Siting Zhao

Generation of CD34+ Cells from CCR5-Disrupted Human Embryonic and Induced Pluripotent Stem Cells

C-C chemokine receptor type 5 (CCR5) is a major co-receptor for the entry of human immunodeficiency virus type-1 (HIV-1) into target cells. Human hematopoietic stem cells (hHSCs) with naturally occurring CCR5 deletions (Δ32) or artificially disrupted CCR5 have shown potential for curing acquired immunodeficiency syndrome (AIDS). However, Δ32 donors are scarce, heterologous bone marrow transplantation is not exempt of risks, and genetic engineering of autologous hHSCs is not trivial. Here, we have disrupted the CCR5 locus of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) using specific zinc finger nucleases (ZFNs) combined with homologous recombination. The modified hESCs and hiPSCs retained pluripotent characteristics and could be differentiated in vitro into CD34(+) cells that formed all types of hematopoietic colonies. Our results suggest the potential of using patient-specific hHSCs derived from ZFN-modified hiPSCs for treating AIDS.

Transition of tumor-associated macrophages from MHC class IIhi to MHC class IIlow mediates tumor progression in mice

BackgroundTumor-associated macrophages (TAMs) are the most abundant immune cells within the tumor stroma and play a crucial role in tumor development. Although clinical investigations indicate that high levels of macrophage (MΦ) infiltration into tumors are associated with a poor prognosis, the exact role played by TAMs during tumor development remains unclear. The present study aimed to investigate dynamic changes in TAM major histocompatibility complex (MHC) class II expression levels and to assess the effects of these changes on tumor progression.ResultsSignificant inhibition of tumor growth in the murine hepatocellular carcinoma Hepa1-6 model was closely associated with partial TAM depletion. Strikingly, two distinct TAM subsets were found to coexist within the tumor microenvironment during Hepa1-6 tumor development. An MHC class IIhi TAM population appeared during the early phase of tumor development and was associated with tumor suppression; however, an MHC class IIlow TAM population became increasingly predominant as the tumor progressed.ConclusionsTumor progression was positively correlated with increasing infiltration of the tumor tissues by MHC class IIlow TAMs. Thus, targeting the transition of MΦ may be a novel strategy for drug development and immunotherapy.

Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs

BackgroundZinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However, the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed.ResultsIn this study, we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN, respectively. However, TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5, a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.ConclusionsTAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology.

Antitumor Effect of Malaria Parasite Infection in a Murine Lewis Lung Cancer Model through Induction of Innate and Adaptive Immunity

Background Lung cancer is the most common malignancy in humans and its high fatality means that no effective treatment is available. Developing new therapeutic strategies for lung cancer is urgently needed. Malaria has been reported to stimulate host immune responses, which are believed to be efficacious for combating some clinical cancers. This study is aimed to provide evidence that malaria parasite infection is therapeutic for lung cancer. Methodology/Principal Findings Antitumor effect of malaria infection was examined in both subcutaneously and intravenously implanted murine Lewis lung cancer (LLC) model. The results showed that malaria infection inhibited LLC growth and metastasis and prolonged the survival of tumor-bearing mice. Histological analysis of tumors from mice infected with malaria revealed that angiogenesis was inhibited, which correlated with increased terminal deoxynucleotidyl transferase-mediated (TUNEL) staining and decreased Ki-67 expression in tumors. Through natural killer (NK) cell cytotoxicity activity, cytokine assays, enzyme-linked immunospot assay, lymphocyte proliferation, and flow cytometry, we demonstrated that malaria infection provided anti-tumor effects by inducing both a potent anti-tumor innate immune response, including the secretion of IFN-γ and TNF-α and the activation of NK cells as well as adaptive anti-tumor immunity with increasing tumor-specific T-cell proliferation and cytolytic activity of CD8+ T cells. Notably, tumor-bearing mice infected with the parasite developed long-lasting and effective tumor-specific immunity. Consequently, we found that malaria parasite infection could enhance the immune response of lung cancer DNA vaccine pcDNA3.1-hMUC1 and the combination produced a synergistic antitumor effect. Conclusions/Significance Malaria infection significantly suppresses LLC growth via induction of innate and adaptive antitumor responses in a mouse model. These data suggest that the malaria parasite may provide a novel strategy or therapeutic vaccine vector for anti-lung cancer immune-based therapy.

