Biomedical subjects

Bing Chen

Publications and source records attributed to Bing Chen.

At least 19 recordsLinked to original sources

PubMed2007

Activation of demineralized bone matrix by genetically engineered human bone morphogenetic protein-2 with a collagen binding domain derived from von Willebrand factor propolypeptide.

There is a large demand for new bone regeneration to restore the function during bone injuries. Bone filling materials are important in bone tissue restoration. In this study, the demineralized bone matrix (DBM) was activated with the engineering human bone morphogenetic protein-2 (BMP-2). To enhance the binding of BMP-2 to the DBM scaffolds, a collagen-binding peptide was fused to the N-terminal of BMP-2. The in vitro results showed that the engineered collagen-targeted BMP-2 (rhBMP2-v) bound to DBM scaffolds specifically and the rhBMP2-v had increased alkaline phosphatase activity in C2C12 cells. In vivo, the DBM scaffolds impregnated with rhBMP2-v showed greater effect on ectopic bone formation. Our data suggested that the collagen-based BMP-2 targeting bone repair system had greater bone inducing ability than DBM loaded with regular BMP-2.

Animals↗

PubMed2006

Novel nerve guidance material prepared from bovine aponeurosis.

Spinal cord injury (SCI) creates an adverse environment for axon regeneration. As a result, the axons at the injury sites begin to be atrophy, retract and lose their functions. Several strategies to promote axon regeneration at the injury site have been tested, but the progress is very limited. One of the major reasons is that the regenerated axons often extend randomly and do not reach the proper place. Fabricating linearly ordered materials as nerve guidance would be important to solve such problems. In this study, a novel type of nerve guidance material was prepared from the bovine aponeurosis, which mainly consisted of ordered collagen fibers. The processed material showed good cell compatibility and low immunogenisity. Moreover, the processed material guided the neurites outgrowth of in vitro cultured cortical neurons along its fibers. The results suggested that the processed aponeurosis would be a proper nerve guidance biomaterial for SCI repair.

Animals↗

PubMed2006

Homogeneous osteogenesis and bone regeneration by demineralized bone matrix loading with collagen-targeting bone morphogenetic protein-2.

Considerable research has been focused on the development of bone morphogenetic protein-2 (BMP-2) delivery system for homologous and efficient bone regeneration. The aim of the present study was to develop a collagen-based targeting bone repair system. A collagen-binding domain (CBD) was added to the N-terminal of native BMP-2 to allow it bind to collagen specifically. We showed that the collagen-binding bone morphogenetic protein-2 (named bone morphogenetic protein2-h, BMP2-h) had maintained the full biological activity as compared to rhBMP2 lacking the CBD. In vitro functional study also demonstrated that collagen matrix could maintain higher bioactivity of BMP2-h than native BMP-2. When demineralized bone matrix (DBM) impregnated with BMP2-h was implanted subcutaneously in rats, homogeneous bone formation was observed. Moreover, in a rabbit mandible defect model, surgical implantation of collagen matrix loaded with BMP2-h exhibited remarkable osteoinductive properties and excellent homogeneous bone formation. Our studies suggested that this novel collagen-based BMP-2 targeting bone repair system induced better bone formation not only in quantity but also in quality. Similar approaches may also be used for the repair of other tissue injuries.

Animals↗

PubMed2006

Characterization of the protective T-cell response generated in CD4-deficient mice by a live attenuated Mycobacterium tuberculosis vaccine.

