Immunological response against allogeneic chondrocytes transplanted into joint surface defects in rats

Rat chondrocytes isolated from the articular-epiphyseal cartilage complex were transplanted into defects prepared in articular cartilage and subchondral bone. Transplants were taken for examination after 3 and 8 wk. Cartilage formed by syngeneic chondrocytes did not evoke formation of infiltrations. Contrary to that, in the vicinity of cartilage produced by allogeneic chondrocytes numerous infiltrating cells were present and cartilage resorption could be observed. Cyclosporine-A (CsA) treatment of recipients of allogeneic chondrocytes only partially suppressed accumulation of infiltrating cells and matrix resorption. Antichondrocyte immune response of chondrocyte graft recipients was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocytechondrocyte cultures and by evaluation of antichondrocyte cytotoxic antibodies. No difference in stimulation of SMC from intact rats by syngeneic and allogeneic chondrocytes was observed. Stimulation by allogeneic chondrocytes was slightly but significantly higher in recipients of syngeneic grafts. SMC of allogenic chondrocyte recipients were strongly stimulated by allogeneic chondrocytes. This response was absent in recipients treated with CsA. Spontaneous antichondrocyte cytotoxic antibody activity was detected in intact rats and in recipients of syngeneic grafts. In recipients of allogeneic chondrocytes the antibody response against allogeneic chondrocytes was raised but was statistically not significant owing to the considerable variation in the level of spontaneously occurring antichondrocyte antibodies.     

Mechanisms of humoral immune response against Pseudomonas aeruginosa biofilm infection in cystic fibrosis

P. aeruginosa chronic lung infection is the major cause of morbidity and mortality in patients with cystic fibrosis (CF), and is characterized by a biofilm mode of growth, increased levels of specific IgG antibodies and immune complex formation. However, despite being designed to combat this infection, such elevated humoral response is not associated with clinical improvement, pointing to a lack of anti-pseudomonas effectiveness. The mode of action of specific antibodies, as well as their structural features, and even the background involving B-cell production, stimulation and differentiation into antibody-producing cells in the CF airways are poorly understood. Thus, the aim of this review is to discuss studies that have addressed the intrinsic features of the humoral immune response and provide new insights regarding its insufficiency in the CF context.     

海藻酸钠-成年软骨细胞培养移植修复成年兔关节软骨缺损的研究

目的 利用海藻酸纳-成年软骨细胞复合构建工程化软骨,并观察应用该工程软骨移植修复成年兔关节软骨缺损的长期效果。方法 取34周龄成年新西兰兔双膝关节软骨,分离软骨细胞.与海藻酸钠混合,细胞密度为4×10~6/ml,通过硅胶模型制成圆形柱状海藻酸纳-软骨细胞盘(20μl/个),体外培养。分别于2、4、6、8、10周取细胞盘,行苏木素-伊红(HE)、AB-PAS染色、免疫组织化学分析。同时选用成年新西兰兔28只,两侧股骨内髁造成全层软骨缺损模型,缺损处随机植入体外培养4周的海藻酸钠一软骨细胞盘(实验组)及无细胞的海藻酸钠载体(对照组),分别于术后6、12、24、48周取材,观察软骨缺损修复情况。结果 成年软骨细胞在海藻酸钠中呈丛状或球状增殖,4周时达增殖高峰,培养期内软骨细胞始终呈圆形或椭圆形,免疫组织化学显示盘中Ⅱ型胶原含量丰富,无Ⅰ型胶原产生。动物实验实验组软骨缺损以透明样软骨修复为主,对照组则以纤维组织填充为主,48周时两组修复组织均有一定程度退变。结论 成年软骨细胞在海藻酸钠中体外培养可维持良好的细胞表型,并形成工程化软骨。用此工程化软骨修复关节软骨损伤经48周观察有透明样软骨修复。     

Detection of human glioma-associated antigen by rat monoclonal antibody raised against syngeneic rat glioma cells

