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1.
Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients.  相似文献   

2.
Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation of adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.  相似文献   

3.
Successful cell therapy will depend on the ability to monitor transplanted cells. With cell labeling, it is important to demonstrate efficient long term labeling without deleterious effects on cell phenotype and differentiation capacity. We demonstrate long term (7 weeks) retention of superparamagnetic iron oxide particles (SPIO) by mesenchymal stem cells (MSCs) in vivo, detectable by MRI. In vitro, multilineage differentiation (osteogenic, chondrogenic and adipogenic) was demonstrated by histological evaluation and molecular analysis in SPIO labeled and unlabeled cells. Gene expression levels were comaparable to unlabeled controls in adipogenic and chondrogenic conditions however not in the osteogenic condition. MSCs seeded into a scaffold for 21 days and implanted subcutaneously into nude mice for 4 weeks, showed profoundly altered phenotypes in SPIO labeled samples compared to implanted unlabeled control scaffolds, indicating chondrogenic differentiation. This study demonstrates long term MSC traceability using SPIO and MRI, uninhibited multilineage MSC differentiation following SPIO labeling, though with subtle but significant phenotypical alterations.  相似文献   

4.
The aim of the present study is to investigate the causal relationship between membrane-damaging activity and bactericidal activity of Naja nigricollis toxin γ. Toxin γ showed a similar inhibitory activity on the growth of Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria). Antibacterial activity of toxin γ correlated positively with increase in membrane permeability of bacterial cells. Morphological examination showed that toxin γ disrupted the integrity of bacterial membrane. Toxin γ showed similar binding capability with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and destabilization of LPS layer and inhibition of LTA biosynthesis on cell wall increased bactericidal effect of toxin γ on E. coli and S. aureus, respectively. Although the potency of toxin γ on permeabilzing model membrane of E. coli and S. aureus was similar, the mode of interaction between toxin γ and model membrane of E. coli and S. aureus differed. Membrane-damaging activity of toxin γ was inhibited by either LPS or LTA. Nevertheless, LPS and LTA altered differently membrane-bound conformation of toxin γ. Taken together, our data suggest that bactericidal activity of toxin γ depends on its ability to induce membrane permeability, and that LPS and LTA structurally suppresses bactericidal effect of toxin γ.  相似文献   

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6.
The oligo-acyl-lysyl, C12(ω7)K-β12, is comprised of only three Lys residues. Despite its small size, it exhibits potent bacteriostatic activity against Gram-positive bacteria, but it is ∼10-fold less potent against Gram-negative bacteria. We followed the interactions of C12(ω7)K-β12 from its initial contact with the bacterial surface across the cell wall down to the cytoplasmic membrane. Binding to anionic lipids, as well as to negatively charged LPS and LTA, occurs with very high affinity. The C12(ω7)K-β12 does not cross the outer membrane of Gram-negative bacteria; rather, it achieves its action by depositing on the LPS layer, promoting surface adhesion and blocking passage of solutes. In Gram-positive bacteria, the thick peptidoglycan layer containing LTA allows passage of C12(ω7)K-β12 and promotes its accumulation in the small periplasm. From that location it is then driven to the membrane by strong electrostatic interactions. Despite its high potency against Gram-positive bacteria, this agent is not capable of efficiently breaking down the permeability barrier of the cytoplasmic membrane or of reaching an intracellular target, as suggested by the fact that it does not interact with DNA.  相似文献   

