Abrogation of proliferation and generation of cytotoxic T cells in human mixed lymphocyte culture reactions by modification of the cell surface with mitogenic oxidizing agents

Multiple lectins with specificity for cell surface glyco-proteins inhibit cellular and humoral immune responses and induce transplantation tolerance. Because cell surface glycoproteins play a significant role in various immune events involving cell to cell interactions and because the mixed lymphocyte culture (MLC) reaction is a prototype of immune phenomenon involving cell to cell interactions as well as an in vitro analogue of graft-destructive immune events, the effect of modification of the cell surface with oxidizing mitogens was investigated. Treatment of responder or stimulator cells with neuraminidase and galactose oxidase (NAGO) or with sodium periodate (IO-4) resulted in marked suppression of alloantigen-induced proliferation and in vitro generation of primary cytotoxic T cells (CTLs) in human MLCs. A prominent coupling of mitogen-induced proliferation to abrogation of MLC was consistently observed with modification of stimulator or responder cell surface with either NAGO or IO-4. The possibility that destruction of receptor sites and/or stimulatory units was responsible for the suppression of MLCs was excluded by restoring both proliferation and generation of primary CTLs by reduction of mitogen-oxidized cell surfaces with sodium borohydride. The ability of polyclonal activators to inhibit antigen-specific responses might be useful in abrogating unfavorable alloimmune responses.     

Prolongation of skin graft survival by modulation of the alloimmune response with alternatively activated dendritic cells

BACKGROUND: Activation of immature dendritic cells (DC) in the presence of the glucocorticoid hormone dexamethasone (DEX) results in alternatively matured DC that present antigen in the absence of a proper co-stimulatory context. This maturation process is irreversible, making these cells an attractive potential tool for the induction of antigen-specific T-cell tolerance in vivo. The authors explored the possibility of using these DC for the induction of transplantation tolerance in a fully allogeneic setting in mice. METHODS: Immature dendritic cells (D1, an immature splenic DC line derived from B6 mice) were pretreated with DEX for 24 hr, after which lipopolysaccharide or nothing was added to the culture for another 48 hr. These cells were analyzed for their in vitro and in vivo stimulating or tolerizing capacities. RESULTS: In line with their phenotype, including decreased interleukin (IL)-12 production, in vitro co-culture of alternatively matured D1 (B6 origin; H-2b) with completely allogeneic T cells of BALB/c origin led to a significant decrease in the alloreactive T-cell response. A single injection of 1 x 10(6) alternatively matured H-2b DC into BALB/c mice induced a different alloimmune response compared with mature DC. The responding T cells showed a lower proliferation rate and a lower interferon-gamma production, whereas a significantly higher proportion of the cells produced IL-10 as measured ex vivo by enzyme-linked immunospot assay. Furthermore, injection with alternatively matured DC, followed by transplantation of fully mismatched skin grafts (C57BL/6), led to a significantly prolonged survival compared with that of mature DC-pretreated mice or untreated mice. The immunomodulatory effect was antigen specific, as third-party reactive alloresponses were not affected. CONCLUSIONS: The authors' data constitute the first direct demonstration that DC alternatively matured in the presence of glucocorticoid hormones can be exploited for the specific suppression of the alloreactive Th1 response, resulting in a delayed skin graft rejection in a complete major histocompatibility complex-incompatible strain combination.     

Improved in vivo endothelialization of prosthetic grafts by surface modification with fibronectin

Endothelial cell growth in vitro is enhanced by coating with fibronectin the surface on which cells grow. Similar coating of prosthetic arterial grafts may promote in vivo graft endothelialization if graft patency is not adversely affected. In each of 15 dogs, two fibronectin-coated polytetrafluoroethylene grafts and two grafts that were not coated were implanted. One graft in each pair was seeded with autologous endothelial cells, so that four different grafts were studied in each animal: a coated, seeded graft; a coated graft that was not seeded; a seeded graft that was not coated; a graft that was neither coated nor seeded. At 2, 4, and 8 weeks, grafts from five animals were examined for patency, surface endothelialization, and indium 111 platelet reactivity. After seeding, surface coverage by endothelium of coated grafts was more complete and more rapid than in uncoated grafts (64% ± 23% vs 31% ± 13% at 4 weeks, p < 0.05). Without seeding, coated grafts also appeared to have increased endothelial cell ingrowth compared with plain grafts (48.8% ± 15.1% vs 37.6% ± 1.5% at 8 weeks). Early (2-week) platelet reactivity of coated grafts was increased (p = 0.06), but patency was not adversely affected. Thus fibronectin coating of prosthetic grafts promotes surface endothelialization in vivo without altering graft patency. (J VASC SURG 1988;8:476-82.)     

