Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.     

含GDNF基因的慢病毒载体的构建和其在人胚胎神经干细胞中的表达

利用含胶质源性神经营养因子(Glial cell derived neurotrophic factor, GDNF)基因的慢病毒(Lentivirus)载体转染了人胚胎来源的神经干细胞, 探讨了转染后GDNF在神经干细胞中的体外表达水平及其影响因素。首先GDNF基因被克隆入慢病毒载体, 通过瞬时转染法包装出病毒上清, 经滴度鉴定后分别按拷贝数分别为 1、2.5、5、10转染神经干细胞。转染后细胞经过潮霉素筛选得到均一表达GDNF的神经干细胞体系。其后分别利用酶联免疫吸附(ELISA)方法和Real-time PCR方法测定不同转染组细胞在不同时间点GDNF的蛋白分泌水平和基因表达水平。实验中构建了表达GDNF基因的慢病毒载体, 包装出的病毒上清在体外培养条件下成功转染了神经干细胞, 经潮霉素筛选可以得到均一的持续表达分泌GDNF的人胚胎皮层神经干细胞体系。实验结果表明转染拷贝数可以影响GDNF的分泌水平, 相同条件下转染拷贝数越高, GDNF分泌量越多, 其基因表达水平越高。因此, 含GDNF的慢病毒载体可以成功转染人胚胎来源的神经干细胞, 使其持续表达GDNF, 转染过程中可以通过拷贝数在一定水平上控制GDNF的蛋白分泌水平和基因表达水平。     

纤维顶球改造增强了人5型腺病毒载体对造血细胞的感染

基于人5型腺病毒(Human adenovirus type 5,HAdV-5)的腺病毒载体对造血细胞的基因转导效率低,将病毒fiber基因替换为HAdV-11p的同源基因F11p后,载体对造血细胞的感染效率增强.本研究拟在F11p纤维顶球(knob)结构域添加RGD4C多肽或HIV包膜糖蛋白(gp120)的V3结构域,观察重组HAdV-5对造血细胞感染效率的变化.在前期构建的pKAd5f11p153R-EPG腺病毒质粒基础上,结合限制性酶切和DNA组装技术,在F11p 153 aa后(knob AB loop,153位)、228位(FG loop)以及300位(IJ loop)插入 RGD4C 肽或者 gp120的 V3肽,构建了共6种重组腺病毒载体(F153RGD-EG、F228RGD-EG、F300RGD-EG、F153CV-EG、F228CV-EG和 F300CV-EG),以fiber未改造的HAdV5-EG和改造为F11p的F11p-EG病毒作对照,观察了其对4种造血细胞系U937、K562、Jurkat和HL60以及人原代T细胞的感染效率.结果显示,对于U937细胞,当感染复数(MOI,vp/cell)为100时,HAdV5-EG感染效率最低,为2％;其次为F228CV-EG,感染率为45％;F300RGD-EG、F153CV-EG和F300CV-EG感染率为85％～90％;F153RGD-EG、F228RGD-EG高于阳性对照病毒F11p-EG,三者分别为99％、99％、95％.各病毒对于Jurkat细胞的感染率均较高,但HAdV5-EG明显低于F11p-EG、F153RGD-EG和F228RGD-EG,当MOI为100时分别为75％、93％、93％和96％.感染K562细胞的情况与U937细胞类似.各病毒对于HL60细胞感染效率最低,MOI为500时,F300RGD-EG和F300CV-EG的转导效率为28％和33％,是F11p-EG的10倍.对于人原代T细胞,F153RGD-EG和F228RGD-EG优于F11p-EG,当MOI为1000时,感染率分别为87％、90％和84％.研究结果表明,F11p knob插入RGD4C比单独F11p替换的HAdV-5对造血细胞的感染效率高,同时,本研究还发现HAdV-11p fiber knob的AB、FG或IJ loop可插入外源多肽,为腺病毒嗜向性改造增加了新靶点.     

