构建肿瘤坏死因子-α免疫微球的实验研究

前瞻性构建肿瘤坏死因子-α免疫微球,以期为将来进一步研究能应用于特异性清除TNF-α的血液净化方法,奠定实验基础。采用以PSM为载体,依次包被PLL及rHTNF-αM cA b的方法而制备的免疫微球,分别以异硫氰酸荧光素标记,应用倒置显微镜及荧光显微镜观察、分光光度计测定等方法,探讨吸附微球的包被条件。结果显示,20℃、pH 9.5、60 m in三者为PLL包被PSM的最佳条件。包被后的洗脱液检测结果显示PLL含量无明显变化,说明PLL在PSM表面包被较为牢固。在相同温度及包被时间内,应用浓度为0.2%的戊二醛溶液进行的rHTNF-αM cA b对PLL的包被结合牢固。实验表明,所构建的肿瘤坏死因子-α免疫微球,达到了预期的目的,可以作为一种新颖的免疫吸附材料。该方法简单,价格便宜,为相关实验研究提供了一种崭新方法。     

Preparation and characterization of porous beta-tricalcium phosphate/collagen composites with an integrated structure

Porous beta-tricalcium phosphate (TCP)/collagen composites with different beta-TCP/collagen weight ratio were prepared. The influences of the preparation conditions on the microstructure of porous composite and the joint status of beta-TCP particles with collagen fibrils were characterized by X-ray diffractometer, scanning electron microscopy and transmission electron microscopy. The results showed: (1) an acid treatment could effectively disassemble collagen fibrils; (2) in the resulting porous composites, beta-TCP particles homogenously existed on the skeleton of the collagen fibril network and bonded tightly to both the fibrils and themselves. The tight bonding formation could be due to the reaction between Ca ions in the particles and carboxyl groups in collagen polypeptide chains and due to the reprecipitation of partially dissolved beta-TCP during synthesis. The tight bonding between beta-TCP particles and collagen fibrils in the composites demonstrated an integrated structure, which was reproducible when beta-TCP/collagen ratio ranged from 2 to 4. Such integrated structure would make significant contributions in reliably tailoring properties of the porous composites by varying beta-TCP content. In addition, the porous composites had large porosity (approximately 95%) and appropriate pore size (approximately 100 microm), showed no negative impact in cytotoxicity assay and complete bone tissue regeneration after 12 weeks in animal test.     

Associations between total serum IgE levels and the 6 potentially functional variants within the genes IL4, IL13, and IL4RA in German children: the German Multicenter Atopy Study

BACKGROUND: Increased total serum IgE levels are a common characteristic of atopic disorders. Six potentially functional variants, including C-590T in the IL4 gene, C-1055T and Arg130Gln in the IL13 gene, and Ile50Val, Ser478Pro, and Gln551Arg in the IL4RA gene, have been evaluated for their involvement in the control of total serum IgE levels and related atopic disorders, but the results of these studies have been inconsistent. OBJECTIVE: We examined whether these 6 variants had genotypic effects on total serum IgE levels in 823 unrelated German children from a large infant cohort, the German Multicenter Atopy Study. METHODS: Marginal effect models were used for the analyses of the repeated IgE measurements. Weighted linear regression and family-based tests of association were performed to minimize the possibility of spurious effects caused by selection bias or confounding on the basis of ethnic background. RESULTS: There are significant associations between increased total serum IgE levels and 2 variants in the IL13 gene (P <.005 and.0002 for Arg130Gln and C-1055T, respectively). These genetic effects are unlikely to be due to solely linkage disequilibrium between 2 polymorphisms, population stratification, or nonrepresentative samples. In addition, exposure to maternal smoking appears to modify the above effects on total serum IgE levels. However, no statistical association was observed between this quantitative phenotype and the other 4 variants examined. CONCLUSION: These findings suggest that variants C-1055T and Arg130Gln of the IL13 gene might play an important role on total serum IgE production in this study population.     

