Biomedical subjects

Dong Yu

Publications and source records attributed to Dong Yu.

At least 37 records · Page 2Linked to original sources

PubMed2004

Immunopharmacological and antitumor effects of second-generation immunomodulatory oligonucleotides containing synthetic CpR motifs.

Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts (CpG DNA) activate the vertebrate immune system and produce potent Th1 immune responses. Recently, we reported immunomodulatory oligonucleotides (IMOs) containing 3'-3'-attached novel structures (immunomers) and synthetic immunostimulatory CpR (R=2'-deoxy-7-deazguanosine) dinucleotides show potent stimulatory activity with distinct cytokine secretion profiles. In the present study, we evaluated in vivo immunopharmacological and antitumor properties of second-generation immunomodulatory oligonucleotides (IMOs) either alone or in combination with chemotherapeutic agents. Repeated peritumoral administration of IMOs at 1 mg/kg to mice bearing established subcutaneous CT26 colon tumor or B16.F0 melanoma resulted in complete regression or strong inhibition of tumor growth. Direct peritoneal injection of IMOs at 2.5 mg/kg to mice bearing peritoneally implanted ascites CT26 or B16.F0 tumors completely eradicated or inhibited tumor growth. Treatment of mice bearing beta-gal expressing CT26.CL25 tumor with IMOs resulted in a significant tumor-specific CTL responses compared with treatment with a control non-CpG DNA or PBS. These responses correlated with IFN-gamma, but not IL-4 secreted in IMO, treated mice. A 5-fold increase in beta-gal specific IgG2a antibodies was found in mice, significantly increasing the IgG2a/IgG1 ratio. IMOs showed similar antitumor activity in both wt and IL-6 knockout (ko) C57BL/6 mice but failed to elicit activity in IL-12 ko C57BL/6 mice. Tumor-free mice from the IMO treatment group rejected the same tumor cell rechallenge, suggesting an adaptive immune response against these cells. Moreover, naïve mice quickly developed specific antitumor response without IMO treatment following adoptive transfer of splenocytes obtained from tumor free mice from the IMO treated group. Additionally, the co-administration of IMOs with chemotherapeutic agents, docetaxel and doxorubicin, resulted in synergistic antitumor effects in both B16.F0 melanoma and 4T1 breast carcinoma models. These results demonstrate potent antitumor activity of second-generation IMO compounds containing a synthetic CpR stimulatory motif in a broad spectrum of tumor models through induction of strong Th1 immune responses. IMO treatment resulted in the development of tumor-specific memory immune responses. No treatment-related toxicity was observed in mice at the doses and treatment schedules studied.

Adoptive Transfer↗

PubMed2004

[Effect of selenium on liver type I deiodinase in iodine-excess mice].

OBJECTIVE: To investigate the effect of selenium on the activities of liver type I deiodinase (IDI) in iodine-excess mice. METHODS: Forty female weanling Balb/c mice of 18-22 body weight were randomly divided into 5 groups: normal control group (N, deionized water), iodine-excess group I (IE I, 3 mg/L Iodine water), IE I + Se group (+ 1 mg/L Se water), iodine-excess group II (IE II, 5 mg/L Iodine water) and IE II + Se group (+ 1 mg/L Se water), and they were fed with standard diets and different level of iodine and selenium water for 12 weeks. RESULTS: Iodine-excess groups show diffused colloid goiter; Serum TT4 of two iodine-excess groups were significantly higher than Normal control, and TT3 of IE II decreased significantly; The selenium level and IDI activity of mice liver was decreased significantly in iodine-excess group, but no difference between IE I and IE II. CONCLUSION: Iodine-excess intake can decrease the selenium level and IDI activity of mice liver, which maybe is the reason to increase the serum TT4 level and lead to goiter, and selenium can alleviate that.

Animals↗

PubMed2003

Coding potential of laboratory and clinical strains of human cytomegalovirus.

