Genetic regulation of thymic involution

In this review, we have summarized our work using combined complex statistical genetics, bioinformatics, and functional genomics to determine the genetic basis of the age-related thymic involution in C57BL/6J X DBA/2J recombinant inbred mice and the parental B6 and D2 mice. We have shown that these mice provided a valuable genetic model that can permit resampling of thymuses from different aged but genetically identical animals and determination of the relative significance of age-associated changes in the thymus. Our results suggest that the quantitative trait loci (QTL) regulating the Con A-induced thymocyte proliferative response were mapped to mouse chromosome Chr 11 (D11Mit51 at 18 cM), a region that harbors the IL-12b gene. The importance of IL-12b in maintaining thymic integrity and function during the aging process was confirmed by a more rapid involution of the thymus in IL-12b knockout (IL-12b-/-) mice compared to wild-type (WT) mice. Functionally, IL-12 provided a strong synergistic effect to augment the IL-7 or IL-2 induced thymocyte proliferative response, especially in both aged WT and IL-12b-/- mice, but not in normal young mice. In contract to the proliferative response, the age-related decline in the total number of thymocytes was determined at different age, and mapped to loci on Chr 9, 62 cM and Chr 10, 32 cM. Using matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS), increased expression of peroxiredoxin was found to be correlated with thymic involution. Our results suggest the possibility to identify the complex molecular network that can be associated with the regulation of thymic involution in aged mice using a high-dimensional functional genomics approach.     

Seeligeriolysin O, a protein toxin of Listeria seeligeri, stimulates macrophage cytokine production via Toll-like receptors in a profile different from that induced by other bacterial ligands

Seeligeriolysin O (LSO), a member of cholesterol-dependent cytolysins of Listeria seeligeri, exhibits cytokine-inducing activity. In this study, we examined the profile of cytokines expressed in macrophages of mice after stimulation with full-length form of recombinant LSO (rLSO530), C-terminal-truncated protein (rLSO483) and two authentic cytokine-inducing Toll-like receptor (TLR) ligands from bacteria, peptidoglycan (PGN) and LPS. Both rLSO530 and rLSO483 were able to induce IL-12 p40 and IL-12 p70 more strongly in macrophages than PGN or LPS. In contrast, IFN-beta and nitric oxide were induced by LPS but not by rLSO530, rLSO483 or PGN. In the presence of exogenously added IFN-beta, IL-12 p40 and IL-12 p70 production was inhibited after LSO stimulation, but IL-12 p70 production was enhanced after PGN stimulation. Although LSO signaling appeared to be associated with both TLR2 and TLR4, the profile of cytokine production by LSO stimulation was distinct from those by stimulation with PGN or LPS. Thus, it was shown that LSO is a unique bacterial ligand that induces macrophage cytokine production in a manner different from PGN or LPS.     

Monocyte-derived RANTES is intrinsically elevated in periodontal disease while MCP-1 levels are related to inflammation and are inversely correlated with IL-12 levels

Bacteria colonizing tooth surfaces are essential in the induction of an inflammatory response in the periodontal tissues, but do not cause periodontitis in everyone, implicating differences in the host immune response. These possible differences were studied using lipopolysaccharide (LPS)-stimulated whole blood cell cultures (WBCC), which revealed a down regulation of monocyte derived interleukin-12 (IL-12p70) in untreated periodontitis patients and an up regulation after therapy. IL-12p70 is a crucial factor in the differentiation of Th1 cell responses. Since CC chemokines are able to influence the T cell differentiation via cytokine secretion in antigen-presenting cells, the production of CC chemokines in periodontitis was evaluated. Therefore WBCC were stimulated with LPS from Escherichia coli for 18 h and the levels of IL-12p70 and CC chemokines were measured in the supernatants by ELISA. Untreated periodontitis patients released 2 fold more RANTES (regulated on activation normal T cell expressed and secreted) (P = 0.01) and lower levels of IL-12p70 in comparison to controls (P < 0.05). A trend towards higher levels of macrophage chemoattractant protein-1 (MCP-1) (P = 0.07) was also seen in untreated periodontitis patients; while similar levels of monocyte derived chemokine (MDC) and macrophage inflammatory proteins-1 alpha and -1 beta (MIP-1 alpha and -1 beta) were found. After periodontal therapy no changes were seen with regard to MDC, MIP-1 alpha, MIP-1 beta and RANTES, whereas the MCP-1 levels decreased (P < 0.05) and the IL-12p70 levels strongly increased (P < 0.01). The data showed a consistent inverse correlation between the levels of MCP-1 and IL-12p70, and their proportional changes after therapy correlated with the clinical inflammatory response after therapy. This indicates that the disease state regulates the release of IL-12p70 and MCP-1 in E. coli LPS-stimulated WBCC. In contrast, the persistent augmented levels of RANTES after therapy are suggestive for an intrinsic behaviour.     

