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Biomedical subjects

M Wang

Publications and source records attributed to M Wang.

At least 73 records · Page 4Linked to original sources
PubMed2004

T-RFLP combined with principal component analysis and 16S rRNA gene sequencing: an effective strategy for comparison of fecal microbiota in infants of different ages.

The fecal microbiota of two healthy Swedish infants was monitored over time by terminal restriction fragment length polymorphism (T-RFLP) analysis of amplified 16S rRNA genes. Principal component analysis (PCA) of the T-RFLP profiles revealed that the fecal flora in both infants was quite stable during breast-feeding and a major change occurred after weaning. The two infants had different sets of microbiota at all sampling time points. 16S rDNA clone libraries were constructed and the predominant terminal restriction fragments (T-RFs) were identified by comparing T-RFLP patterns in the fecal community with that of corresponding 16S rDNA clones. Sequence analysis indicated that the infants were initially colonized mostly by members of Enterobacteriaceae, Veillonella, Enterococcus, Streptococcus, Staphylococcus and Bacteroides. The members of Enterobacteriaceae and Bacteroides were predominant during breast-feeding in both infants. However, Enterobacteriaceae decreased while members of clostridia increased after weaning. T-RFLP in combination with PCA and 16S rRNA gene sequencing was shown to be an effective strategy for comparing fecal microbiota in infants and pointing out the major changes.

Base Sequence↗
PubMed2004

Conservation of the genomic structure and receptor-mediated signaling between human and rat IL-24.

IL-24/MDA-7 is a new member of the IL-10 family of cytokines, which signals through two heterodimeric receptor complexes (IL-20R1/IL-20R2 and IL-22R/IL-20R2). Previously, we identified a rat gene named mob-5, which encodes a secreted protein that shares a high degree of homology with human IL-24. Expression of mob-5 and its putative cell surface receptors was shown to be upregulated by oncogenic ras. Here we show that not only do rat mob-5 and human IL-24 share a strikingly similar genomic structure but also that the rat MOB-5 protein can bind to and signal through the human IL-24 receptors. Like human IL-24, binding of the rat MOB-5 protein to the human IL-24 receptors leads to activation of the JAK/STAT pathway, which in turn supports receptor-dependent survival and proliferation of Ba/F3 cells. Furthermore, using human colon cancer cell lines with somatic knockout of either the mutant or the wild-type k-ras allele, we demonstrate that the human IL-24 receptors also are upregulated by oncogenic ras. Taken together, these results provide strong experimental evidence that MOB-5 is indeed the rat homolog of human IL-24.

Amino Acid Sequence↗
PubMed2004

Transcriptional targeting of adenoviral vector through the CXCR4 tumor-specific promoter.

Adenoviral vectors are considered to be good gene delivery vectors for cancer gene therapy due to their wide host tissue range and cell cycle-independent infectivity. However, the disadvantages include the lack of specificity for cancer cells and the high liver accumulation in vivo. The human CXCR4 gene is expressed at high levels in many types of cancers, but is repressed in the liver. We explored the CXCR4 promoter as a candidate to restrict adenoviral transgene expression to tumor cells with a low expression in host tissues. The luciferase activities in multiple cancer cell lines infected with recombinant adenovirus reAdGL3BCXCR4 or the control vector reAdGL3BCMV revealed that the CXCR4 promoter exhibited relatively high transcriptional activity in a breast cancer cell line, MDA-MB-361, and two ovarian cancer cell lines, OVCAR-3 and SKOV3. ip1, 65% (P=0.0087), 16.7% (P=0.1) and 20% (P=0.0079) compared to that of the CMV promoter, respectively, and low expression, 4.9 and 0.1%, respectively, in both normal cell lines HFBC and HMEC. In addition, CXCR4 had a low expression of luciferase (0.32%) compared to that of the CMV promoter in mouse liver in vivo. The data also revealed that the CXCR4 promoter was a stronger tumor-specific promoter (TSP) than the Cox-2M promoter in primary melanomas obtained from two patients. The CXCR4 promoter is shown to have a 'tumor-on' and 'liver-off' status in vitro and in vivo, and CXCR4 may prove to be a good candidate TSP for cancer gene therapy approaches for melanoma and breast cancers.

Adenoviridae↗
PubMed2004

Stable transfection of MSCs by electroporation.