Six strains of human cytomegalovirus have been sequenced, including two laboratory strains (AD169 and Towne) that have been extensively passaged in fibroblasts and four clinical isolates that have been passaged to a limited extent in the laboratory (Toledo, FIX, PH, and TR). All of the sequenced viral genomes have been cloned as infectious bacterial artificial chromosomes. A total of 252 ORFs with the potential to encode proteins have been identified that are conserved in all four clinical isolates of the virus. Multiple sequence alignments revealed substantial variation in the amino acid sequences encoded by many of the conserved ORFs.

Chromosomes, Artificial, Bacterial↗

PubMed2003

A dinucleotide motif in oligonucleotides shows potent immunomodulatory activity and overrides species-specific recognition observed with CpG motif.

Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system through Toll-like receptor 9 (TLR9). In the present study, we used a synthetic nucleoside with a bicyclic heterobase [1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; R] to replace the C in CpG, resulting in an RpG dinucleotide. The RpG dinucleotide was incorporated in mouse- and human-specific motifs in oligodeoxynucleotides (oligos) and 3'-3-linked oligos, referred to as immunomers. Oligos containing the RpG motif induced cytokine secretion in mouse spleen-cell cultures. Immunomers containing RpG dinucleotides showed activity in transfected-HEK293 cells stably expressing mouse TLR9, suggesting direct involvement of TLR9 in the recognition of RpG motif. In J774 macrophages, RpG motifs activated NF-kappa B and mitogen-activated protein kinase pathways. Immunomers containing the RpG dinucleotide induced high levels of IL-12 and IFN-gamma, but lower IL-6 in time- and concentration-dependent fashion in mouse spleen-cell cultures costimulated with IL-2. Importantly, immunomers containing GTRGTT and GARGTT motifs were recognized to a similar extent by both mouse and human immune systems. Additionally, both mouse- and human-specific RpG immunomers potently stimulated proliferation of peripheral blood mononuclear cells obtained from diverse vertebrate species, including monkey, pig, horse, sheep, goat, rat, and chicken. An immunomer containing GTRGTT motif prevented conalbumin-induced and ragweed allergen-induced allergic inflammation in mice. We show that a synthetic bicyclic nucleotide is recognized in the C position of a CpG dinucleotide by immune cells from diverse vertebrate species without bias for flanking sequences, suggesting a divergent nucleotide motif recognition pattern of TLR9.

Adjuvants, Immunologic↗

PubMed2003

Roles of the insulin-like growth factor I receptor C-terminus in cellular radioresistance.

Available evidence suggests that insulin-like growth factor I receptor (IGF-IR) expression leads to increased cellular radioresistance. The most direct explanation of these findings predicts that IGF-IR is the source of survival signals in resistant cells. Mutational analysis revealed that protein truncated at amino acid 1245 in the C-terminus retained the ability of IGF-IR to confer radioresistance whereas point mutations at both Tyr-1250 and Tyr-1251 abrogated this effect using IGF-IR-deficient mouse embryo fibroblasts (R-) as a recipient. In cells expressing the latter mutant receptors, both phosphatidylinositol-3(') kinase (PI3-K) and mitogen-activated protein kinase (MAPK) signaling pathways remained intact, and addition of exogenous IGF-I could not change the radiosensitivity of these cells. Further analysis indicated that the abrogation of radioresistance required the presence of His-1293 and Lys-1294. These results suggest a novel regulatory role of the C-terminus of IGF-IR in mediating cellular radioresistance that may be independent of survival signals transmitted through this receptor.

Amino Acid Sequence↗

PubMed2003

Self-stabilized CpG DNAs optimally activate human B cells and plasmacytoid dendritic cells.

We recently showed that 5'-terminal secondary structures in CpG DNA affect activity significantly more than those at the 3'-end [Biochem. Biophys. Res. Commun. 306 (2003) 948]. The need for an accessible 5'-end of CpG DNA for activity suggested that the receptor reads the DNA sequence from this end. In continuation of these studies, we have designed immunomodulatory oligonucleotides (IMOs), consisting of a nine-mer stimulatory domain, containing a CpG motif and a hairpin-loop structure at the 3'-end, referred to as self-stabilized CpG DNAs. We studied the ability of self-stabilized CpG DNAs to stimulate human B-cell proliferation and interferon-alpha (IFN-alpha) secretion in plasmacytoid dendritic cell (pDC) culture assays. Self-stabilized CpG DNAs activated human B cells and induced plasmacytoid dendritic cells to secrete high levels of IFN-alpha. While both stimulatory and secondary structures in CpG DNAs were required for pDC activation, CpG motifs were sufficient to activate B cells. Interestingly, CpG motifs were not required for activity in the hairpin duplex region. Further modifications of the hairpin duplex region with a mixture of oligodeoxynucleotides and oligo-2'-O-methylribonucleotides in a heteroduplex formation permitted activation of both human B cells and pDCs.