The global epidemic of tuberculosis, fuelled by acquired immune-deficiency syndrome, necessitates the development of a safe and effective vaccine. We have constructed a DeltaRD1DeltapanCD mutant of Mycobacterium tuberculosis (mc(2)6030) that undergoes limited replication and is severely attenuated in immunocompromised mice, yet induces significant protection against tuberculosis in wild-type mice and even in mice that completely lack CD4(+) T cells as a result of targeted disruption of their CD4 genes (CD4(-/-) mice). Ex vivo studies of T cells from mc(2)6030-immunized mice showed that these immune cells responded to protein antigens of M. tuberculosis in a major histocompatibility complex (MHC) class II-restricted manner. Antibody depletion experiments showed that antituberculosis protective responses in the lung were not diminished by removal of CD8(+), T-cell receptor gammadelta (TCR-gammadelta(+)) and NK1.1(+) T cells from vaccinated CD4(-/-) mice before challenge, implying that the observed recall and immune effector functions resulting from vaccination of CD4(-/-) mice with mc(2)6030 were attributable to a population of CD4(-) CD8(-) (double-negative) TCR-alphabeta(+), TCR-gammadelta(-), NK1.1(-) T cells. Transfer of highly enriched double-negative TCR-alphabeta(+) T cells from mc(2)6030-immunized CD4(-/-) mice into naive CD4(-/-) mice resulted in significant protection against an aerosol tuberculosis challenge. Enriched pulmonary double-negative T cells transcribed significantly more interferon-gamma and interleukin-2 mRNA than double-negative T cells from naive mice after a tuberculous challenge. These results confirmed previous findings on the potential for a subset of MHC class II-restricted T cells to develop and function without expression of CD4 and suggest novel vaccination strategies to assist in the control of tuberculosis in human immunodeficiency virus-infected humans who have chronic depletion of their CD4(+) T cells.

Adoptive Transfer↗

PubMed2006

Restraining the conformation of HIV-1 gp120 by removing a flexible loop.

The trimeric HIV/SIV envelope glycoprotein, gp160, is cleaved to noncovalently associated fragments, gp120 and gp41. Binding of gp120 to viral receptors leads to large structural rearrangements in both fragments. The unliganded gp120 core has a disordered beta3-beta5 loop, which reconfigures upon CD4 binding into an ordered, extended strand. Molecular modeling suggests that residues in this loop may contact gp41. We show here that deletions in the beta3-beta5 loop of HIV-1 gp120 weaken the binding of CD4 and prevent formation of the epitope for monoclonal antibody (mAb) 17b (which recognizes the coreceptor site). Formation of an encounter complex with CD4 binding and interactions of gp120 with mAbs b12 and 2G12 are not affected by these deletions. Thus, deleting the beta3-beta5 loop blocks the gp120 conformational change and may offer a strategy for design of restrained immunogens. Moreover, mutations in the SIV beta3-beta5 loop lead to greater spontaneous dissociation of gp120 from cell-associated trimers. We suggest that the CD4-induced rearrangement of this loop releases structural constraints on gp41 and thus potentiates its fusion activity.

Antibodies, Monoclonal↗

PubMed2006

Aberrant transcriptional regulation of the MLL fusion partner EEN by AML1-ETO and its implication in leukemogenesis.

The EEN (extra eleven nineteen) gene, located on chromosome 19p13, was cloned as a fusion with MLL from a patient with acute myeloid leukemia (AML) with translocation t(11;19)(q23;p13). In this study, we characterized the genomic structure of the EEN gene, including its 5' regulatory region and transcription start site (TSS). We found that Sp1 could bind to the guanine-cytosine (GC)-stretch of the EEN promoter and was critical for the normal EEN expression, whereas the leukemia-associated fusion protein AML1-ETO could aberrantly transactivate the EEN gene through an AML1 binding site. Of note, overexpressed EEN showed oncogenic properties, such as transforming potential in NIH3T3 cells, stimulating cell proliferation, and increasing the activity of transcriptional factor AP-1. Retroviral transduction of EEN increased self-renewal and proliferation of murine hematopoietic progenitor cells. Moreover, Kasumi-1 and HL60-cell growth was inhibited with down-regulation of EEN by RNAi. These findings demonstrate that EEN might be a common target in 2 major types of AML associated with MLL or AML1 translocations, and overexpression of EEN may play an essential role in leukemogenesis.

Acute Disease↗

PubMed2006

Small molecules that bind the inner core of gp41 and inhibit HIV envelope-mediated fusion.

