Estudo do perfil salivar proteico de crianças obesas pré-púberes com foco nas alterações de mediadores da metainflamação

O objetivo deste estudo foi investigar na saliva de crianças pré-púberes obesas, em comparação com crianças eutróficas, a presença de possíveis diferenças na expressão de mediadores proteicos relacionados à metainflamação através de um estudo observacional transversal. Foram selecionadas 105 crianças pré-púberes com idade entre 5 e 9 anos, sem quaisquer outros comprometimentos sistêmicos ou bucais sendo realizada a mensuração do comprimento da circunferência da cintura (CC), além do peso e estatura para cálculo do IMC e seu escore Z (zscore IMC), de forma a compor 3 grupos: EU - eutrofia (-2SD≤ zscore IMC≤1SD), SP - sobrepeso (1SD < zscore IMC < 2SD) e OB obesidade (zscore IMC ≥ 2SD). Após o exame médico e odontológico foi feita a coleta de sangue e de saliva total não estimulada, de forma protocolada. Amostras séricas e salivares foram analisadas, individualmente, para a dosagem de IL1β, IL6, IL8. IL10, IL12p70, TNFα, MCP1, leptina, grelina e insulina, por Multiplex e adiponectina total e de alto peso molecular (adipoHMW), por ELISA. Além disso, as amostras séricas foram utilizadas para o delineamento hemodinâmico dos pacientes. As amostras salivares de EU e OB foram também analisadas em pools por espectrometria de massa (MS) e a presença de algumas proteínas foi validada por imunoblotting, para a comparação do perfil salivar proteico. As concentrações dos analitos foram comparadas tanto entre os grupos (teste de Kruswall-Wallis), como em relação ao zscore IMC e ao CC (Correlação de Spearman ou Pearson). Dos 12 analitos, somente a adipoHMW não foi detectada em nenhuma das amostras salivares. Na comparação OBxEU houve aumento na concentração total de proteínas salivares, da insulina e leptina sérica e salivar e do MCP1, além de diminuição da adiponectina sérica total e de adipoHMW e uma menor relação adiponectina/leptina (A/L) no grupo OB. As principais correlações obtidas com o zscore IMC foram com as concentrações séricas e salivares de insulina e leptina (positivas) e com a relação A/L (negativa), observando-se também a correlação negativa com a adiponectina total e adipoHMW séricas. Na comparação entre as concentrações séricas e salivares, foi possível detectar correlação positiva entre as dosagens de insulina e leptina, assim como com os valores da relação A/L. Na análise proteômica por MS foram identificadas 670 proteínas, sendo 163 delas com expressão diferenciada na saliva de OB em relação a EU. Dentre estas, encontram-se alteradas proteínas relacionadas à resposta inflamatória humoral, em especial com a via alternativa do sistema complemento e do metabolismo redox. Foram selecionadas três destas proteínas para validação por imunoblotting, que confirmou o aumento do Fator H e a diminuição do Fator B e da tioredoxina na saliva de OB em relação a EU. Considerando os resultados, podemos verificar que embora com menores concentrações absolutas dos mediadores, a saliva mostrou praticamente as mesmas associações observadas no sangue, em especial para a insulina, leptina e relação A/L, sendo possível admitir que a análise salivar venha a ser um bom método diagnóstico não invasivo para a obesidade infantil.

Antihemostatic molecules from saliva of blood-feeding arthropods

The ability to feed on vertebrate blood has evolved many times in various arthropod clades. Consequently, saliva of blood-feeding arthropods has proven to be a rich source of antihemostatic molecules. A variety of platelet aggregation inhibitors antagonize platelet responses to wound-generated signals, including ADP, thrombin, and collagen. Anticoagulants disrupt elements of both the intrinsic and extrinsic pathways. Vasodilators include nitrophorins (nitric oxide storage and transport heme proteins), a variety of peptides that mimic endogenous vasodilatory neuropeptides, and proteins that catabolize or sequester endogenous vasoconstrictors. Multiple salivary proteins may be directed against each component of hemostasis, resulting in both redundancy and in some cases cooperative interactions between antihemostatic proteins. The complexity and redundancy of saliva ensures an efficient blood meal for the arthropod, but it also provides a diverse array of novel antihemostatic molecules for the pharmacologist.

