Simian Immunodeficiency Virus Infection Alters Chemokine Networks in Lung Tissues of Cynomolgus Macaques : Association with Pneumocystis carinii Infection

Infection by HIV-1 frequently leads to pulmonary complications, including alterations to local immune environments. To better understand these alterations, we have examined in detail the patterns and levels of expression of chemokine, cytokine, and chemokine receptor mRNAs in lung tissues from 16 uninfected or simian immunodeficiency virus (SIV)/DeltaB670 infected cynomolgus macaques at different stages of infection. Among the most up-regulated immune genes were interferon (IFN)-γ, IFN-γ-inducible CXCR3 ligands, and CCR5 ligands, as well as the cognate chemokine receptors. These changes were greatest in animals with clear Pneumocystis carinii coinfection. Immunohistochemistry and in situ hybridization revealed monocytes/macrophages to be the predominant type of cell infiltrating into lung tissues and serving as the major cellular source of chemokines. To explore the causes of chemokine alterations, we treated macaque lung cells with IFN-γ, lipopolysaccharide, Poly(I:C), and P. carinii in vitro, and results revealed that these stimuli can induce the expression of CXCR3 ligand and/or CCR5 ligand mRNAs. Taken together, these studies provide a comprehensive definition of the chemokine networks available to modulate cellular recruitment to lung tissues during SIV infection and implicate both cytokines (IFN-γ) and pathogens (SIV and P. carinii) as contributors to increased expression of pro-inflammatory chemokines.Pulmonary diseases are frequent complications of HIV-1 infection, affecting 75% to 85% of patients with AIDS.¹ In addition, Pneumocystis carinii (now called Pneumocystis jirovecii in humans) pneumonia (PCP) is one of most frequent pulmonary opportunistic infections in HIV-1 infected individuals. However, little is known about the mechanisms leading to changes in pulmonary immune function and how these changes ultimately contribute to pathogenesis. Identifying alterations of overall immune function and the local mediators of these changes in the lungs during HIV-1 infection and AIDS will be useful for understanding and treating HIV-1-associated pulmonary complications.Chemokines are soluble immune factors that recruit cells bearing appropriate receptors into local environments as part of homeostatic immune cell trafficking and inflammatory reactions. There are approximately 50 known chemokines and 18 chemokine receptors.² Chemokines play an important role in the control of infectious agents by recruiting effector cells to local inflammatory sites of infection and by serving directly as antimicrobial peptides.³ During HIV-1 infection, chemokines and their receptors have important roles in virus/host interactions because a subset of chemokine receptors act as viral entry co-receptors⁴ and natural and modified ligands for these receptors can inhibit viral infection.^5,6 Chemokines mediate immunity and host defense, but when produced in inappropriate amounts or in inappropriate contexts they may also contribute to inflammation-associated pathology. Either the absence or excess of specific chemokines might affect immune responses and thereby affect susceptibility to infection or subsequent disease progression.⁷ Because inflammatory chemokines play both beneficial and harmful roles in infectious diseases, there is a need for further study of the interactions between microbes, infected host cells, and other cells of the immune system to determine the relations between direct and indirect effects of the microbes on changing local chemokine expression and inflammation.Simian immunodeficiency virus (SIV) infection can lead to immune dysfunction in multiple species of nonhuman primates, and macaques with AIDS often are infected with opportunistic pathogens such as P. carinii. Therefore, we hypothesized that SIV and/or P. carinii infections in nonhuman primate model systems might broadly alter chemokine networks in lung tissues, and thereby contribute to SIV-associated pulmonary pathogenesis. To address this issue, we used the SIV-infected cynomolgus macaque (Macaca fascicularis) model to investigate the relations between systemic SIV infection, local SIV and/or P. carinii infections, and changes in chemokine, chemokine receptor, and cytokine expression. We present here a comprehensive analysis of the expression of chemokines and their receptors in lung tissues during SIV infection by using real-time RT-PCR and in situ hybridization (ISH). We also used simultaneous detection strategies to define infiltrating cell types and cellular sources of chemokines in lung tissues. In addition, we measured the expression of Toll-like receptors (TLRs) 1 to 10 in macaque lung tissues to determine the overall pathogen-sensing capabilities of this large mucosal organ. Altogether, our findings identify associations between local levels of SIV and P. carinii, and increased expression of inflammatory chemokines.     