Human marrow stromal cells (hMSCs) are an attractive source of adult stem cells for autologous cell and gene therapy. To transfect hMSCs without the use of viruses, we developed improved conditions for stable transfection of the cells by electroporation. hMSCs were isolated by adherence to plastic, and were electroporated at 600 V and 100 micros in a 2-mm gap cuvette with a plasmid containing enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase gene (neo(r)). After electroporation of 10(6) cells with 10 microg of the linearized plasmid DNA, hMSCs with stable DNA integration were selected by culturing with 200 microg/ml G418. The transfected hMSCs were expanded another 300-fold in 14 days to obtain 89 million cells, of which 98% expressed EGFP. Chloroquine increased the number of hMSCs transiently expressing EGFP from 12% to over 50%, but decreased stable integration. Stable integration of plasmid DNA into rat MSCs by electroporation was also successful. The transfected MSCs retained their capacity to differentiate into both adipocytes and osteoblasts. Thus, MSCs were stably transfected with plasmid DNA and retained their differentiation capacity after expansion.

Adipocytes↗
PubMed2004

Combining high selectivity of replication with fiber chimerism for effective adenoviral oncolysis of CAR-negative melanoma cells.

Oncolytic adenoviruses constitute a new and promising tool for cancer treatment that has been rapidly translated into clinical trials. However, minimal or absent expression of the adenovirus serotype 5 (Ad5) receptor CAR (coxsackievirus and adenovirus receptor) on cancer cells represents a major limitation for Ad5-based oncolysis. Here, we report on the resistance of CAR-negative primary melanoma cells to cell killing by wild-type Ad5 (Ad5wt) even after high titer infection, thus underlining the need for tropism-modification of oncolytic adenoviruses. We engineered a new generation of oncolytic adenoviruses that exhibit both efficient target cell infection by swapping Ad5 fiber domains with those of Ad serotype 3, which binds to a receptor distinct from CAR, and targeted virus replication. Fiber chimerism resulted in efficient cytopathicity to primary melanoma cells, which was at least 10(4)-fold increased relative to Ad5wt. Since viral infectivity mediated by such modified viral capsids was not cell type-specific, it was pivotal to carefully restrict adenoviral replication to target cells. Towards this end, we replaced both E1A and E4 promoters of fiber chimeric viruses by tyrosinase enhancer/promoter constructs. The resulting viruses showed melanoma-specific expression of E1A and E4 and combined efficient virus replication and cell killing in melanoma cell lines and primary melanoma cells with a remarkable specificity profile that implements strong attenuation in nonmelanoma cells, including normal fibroblasts and keratinocytes.

Adenoviridae↗
PubMed2004

The interferon-alpha genes from three chicken lines and its effects on H9N2 influenza viruses.

The interferon-alpha genes from three chicken lines were cloned by a direct PCR technique, and the effects of recombinant protein expressed in a prokaryotic system on highly pathogenic H9N2 influenza viruses were investigated. The cloned ChIFN-alpha gene encoded a protein of 193 amino acids with a signal sequence of 31 amino acids and mature peptides of 162 amino acids. Comparison of ChIFN-alpha sequences, detected six amino acids substitutions at positions 50, 58, 65, 81, 181, and 183. Homology analysis indicated that ChIFN-alpha genes could be subdivided into two lineages, SH-ChIFN-alpha and WJ-ChIFN-alpha. In addition, both SH-ChIFN-alpha and WJ-ChIFN-alpha were expressed with the N-terminal 6 consecutive histidine residues in a high-level prokaryotic expression system. Recombinant chicken interferon-alpha (rChIFN-alpha) protein has anti-VSV activity of more than 1 x 10(8) U/mg. Moreover, High concentration (10,000U) of rSH-ChIFN-alpha resulted in over 40% inhibition of the H9N2 virus infection in chicken embryos (Ovo), and 100% inhibition from one day-old to five day-old chickens (Vivo). The results suggested that rChIFN-alpha is a potential agent against many Chicken viral strains.

Amino Acid Sequence↗
PubMed2004

Mechanism of apatite formation on pure titanium treated with alkaline solution.

The mechanism of bone-like apatite formation on the surface of pure titanium pretreated with NaOH solution is still being investigated. The apatite formation may depend on the solution that is used. In the present study, several types of solutions such as simulated body fluid (SBF), calcium aqueous solution (CAS), and phosphate aqueous solution (PAS) were used to investigate bone-like apatite formation on alkali-treated titanium. In order to observe the effect of hydrolysis on the apatite formation, experiments of pretreated titanium immersed in distilled water before the immersion in SBF were also conducted. The results showed that the mechanism of apatite formation was the hydrolysis reaction of sodium titanate which induced the apatite formation. The pre-precipitation of either calcium or phosphate could prevent the apatite formation on the surface of alkaline treated titanium.

Adsorption↗
PubMed2004

Probing Norwalk-like virus presence in shellfish, using artificial neural networks.