A redesigned CRISPR/Cas9 system for marker-free genome editing in Plasmodium falciparum

Background A highly efficient CRISPR/Cas9-based marker-free genome editing system has been established in Plasmodium falciparum (Pf). However, with the current methods, two drug-selectable markers are needed for episome retention, which may present hurdles for consecutive genome manipulations due to the limited number of available selectable markers. The loading capacity of donor DNA is also unsatisfactory due to the large size of the Cas9 nuclease and sgRNA co-expression system, which limits the size of knock-in DNA fragments. Because of the inefficient end joining (EJ) DNA repair mechanism of Pf, a suicide-rescue approach could be used to address the challenges. Cas9 nuclease and sgRNA were co-expressed from a single plasmid (suicide vector) with one selectable marker, and the donor DNA was ligated into the other plasmid (rescue vector) containing only the ampicillin-resistance gene (AmpR) and a ColEl replication origin (ori). Nonetheless, whether this approach can mediate even the regular gene editing in Pf remains unknown. This study aimed to demonstrate the basic gene editing function of this Cas9-mediated suicide-rescue system. Findings The suicide and rescue vectors were constructed and co-transfected into Pf3D7. This system worked as expected when used to disrupt the Pfset2 gene and to insert a green fluorescent protein-renilla luciferase (gfp-ruc) fusion gene cassette of 3334 base pairs (bp) into the Pf47 locus, demonstrating that the suicide vector actually induced double-strand breaks (DSBs) and that the rescue vector functioned without maintenance via drug selection. Conclusions The adapted marker-free CRISPR/Cas9 system with only a single episome-selectable marker performs well as the current systems for general gene editing which lays a solid foundation for further studies including consecutive gene manipulations and large gene knock-ins. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1487-4) contains supplementary material, which is available to authorized users.BackgroundA highly efficient CRISPR/Cas9-based marker-free genome editing system has been established in Plasmodium falciparum (Pf). However, with the current methods, two drug-selectable markers are needed for episome retention, which may present hurdles for consecutive genome manipulations due to the limited number of available selectable markers. The loading capacity of donor DNA is also unsatisfactory due to the large size of the Cas9 nuclease and sgRNA co-expression system, which limits the size of knock-in DNA fragments. Because of the inefficient end joining (EJ) DNA repair mechanism of Pf, a suicide-rescue approach could be used to address the challenges. Cas9 nuclease and sgRNA were co-expressed from a single plasmid (suicide vector) with one selectable marker, and the donor DNA was ligated into the other plasmid (rescue vector) containing only the ampicillin-resistance gene (AmpR) and a ColEl replication origin (ori). Nonetheless, whether this approach can mediate even the regular gene editing in Pf remains unknown. This study aimed to demonstrate the basic gene editing function of this Cas9-mediated suicide-rescue system.FindingsThe suicide and rescue vectors were constructed and co-transfected into Pf3D7. This system worked as expected when used to disrupt the Pfset2 gene and to insert a green fluorescent protein-renilla luciferase (gfp-ruc) fusion gene cassette of 3334 base pairs (bp) into the Pf47 locus, demonstrating that the suicide vector actually induced double-strand breaks (DSBs) and that the rescue vector functioned without maintenance via drug selection.ConclusionsThe adapted marker-free CRISPR/Cas9 system with only a single episome-selectable marker performs well as the current systems for general gene editing which lays a solid foundation for further studies including consecutive gene manipulations and large gene knock-ins.

Dynamic Balance of pSTAT1 and pSTAT3 in C57BL/6 Mice Infected with Lethal Or Nonlethal Plasmodium yoelii

Signal transducer and activator of transcription (STAT) proteins play an important role in cytokine signaling pathways and regulation of immune responses. The balance of the phosphorylated (activated) STAT1 (pSTAT1) and STAT3 (pSTAT3) has been documented in cancer immunology. In this study, we investigated the dynamic balance of pSTAT1 and pSTAT3 in C57BL/6 mice infected with either a nonlethal (Py17XNL) or lethal (Py17XL) strain of Plasmodium yoelii. Both Py17XNL and Py17XL infections induced a maximum activation of STAT1 and STAT3 on the first day after parasite inoculation. Additionally, the Py17XNL infection induced a pSTAT1-dominant response in mice during the early stage of infection, with the resolution of parasitemia. In contrast, Py17XL infection induced a pSTAT3-dominant response during the early phase of infection, with the death of the animals. Our results indicated that maximum activation of STAT1 and STAT3 occurred much earlier than the peak levels of cytokines induced by Plasmodium yoelii infection based on previous reports and that infection with Py17XNL and Py17XL induced different dynamic patterns of pSTAT1 and pSTAT3 balance.

