Biomedical subjects

Zhiren Zhang

Publications and source records attributed to Zhiren Zhang.

11 recordsLinked to original sources

PubMed2006

Polyguanosine motif increases cellular oligonucleotide uptake in the brain cortex.

Cellular uptake and localization of fluorescein isothiocyanate-labelled or biotin-labelled CpG-oligonucleotides (oligonucleotides containing unmethylated CpG dinucleotides within specific flanking bases) with or without 3'-end continuous guanosines (polyG motif) were studied in the rat cortex. Twenty minutes after intracerebral microinjection, labelled CpG-oligonucleotide accumulated in both nuclear and cytoplasmic compartments of neuronal and nonneuronal cells localized near the injection site. In the brain, polyG motif significantly increased the cellular uptake of phosphodiester CpG-oligonucleotide (P < 0.05) but not phosphothioate CpG-oligonucleotide (P > 0.05). These data not only provide in-vivo evidence for the local uptake and distribution of oligonucleotides in the brain cortex but also indicate that phosphodiester CpG-oligonucleotides containing a 3'-end polyG motif show an increased cellular uptake and thus might be promising alternate analogues for application to the brain.

Amino Acid Motifs↗

PubMed2006

siRNA binding proteins of microglial cells: PKR is an unanticipated ligand.

Small interfering RNA (siRNA), double-stranded RNA (dsRNA) 21-23 nucleotides (nt) long with two nt 3' overhangs, has been shown to mediate powerful sequence-specific gene silence in mammalian cells through RNA interference (RNAi). Due to its high efficiency and high specificity siRNA has been used as a powerful post genomic tool and a potent therapeutic candidate. However, there is still a lot to learn about the mobility of siRNA inside cells and the cellular factors that might interfere with the specificity and activity of siRNA. Microglia are the brain's effector cells of the innate immune system and suitable targets in the development of novel therapeutic strategies. Here, we show the cellular uptake and intracellular distribution of siRNA in murine microglial N9 cells. siRNA was internalized by microglial N9 cells without transfection reagent and mainly localized to the endosomes However, no significant gene silencing effects were observed. Its cellular uptake and cellular distribution pattern were similar with that of a same length single stranded DNA (ssDNA). Further, cellular binding proteins of siRNA were purified and identified by mass spectrometry. Negative control siRNA and siRNA targeted to beta-actin were used in this part of experiment. Most of the siRNA binding proteins for negative control siRNA and siRNA targeted to beta-actin were dsRNA-binding proteins, such as dsRNA-dependent protein kinase R (PKR). Furthermore, both control siRNA and siRNA targeted to beta-actin activated PKR in N9 cells, which suggest that siRNA might cause off-target effects through activation of PKR.

Animals↗

PubMed2005

Lesional accumulation of P2X4 receptor+ monocytes following experimental traumatic brain injury.

P2X4 receptor (P2X4R) is an ATP-gated ion channel. ATP is an important messenger in traumatic brain injury. Here, we report expression of P2X4R in rat traumatic brain injury with focus on the early phase, most amenable to therapy. Accumulation of P2X4R+ cells was observed as early as 6 h after injury and continued to increase 4 days post-injury at the lesion and remote areas. Double staining revealed that most P2X4R+ cells co-expressed ED-1, a marker for reactive microglia/macrophages, but not nestin or W3/13. Our data suggest that P2X4R expression defines a subtype of activated microglia/macrophages involved in the early processes following traumatic brain injury.

Animals↗

PubMed2005

Spinal cord glia activation following peripheral polyinosine-polycytidylic acid administration.

Effects of repeated intraperitoneal injections of polyinosine-polycytidylic acid on spinal cord cells were analysed. After each injection, the number of ED-1 microglia significantly increased in rat spinal cords. Expression of endothelial monocyte-activating polypeptide II, however, was not observed. The morphology of microglia indicated an incomplete activation state even after three repeated polyinosine-polycytidylic acid injections. Astrocyte activation was observed after the first injection using glial fibrillary acidic protein staining. Simultaneously with glia activation, hyperalgesia was observed, but the expression of P2X4 receptor, which is considered to be closely associated with hyperalgesia, on microglia was not detected. In sum, our data suggest that repeated peripheral injections of polyinosine-polycytidylic acid might alert the central nervous system through limited activation of microglia and astrocytes.

Animals↗

PubMed2005

Uptake, intracellular distribution, and novel binding proteins of immunostimulatory CpG oligodeoxynucleotides in microglial cells.

