In vitro production of interleukin-1 receptor antagonist in IgG- mediated red cell incompatibility

BACKGROUND: Recently, proinflammatory cytokines including interleukin 1 (IL-1) have been implicated in the pathophysiology of immune-mediated hemolysis. Little is known, however, about the mechanisms by which inflammatory events in these reactions may be downregulated. IL-1 receptor antagonist (IL-1ra) inhibits the actions of IL-1 by competition for cellular receptors, and thus it may regulate the biologic actions of IL-1 during immune-mediated hemolysis. The production of IL-1ra by peripheral blood mononuclear cells (PBMNCs) in response to IgG-coated red cells in vitro was investigated. STUDY DESIGN AND METHODS: Fresh PBMNCs were obtained by density gradient separation of heparinized whole blood. PBMNCs were cultured in the presence of anti-D-coated, Rh-positive red cells or uncoated Rh- negative red cells under nonadherent conditions. IL-1ra concentrations in the culture media were measured by enzyme-linked immunosorbent assay. IL-1ra and IL-1 beta gene expression was assessed by Northern blot analysis of PBMNC mRNA. RESULTS: IL-1ra production was evident 4 hours after stimulation with IgG-coated red cells and increased progressively over 24 hours. Gene expression of IL-1ra was first detected at 2 hours, lagging behind that of IL-1 beta. IL-1ra gene expression was not inhibited by neutralizing polyclonal antibodies to IL-1. Immunocytochemical staining to determine the cellular source localized IL-1ra production to monocytes engaged in erythrophagocytosis. IL-1ra production was inhibited by dexamethasone (10(-7) M). CONCLUSION: These results suggest that IL-1ra production may partly account for the variable pathophysiologic events seen in IgG- mediated autoimmune hemolysis and hemolytic transfusion reactions. Steroid treatment may also downregulate anti-inflammatory cytokine production in immune hemolysis.     

纳米羟基磷灰石对人骨髓基质干细胞体外成骨活性的影响

目的：观察人骨髓基质干细胞在纳米羟基磷灰石材料上的生长、分化特点以及成骨细胞形态. 方法：实验于2004-03/2005-07在中南大学湘雅医院医学实验室完成.①实验方法：采用密度梯度离心的方法分离人骨髓基质干细胞,加入含体积分数为0.1的新生牛血清的低糖DMEM完全培养基中作原代培养,细胞长至90%汇合时传代,取培养第3代细胞接种于纳米羟基磷灰石（卫生部肝胆肠研究中心纳米课题组提供）表面,并加入含1 mmol/L地塞米松、50 mg/L维生素C、10 mmol/L β-甘油磷酸钠的培养基中诱导分化、培养.②观察指标：复合培养第2,4,6,8天后采用MTT法检测细胞增殖情况;复合培养7 d后行钙-钴法成骨细胞染色观察细胞分化情况;培养8 d后应用扫描电子显微镜观察细胞在材料上的生长情况. 结果：MTT法检测出的吸光值随着培养时间的增加而增大（P＜0.05）;经加入特定的成骨诱导体系培养7 d后,附着于材料的骨髓基质干细胞具有典型的成骨细胞形态,经钙-钴法染色后细胞胞浆中可见灰黑色颗粒或块状沉淀的阳性反应,细胞形态也多呈多角形或短矩形;培养8 d后扫描电镜可观察到材料孔隙内有细胞长入. 结论：人骨髓基质干细胞在纳米羟基磷灰石材料上生长良好,提示纳米羟基磷灰石适于骨髓基质干细胞的生长、分化,可作为骨组织工程的细胞外支架材料.     

Abnormal spectrin in hereditary elliptocytosis

An abnormal alpha subunit of erythrocyte spectrin has been described in hereditary pyropoikilocytosis (HPP), a rare hemolytic anemia characterized by erythrocyte budding and fragmentation. In HPP spectrin, the N terminal domain of the alpha subunit (alpha I T80) shows increased susceptibility to tryptic digestion, resulting in cleavage to a 50,000-d peptide, presumably due to a change in primary structure of the alpha I domain which alters conformation and generates the new cleavage site. The functional result of this conformational alteration is marked impairment of spectrin oligomer formation in vitro, consistent with the established role of alpha I T80 in spectrin self-association. In the present study, we demonstrate an abnormal spectrin alpha subunit in two kindreds with hereditary elliptocytosis (HE) that is qualitatively identical to HPP spectrin. Clinical expression of HE in these families ranges from mild elliptocytosis without hemolysis to severe poikilocytic hemolytic anemia clinically resembling HPP. In all affected individuals, a fraction of alpha I T80 is abnormal, as shown by its cleavage during mild tryptic digestion to the 50 kd peptide described in HPP; the fraction of alpha I T80 affected is directly proportional to the severity of clinical expression of HE. Spectrin oligomer formation is likewise impaired to a degree which correlates with hematologic disease. One of the HE kindreds studied demonstrated polymorphism in the spectrin alpha II domain, previously described as a frequent occurrence in blacks. This family also demonstrates a variant alpha III domain in spectrin that has not previously been described. We conclude that the abnormality in the alpha I domain originally described in HPP spectrin is shared by a subset of patients with HE; the severity of clinical expression, ranging from mild nonhemolytic HE to poikilocytic hemolytic anemia, is related to the fractional quantity of the alpha subunit that is affected.     