Evaluation of spiropiperidine hydantoins as a novel class of antimalarial agents.

Given the rise of parasite resistance to all currently used antimalarial drugs, the identification of novel chemotypes with unique mechanisms of action is of paramount importance. Since Plasmodium expresses a number of aspartic proteases necessary for its survival, we have mined antimalarial datasets for drug-like aspartic protease inhibitors. This effort led to the identification of spiropiperidine hydantoins, bearing similarity to known inhibitors of the human aspartic protease β-secretase (BACE), as new leads for antimalarial drug discovery. Spiropiperidine hydantoins have a dynamic structure-activity relationship profile with positions identified as being tolerant of a variety of substitution patterns as well as a key piperidine N-benzyl phenol pharmacophore. Lead compounds 4e (CWHM-123) and 12k (CWHM-505) are potent antimalarials with IC50 values against Plasmodium falciparum 3D7 of 0.310 μM and 0.099 μM, respectively, and the former features equivalent potency on the chloroquine-resistant Dd2 strain. Remarkably, these compounds do not inhibit human aspartic proteases BACE, cathepsins D and E, or Plasmodium plasmepsins II and IV despite their similarity to known BACE inhibitors. Although the current leads suffer from poor metabolic stability, they do fit into a drug-like chemical property space and provide a new class of potent antimalarial agents for further study.

Antimalarial Effects of Human Immunodeficiency Virus Protease Inhibitors in Rhesus Macaques

ABSTRACT The antimalarial activity of the human immunodeficiency virus protease inhibitors indinavir and saquinavir was evaluated in rhesus macaques for the first time. Indinavir effectively suppressed the growth of Plasmodium cynomolgi and Plasmodium knowlesi in vivo after a 7- or 3-day treatment, respectively, with clinically relevant doses, whereas saquinavir showed only weak activity against P. cynomolgi.

Exosomes from Plasmodium -infected hosts inhibit tumor angiogenesis in a murine Lewis lung cancer model

Previous research to investigate the interaction between malaria infection and tumor progression has revealed that malaria infection can potentiate host immune response against tumor in tumor-bearing mice. Exosomes may play key roles in disseminating pathogenic host-derived molecules during infection because several studies have shown the involvement and roles of extracellular vesicles in cell–cell communication. However, the role of exosomes generated during Plasmodium infection in tumor growth, progression and angiogenesis has not been studied either in animals or in the clinics. To test this hypothesis, we designed an animal model to generate and isolate exosomes from mice which were subsequently used to treat the tumor. Intra-tumor injection of exosomes derived from the plasma of Plasmodium-infected mice provided significantly reduced Lewis lung cancer growth in mice. We further co-cultured the isolated exosomes with endothelial cells and observed significantly reduced expression of VEGFR2 and migration in the endothelial cells. Interestingly, high level of micro-RNA (miRNA) 16/322/497/17 was detected in the exosomes derived from the plasma of mice infected with Plasmodium compared with those from control mice. We observed that overexpression of the miRNA 16/322/497/17 in endothelial cell corresponded with decreased expression of VEGFR2, inhibition of angiogenesis and inhibition of the miRNA 16/322/497/17 significantly alleviated these effects. These data provide novel scientific evidence of the interaction between Plasmodium infection and lung cancer growth and angiogenesis.

Plasmodium infection reduces the volume of the viral reservoir in SIV-infected rhesus macaques receiving antiretroviral therapy

BackgroundPrevious studies indicated that Plasmodium infection activates the immune system, including memory CD4+ T cells, which constitute the reservoir of human immunodeficiency virus type-1 (HIV-1). Therefore, we postulated that co-infection with malaria might activate the reservoir of HIV-1. To test this hypothesis, we used a rhesus macaque model of co-infection with malaria and simian immunodeficiency virus (SIV), along with antiretroviral therapy (ART).ResultsOur results showed that Plasmodium infection reduced both the replication-competent virus pool in resting CD4+ T cells and the integrated virus DNA (iDNA) load in peripheral blood mononuclear cells in the monkeys. This reduction might be attributable to malaria-mediated activation and apoptotic induction of memory CD4+ T cells. Further studies indicated that histone acetylation and NF-kappaB (NF-κB) activation in resting CD4+ T cells may also play an important role in this reduction.ConclusionsThe findings of this work expand our knowledge of the interaction between these two diseases. As more HIV-1-infected individuals in malaria-endemic areas receive ART, we should explore whether any of the patients co-infected with Plasmodium experience virologic benefits.