A horsefly saliva antigen 5-like protein containing RTS motif is an angiogenesis inhibitor

The Ag5 proteins are the most abundant and immunogenic proteins in the venom secretory ducts of stinging insects. An antigen 5-like protein (named tabRTS) composed of 221 amino acid residues was purified and characterized from the salivary glands of the horsefly, Tabanus yao (Diptera, Tabanidae). Its cDNA was cloned from the cDNA library of the horsefly's salivary gland. TabRTS containing the SCP domain (Sc7 family of extracellular protein domain) was found in insect antigen 5 proteins. More interestingly, there is an Arg-Thr-Ser (RTS) disintegrin motif at the C-terminus of tabRTS. The RTS motif is positioned in a loop bracketed by cysteine residues as those found in RTS-disintegrins of Crotalidae and Viperidae snake venoms, which act as angiogenesis inhibitors. Endothelial Cell Tube formation assay in vitro and chicken chorioallantoic membrane (CAM) angiogenesis assay in vivo were performed as to investigate the effect of tabRTS on angiogenesis. It was found that tabRTS could significantly inhibit angiogenesis in vitro and in vivo. Anti-alpha(1)beta(1) monoclonal antibody could dose-dependently inhibit the anti-angiogenic activity of tabRTS. This result indicated that tabRTS possibly targets the alpha(1)beta(1) integrin to exert the anti-angiogenic activity as snake venom RTS-/KTS-disintegrins do. The current work revealed the first angiogenesis inhibitor protein containing RTS motif from invertebrates, a possible novel type of RTS-disintegrin. (C) 2009 Elsevier Ltd. All rights reserved.

Circulating immunoglobulin A- and immunoglobulin G-secreting hybridoma cells in peripheral blood preferably migrate to female genital tracts. The role of sex hormones

Antigen-specific circulating immunoglobulin-secreting cells (ISC) migrate to various secondary and tertiary lymphoid tissues. To understand the migration of the cells into the genital tract and its regulation by sex hormones, spleen-derived SG2 hybridoma cells secreting immunoglobulin G2b (IgG2b) and Peyer's patch-derived PA4 hybridoma cells secreting polymer IgA were labelled with (3) H-TdR, and intravenously injected into syngeneic mice of both sexes. Using flow cytometry, surface molecular markers of plasma cells, CD38 and CD138, and adhesion molecules, CD49d, CD162, and CD11a were found to be positive in SG2 and PA4 cells, but CD62L, alpha4beta7 and CD44 were not expressed on these cells. The relative distribution indexes (RDIs) of the cells in genital tract and other tissues were measured. The means of RDIs of SG2 and PA4 cells in female genital tissues were 6.5 and 4.5 times as many as the means in male genital tissues, respectively. The treatment of ovariectomized mice with beta-oestradiol significantly increased the RDIs of PA4 cells in cervix and vagina, but decreased the RDIs of SG2 cells in vagina, horn of uterus, uterus and rectum (P <0.05). Progesterone treatment increased the RDIs of PA4 cells in vagina and rectum (P <0.05). The treatment with testosterone significantly increased the RDIs of SG2 and PA4 cells in epididymis and accessory sex glands (P <0.05). These results demonstrate that the female genital tract is the preferable site for the migration of circulating hybridoma cells to the male genital tract, and sex hormones play an important role in regulation of the migration of circulating ISC to genital tracts.