Microglial cells are central components of the innate immune system of the brain and contribute to inflammatory and degenerative processes. DNA with unmethylated CpG dinucleotides is a potent stimulant of microglial cells. We have analyzed uptake, intracellular distribution, and cellular binding proteins of CpG oligdeoxynucleotides (ODNs) by the microglial cell line N9. The uptake of CpG-ODN is concentration-, time-, and temperature-dependent, but, interestingly, independent of the CpG dinucleotides. After internalisation, CpG-ODN localized to the cytoplasm and showed a typical speckled distribution pattern. We further purified the cellular binding proteins of CpG-ODN and identified several binding proteins by tryptic digestion and mass spectrometry. Most of the CpG-ODN binding proteins are RNA processing enzymes, which are important for RNA splicing, export, and stability. Further, we identified a protein, pigpen, which has not been observed in microglial cells, so far. These proteins apparently bind CpG-ODN with low selectivity, as binding is independent of CpG dinucleotides. Interference of immunostimulatory and therapeutic oligonucleotides with proteins and enzymes of RNA transport and processing has not been described so far and might affect the physiological functions of these proteins and also might influence cellular localization of therapeutic ODN. These findings are helpful in understanding the cellular fate of ODN and the nonsequence-specific effects of ODN and for rational design and evaluation of ODN-based therapeutic strategies.

ADP Ribose Transferases↗

PubMed2005

Uptake, cellular distribution and novel cellular binding proteins of immunostimulatory CpG oligodeoxynucleotides in glioblastoma cells.

Glioblastomas are the most malignant and most frequent brain tumors and exciting targets of gene and immunotherapy. Despite rapid development of experimental therapy little is known about the cellular behaviour of therapeutic oligodeoxynucleotides (ODNs). Here we designed uptake, cellular distribution and cellular binding proteins of immunostimulatory CpG-ODNs in glioblastoma cells by flow cytometry, fluorescence microscopy and mass spectrometry. Our data show that the phosphorothioate (PS) CpG-ODNs uptake in T98G and C6 cells is dose-, time-, temperature-dependent and independent of the CpG dinucleotides. Uptake can be inhibited by sodium azide, polyanions but not by chloroquine. After internalisation FITC labelled CpG-ODNs showed a spotted distribution in cytoplasm. Dozens of cellular binding proteins were identified using mass spectrometry. The binding of ODNs to proteins is dependent on modification and sequence but independent on CpG motif. ODNs bind to cellular proteins that are important for RNA processing and transport. Furthermore, three novel membrane proteins were identified, which might contribute to uptake of ODNs. ODNs binding to these proteins might interfere with the physiological function and thus might cause unwanted effects. Such binding also might influence the uptake efficiency or cellular distribution of therapeutic ODNs.

Adjuvants, Immunologic↗

PubMed2005

The immunostimulatory activity of CpG oligonucleotides on microglial N9 cells is affected by a polyguanosine motif.

Oligonucleotides (ODN) with hexameric motifs containing central unmethylated CpG dinucleotides are immunostimulatory. Also ODN with continuous guanosines (polyG motif) show a wide range of immunological activity. Depending on the position, the chemical property of the ODN backbone and the cell type, polyG motifs have either an enhancing or a suppressing effect on the immunostimulatory activity of the CpG-ODN. Microglial cells are central components of the innate immune system of the brain and are activated by CpG-ODN in vitro and in vivo. Here we present the analysis of the immunomodulatory effects of CpG-ODN carrying a polyG motif on the microglial cell line N9. Our data show that N9 cells express Toll-like receptor 9 (TLR9) and are activated by CpG-ODN, which leads to expression of interleukin-12p40 (IL12p40), tumor necrosis factor-alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS). A 3'-end polyG motif inhibits phosphothioate (PS) CpG-ODN immunostimulatory activity but enhances the immunostimulatory activity of phosphodiester (PE) CpG-ODN. Correspondingly, a 3'-end polyG motif improves the cellular uptake of PE CpG-ODN but does not change their cellular distribution pattern. Furthermore, PE CpG-ODN with a 3'-end polyG motif interact with a much higher number of cellular proteins than PE CpG-ODN. These data indicate that the 3'-end polyG motif could enhance the immunostimulatory activity of PE CpG-ODN in microglial N9 cells through increasing interaction with cellular proteins. Therefore PE CpG-ODN containing a 3'-end polyG motif resulting in increased immunostimulatory activity might be promising alternate analogues for studies in the central nervous system.