Replication-Defective Adenovirus Vectors with Multiple Deletions Do Not Induce Measurable Vector-Specific T Cells in Human Trials

The magnitude and character of adenovirus serotype 5 (Ad5)-specific T cells were determined in volunteers with and without preexisting neutralizing antibodies (NAs) to Ad5 who received replication-defective Ad5 (rAd5)-based human immunodeficiency virus vaccines. There was no correlation between T-cell responses and NAs to Ad5. There was no increase in magnitude or activation state of Ad5-specific CD4⁺ T cells at time points where antibodies to Ad5 and T-cell responses to the recombinant gene products could be measured. These data indicate that rAd5-based vaccines containing deletions in the E1, E3, and E4 regions do not induce appreciable expansion of vector-specific CD4⁺ T cells.Replication-defective adenoviruses (rAd) have been engineered to provide high levels of expression of foreign inserts with minimum expression of adenovirus proteins, making them excellent candidates for vaccine and gene therapy applications (3, 16). Despite promising immunogenicity, a prophylactic vaccine trial of a serotype 5 rAd (rAd5) vector expressing human immunodeficiency virus (HIV) Gag, Pol, and Nef genes (Step trial) was recently halted due to an increase in HIV infections among volunteers who had preexisting neutralizing antibodies (NAs) to Ad5 (7). This finding raises the possibility that the presence of Ad5-specific T-cell responses (specifically CD4⁺ T-cell responses) in subjects with preexisting Ad5 NAs could be boosted by rAd5 vaccines, thereby providing an expanded susceptible target cell population that could be more easily infected by HIV. If this mechanism were operative, it would have broad implications for the future use of rAd viruses, and indeed other virus vectors, as vaccines or therapeutic agents within HIV-susceptible populations (2, 12, 15). We therefore measured the frequency, magnitude, and activation status of rAd5-specific T cells in HIV-uninfected volunteers who had received rAd5-based HIV vaccines in the presence or absence of preexisting NAs to Ad5.We studied 31 volunteers enrolled in two NIAID Institutional Review Board-approved phase I clinical trials of rAd5-based HIV vaccines. VRC 006 was a dose escalation study evaluating a single inoculation of a rAd5 mixture expressing EnvA, EnvB, EnvC, and fusion protein Gag/PolB at 10⁹, 10¹⁰, and 10¹¹ total particle units (10). VRC 008 evaluated DNA priming by needle and syringe or Biojector, followed by rAd5 boosting. Both studies enrolled healthy, HIV-uninfected adults; used the same rAd5 products; and evaluated immunogenicity on the day of and 4 weeks after rAd5 immunization. Both of these trials involved rAd5 products that contained deletions in the E1, E3, and E4 regions (8, 10).NAs to Ad5 were determined for all volunteers as previously described (19). A 90% NA titer of 12 or more was considered positive and taken as evidence of preexisting humoral immunity to Ad5. Volunteers were chosen for assessment of Ad5-specific T-cell responses based upon the availability of peripheral blood mononuclear cell samples at key time points and the presence or absence of preexisting NAs to Ad5. Only volunteers who received the vaccine (not the placebo) were included. Table ​Table11 lists the volunteers who were tested for Ad5-specific T-cell responses and their NA titers to Ad5 before and after rAd5 vaccination. All volunteers, except for one (volunteer 12) who had a less-than-maximum NA titer to Ad5 before vaccination, had an increase in titer by 4 weeks after vaccination, indicating the successful “take” of the rAd5-based vaccine. There was no correlation between rAd5 dose and increase in Ad5 NA titer.

TABLE 1.

Ad5 serostatus before and after vaccination

Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should impact successful gene delivery in CF patients.     

Hypoxia-Inducible Factors Have Distinct and Stage-Specific Roles during Reprogramming of Human Cells to Pluripotency

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Alternative Serotype Adenovirus Vaccine Vectors Elicit Memory T Cells with Enhanced Anamnestic Capacity Compared to Ad5 Vectors

