参考文献

Pneumococcal colonization and disease is often associated with biofilm formation,in which the bacteria exhibit elevated resistance both to antibiotics and to host defense systems,often resulting in infections that are persistent and difficult to treat. We evaluated the effect of sinefungin,a nucleoside analogue of S-adenosylmethionine,on pneumococcal in vitro biofilm formation and in vivo colonization. Sinefungin is bacteriostatic to pneumococci and significantly decreased biofilm growth and inhibited proliferation and structure of actively growing biofilms but did not alter growth or the matrix structure of established biofilms. Sinefungin significantly reduced pneumococcal colonization in rat middle ear. The quorum sensing molecule (autoinducer-2) production was significantly reduced by 92% in sinefungin treated samples. The luxS,pfs,and speE genes were downregulated in biofilms grown in the presence of sinefungin. This study shows that sinefungin inhibits pneumococcal biofilm growth in vitro and colonization in vivo,decreases AI-2 production,and downregulates luxS,pfs,and speE gene expressions. Therefore,the S-adenosylmethionine (SAM) inhibitors could be used as lead compounds for the development of novel antibiofilm agents against pneumococci. View Publication

Declined production of collagen by fibroblasts is one of the major causes of aging appearance. However,only few of compounds found in cosmetic products are able to directly increase collagen synthesis. A novel small heterocyclic compound called kartogenin (KGN) was found to stimulate collagen synthesis of mesenchymal stem cells (MSCs). So,we hypothesized and tested that if KGN could be applied to stimulate the collagen synthesis of fibroblasts. Human dermal fibroblasts in vitro were treated with various concentrations of KGN,with dimethyl sulfoxide (DMSO) serving as the negative control. Real-time reverse-transcription polymerase chain reaction,Western blot,and immunofluorescence analyses were performed to examine the expression of collagen and transforming growth factor beta (TGF-β) signaling pathway. The production of collagen was also tested in vivo by Masson's trichrome stain and immunohistochemistry in the dermis of mice administrated with KGN. Results showed that without obvious influence on fibroblasts' apoptosis and viability,KGN stimulated type-I collagen synthesis of fibroblasts at the mRNA and protein levels in a time-dependent manner,but KGN did not induce expression of α-skeletal muscle actin (α-sma) or matrix metallopeptidase1 (MMP1),MMP9 in vitro. Smad4/smad5 of the TGF-β signaling pathway was activated by KGN while MAPK signaling pathway remained unchanged. KGN also increased type-I collagen synthesis in the dermis of BALB/C mice. Our results indicated that KGN promoted the type-I collagen synthesis of dermal fibroblasts in vitro and in the dermis of mice through activation of the smad4/smad5 pathway. This molecule could be used in wound healing,tissue engineering of fibroblasts,or aesthetic and reconstructive procedures. View Publication

BACKGROUND Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) are in the foreground as a preferable application for treating diseases. However,the safety of hUC-MSCs after long-term culturing in vitro in serum-free medium remains unclear. METHODS hUC-MSCs were separated by adherent tissue culture. hUC-MSCs were cultured in serum-free MesenCult-XF medium and FBS-bases DMEM complete medium. At the 1st,3rd,5th,8th,10th,and 15th passage,the differentiation of MSCs into osteogenic,chondrogenic,and adipogenic cells was detected,and MTT,surface antigens were measured. Tumorigenicity was analyzed at the 15th passage. Conventional karyotyping was performed at passage 0,8,and 15. The telomerase activity of hUC-MSCs at passage 1-15 was analyzed. RESULTS Flow cytometry analysis showed that very high expression was detected for CD105,CD73,and CD90 and very low expression for CD45,CD34,CD14,CD79a,and HLA-DR. MSCs could differentiate into osteocytes,chondrocytes,and adipocytes in vitro. There was no obvious chromosome elimination,displacement,or chromosomal imbalance as determined from the guidelines of the International System for Human Cytogenetic Nomenclature. Telomerase activity was down-regulated significantly when the culture time was prolonged. Further,no tumors formed in rats injected with hUC-MSCs (P15) cultured in serum-free and in serum-containing conditions. CONCLUSION Our data showed that hUC-MSCs met the International Society for Cellular Therapy standards for conditions of long-term in vitro culturing at P15. Since hUC-MSCs can be safely expanded in vitro and are not susceptible to malignant transformation in serum-free medium,these cells are suitable for cell therapy. View Publication