7.
Lipoteichoic acid (LTA) is a structural component of the cell walls of Gram-positive bacteria. Similar to lipopolysaccharide (LPS) which is expressed in Gram-negative bacteria, LTA exhibits immunostimulatory properties. Frequently observed positive response of LTA in the Limulus amebocyte lysate (LAL) assay has been interpreted as a sign of LPS contamination, raising doubts about the intrinsic immune activities of LTA. Regarding many similarities in immunobiological and physicochemical properties of LTA and LPS, we hypothesized that similar to LPS, the LAL reactivity of LTA might be due to its ability to bind to LAL. Our data confirm the positivity of Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis and Streptococcus pyogenes LTAs in the LAL test. The estimates of suspected LPS content were 605, 10.3, 6.2 and 127 pg/μg LTA, respectively. The effectiveness of LTAs to induce the NO production in rat peritoneal cells was remarkably higher than that of equivalent concentrations of reference LPS (Escherichia coli). The LPS-induced NO was inhibited by polymyxin B (PMX), the IC50 of PMX:LPS concentration ratio (pg:pg) being 1050:1. Many fold higher concentrations of PMX were needed to partially suppress the NO-augmenting effects of LTAs, applied at concentrations representing the equivalents of LPS. Transposed to the concentrations of LTAs per se, the IC50s of the PMX:LTA ratios (μg:μg) ranged from 0.3:1 (S. aureus) to 7.5:1 (B. subtilis). It is concluded that LTA is not necessarily contaminated with LPS. The results prove the intrinsic immunostimulatory properties of LTAs of Gram-positive bacteria. The positive response of LTA in the LAL assay results from its capacity to bind to LAL. In addition, LTA binds with high affinity to PMX.  相似文献   

8.
Many receptors that are employed for the engulfment of apoptotic cells are also used for the recognition and phagocytosis of bacteria. Tyro3, Axl, and Mertk (TAM) are important in the phagocytosis of apoptotic cells by macrophages. Animals lacking these receptors are hypersensitive to bacterial products. In this report, we examine whether the TAM receptors are involved in the phagocytosis of bacteria. We found that macrophages lacking Mertk, Axl, Tyro3 or all three receptors were equally efficient in the phagocytosis of Gram-negative E. coli. Similarly, the phagocytosis of E. coli and Gram-positive S. aureus bioparticles by macrophages lacking TAM receptors was equal to wild-type. In addition, we found that Mertk did not play a role in killing of extracellular E. coli or the replication status of intracellular Francisella tularensis. Thus, while TAM receptors may regulate signal transduction to bacterial components, they are not essential for the phagocytosis and killing of bacteria.  相似文献   

9.
Advances in bioengineering, material chemistry, and developmental biology have led to the design of three-dimensional (3D) culture systems that better resemble the surrounding structure and chemistry of the in situ niches of cells in tissues. This study was designed to characterize and compare porcine adipose-derived stem cells (ADSC) and bone-marrow-derived stem cells (BMSC) induced to differentiate toward osteogenic and adipogenic lineages in vitro by using a 3D alginate hydrogel. The morphology and gene expression of the two cell populations during differentiation were analyzed. Both ADSC and BMSC showed morphological evidence of osteogenic and adipogenic differentiation. Expression patterns of genes characteristic of the onset of osteogenic differentiation (ALP, COL1A1, SPARC, SPP1) were low at the beginning of culture and generally increased during the period of differentiation up to 28 days in culture. Expression of genes associated with adipogenic differentiation (ACSL1, ADFP, ADIPOQ, CD36, DBI, DGAT2, PPARG, SCD) was consistently increased in ADSC cultured in alginate hydrogel relative to the start of differentiation. However, adipogenic gene expression of BMSC cultured in alginate hydrogel was more limited when compared with that of ADSC. Evaluation of cell numbers (via the MTT staining assay) suggested a greater viability of BMSC under osteogenic conditions in alginate hydrogels than under adipogenic conditions, whereas ADSC had greater viability under adipogenic conditions than under osteogenic conditions. This study thus provides an important initial evaluation of ADSC and BMSC seeded and differentiated toward the osteogenic and adipogenic cell lineages in a 3D alginate hydrogel in vitro.  相似文献   

