Isolation and characterization of a monoclonal anti-P30 antibody resistant mutant of Toxoplasma gondii

Of the possible iodine-labelled Toxoplasma gondii surface proteins, P30 (apparent Mr 30,000) is the principal one recognized by acute and convalescent anti-toxoplasma sera. This protein which comprises from 3 to 5% of the total parasite protein was used to raise a panel of parasiticidal monoclonal anti-P30 antibodies. One of these monoclonal antibodies was able to select a resistant mutant from a large population of chemically mutagenized wild-type P strain parasites. This mutant retained the wild type sensitivity to other non-P30 parasiticidal monoclonal antibodies as well as polyclonal anti-P30 rabbit sera. Analysis of surface radioiodinated wild type and mutant parasites showed that the mutant had a quantitative reduction in the amount of P30. A comparison of surface biotin labelled wild type and resistant parasites by two dimensional electrophoresis showed that the mutant lacked one and possibly two of several proteins that make up wild type P30. Western blot analysis indicated that the mutant was devoid of antigenically reactive P30. These findings further support the hypothesis that antigenic variants of T. gondii can be induced and may involve the major surface membrane antigens of the parasite.     

In vivo anti-melanoma activities of the Melan-A/MART-1101–115 T CD4+ cell peptide

Malignant melanoma causes significant health problems. The identification of tumour-associated antigens has led to novel approaches to increase T cell mediated anti-tumour immune response. Melan-A/MART-1 has been use as target antigen for several T cell based immunotherapeutic treatments. More recently, the critical role of CD4+ T cells in inducing and maintaining anti-tumour immunity has been increasingly recognized. In order to optimize tumour immunotherapy, greater efforts have been concentrated on the identification of tumour antigens presented by MHC class II molecules to CD4+ T cells. In a publication, Tiwari et al. (2004) [1] have identified by a computational approach the 15-mer amino-acid sequence 101–115 (PPAYEKLSAEQSPPP) of the Melan-A/MART-1 as a good target for a vigorous and safe immunotherapy. Therefore, we have investigated the in vivo anti-tumour activity of this peptide in a murine melanoma model. For the prophylactic treatment, 20 μg or 50 μg peptide was subcutaneously injected in mice once a week during 3 weeks before tumour induction. Treatment with 50 μg peptide significantly affected tumour development. Thus, our preliminary data demonstrate potential in vivo prophylactic activity of the 101–115 peptide-based vaccine to control melanoma growth.     

Human CD4+CD25+ Regulatory T Cells Suppress Anti-Porcine Xenogeneic Responses

Due to the shortage of human organs, xenotransplantation is being explored as an alternative to allotransplantation, but immune rejection remains a major hurdle to its implementation. We tested the ability of human CD4+CD25+ T cells (Treg cells) to suppress CD4+ T cell-mediated anti-porcine xenoresponses usingin vitroassays. Human Treg cells were hyporesponsive to porcine cell stimulation and suppressed the proliferative response of CD4+CD25- T cells in a dose-dependent manner, and comparison of the allo- and xenoresponses indicated that more Treg cells might be required to suppress the xenogeneic response than the allogeneic response. Stimulation of CD4+CD25- T cells with porcine cells resulted in secretion of IFN-gamma, TNF-alpha, IL-10, IL-6 and IL-2, and Treg cells suppressed the secretion of these cytokines, as well as the CD4+CD25- T-cell cytolytic response against porcine cells. These results suggest a potential role for Treg cells in promoting xenograft survival.     

Expression of the T-cell markers CD2 and CD28 in healthy and atopic children during the first 18 months of life

Atopy may be associated with a reduced T-cell function early in life, particularly regarding maturation of Th1 responses. The T-cell surface molecules CD2 and CD28 are involved in important T-cell activation pathways. Stimulation via the CD2 receptor increases the responsiveness to interleukin (IL)-12, which is a potent inducer of Th1 responses, whereas CD28 stimulation is critical for Th2 differentiation. Our aim was to prospectively study the expression of the cell-surface markers CD2 and CD28 on T-cells in relation to development of atopic disease. Children (n = 172) were followed from birth to 18 months and the cumulative history of atopic disease was recorded. Blood samples were obtained at birth and at 18 months, and in a subgroup of 78 infants also at 3, 6 and 12 months. Flow cytometry was used to analyze the T-cell markers CD2 and CD28, the latter also within the subsets of T-helper (CD4+) and T-cytotoxic (CD8+) cells. At 18 months, 31 children had and 118 did not have atopic symptoms. At this age, skin prick test (SPT) positive children with atopic symptoms with or without an atopic family history (AFH) showed a lower expression of CD2 mode fluorescence intensity (FI) as well as a lower proportion of CD2+ cells, as compared with non-sensitized children with neither atopic symptoms nor AFH. This was accompanied by a higher expression of CD28 FI on CD2+CD8+CD28+ cells. No significant differences were seen at time points before 18 months, although the proportion of CD2+ tended to be low also earlier in life. In conclusion, the observed reduced expression of CD2 in atopic infants may support previous findings that atopy is associated with a reduced CD2 function. The high CD28 FI in SPT positive children with atopic symptoms may possibly be a consequence of a TH2-skewed immune system.     

