CD3单抗诱导幼龄小鼠胸腺细胞凋亡研究

用抗小鼠ＣＤ３单抗刺激幼龄小鼠胸腺细胞,培养不同时间后,检测小鼠胸腺细胞的凋亡情况。结果表明,胸腺细胞呈现了凋亡的典型形态学改变。流式细胞仪检测可见凋亡细胞特有的ＡＰ峰,ＣＤ３单抗刺激未成熟胸腺细胞可以通过内源性的凋亡途径引起细胞死亡。     

人NK细胞对同种和异种内皮细胞MHCⅠ类分子的差异识别

以HUVEC和PAEC为靶细胞,人PBNK和NK92为效应细胞,研究了人NK细胞对同种和异种内皮细胞杀伤的差异性;并通过酸处理内皮细胞及抗体封闭MHCⅠ类分子、CD94和KIR(NKB1),研究了MHCⅠ类分子对人NK细胞杀伤HUVEC和PAEC的影响. 结果表明,PBNK和NK92对PAEC的杀伤均大于对HUVEC的杀伤. 酸处理后,两种NK细胞对HUVEC杀伤的增加幅度大于对PAEC的;此外,抗CD94单抗未能改变NK的杀伤效应;KIR(NKB1)封闭使PBNK杀伤HUVEC增加95%,杀伤PAEC仅增加29%. 以上结果提示:NK细胞对异种内皮细胞MHCⅠ类分子的差异识别作用可能是NK细胞杀伤PAEC的主要原因,而KIR很可能是NK杀伤内皮细胞时主要的传递抑制信号的受体.     

Expression and regulation of protein kinase CK2 during the cell cycle

There are indications from genetic, biochemical and cell biological studies that protein kinase CK2 (formerly casein kinase II) has a variety of functions at different stages in the cell cycle. To further characterize CK2 and its potential roles during cell cycle progression, one of the objectives of this study was to systematically examine the expression of all three subunits of CK2 at different stages in the cell cycle. To achieve this objective, we examined levels of CK2, CK2 and CK2 on immunoblots as well as CK2 activity in samples prepared from: (i) elutriated populations of MANCA (Burkitt lymphoma) cells, (ii) serum-stimulated GL30-92/R (primary human fibroblasts) cells and (iii) drug-arrested chicken bursal lymphoma BK3A cells. On immunoblots, we observed a significant and co-ordinate increase in the expression of CK2 and CK2 following serum stimulation of quiescent human fibroblasts. By comparison, no major fluctuations in CK2 activity were detected during any other stages during the cell cycle. Furthermore, we did not observe any dramatic differences between the relative levels of CK2 to CK2 during different stages in the cell cycle. However, we observed a significant increase in the amount of CK2 relative to CK2 in cells arrested with nocodazole. We also examined the activity of CK2 in extracts or in immunoprecipitates prepared from drug-arrested cells. Of particular interest is the observation that the activity of CK2 is not changed in nocodazole-arrested cells. Since CK2 is maximally phosphorylated in these cells, this result suggests that the phosphorylation of CK2 by p34cdc2 does not affect the catalytic activity of CK2. However, the activity of CK2 was increased by incubation with p34cdc2 in vitro. Since this activation was independent of ATP we speculate that p34cdc2 may have an associated factor that stimulates CK2 activity. Collectively, the observations that relative levels of CK2 increase in mitotic cells, that CK2 and CK2 are phosphorylated in mitotic cells and that p34cdc2 affects CK2 activity in vitro suggest that CK2 does have regulatory functions associated with cell division.     