A monoclonal antibody termed "FR77" was obtained from a hybridoma clone established by fusion between P3x63Ag8.653 mouse myeloma cells and spleen cells of a Fischer F344 rat hyperimmune to syngeneic 9L/R3 glioma cells. Immunoperoxidase staining of various cultured cells showed that FR77 was reactive to both rat and human glioma cells, but was not reactive with other nonglioma cells. Immunohistochemical examination of paraffin-embedded or cryostat-frozen sections of various human tissues revealed that FR77 was strongly reactive with glioblastoma, grade III astrocytoma, and craniopharyngioma; partially reactive with intracerebral primitive neuroectodermal tumor, pineoblastoma, and desmoplastic medulloblastoma; and weakly reactive with low-grade astrocytoma. It was not reactive with other types of brain tumors and normal human tissues tested. The FR77-defined antigen was observed to be predominantly localized in the cytoplasm of antigen-bearing cells as suggested by the immunostaining pattern, but part of it was also expressed on the cell surface of glioma cells as demonstrated by a complement-mediated cytotoxic test. Fractionation of the antigenic component and periodic acid treatment of tumor tissue bearing the FR77-defined antigen indicated that the antigen is of a neutral glycolipid nature and that the antigenic determinant to FR77 is present on its sugar portion.     

ECM修复兔桡骨缺损的实验研究

[目的]研究自制的兔耳廓脱细胞软骨基质（extracellular cartilage matrix,ECM）修复骨缺损的特点及机理。[方法]采用新西兰大白兔共40只,随机分A、B两组,在其两侧桡骨干中段制作5 mm的骨缺损模型。右侧作为实验侧,缺损区A组植入脱细胞软骨基质复合自体培养骨髓干细胞,B组仅植入脱细胞软骨基质,左侧为空白对照。分别于2、4、6、10周处死动物,标本行放射学及组织学检查。[结果]X线及组织切片均证实复合体在6周时已有致密骨组织形成,10周时已有新生髓腔生成。[结论]表明兔耳廓脱细胞软骨基质与自体骨髓干细胞复合体有良好的成骨能力,有引导组织再生、防止骨不连作用。     

Treatment of growth plate injury with autogenous chondrocytes: a study in rabbits

We designed this study to investigate transplantation of autogenous chondrocytes cultured in atelocollagen gel to treat the injured growth plate. An experimental model of growth arrest was made by resecting the medial two thirds of the left proximal tibial physis in 8-10-week-old Japanese white rabbits. Autogenous chondrocytes, which had been harvested from cartilage of the knee joints, embedded in atelocollagen gel, and cultured for a week, were transplanted into the defect in the growth plate. In two other experimental groups, we transplanted autogenous fat tissue into the same defects, or left them empty. Histological and radiographic examinations were done before and after transplantation at various times up to 52 weeks postoperatively. The histological study showed that grafted chondrocytes synthesized extracellular matrix and prevented early ossification and closure of the growth plate, which occurred in the Fat and Defect groups. Angular deformity and length discrepancy in the transplanted group were less than in the control group. Our findings suggest that transplantation of autogenous chondrocytes, cultured in atelocollagen gel, may improve treatment of the injured growth plate.     

The genetic control of the antibody response to the Pa antigen in the rat

The antibody response to the pregnancy-associated (Pa) antigen in the pregnant female segregated with the major histocompatibility complex (MHC) when the female progeny in the (PVG x WF)F1 x PVG testcross, in which the PVG strain is a low responder to Pa and the WF strain is a high responder, were mated to DA male rats. These results show that non-MHC immune response genes do not play a role, or at least any significant role, in the immune response to the paternal component of the placental antigens: the response approximates a unigenic trait with simple Mendelian segregation. Serological analysis showed that the antibodies formed by both the high responder (GMT 15, titer 1:32,768) and low responder (GMT 3, titer 1:8) female rats were directed against the Pa antigen. These findings show that the genetic control mechanism and the specificity of the antibody elicited by the placental antigen Pa are different from those in the responses to organ grafts and to soluble antigens.     

The humoral responses of haemodialysis patients to antigen challenge.