The effect of folate analogues and vitamin B12 on provision of thymine nucleotides for DNA synthesis in megaloblastic anemia

The role of vitamin B12 in the folate dependent biosynthesis of thymidine nucleotides is controversial. In an attempt to clarify this, three methods have been used to assess the relative efficacy of vitamin B12 (hydroxocobalamin) and various folate analogues in titrated concentrations at correcting 'de novo' thymidylate synthesis by megaloblastic human marrow cells: (1) The deoxyuridine (dU) suppression test which analyses the reduction in (3H)-thymidine labeling of DNA by unlabeled dU. Marrow cells were also labeled with (6-3H)-dU with assessment of (2) its incorporation into DNA and (3) the accumulation of (6-3H)-deoxyuridine monophosphate (3H-dUMP). The three methods gave similar results. In both, N6-formyl tetrahydrofolate (formyl-FH4) was the most effective agent at correcting thymidylate synthesis in megaloblastic anemia due to vitamin B12 or folate deficiency. Vitamin B12 corrected the lesion in vitamin B12 deficiency but not in folate deficiency. Tetrahydrofolate (FH4) and folic acid were effective in deficiency of vitamin B12 or folate, although in both deficiencies they were less effective than formyl-FH4. Methyl-FH4 was effective in folate deficiency but not in vitamin B12 deficiency. These results confirm the failure of methyl-FH4 utilisation in vitamin B12 deficiency. They suggest that if vitamin B12 is needed in the formylation of FH4, this is a minor role in provision of the correct coenzyme for thymidylate synthesis compared with its major role of provision of FH4 from methyl- FH4.     

慢病毒介导碱性成纤维细胞生长因子基因转染促进半月板纤维软骨细胞的增殖与基质合成

目的:碱性成纤维细胞生长因子是一种作用广泛的修复始动因子,能有效地促进多种细胞的增殖和基质合成功能。借助慢病毒载体进行碱性成纤维细胞生长因子基因转染半月板纤维软骨细胞,观察半月板纤维软骨细胞的增殖与基质合成情况。方法:实验于2004-05/2005-11在上海市四肢显微外科实验室完成。①实验材料:ViraPower慢病毒载体系统;3个月龄新西兰大白兔3只。②实验过程和分组:分离培养兔半月板纤维软骨细胞,待培养的第1代细胞生长至60%融合时,将细胞与克隆有碱性成纤维细胞生长因子基因的重组慢病毒载体悬液共培养,定义为实验组细胞;同时设立对照组细胞,与不携带任何基因的慢病毒悬液共培养;空白组细胞,未接受外加处理。③转染后48h用酶联接免疫吸附剂测定方法检测3组半月板细胞培养液中碱性成纤维细胞生长因子的表达;激光流式细胞仪检测细胞周期,3H-脯氨酸摄入法检测胶原合成;转染后第2,4,6,8天用四甲基偶氮唑盐法检测细胞增殖。结果:①与重组慢病毒共培养48h后,在实验组细胞培养液中检测到碱性成纤维细胞生长因子的表达,而对照组和空白组细胞的培养液中未能检测到碱性成纤维细胞生长因子的表达;共培养6d后实验组细胞吸光度高于对照组和空白组(P<0.01)。②实验组细胞DNA合成前期、DNA合成期、分裂前期及分裂期的时间较对照组和空白组缩短(P<0.05)。③实验组细胞胶原高于对照组和空白组。结论:利用慢病毒转基因技术能有效地将碱性成纤维细胞生长因子基因转染入半月板纤维软骨细胞,继而促进半月板细胞的增殖和基质合成,有可能为治疗半月板损伤提供新的方法。     

Deletions within the LMP1 oncogene of Epstein-Barr virus are clustered in Hodgkin's disease and identical to those observed in nasopharyngeal carcinoma

This study of 52 European patients with Hodgkin's disease (HD) expressing the latent membrane protein 1 (LMP1) oncogene within diagnostic Hodgkin and Reed-Sternberg (HRS) cells was performed to detect LMP1 isolates carrying deletions and to characterize them at a molecular and histologic level. Deletions were identified in 5 cases, clustered near the 3' end of the LMP1 gene, and histologically associated with numerous HRS cells. DNA sequencing showed homology with the deletions seen in the Asian nasopharyngeal carcinoma (NPC) isolates CAO and 1510. Our findings suggest that partial deletions of the LMP1 oncogene, associated with aggressive behavior in NPC CAO and NPC 1510, occur at a particular localization and confer a proliferative phenotype to lymphoid cells in HD.     

2-(E)-取代苯亚甲基环戊酮及其Mannich碱盐酸盐类化合物的合成和抗炎、抗癌活性研究

本文设计,合成了2-(E)-取代苯亚甲基环戊酮(Ⅰ)类化合物18个及其Mannich碱盐酸盐(Ⅱ)类化合物11个,其中22个为新化合物。初步药理结果显示:对于角叉菜胶诱发的大鼠足趾水肿,Ⅰ_4,Ⅰ_(12)及Ⅰ_(13)口服有较显著的抑制作用,水溶性Ⅱ类化合物皮下注射有极强的抑制作用,其中Ⅱ_3于50,25和12.5 mg/kg剂量下抑制率分别为95.8%,70.3%和44.2%,它与布洛芬效力(25 mg/bg抑制率72.9%)相当.Ⅱ类化合物体外对L1210细胞及体内对荷艾氏腹水癌小鼠均有一定抗癌活性。     

Perceptions of genetic counseling services in direct‐to‐consumer personal genomic testing