Inactivation of Friend erythroleukemia virus and Friend virus- transformed cells by merocyanine 540-mediated photosensitization

The Friend virus complex was used as a model to study the effects of merocyanine 540 (MC 540)-mediated photosensitization on enveloped viruses. Simultaneous exposure to the lipophilic dye MC 540 and white light inactivated cell-free virus, cell-associated virus, and virus- transformed cells. When used under experimental conditions that are known to preserve most mature blood cells, at least some coagulation factors, and a significant portion of the pluripotent hematopoietic stem cell compartment, MC 540-mediated photosensitization reduced virus titers by greater than or equal to 4 log and the concentration of in vitro clonogenic erythroleukemia cells by greater than or equal to 5 log. Animals that received a single intravenous injection of photosensitized virus were resistant to a subsequent challenge with live virus. High sensitivity to MC 540-mediated photosensitization appears to be a property that is shared by other enveloped viruses. Thus, photosensitization mediated by MC 540 may be of benefit in the sterilization of blood products (in particular, cellular products), the production of vaccines, and selected areas of antiviral therapy.     

脐血干细胞转化为肝细胞的研究进展

自进行第1例脐血干细胞移植以来,各国科研机构相继开展对于脐血干细胞的研究,脐血库也纷纷建立．随着再生医学的发展,众多国内外学者在尝试进行非肝源性细胞向肝细胞分化方面的研究,并已经证实脐血干细胞在特定的微环境下可以在体内和体外转化为肝样细胞,为脐血干细胞在肝脏疾病中的应用奠定了基础．本文就脐血干细胞向肝细胞转化的最新研究进展作一综述．     

Cell-type-specific cytotoxicity of anti-CD4 and anti-CD8 ricin immunotoxins against human alloreactive T-cell clones

Potent T-cell subset-directed immunotoxins (ITs) were generated by conjugating the anti-CD4 monoclonal antibody (MoAb) G17-2 and the anti- CD8 MoAb G10.1 to the ribosome-inhibitory protein, ricin. The cell-type- specific cytotoxicities of the generated ITs were evaluated at the clonal level using human alloreactive T-cell clones. The kinetics of anti-CD4 ricin-induced inactivation of protein synthesis in target CD4+ cloned T-cells was first order with no detectable lag period and a maximum rate of 0.07 logs per hour (t10 = 13.6 hours; first-order rate constant/K = 0.17 hr-1). The alloantigen specific lytic function of the CD4+ cytolytic T-cell clone JMAC28 was acutely sensitive to anti-CD4 ricin, and no residual lytic activity against allogeneic targets was detectable 24 hours after treatment with as little as 0.5 mmol/L anti- CD4 ricin. Notably, both anti-CD4 ricin and anti-CD8 ricin elicited a selective and dose-dependent inhibition of clonal proliferation of target T-cell clones with a maximum kill of greater than 3 logs at 5 nmol/L. No significant "bystander effects" were observed for non-target cells. Bone marrow progenitor cells CFU-GM, BFU-E, and CFU-GEMM were only minimally affected by either IT. We conclude that these ITs show considerable potential for effective depletion of T-cell subpopulations from allogeneic donor marrow grafts for clinical graft-versus-host disease (GVHD) prophylaxis.     

Antigen expression and polymerase chain reaction amplification of mantle cell lymphomas

Flow immunophenotyping, DNA content analysis, and polymerase chain reaction (PCR) amplification for t(11;14) and t(14;18) were performed on 11 cases of typical mantle cell lymphoma (MCL), 5 cases of apparent MCL with proliferation centers (MCL-PC), and 5 cases of small lymphocytic lymphoma (SLL). Immunophenotyping showed IgM (P < .001), Ig light (P < .001), and CD20 (P < .001) expression to be more intense in MCL than in SLL. In MCL-PC, the mean intensity of IgM, Ig light chain, and CD20 expression was intermediate to the intensities observed in MCL and SLL. Furthermore, in contrast to SLL, all MCL and 4 of 5 MCL-PC cases exhibited stronger CD20 than CD19 expression. CD10 expression was not observed in any case and CD5 expression was present in all SLL and MCL-PC cases and in 9 of 11 MCL cases. DNA content analysis showed an S- phase fraction of less than 3% in all cases studied and, except for 1 MCL case, all lymphomas were DNA diploid. The t(11;14) breakpoint junctions involving the bcl-1 major translocation cluster were amplified by PCR in 4 of 11 (36%) MCL cases and in none of the MCL-PC or SLL cases. The t(14;18) involving the bcl-2 major breakpoint region was not identified by PCR in any case. We conclude that the level of expression of surface antigens and the rapid detection of t(11;14) by PCR are potentially useful for distinguishing MCL and SLL in the clinical setting. Further investigations as to the biologic relationship between MCL, MCL-PC, and SLL, and the utility of t(11;14) PCR in these lymphomas are warranted.     

