Mucosal transmission of R5 and X4 tropic HIV-1 via vaginal and rectal routes in humanized Rag2-/- gammac -/- (RAG-hu) mice

Studies on HIV-1 mucosal transmission to evaluate early events in pathogenesis and the development of effective preventive/prophylactic methods have thus far been hampered by the lack of a suitable animal model susceptible to HIV-1 infection by either vaginal and/or rectal routes. In this regard, while primate-SIV/SHIV and cat-FIV models provided useful surrogate platforms to derive comparative data, these viruses are distinct and different from that of HIV-1. Therefore an optimal model that permits direct study of HIV-1 transmission via mucosal routes is highly desirable. The new generation of humanized NOD/SCID BLT, NOD/SCIDgammac(-/-), and Rag2(-/-)gammac(-/-) mouse models show great promise to achieve this goal. Here, we show that humanized Rag2(-/-)gammac(-/-) mice (RAG-hu) engrafted with CD34 hematopoietic progenitor cells harbor HIV-1-susceptible human cells in the rectal and vaginal mucosa and are susceptible to HIV-1 infection when exposed to cell-free HIV-1 either via vagina or rectum. Infection could be established without any prior hormonal conditioning or mucosal abrasion. Both R5 and X4 tropic viruses were capable of mucosal infection resulting in viremia and associated helper T cell depletion. There was systemic spread of the virus with infected cells detected in different organs including the intestinal mucosa. R5 virus was highly efficient in mucosal transmission by both routes whereas X4 virus was relatively less efficient in causing infection. HIV-1 infection of RAG-hu mice by vaginal and rectal routes as shown here represents the first in vivo model of HIV-1 transmission across intact mucosal barriers and as such may prove very useful for studying early events in HIV-1 pathogenesis in vivo, as well as the testing of microbicides, anti-HIV vaccines/therapeutics, and other novel strategies to prevent HIV-1 transmission.     

IRF-1基因在白血病中的表达及其临床意义

目的 :初步分析了白血病细胞IRF 1基因表达水平及其临床意义。方法 :采用逆转录 聚合酶链反应扩增IRF 1基因 ,以 β actin基因作为对照 ,结合凝胶图象扫描 ,以IRF 1/ β actin作为IRF 1相对表达量进行分析。分析白血病 77例 ,难治性贫血RAEB 2例及 4个白血病细胞系。结果 :急性白血病 18例未检测到IRF 1基因 ,急白组及慢粒组IRF 1表达低于正常组(P <0 0 5 )。HL6 0 ,K5 6 2 ,U9373个白血病细胞系IRF 1表达明显降低。慢粒患者应用IFN治疗后IRF 1表达量增加。结论 :IRF 1基因是影响白血病发病的一个重要的抑癌基因 ,在白血病可能存在缺失或部分失活的异常改变 ,对白血病的诊治有参考价值。     

Experimental autoimmune thyroiditis in nonobese diabetic mice lacking interferon regulatory factor-1

Interferon regulatory factor-1 (IRF-1) is pivotal in the regulation of interferon (IFN)-mediated immune reactions, and studies suggest that IRF-1 is involved in the development of autoimmune diseases. IRF-1+/+, +/-, and -/- nonobese diabetic (NOD) mice were immunized with mouse thyroglobulin (mTg) to determine whether IRF-1 is required in experimental autoimmune thyroiditis (EAT), a murine model for Hashimoto's thyroiditis (HT). IRF-1-deficient mice developed EAT and anti-mTg antibodies comparable to IRF-1+/+ and +/- mice. Whereas both CD4+ and CD8+ T cells were found in thyroids of IRF-1+/+ mice, the latter was not in IRF-1-/- mice. Major histocompatibility complex class II antigen was comparably expressed in thyroids of IRF-1+/+ and -/- mice. Lack of IRF-1 resulted in decreased CD8+ T cell number in the spleen and reduced IFNgamma production by splenocytes. Our results suggest that IRF-1 is not pivotal in EAT in NOD mice.     