Memory B cells (MBCs) are long-lived sources of rapid,isotype-switched secondary antibody-forming cell (AFC) responses. Whether MBCs homogeneously retain the ability to self-renew and terminally differentiate or if these functions are compartmentalized into MBC subsets has remained unclear. It has been suggested that antibody isotype controls MBC differentiation upon restimulation. Here we demonstrate that subcategorizing MBCs on the basis of their expression of CD80 and PD-L2,independently of isotype,identified MBC subsets with distinct functions upon rechallenge. CD80(+)PD-L2(+) MBCs differentiated rapidly into AFCs but did not generate germinal centers (GCs); conversely,CD80(-)PD-L2(-) MBCs generated few early AFCs but robustly seeded GCs. The gene-expression patterns of the subsets supported both the identity and function of these distinct MBC types. Hence,the differentiation and regeneration of MBCs are compartmentalized. View Publication

Despite the fact that glucocorticoids and long acting beta agonists are effective treatments for asthma,their effects on human mast cells (MC) appear to be modest. Although MC are one of the major effector cells in the underlying inflammatory reactions associated with asthma,their regulation by these drugs is not yet fully understood and,in some cases,controversial. Using a human immortalized MC line (LAD2),we studied the effects of fluticasone propionate (FP) and salmeterol (SM),on the release of early and late phase mediators. LAD2 cells were pretreated with FP (100 nM),SM (1 µM),alone and in combination,at various incubation times and subsequently stimulated with agonists substance P,C3a and IgE/anti-IgE. Degranulation was measured by the release of β-hexosaminidase. Cytokine and chemokine expression were measured using quantitative PCR,ELISA and cytometric bead array (CBA) assays. The combination of FP and SM synergistically inhibited degranulation of MC stimulated with substance P (33% inhibition compared to control,n = 3,P>05). Degranulation was inhibited by FP alone,but not SM,when MC were stimulated with C3a (48% inhibition,n = 3,P>05). As previously reported,FP and SM did not inhibit degranulation when MC were stimulated with IgE/anti-IgE. FP and SM in combination inhibited substance P-induced release of tumor necrosis factor (TNF),CCL2,and CXCL8 (98%,99% and 92% inhibition,respectively,n = 4,P>05). Fluticasone and salmeterol synergistically inhibited mediator production by human MC stimulated with the neuropeptide substance P. This synergistic effect on mast cell signaling may be relevant to the therapeutic benefit of combination therapy in asthma. View Publication

Vascular endothelial growth factor (VEGF) was purified from media conditioned by bovine pituitary folliculostellate cells (FC). VEGF is a heparin-binding growth factor specific for vascular endothelial cells that is able to induce angiogenesis in vivo. Complementary DNA clones for bovine and human VEGF were isolated from cDNA libraries prepared from FC and HL60 leukemia cells,respectively. These cDNAs encode hydrophilic proteins with sequences related to those of the A and B chains of platelet-derived growth factor. DNA sequencing suggests the existence of several molecular species of VEGF. VEGFs are secreted proteins,in contrast to other endothelial cell mitogens such as acidic or basic fibroblast growth factors and platelet-derived endothelial cell growth factor. Human 293 cells transfected with an expression vector containing a bovine or human VEGF cDNA insert secrete an endothelial cell mitogen that behaves like native VEGF. View Publication

BACKGROUND: The instant blood-mediated inflammatory response (IBMIR) has been shown as a major factor that causes damage to transplanted islets. Withaferin A (WA),an inhibitor of nuclear factor (NF) κB,was shown to suppress the inflammatory response in islets and improve syngeneic islet graft survival in mice. We investigated how treating islets with NF-κB inhibitors affected IBMIR using an in vitro human autologous blood islet model. METHODS: Human islets were pretreated with or without NF-κB inhibitors WA or CAY10512 before mixing autologous blood in a miniaturized in vitro tube model. Plasma samples were collected at multiple time points and used for the measurement of C-peptide,proinsulin,thrombin-antithrombin (TAT) complex,and a panel of proinflammatory cytokines. Infiltration of neutrophils into islets was analyzed using immunohistochemistry. RESULTS: Rapid release of C-peptide and proinsulin was observed 3 hr after mixing islets and blood in the control group,but not in the NF-κB inhibitor-treated groups,whereas TAT levels were elevated in all three groups with a peak at 6 hr. Significant elevation of proinflammatory cytokines was observed in the control group after 3 hr,but not in the treatment groups. Significant inhibition of neutrophil infiltration was also observed in the WA group compared with the control (Ptextless0.001) and CAY10512 (Ptextless0.001) groups. CONCLUSIONS: A miniaturized in vitro tube model can be useful in investigating IBMIR. The presence of NF-κB inhibitor could alleviate IBMIR,thus improving the survival of transplanted islets. Protection of islets in the peritransplant phase may improve long-term graft outcomes. View Publication