B-Lymphocytes↗

PubMed2003

Functional map of human cytomegalovirus AD169 defined by global mutational analysis.

Human cytomegalovirus has a complex double-stranded DNA genome of approximately 240,000 bp that contains approximately 150 ORFs likely to encode proteins, most of whose functions are not well understood. We have used an infectious bacterial artificial chromosome to introduce 413 defined insertion and substitution mutations into the human cytomegalovirus AD169 genome by random and site-directed transposon mutagenesis. Mutations were produced in all unique ORFs with a high probability of encoding proteins for which mutants have not been previously documented and in many previously characterized ORFs. The growth of selected mutants was assayed in cultured human fibroblasts, and we now recognize 41 essential, 88 nonessential, and 27 augmenting ORFs. Most essential and augmenting genes are located in the central region, and nonessential genes generally cluster near the ends of the viral genome.

Cells, Cultured↗

PubMed2003

Secondary structures in CpG oligonucleotides affect immunostimulatory activity.

Oligodeoxynucleotides containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system. Our previous studies showed that the 5(')-end of a CpG oligonucleotide should be accessible for receptor recognition and subsequent immune stimulation. Activity is abrogated if this end is blocked by joining two CpG oligos through 5(')-5(') linkage. It was not known whether a similar effect would arise from secondary structures at either end of a CpG oligo, such as hairpin loops or terminal dimers. In the present study we found that 5(')-terminal secondary structures affect activity significantly more than those at the 3(')-end. The need for an open 5(')-end suggests that the receptor responsible for immune stimulation reads the DNA sequence from this end. These results may also provide insights to place CpG motifs appropriately in DNA vaccines to induce additional Th1 type responses.

Animals↗

PubMed2003

n-Type conducting CdSe nanocrystal solids.

A bottleneck limiting the widespread application of semiconductor nanocrystal solids is their poor conductivity. We report that the conductivity of thin films of n-type CdSe nanocrystals increases by many orders of magnitude as the occupation of the first two electronic shells, 1Se and 1Pe, increases, either by potassium or electrochemical doping. Around half-filling of the 1Se shell, a peak in the conductivity is observed, indicating shell-to-shell transport. Introducing conjugated ligands between nanocrystals increases the conductivities of these states to approximately 10(-2) siemens per centimeter.

Journal Article↗

PubMed2003

Divergent synthetic nucleotide motif recognition pattern: design and development of potent immunomodulatory oligodeoxyribonucleotide agents with distinct cytokine induction profiles.

Unmethylated CpG dinucleotides present within certain specific sequence contexts in bacterial and synthetic DNA stimulate innate immune responses and induce cytokine secretion. Recently, we showed that CpG DNAs containing two 5'-ends, immunomers, are more potent in both regards. In this study, we show that an immunomer containing a synthetic CpR motif (R = 2'-deoxy-7-deazaguanosine) is a potent immunostimulatory agent. However, the profile of cytokine induction is different from that with immunomers containing a natural CpG motif. In general, a CpR immunomer induced higher interleukin (IL)-12 and lower IL-6 secretion. Compared with conventional CpG DNAs, both types of immunomers showed a rapid and enhanced activation of the transcription factor NF-kappaB in J774 cells. NF-kappaB activation by CpG DNA corresponded to degradation of IkappaBalpha in J774 cells. All three immunostimulatory oligonucleotides activated the p38 mitogen-activated protein kinase pathway as expected. Immunomers containing CpG and CpR motifs showed potent reversal of the antigen-induced Th2 immune response towards a Th1 type in antigen-sensitized mouse spleen cell cultures. Immunomers containing a CpR motif showed significant antitumor activity in nude mice bearing MCF-7 human breast cancer and U87MG glioblastoma xenografts. These studies suggest the ability for a divergent synthetic nucleotide motif recognition pattern of the receptor involved in the immunostimulatory pathway and the possibility of using synthetic nucleotides to elicit different cytokine response patterns.

