THE CHARACTERIZATION OF MYCOBACTERIAL STRAINS BY THE COMPOSITION OF THEIR LIPIDE EXTRACTS

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72212/1/j.1749-6632.1957.tb49655.x.pd

TWIST1 controls cellular senescence and energy metabolism in mesenchymal stem cells

Mesenchymal stem cells (MSCs) are promising cells for regenerative medicine therapies because they can differentiate towards multiple cell lineages. However, the occurrence of cellular senescence and the acquiring of the senescence-associated secretory phenotype (SASP) limit their clinical use. Since the transcription factor TWIST1 influences expansion of MSCs, its role in regulating cellular senescence was investigated. The present study demonstrated that silencing of TWIST1 in MSCs increased the occurrence of senescence, characterised by a SASP profile different from irradiation-induced senescent MSCs. Knowing that senescence alters cellular metabolism, cellular bioenergetics was monitored by using the Seahorse XF apparatus. Both TWIST1-silencing-induced and irradiation-induced senescent MSCs had a higher oxygen consumption rate compared to control MSCs, while TWIST1-silencing-induced senescent MSCs had a low extracellular acidification rate compared to irradiation-induced senescent MSCs. Overall, data indicated how TWIST1 regulation influenced senescence in MSCs and that TWIST1 silencing-induced senescence was characterised by a specific SASP profile and metabolic state

Angiogenic Potential of Tissue Engineered Cartilage From Human Mesenchymal Stem Cells Is Modulated by Indian Hedgehog and Serpin E1

With rising demand for cartilage tissue repair and replacement, the differentiation of mesenchymal stem cells (BMSCs) into cartilage tissue forming cells provides a promising solution. Often, the BMSC-derived cartilage does not remain stable and continues maturing to bone through the process of endochondral ossification in vivo. Similar to the growth plate, invasion of blood vessels is an early hallmark of endochondral ossification and a necessary step for completion of ossification. This invasion originates from preexisting vessels that expand via angiogenesis, induced by secreted factors produced by the cartilage graft. In this study, we aimed to identify factors secreted by chondrogenically differentiated bone marrow-derived human BMSCs to modulate angiogenesis. The secretome of chondrogenic pellets at day 21 of the differentiation program was collected and tested for angiogenic capacity using in vitro endothelial migration and proliferation assays as well as the chick chorioallantoic membrane (CAM) assay. Taken together, these assays confirmed the pro-angiogenic potential of the secretome. Putative secreted angiogenic factors present in this medium were identified by comparative global transcriptome analysis between murine growth plate cartilage, human chondrogenic BMSC pellets and human neonatal articular cartilage. We then verified by PCR eight candidate angiogenesis modulating factors secreted by differentiated BMSCs. Among those, Serpin E1 and Indian Hedgehog (IHH) had a higher level of expression in BMSC-derived cartilage compared to articular chondrocyte derived cartilage. To understand the role of these factors in the pro-angiogenic secretome, we used neutralizing antibodies to functionally block them in the conditioned medium. Here, we observed a 1.4-fold increase of endothelial cell proliferation when blocking IHH and 1.5-fold by Serpin E1 blocking compared to unblocked control conditioned medium. Furthermore, endothelial migration was increased 1.9-fold by Serpin E1 blocking and 2.7-fold by IHH blocking. This suggests that the pro-angiogenic potential of chondrogenically differentiated BMSC secretome could be further augmented through inhibition of specific factors such as IHH and Serpin E1 identified as anti-angiogenic factors

Reduced nasal IL-10 and enhanced TNFalpha responses during rhinovirus and RSV-induced upper respiratory tract infection in atopic and non-atopic infants

