The product of the phosphatidylinositol glycan complementation group A gene (Pig-A) is involved in the synthesis of glycosylphosphatidylinositol (GPI) anchors that link various protein markers to the surface of several types of mammalian cells, including hematopoietic cells. Previous observations indicate that Pig-A mutation results in the lack of GPI synthesis and the absence of GPI-anchored proteins on the cell surface. As a first step in designing a rapid assay for measuring Pig-A mutation in the rat, we developed flow cytometry (FCM) strategies for detecting GPI-negative cells in rat peripheral blood and spleen. Anti-CD59 was used to detect GPI-anchored proteins on red blood cells (RBCs), and anti-CD48 was used to detect GPI-anchored proteins on spleen T-cells. The spontaneous frequency of CD59-negative RBCs in five male F344 rats ranged from 1 x 10(-6) to 27 x 10(-6). In contrast, treatment of five rats with three doses of 40 mg/kg N-ethyl-N-nitrosourea (ENU) increased the frequency of CD59-negative RBCs to 183 x 10(-6) to 249 x 10(-6) at 2 weeks and to 329 x 10(-6) to 413 x 10(-6) at 4 weeks after dosing. In the same 4-week posttreatment rats, the frequency of CD48-negative T-cells was 11 x 10(-6) to 16 x 10(-6) in control rats and 194 x 10(-6) to 473 x 10(-6) in ENU-treated rats. The frequencies of GPI-deficient cells were similar for RBCs and spleen T-cells. These results indicate that FCM detection of GPI-linked markers may form the basis for a rapid in vivo mutation assay. Although RBCs may be useful for a minimally invasive assay, T-cells are a promising tissue for both detecting GPI-deficient cells and confirming that Pig-A gene mutation is the cause of the phenotype.
Chemokines have been shown to play an important role in leukocyte infiltration into ischemic lesions. Recently, the increased expression of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant (CINC) was observed in experimental stroke models where infiltrated leukocytes were supposed to induce tissue injury, however, the protein level and time course of these chemokines have not been fully elucidated. Therefore, we analyzed the timedependent production of MCP-1 and CINC in the rat brain after transient middle cerebral artery occlusion (MCAO) by means of specific enzymelinked immunosorbent assay systems. The MCP-1 levels in the ipsilateral hemispheres increased from 6 h, peaked at 2 days, and thereafter gradually decreased. The peak MCP-1 concentration was 89.2 ؎ 28.2 ng/g tissue wet weight (mean ؎ SEM, n ؍ 5, 49.3-fold greater than the contralateral value at the same time, P F 0.05), which is supposed to be high enough to exert its biological effects. In contrast, the maximum CINC concentration that corresponded to 2.9 ؎ 0.7 ng/g tissue wet weight (mean ؎ SEM, n ؍ 5, 55.0-fold greater than the contralateral value at the same time, P F 0.05), was observed at 6 h. In addition, we confirmed the temporal profile of leukocyte subtypes that infiltrated into the ischemic brain, thus, neutrophil infiltration occurred at early stages (1-3 days), followed by massive infiltration of macrophages at later stages (2-7 days). These studies suggest that MCP-1 in cerebral ischemia actually plays a significant role in the migration of macrophages into the lesion and that the differential temporal production of these chemokines contributes to the regulation of infiltrated leukocyte subtypes. J. Leukoc. Biol. 65: 744-749; 1999.
Infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) causes hemorrhagic colitis and hemolytic uremic syndrome. TMA-15 is a humanized monoclonal antibody against Stx2, a major pathogenic factor. In a mouse infection model that used B2F1, a virulent STEC strain, the efficacy of TMA-15 was assessed when it was administered after bacterial and toxin exposure. In this model, a time-course analysis of the serum Stx2 level showed that the toxin was detectable from 24 h after infection. In an evaluation of the time-dependent efficacy, treatment with TMA-15 up to 24 h after infection ameliorated the lethal challenge, although treatment at 48 h showed no efficacy. To determine the effective dose, escalating doses were administered at 24 h after infection. The number of mice that survived after doses of 0, 0.25, 0.5, 1.0, and 2.0 mg/kg were 0/20, 11/20, 17/20, 20/20, and 20/20, respectively. These findings suggest that TMA-15 shows potential for prevention of severe complications associated with STEC infection.
We previously reported that rat spleen T-cells and peripheral red blood cells that are deficient in glycosylphosphatidylinositol (GPI) synthesis [presumed mutants for the phosphatidylinositol glycan complementation group A gene (Pig-A)] could be detected by flow cytometry (FCM) as cells negative for GPI-linked markers (CD48 and CD59, respectively). To establish this procedure as a rapid in vivo gene mutation assay, we have examined the Pig-A gene of GPI-deficient rat spleen T-cells for DNA sequence alterations. Splenocytes were isolated from male F344 rats, primed with ionomycin and phorbol-12-myristate-13-acetate, and seeded at limiting-dilution into 96-well plates. To select for GPI-deficient T-cells, the cells were cultured for 10 days in a medium containing rat T-STIM and 2 nM proaerolysin (ProAER). The frequency of ProAER-resistant (ProAER(r)) spleen T-cells from control rats ranged from 1.3 x 10(-6) to 4.8 x 10(-6), while administration of three doses of 40 mg/kg N-ethyl-N-nitrosourea increased the frequency of ProAER(r) T-cells 100-fold at 4 weeks after dosing. FCM analysis of the cells in ProAER(r) clones revealed that they were CD48-negative, and thus presumably GPI-deficient. Sequencing of Pig-A cDNA from six ProAER(r) clones indicated that they all contained alterations in the Pig-A protein coding sequence; five had base pair substitutions and one had multiple exons deleted. These results indicate that GPI-deficient spleen T-cells are Pig-A gene mutants and support the use of FCM analysis of GPI-deficient cells as a rapid assay for measuring in vivo gene mutation.
Results suggest that young STEC-positive children with bloody diarrhea and exhibiting neutrophilic leukocytosis in the early course of their diarrhea are at risk for HUS progression. The observation of measurable concentrations of Stx-2 levels in the early post-bloody-diarrhea period and rapid dissipation at the time of HUS onset requires further evaluation.
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