Phagocytosis of antibody-sensitized Trypanosoma brucei in vitro by bovine peripheral blood monocytes.

Cells separated by the fluorescence activated cell sorter on the basis of their surface IgD (sIgD) phenotype have been examined for responsiveness to thymus-dependent and thymus-independent antigens. The ability of monoclonal anti-IgD alloantibodies to inhibit responses in vitro to the various classes of antigen has also been investigated. Evidence is presented indicating that both sIgD positive and sIgD negative cells can respond to all types of antigen tested. However, although the presence of sIgD was necessary for the response of sIgD positive cells to thymus-dependent antigens, the presence of this isotype was not obligatory for the response of the sIgD positive population to thymus independent antigens. The possible role of sIgD as the obligatory purveyor of a B-cell activation signal is discussed in the light of these findings.     

Trypsin-stimulated transformation of Trypanosoma brucei gambiense bloodstream forms to procyclic forms in vitro

The effect of trypsin treatment on the transformation of monomorphic Trypanosoma b. gambiense (Wellcome strain) bloodstream forms to procyclic forms was studied in HEPES-buffered RPMI 1640 medium supplemented with 20% inactivated fetal calf serum in the presence of GA-1 cells as feeder layers at 27 C. In this system, 35%–40% of the bloodstream forms transformed to procyclic forms within 24 h, and over 95% of the trypanosomes changed into procyclic forms by day 3 after initiation of the culture. Established cultures of procyclic forms yielded up to 1.5–2×10⁷ trypanosomes/ml. However, transformation of nontreated and inhibited trypsin-treated bloodstream forms were prolonged compared to trypsin-treated populations. In this experiment, the first procyclic forms could be detected on day 7 after initiation of the culture and transformation was complete within 15 days. The transformation of T. b. gambiense from bloodstream to procyclic forms required the living GA-1 cells as feeder layer cells, but established cultures of procyclic forms could be maintained in the culture medium without feeder cells for more than 300 days.     

High and low responsiveness of bovine lymphocytes to Trypanosoma brucei in vitro: lack of correlation with resistance to trypanosomiasis.

Bovine peripheral blood lymphocytes (PBL) were stimulated to proliferate in vitro by live, irradiated or freeze-thawed Trypanosoma brucei, but not by the isolated variant surface glycoprotein. The optimal dose was 10(5) trypanosomes per 5 X 10(5) lymphocytes in 0.2 ml. Maximal proliferation was at day 5. Of the 98 cattle tested, 36 were high-responders (stimulation indexes 20-104), 49 were low or non-responders (SI 1-10) and 13 were intermediate. The responder status of individual animals did not change over a period of 1 year, nor did it alter following deliberate trypanosome infection. The stimulation was dependent on macrophage/monocyte type accessory cells, and this co-operation did not seem to be MHC restricted. Lack of stimulation of non-responder PBL did not appear to be due to the activation of suppressor cells. Accessory cells from non-responder animals could complement PBL from responders, but accessory cells from responders could not complement non-responder PBL. Responsiveness is therefore a characteristic of lymphocytes. Analysis of the surface markers of these lymphocytes or the blast cells generated in culture showed that they were a subpopulation of T cells, possibly TH cells. Analysis of PBL from 98 animals, which had been selected for trypanoresistance or trypanosensitivity under natural tsetse fly challenge, failed to establish a correlation between resistance and level of lymphocyte stimulation by trypanosomes in vitro.     

抗转化生长因子β1的U1 snRNA嵌合型核酶体外剪切作用

目的： 研究抗转化生长因子β₁ U1snRNA嵌合型锤头状核酶的细胞外切割活性。方法： 通过计算机设计针对TGFβ₁的锤头状核酶，然后把合成的核酶片段克隆入含有U1 snRNA启动子/增强子和终止子的U1 snRNA核酶载体中。通过RT-PCR扩增获得TGFβ₁的部分基因片段，将其克隆入T载体中T7启动子的下游，体外转录获得核酶和靶RNA，转录过程中掺入同位素，通过变性的聚丙烯酰胺凝胶电泳纯化回收。［³²P］标记的核酶与靶RNA在不同条件下进行切割反应，变性PAGE电泳，放射自显影，分析反应结果。结果： 活性的U1 snRNA嵌合型核酶（U1Rz803）在生理温度下具有良好特异的切割活性； 而点突变型核酶U1Rz803m没有切割活性，因此这些结果显示U1Rz803设计是正确的。结论： 本研究中制备的U1Rz803具有良好的特异催化切割活性。U1 snRNA嵌合型核酶U1Rz803有望在胞内抑制TGFβ₁的表达，为研究转化生长因子（TGF）β₁在造血调控中的作用机制提供有效工具。