Expression of heme oxygenase-1 is associated with abortion caused by Brucella abortus infection in pregnant mice

Brucella abortus is a facultative intracellular pathogen that can survive inside macrophages and trophoblast giant (TG) cells, and the causative agent of brucellosis. In the present study, we found that expression of heme oxygenase-1 (HO-1) in TG cells is correlated with abortion induced by B. abortus infection in pregnant mice. Expression of HO-1 in the placenta was decreased by B. abortus infection and treatment with cobalt-protoporphyrin (Co-PP), which is known to up-regulate HO-1 expression, inhibited abortion due to the bacterial infection. In TG cells, treatment with Co-PP was shown to up-regulate HO-1, whereas its expression was decreased by B. abortus infection. Such down-regulation of HO-1 in the TG cells was enhanced by IFN-gamma treatment. HO-1 down-regulation in TG cells due to knockdown or IFN-gamma treatment served to induce cell death caused by B. abortus infection. These results suggest that down-regulation of HO-1 in TG cells due to B. abortus infection is an important event in infectious abortion.     

The BMEI0216 gene of Brucella melitensis is required for internalization in HeLa cells

Brucella is an intracellular facultative bacterium able to survive and multiply in professional and non-professional phagocytes. However, its adhesion and invasion mechanisms have not been elucidated yet. In this work, we assess the interruption of a BMEI0216 gene of Brucella melitensis, by using HeLa epithelial cells and murine macrophages for invasion and replication assays. The mutation did not affect survival or multiplication within macrophages. Likewise, invasion assays with HeLa cells revealed no differences at 30 and 45 min, whereas, at 1 and 2h, the infection ability of the mutant was drastically reduced. These results suggest that the BMEI0216 gene is required for B. melitensis internalization.     

快速老化系小鼠与昆明系小鼠AD相关指标的比较

目的： 观察6月龄的快速老化系SAM-P/8、SAM-R/1小鼠与昆明系小鼠的AD相关指标的变化。方法： 取健康（20±5）g 6月龄SAM-P/8小鼠、SAM-R/1小鼠和昆明系小鼠各14只，雌雄各半，随机分成：SAM-P/8小鼠（AD疾病模型小鼠）、SAM-R/1小鼠对照组和昆明系小鼠对照组，观察上述3组小鼠行为学、神经生化、超微结构、基因表达情况。结果： 6月龄SAM-P/8小鼠1-4d学习成绩、第5 d记忆成绩低于6月龄SMA-R小鼠和昆明系小鼠（P<0.05），真性胆碱酯酶活性则高于6月龄SMA-R/1小鼠和昆明系小鼠（P<0.05）；SAM-P/8小鼠海马神经元超微结构显示明显纤维化，而SAM-R/1小鼠和昆明系小鼠无明显纤维化；SAM-P/8小鼠脑神经细胞的凋亡相关基因表达有明显上调达2倍以上，而SAM-R/1小鼠和昆明系小鼠则未见这些基因有明显上调。SAM-R/1小鼠和昆明系小鼠比较，除基因表达略有差异之外，其它指标无明显差异。结论： 6月龄SAM-P/8小鼠在很多方面已经具备了AD自然发病模型典型特征。而用于正常对照的6月龄SAM-R/1与昆明系小鼠的行为学、TchE活性、超微结构则无显著差异；凋亡相关基因表达差异不明显，而部分基因表达则有一定差异。     

HIV-1 accessory proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation

The activation of IRF-3 during the early stages of viral infection is critical for the initiation of the antiviral response; however the activation of IRF-3 in HIV-1 infected cells has not yet been characterized. We demonstrate that the early steps of HIV-1 infection do not lead to the activation and nuclear translocation of IRF-3; instead, the relative levels of IRF-3 protein are decreased due to the ubiquitin-associated proteosome degradation. Addressing the molecular mechanism of this effect we show that the degradation is independent of HIV-1 replication and that virion-associated accessory proteins Vif and Vpr can independently degrade IRF-3. The null mutation of these two genes reduced the capacity of the HIV-1 virus to down modulate IRF-3 levels. The degradation was associated with Vif- and Vpr-mediated ubiquitination of IRF-3 and was independent of the activation of IRF-3. N-terminal lysine residues were shown to play a critical role in the Vif- and Vpr-mediated degradation of IRF-3. These data implicate Vif and Vpr in the disruption of the initial antiviral response and point to the need of HIV-1 to circumvent the antiviral response during the very early phase of replication.     

