点突变p53肽诱导肽特异性CTL的研究

目的探索点突变ｐ５３肽诱导细胞免疫应答的可能性,为其作为肽疫苗用于肿瘤免疫治疗提供实验依据。方法人工合成小鼠点突变ｐ５３肽Ｐ１３２Ｆ（ＬＮＫＬＦＦＱＬ,１３２Ｃｙｓ→Ｐｈｅ突变）,与不完全弗氏佐剂（ＩＦＡ）或ＲＩＢＩ佐剂（ＲＡＳ）混合,皮下免疫Ｃ５７ＢＬ／６小鼠,分离脾细胞体外用肽再刺激,诱导细胞毒Ｔ淋巴细胞（ＣＴＬ）,并以５１Ｃｒ释放法检测其杀伤活性。结果Ｐ１３２Ｆ肽加上ＩＦＡ或ＲＡＳ免疫小鼠,均能诱导肽特异性ＣＤ８＋ＣＴＬ;低剂量重组白细胞介素２（ｒＩＬ－２）能增强肽免疫效果。结论所用突变的ｐ５３蛋白具有免疫原性,提示来源于突变ｐ５３的抗原肽有可能作为肽疫苗用于临床肿瘤免疫治疗     

Induction of DNA synthesis in lymphocytes from ALG-treated patients

Lymphocytes from kidney-grafted patients treated with anti-lymphocytic-globulin were found to be stimulated normally by phytohaemagglutinin to increased DNA synthesis, but to have lost their responsiveness to anti-lymphocyte serum. Lymphocytes from kidney-grafted patients, who were not treated with anti-lymphocytic globulin, and uraemic patients prior to transplantation, responded more or less normally to both phytohaemagglutinin and anti-lymphocyte serum. It is suggested that these findings cannot be explained by a shift in the cell populations as a consequence of anti-lymphocytic-globulin treatment, but rather by modulation of lymphocyte responsiveness to various triggering mechanisms.     

Induction of protective CD8+ T lymphocytes by an attenuated Listeria monocytogenes actA mutant.

We tested the ability of an attenuated actA mutant of Listeria monocytogenes to induce protective immunity in mice. This mutant can enter and multiply in the cytosol of the infected host cell, but is deficient in actin-dependent cell-to-cell spread. It was found to be of attenuated virulence for inbred C3H mice: the LD50 after i.v. injection was 1000-fold higher than that of the wild-type strain. Mutant bacteria multiplied up to the fourth day in the liver, but only for 1 day in the spleen. A single infection with the maximum sublethal dose of the actA mutant induced long-lasting immunity; the LD50 of virulent wild-type L. monocytogenes increased 100-fold and growth of wild-type L. monocytogenes was controlled in liver and spleen of these mice. The presence of Listeria-reactive T cells in spleen of C3H mice infected 7 days previously with the actA mutant was monitored, through their ability to protect naive syngeneic recipients against wild-type L. monocytogenes. Protection was mainly conferred by Thy-1+ CD8+ T lymphocytes; depletion of CD4+ T cells had no significant effect on the level of transferred protection. Such attenuated mutants may be used to develop live vector vaccines for delivery of heterologous proteins into the cytosol, thereby favoring the induction of a CD8+ T cell response.     

Induction of cyclooxygenase-2 expression by allergens in lymphocytes from allergic patients

Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up-regulation of COX-2 expression is responsible for increased PG release during inflammatory conditions and is thought to be also involved in allergic states. In this study, we demonstrate that in human T, B and natural killer lymphocytes from allergic patients, COX-2 expression became induced upon cell challenge with specific allergens and that this process is presumably IgE dependent and occurs after CD23 receptor ligation. This induction took place at both mRNA and protein levels and was accompanied by PGD2 release. IgE-dependent lymphocyte treatment elicited, in parallel, an activation of the MAPK p38 and extracellular signal-regulated kinase 1/2, an enhancement of calcineurin (CaN) activity, and an increase of the DNA-binding activity of the nuclear factor of activated T cells and of NF-kappaB, with a concomitant decrease in the levels of the cytosolic inhibitor of kappaB, IkappaB. In addition, specific chemical inhibitors of MAPK, such as PD098059 and SB203580, as well as MG-132, an inhibitor of proteasomal activity, abolished allergen-induced COX-2 up-regulation, suggesting that this process is mediated by MAPK and NF-kappaB. However, induction of COX-2 expression was not hampered by the CaN inhibitor cyclosporin A. We also examined the effect of a selective COX-2 inhibitor, NS-398, on cytokine production by human lymphocytes. Treatment with NS-398 severely diminished the IgE-dependently induced production of IL-8 and TNF-alpha. These results underscore the relevant role of lymphocyte COX-2 in allergy and suggest that COX-2 inhibitors may contribute to the improvement of allergic inflammation through the reduction of inflammatory mediator production by human lymphocytes.     

Induction of micronuclei in murine lymphocytes by morphine

Although individuals who abuse drugs are prone to an increased risk of malignancy, the mutagenic and carcinogenic potential of these agents has received relatively little attention. We report here on the potential of morphine to induce micronuclei in murine lymphocytes. Following a single intraperitoneal injection of 20 mg/kg morphine, the frequency of micronucleated binuclear (cytochalasin-blocked) murine T- and B-splenocytes was elevated from 1 2–36 hr after treatment. The maximum frequencies seen 24 hr after injection were 6.3– and 4.9–fold greater than the respective controls. A dose-dependent induction of micronuclei was observed from 5–20 mg/kg morphine, with no further increases in frequency produced by higher doses. In contrast, incubation of mitogen-stimulated splenocytes with 10-⁷-10-⁴ M morphine in vitro produced no change in frequency of micronucleated cells relative to controls. Treatment with the narcotic antagonist naloxone (5 mg/kg) alone had no effect on the frequency of micronuclei, but reduced the clasto-genic response of a subsequently administered dose of morphine (20 mg/kg). Thus, in murine lymphocytes morphine indirectly produces genetic damage, which is at least in part opioid receptor-mediated. © 1995 Wiley-Liss, Inc.     

Induction of sarcomas by mutant IDH2

More than 50% of patients with chondrosarcomas exhibit gain-of-function mutations in either isocitrate dehydrogenase 1 (IDH1) or IDH2. In this study, we performed genome-wide CpG methylation sequencing of chondrosarcoma biopsies and found that IDH mutations were associated with DNA hypermethylation at CpG islands but not other genomic regions. Regions of CpG island hypermethylation were enriched for genes implicated in stem cell maintenance/differentiation and lineage specification. In murine 10T1/2 mesenchymal progenitor cells, expression of mutant IDH2 led to DNA hypermethylation and an impairment in differentiation that could be reversed by treatment with DNA-hypomethylating agents. Introduction of mutant IDH2 also induced loss of contact inhibition and generated undifferentiated sarcomas in vivo. The oncogenic potential of mutant IDH2 correlated with the ability to produce 2-hydroxyglutarate. Together, these data demonstrate that neomorphic IDH2 mutations can be oncogenic in mesenchymal cells.     

Induction of suppressor cell activities in normal lymphocytes by sera from gastric cancer patients.

Inductions of suppressor cell activities in normal lymphocytes by sera from 154 patients with primary gastric cancer were investigated. Sera from patients with advanced cancer induced suppressor cells which significantly depressed the proliferative responses of autologous responder lymphocytes, while those from early cancer did not. An increase in OKT8 reactive T cell populations in normal lymphocytes occurred rather than a decrease in OKT4 reactive T cells after stimulation with sera as well as concanavalin A in relation to the increase of suppressor cell activities. Furthermore, sera-induced suppressor cell activities apparently correlated with spontaneous suppressor cell activities in cancer donors and both of these activities declined after plasma exchange. These results suggest that sera from gastric cancer patients may contain factors which can induce such suppressor cell activities and that serum factors may activate the suppressor precursors to be spontaneous suppressor cells in vivo in a manner similar to that seen in vitro.     