Animals↗

PubMed2003

Experimental therapy of human prostate cancer by inhibiting MDM2 expression with novel mixed-backbone antisense oligonucleotides: in vitro and in vivo activities and mechanisms.

BACKGROUND: MDM2 oncogene is overexpressed in many human cancers including prostate cancer and MDM2 levels are associated with poor prognosis. This study was undertaken to investigate the functions of MDM2 oncogene in prostate cancer growth and the value of MDM2 as a drug target for prostate cancer therapy by inhibiting MDM2 expression. METHODS: Antisense anti-human-MDM2 mixed-backbone oligonucleotide and its mismatch control were tested in in vitro and in vivo human prostate cancer models (LNCaP, DU 145, and PC-3) for anti-tumor activity. Targeted gene products and related proteins were analyzed and the anti-tumor activity was determined when the oligonucleotides were used alone or in combination with cancer therapeutics. RESULTS: The antisense oligonucleotide specifically inhibited MDM2 expression in a dose- and time-dependent manner, resulting in significant anti-tumor activity in vitro and in vivo. In LNCaP cells, p53 and p21 levels were elevated. The antisense oligonucleotide also potentiated the effects of p53 activation and p21 induction by chemotherapeutic agents 10-hydroxycamptothecin, adriamycin, 5-fluorouracil, and paclitaxel. In DU145 cells, following inhibition of MDM2 expression, p21 levels were elevated although p53 levels remained unchanged. In both cell lines, the antisense oligonucleotide inhibited tumor cell growth and induced apoptosis in vitro. In a dose-dependent manner, the antisense oligonucleotide showed anti-tumor activity in nude mice bearing DU145 or PC-3 xenografts. It significantly increased therapeutic effectiveness of the chemotherapeutic agent irinotecan and slightly improved the effects of paclitaxel and Rituxan. CONCLUSIONS: These results indicate that MDM2 has a role in prostate tumor growth through both p53-dependent and p53-independent mechanisms, indicating that MDM2 inhibitors have a broad spectrum of anti-tumor activities in human prostate cancers regardless of p53 status.

Animals↗

PubMed2003

Requirement of nucleobase proximal to CpG dinucleotide for immunostimulatory activity of synthetic CpG DNA.

Synthetic oligodeoxyribonucleotides containing CpG dinucleotides exhibit potent immunostimulatory activity in vertebrates. Although the molecular mechanisms of recognition and interaction of CpG DNA sequences with receptors are not well understood, the current evidence suggests that the receptor shows considerable selectivity for CpG DNA sequences with different preferences in mouse (GACGTT) and human (GTCGTT) species. In our continued effort to understand the chemical and structural characteristics of CpG DNA required for the immunostimulatory activity and thereby for the recognition of receptors in the immunostimulatory pathway, we examined the requirement of nucleobases in the two adjacent nucleotide positions on the 5'- and the 3'-side to the CpG dinucleotide (P(1)P(2)CGP(3)P(4)) for the immunostimulatory activity. These studies, in which a natural nucleoside is substituted with an abasic nucleoside (X), suggest that a nucleobase is absolutely required in C, G, P(3), and P(4) positions for immunostimulatory activity. Surprisingly, an abasic nucleoside is permitted at either P(1) or P(2) depending on the neighboring base. It was found that 'GXCGTT' motif has an intermediate immunostimulatory activity between those of 'GACGTT' and 'GTCGTT' in the mouse cells.

3' Flanking Region↗

PubMed2003

CpG penta- and hexadeoxyribonucleotides as potent immunomodulatory agents.