用分子克隆技术构建D12S391基因座等位基因分型标准物及群体遗传学研究

目的 解决长期困扰短串联重复序列（short tandem repeat,STR）分型上存在的准确性和标准化问题。方法 先用PCR扩增出D12S391基因座的9个等位基因片段，将其插入pUC重组质粒中，经DNA测序分析证实插入片段的结构及大小，用国际标准将插入的等位基因片段进行命名，最后经转染、扩大培养、扩增及再鉴定后，制备出标准的D12S391等位基因分型标准物。结果 应用此法制备出大量的D12S391基因座等位基因分型标准物，并将其用于调查该基因座在德国Mainz地区、日本Miyazaki地区及中国成都汉族、北京汉族、新疆维吾尔族和甘肃回族6个群体中的基因型分布频率。D12S391基因座在各群体中均有较高的多态性，其非父排除概念及个人识别能力分别为0.609-0.786和0.940-0.952。结论 该法制备的STR基因座等位基因分型标准物在法医科学实践中应用价值极高，D12S391基因座是一个非常适合于群体遗传学研究和法医科学应用的遗传标记。     

Establishment of human osteosarcoma cell lines with high metastatic potential to lungs and their utilities for therapeutic studies on metastatic osteosarcoma

Relevant animal models for metastasis of osteosarcoma is needed to understand the biology and to develop the treatment modality of metastasis of human osteosarcoma. Therefore, we screened six human osteosarcoma cell lines for metastatic ability in nude mice. The HuO9 cell line was identified as being metastatic to the lung after intravenous injection. We established two sublines, HuO9-M112 and HuO9-M132, with high metastatic potential to the lung from the parental HuO9 cells by in vivo selection. There were no differences between these two sublines and the parental cells in the growth rate in vitro and the tumorigenicity after subcutaneous injection in nude mice, however, mice injected with the metastatic sublines became moribund earlier than mice injected with the parental HuO9 cells did. Thus, adriamycin (ADR) and recombinant interleukin-12 (IL-12) were administered to mice injected with the HuO9-M112 subline to suppress experimental lung metastases. Production of lung colonies was significantly suppressed and the prognoses of mice were significantly improved by both ADR and IL-12 treatments. These results indicate that both ADR and IL-12 are effective agents against pulmonary metastatic osteosarcoma, and that these sublines are useful for studies on the biological behavior and treatment of pulmonary metastatic osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

IL-12家族新成员及其在免疫应答中的重要调节作用

白细胞介素-12(Interleukin-12, IL-12)家族成员IL-12、 IL-23、 IL-27三者及其各自受体在结构上的相似性, 使得它们在发挥调节NK细胞活性、 T细胞增殖和细胞因子产生以及抗体类别转换等方面的功能相互重叠但又不完全相同.而2007年发现的该家族的另一新成员IL-35, 结构上虽与其他3个成员同源, 但却具有独特的生物学功能, 是一种主要由调节性T细胞(Treg)分泌的抑制性细胞因子.IL-12家族各成员以及同其他细胞因子之间存在着相互协同、相互拮抗的网络, 不仅在细胞内感染及炎症过程中起着重要的调节作用, 而且与银屑病、多发性硬化症、 Crohn' s病等多种临床疾病的发病密切相关.IL-12家族相关生物制品在自身免疫性疾病、感染性疾病以及肿瘤的治疗中有着广阔的应用前景.     

Immunomodulation by vitamin B12: augmentation of CD8+ T lymphocytes and natural killer (NK) cell activity in vitamin B12-deficient patients by methyl-B12 treatment

It has been suggested that vitamin B12 (vit.B12) plays an important role in immune system regulation, but the details are still obscure. In order to examine the action of vit.B12 on cells of the human immune system, lymphocyte subpopulations and NK cell activity were evaluated in 11 patients with vit.B12 deficiency anaemia and in 13 control subjects. Decreases in the number of lymphocytes and CD8+ cells and in the proportion of CD4+ cells, an abnormally high CD4/CD8 ratio, and suppressed NK cell activity were noted in patients compared with control subjects. In all 11 patients and eight control subjects, these immune parameters were evaluated before and after methyl-B12 injection. The lymphocyte counts and number of CD8+ cells increased both in patients and in control subjects. The high CD4/CD8 ratio and suppressed NK cell activity were improved by methyl-B12 treatment. Augmentation of CD3-CD16+ cells occurred in patients after methyl-B12 treatment. In contrast, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, lectin-stimulated lymphocyte blast formation, and serum levels of immunoglobulins were not changed by methyl-B12 treatment. These results indicate that vit.B12 might play an important role in cellular immunity, especially relativing to CD8+ cells and the NK cell system, which suggests effects on cytotoxic cells. We conclude that vit.B12 acts as an immunomodulator for cellular immunity.     