The failure of the adenovirus serotype 5 (Ad5) vector-based human immunodeficiency virus type 1 (HIV-1) vaccine in the STEP study has led to the development of adenovirus vectors derived from alternative serotypes, such as Ad26, Ad35, and Ad48. We have recently demonstrated that vaccines using alternative-serotype Ad vectors confer partial protection against stringent simian immunodeficiency virus (SIV) challenges in rhesus monkeys. However, phenotypic differences between the T cell responses elicited by Ad5 and those of alternative-serotype Ad vectors remain unexplored. Here, we report the magnitude, phenotype, functionality, and recall capacity of memory T cell responses elicited in mice by Ad5, Ad26, Ad35, and Ad48 vectors expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP). Our data demonstrate that memory T cells elicited by Ad5 vectors were high in magnitude but exhibited functional exhaustion and decreased anamnestic potential following secondary antigen challenge compared to Ad26, Ad35, and Ad48 vectors. These data suggest that vaccination with alternative-serotype Ad vectors offers substantial immunological advantages over Ad5 vectors, in addition to circumventing high baseline Ad5-specific neutralizing antibody titers.     

人Hes1-shRNA，Hes5-shRNA慢病毒表达载体的构建、包装及鉴定

目的构建人Hesl-shRNA和Hes5-shRNA慢病毒表达载体,为Notch—Hes信号通路的相关研究奠定基础。方法根据人Hes1,Hes5基因mRNA序列分别设计、合成多对互补的DNA单链寡核苷酸,退火后克隆至pENTR／U6入门载体。通过入门载体瞬时转染神经胶质瘤U251细胞筛选有效干扰序列。将含有效干扰序列的入门载体与pLenti6／BLOCK—iT—DEST载体进行LR重组构建Hesl—shRNA和Hes5-shRNA慢病毒表达载体,经脂质体介导入293FT细胞,包装成慢病毒。用该慢病毒感染U251细胞,Western印迹法分别检测Hes1,Hes5蛋白的表达。结果分别构建了针对Hes1和Hes5基因的特异性shRNA慢病毒表达载体,其包装获得慢病毒可有效感染U251细胞并分别对HeM,Hes5蛋白的表达有显著抑制作用。结论成功构建了Hesl—shRNA和Hes5-shRNA慢病毒表达载体。     

Stress-Induced In Vivo Recruitment of Human Cytotoxic Natural Killer Cells Favors Subsets with Distinct Receptor Profiles and Associates with Increased Epinephrine Levels

Background

Acute stress drives a ‘high-alert’ response in the immune system. Psychoactive drugs induce distinct stress hormone profiles, offering a sought-after opportunity to dissect the in vivo immunological effects of acute stress in humans.

Methods

3,4-methylenedioxymethamphetamine (MDMA), methylphenidate (MPH), or both, were administered to healthy volunteers in a randomized, double-blind, placebo-controlled crossover-study. Lymphocyte subset frequencies, natural killer (NK) cell immune-phenotypes, and changes in effector function were assessed, and linked to stress hormone levels and expression of CD62L, CX3CR1, CD18, and stress hormone receptors on NK cells.

Results

MDMA/MPH > MDMA > MPH robustly induced an epinephrine-dominant stress response. Immunologically, rapid redistribution of peripheral blood lymphocyte-subsets towards phenotypically mature NK cells occurred. NK cytotoxicity was unaltered, but they expressed slightly reduced levels of the activating receptor NKG2D. Preferential circulation of mature NK cells was associated with high epinephrine receptor expression among this subset, as well as expression of integrin ligands previously linked to epinephrine-induced endothelial detachment.

Conclusion

The acute epinephrine-induced stress response was characterized by rapid accumulation of mature and functional NK cells in the peripheral circulation. This is in line with studies using other acute stressors and supports the role of the acute stress response in rapidly mobilizing the innate immune system to counteract incoming threats.     