Granulocyte colony-stimulating factor "mobilized" peripheral blood progenitor cells accelerate granulocyte and platelet recovery after high-dose chemotherapy

Hematopoietic growth factors have been used to accelerate engraftment after bone marrow transplantation and to "mobilize" peripheral blood progenitor cells (PBPC). We report on the data in 85 consecutive patients with Hodgkin's disease who were treated in a single institution using different methods to obtain PB progenitor cells. Use of granulocyte colony-stimulating factor for mobilization resulted in a significantly accelerated time to recovery of granulocytes (10 days v 12 days, P < .01) when compared with "nonmobilized" PBPC recipients. Similarly, use of mobilized PBPC resulted in a significantly accelerated time to platelet engraftment (13 days v 30 days, P < .001) when compared with "nonmobilized" recipients. Moreover, there was a statistically significant difference in total costs in favor of the group receiving "mobilized" PBPC.     

Cytogenetic features and serum lactic dehydrogenase level predict a poor treatment outcome for children with pre-B-cell leukemia

Leukemic cells from 89 (24%) of 369 children with newly diagnosed acute lymphoblastic leukemia (ALL) were found to have a pre-B immunophenotype. By comparison with blasts having the common ALL phenotype, the pre-B cells were more likely to have a DNA index less than 1.16 (P = 0.02), a pseudodiploid karyotype (P less than 0.001), and a chromosomal translocation (P = 0.001). Increased serum lactic dehydrogenase levels (P = 0.001) were also characteristic of pre-B ALL; otherwise, the clinical and laboratory features of the two groups were similar. A nonrandom chromosomal translocation, t(1;19)(q23;p13.3), was identified in blast cells from 16 (23%) of the 70 patients with pre-B ALL and adequate chromosome banding studies; different translocations were found in 11 of the remaining patients. The presence of any chromosomal translocation in the pre-B group was significantly related to a higher leukocyte count, an increased level of serum lactic dehydrogenase, an increased percentage of S-phase cells, black race, and a blast cell DNA index less than 1.16. Four presenting features were found to confer an increased risk of treatment failure among pre-B patients: pseudodiploidy, chromosomal translocation, black race, and higher serum lactic dehydrogenase level. In a multivariate analysis, pseudodiploidy emerged as the strongest factor for predicting relapse in pre-B ALL. The frequent association of chromosomal abnormalities of known adverse prognostic significance and high serum lactic dehydrogenase levels with pre-B-cell ALL explains, at least in part, the poor treatment outcome reported for children with this subtype of leukemia.     

Interleukin-1 receptor antagonist blocks chemokine production in the mixed lymphocyte reaction

The mixed lymphocyte reaction (MLR) has previously been used to elucidate pathways of cytokine activation and T-lymphocyte proliferation and is regarded as a model that simulates responses in allograft rejection. Studies have indicated that interleukin-1 (IL-1), a potent inflammatory cytokine, may have an important activating role in the MLR response. The discovery of a naturally occurring IL-1 receptor antagonist protein (IRAP) has renewed interest in control of IL-1--dependent responses both in vitro and in vivo. MLR cultures were used to study the role of IL-1 and IRAP in the regulation of subsequent cytokines during a T-lymphocyte-mediated alloantigen response. The temporal expression of IL-1 and IRAP during 5-day one-way MLR assays suggested antagonistic production of the two cytokines. IL-1 was produced early in the response, peaking at 4 hours through day 2, subsequently declining to near-background levels on day 5 of culture. In contrast, production of IRAP was delayed until day 2, steadily increased on days 3 and 4, and peaked on day 5 of culture, which correlated with the declining levels of IL-1. The addition of graded doses of IRAP (25 to 1,000 ng/mL) to MLR cultures decreased IL-1 production but had no effect on T-lymphocyte proliferative response. In addition, IRAP had little effect on the production of either IL-2 or tumor necrosis factor. The addition of 25 ng/mL of IRAP to MLR assays showed significantly decreased levels of two potent chemotactic cytokines, IL-8 and macrophage inflammatory protein-1 alpha (MIP-1 alpha), at peak chemokine production on day 5 of culture. The levels of IL-8 and MIP-1 alpha could be restored by the addition of IL-1 to the IRAP-treated cultures. IL-8 and MIP-1 alpha represent the two different families of chemotactic cytokines, C-X-C (IL-8) and C-C (MIP-1 alpha), and potentially play important roles in the recruitment of leukocytes to a site of immune allogeneic response. These studies indicate that regulation of IL-1 by IRAP does not significantly reduce T-lymphocyte activation but can regulate the production of chemokines involved in leukocyte recruitment.