10.
Hepcidin is an antimicrobial peptide and iron-regulatory molecule with highly conserved disulfide bridges among vertebrates, but structural insights into the function in fish remains largely missing. We demonstrate here that recombinant hepcidin-2 from zebrafish is capable of inhibiting the growth of the Gram-negative bacteria Escherichia coli and Vibrio anguillarum, and the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with minimum inhibitory concentrations (MICs) of 18, 15, 13 and 9 μM, respectively. We also show by TEM examination that recombinant hepcidin-2 is directly cidal to the cells of E. coli and S. aureus. Moreover, we find that hepcidin-2 displays affinity to LPS, LTA and PGN. All these data indicate that hepcidin-2 is both a pattern recognition molecule, capable of identifying LPS, LTA and PGN, and an antibacterial effector, capable of inhibiting the growth of bacteria. The data also show that the antibacterial activity of hepcidin-2 depends upon the disulfide bridges.  相似文献   

11.
ADSCs (adipose‐derived mesenchymal stem cells) are candidate adult stem cells for regenerative medicine. Notch signalling participates in the differentiation of a heterogeneous ADSC population. We have isolated, human adipose tissue‐derived single‐cell clones using a cloning ring technique and characterized for their stem cell characteristics. The role of Notch signalling in the differentiation capacity of these adipose‐derived single‐cell‐clones has also been investigated. All 14 clones expressed embryonic and mesenchymal stem cell marker genes. These clones could differentiate into both osteogenic and adipogenic lineages. However, the differentiation potential of each clone was different. Low adipogenic clones had significantly higher mRNA expression levels of Notch 2, 3 and 4, Jagged1, as well as Delta1, compared with those of high adipogenic clones. In contrast, no changes in expression of Notch signalling component mRNA between low and high osteogenic clones was found. Notch receptor mRNA expression decreased with the adipogenic differentiation of both low and high adipogenic clones. The γ‐secretase inhibitor, DAPT (N‐[N‐(3,5‐difluorophenacetyl)‐l ‐alanyl]‐(S)‐phenylglycine t‐butyl ester), enhanced adipogenic differentiation. Correspondingly, cells seeded on a Notch ligand (Jagged1) bound surface showed lower intracellular lipid accumulation. These results were noted in both low and high adipogenic clones, indicating that Notch signalling inhibited the adipogenic differentiation of adipose ADSC clones, and could be used to identify an adipogenic susceptible subpopulation for soft‐tissue augmentation application.  相似文献   

12.
A theoretical inverse relationship has long been postulated for osteogenic and adipogenic differentiation (bone versus adipose tissue differentiation). This inverse relationship in theory at least partially underlies the clinical entity of osteoporosis, in which marrow mesenchymal stem cells (MSCs) have a predilection for adipose differentiation that increases with age. In the present study, we assayed the potential anti-adipogenic effects of Nell-1 protein (an osteoinductive molecule). Using 3T3-L1 (a human preadipocyte cell line) cells and human adipose-derived stromal cells (ASCs), we observed that adenoviral delivered (Ad)-Nell-1 or recombinant NELL-1 protein significantly reduced adipose differentiation across all markers examined (Oil red O staining, adipogenic gene expression [Pparg, Lpl, Ap2]). In a prospective fashion, Hedgehog signaling was assayed as potentially downstream of Nell-1 signaling in regulating osteogenic over adipogenic differentiation. In comparison to Ad-LacZ control, Ad-Nell-1 increased expression of hedgehog signaling markers (Ihh, Gli1, Ptc1). These studies suggest that Nell-1 is a potent anti-adipogenic agent. Moreover, Nell-1 signaling may inhibit adipogenic differentiation via a Hedgehog dependent mechanism.  相似文献   

13.
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15.
Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46-94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn2+-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection.  相似文献   

16.
Effects of partial oxygen pressure in a gas mixture surrounding the culture medium on survival, proliferation and differentiation of mesenchymal stem cells (MSC) isolated from mouse bone marrow was studied. It was found that 3% oxygen increased the survival of cells seeded at low density; the rate and duration of the MSC proliferation were also elevated. Effect of oxygen concentration on replicative activity of MSC was manifested as early as the first few days after the onset of cell growth. Studies of colony formation revealed that preincubation of cells with 21% oxygen had a negative effect on cell growth under 3% oxygen, while preincubation with 3% oxygen stimulated MSC proliferation in the presence of 21% oxygen. Notwithstanding, this effect of oxygen cannot be interpreted as unequivocally deleterious. Low partial oxygen pressure inhibited osteogenic differentiation of MSC, but adipogenic differentiation was insensitive to oxygen concentration. It is concluded that proliferation and differentiation of MSC depend critically on oxygen content in the culture medium.  相似文献   