人SPRED2 基因重组腺病毒载体的制备及其对K562 细胞

目的:构建携带SPRED2的质粒载体与重组腺病毒载体,并观察其在K562细胞的表达及对ERK信号通路的作用,为Spred2在造血细胞中的作用的研究奠定基础。方法:以HepG2细胞cDNA为模板,RT-PCR克隆SPRED2全长CDS序列,并亚克隆到pCDNA3.0和pshuttle-CMV质粒载体,构建携带SPRED2的真核表达载体pCDNA3.0-Spred2与穿梭载体pshuttle-CMV-Spred2;将线性化pshuttle-CMV-Spred2与腺病毒骨架质粒Adf11p在感受态细胞BJ5183中进行同源重组,产生重组质粒Adf11p-Spred2;后者经线性化后转染至HEK293细胞进行病毒包装;在HEK293细胞扩增病毒颗粒,以CsCl密度梯度离心法进行纯化,TCID50法测定病毒滴度;将病毒颗粒以100MOI感染K562细胞,Western blot检测Spred2过表达情况及Spred2对细胞ERK的影响。结果:经酶切、DNA测序、Western blot检测等方法鉴定,证明pCDNA3.0-Spred2与Adf11p-Spred2携带Spred2序列正确,能够在HEK293细胞、K562细胞正确表达,Spred2过表达能够显著抑制K562细胞ERK活性。结论:成功构建对K562细胞有高感染效率的SPRED2重组腺病毒载体,且Spred2对K562细胞ERK信号通路有显著抑制作用。     

Cell-Type-Specific Gene Transfer into Human Cells with Retroviral Vectors That Display Single-Chain Antibodies

The successful application of human gene therapy protocols on a broad clinical basis will depend on the availability of in vivo cell-type-specific gene delivery systems. We have developed retroviral vector particles, derived from spleen necrosis virus (SNV), that display the antigen binding site of an antibody on the viral surface. Using retroviral vectors derived from SNV that displayed single-chain antibodies (scAs) directed against a carcinoembryonic antigen-cross-reacting cell surface protein, we have shown that an efficient, cell-type-specific gene delivery can be obtained. In this study, we tested whether other scAs displayed on SNV vector particles can also lead to cell-type-specific gene delivery. We displayed the following scAs on the retroviral surface: one directed against the human cell surface antigen Her2neu, which belongs to the epidermal growth factor receptor family; one directed against the stem cell-specific antigen CD34; and one directed against the transferrin receptor, which is expressed on liver cells and various other tissues. We show that retroviral vectors displaying these scAs are competent for infection in human cells which express the antigen recognized by the scA. Infectivity was cell type specific, and titers above 10⁵ CFU per ml of tissue culture supernatant medium were obtained. The density of the antigen on the target cell surface does not influence virus titers in vitro. Our data indicate that the SNV vector system is well suited for the development of a large variety of cell-type-specific targeting vectors.     

Long-Term Reproducible Expression in Human Fetal Liver Hematopoietic Stem Cells with a UCOE-Based Lentiviral Vector

Hematopoietic Stem Cell (HSC) targeted gene transfer is an attractive treatment option for a number of hematopoietic disorders caused by single gene defects. However, extensive methylation of promoter sequences results in silencing of therapeutic gene expression. The choice of an appropriate promoter is therefore crucial for reproducible, stable and long-term transgene expression in clinical gene therapy. Recent studies suggest efficient and stable expression of transgenes from the ubiquitous chromatin opening element (UCOE) derived from the human HNRPA2B1-CBX3 locus can be achieved in murine HSC. Here, we compared the use of HNRPA2B1-CBX3 UCOE (A2UCOE)-mediated transgene regulation to two other frequently used promoters namely EF1α and PGK in human fetal liver-derived HSC (hflHSC). Efficient transduction of hflHSC with a lentiviral vector containing an HNRPA2B1-CBX3 UCOE-eGFP (A2UCOE-eGFP) cassette was achieved at higher levels than that obtained with umbilical cord blood derived HSC (3.1x; p<0.001). While hflHSC were readily transduced with all three test vectors (A2UCOE-eGFP, PGK-eGFP and EF1α-eGFP), only the A2-UCOE construct demonstrated sustained transgene expression in vitro over 24 days (p<0.001). In contrast, within 10 days in culture a rapid decline in transgene expression in both PGK-eGFP and EF1α-eGFP transduced hflHSC was seen. Subsequently, injection of transduced cells into immunodeficient mice (NOD/SCID/Il2rg^-/-) demonstrated sustained eGFP expression for the A2UCOE-eGFP group up to 10 months post transplantation whereas PGK-eGFP and EF1α-eGFP transduced hflHSC showed a 5.1 and 22.2 fold reduction respectively over the same time period. We conclude that the A2UCOE allows a more efficient and stable expression in hflHSC to be achieved than either the PGK or EF1α promoters and at lower vector copy number per cell.     

