Toxoplasma gondii by using quantitative competitive PCR.

The kinetics of changes in Toxoplasma gondii abundance were evaluated with a quantitative competitive (QC)-polymerase chain reaction (PCR) assay at various sites in both C57BL/6 and BALB/c mice. Higher mortality was apparent in C57BL/6 mice than in BALB/c mice when infected with a high dose of cysts. There were significant differences in cyst number when infected with a low dose of cysts, although there was no significant difference in mortality between the 2 mouse strains. One day after infection with a low dose of an avirulent Fukaya strain, T. gondii was detected in peripheral blood, mesenteric lymph nodes, spleen, lungs, and brain. Two weeks after infection, the number of T. gondii in the brain greatly increased in C57BL/6 mice but not in BALB/c mice. Thus, it would appear that the first to second week after infection is a critical period in determining T. gondii abundance. QC PCR allows the detection of low numbers of T. gondii at an early stage of infection in the murine model. This is useful for the early diagnosis of toxoplasmosis and to understand reactivation of toxoplasmosis.

Correlation between direct binding ability of synthetic T. gondii SAG1 peptides to HLA-A2 measured by a sensor for surface plasmon resonance and antigenicity of the peptides for T. gondii-infected cell-specific CTL.

Toxoplasma gondii (T. gondii) -infected B lymphoma cells present T. gondii antigens in the context of major histocompatibility complex molecules to T. gondii-specific CD8+ cytotoxic T cells (CTL). HLA-A2 molecules of T. gondii-infected human cells have been shown to be used in presenting T. gondii antigens to CD8+ CTL. SAG1, one of the major antigenic molecules of T. gondii, is an antigen for T. gondii-specific CTL, and represents a possible basis for vaccines. The direct binding of nonamer SAG1 peptides to HLA-A2 was assayed here using an automated biosensor system with a sensor for surface plasmon resonance detection. The antigenicity of synthetic SAG1 peptides to T. gondii-specific CD8+ CTL also was assayed. The present study found a high correlation between the binding ability of synthetic SAG1 peptides to HLA-A2 and the antigenicity of peptides to T. gondii-infected cell-specific CD8+ CTL.

Heat shock cognate protein 71-associated peptides function as an epitope for Toxoplasma gondii-specific CD4+ CTL.

HLA-DR-restricted CD4+ cytotoxic T-lymphocyte (CTL) lines specific for Toxoplasma gondii (T. gondii)-infected melanoma cells have been established from peripheral blood lymphocytes (PBLs) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T. gondii-infected melanoma cells to these CD4+ CTL lines was investigated. A human melanoma cell line (P36) pulsed with T. gondii-infected P36 cell-derived HSC71 was lysed by a T. gondii-specific CD4+ CTL line (Tx-HSC-1). The Tx-HSC-1 also killed T. gondii-infected P36 cells. The lytic activity of Tx-HSC-1 against P36 cells pulsed with T. gondii-infected P36 cell-derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti-human leukocyte antigen (HLA)-DR mAb also partially blocked the lytic activity, whereas anti-HLA-A,B,C mAb did not block the lytic activity. In addition, a flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T. gondii-infected P36 cells as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line P36, and that HSC71 may play a potential role in Ag presentation and processing of T. gondii-infected P36 cells to CD4+ CTL.

Differential regulation of HLA-DR expression and antigen presentation in Toxoplasma gondii-infected melanoma cells by interleukin 6 and interferon gamma.

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 gamma (IFN-gamma) 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 T. gondii-infected P36 cells were upregulated by treatment with both recombinant IL-6 (rIL-6) and recombinant IFN-gamma (rIFN-gamma). The antigen-presenting ability of T. gondii-infected P36 cells to T. gondii-infected cell-specific CTL was enhanced by rIFN-gamma 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-gamma.

Enhanced cytotoxicity of IFN-gamma-producing CD4+ cytotoxic T lymphocytes specific for T. gondii-infected human melanoma cells.

CD4+ lines specific for Toxoplasma gondii-infected human melanoma P36 cells were established from PBL of a patient with chronic toxoplasmosis. CD4+ CTL lines were obtained by weekly in vitro stimulation with T. gondii-infected P36 cells that shared HLA-DR4 molecules with the patient. The lytic activity of CD4+ CTL lines against T. gondii-infected P36 or T. gondii-infected autologous EBV-transformed B lymphoma (EBV-Ya) was inhibited by anti-HLA-DR mAb, whereas anti-HLA-A, B, C mAb failed to block the lytic activity. Thus, the cytotoxicity of CD4+ CTL lines against T. gondii-infected P36 was restricted by HLA-DR molecules. In response to Ag-specific stimulation, CD4+ CTL lines produced significant levels of IFN-gamma. Exogenously added IFN-gamma up-regulated the surface expression of MHC class II, but not of class I in T. gondii-infected P36 cells. In addition, the CTL activity against T. gondii-infected P36 cells was augmented when target cells were co-cultured with IFN-gamma. These data indicate that CD4+ CTL-mediated cytotoxicity against T. gondii-infected melanocytes is enhanced by the autocrine production of IFN-gamma. Further, CD4+ CTL may play a role in the manifestation of toxoplasmic retinochoroiditis by killing T. gondii-infected melanocytes.

Isolation of naturally processed peptides from a Toxoplasma gondii-infected human B lymphoma cell line that are recognized by cytotoxic T lymphocytes.

Naturally processed peptides derived from Toxoplasma gondii (T. gondii) were acid extracted from T. gondii-infected cells and detected by cytotoxic T lymphocytes (CTL) derived from peripheral blood lymphocytes of a patient with chronic toxoplasmosis. The CTL lines were obtained by weekly in vitro stimulation with a T. gondii-infected human B cell lymphoma line, ARH, which shares HLA-A2 and -Cw4 determinants with the patient. The lytic activity of these CTL lines against T. gondii-infected ARH and ARH pulsed with fraction 29 of a reversed-phase high-performance liquid chromatography (HPLC) extract from T. gondii-infected ARH was inhibited by an anti-HLA-A, B, C monoclonal antibody (mAb) and an anti-HLA-A2 mAb. Anti-HLA-DR mAb failed to block the lytic activity. Thus, the presentation of peptides by T. gondii-infected cells for CTL is mediated by HLA-A2 molecules. Interestingly, antigen presentation of ARH pulsed with naturally processed HPLC fraction 29 peptides was not inhibited by treatment with brefeldin A. The amino acid sequence of the HLA-A2-bound peptide in fraction 29 was in part consistent with the predictive algorithm of HLA-A2-binding peptide motifs.

Is CD8 dependence a true reflection of TCR affinity for antigen?

Cytotoxic T lymphocytes (CTL) are generally specific for class I MHC proteins plus antigen and express CD8 co-receptor molecules. The effector function of some CTL can be blocked by antibodies to CD8 (CD8 dependent CTL), whereas that of others is resistant to blocking (CD8 independent CTL). This difference in sensitivity to antibody-mediated inhibition is assumed to reflect variations in affinity of particular TCR for antigen. However, we have found that a major difference between CD8 independent and CD8 dependent T cells lies in their sensitivity to stimulation, the former responding to lower concentrations of anti-CD3 antibody than the latter. Thus the contribution to cell signalling provided by the co-association of p56lck and CD8 is particularly relevant for CD8 dependent cells. These data challenge the notion that the affinity of an individual TCR for antigen is related to the sensitivity of a cell to inhibition by anti-CD8 antibodies. Furthermore we show that antibodies to co-receptor molecules have multiple distinct effects on T cell activation, only some of which may be related to T cell affinity.