Twenty-four patients on regular haemodialysis had repeated serum samples tested for lymphocyte cytotoxicity and for factors which inhibit the mixed lymphocyte culture (MLC). It was found that serum with a high urea content did not increase the incidence of inhibition of MLC. In 16 patients on haemodialysis there was inhibition of MLC by autologous serum and the same combinations of lymphocytes were inhibited in repeated tests. The serum from 10 of these patients was also cytotoxic for lymphocytes. Serum from 12 patients was not cytotoxic but 6 of these caused inhibition MLC. It is suggested that inhibition of MLC may be produced by factors which are not specific for HLA. Serum from patients who previously rejected kidney transplants or who had pregnancies or multiple blood transfusions showed increased cytotoxicity, but these factors did not increase the frequency of serum inhibition of MLC. The significance of serum inhibitory factors and cytotoxic antibodies in the early clinical course of 12 cadaver transplants is discussed.     

不同强度持续静态压力对兔腰椎小关节软骨细胞活性影响的实验研究

目的 探讨不同强度持续静态压力对体外培养的兔腰椎小关节软骨细胞活性的影响. 方法 分离新西兰白兔腰椎小关节软骨细胞,体外培养.将培养的第3代软骨细胞分为4组:对照组、30 kPa组、60 kPa组和90 kPa持续静态加压组.显微镜下观察其形态,分别用免疫组化、噻唑蓝(MTT)、酶联免疫吸附法(ELISA)法鉴定软骨细胞,检测细胞增殖及Ⅱ型胶原的合成情况. 结果 第3代软骨细胞Ⅱ型胶原免疫组织化学染色,显示为阳性.软骨细胞增殖OD值,1d除了对照组与30 kPa组相比差异无统计学意义外,其他各组两两比较差异均有统计学意义(P ＜0.05);2～4 d:除了 30 kPa组与60 kPa组相比差异无统计学意义外,其他各组两两比较差异均有统计学意义(P＜0.05);5～10 d:各组间两两比较差异均有统计学意义(P＜0.05).空白对照组的Ⅱ型胶原含量最高[(7.517±0.328)μg/L],其次是60 kPa组[(6.035±0.075) μg/L],90 kPa组含量最低[(2.873±0.127) μg/L],30 kPa组的Ⅱ型胶原含量为(4.846±0.093)μg/L,各组间两两比较差异均有统计学意义(P＜0.05).结论 兔腰椎小关节软骨细胞在持续静态压力下容易发生退变,细胞增殖率和Ⅱ型胶原含量均下降.     

Incomplete tumour control following DNA vaccination against rat gliomas expressing a model antigen

Background

Vaccination against tumour-associated antigens is one approach to elicit anti-tumour responses. We investigated the effect of polynucleotide (DNA) vaccination using a model antigen (E. coli lacZ) in a syngeneic gliosarcoma model (9L).

Methods

Fisher 344 rats were vaccinated thrice by intramuscular injection of a lacZ-encoding or a control plasmid in weekly intervals. One week after the last vaccination, lacZ-expressing 9L cells were implanted into the striatum.

Results

After 3 weeks, in lacZ-vaccinated animals the tumours were significantly smaller than in control-vaccinated animals. In cytotoxic T cell assays lysis rates of >50 % could only be observed in a few of the lacZ-vaccinated animals. This response was directed against lacZ-expressing and parental 9L cells but not against syngeneic MADB 106 adenocarcinoma cells. In Elispot assays interferon-γ production was observed upon stimulation with 9LlacZ and 9L wild-type but not MADB 106 cells. This response was higher for lacZ-immunized animals. All animals revealed dense infiltrates with CD8+ lymphocytes and, to a lesser extent, with NK cells. CD25-staining indicated cells possibly associated with the maintenance of peripheral tolerance to self-antigens. All tumours were densely infiltrated by microglia consisting mostly of ramified cells. Only focal accumulation of macrophage-like cells expressing ED1, a marker for phagocytic activity, was observed.

Conclusion

Prophylactic DNA vaccination resulted in effective but incomplete suppression of brain tumour formation. Mechanisms other than cytotoxic T cell responses as measured in the generally used in vitro assays appear to play a role in tumour suppression.