Frequent Detection of Human Adenovirus from the Lower Gastrointestinal Tract in Men Who Have Sex with Men

Background

The association between baseline seropositivity to human adenovirus (HAdV) type 5 and increased HIV acquisition in the Step HIV Vaccine Study has raised questions concerning frequency of acquired and/or persistent Adenovirus infections among adults at high risk of HIV-1 infection.

Methodology

To evaluate the frequency and pattern of HAdV shedding from the lower GI tract, we retrospectively tested rectal swabs for HAdVs in a cohort of 20 HSV-2 positive HIV-positive Peruvian men who have sex with men (MSM) undergoing rectal swabbing three times/week for 18 consecutive weeks, in a prospective study of HSV-2 suppression in HIV infection. Viral DNA was extracted and amplified using a sensitive multiplex PCR assay that detects all currently recognized HAdV types. Molecular typing of viruses was performed on selected samples by hexon gene sequencing. Baseline neutralizing antibody titers to HAdVs −5, −26, −35 and −48 were also assessed.

Principal Findings

15/20 individuals had HAdV detected during follow up. The median frequency of HAdV detection was 30% of samples (range 2.0% to 64.7%). HAdV shedding typically occurred on consecutive days in clustered episodes lasting a median of 4 days (range 1 to 9 days) separated by periods without shedding, suggesting frequent new infections or reactivation of latent infections over time. 8 of the 15 shedders had more than one type detected in follow-up. 20 HAdV types from species B, C, and D were identified, including HAdV-5, −26 and −48, HAdV types under development as potential vaccine candidates. 14/20 subjects were seropositive for HAdV-5; 15/20 for HAdV-26; 3/20 for HAdV-35; and 2/20 for HAdV-48. HAdV shedding did not correlate with CD4 count, plasma HIV-1 viral load, or titers to HAdV-5 or HAdV-35. The sole individual with HAdV-5 shedding was HAdV-5 seropositive.

Conclusions

HAdV shedding was highly prevalent and diverse, including types presently under consideration as HIV vaccine vectors. Subclinical HAdV infection of the GI tract is common among MSM in Peru; the prevalence of HAdV in the enteric tract should be evaluated in other populations. The association between ongoing recent enteric HAdV and the immune response to recombinant HAdV vaccines should be evaluated.     

Immunization of Mice with Lentiviral Vectors Targeted to MHC Class II+ Cells Is Due to Preferential Transduction of Dendritic Cells In Vivo

Gene transfer vectors such as lentiviral vectors offer versatile possibilities to express transgenic antigens for vaccination purposes. However, viral vaccines leading to broad transduction and transgene expression in vivo, are undesirable. Therefore, strategies capable of directing gene transfer only to professional antigen-presenting cells would increase the specific activity and safety of genetic vaccines. A lentiviral vector pseudotype specific for murine major histocompatibilty complex class II (LV-MHCII) was recently developed and the present study aims to characterize the in vivo biodistribution profile and immunization potential of this vector in mice. Whereas the systemic administration of a vector pseudotyped with a ubiquitously-interacting envelope led to prominent detection of vector copies in the liver of animals, the injection of an equivalent amount of LV-MHCII resulted in a more specific biodistribution of vector and transgene. Copies of LV-MHCII were found only in secondary lymphoid organs, essentially in CD11c+ dendritic cells expressing the transgene whereas B cells were not efficiently targeted in vivo, contrary to expectations based on in vitro testing. Upon a single injection of LV-MHCII, naive mice mounted specific effector CD4 and CD8 T cell responses against the intracelllular transgene product with the generation of Th1 cytokines, development of in vivo cytotoxic activity and establishment of T cell immune memory. The targeting of dendritic cells by recombinant viral vaccines must therefore be assessed in vivo but this strategy is feasible, effective for immunization and cross-presentation and constitutes a potentially safe alternative to limit off-target gene expression in gene-based vaccination strategies with integrative vectors.