单细胞凝胶电泳技术在生殖细胞DNA损伤检测中的应用

单细胞凝胶电泳技术(SCGE)是一种快速检测单个细胞DNA损伤的实验技术,在生殖细胞DNA损伤的检测中广泛应用。本综述系统介绍了SCGE在睾丸生精细胞、支持细胞、间质细胞、卵巢细胞以及卵母细胞等生殖细胞DNA损伤检测中的应用现状,并对SCGE在生殖毒性检测中的发展提出了展望。     

The mitogenic activity of peritoneal tissue repair cells: control by growth factors

The purpose of this study was to determine the proliferative activity of tissue repair fibroblasts recovered directly from injured peritoneum at various times after surgery and to test the mitogenic response of tissue repair cells (TRC) to growth factors. Rabbits underwent bilateral peritoneal abrasion (5 X 5 cm) with sterile gauze until punctate bleeding developed. Postsurgical (Days 2, 5, 7, and 10) tissue repair cells were recovered from the injured peritoneum by scraping with a scalpel blade. Although tissue repair cells consisted of a mixed cell type after 4 days in culture, recovered cells were essentially fibroblasts. These TRC were then pulsed with [3H]thymidine after 4 days in culture. The incorporation of thymidine into Postsurgical Day 5 TRC increased significantly compared to that of Day 2 TRC (P less than 0.05). Incorporation then decreased with time following surgery. Fibroblast growth factor (FGF) and epidermal growth factor (EGF) stimulated the incorporation of thymidine into TRC. However, the response of Postsurgical Day 7 and 10 TRCs to 1 microgram/ml EGF was significantly greater than those of Postsurgical Day 2 and 5 TRCs (Day 2 TRC, 166 +/- 7.4; Day 10 TRC, 420 +/- 96% of control cells without EGF, P less than 0.05). Platelet-derived growth factor (PDGF, 10 ng/ml) also stimulated the incorporation of thymidine into Day 10 TRCs, but this stimulatory activity (129.9 +/- 8.5% of control) was less than EGF or FGF. IL-1 alpha and IL-2 did not stimulate the incorporation of thymidine into TRC at a concentration of 100 pg/ml, but these cytokines did stimulate protein synthesis by TRC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of the liver in alloimmune response following inoculation of donor spleen cells

The liver is thought to be an immunologically privileged organ in the response to inoculated antigens. We previously demonstrated that it is possible to localize inoculated antigens to the liver alone or only to extrahepatic tissue using orthotopic syngeneic liver transplantation (OSLT). In this study, we analyzed more detailed mechanisms of the anti-alloimmune response in the liver. DA rat spleen cells were systemically injected into WS rats (donor spleen cell inoculation, DSI). In the sensitized liver-grafted (SLG) group, after DSI, liver grafts were retrieved from sensitized WS rats, then transplanted into naive WS rats. In the sensitized liver-removed (SLR) group, after DSI, WS rats were totally hepatectomized and given livers transplanted from naive WS rats. All the rats were challenged with heterotopic heart grafts 10 days after DSI. Mean heart graft survival in the control, DSI, SLG, and SLR groups were 11.6+/-1.6, 10.7+/-2.4, 4.4+/-1.0, and 24.6+/-6.3 days, respectively. Accelerated rejection in the SLG group as well as graft prolongation in the SLR group disappeared when OSLT was performed 2 days after DSI or later. Irradiation of DA splenocytes before inoculation did not alter graft survival in SLG. However, pretreatment with gadolinium chloride prior to DSI attenuated the antidonor response in the SLG group. In conclusion, a vigorous antidonor response occurred in the liver after systemic inoculation of spleen cells. It peaked I day after DSI and disappeared rapidly. Kupffer cells seemed to play an important role in this phenomenon.     

壳聚糖表面改性钛合金材料的研究进展

钛合金材料生物性能良好,是骨科常用的内植入材料,但其骨整合性及抗菌性能较差,需进行表面改性以弥补其不足之处。壳聚糖具有良好的生物相容性及成膜性,且可作为载体将目标药物引入钛合金表面,可有效改善钛合金材料的生物学性能,增加其使用范围。本文对近几年壳聚糖表面改性钛合金材料的相关研究进行归纳总结,从壳聚糖涂层改性的方式、钛合金材料成骨性及抗菌性的改善3个方面展开论述,以期为钛合金材料涂层改性在临床中的应用提供指导依据。     

Detection of cell death of cultured mouse mesangial cells induced by oxidized low-density lipoprotein