A database was examined using artificial neural network (ANN) models to investigate the efficacy of predicting PCR-identified Norwalk-like virus presence and absence in shellfish. The relative importance of variables in the model and the predictive power obtained by application of ANN modelling methods were compared with previously developed logistic regression models. In addition, two country-specific datasets were analysed separately with ANN models to determine if the relative importance of the input variables was similar for geographically diverse regions. The results of this analysis found that ANN models predicted Norwalk-like virus presence and absence in shellfish with equivalent, and better, precision than logistic regression models. For overall classification performance, ANN modelling had a rate of 93%, vs 75% for the logistic regression. ANN models were able to illuminate the site-specific relationships between indicators and pathogens.

Databases, Factual↗
PubMed2003

A novel IL-10 signalling mechanism regulates TIMP-1 expression in human prostate tumour cells.

We have previously reported that interleukin 10 (IL-10) signalling stimulated activation of a specific enhancer element, termed HTE-1, to promote tissue inhibitor of matrix metalloproteinase1 (TIMP-1) expression in human bone metastatic PC-3 subclone (PC-3 ML) cells. Recently, we have identified an IL-10 responsive signal molecule, termed IL-10E1, which binds the HTE-1 element and cloned the gene encoding for the 22 kDa protein. In this paper, we have examined the mechanism of IL-10/IL-10 receptor signalling in two distinct human prostate cell lines, a 'normal' prostate epithelial cell line, termed NPTX-1532 and highly metastatic PC-3 ML tumour cells. Signalling cascade studies revealed that IL-10 stimulated tyrosine phosphorylation of JAK1 and TYK2 receptor kinases and tyrosine phosphorylation of IL-10E1. Phosphorylation, triggered IL-10E1's rapid translocation to the nucleus by 10-30 min. Deletion analysis combined with transient transfection experiments revealed that the n-terminal domain (approximately 74 a.a.) of the IL-10E1 protein, the nt-nls peptide, was stimulated by IL-10 to translocate to the nucleus and induce TIMP-1 expression. Site-directed mutagenesis further showed that phosphorylation of two tyrosine moieties (Y57 and Y62) of the nt-nls peptide was required for IL-10 activation of signalling and TIMP-1 expression. The data demonstrate, for the first time, that IL-10 receptor signalling of TIMP-1 expression is regulated by tyrosine phosphorylation of a novel gene, IL-10E1, in human prostate cells.

Amino Acid Sequence↗
PubMed2003

Interaction of metal salts with cytoskeletal motor protein systems.

Interactions of chemicals with the microtubular network of cells may lead to genotoxicity. Micronuclei (MN) might be caused by interaction of metals with tubulin and/or kinesin. The genotoxic effects of inorganic lead and mercury salts were studied using the MN assay and the CREST analysis in V79 Chinese hamster fibroblasts. Effects on the functional activity of motor protein systems were examined by measurement of tubulin assembly and kinesin-driven motility. Lead and mercury salts induced MN dose-dependently. The no-effect-concentration for MN induction was 1.1 microM PbCl(2), 0.05 microM Pb(OAc)(2) and 0.01 microM HgCl(2). The in vitro results obtained for PbCl(2) correspond to reported MN induction in workers occupationally exposed to lead, starting at 1.2 microM Hg(II) (Vaglenov et al., 2001, Environ. Health Perspect. 109, 295-298). The CREST Analysis indicate aneugenic effects of Pb(II) and aneugenic and additionally clastogenic effects of Hg(II). Lead (chloride, acetate, and nitrate) and mercury (chloride and nitrate) interfered dose-dependently with tubulin assembly in vitro. The no-effect-concentration for lead salts in this assay was 10 microM. Inhibition of tubulin assembly by mercury started at 2 microM. The gliding velocity of microtubules along immobilised kinesin molecules was affected by 25 microM Pb(NO(3))(2) and 0.1 microM HgCl(2) in a dose-dependent manner. Our data support the hypothesis that lead and mercury genotoxicity may result, at least in part, via disturbance of chromosome segregation via interaction with cytoskeletal proteins.

Animals↗
PubMed2003

Effects of the nitric oxide donor, DEA/NO on cortical spreading depression.