Acute lymphoblastic leukemia in children: correlation of musculoskeletal manifestations and immunophenotypes

Purpose Studies on musculoskeletal manifestations (MSM) of childhood acute lymphoblastic leukemia (ALL) have yielded variable findings with regard to their clinical impact. We investigated the significance for differential diagnosis, treatment and outcome of musculoskeletal complaints as presenting symptoms of ALL, and their correlation with leukemia immunophenotypes, for which data is lacking. Methods Data on 783 children in the national study for childhood ALL between 1984 and 2003 were reviewed retrospectively. Statistical analysis examined possible relationships between MSM at the time of diagnosis and demographic and clinical data, biological features of leukemia (peripheral blood counts, immunophenotype and main cytogenetic aberration), response to initial prednisone treatment, and outcome. Results Of 765 children with data on orthopaedic complaints, 240 presented with MSM (31.4%). Among these children, B cell precursor (BCP) was much more common (209/576, 36.3%) than T cell ALL (25/176, 14.2%). Patients with MSM had lower white blood cell counts (WBC) (median of 9 vs. 20 × 10⁹/L, P < 0.001) and percentage of blast cells in the peripheral blood at diagnosis compared to those without (median of 27 vs. 53%, P < 0.001). Hepatomegaly and splenomegaly were less common in MSM group (67 vs. 53% <3 cm, P < 0.001, and 63 vs. 50% <3 cm, P < 0.001, respectively). Poor response to initial treatment with prednisone was recorded in 7.1% of patients with MSM versus 11.5% of those without (P = 0.086). The analysis revealed no independent effect of MSM on event-free survival (EFS), after correcting for differences in EFS related to immunophenotype or initial WBC. Conclusions MSM occur mostly in children with BCP ALL who present with less involvement of extramedullary organs, low peripheral blood blasts and white blood cells counts. These findings highlight the importance of including ALL in the differential diagnosis of MSM even in the presence of an apparently normal peripheral blood count. Our study also suggests that MSM are caused by leukemic cells with enhanced biological propensity to remain relatively confined within the intramedullary bone-marrow space.     

自制苏芪合剂联合治疗Ⅲ～Ⅳ级IgA肾病的临床疗效观察

目的 探讨自制苏芪合剂对Ⅲ～Ⅳ级IgA肾病患者的疗效及患者T细胞功能的改善.方法 28例Ⅲ～Ⅳ级IgA肾病患者随机分为两组,对照组:醋酸泼尼松-日1mg,kg,隔日晨起顿服,盐酸苯那普利-日10mg;治疗组:在上述用药的基础上加用自制苏芪合剂.8周后,观察患者缓解率、T细胞功能及不良反应.结果 治疗组患者在缓解率、T细胞功能改善等方面优于对照组,且因糖皮质激素的使用而出现的不良反应少于对照组.结论 苏芪合剂联合治疗Ⅲ～Ⅳ级IgA肾病疗效显著并能有效改善T细胞功能.     

In Vivo Anergized T Cells Form Altered Immunological Synapses In Vitro

T cells contact allogeneic antigen presenting cells (APCs) and assemble, at their contact interface, a molecular platform called the immunological synapse. Synapse-based molecules provide directional signals for the T cell--either positive signals, resulting in T-cell activation, or negative signals causing T-cell inactivation or anergy. To better understand the molecular basis of in vivo T-cell anergy we analyzed the contacts made between in vivo anergized T cells and APCs, and determined which signaling molecules were included or excluded from their immunological synapses. Anergy was induced in TCR transgenic mice by the intravenous injection of semiallogeneic donor spleen cells. T cells from anergized mice were mixed with APCs, the T-cell/APC synapses imaged using deconvolution microscopy, and their molecular compositions were determined. T cells from anergic mice formed unstable immunological synapses in vitro with allogeneic APCs and failed to recruit the signaling proteins necessary to initiate T-cell activation. These findings suggest that T-cell anergy induced by exposure to semiallogeneic donor cells is associated with defects in the earliest events of T-cell activation, immunological synapse formation and recruitment of TCR-mediated signaling proteins.     