The objectives of the present study using cultured mouse mesangial cells (MMC) were (1) to evaluate the type of cytotoxicity induced by oxidized (ox) LDL, i.e. apoptosis, necrosis and types of other cell death and (2) to investigate the pathway of cell death under incubation with antioxidants or scavenger receptor (SR) antagonists. LDH release and a morphological examination were used in this study. Trypan blue staining of MMC was performed to detect dead cells in culture. Cytotoxicity of ox-LDL in MMC was found to be dose- and time-dependent. In the morphological study of electron microscopy, three different types of cell death in ox-LDL-treated MMC were identified. In the morphological study with semithin sections, these three types of dead cells were identified at different dosages of ox-LDL. Type 1 or type 2 dead cells were observed in low dose ox-LDL or in middle-dose ox-LDL-treated MMC, respectively. Type 3 dead cells were marked in high dose ox-LDL-treated MMC. It appears that the cells were apoptotic (type 1), necrotic (type 3) and other types (type 2). The cytotoxicity of ox-LDL was not mediated by cellular internalization of ox-LDL via SRs. On the other hand, the cytotoxicity of ox-LDL was inhibited by antioxidants such as alpha-tocopherol, probucol, N-acetyl-cysteine or glutathione ethyl ester. It is indicated that the pathways of ox-LDL induced cell death were distinct from the pathway via SRs.     

人骨髓间充质干细胞表面抗原测定

目的 :研究体外培养的各代间充质干细胞 (MSCs)及其成骨分化后的部分细胞表面抗原变化。方法 :将体外普通培养的骨髓间充质干细胞 ,每隔 1代在流式细胞仪上检测CD48、CD5 6、CD71和CD90阳性表达率。另外 ,为促进成人MSCs体外成骨性分化 ,第 1传代培养时加入成骨性添加剂 ,培养第 6、 12、 18、 2 4、 3 0d时 ,也分别用流式细胞仪分析上述MSCs表面抗原的表达 ,并用碱性磷酸酶组化染色和VonKossa染色。结果 :( 1)在普通培养条件下 ( 10 %DMEM ) ,从第 1代起 ,MSC的表面抗原CD48、CD5 6、CD71、CD90都呈阳性表达。 ( 2 )诱导培养的MSCs表面抗原CD48、CD90阳性表达率在诱导第 12、 18、 2 4及 3 0d时 ,分别与第 6d相比都有显著差异。 ( 3 )在成骨诱导条件下 ,MSCs表现出AKP染色增强和VonKossa染色可见钙化的基质沉积。当MSC开始向成骨细胞分化后 ,CD71阳性率下降最明显。结论 :( 1)目前采用的体外普通培养条件下 ,培养、传代方法对MSCs表面抗原CD48,CD5 6,CD71及CD90表达并无明显影响。 ( 2 )诱导成骨培养的MSCs的CD71从高阳性率转变为极低阳性率可能与MSCs向成骨细胞系的分化成熟相对应。 ( 3 )CD90及CD48也可能与MSCs的细胞分化状态特别是向成骨细胞系的改变有关     

Donor non-specific IFN-gamma production by primed alloreactive cells as a potential screening test to predict the alloimmune response

In order to devise an in vitro experimental system that predicts the in vivo generation of T lymphocytes capable of initiating the rejection process and thereby to individualize the immunosuppressive strategy in a rational way, we studied the in vitro strength of alloresponses to non-specific donors as a surrogate tool to identify patients with heightened alloimmunity. We measured interferon gamma (IFN-gamma) produced by primed alloactivated peripheral blood lymphocytes (p-allo-PBL) against third party stimulator PBLs in four groups by the enzyme linked immunospot (Elispot) assay: 16 with excellent renal transplant function (group 1); nine with chronic rejection (group 2); 11 allo-sensitized (PRA>60%) by graft loss on dialysis (group 3) and 36 normal controls (group 4). The Elispot assay was performed using 10(6) irradiated stimulator PBLs and 10(5) responder PBLs for 24 and 48 h. Each responder was challenged by 2-4 independent stimulators. RESULTS: At 24 h, mean+/-S.D. and 95% confidence intervals (CI) of spots/10(5) responder cells were 12.8+/-8.7 (10-15.5); 57.8+/-116 (11.9-103.7); 77.5+/-91.3 (39.8-115.2); and 20.7+/-17.9 (17.4-24.1) in groups 1-4, respectively. P<0.01 between groups 1 or 4 vs. 2 or 3. An arbitrary spot level of >or=30 has positive and negative predictive values of 58% and 95%, respectively, sensitivity of 94% and specificity of 65% to identify patients with enhanced immunity. CONCLUSION: Chronic allogeneic stimulation is associated with enhanced p-allo-PBL. IFN-gamma producing frequencies against third party stimulators. Significant variation in IFN-gamma spots produced by p-allo-PBL may be useful to choose less allogeneic donors. Diminished p-allo-PBL alloresponse to third party stimulators may predict transplant patients with decreased alloresponses who may benefit from lesser immunosuppressive drugs.