We demonstrate a new design for immunomodulatory CpG DNA containing two sequences each with as few as five or six-nucleotides joined together via 3(')-3(') linkers. These do not require the -PuPu(Py)CGPyPy- hexameric motif generally found essential for CpG DNA immune stimulation. These novel, short-immunomers show potent immunostimulatory activity manifested by IL-12 and IL-6 secretion in murine spleen cell and PBMC cultures and splenomegaly in vivo. Short-immunomers show strong activation of NF-kappaB and stress-activated signaling pathways and induce cytokines in J774 cell cultures. The same sequences also induce cytokines in healthy human PBMC cultures whereas conventional CpG DNA requires different optimal sequences for murine and human immune cells. Additionally, short-immunomers inhibit IL-5 secretion and induce IFN-gamma secretion in conalbumin-sensitized mouse spleen cell cultures, suggesting reversal of established Th2 responses to Th1 type responses. Short-immunomer also inhibits growth of MCF-7 human tumor xenograft in nude mice. This is the first report of activity with such short DNA sequences and also of sequences lacking hexameric motifs proposed in earlier studies.

Adjuvants, Immunologic↗

PubMed2003

Point mutations in exon I of the herpes simplex virus putative terminase subunit, UL15, indicate that the most conserved residues are essential for cleavage and packaging.

The herpes simplex virus UL15 and UL28 genes are believed to encode two subunits of the terminase involved in cleavage and packaging of viral genomes. Analysis of the UL15 protein sequence and its herpesvirus homologues revealed the presence of 20 conserved regions. Twelve of the twenty regions conserved among herpesviruses are also conserved in terminases from DNA bacteriophage. Point mutations in UL15 were designed in four conserved regions: L120N (CR1), Q205E (CR2), Q251E (CR3), G263A (CR3), and Y285S (CR4). Transfection experiments indicated that each mutant gene could produce stable UL15 protein at wild-type levels; however, only one mutant (Q251E) was able to complement the UL15-null virus. Each mutation was introduced into the viral genome by marker transfer, and all mutants except Q251E were unable to form plaques on Vero cells. Furthermore, failure to form plaques on Vero cells correlated with a defect in cleavage and packaging. Immunofluorescence experiments indicated that in cells infected with all mutant viruses the UL15 protein could be detected and was found to localize to replication compartments. Although wild-type and mutant Q251E were able to produce A, B, and C capsids, the rest of the mutants were only able to produce B capsids, a finding consistent with their defects in cleavage and packaging. In addition, all mutant UL15 proteins retained their ability to interact with B capsids. Therefore, amino acid residues 120, 205, 263, and 285 are essential for the cleavage and packaging process rather than for association with capsids or localization to replication compartments.

Amino Acid Sequence↗

PubMed2002

Redundancy of radioresistant signaling pathways originating from insulin-like growth factor I receptor.

The insulin-like growth factor I receptor (IGF-IR) has the ability to confer clonogenic radioresistance following ionizing irradiation. We attempted to determine the downstream pathways involved in IGF-IR-mediated radioresistance and used mouse embryo fibroblasts deficient in endogenous IGF-IR (R-) as recipients for a number of mutant IGF-IRs. Mutational analysis revealed that the tyrosine at residue 950 (Tyr-950) of IGF-IR, as well as the C-terminal domain, are required for radioresistance and that both domains must be mutated to abrogate the phenotype. Furthermore, the contribution of downstream pathways was analyzed by combining the use of wild-type or Tyr-950 and C-terminal mutants with specific inhibitors of phosphatidylinositol 3'-kinase (PI3-K) or mitogen-activated protein extracellular signal-regulated kinase (ERK) kinase (MEK). Radioresistance could be induced by IGF-IR as long as the ability of the receptor to stimulate the MEK/ERK pathway was retained. This was confirmed by the expression of constitutively active MEK in R- cells. The ability to stimulate the PI3-K pathway alone was not sufficient, but PI3-K activation coupled with MEK/ERK pathway-independent signals from the C terminus was able to induce radioresistance. Taken together, these results indicate that the IGF-IR-mediated radioresistant signaling mechanism progresses through redundant downstream pathways.

Animals↗

PubMed2002

'Immunomers'--novel 3'-3'-linked CpG oligodeoxyribonucleotides as potent immunomodulatory agents.