Highly conserved antigen-presenting function of CD1d molecules

 The lack of polymorphism of nonclassical antigen-presenting molecules has led to the proposal that they may carry out some conserved and essential antigen-presenting function. Although this is a plausible hypothesis, the major histocompatibility complex has undergone dramatic expansions and contractions through evolution, and there is surprisingly little evidence for interspecies conservation of nonclassical class I molecules. The CD1d molecule, by contrast, shows an extremely high degree of functional conservation between mice and humans, with regard to its interaction with the relatively invariant TCRs that are expressed by NK T cells. This conservation for CD1d recognition is observed either in the absence of exogenous Ag or together with a lipoglycan antigen. The close functional and phenotypic conservation of NK T cells, in mammalian species separated by approximately 50 million years, suggests an essential role in the immune system for CD1d recognition by NK T cells.     

Modulation of CD4 cell cytokine production by colon cancer-associated mucin

Mucins have been implicated in tumor-associated immunosuppression. The possibility that colon cancer mucin (CCM) may modulate T-helper 1 (TH1) activity was evaluated by investigating its effect on the production of interleukin-2 (IL-2) by CD4⁺ cells, a process that requires antigen-specific and costimulatory signals. Methods: CCM was purified from human colorectal cancer cells by gel-exclusion fast-pressure liquid chromatography. Cytokine production of purified CD4⁺ cells was evaluated at the protein and gene level in the presence of a phorbol ester or an anti-CD3 monoclonal antibody (mAb) plus mAb against the CD28 costimulatory receptor to mimic two-signal activation. Results: Soluble CCM, which contains mucins MUC2 as well as MUC1, inhibited IL-2 mRNA expression and secretion of CD4⁺ stimulated with a phorbol ester or an anti-CD3 mAb plus anti-CD28 mAb. Pretreatment of CD4⁺ cells with anti-CD28 mAb abrogated the suppressive effects of CCM on IL-2 production, and flow cytometry showed decreased binding of anti-CD28 mAb to its receptor in the presence of mucin. In addition, Ca²⁺ mobilization after T cell receptor cross-linking with anti-CD3 mAb was maintained in the presence of CCM. Although interferon γ production was also diminished, CCM did not induce a general inhibition of cytokine production, nor did it decrease cell viability. Macrophage inflammatory protein 1α production was up-regulated; the production of IL-10 and transforming growth factor β was unchanged. Conclusions: The results indicate that CCM can alter TH1 activity and suggest that the modulation of costimulatory interactions is involved. They provide another mechanism of immunosuppression mediated by these highly expressed tumor products. Received: 23 March 1999 / Accepted: 3 August 1999     

Complement-mediated lysis of cultured osteosarcoma cell lines using chimeric mouse/human TP-1 IgG1 and IgG3 antibodies

Osteosarcoma is the commonest malignant tumour of the bones. The presence of micrometastases at the time of primary diagnosis is associated with poor prognosis. Despite developments in surgery and aggressive chemotherapy, about 50% of the patients still succumb to the disease. Thus, there is a need to develop alternative treatment modalities. One such strategy is to use antibodies with improved effector functions. The two monoclonal antibodies, TP-1 and TP-3, recognize a tumour-associated antigen on human osteosarcoma cells. In the present study, we describe the cloning of the TP-1 variable genes, and the production of complete chimeric mouse/human monoclonal antibodies. Constructs containing the constant genes from human IgG1, IgG3 or a mutant IgG3 with a shortened hinge region, called m15, were expressed in the mouse myeloma cell line, NS0. The m15 mutant has been shown to be very potent in triggering complement-mediated lysis. Our goal was to investigate whether this mutant could overcome the complement protection on human osteosarcoma cells, which is generally present on all human cells. We found that the target cells expressed several membrane-bound complement inhibitors, and that masking of these inhibitors rendered the cells sensitive to lysis. The m15 mutant exhibited greater lytic activity than both IgG3 and IgG1, although it could not cause extensive killing of the target cells alone. Received: 21 January 1999 / Accepted: 3 June 1999     

Expression of zeta molecules is decreased in NK cells from HIV-infected patients

Cytolysis by natural killer (NK) cells is impaired in HIV infection. We investigated whether the expression of zeta (zeta) molecules, essential elements of signalling initiated upon ligation of, e.g., CD16, is reduced and if so, whether this reduction could be involved in defective cytolysis. FACS analysis revealed significantly lower levels of zeta in NK cells from AIDS patients compared to cells from patients without AIDS and healthy controls. CD16-dependent cytolysis by NK cells correlated with expression of zeta molecules and CD16, the latter possibly related to zeta expression. No correlation was observed between CD16-independent cytolysis and zeta expression. Reduced expression of zeta molecules by NK cells from HIV-infected patients thus correlates with disease progression and may, in part, explain the defective cytolysis by these cells.     

Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation: a calorimetric and spectroscopic study

The effect of methylurea, N,N'-dimethylurea, ethylurea, and butylurea as well as guanidine hydrochloride (GuHCl), urea and pH on the thermal stability, structural properties, and preferential solvation changes accompanying the thermal unfolding of ribonuclease A (RNase A) has been investigated by differential scanning calorimetry (DSC), UV, and circular dichroism (CD) spectroscopy. The results show that the thermal stability of RNase A decreases with increasing concentration of denaturants and the size of the hydrophobic group substituted on the urea molecule. From CD measurements in the near- and far-UV range, it has been observed that the tertiary structure of RNase A melts at about 3 degrees C lower temperature than its secondary structure, which means that the hierarchy in structural building blocks exists for RNase A even at conditions at which according to DSC and UV measurements the RNase A unfolding can be interpreted in terms of a two-state approximation. The far-UV CD spectra also show that the final denatured states of RNase A at high temperatures in the presence of different denaturants including 4.5 M GuHCl are similar to each other but different from the one obtained in 4.5 M GuHCl at 25 degrees C. The concentration dependence of the preferential solvation change delta r23, expressed as the number of cosolvent molecules entering or leaving the solvation shell of the protein upon denaturation and calculated from DSC data, shows the same relative denaturation efficiency of alkylureas as other methods.     

Dissociable and nondissociable forms of platelet-activating factor acetylhydrolase in human plasma LDL: implications for LDL oxidative susceptibility

Platelet-activating factor acetylhydrolase (PAF-AH) is transported by lipoproteins in plasma and is thought to possess both anti-inflammatory and anti-oxidative activity. It has been reported that PAF-AH is recovered primarily in small, dense LDL and HDL following ultracentrifugal separation of lipoproteins. In the present studies, we aimed to further define the distribution of PAF-AH among lipoprotein fractions and subfractions, and to determine whether these distributions are affected by the lipoprotein isolation strategy (FPLC versus sequential ultracentrifugation) and LDL particle distribution profile. When lipoproteins were isolated by FPLC, the bulk (～85%) of plasma PAF-AH activity was recovered within LDL-containing fractions, whereas with ultracentrifugation, there was a redistribution to HDL (which contained ～18% of the activity) and the d>1.21 g/ml fraction (which contained ～32%). Notably, re-ultracentrifugation of isolated LDL did not result in any further movement of PAF-AH to higher densities, suggesting the presence of dissociable and nondissociable forms of the enzyme on LDL. Differences were noted in the distribution of PAF-AH activity among LDL subfractions from subjects exhibiting the pattern A (primarily large, buoyant LDL) versus pattern B (primarily small, dense LDL) phenotype. In the latter group, there was a relative depletion of PAF-AH activity in subfractions in the intermediate to dense range (d=1.039–1.047 g/ml) with a corresponding increase in enzyme activity recovered within the d>1.21 g/ml ultracentrifugal fraction. Thus, there appears to be a greater proportion of the dissociable form of PAF-AH in pattern B subjects. In both populations, most of the nondissociable activity was recovered in a minor small, dense LDL subfraction. Based on conjugated dienes as a measure of lipid peroxidation, variations in PAF-AH activity appeared to contribute to variations in oxidative behavior among ultracentrifugally isolated LDL subfractions. The physiologic relevance of PAF-AH dissociability and the minor PAF-AH-enriched oxidation-resistant LDL subpopulation remains to be determined.