Detection of a trigger zone of bradykinin-induced fast calcium waves in PC12 neurites

 Bradykinin and caffeine were used as two different agonists to study inositol 1,4,5-trisphosphate (IP₃)-sensitive and caffeine/ryanodine-sensitive intracellular Ca²⁺ release in the outgrowing neurites of nerve-growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Changes in neuritic intracellular free Ca²⁺ ([Ca²⁺]_i) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) ester of the Ca²⁺-sensitive dyes Fura-red and Fluo-3, in combination with confocal microscopy. Bradykinin-induced Ca²⁺ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca²⁺ release was very low in neurites at rest. It increased after the cells were preloaded with Ca²⁺. The Ca²⁺ signal evoked at high concentrations of bradykinin (>500 nM) arose from a trigger zone in the proximal part of the neurite, as a bi-directional wave towards the growth cone and cell body. The speed of neuritic Ca²⁺ waves was reduced in cells loaded with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-tetraacetic acid/AM. Preloading of Ca²⁺ stores led to increased bradykinin-induced Ca²⁺ release, as seen for caffeine, and faster Ca²⁺ wave speeds. Caffeine evoked a simultaneous [Ca²⁺]_i rise along the neurites of Ca²⁺ preloaded cells. Higher Ca²⁺ signal amplitudes and faster Ca²⁺ wave speeds, but no longer-lasting IP₃-induced [Ca²⁺]_i signals, correlated with increased caffeine-induced Ca²⁺ release in the neurites. At low concentrations of bradykinin (<1.0 nM), the Ca²⁺ signals ceased to propagate as complete Ca²⁺ waves. Instead, repetitive stochastic Ca²⁺ release events (neuritic Ca²⁺ puffs) were observed. Neuritic Ca²⁺ puffs spread across only a few microns, at a slower speed than neuritic Ca²⁺ waves. These Ca²⁺ puffs represent elementary Ca²⁺ release units, whereby the released Ca²⁺ ions form these elementary events into the shape of a Ca²⁺ wave. Received: 16 April 1996 / Received after revision and accepted: 13 May 1996     

Doxorubicin, an RNA synthesis inhibitor, prevents vasoconstriction and inhibits aberrant expression of endothelin-1 in the cerebral vasospasm model of the rat

The present study investigates the effects of bis(7)-tacrine, a novel dimeric acetylcholinesterase inhibitor, on hydrogen peroxide(H₂O₂)-induced cell injury with comparison to the corresponding monomer, tacrine. Exposure of rat pheochromocytoma line PC12 cells to H₂O₂ induced significant cell damage. This reagent also caused redox desequilibrium as indicated by a decrease in activities of intracellular antioxidant enzymes such as glutathione peroxidase as well as catalase and an accumulation of malondialdehyde, a product of lipid peroxidation. Pretreatment of cells with bis(7)-tacrine or tacrine attenuated H₂O₂-induced cell toxicity, and bis(7)-tacrine demonstrated higher potency than tacrine in improving redox desequilibrium. These results suggest that bis(7)-tacrine and tacrine significantly protect against H₂O₂ insult, which might be beneficial for their potential usage in the prevention and treatment of Alzheimer's disease.     

Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-{gamma} production by anti-tumor T cells

Unfractionated spleen cells taken from tumor-bearing mice 2weeks after tumor implantation contained tumor-primed T cellswhich produced cytokines including IL-2 and IFN- when culturedin vitro. With progressive tumor growth this initial lymphokine-producingcapacity decreased. Here, we investigated the ability of IL-12to (I) restore suppressed IFN- production, (II) cause tumorregression and (II) induce anti-tumor protective immunity. Additionof rIL-12 to spleen cell cultures from 4- to 10-week-old tumor-bearingmice resulted in a striking enhancement in the production ofIFN- compared with cultures of these cells in the absence ofrIL-12 or of normal spleen cells in the presence of rIL-12.Five I.p. injections of rIL-12 into mice bearing s.c. tumorsinduced complete tumor regression. This was found when rIL-12was given at early (1–2 weeks), intermediate (4–5weeks) or even late (7 weeks) stages of tumor growth. Furthermore,IL-12-treated mice which rejected the primary tumor exhibitedcomplete resistance to a rechallenge with the same tumor butdid not reject a second syngenetic tumor. Immunohistochemicalanalyses following IL-12 treatment revealed that CD4⁺ and CD8⁺T cells infiltrate the tumor. More importantly, IFN- mRNA expressionwas observed in fresh tumor masses from tumor-bearing mice receivingIL-12 treatment The importance of IFN- was further demonstratedby the observation that the systemic administration of anti-IFN-mAb prior to IL-12 treatment completely abrogated the anti-tumoreffect of IL-12. Thus, these results indicate that administrationof modest levels of rIL-12 to tumor-bearing mice results intumor regression through mechanisms involving reversal of suppressedIFN- production by anti-tumor T cells and the establishmentof a tumor-specific protective immune response.