Animals↗

PubMed2005

Microglia activation in rat spinal cord by systemic injection of TLR3 and TLR7/8 agonists.

Here we describe activation of microglia in the rat spinal cord by systemic injections of toll-like receptor agonist polyinosine-polycytidylic acid (poly(I:C), a TLR3 ligand) and R848 (a TLR 7/8 ligand). A significant but transient increase of ED-1+ spinal cord microglia was observed 4 days after a single intraperitoneal (i.p.) injection. Immunostainings by different microglial markers, AIF-1, EMAPII, OX6, P2X(4) receptor (P2X4R), indicated that microglia were not fully activated and tracing of cell proliferation by 5-bromo-2 -deoxyuridine revealed that only a small fraction of proliferating cells were microglia (less than 5%). Thus, these stimulators of the innate immune system have, after peripheral administration, clearly effects on the innate immune system of the spinal cord. This should be considered in the design of clinical trials, as both TLR ligands have been used in patients. As injections of TLR ligands can be used to modulate immune activity in the spinal cord, such agents might be tools to modulate local regenerative processes in the spinal cord.

Analysis of Variance↗

PubMed2005

TLR and NOD2 ligands induce cell proliferation in the rat intact spinal cord.

We demonstrate, by 5-bromo-2'-deoxyuridine (BrdU) tracing, the effects of peripheral administration of toll-like receptor (TLR) and NOD2 ligands (stimulators of the innate immune system) on the proliferation of spinal cord cells. Bolus injection of phosphorothioate oligonucleotides containing CpG motifs had no prominent effects on spinal cord neural progenitor cell proliferation, whereas single intraperitoneal injection of polyinosine-polycytidylic acid (Poly I:C, TLR3 ligand), lipopolysaccharide (LPS, TLR4 ligand), R848 (TLR7/8 ligand), or N-acetylmuramyldipeptide (MDP, Nod2 ligand) temporarily increased the number of BrdU(+) cells in the spinal cord. For Poly I:C- or R848-treated groups, the density of BrdU cells reached maximal levels on days 2 to 3 postinjection and then rapidly declined to baseline levels. Only a few of the proliferating cells were of microglial origin, but BrdU(+)/nestin(+) cells were found, suggestive of a proliferation of local progenitor cells. In addition, stimulation of cell proliferation correlated with activation of the innate immune system, that is, microglial cells. Interestingly, activation and cell proliferation was inhibited by corticosteroid dexamethasone but not by indomethacin. These findings suggest an intricate interaction of phylogenetically ancient cellular processes of the innate immune system and regeneration.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMedDate not supplied

Nucleic acid aptamers in human viral disease.

Nucleic acid aptamers are short, single-stranded oligonucleotides or their modified analogues which avidly and specifically interact with targeted ligands through their 3-dimensional structure. Aptamers can be selected out of a large combinatorial oligonucleotide library through an in vitro evolution process termed SELEX. Since 1990, a wide variety of aptamers targeted to ligands ranging from small molecules to complex mixtures have been isolated. Most selected aptamers have shown high specificity to and affinity for their ligands and are potential detection and/or diagnostic reagents. Furthermore, some aptamers specifically inhibit biological functions of targeted proteins, resulting in potent therapeutic candidates in disease models. Some recent advances to increase the stability of aptamers, extend their in vivo circulation time and their in vivo expression have pushed aptamers closer to therapeutic applications. This review presents recent developments in the field of aptamer research and focuses on their applications to human viral diseases, particularly HIV induced diseases.

HIV Infections↗

PubMedDate not supplied

Nanovesicular vaccines: exosomes.

Exosomes are small membrane vesicles derived from late endosome. They are about 30--100 nm in diameter. The secretion of exosomes is a process in which multivesicular bodies fuse with the cell membrane, and all cells that contain multivesicular endocytic compartments could theoretically secrete exosomes. The surprising biological functions of exosomes are only slowly being unveiled, but it is already clear that they serve to remove obsolete membrane proteins and act as messages of inter-cellular communication. Exosomes derived from tumor or antigen-presenting cells have been extensively investigated. They are released into the extracellular environment and fuse with the membranes of neighboring cells, delivering membrane and cytoplasmic proteins from one cell to another. Exosomes carry immunorelevant structures which play important roles in immune response, such as MHC molecules, costimulatory molecules, heat shock proteins, and naive tumor antigens. Therefore they have been suggested as potential vaccines. Consequently, exosomes have shown considerable anti-tumor effect in several studies and are in phase I clinical trials.

Animals↗