HIV-1 enters cells by membrane fusion, mediated by the trimeric viral envelope glycoprotein gp160, which is processed by a single proteolytic cleavage into stably associated gp120 and gp41. The gp120/gp41 trimer can be triggered to undergo an irreversible conformational change. Using a protein-based assay designed to mimic the gp41 conformational change, we screened for small molecules that prevent the formation of postfusion gp41. Several compounds were identified. One set of structurally related molecules inhibited formation of a postfusion-like assembly with an IC50 of approximately 5 microM. The compounds also inhibited envelope-mediated membrane fusion in both cell-cell fusion and viral infectivity assays. Thus, our screen identifies effective fusion inhibitors. Tested against a panel of envelope proteins from primary HIV-1 isolates, the compounds inhibited fusion across a broad range of clades, including both M and T tropic strains. They bind in a highly conserved, hydrophobic pocket on the inner core of the gp41 trimer, a region previously identified as a potential inhibitor site.

Amino Acid Sequence↗

PubMed2006

Heat-shock promoters: targets for evolution by P transposable elements in Drosophila.

Transposable elements are potent agents of genomic change during evolution, but require access to chromatin for insertion-and not all genes provide equivalent access. To test whether the regulatory features of heat-shock genes render their proximal promoters especially susceptible to the insertion of transposable elements in nature, we conducted an unbiased screen of the proximal promoters of 18 heat-shock genes in 48 natural populations of Drosophila. More than 200 distinctive transposable elements had inserted into these promoters; greater than 96% are P elements. By contrast, few or no P element insertions segregate in natural populations in a "negative control" set of proximal promoters lacking the distinctive regulatory features of heat-shock genes. P element transpositions into these same genes during laboratory mutagenesis recapitulate these findings. The natural P element insertions cluster in specific sites in the promoters, with up to eight populations exhibiting P element insertions at the same position; laboratory insertions are into similar sites. By contrast, a "positive control" set of promoters resembling heat-shock promoters in regulatory features harbors few P element insertions in nature, but many insertions after experimental transposition in the laboratory. We conclude that the distinctive regulatory features that typify heat-shock genes (in Drosophila) are especially prone to mutagenesis via P elements in nature. Thus in nature, P elements create significant and distinctive variation in heat-shock genes, upon which evolutionary processes may act.

Animals↗

PubMed2006

The effect of collagen-targeting platelet-derived growth factor on cellularization and vascularization of collagen scaffolds.

Platelet-derived growth factor BB (PDGF-BB) was an important inductive factor during wound healing, but the lack of efficient delivery system limited its clinical application. Here, a peptide of seven amino acids was firstly utilized to engineer PDGF-BB to construct a collagen-targeting system. This peptide functioned as collagen-binding domain (CBD) to specially target the PDGF-BB to the collagen and restrict its diffusion. In our study, in vitro activity assay showed that the CBD-fused PDGF (CBD-PDGF) and native PDGF (NAT-PDGF) possessed similar activity to stimulate the human fibroblast proliferation. As expected, this peptide promoted the binding of PDGF to collagen scaffolds, and CBD-PDGF caused more cells to proliferate on the collagen gel than NAT-PDGF when the same amounts of PDGF were added. In the in vivo experiment, CBD-PDGF-loaded collagen scaffolds were uniformly cellularized and vascularized compared to that of NAT-PDGF-loaded scaffolds. Our study demonstrated that the CBD targeted PDGF to the collagen scaffold to exert its functions, and it suggested this could be an effective system for targeting tissue regeneration and wound repair.

Animals↗

PubMed2006

Expression of T-STAR gene is associated with regulation of telomerase activity in human colon cancer cell line HCT-116.