TLR途经负性调节因子A20、IRF-4和TRAF4在川崎病免疫发病中的变化

目的 探讨Toll样受体（TLR）信号途径负性调控因子在川崎病（KD）免疫发病机制中的作用。方法 急性期KD患儿48例，正常同年龄对照组16例，感染性疾病对照组（ID）16例。采用逆转录．聚合酶链反应（RT．PcR）及荧光定量PCR检测外周血单核，巨噬细胞（MC）TLR4信号途径传导分子、负性调节因子及前炎症细胞因子mRNA的表达。结果 （1）急性期KD患儿TLR4、MD-2、MyD88、IRAK-4、TRAF6、TAK1、TAB1及TAB2mRNA表达明显高于正常同年龄对照组（P〈0．01）；（2）KD及ID患儿A20、IRF-4、TRAF4基因表达上调（P〈0．01）；KD患儿除．A20表达水平高于ID对照组外，IRF-4、TRAF4表达水平明显低于ID对照组（P〈0．01）；KD患儿前炎症细胞因子表达明显高于ID组（P〈0．01）；（3）经细菌脂多糖（LPS）刺激后，KD患儿及正常对照组A20、IRF-4及TRAF4表达明显增高（P〈0．01），但KD患儿3种负性调节因子基因表达显著低于正常对照组（P〈0．01）；（4）KD合并冠状动脉损伤组（KD-CAL^＋）MC负性调节因子IRF-4及TRAF4明显低于无冠状动脉损伤组（KD-CAL^-），A20表达则显著高于后者；KD-CAL^＋组前炎症细胞因子IL-1β、IL-6、TNF-α均高于KD-CAL^-组（P〈0．01）。结论 急性期KD患儿TLR信号途径负性调节因子相对表达不足可能与KD的免疫发病机制有关。     

HMGB1, an innate alarmin, in the pathogenesis of type 1 diabetes

HMGB1, an evolutionarily conserved chromosomal protein, was recently re-discovered to act as a “danger signal” (alarmin) to alert the innate immune system for the initiation of host defense or tissue repair. Extracellular HMGB1 can be either passively released from damaged/necrotic cells or secreted by activated immune cells. Upon stimulation, dendritic cells (DCs), macrophages and natural killer (NK) cells secrete high levels of HMGB1 into the intercellular milieu. HMGB1 is potent to target DCs, macrophages, neutrophils and CD4⁺ T cells. It also upregulates the expression of BCL-XL by which it may prevent the elimination of activated immune cells. As a result, HMGB1 has been suggested to be implicated in the pathogenesis of autoimmune disorders such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and experimental allergic encephalomyelitis (EAE). Given the similarities of autoimmune response against beta cell self-antigens in type 1 diabetes (T1D), in this view we will discuss the possible implications of HMGB1 in T1D pathogenesis. Specifically, we will summarize and update the advancement of HMGB1 in the pathogenesis of autoimmune initiation and progression duringT1D development, as well as islet allograft rejection of diabetic patients after islet transplantation. Elucidation of the role for HMGB1 in T1D pathogenesis would not only enhance the understanding of disease etiology, but also have the potential to shed new insight into the development of therapeutic strategies for prevention or intervention of this disorder.     

Dengue virus infection and immune response in humanized RAG2(-/-)gamma(c)(-/-) (RAG-hu) mice

Dengue viral (DENV) pathogenesis and vaccine studies are hampered by the lack of an ideal animal model mimicking human disease and eliciting an adaptive human immune response. Although currently available animal models have been very useful in dissecting some key aspects of disease pathogenesis, a major limitation with these is the lack of a human immune response. In this study, we sought to overcome this difficulty by utilizing a novel mouse model that permits multi-lineage human hematopoiesis and immune response following transplantation with human hematopoietic stem cells. To generate immunocompetent humanized mice, neonatal RAG2(-/-)gamma(c)(-/-) mice were xenografted with human CD34+ hematopoietic stem cells, resulting in de novo development of major functional cells of the human adaptive immune system. To evaluate susceptibility to dengue viral infection, humanized mice were challenged with DEN-2 serotype. Viremia lasting up to 3 weeks was detected in infected mice with viral titers reaching up to 10(6.3) RNA copies/ml. Fever characteristic of dengue was also noted in infected mice. Presence of human anti-dengue antibodies was evaluated using an antibody capture ELISA. Anti-dengue IgM was first detected by 2 weeks post-infection followed by IgG at 6 weeks. Sera from some of the infected mice were also found to be capable of dengue virus neutralization. Infected mouse sera showed reactivity with the viral envelope and capsid proteins in immunoprecipitation assay. These results demonstrate for the first time that humanized mice are capable of dengue viral primary human immune responses thus paving the way for new dengue immunopathogenesis and vaccine studies.     