17.
Ribsome and messenger specificity in protein synthesis by bacteria   总被引:3,自引:0,他引:3  
Ribosomes from Escherichia coli and Pseudomonas fluorescens, two Gram-negative bacteria, translated messenger RNA preparations from all bacteria tested (3 Gram-negative species, and 6 Gram-positive species) as well as f2 RNA and T4 early messenger RNA. Ribosomes from Clostridium pasteurianum, Streptococcus faecalis, and Bacillus subtilis, three Gram-positive organisms, did not translate messenger RNA preparations derived from any of the Gram-negative organisms, the f2 RNA, or the T4 early messenger RNA but did translate messenger preparations from all 6 Gram-positive bacterial species tested.  相似文献   

18.
Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity.  相似文献   

19.
Three pathogens, Riemerella anatipestifer, Escherichia coli, and Salmonella enterica, are leading causes of bacterial fibrinous pericarditis and perihepatitis in ducks in China and worldwide. It is difficult to differentiate these pathogens when obtaining a diagnosis on clinical signs and pathological changes. The aim of this research was to develop a multiplex polymerase chain reaction (m-PCR) that could discriminate R. anatipestifer, E. coli, and S. enterica rapidly in field isolates, or detect the three bacteria in clinical samples from diseased ducks. We selected the DnaB helicase (dnaB) gene of R. anatipestifer, alkaline phosphatase (phoA) gene of E. coli and invasion protein (invA) gene of S. enterica as target genes. In optimized conditions, the limitation of detection was approximately 103 colony forming units (CFU) of each of these three bacterial pathogens per PCR reaction tube. The m-PCR method showed specific amplification of respective genes from R. anatipestifer, E. coli, and S. enterica. Using the m-PCR system, bacterial strains isolated from diseased ducks in our laboratory were categorized successfully, and the pathogens could also be detected in clinical samples from diseased ducks. Therefore, the m-PCR system could distinguish the three pathogens simultaneously, for identification, routine molecular diagnosis and epidemiology, in a single reaction.  相似文献   

20.
The various functions of gelsolin in extracellular compartments are not yet clearly defined but include actin scavenging and antiinflammatory effects. Gelsolin was recently reported to bind endotoxin (LPS) from various Gram-negative bacteria with high affinity. In this study we investigate whether gelsolin also interacts with bacterial wall molecules of Gram-positive bacteria such as lipoteichoic acid (LTA) and whether gelsolin's interaction with bacterial lipids from Gram-negative or Gram-positive bacteria affects their cellular inflammatory responses. A peptide based on the PPI binding site of gelsolin (160-169) binds purified LTA at the same molecular ratio that it binds phosphatidylinositol 4,5-bisphosphate. The OD of recombinant human plasma gelsolin was found to decrease following the addition of purified LTA, and the binding of gelsolin to LTA inhibits F-actin depolymerization by gelsolin. Simultaneously, the ability of LTA to activate translocation of NF-kappaB, E-selectin expression, and adhesion of neutrophils to LTA-treated human aortic endothelial cells was compromised by gelsolin. Gelsolin was able to partially inhibit LPS- or LTA-induced release of IL-8 from human neutrophils but was unable to prevent Gram-positive Bacillus subtilis or Gram-negative Pseudomonas aeruginosa growth and had no effect on the antibacterial activity of the cathelicidin-derived antibacterial peptide LL37. These data suggest that extracellular gelsolin is involved in the host immune recognition of LTA or LPS following release of these molecules from the bacterial outer membrane during cell division or attack by drugs and immune components.  相似文献   

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