AIM: To investigate the effects on telomerase activity of transfection of human T-STAR gene full-length sense cDNA or partial antisense cDNA into human colon cancer cell line HCT-116. METHODS: mRNA and protein expression levels of T-STAR gene were determined by RT-PCR and western blot, and telomerase activity was measured by PCR-ELISA, after transfection of T-STAR sense or antisense gene into HCT-116 cells with lipofectamine. RESULTS: T-STAR gene expression was enhanced or knocked down both at mRNA and protein levels, and telomerase activity was significantly increased or decreased. CONCLUSION: The T-STAR gene may participate in regulation of telomerase activity in human colon cancer HCT-116 cells in a parallel fashion.

DNA, Antisense↗

PubMed2006

Expression of active hBMP2 in transgenic tobacco plants.

Bone morphogenetic protein 2 (BMP2) is important for bone tissue repair. The goal of this research is to construct a high level human BMP2 (hBMP2) expression system using transgenic tobacco plants as a bioreactor. Cauliflower mosaic virus (CaMV) 35S promoter, alfalfa mosaic virus (AMV) enhancer, tobacco mosaic virus (TMV) enhancer, matrix attachment regions (MARs) sequence, and "Kozak" sequence were used to construct recombinant expression vectors and the high-expression vectors were screened out through GUS-fusions assay. The promoter is the most important factor; double-CaMV 35S promoter is more effective than single promoter. The AMV or TMV enhancer is able to promote the foreign protein expression. After four-step purification, the activated hBMP2 (0.02% total soluble protein) was obtained. Our results suggested that the transgenic tobacco has great potential to be used as a bioreactor to produce hBMP2.

Animals↗

PubMed2006

Using multifocal ERG responses to discriminate diabetic retinopathy.

PURPOSE: Increase the accuracy in classifying subjects with or without early diabetic retinopathy, by analyzing the multifocal electroretinogram (mfERG) responses. METHOD: The mfERG was recorded for 14 control subjects (Normal) and 26 non-insulin dependent diabetic patients, 16 of the patients had no apparent retinopathy (NDR), the other 10 had mild to moderate non-proliferative retinopathy (NPDR). The first order kernel (K1) and the first slice of second order kernel (K21) of the mfERG were summed across the field and exported as trace arrays. The feature subset was selected from the kernel arrays by criterion of inter-intra distance and sequential forward searching strategy. Based on the selected features, Fisher's linear classifiers were trained and the classification error rates were given. RESULT: With one feature selected, the error rates of the classifiers were bigger than 23% between the NDR and NPDR subjects. When six features were selected to train the classifiers, the classification error rate decreased to 6.5% between Normal and NDR, 0 between Normal and NPDR, and 9.6% between NDR and NPDR. The criterion function of inter-intra distance was calculated for each feature in the K1 and K21 arrays. According to rank of the function value, during early DR, deformations should be easier to find at the front limb of peaks N2 and P1 on the K1 trace, and at the front limb of peak N2 on the K21 trace. As a conclusion, the multifocal ERG responses have great potentials in exploring diabetic retinopathy, assisted with the pattern classification methods.

Adult↗

PubMed2006

Mycobacterium tuberculosis DeltaRD1 DeltapanCD: a safe and limited replicating mutant strain that protects immunocompetent and immunocompromised mice against experimental tuberculosis.

The global epidemic of tuberculosis (TB), fueled by the growing HIV pandemic, warrants the development of a safe and effective vaccine against TB. We report the construction and characterization of an unlinked double deletion mutant of Mycobacterium tuberculosis H37Rv that deletes both the primary attenuating mutation of BCG (DeltaRD1) and two genes required for the synthesis of pantothenate (DeltapanCD). The M. tuberculosis DeltaRD1 DeltapanCD (mc(2)6030) mutant undergoes limited replication in mice, and yet is both significantly safer than BCG in immunocompromised mice and also safe in guinea pigs. Additionally, the mc(2)6030 strain does not reactivate in a mouse chemo-immunosuppression model. Importantly, long-lived protective immune responses following immunization with the mc(2)6030 strain prolong the survival of wild type mice, and CD4-deficient mice against an aerosol challenge with virulent M. tuberculosis. Given its overall safety and effectiveness, the mc(2)6030 live attenuated strain should be considered as a human vaccine candidate for protecting both healthy and HIV-infected individuals against TB.