Effects of testosterone undecanoate as a male contraceptive candidate on rat immunological features

Testosterone undecanoate (TU) is under phase III clinical trial as a hormonal male contraceptive in China. Sex hormones can modulate the immune system. Female hormonal contraceptives may affect SIV/HIV-1 transmission. To evaluate the safety of TU and to understand whether long-term use of TU for a male contraceptive affects users' immunological features, adult male rats were treated for a 32-week TU-treated phase at the dose of 20 mg TU/kg body weight and a 24-week recovery phase. The reproductive and immunological parameters of 4-6 rats in each subgroup were examined at the stated time point. The mean sperm count and viability in the treated rats were significantly suppressed (p < 0.01). In the TU-treated group: the mean blood leukocyte and lymphocyte counts; the proliferation indexes of T cells from peripheral blood mononuclear cells (PBMC) and spleen; and, of B cells from spleen, as well as the mean counts of blood T, NK, and B cells decreased in comparison with those of control group. These decreases were not significant (p > 0.01). Similarly, the mean serum IgM, IgG, and IgA levels and complement activity in TU-treated rats were lower than those in control group (p > 0.01), and the changes in the antibody levels of the examined genital secretions were not significant (p > 0.01). The changes in the thickness of urethra epithelium, and in secretory component (SC) expression in genitals were not observed in the treated group. These results demonstrated that long-term supraphysiological TU injection did not obviously affect the examined rat immunological parameters.

Migration of mouse antibody-secreting hybridoma cells from blood to genital tract and its regulation by sex hormones are associated with the differential expression patterns of adhesion molecules and chemokines in the tract rather than in the antibody-secreting cells

To understand better the molecular mechanisms of differential migration of antibody-secreting cells (ASCs) into mouse genital tracts, and regulation by sex hormones, surface markers, hormone receptors and adhesion molecules in mouse SG2 and PA4 hybridoma cells, respectively, secreting IgG2b and polymeric IgA antibody were detected by flow cytometry or RT-PCR. Semiquantitative RT-PCR was also used for measuring mRNA expression of adhesion molecules and chemokines (VCAM-1, ICAM-1, P-selectin, JAM-1 and CXCL12) in genital tracts of various adult mouse groups. The mRNAs of androgen receptor, estrogen receptor beta and CXCR4 were expressed in the ASCs. Sex hormones had no effect on expression of these molecules in ASCs. Except for VCAM-1, mRNA of all examined genes was expressed in normal mouse genital tracts. The mean of relative amounts of ICAM-1 and CXCL12 mRNA in all examined organs of females were higher (2.1- and 1.9-fold) than those in males. After orchiectomy or ovariectomy, the expression of ICAM-1, CXCL12 and P-selectin mRNA in the examined organs increased, except JAM-1 in male and CXCL12 in female. Sex hormone treatment recovered the changes to normal levels of mRNA expression in many examined genital tissues. In combination with our previous work, preferential migration of ASCs into female genital tract and regulation of migration by sex hormones are associated with expression patterns of adhesion molecules and chemokines in genital tract rather than in ASCs. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

Live Attenuated S. Typhimurium Vaccine with Improved Safety in Immuno-Compromised Mice

Live attenuated vaccines are of great value for preventing infectious diseases. They represent a delicate compromise between sufficient colonization-mediated adaptive immunity and minimizing the risk for infection by the vaccine strain itself. Immune defects can predispose to vaccine strain infections. It has remained unclear whether vaccine safety could be improved via mutations attenuating a vaccine in immune-deficient individuals without compromising the vaccine's performance in the normal host. We have addressed this hypothesis using a mouse model for Salmonella diarrhea and a live attenuated Salmonella Typhimurium strain (ssaV). Vaccination with this strain elicited protective immunity in wild type mice, but a fatal systemic infection in immune-deficient cybb-/-nos2-/- animals lacking NADPH oxidase and inducible NO synthase. In cybb-/-nos2-/- mice, we analyzed the attenuation of 35 ssaV strains carrying one additional mutation each. One strain, Z234 (ssaV SL1344_3093), was >1000-fold attenuated in cybb-/-nos2-/- mice and ≈100 fold attenuated in tnfr1-/- animals. However, in wt mice, Z234 was as efficient as ssaV with respect to host colonization and the elicitation of a protective, O-antigen specific mucosal secretory IgA (sIgA) response. These data suggest that it is possible to engineer live attenuated vaccines which are specifically attenuated in immuno-compromised hosts. This might help to improve vaccine safety. © 2012 Periaswamy et al.