Rhinovirus and respiratory syncytial virus (RSV) are the most prevalent inducers of upper respiratory tract infections (URTI) in infants and may stimulate immune maturation. To estimate the amount of immune stimulation, nasal immune responses were examined during rhinovirus and RSV-induced URTI in infants. Nasal brush samples were taken from infants (2-26 months; 57% atopic family) with rhinovirus-induced URTI (N=20), with RSV-induced URTI (N=7), and with rhinovirus-induced rhinitis (N=11), from children with asymptomatic rhinovirus infection (N=7) and from eight non-infected children. Numbers of nasal brush cells positive for Th1-, Th2-, regulatory and proinflammatory cytokines were measured by immunohistochemistry or by measuring protein levels using a cytometric bead array analysis. During rhinovirus and RSV-induced URTI, fewer regulatory cytokine IL-10 positive cells were found compared to non-infected children. This fall was accompanied by an increase in levels of the Th1 cytokine TNFalpha. IL-10 responses were inversely related to TNFalpha responses. No enhanced responses were observed for IFNgamma, IL-12 and IL-18. Cytokine responses were comparable in children with rhinovirus-induced URTI and in children with rhinitis, while responses in asymptomatic rhinovirus-infected children were located between those for symptomatic and asymptomatic rhinovirus-infected children. Cytokine responses did not depend on the age of the child or atopy in the family. In conclusion, reduced nasal IL-10 responses during URTI in infants could facilitate the induction of a TNFalpha response. TNFalpha in turn could replace the immature production of IL-12, IL-18 and IFNgamma during URTI to induce an effective clearance of the viral infection and which could stimulate the maturation of Th1 cytokine production in infanc

Differences in cartilage-forming capacity of expanded human chondrocytes from ear and nose and their gene expression profiles

The aim of this study was to evaluate the potential of culture-expanded human auricular and nasoseptal chondrocytes as cell source for regeneration of stable cartilage and to analyze the differences in gene expression profile of expanded chondrocytes from these specific locations. Auricular chondrocytes in monolayer proliferated less and more slowly (two passages took 26.7 ± 2.1 days and were reached in 4.37 ± 0.30 population doublings) than nasoseptal chondrocytes (19.3 ± 2.5 days; 5.45 ± 0.20 population doublings). However, auricular chondrocytes produced larger pellets with more cartilage-like matrix than nasoseptal chondrocytes (2.2 ± 0.71 vs. 1.7 ± 0.13 mm in diameter after 35 days of culture). Although the matrix formed by auricular and nasoseptal chondrocytes contained collagen X, it did not mineralize in an in vitro model or after in vivo subcutaneous implantation. A DNA microarray study on expanded auricular and nasoseptal chondrocytes from the same donors revealed 1,090 differentially expressed genes. No difference was observed in the expression of known markers of chondrogenic capacity (e.g., collagen II, FGFR3, BMP2, and ALK1). The most striking differences were that the auricular chondrocytes had a higher expression of anabolic growth factors BMP5 and IGF1, while matrix-degrading enzymes MMP13 and ADAMTS5 were higher expressed in nasoseptal chondrocytes. This might offer a possible explanation for the observed higher matrix production by auricular chondrocytes. Moreover, chondrocytes isolated from auricular or nasoseptal cartilage had specific gene expression profiles even after expansion. These differently expressed genes were not restricted to known characterization of donor site subtype (e.g., elastic), but were also related to developmental processe

Constitutively active (ca) caALK5 and caALK1 receptors do not induce BMSC chondrogenesis as efficiently as TGFβ does.

<p>Human fetal BMSCs (donor F1) transduced with adenoviral caALK5, caALK1 or LacZ as control were pellet-cultured in chondrogenic medium for 7 days. Only LacZ-transduced pellets were exposed to TGFβ to compare if either caALK5 or caALK1 induced chondrogenesis as efficiently as induced by TGFβ. Chondrogenesis was evaluated by cartilage-specific gene expression analysis of <i>ACAN</i> <b>(A)</b> and <i>COL2A1</i> <b>(B)</b> and by staining proteoglycans with Safranin O/Fast Green <b>(C; upper panel)</b> and collagen type II with immunohistochemistry <b>(C; lower panel)</b>. Representative images of consecutive pellet sections per condition are shown and the scale bar represents 500 μm. Gene expression was normalized to reference gene <i>RPS27a</i> and data are expressed as % relative to normalized gene levels in LacZ-transduced pellets stimulated with TGFβ. Bars represent mean ± SD from quadruplet pellets of 1 representative experiment (out of 3), ***p<0.001.</p