多发性骨髓瘤患者树突状细胞介导的特异性抗瘤活性的体外诱导

目的 :观察U2 6 6 细胞及其可溶性抗原激发的多发性骨髓瘤 (MM)患者树突状细胞 (DC)体外诱导U2 6 6 特异性CTL的作用。方法 :将MM患者外周血来源的单核细胞在rhGM CSF 80 0U ml与IFNα 6 0 0U ml条件下利用无血清技术培养生成DC ,应用丝裂霉素C处理的U2 6 6 细胞及用U2 6 6 细胞制备的可溶性抗原预刺激DC ,然后与自体淋巴细胞共同孵育 5～ 7d以诱生特异性CTL ,采用MTT法检测对U2 6 6 细胞的特异性杀伤效果。结果 :MM患者外周血单核细胞在GM CSF IFNα条件下培养 8d后生成具有典型特征的DC ,高度表达CD86、CD5 4及MHCII类分子HLA DR。应用MTT法检测U2 6 6 细胞及其可溶性抗原激发的DC诱导特异性CTL对靶细胞U2 6 6 的杀伤率分别为 2 1 2 %± 5 4 %和 2 8 0 %± 7 6 % ,对照组未用抗原刺激组都为 11 7%±4 3%。而以抗原直接刺激自体淋巴细胞组为 15 6 %± 4 8%和 13 1%± 5 5 % (P <0 0 1)。结论 :U2 6 6 细胞及其可溶性抗原激发的DC与自体淋巴细胞孵育能诱导抗U2 6 6 特异性CTL。     

Induction of IgE receptors on human lymphocytes

A mouse myeloma IgE protein was cross-linked with dimethylsuberimidate to induce polymerization and incubated with normal human peripheral blood T lymphocytes in tissue culture. After 24 hours at 37 degrees C approximately 30% of the cells formed rosettes with IgE sensitized red cells as compared with much lower levels in the controls. A variety of controls established that the cellular receptors involved in rosette formation were specific for IgE. Monomeric mouse IgE failed to induce a response.     

Induction of delayed hypersensitivity by dinitrophenylated lymphocytes

Erythrocytes, serum proteins and both living and killed lymphocytes from the peripheral blood of guinea-pigs were dinitrophenylated and then injected intraperitoneally into either the donor or other guinea-pigs. Animals sensitized by intradermal DNCB and unsensitized normal guinea-pigs served as positive and negative controls respectively. Eleven to 14 days after injection, the animals were tested with topically applied DNCB and the intensity of the reactions assessed by the local exudation of bovine serum albumin labelled with radio-iodine ([¹³¹I]BSA.)

Sensitization was achieved with dinitrophenylated living autologous lymphocytes, to a lesser degree with homologous lymphocytes, but not with erythrocytes, serum proteins or killed lymphocytes.

     

Measurement of in vivo mutant frequency in lymphocytes in the mouse

A limiting-dilution cloning technique for quantifying in vivo mutations at the hypoxanthine phosphoribosyl transferase locus in mouse splenocytes was developed. Mouse splenocytes were cultured in round-bottom microwells with irradiated feeder cells, concanavalin A, and a source of interleukin 2 at five cells/well in the absence of thioguanine, and at 5 X 10(4) cells/well in the presence of 2.5 micrograms/ml thioguanine; mutant frequency was calculated as the ratio of the cloning efficiencies with or without thioguanine. The geometric mean (95% range) for the mutant frequency in 20 mice was 1.54 X 10(-6) (4.7 X 10(-7) -2.6 X 10(6)) and whole-body X-irradiation resulted in a dose-related increase in mutant frequency of up to approximately 20 times the baseline level. The in vivo murine mutation assay should be a useful system for genotoxicity testing and may be of particular value in establishing risk estimates for human populations exposed to genotoxins.     