Anti-CD3 mAb treatment cures PDL1-/-.NOD mice of diabetes but precipitates fatal myocarditis

Anti-CD3 mAb is an effective therapy that can reverse diabetes in NOD mice and has therapeutic potential in patients with type 1 diabetes (T1D). We administered anti-CD3 to PDL1-/-.NOD mice in order to determine whether this treatment would reverse the development of diabetes in these mice. Mice injected with anti-CD3 mAb neonatally were protected from T1D. However, all of these anti-CD3 mAb treated PDL1-/-.NOD mice developed a wasting disease between 12 and 20 weeks of age with sudden deterioration and weight loss, leading to death within 3-5 days of development of illness. Histology revealed severe inflammation in the heart and skeletal muscles. These results suggest that deficiency of PDL1 in NOD background has the potential to lead to immune-mediated tissue damage in organs other than the pancreas, but this cannot be appreciated in PDL1-/-.NOD mice as the mice develop T1D at an early age and die from diabetes prior to manifesting other autoimmune diseases.     

Type 1 diabetes pathogenesis is modulated by spontaneous autoimmune responses to endogenous retrovirus antigens in NOD mice

Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and T cells, causing Type 1 Diabetes in non‐obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well‐characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus‐like particles produced by co‐expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes‐resistant mice developed anti‐Env autoantibodies that increase in titer as disease progresses. A lentiviral‐based RNA interference knockdown of Gag revealed that Gag contributes to the MV‐induced T‐cell response, whose diabetogenic function can be demonstrated via cell‐transfer into immune‐deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet‐derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes‐resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti‐retroviral responses to trigger autoimmunity.     

布氏杆菌pCDNA3.1-L7/L12核酸疫苗的构建及其免疫学评价

目的 获得布氏杆菌保护性抗原L2／L12重组蛋白及pCDNA3．1-L7／L12重组质粒，并比较其诱导特异性免疫应答的能力。方法 PCR扩增布氏杆菌核蛋白L7／L12基因分别构建至原核表达载体PET32a( )和真核表达载体pCDNA3.1( )中；pET32a-L7／L12重组质粒转化BL21(DE3)，所表达蛋白经SDS-PAGE、免疫印迹分析、纯化后免疫小鼠；pCDNA3.1-12／L12重组质粒配以GM-CSF同时肌肉注射免疫小鼠，3次免疫后测定免疫功能进行免疫效果的评价。结果ELISA、Western blot检测到免疫鼠体内有特异性抗体产生，蛋白苗所诱导的抗体效价远远高于DNA疫苗；通过淋巴细胞增殖实验、细胞因子和CD分子测定表明DNA疫苗以诱发TH1型免疫为主。结论 所构建的布氏杆菌DNA疫苗和蛋白苗均具有诱导特异性细胞和体液免疫应答的能力，DNA疫苗诱导产生的细胞免疫反应强于蛋白苗，可作为潜在的布氏菌新型疫苗，有进一步研究的意义。     

Behavior genetics of audiogenic seizures in DBA/2J and Rb-1 mice

Susceptibility to audiogenic seizures (AGS) was studied in DBA/2J (D), Rb-1 (Rb), F ₁ , F ₂ , D×F ₁ , and Rb×F ₁ mice at selected ages from 18 to 60 days of age. Sixty F ₁ mice were tested for AGS at varying ages from 18 to 40 days. None showed an AGS. Approximately 25% of the F ₂ , 50% of the Rb×F ₁ backcross, and 5% of the D×F ₁ backcross mice were susceptible at all ages tested. Data for the Rb-1 mice are in agreement with a single-gene model of susceptibility; no simple model fits the DBA data.