HIV-1 Gag-specific immunity induced by a lentivector-based vaccine directed to dendritic cells

Lentivectors (LVs) have attracted considerable interest for their potential as a vaccine delivery vehicle. In this study, we evaluate in mice a dendritic cell (DC)-directed LV system encoding the Gag protein of human immunodeficiency virus (HIV) (LV-Gag) as a potential vaccine for inducing an anti-HIV immune response. The DC-directed specificity is achieved through pseudotyping the vector with an engineered Sindbis virus glycoprotein capable of selectively binding to the DC-SIGN protein. A single immunization by this vector induces a durable HIV Gag-specific immune response. We investigated the antigen-specific immunity and T-cell memory generated by a prime/boost vaccine regimen delivered by either successive LV-Gag injections or a DNA prime/LV-Gag boost protocol. We found that both prime/boost regimens significantly enhance cellular and humoral immune responses. Importantly, a heterologous DNA prime/LV-Gag boost regimen results in superior Gag-specific T-cell responses as compared with a DNA prime/adenovector boost immunization. It induces not only a higher magnitude response, as measured by Gag-specific tetramer analysis and intracellular IFN-γ staining, but also a better quality of response evidenced by a wider mix of cytokines produced by the Gag-specific CD8^+ and CD4^+ T cells. A boosting immunization with LV-Gag also generates T cells reactive to a broader range of Gag-derived epitopes. These results demonstrate that this DC-directed LV immunization is a potent modality for eliciting anti-HIV immune responses

The Effect of Safflower Yellow on Spinal Cord Ischemia Reperfusion Injury in Rabbits

Safflower yellow (SY) is the safflower extract and is the one of traditional Chinese medicine. The aim of the present work was to investigate the effect of SY on spinal cord ischemia reperfusion injury (SCIRI) in rabbits. The models of spinal cord ischemia reperfusion (SI/R) were constructed, and the degree of the post-ischemic injury was assessed by means of the neurological deficit scores and plasma levels of lipid peroxidation reactioin and neuronal morphologic changes. SCIRI remarkably affected the functional activities of the hind limbs and activated lipid peroxidation reaction. SY could attenuate apoptosis and SCIRI by enhancing Bcl-2 expression and inhibiting Bax and caspase-3 activation

FoxM1B transcriptionally regulates vascular endothelial growth factor expression and promotes the angiogenesis and growth of glioma cells.

We previously found that FoxM1B is overexpressed in human glioblastomas and that forced FoxM1B expression in anaplastic astrocytoma cells leads to the formation of highly angiogenic glioblastoma in nude mice. However, the molecular mechanisms by which FoxM1B enhances glioma angiogenesis are currently unknown. In this study, we found that vascular endothelial growth factor (VEGF) is a direct transcriptional target of FoxM1B. FoxM1B overexpression increased VEGF expression, whereas blockade of FoxM1 expression suppressed VEGF expression in glioma cells. Transfection of FoxM1 into glioma cells directly activated the VEGF promoter, and inhibition of FoxM1 expression by FoxM1 siRNA suppressed VEGF promoter activation. We identified two FoxM1-binding sites in the VEGF promoter that specifically bound to the FoxM1 protein. Mutation of these FoxM1-binding sites significantly attenuated VEGF promoter activity. Furthermore, FoxM1 overexpression increased and inhibition of FoxM1 expression suppressed the angiogenic ability of glioma cells. Finally, an immunohistochemical analysis of 59 human glioblastoma specimens also showed a significant correlation between FoxM1 overexpression and elevated VEGF expression. Our findings provide both clinical and mechanistic evidence that FoxM1 contributes to glioma progression by enhancing VEGF gene transcription and thus tumor angiogenesis

A TLR4 agonist synergizes with dendritic cell-directed lentiviral vectors for inducing antigen-specific immune responses