Induction of apoptosis by bleomycin in resting and cycling human lymphocytes

Bleomycin induces DNA and chromosome breakage. The differentialsensitivity to the drug has been used in vitro to identify individualsat high risk of developing tumours.However, there are limitedreports on the ability of bleomycin to induce apoptosis. Inthis study we tested induction of apoptosis in human peripherallymphocytes by bleomycin at different concentrations and differentculture times using various parameters, such as nuclear fragmentationand DNA fragmentation, evaluated either in situ with terminaltransferase and labelled nucleotides (TUNEL) or by flow cytometryanalysis. We demonstrate that bleomycin induces apoptosis withoutprevious permeabilization of the cell membrane. Cell death occursmainly by apoptosis and not by necrosis, with significant alterationof membrane lipoperoxidation (evaluated by luminescence). ⁴whom correspondence should be addressed. Tel: +39 6 72596060; Fax: + 39 6 72596053     

Induction of apoptosis in cord blood lymphocytes by HHV-6

Apoptosis induced by human herpesvirus 6 (HHV-6) in cord blood lymphocytes was investigated. Cord blood mononuclear cells (CBMC) prestimulated with phytohemagglutinin (PHA) were infected with HHV-6 and cultured with interleukin 2 (IL-2) for 5 days. Apoptosis was investigated by cell cycle analysis, terminal deoxytransferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, and staining with monoclonal antibody APO2.7 reacting with 7A6 antigen. The percentage of the hypodiploid fraction by cell cycle analysis and the percentage of apoptosis determined by TUNEL assay were significantly higher in HHV-6-infected CBMC compared with uninfected CBMC. 7A6 antigen, induced on the mitochondria membrane in apoptotic cells, were mainly expressed in CD4+ cells. 7A6 antigen was also detected in HHV-6-infected cells determined by monoclonal antibody OHV-3 reacting with HHV-6 glycoprotein. These data indicated that HHV-6 induced apoptosis in HHV-6-infected cells after stimulation with IL-2 for 5 days. The addition of anti-Fas antibody, anti-Fas ligand antibody, and anti-TNF-alpha antibody did not affect the induction of apoptosis by HHV-6, indicating that the Fas-Fas ligand pathway and TNF pathway did not contribute to the apoptosis induced by HHV-6.     

Induction of suppressor cells in normal lymphocytes by uremic serum

Sera of patients on chronic hemodialysis induced suppressor cell activity (SCA) in normal peripheral blood mononuclear cells, which significantly impaired blastogenic response to PHA. This SCA is statistically not different from Con A induced SCA. Both SCAs are however additive. Speculations concerning the modes of action of this induced SCA are discussed.     

Induction of immunoglobulin-producing human peripheral blood lymphocytes in serum-free medium

Induction of immunoglobulin-secreting cells from human peripheral blood lymphocytes in a serum-free culture medium was studied. Albumin, transferrin, insulin and fibronectin can replace serum entirely for support of pokeweed mitogen (PWM)-stimulated B lymphocytes, measured by a reverse hemolytic plaque assay using protein A-coated red cells. In this serum-free system, growth and maturation to IgM and IgG secretion occur at the same or higher efficiency as in conventional serum-containing medium, with maximum numbers of plaque-forming cells on day 6 at optimal dose of PWM, 0.5 ～ 5 μg/ml. This system can be used to avoid the interference from undefined serum components.     