Animals↗

PubMed2006

Expansion of canine bone marrow-derived endothelial progenitor cells and dynamic observation.

We cultured bone marrow-derived endothelial progenitor cells by a simple ex vivo expansion method and observed the expansion efficacy. Bone marrow mononuclear cells from five mongrel adult dogs were cultured with EGM-2MV medium in culture flasks coated with fibronectin. Morphology was observed with phase contrast microscopy, and a growth curve was constructed to evaluate the efficacy of expansion. Incorporation of Dil-ac-LDL was tested to evaluate the function. At different time points, immunocytochemical staining for flk-1, CD133, and factor VIII-related antigens was done and compared to staining of endothelial cells and mesenchymal stem cells and percentages of CD133, vascular endothelial growth factor receptor 2 (VEGFR-2), and the double-positive cells measured with flow cytometry to identify the quality and efficacy of expansion. Cluster-like attached cells grew to confluence at an average time of 10 days, and the mean number of cells harvested from 1 mL of bone marrow was (1.3 +/- 0.3) x 10(6). The cells presented a cobblestone-like appearance and took up Dil-ac-LDL. Immunocytochemistry showed that flk-1, CD133, and factor VIII-related antigens were positive. Flow cytometry showed that VEGFR-2 and CD133 double-positive cells augmented 242-fold at the tenth day. Ex vivo expansion can effectively proliferate endothelial progenitor cells from bone marrow; the expansion efficacy could meet the requirements for tissue engineering of blood vessels.

AC133 Antigen↗

PubMed2006

NOTCH1 mutations in T-cell acute lymphoblastic leukemia: prognostic significance and implication in multifactorial leukemogenesis.

PURPOSE: NOTCH signaling pathway is essential in T-cell development and NOTCH1 mutations are frequently present in T-cell acute lymphoblastic leukemia (T-ALL). To gain insight into its clinical significance, NOTCH1 mutation was investigated in 77 patients with T-ALL. EXPERIMENTAL DESIGN: Detection of NOTCH1 mutation was done using reverse transcription-PCR amplification and direct sequencing, and thereby compared according to the clinical/biological data of the patients. RESULTS: Thirty-two mutations were identified in 29 patients (with dual mutations in 3 cases), involving not only the heterodimerization and proline/glutamic acid/serine/threonine domains as previously reported but also the transcription activation and ankyrin repeat domains revealed for the first time. These mutations were significantly associated with elevated WBC count at diagnosis and independently linked to short survival time. Interestingly, the statistically significant difference of survival according to NOTCH1 mutations was only observed in adult patients (>18 years) but not in pediatric patients (< or = 18 years), possibly due to the relatively good overall response of childhood T-ALL to the current chemotherapy. NOTCH1 mutations could coexist with HOX11, HOX11L2, or SIL-TAL1 expression. The negative effect of NOTCH1 mutation on prognosis was potentiated by HOX11L2 but was attenuated by HOX11. CONCLUSION: NOTCH1 mutation is an important prognostic marker in T-ALL and its predictive value could be even further increased if coevaluated with other T-cell-related regulatory genes. NOTCH pathway thus acts combinatorially with oncogenic transcriptional factors on T-ALL pathogenesis.

Adolescent↗

PubMed2006

Phenotypical analysis of adult rat olfactory ensheathing cells on 3-D collagen scaffolds.