TLR4 agonists can be used as adjuvants to trigger innate immune responses of antigen-presenting cells (APCs) such as dendritic cells (DCs) to enhance vaccine-specific immunity. Adjuvant effects of TLR4 agonists are mediated by downstream signaling controlled by both MyD88 and TRIF adapter proteins. In this study, we investigated the adjuvanting capacity of glucopyranosyl lipid A (GLA), a chemically synthesized TLR4 agonist, to boost antigen-specific immunity elicited by DC-directed lentiviral vectors (DC-LV). We found that stimulation by this agonist in vitro can activate DCs in a TLR4-dependent manner. The agonist can significantly boost DC-LV-induced humoral and cellular immune responses, resulting in better antitumor reactions in response to tumor challenges. We observed that the adjuvant-mediated enhancement of cytotoxic CD8+ T cell responses is CD4+ T cell-dependent and determined that in vitro the agonist stimulation involves the participation of both MyD88 and TRIF pathways to activate DCs. In vivo immunization study however revealed that adjuvant effects depend more on the MyD88 signaling as TRIF^(−/−) mice but not MyD88^(−/−) mice were able to maintain the enhanced CD8+ T cell responses upon DC-LV immunization. Thus, our study supports the use of this TLR4 agonist as a potent adjuvant candidate for boosting DC-LV immunization

FoxM1B regulates NEDD4-1 expression, leading to cellular transformation and full malignant phenotype in immortalized human astrocytes.

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms

Exosomes from embryonic mesenchymal stem cells alleviate osteoarthritis through balancing synthesis and degradation of cartilage extracellular matrix

Abstract Background Mesenchymal stem cell therapy for osteoarthritis (OA) has been widely investigated, but the mechanisms are still unclear. Exosomes that serve as carriers of genetic information have been implicated in many diseases and are known to participate in many physiological processes. Here, we investigate the therapeutic potential of exosomes from human embryonic stem cell-induced mesenchymal stem cells (ESC-MSCs) in alleviating osteoarthritis (OA). Methods Exosomes were harvested from conditioned culture media of ESC-MSCs by a sequential centrifugation process. Primary mouse chondrocytes treated with interleukin 1 beta (IL-1β) were used as an in vitro model to evaluate the effects of the conditioned medium with or without exosomes and titrated doses of isolated exosomes for 48 hours, prior to immunocytochemistry or western blot analysis. Destabilization of the medial meniscus (DMM) surgery was performed on the knee joints of C57BL/6 J mice as an OA model. This was followed by intra-articular injection of either ESC-MSCs or their exosomes. Cartilage destruction and matrix degradation were evaluated with histological staining and OARSI scores at the post-surgery 8 weeks. Results We found that intra-articular injection of ESC-MSCs alleviated cartilage destruction and matrix degradation in the DMM model. Further in vitro studies illustrated that this effect was exerted through ESC-MSC-derived exosomes. These exosomes maintained the chondrocyte phenotype by increasing collagen type II synthesis and decreasing ADAMTS5 expression in the presence of IL-1β. Immunocytochemistry revealed colocalization of the exosomes and collagen type II-positive chondrocytes. Subsequent intra-articular injection of exosomes derived from ESC-MSCs successfully impeded cartilage destruction in the DMM model. Conclusions The exosomes from ESC-MSCs exert a beneficial therapeutic effect on OA by balancing the synthesis and degradation of chondrocyte extracellular matrix (ECM), which in turn provides a new target for OA drug and drug-delivery system development

Combination Treatment with MEK and AKT Inhibitors Is More Effective than Each Drug Alone in Human Non-Small Cell Lung Cancer In Vitro and In Vivo