Cortical spreading depression (CSD) is a transient disruption of local ionic homeostasis that may promote migraine attacks and the progression of stroke lesions. We reported previously that the local inhibition of nitric oxide (NO) synthesis with Nomega-nitro-L-arginine methyl ester (L-NAME) delayed markedly the initiation of the recovery of ionic homeostasis from CSD. Here we describe a novel method for selective, controlled generation of exogenous NO in a functioning brain region. It is based on microdialysis perfusion of the NO donor, 2-(N,N-diethylamino)-diazenolate-2-oxide (DEA/NO). As DEA/NO does not generate NO at alkaline pH, and as the brain has a strong acid-base buffering capacity, DEA/NO was perfused in a medium adjusted at alkaline (but unbuffered) pH. Without DEA/NO, such a microdialysis perfusion medium did not alter CSD. DEA/NO (1, 10 and 100 microM) had little effect on CSD by itself, but it reversed in a concentration-dependent manner the effects of NOS inhibition by 1 mM L-NAME. These data demonstrate that increased formation of endogenous NO associated with CSD is critical for subsequent, rapid recovery of cellular ionic homeostasis. In this case, the molecular targets for NO may be located either on brain cells to suppress mechanisms directly involved in CSD genesis, or on local blood vessels to couple flow to the increased energy demand associated with CSD.

Animals↗
PubMed2003

Nucleation and growth of apatite on chemically treated titanium alloy: an electrochemical impedance spectroscopy study.

Bone-like apatite formed on the surface of Ti6Al4V pretreated with NaOH solution after having been immersed in simulated body fluid (SBF), while no apatite formed on the surface of untreated Ti6Al4V. In the present study, electrochemical impedance spectroscopy (EIS) measurement was used to investigate the nucleation and growth of apatite on chemically treated Ti6Al4V immersed in the SBF solution, and the difference between the behaviors of treated and untreated Ti6Al4V. Appropriate equivalent circuit models were constructed to describe the nucleation and growth of apatite, and thin oxide film formed on the surface of untreated Ti6Al4V. It was found that EIS is a useful method for investigating the nucleation and growth of bone-like apatite on Ti6Al4V pretreated with NaOH solution.

Alloys↗
PubMed2003

Coupling of oral human or porcine insulin to the B subunit of cholera toxin (CTB) overcomes critical antigenic differences for prevention of type I diabetes.

Our earlier investigations have demonstrated a critical difference in the efficacy of orally administered porcine compared to human or mouse insulin (no effect) in preventing type I diabetes in two distinct experimental models. Based on these findings one has to assume that certain insulins might not be suitable for the induction of oral 'tolerance'/bystander suppression, which might be one cause for recent failures in human oral antigen trials. Here we demonstrate that coupling to the non-toxic subunit of cholera toxin (CTB) can abolish these differences in efficacy between human and porcine insulin. As expected, an added benefit was the much smaller oral antigen dose required to induce CD4+ insulin-B specific regulatory cells that bystander-suppress autoaggressive responses. Mechanistically we found that uptake or transport of insulin-CTB conjugates in the gut occurs at least partially via binding to GM-1, which would explain the enhanced clinical efficacy. Both B chains bound well to major histocompatibility complex (MHC) class II, indicating comparable immunological potential once uptake and processing has occurred. Thus, our findings delineate a pathway to overcome issues in oral antigen choice for prevention of type I diabetes.

Administration, Oral↗
PubMed2003

The cholera toxin B subunit is a mucosal adjuvant for oral tolerance induction in type 1 diabetes.

When conjugated to various proteins, the nontoxic B-chain of cholera toxin (CTB) significantly increases the ability of these proteins to induce immunological tolerance after oral administration. Here, we investigated if a nonconjugated form of CTB enhances the induction of immune tolerance after oral insulin administration. Induction of immunological tolerance was studied after oral administration of insulin preparations in three mouse models; an insulin/ovalbumin coimmunization model, a model of virus-induced diabetes in transgenic RIP-LCMV-NP mice and in nonobese diabetic (NOD) mice serving as a model of spontaneous diabetes. In the immunization model, we demonstrate that mixing with CTB increases the tolerogenic potential of insulin, approximately 10 fold. Titration of the CTB concentration in this system revealed that an insulin : CTB ratio of 100 : 1 was optimal for the induction of bystander suppression. Further studies revealed that this insulin : CTB ratio also was optimal for the prevention of diabetes in a virus-induced, transgenic diabetes model. In addition, the administration of this optimal insulin-CTB preparation significantly prevented the onset of diabetes in old NOD mice with established islet infiltration. The data presented here demonstrate that CTB, even in its unconjugated form, functions as a mucosal adjuvant, increasing the specific tolerogenic effect of oral insulin.

Adjuvants, Immunologic↗
PubMed2003

The cholera toxin B subunit directly costimulates antigen-primed CD4+ T cells ex vivo.