To describe consumers' perceptions of genetic counseling services in the context of direct‐to‐consumer personal genomic testing is the purpose of this research. Utilizing data from the Scripps Genomic Health Initiative, we assessed direct‐to‐consumer genomic test consumers' utilization and perceptions of genetic counseling services. At long‐term follow‐up, approximately 14 months post‐testing, participants were asked to respond to several items gauging their interactions, if any, with a Navigenics genetic counselor, and their perceptions of those interactions. Out of 1325 individuals who completed long‐term follow‐up, 187 (14.1%) indicated that they had spoken with a genetic counselor. The most commonly given reason for not utilizing the counseling service was a lack of need due to the perception of already understanding one's results (55.6%). The most common reasons for utilizing the service included wanting to take advantage of a free service (43.9%) and wanting more information on risk calculations (42.2%). Among those who utilized the service, a large fraction reported that counseling improved their understanding of their results (54.5%) and genetics in general (43.9%). A relatively small proportion of participants utilized genetic counseling after direct‐to‐consumer personal genomic testing. Among those individuals who did utilize the service, however, a large fraction perceived it to be informative, and thus presumably beneficial.     

Chronic mercury exposure impairs the sympathovagal control of the rat heart

Mercury is known to cause harmful neural effects affecting the cardiovascular system. Here, we evaluated the chronic effects of low‐dose mercury exposure on the autonomic control of the cardiovascular system. Wistar rats were treated for 30 days with HgCl₂ (1st dose 4.6 μg/kg followed by 0.07 μg/kg per day, intramuscular) or saline. The femoral artery and vein were then cannulated for evaluation of autonomic control of the hemodynamic function, which was evaluated in awake rats. The following tests were performed: baroreflex sensitivity, Von Bezold‐Jarisch reflex, heart rate variability (HRV) and pharmacological blockade with methylatropine and atenolol to test the autonomic tone of the heart. Exposure to HgCl₂ for 30 days slightly increased the mean arterial pressure and heart rate (HR). There was a significant reduction in the baroreflex gain of animals exposed to HgCl₂. Moreover, haemodynamic responses to the activation of the Von Bezold‐Jarisch reflex were also reduced. The changes in the spectral analysis of HRV suggested a shift in the sympathovagal balance toward a sympathetic predominance after mercury exposure, which was confirmed by autonomic pharmacological blockade in the HgCl₂ group. This group also exhibited reduced intrinsic HR after the double block suggesting that the pacemaker activity of the sinus node was also affected. These findings suggested that the autonomic modulation of the heart was significantly altered by chronic mercury exposure, thus reinforcing that even at low concentrations such exposure might be associated with increased cardiovascular risk.     

椎体复位器治疗胸腰椎骨折过程中的生物力学参数变化

目的:对胸腰椎压缩性骨折的复位目前主要是通过Harrington棒的撑开或Dick等器械压缩,间接地使压缩的椎体得到复位.但复位的力量不易掌握,效果往往也不够理想.因此,需要了解和掌握复位器未发生变形时所能承受的最大应力及椎体复位器在椎体内撑开复位的应力与位移的关系.方法:实验于1998-10/2003-04在上海市材料力学研究所、上海市第九人民医院生物力学研究室及上海第二医科大学解剖教研室市生物力学研究室完成.自行设计的椎体复位器为圆柱形,顶端的上下开启由尾端的圆形手柄控制.实验方法:①椎体复位器的抗压力实验:将椎体复位器撑开到最大后进行轴向压缩实验,当复位器刚好发生形变时的轴向压应力,即是复位器进行轴向撑开时所能承受最大应力.②新鲜小牛骨椎体抗轴向压力实验:在36个新鲜小牛骨椎体上制作压缩性骨折模型时,了解椎体形变时的应力形变的关系.实验前测出每个椎体的高度值,发生压缩形变后,再测量每个椎体的高度值,并在机器上测出相应的压应力值.③椎体复位器复位压缩性小牛骨椎体的生物力学实验:将20个新鲜小牛骨胸腰椎椎体分成2组,每组10个椎体.在轴向载荷下压缩椎体,制作压缩性骨折的模型,1/2椎体高度压缩组其压缩后高度接近原有椎体高度的1/2,2/3椎体高度压缩组其压缩后高度接近原有椎体高度的2/3.从椎体椎弓根两侧放入复位器,将压缩的椎体予以复位.了解复位器在椎体内的撑开应力与复位时位移关系.结果:①复位器未发生变形时所能承受的最大力为281 N.②36个新鲜小牛骨椎体在承受轴向载荷发生变形时屈服点的平均应力为74.96 kPa,各数据符合正态分布(t=16.044,P＜0.01).③1/2椎体高度压缩组和2/3椎体高度压缩组复位器在椎体内撑开后应力值差异无显著性意义(t=0.835,P＞0.05).结论:椎体压缩程度与椎体内撑开复位的力量没有直接关系,自行设计的椎体复位器在椎体内撑开复位的力量远小于复位器的屈服点应力.