IgM, IgD and IgY and their expression pattern in the Chinese soft-shelled turtle Pelodiscus sinensis

Three Ig isotypes, IgM, IgD, and IgA, were previously known in reptiles. Here, in this report we describe IgM, IgD and a novel immunoglobulin heavy-chain isotype upsilon (IgY) in Chinese soft-shelled turtle (Pelodiscus sinensis). The IgM and IgY constant domains are characteristically similar to their counterparts described in other vertebrates. The expression of IgM and IgD were detected at mRNA level early during embryonic development, and their expression increased during further development. However, the IgY expression was not detected in larval turtles until 90 days after hatching-out. The increase in the transcription of these three Ig molecules was analyzed by using real-time PCR in spleen, kidney and blood following the injection of inactivated Aeromonas hydrophila. The primary increase in the expression of these three Igs was observed I week after the first injection, although not statistically significant, and the second injection 2 weeks after the first injection provoked a significant increase in the expression of these Igs, revealing a pattern of primary and secondary antibody response in the turtle. The present study represents the first report on reptile IgY and the pattern of IgM, IgD and IgY transcription in reptiles. (C) 2009 Elsevier Ltd. All rights reserved.

姚虻唾液腺三类抗血栓活性蛋白的结构和功能研究

血栓栓塞性疾病严重影响人类的健康，是导致死亡率最高的病因之一。在过去 的几十年中，已经开发了针对血液凝固、血小板激活和聚集、血栓溶解的不同步骤而 起作用的基础和临床药物，但是许多抗血栓药物经常被包括低血压、出血等系统性的 副作用所限制，因此，需要开发新型的更具潜力和特异性的抗血栓药物。 吸血节肢动物与宿主相互作用的分子机制研究是目前国际研究的热点之一，通过 该类研究可以发现大量的具有药用前景的先导分子和提供节肢动物控制策略。我们通 过生物化学、分子生物学以及药理学研究手段首次从中国特有的姚虻（Tabanus yao Macquart）唾液腺发现了同时具有水解纤维蛋白原和抑制血小板聚集的双功能的 丝氨酸蛋白酶Tablysin、纤维蛋白水解酶TY6和腺苷三磷酸双磷酸酶Tabapy等3个家 族的抗血栓活性成分。并研究了这3类因子在牛虻唾液腺中的分子多样性、它们的结 构基础及发挥抗血栓功能的作用机制，通过实验动物模型研究体内外抗血栓能力，深 入解析了牛虻作为传统抗血栓中药的物质基础，为发掘具有良好抗血栓功能的新型候 选药物分子，为传统活血化瘀中药牛虻的现代化开发提供了广阔的思路和打下了坚实 的基础。 研究表明，姚虻纤维蛋白原水解酶Tablysin为一种单链蛋白。其表观分子量为 27kDa。此酶为一种丝氨酸蛋白酶家族的新成员，其活性不受金属离子螯合剂EDTA 和还原剂DTT的影响。血浆蛋白酶抑制剂Aprotinin，α-macroglobulin能部分抑制该 酶活性。此酶适应的pH范围较广（pH 2.4-10.0），但对热变化不具很好的耐受性。 Edman降解法进行氨基酸测序分析发现，此酶的N-末端有封闭现象，后经Q-TOF质 谱分析，确定此酶的部分氨基酸序列。此外，我们成功构建了具有完整性的姚虻唾液 腺cDNA文库，获得2×106 pfu个重组子，进一步的筛选了Tablysin的编码基因。 对此酶的溶栓机制进行分析发现，其具有直接水解纤维蛋白原的活性，不具有纤 溶酶原激活剂的活性。此酶能明显地水解纤维蛋白原的α-链，而对β-链的水解活性较 低，不能水解γ链（α > β > γ）。此酶不能有效地水解纤维蛋白，不会水解层粘连蛋白 （Laminin）和纤维粘连蛋白（Fibronectin）等基质蛋白。不具有出血活性。进 一步探讨此酶的作用机制，发现其能以剂量依赖的方式抑制ADP诱导的血小板聚 集。利用流式细胞仪检测到该酶能与血小板膜糖蛋白受体GPIIb / IIIa结合，因此推测其可能是通过抑制血小板和纤维蛋白原结合的作用机制来抑制血小板 的聚集。 我们通过将目的基因连接到pET-32a+载体，并在大肠杆菌表达体系中得到 了高效表达。体外活性检测表明重组蛋白质与天然产物活性相当，用其检测了 其对角叉菜胶致小鼠尾静脉血栓模型体内血栓形成的影响， 实验结果表明 Tablysin具有明显的抗静脉血栓作用。该工作对有潜力开发成为新型单组分抗血栓 药物的姚虻活性蛋白进行了较为系统的理化性质研究，为该分子的进一步开发和研究 奠定了基础。 我们从姚虻唾液腺中分离纯化到一组具有水解纤维蛋白原α-链活性的酶， 命名为TY6家族。对TY6的生化性质进行研究，表明此酶为一种单链的丝氨酸蛋白 酶，其表观分子量为27 kDa，其活性不受金属离子螯合剂EDTA的影响，但能被PMSF 所抑制。Molish反应显示为阴性，说明此酶不是一种糖蛋白。从姚虻唾液腺cDNA 文库中克隆得到其编码序列，发现该酶家族的编码基因在一级结构上表现出丰 富的多样性。通过Western blotting检测到其能与牛虻叮咬后过敏患者血清特异性IgE 结合。推测该基因的多样性是吸血昆虫适应吸血寄生生活而采取的进化策略。 该工作为牛虻唾液腺抗血栓功能基因的继续筛选和分析奠定了基础，并为牛虻的生物 防治提供了思路和对策。 我们从姚虻唾液腺中分离纯化到一个具有水解ADP活性的酶（Apyrase）， 命名为Tabapy。活性检测发现Tabapy具有明显的水解ADP的活性，并能以剂量 依赖的方式抑制ADP诱导的血小板聚集。通过Western blotting检测到Tabapy能与 牛虻叮咬后过敏患者血清特异性IgE结合。用PCR方法从姚虻唾液腺cDNA文库中 克隆得到编码序列，经BLAST分析表明，该酶与来源于斑虻唾液腺的血小板聚 集抑制剂Chrysoptin具有90％的序列相似性，并经过Q-TOF分析确证，有5个肽段 的氨基酸残基与该cDNA序列推导的多肽链匹配。该工作为研究吸血节肢动物 吸血生理及进一步研究Apyrase的结构和功能打下了基础。