The nontoxic B subunit of cholera toxin (CTB) has been used as an adjuvant in experimental systems of mucosal vaccination. However, the mechanisms behind its adjuvant effects remain unclear. Here, we have used an ex vivo system to elucidate these mechanisms in antigen-specific T cells. Using splenocytes from keyhole limpet haemocyanin (KLH)-immunized mice, initial experiments showed that recombinant CTB (rCTB) did not affect the KLH-specific proliferation of splenocytes isolated from mice immunized 2 weeks earlier. However, rCTB strongly enhanced the KLH-specific proliferation of splenocytes from mice immunized with KLH 4 weeks prior. This adjuvant effect was dose-dependent, with maximum at 30-300 ng/ml rCTB. At higher doses of CTB this effect declined because of the induction of apoptosis. Using antibody depletion and coculture systems, we show that rCTB directly costimulates KLH-specific CD4+ and CD8+ T-cell proliferation but not B cells. Enzyme-linked immunospot (ELISPOT) assays revealed that rCTB also enhanced the KLH-specific CD4+ T-cell-mediated production of interleukin-2 (IL-2), IL-4 and interferon-gamma(IFN-gamma) by four to fivefold. Characterizing the adjuvant effect of rCTB in vivo confirmed the results above, i.e. rCTB mediated a twofold increase in the ex vivo T-cell response when used as a classical adjuvant in a secondary, but not in a primary KLH-immunization regimen. Together these data show that rCTB can act as a strong adjuvant, by directly costimulating antigen-primed CD4+ and CD8+ T cell in a dose-dependent manner. This new insight might be valuable in the future rational design of bacterial toxin-based vaccines.

Adjuvants, Immunologic↗
PubMed2003

Transcription factor 1 and beta-cell function in glucose-tolerant subjects.

AIMS: Although beta-cell dysfunction and insulin resistance are observed in patients with Type 2 diabetes, beta-cell dysfunction plays a crucial role in the development of the disease. Mutations of transcription factor 1 (TCF1, or hepatocyte nuclear factor-1alpha) affect beta-cell function. We examined the impact of amino acid polymorphisms of this gene on beta-cell function in 60 glucose-tolerant Caucasians. METHODS: Insulin sensitivity index (ISI) and 1st and 2nd phase insulin responses (1stIR, 2ndIR) were measured using the hyperglycaemic clamp. The genotypes were determined from genomic DNA and their impact on beta-cell function was investigated. RESULTS: Among three polymorphisms (I27L, A98V, and S487N), significant impact on beta-cell function was found in the I27L polymorphism. Univariate analyses revealed differences for the I27L polymorphism in both 1stIR (P = 0.0052) and 2ndIR (P = 0.0432). Multivariate analysis revealed that the I27L polymorphism (P = 0.0124) with ISI accounted for 32% of the variation in 1stIR and had a marginal impact on 2ndIR (P = 0.0343), accounting for 52% of the variation with ISI and age. By dissecting out the impact of other covariate(s), the I27L polymorphism independently explained 12% of the variation in 1stIR and 6% of the variation in 2ndIR. CONCLUSION: The I27L polymorphism of TCF1 is an independent determinant of beta-cell function by affecting both 1st and 2nd phase insulin response. This polymorphism could play a role in the pathogenesis of Type 2 diabetes. A large-scale association study (approx. 2000 subjects) will be required to demonstrate the genetic susceptibility of this polymorphism to Type 2 diabetes.

Adult↗
PubMed2003

Defining "early dementia" and monitoring intervention: what measures are useful in family caregiving?

Measures of cognition are often used to define and measure the progress of dementia and outcomes of intervention. This paper examines whether measures of psychosocial disability used with those of cognition are more useful than measures of cognition alone, particularly in early dementia. A measure of cognition and two instruments of caregiver burden, used as routine clinical outcome measures of three types of Old Age Psychiatry dementia services, were examined. All cases with dementia in a memory clinic (MC; n = 149), a community mental health service for older people (CMHT; n = 120) and a specialist dementia day hospital (DH; n = 118), in one NHS district were followed up at 12 months. Measures of cognition (MMSE), behaviour, caregiver coping (Problem Checklist; PC) and caregiver mood (Hospital Anxiety and Depression Scale; HAD) were taken at baseline (MC, n = 48; CMHT, n = 113; DH, n = 55) and at follow-up (MC, n = 35; CMHT, n = 34; DH, n = 23). At baseline, all three groups had an average MMSE score of "mild impairment" but measures of behaviour and caregiver burden showed subtle between-group differences. At the 12-month follow-up, cognition remained stable in all groups, but the frequency of day-to-day problems increased and caregiver mood deteriorated in families receiving DH support. The use of psychosocial measures of disability in conjunction with those of cognition, are important in the definition and longitudinal measurement of intervention and support in early dementia.

Adaptation, Psychological↗