INTRODUCTION: We previously demonstrated that the lifespan of primary human keratinocytes could be extended indefinitely by culture in the presence of the Rho kinase (ROCK) inhibitor Y-27632. This technique has proven to be very useful in diverse areas of basic and clinical research. METHODS: In this follow-up study we determine whether the continual presence of Y-27632 is required for sustained proliferation. We also test whether different ROCK inhibitors can be used for this technique and whether it can also promote indefinite proliferation of animal keratinocytes. We measure keratinocyte gene expression,proliferation,behaviour and lifespan in the presence and absence of Y-27632. RESULTS: We demonstrate that the extension of lifespan observed by culture of keratinocytes in the presence of fibroblast feeders and a ROCK inhibitor is reversible and that cells senesce gradually when the inhibitor is removed from the medium. Conversely,keratinocytes that are close to the end of their replicative life span can be revived by ROCK inhibition. We demonstrate that different inhibitors of ROCK can also efficiently extend the lifespan of human keratinocytes and that ROCK inhibition extends the lifespan of animal keratinocytes derived from mouse and bovine epithelia. Gene expression analysis of human epidermal keratinocytes cells grown in the presence of Y-27632 demonstrates that ROCK inhibition primarily inhibits keratinocyte differentiation. Live-imaging of keratinocytes cultured with ROCK inhibitors show that the effect of ROCK inhibition on cellular proliferation is immediate and ROCK inhibited cells proliferate rapidly without differentiation or stratification. CONCLUSIONS: ROCK inhibition rapidly and conditionally induces indefinite proliferation of keratinocytes. This method has far-reaching applications for basic research,as well as for regenerative and personalized medicine. View Publication

A two-step separation procedure is described for the positive selection of cells based on their reactivity with mouse monoclonal antibodies. In the first step cells are specifically cross-linked to hapten-modified glass beads using tetrameric monoclonal antibody complexes. In the second step bound cells are selectively eluted by reductive cleavage of the tetrameric antibody complexes. The latter are comprised of two mouse IgG1 monoclonal antibodies (one recognizing a cell surface antigen on target cells and the other a hapten coupled to the glass beads) bound together by two F(ab')2 fragments of rat anti-mouse IgG1 monoclonal antibody. The complexes provide a specific cleavable cross-link between cell and bead because the disulfide bonds between the two Fab' arms of the F(ab')2 fragments can be broken under relatively mild conditions using dithiothreitol. This specific cleavage of the cross-linker allows elution of the specifically absorbed cells without co-elution of non-specifically bound cells. This is shown in the purification of CD3+ T cells from human peripheral blood,where the removed fractions were over 90% pure and approximately 50% of the positive cells were recovered. Separation of cells labelled with limiting amounts of tetrameric antibody complexes demonstrated that this separation technique was also effective for the purification of cells expressing low amounts of antigens. This was confirmed by the purification of CD34-positive cells from human bone marrow. With this approach,colony-forming cells were enriched 15-24-fold over density separated marrow. View Publication

Antiserum to epithelial membrane antigen and three monoclonal antibodies (MAb) to milk-fat globule membranes immunocytochemically stain only epithelial cells,whereas a fourth reacts also with myoepithelial cells in inter- and intralobular ducts of human breast. Staining with peanut lectin shows a gradual increase for epithelial cells,from little or no staining in ducts through variable staining in ductules to intense staining in secretory alveoli. Antisera and MAb to vimentin,smooth-muscle actin,MAb to the common acute lymphoblastic leukemia antigen and to a glycoprotein of 135 KD stain myoepithelial cells in main ducts,but this staining is reduced in inter- and intralobular ducts and ductules. MAb to epithelial-specific keratin 18 stain a minor population of ductal epithelial cells,the major population of epithelial cells in interlobular (ILD) and extralobular terminal ducts (ETD),and epithelial cells in a minority of ductules. In lactating glands most epithelial cells in ductules are stained,but the alveolar and myoepithelial cells are unstained. Keratin MAb PKK2 and LP34 strongly stain myoepithelial cells,but only a minor population of epithelial cells in main ducts. However,these MAb stain principally the epithelial cells in ILD,ETD,and a minority of ductules. In lactating glands most epithelial cells are stained in ductules,but the myoepithelial and not the alveolar cells are stained intensely in secretory lobules. It is suggested that the unusual staining pattern of cells found principally in the ILD,ETD,and some ductules may represent regions of growth and/or subpopulation(s) of cells intermediate between epithelial and myoepithelial cells. View Publication