AZD6244 and MK2206 are targeted small-molecule drugs that inhibit MEK and AKT respectively. The efficacy of this combination in lung cancer is unknown. Our previous work showed the importance of activated AKT in mediating resistance of non-small cell lung cancer (NSCLC) to AZD6244. Thus we hypothesized that dual inhibition of both downstream MEK and AKT pathways would induce synergistic antitumor activity. In this study, we evaluated the efficacy of AZD6244 and MK2206 individually on a large panel of lung cancer cell lines. Then, we treated 28 human lung cancer cell lines with a combination of AZD6244 and MK2206 at clinically applicable drug molar ratios. The AZD6244-MK2206 combination therapy resulted in a synergistic effect on inhibition of lung cancer cell growth compared to the results of single drug treatment alone. MK2206 enhanced AZD6244-induced Bim overexpression and apoptosis in A549 and H157 cells. When we tested the combination of AZD6244 and MK2206 at ratios of 8∶1, 4∶1, 2∶1, and 1∶8, we found that the synergistic effect of the combination therapy was ratio-dependent. At ratios of 8∶1, 4∶1, and 2∶1, the drug combination consistently demonstrated synergy, whereas decreasing the ratio to 1∶8 resulted in a loss of synergy and produced an additive or antagonistic effect in most cell lines. Furthermore, the AZD6244-MK2206 combination therapy showed synergy in the suppression of A549 and H157 xenograft tumor growth and increased mean animal survival time. The AZD6244-MK2206 combination therapy resulted in effective inhibition of both p-ERK and p-AKT expression in tumor tissue. In addition, a significant increase of apoptosis was detected in tumor tissue from mice treated with AZD6244-MK2206 compared with that from the single agent treated mice. Our study suggests that the combination of AZD6244 and MK2206 has a significant synergistic effect on tumor growth in vitro and in vivo and leads to increased survival rates in mice bearing highly aggressive human lung tumors

Dendritic cell–targeted lentiviral vector immunization uses pseudotransduction and DNA-mediated STING and cGAS activation

Dendritic cell (DC) activation and antigen presentation are critical for efficient priming of T cell responses. Here, we study how lentiviral vectors (LVs) deliver antigen and activate DCs to generate T cell immunization in vivo. We report that antigenic proteins delivered in vector particles via pseudotransduction were sufficient to stimulate an antigen-specific immune response. The delivery of the viral genome encoding the antigen increased the magnitude of this response in vivo but was irrelevant in vitro. Activation of DCs by LVs was independent of MyD88, TRIF, and MAVS, ruling out an involvement of Toll-like receptor or RIG-I–like receptor signaling. Cellular DNA packaged in LV preparations induced DC activation by the host STING (stimulator of interferon genes) and cGAS (cyclic guanosine monophosphate–adenosine monophosphate synthase) pathway. Envelope-mediated viral fusion also activated DCs in a phosphoinositide 3-kinase–dependent but STING-independent process. Pseudotransduction, transduction, viral fusion, and delivery of cellular DNA collaborate to make the DC-targeted LV preparation an effective immunogen

The prognostic value of whole-genome DNA methylation in response to Leflunomide in patients with Rheumatoid Arthritis

ObjectiveAlthough Leflunomide (LEF) is effective in treating rheumatoid arthritis (RA), there are still a considerable number of patients who respond poorly to LEF treatment. Till date, few LEF efficacy-predicting biomarkers have been identified. Herein, we explored and developed a DNA methylation-based predictive model for LEF-treated RA patient prognosis.MethodsTwo hundred forty-five RA patients were prospectively enrolled from four participating study centers. A whole-genome DNA methylation profiling was conducted to identify LEF-related response signatures via comparison of 40 samples using Illumina 850k methylation arrays. Furthermore, differentially methylated positions (DMPs) were validated in the 245 RA patients using a targeted bisulfite sequencing assay. Lastly, prognostic models were developed, which included clinical characteristics and DMPs scores, for the prediction of LEF treatment response using machine learning algorithms.ResultsWe recognized a seven-DMP signature consisting of cg17330251, cg19814518, cg20124410, cg21109666, cg22572476, cg23403192, and cg24432675, which was effective in predicting RA patient’s LEF response status. In the five machine learning algorithms, the support vector machine (SVM) algorithm provided the best predictive model, with the largest discriminative ability, accuracy, and stability. Lastly, the AUC of the complex model(the 7-DMP scores with the lymphocyte and the diagnostic age) was higher than the simple model (the seven-DMP signature, AUC:0.74 vs 0.73 in the test set).ConclusionIn conclusion, we constructed a prognostic model integrating a 7-DMP scores with the clinical patient profile to predict responses to LEF treatment. Our model will be able to effectively guide clinicians in determining whether a patient is LEF treatment sensitive or not