Generation of functional and mature dendritic cells from cord blood and bone marrow CD34+ cells by two-step culture combined with calcium ionophore treatment

The object of this study is to explore a culture method to generate a large number of functional and mature dendritic cells (DC) from human CD34+ hematopoietic progenitor cells. In the present study, we used a two-step method combined with calcium ionophore to induce DC from cord blood (CB) or normal human bone marrow (BM) CD34+ progenitor cells. The two-step method consists of 10 days of first step culture for the expansion and proliferation of CD34+ hematopoietic progenitor cells in the presence of SCF, IL-3, IL-6, G-CSF, and 7--11 days of second step culture for the induction of DC in the presence of GM-CSF, IL-4 and TNF-alpha. By the two-step culture, total nucleated cells were increased 208+/-66 (+/-SD, n=13), or 94+/-29 (n=5)-fold in the culture of CB or BM cells, respectively, compared with the number of CD34+ cells at the time of starting culture. Out of the total nucleated cells, 23 +/-10.4% of cells in CB cell culture and 25 +/-5% of cells in the BM cell culture acquired DC characteristic phenotypes, which were marked expressions of CD1a, HLA-DR, co-stimulatory molecules such as CD80, CD40, and adhesion molecule such as CD58. In allogeneic mixed leukocyte reaction (MLR), two-step cultured cells showed potent allo-stimulatory capacity. With this two-step culture, the absolute number of CD1a+ cells that co-expressed HLA-DR, CD80, CD40 and CD58 was enhanced approximately 3 times in CB cell culture and 1.9 times in BM cell culture, compared with the commonly used one-step culture method for the generation of DC from CD34+ cells using SCF, GM-CSF and TNF-alpha. However, on these DC generated in the two-step culture, the expressions of co-stimulatory molecule CD86 and mature DC marker CD83 were not sufficient. By the treatment of two-step cultured cells with calcium ionophore agent (A23187), the expression of co-stimulatory molecules such as CD86 and CD80 (especially CD86) was up-regulated. Besides, the expression of mature DC marker CD83 was remarkably induced by treatment with A23187 for a short duration (24 h). Consistent with the up-regulation of surface molecules CD86, CD80 and CD83, the two-step cultured cells treated with A23187 also showed a stronger allo-stimulatory capacity compared with the cells without A23187 treatment. In conclusion, the present study demonstrated that the two-step culture method effectively improved the yield of CD1a+ DC generated from CD34+ cells, and the phenotypes and functions of these CD1a+ DC could be enhanced efficiently by treatment with a calcium ionophore agent.     

An azurocidin-like protein is induced in Trichoplusia ni larval gut cells after bacterial challenge

Trichoplusia ni immune genes up-regulated in response to bacterial infection have been isolated using differential display polymerase chain reaction. Here we report the cloning and characterisation of a gut-specific immune gene encoding an azurocidin-like protein. The deduced protein is 317 amino acid residues long with a hydrophobic C-terminus and a predicted 17-residue signal peptide. The mature T. ni protein shows 30% identity to human azurocidin, an antibacterial protein. Like azurocidin, the T. ni protein contains two amino acid substitutions in the active site triad normally present in serine proteases. The T. ni protein was synthesised with a six-histidine C-terminal extension using the baculovirus expression system. Sequencing of the recombinant azurocidin-like protein confirmed the predicted cleavage of the signal peptide. Northern blots show that T. ni azurocidin-like protein is expressed solely in the larval gut and that expression is up-regulated by injecting or feeding bacteria. Expression reaches its highest level at 10 h after bacteria injection.     

Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa

The objective was to test the hypothesis that the optimal cryoprotective agent for cryopreservation of human spermatozoa would be a solute for which cells have the highest plasma membrane permeability, resulting in the least amount of volume excursion during its addition and removal. To test this hypothesis, theoretical simulations were performed using membrane permeability coefficients to predict optimal procedures for the addition and removal of a cryoprotectant. Simulations were performed using data from four different cryoprotectants: (i) glycerol, (ii) dimethyl sulphoxide, (iii) propylene glycol and (iv) ethylene glycol. Thermodynamic formulations were applied to determine approaches for the addition and removal of 1 M and 2 M final concentrations of cryoprotectant, allowing the spermatozoa to maintain a cell volume within their osmotic tolerance limits. Based on these data, ethylene glycol was predicted to be optimal for minimizing volume excursions among the solutes evaluated. These predictions were then experimentally tested using glycerol as the control cryoprotectant and ethylene glycol as the experimental cryoprotectant. The results indicate that there was a higher (P < 0.05) recovery of motile spermatozoa after cryopreservation when using 1 M ethylene glycol than with 1 M glycerol, supporting the hypothesis that use of the cryoprotectant for which the cell has the highest permeability will result in higher cell survival.      

Responses of reticulospinal neurons in the lateral reticular nucleus area of the rat to cutaneous noxious and non-noxious stimulation

Response properties of reticulospinal neurons in the lateral reticular nucleus (LRN) area to natural cutaneous stimulation were investigated systematically in 45 urethane-anesthetized rats by using extracellular recording techniques. A total of 64 neurons were tested with peripheral stimuli, of which 19 were responsive only to noxious stimuli; 7 responsive to both noxious and non-noxious stimuli; 4 responsive only to non-noxious stimuli; and 34 not responsive to any cutaneous stimuli. Both the noxious and non-noxious receptive fields were large and bilateral. Among the neurons responding to noxious stimuli, the majority (72%) was excited. This study provides evidence that some reticulospinal neurons in the rat LRN area are involved in the mechanisms of nociception.     

Increased mast cell density during the infection with velogenic Newcastle disease virus in chickens

In addition to their well-characterized role in allergic inflammation, recent data confirm that mast cells play a more extensive role in a variety of viral infections. The contribution of mast cells to Newcastle disease pathogenesis has not been investigated. We evaluated mast cell activity after Newcastle disease virus (NDV) infection in specific pathogen free chickens using cytochemical and immunocytochemical analyses. The results were as follows. Severe tissue damage was observed in the proventriculus, duodenum, jejunum and caecal tonsil, and NDV antigens were detected and presented extensively in these tissues. Second, in the NDV-infected group, the mast cell population was increased markedly in the proventriculus, duodenum, jejunum and caecal tonsil at 24, 48, 72 and 96 h after infection (P<0.01). However, very few mast cells were observed in those same tissues in the control. More intriguingly, the greatest number of mast cells was found in the proventriculus, which also showed the greatest level of NDV antigens. Third, the content of tryptase was significantly higher (P<0.01) in the NDV-infected group compared with the control from 24 to 96 h post infection). Furthermore, as an important protease released by mast cells, tryptase had a positive correlation with mast cell distribution. These data indicated that mast cells were involved in the response to NDV. Our results also suggested that the broad range of mast cell mediators might have a role in the pathology of Newcastle disease.     

Ryk-mediated Wnt repulsion regulates posterior-directed growth of corticospinal tract

Guidance cues along the longitudinal axis of the CNS are poorly understood. Wnt proteins attract ascending somatosensory axons to project from the spinal cord to the brain. Here we show that Wnt proteins repel corticospinal tract (CST) axons in the opposite direction. Several Wnt genes were found to be expressed in the mouse spinal cord gray matter, cupping the dorsal funiculus, in an anterior-to-posterior decreasing gradient along the cervical and thoracic cord. Wnts repelled CST axons in collagen gel assays through a conserved high-affinity receptor, Ryk, which is expressed in CST axons. Neonatal spinal cord secretes diffusible repellent(s) in an anterior-posterior graded fashion, with anterior cord being stronger, and the repulsive activity was blocked by antibodies to Ryk (anti-Ryk). Intrathecal injection of anti-Ryk blocked the posterior growth of CST axons. Therefore, Wnt proteins may have a general role in anterior-posterior guidance of multiple classes of axons.