Olfactory ensheathing cell (OEC) transplantation is a promising or potential therapy for spinal cord injury (SCI). However, the effects of injecting OECs directly into SCI site have been limited and unsatisfied due to the complexity of SCI. To improve the outcome, proper biomaterials are thought to be helpful since these materials would allow the cells to grow three-dimensionally and guide cell migration. In this paper, we have studied the behavior of OECs in two-dimensional (2-D) condition as well as on three-dimensional (3-D) collagen scaffolds by analyzing their phenotypes such as cell proliferation, apoptosis, morphology, and gene activities of some neurotrophic factors and myelin proteins. OECs proliferation rate was increased on 3-D collagen scaffolds compared to the 2-D culture condition. OECs on 3-D collagen scaffolds also showed less apoptosis. In addition, OECs on 3-D collagen scaffolds maintained the original spindle-shape morphology and P75NTR gene activity. NGF, BDNF, and PLP were found to be upregulated in OECs cultured on 3-D collagen scaffolds by the semi-quantitative RT-PCR approach. The results suggested that 3-D collagen scaffolds provide suitable environments for the OECs to maintain their morphology as well as several important functional phenotypes and all these could be helpful for the effective treatment of SCI.

Animals↗

PubMed2006

Hexon-chimaeric adenovirus serotype 5 vectors circumvent pre-existing anti-vector immunity.

A common viral immune evasion strategy involves mutating viral surface proteins in order to evade host neutralizing antibodies. Such immune evasion tactics have not previously been intentionally applied to the development of novel viral gene delivery vectors that overcome the critical problem of anti-vector immunity. Recombinant, replication-incompetent adenovirus serotype 5 (rAd5) vector-based vaccines for human immunodeficiency virus type 1 and other pathogens have proved highly immunogenic in preclinical studies but will probably be limited by the high prevalence of pre-existing anti-Ad5 immunity in human populations, particularly in the developing world. Here we show that rAd5 vectors can be engineered to circumvent anti-Ad5 immunity. We constructed novel chimaeric rAd5 vectors in which the seven short hypervariable regions (HVRs) on the surface of the Ad5 hexon protein were replaced with the corresponding HVRs from the rare adenovirus serotype Ad48. These HVR-chimaeric rAd5 vectors were produced at high titres and were stable through serial passages in vitro. HVR-chimaeric rAd5 vectors expressing simian immunodeficiency virus Gag proved comparably immunogenic to parental rAd5 vectors in naive mice and rhesus monkeys. In the presence of high levels of pre-existing anti-Ad5 immunity, the immunogenicity of HVR-chimaeric rAd5 vectors was not detectably suppressed, whereas the immunogenicity of parental rAd5 vectors was abrogated. These data demonstrate that functionally relevant Ad5-specific neutralizing antibodies are focused on epitopes located within the hexon HVRs. Moreover, these studies show that recombinant viral vectors can be engineered to circumvent pre-existing anti-vector immunity by removing key neutralizing epitopes on the surface of viral capsid proteins. Such chimaeric viral vectors may have important practical implications for vaccination and gene therapy.

Adenoviridae↗

PubMed2006

Remarkable site specificity of local transposition into the Hsp70 promoter of Drosophila melanogaster.

Heat-shock genes have numerous features that ought to predispose them to insertional mutagenesis via transposition. To elucidate the evolvability of heat-shock genes via transposition, we have exploited a local transposition technique and Drosophila melanogaster strains with EPgy2 insertions near the Hsp70 gene cluster at 87A7 to produce numerous novel EPgy2 insertions into these Hsp70 genes. More than 50% of 45 independent insertions were made into two adjacent nucleotides in the proximal promoter at positions -96 and -97, and no insertions were into a coding or 3'-flanking sequence. All inserted transposons were in inverse orientation to the starting transposon. The frequent insertion into nucleotides -96 and -97 is consistent with the DNase hypersensitivity, absence of nucleosomes, flanking GAGA-factor-binding sites, and nucleotide sequence of this region. These experimental insertions recapitulated many of the phenotypes of natural transposition into Hsp70: reduced mRNA expression, less Hsp70 protein, and decreased inducible thermotolerance. The results suggest that the distinctive features of heat-shock promoters, which underlie the massive and rapid expression of heat-shock genes upon heat shock, also are a source of evolutionary variation on which natural selection can act.

Animals↗