Oligodeoxyribonucleotides containing CpG dinucleotides (CpG DNAs) are currently being evaluated as novel immunomodulators in clinical trials. Recently, we showed that an accessible 5' end is required for immunostimulatory activity and blocking the 5' end of CpG DNA by conjugation of certain ligands abrogates immunostimulatory activity. Based on these results, we designed and synthesized 3'-3'-linked CpG DNAs that contained two or more identical CpG DNA segments, referred to here as 'immunomers'. The use of solid support bearing diDMT-glyceryl-linker permitted convenient synthesis of immunomers with both segments synthesized simultaneously, giving better yields and purity. The in vitro and in vivo studies suggest that as a result of accessibility to two 5' ends for recognition, immunomers show an enhanced immunostimulatory activity compared with linear CpG DNAs. We also studied the suitability of a number of different linkers for attaching the two segments of immunomers. A C3-linker was found to be optimal for joining the two segments of immunomers. Incorporation of multiple linkers between the two segments of immunomers resulted in different cytokine profiles depending on the nature and number of linkers incorporated. Additionally, the length of immunomer also plays a significant role in inducing immune responses. An immunomer containing 11 nt in each segment showed the highest activity and an 11mer linear CpG DNA failed to stimulate an immune response. These results suggest that immunomers have several advantages over conventional linear CpG DNAs for immunomodulatory activity studies.

Adjuvants, Immunologic↗

PubMed2002

Design, synthesis, and immunostimulatory properties of CpG DNAs containing alkyl-linker substitutions: role of nucleosides in the flanking sequences.

Bacterial and synthetic DNA containing unmethylated CpG dinucleotides activate the innate immune system and promote Th1-like immune responses. Recently, a receptor, TLR9, has been shown to recognize CpG DNA and activate immune cascade. But there have been no reports on the molecular mechanisms of recognition between CpG DNA and the receptor(s). Our earlier studies described a number of the chemical and structural characteristics of CpG dinucleotide and the sequences flanking the CpG dinucleotide that are critical for immunostimulatory activity. In the present study, we examined the effect of the presence and absence of a nucleoside in the flanking sequences by replacing one or two natural deoxyribonucleosides at various positions with one or more alkyl- (C2-C12), branched alkyl- (glyceryl or aminobutyryl-propanediol), or ethyleneglycol- (tri or hexa) linkers. The results suggest that a linker substitution at the first two nucleoside positions adjacent to the CpG dinucleotide on the 5'- or the 3'-side neutralizes the immunostimulatory activity, as determined by in vitro mouse spleen cell proliferation, cytokine secretion, and in vivo mouse spleen enlargement. The same substitutions placed about three to six nucleotides away from the CpG dinucleotide either did not affect or potentiated immunostimulatory activity compared with parent CpG-DNA without modifications. Substitution of deoxyribonucleosides with a C3 or C4 alkyl-linker was found to be optimal for potentiating immunostimulatory activity.

Adjuvants, Immunologic↗

PubMed2002

Potent CpG oligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties.

Bacterial and synthetic DNAs, containing CpG dinucleotides in specific sequence contexts, activate the vertebrate immune system. Unlike phosphorothioate (PS) CpG DNAs, phosphodiester (PO) CpG DNAs require either palindromic sequences and/or poly(dG) sequences at the 3(')-end for activity. Here, we report 'PO-immunomers' having two PO-CpG DNA molecules joined through their 3(')-ends. These PO-imunomers permitted us, for the first time, to assess immunostimulatory properties of PO-CpG DNAs in vitro and in vivo without the need for palindromic and/or poly(dG) sequences. In medium containing 10% fetal bovine serum, PO-immunomers were more resistant than PO-CpG DNAs to nucleases. Compared to PS-CpG DNA in BALB/c and C3H/HeJ mice spleen cell culture assays, PO-immunomers showed increased IL-12 secretion and minimal amounts of IL-6 secretion. PO-immunomers activated NF-kappa B and induced cytokine secretion in J774 cell cultures. In addition, PO-immunomers showed antitumor activity in nude mice bearing human breast (MCF-7) and prostate (DU145) cancer xenografts.

Adjuvants, Immunologic↗