Frequencies of hprt mutant lymphocytes in smokers,non-smokers,and former smokers

Previous work with the autoradiographic mutant lymphocyte assay has provided information about the time-course of development of hprt mutations and the persistence of detectable mutant cells in human subjects following therapeutic exposures to genotoxic agents. These early studies also revealed elevations in frequencies of mutant cells in pretreatment blood samples from patients who were current tobacco smokers, but no information was available on former smokers. In the present study, blood samples were obtained from 21 healthy former tobacco smokers who had quit smoking at least 1 year before sampling, 42 subjects who had never smoked, and 23 tobacco smokers. Plasma from all samples was tested for cotinine, a metabolite of nicotine. Current smokers were categorized as heavy smokers (≥10 cigarettes per day, cotinine ≥90 ng/ml plasma) and light smokers (<10/day, cotinine < 90 ng/ml). Lymphocytes from the blood samples were isolated, cryopreserved, and later thawed and assayed with the autoradiographic hprt assay. The 21 former tobacco smokers had a mean variant (mutant) frequency (Vf ± standard error) of 1.97 (±0.13) per million evaluatable cells. The Vf of 42 subjects who had never smoked was 1.74 (±0.13) × 10^-6, not significantly different from the former smokers. The smokers had Vfs of 8.09 (±0.78) × 10^-6 for 18 heavy smokers and 5.22 (±1.02) × 10^-6 for five light smokers. The two categories of smokers had frequencies of mutant cells significantly different from each other, and each was significantly higher than non-smokers and former smokers (P < 0.05). Vfs were significantly correlated with both cotinine concentrations and the number of cigarettes smoked per day, P < 0.001. This study demonstrates the sensitivity of the autoradiographic hprt assay for detecting mutagenic effects related to chronic low-level exposures to genotoxins, and indicates that this assay is more likely to detect the effects of recent rather than past exposures. Environ. Mol. Mutagen. 30:131–138, 1997. © 1997 Wiley-Liss, Inc.     

Induction of immunological tolerance in immunoglobulin E B lymphocytes in rats

Immunological tolerance has been, induced in 2,4-dinitrophenyl (DNP)-specific bone marrow-derived or B lymphocytes of the IgE and IgG antibody classes by treatment of rats with the DNP derivative of the D-amino acid copolymer, D-glutamic acid, D-lysine (D-GL). The tolerant state is manifested as an inability of treated rats to produce serum anti-DNP antibodies and the failure of peritoneal cells from tolerant animals to release histamine following in vitro antigen challenge. The implications of these and related observations for potential therapeutic measures in clinical hypersensitivity states are discussed.     

Induction of cardiomyopathy in severe combined immunodeficiency mice by transfer of lymphocytes from patients with idiopathic dilated cardiomyopathy

Growing evidence suggests that autoimmune mechanisms play an important role in the pathogenesis of idiopathic dilated cardiomyopathy (DCM). The aim of the study was to evaluate the effects of transfer of lymphocytes from patients with DCM into severe combined immunodeficiency (SCID) mice on the heart structure and function. Thirty CB-17 SCID (6-8 weeks old) mice were used and divided into 3 groups (n = 10). Mice were injected intraperitoneally with up to 25 x 10(6) peripheral blood lymphocytes (PBL) from either patients with DCM which contain human autoantibodies against cardiac beta1-adrenergic receptors and M2-muscarinic receptors (DCM group) or PBL from healthy controls (control-H group). Ten mice did not receive any injections and were used as baseline controls (control-N group). Echocardiography and morphological studies were performed seventy five days after the transfer. Results showed that in DCM group, left ventricle dimensions (LVD) in diastole were increased (4.2 +/- 0.1mm) as compared to both control-H group (3.8 +/- 0.1mm) and control-N group (3.6 +/- 0.1 mm) (p < 0.01). Further, there was a trend for increased LVD in systole. Fractional shortening was not different between groups. Histological evaluation revealed accumulation of human lymphocytes in the capillaries and scarce infiltration of the lymphocytes in the hearts from DCM group. Diffuse fibrosis was significant increased in DCM mice as compared to mice receiving PBL from normal subjects (2.2 +/- 0.3% vs. 0.8 +/- 0.1%, p < 0.01). In conclusion, transfer of the PBL from the patients with DCM was able to induce early stage of heart dilatation in SCID mice. These data provide for the first time the direct evidence supporting that the autoimmune mechanism is important in the pathogenesis of human DCM.