Enhanced biological effect induced by a radioactive C-9-ion beam at the depths around its Bragg peak

To explore the potential of double irradiation source, radioactive C-9-ion beam, in tumor therapy, a comparative study oil the surviving effect of human salivary gland cells at different penetration depths between C-9 and C-12-ion beams has been carried out. The 9C-ion C beam, especially at the distal side of the beam came out more efficient in cell killing at the depths around its Bragg peak than the 12 Bragg peak. Compared to the C-12 beam, an increase in RBE by a factor of up to 2.13 has been observed at the depths distal to the Bragg peak of the 9C beam. The 9C beam showed an enhanced biological effect at the penetration depths around its Bragg peak, corresponding to the stopping region of the incident C-9-ions and where the delayed low-energy particles were emitted. Further analysis revealed that cell lethality by the emitted particles from the stopping C-9-ions is responsible for the excessive biological effect at the penetration depths around the Bragg peak of the C-9 beam.

BaLiF_3∶Ce~(3+)纳米粒子的制备及其光谱特性

BaLiF3属立方钙钛矿型复合氟化物,作为高效闪烁晶体可用于热中子检测[1].由于其能带隙宽,易于实现各种不同价态稀土离子掺杂,可以获得许多可调谐性质,因此它也是比较理想的光学功能材料的基质[2].Ce3+激活的BaLiF3晶体作为紫外发射的短波固体激光材料和光放大材料的研究多有报道[3~5].Ce3+的发射特性强烈依赖于基质晶体结构[6,7].氟化物基质中氧杂质的含量是影响光谱性质的重要因素[8].为了寻找氧含量低、发射波长更短、可调谐范围更宽的激光材料,本工作制备了BaLiF3∶Ce3+纳米晶,获得了一些新的有意义的实验结果.如果将其引入合适的聚合物基体制备出纳米复合体系,有可能为新一代杂化激光材料提供更理想的光学活性组分[9].1实验部分1.1仪器用日本R igaκu D/maxⅡB型X射线衍射仪(XRD)测量样品的晶体结构,辐射源CuKα1(λ=0·154 1 nm);用日本H itachi S-570环境扫描电子显微镜(ESEM)观察样品的形貌和粒径大小;用日立F-4500荧光光谱仪测试样品的激发和发射光谱;用英国VG公司ESCALAB MKⅡX射线电子能谱仪(XPS)检测样品的含氧量.1.2实验过...

基于整车配送的多仓库开路VRPTW问题的研究与实现

以整车销售物流为背景,探讨多仓库带时窗约束的车辆路线安排问题的解决方法.提出了更为复杂的基于现实的细节性要求的多配送中心开路VRPTW问题模型,并将遗传算法产生部分解和评估完整解的优化解决方法和涌现交叉算子MX1引入到带时窗的多仓库VRP问题优化中,实现了快速全局优化.提出的开路混合配送方法有利于提高车辆满载率,降低回程空载率.同时实现了运输资源的优化配置,提高车辆利用率.计算机仿真实验证明了算法的可行性.

8～12岁男孩的性激素与智力、人格的关系

Recently there emerged more and more researhes concerning about sex hormone-related cognitive ability and behaviours. Few were carried out in pre- to early adolescent children., and the objective of the current study is to investigate whether there are covariance between sex hormones, intelligence and personality in 232 pre- to early adolescent boys, including 62 gifted boys. Indexes of sex hormone levels were salivary testosterone and estradiol concentrations, the 2D:4D digit ratio ( a reliable pointer of prenatal sex hormone concentraions). The Cattell Culture Fair Intelligence Test and a Chinese version of Children’s Personality Questionaire was applied in the current study. The main findings are: 1) salivary sex hormone concentrations significantly positively correlated with intelligence performance in 10-year-old boys; 2) salivary testosterone negatively related to intelligence performance in 12-year-old boys; 3) gifted boys bears lower testosterone concentrations in both prenatal period and pre- to early adolescence; 4) for personality, higher salivary estradiol was related to extraversion and digit ratios correlated with several personality factors in 8-year-old boys. In conclusion, results in the current study suggested that for male early maturers, intelligence may be negatively influenced by early coming androgen surge. In contrast, male late matures may benefit from their lately and moderately increasing hormone. Besides, the results suggested that the relationship between 2D:4D digit ratio and personality may also be paid attention to.

心理行为因素、免疫功能与乳腺癌的相关性研究

Present study consists of two experiments. In the first experiment, the relationship between psycho-behavioral factors, immune function and the stage of breast cancer was investigated. Fifty six postoperated hospitalized breast cancer patients with radiotherapy were used as the subjects. EPQ and MAC questionnaires were used to assess the patients' psycho-behavioral factors. Blood NK activation level and saliva IgA level were determined as the assessment of their immune function. It was found that the late stage breast cancer patients were more stable but more anxious in emotion. Patients with extrovert personality had higher NK activation and higher fighting spirit than did the patients with introvert personality. The patients with more fatalism had higher levels of saliva IgA and NK activation than did the patients with less fatalism. It was also found that the higher the level of saliva IgA, the higher the level of fighting spirit. In the second experiment, the effect of psycho-behavioral intervention on the immune function and the emotional state of these patients was studied. It was found that the psycho-behavioral intervention significantly increased the level of NK activation and tended to decrease the level of anxious preoccupation. The number of the patients who had to use medicine to increase peripheral blood white cells during radiotherapy was significantly decreased in patients with psycho-behavioral therapy when compared with the control group. Collectively, the data suggested that the immune function, cancer stage were related to the patients' psycho-behavioral factors and the psycho-behavioral intervention could improve the patients' immune function and emotional state.