Differential Effects of Immunosuppressive Drugs on T-Cell Motility

The best-characterized mechanism of the action of immunosuppressive drugs is to prevent T-cell clonal expansion, thus containing the magnitude of the ensuing immune response. As T-cell recruitment to the inflammatory site is another key step in the development of T-cell-mediated inflammation, we analyzed and compared the effects of two commonly used immunosuppressants, cyclosporin A (CsA) and the rapamycin-related compound SDZ-RAD, on the motility of human CD4+ T cells. We show that CsA, but not SDZ-RAD, inhibits T-cell transendothelial migration in vitro. CsA selectively impaired chemokine-induced T-cell chemotaxis while integrin-mediated migration was unaffected. The inhibition of T-cell chemotaxis correlated with reduced AKT/PKB but not ERK activation following exposure to the chemokine CXCL-12/SDF-1. In addition, CsA, but not SDZ-RAD, prevents some T-cell receptor-mediated effects on T-cell motility. Finally, we show that CsA, but not SDZ-RAD inhibits tissue infiltration by T cells in vivo. Our data suggest a prominent antiinflammatory role for CsA in T-cell-mediated tissue damage, by inhibiting T-cell trafficking into tissues in addition to containing clonal expansion.     

支原体肺炎肺外损伤患儿T细胞亚群、IFNγ、TNFα的变化及意义

目的 探讨支原体肺炎肺外损伤患儿细胞免疫、细胞因子状况和胸腺肽的治疗效果。方法 采用流式细胞仪和酶标仪检测31例支原体肺炎肺外损伤组患儿急性期、恢复期血中CD3CD4、CD8、CD4／CD8干扰素γ（IFNγ）、肿瘤坏死因子α（TNFα水平，并与支原体肺炎组比较。结果 ①急性期支原体肺炎肺外损伤组和支原体肺炎组比较，CD3、CD4显著降低（P〈0．05），TNFα显著升高（P〈0．01），CD8、IFNγ无统计学意义（P〉0．05）；②支原体肺炎肺外损伤组急性期和4周后比较，CD3、CD4、IFNγ升高（P〈0．05），TNFα显著降低（P〈0．01），CD8无变化。用胸腺肽治疗患者以上指标变化更明显。结论 支原体肺炎肺外损伤患儿细胞免疫功能低于支原体肺炎患儿；细胞因子中TNFα早期升高，而IFNγ不明显；恢复期TNFα下降，而IFNγ升高明显。用胸腺肽治疗能缩短病程。     

Arsenic trioxide–loaded, microemulsion-enhanced cytotoxicity on MDAH 2774 ovarian carcinoma cell line

The antiproliferative effect of As(2)O(3)-loaded microemulsion (As(2)O(3)-M) on human MDAH 2774 ovarian cancer cells was compared with a regular solution of the As(2)O(3). We used MDAH 2774 as model cell lines for ovarian cancer. The (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide) (XTT) and trypane blue dye exclusion tests were used to evaluate cytotoxicity. Apoptotic effect of solutions was evaluated using cell death detection kit. Standard microemulsion formulation used in this experiment contains 5 x 10(-6) M As(2)O(3). It was clearly demonstrated that As(2)O(3)-M had a significant cytotoxic effect on MDAH 2774 cell line, and the cytotoxic effect of As(2)O(3)-M was significantly higher than that of regular As(2)O(3) solutions. Even approximately 6000 times diluted microemulsion formulation loaded with 5 x 10(-6) M As(2)O(3) showed a cytotoxic effect. As a result, this diluted concentration (approximately 8 x 10(-10) M) was found to be approximately 6000 times more effective than regular As(2)O(3) solutions (5 x 10(-6) M). Moreover, this diluted concentration resulted in 1.5-fold enhancement of apoptosis. According to the in vitro cytotoxicity studies, we concluded that by incorporating As(2)O(3) into the microemulsion (As(2)O(3)-M), which is a new drug carrier system, it is possible to increase antiproliferative effect of regular As(2)O(3) on MDAH 2774 cells. Translating these results to in vivo conditions would open new windows in the treatment of ovarian cancer.