Optimized Transduction of Canine Paediatric CD34+ Cells Using an MSCV-based Bicistronic Vector

Suter, Steven E.; Gouthro, Terry A.; McSweeney, Peter A.; Nash, Richard A.; Haskins, Mark E.; Felsburg, Peter J.; Henthorn, Paula S.

doi:10.1007/s11259-006-3356-7

Cited by 7 publications

(7 citation statements)

References 91 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Lentiviral infections of B-cells were performed using the centrifugation method 109 . In brief, 7 to 14×10 6 peripheral blood B cells were re-suspended in 5 to 10 ml of lenti-viral supernatant with 8 µg/mL final concentration of polybreme (Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

“…In brief, 7 to 14×10 6 peripheral blood B cells were re-suspended in 5 to 10 ml of lenti-viral supernatant with 8 µg/mL final concentration of polybreme (Sigma-Aldrich). The mixture was centrifuged in 6 well plates for 1.5 h at 2,500g at 32°C 109 in order to achieve close contact between the retroviral particles and the cells. After 24 h, the media was replaced with fresh RPMI complete (at 1×10 6 cells/mL), and cells were cultured at 37°C and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Small-Molecule Inhibitors of the CD40–CD40L Costimulatory Protein–Protein Interaction

et al. 2017

View full text Add to dashboard Cite

Costimulatory interactions are required for T cell activation and development of an effective immune response; hence, they are valuable therapeutic targets for immunomodulation. However, they, as all other protein-protein interactions, are difficult to target by small molecules. Here, we report the identification of novel small-molecule inhibitors of the CD40–CD40L interaction designed starting from the chemical space of organic dyes. For the most promising compounds such as DRI-C21045, activity (IC50) in the low micromolar range has been confirmed in cell assays including inhibition of CD40L-induced activation in NF-κB sensor cells, THP-1 myeloid cells, and primary human B cells as well as in murine allogeneic skin transplant and alloantigen-induced T cell expansion in draining lymph node experiments. Specificity versus other TNF-superfamily interactions (TNF-R1–TNF-α) and lack of cytotoxicity have also been confirmed at these concentrations. These novel compounds provide proof-of-principle evidence for the possibility of small-molecule inhibition of costimulatory protein-protein interactions, establish the structural requirements needed for efficient CD40–CD40L inhibition, and serve to guide the search for such immune therapeutics.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Small-Molecule Inhibitors of the CD40–CD40L Costimulatory Protein–Protein Interaction

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Cells were isolated as described elsewhere (27) and cultured in Dulbecco's modified Eagle's medium (DMEM) with L-glutamine, penicillin/streptomycin, and 10% fetal bovine serum (FBS). Spinfection was performed at 70% confluency with replication-deficient adenovirus expressing Cre recombinase to inactivate Smad3 in cultured chondrocytes from Smad3 fl/fl mice (28). As a negative control, cells were infected with adenovirus expressing green fluorescent protein (GFP).…”

Section: Methodsmentioning

confidence: 99%

Chondrocyte‐intrinsic Smad3 represses Runx2‐inducible matrix metalloproteinase 13 expression to maintain articular cartilage and prevent osteoarthritis

Chen¹,

Thuillier²,

Chin³

et al. 2012

Arthritis & Rheumatism

120

117

View full text Add to dashboard Cite

Objective To identify mechanisms by which Smad3 maintains articular cartilage and prevents osteoarthritis. Methods A combination of in vivo and in vitro approaches was used to test the hypothesis that Smad3 represses Runx2-inducible gene expression to prevent articular cartilage degeneration. Col2-Cre;Smad3fl/fl mice allowed study of the chondrocyte-intrinsic role of Smad3, independently of its role in the perichondrium or other tissues. Primary Smad3fl/fl articular chondrocytes and ATDC5 chondroprogenitors were employed to evaluate Smad3 and Runx2 regulation of matrix metalloproteinase-13 (MMP-13) mRNA and protein expression. Results Chondrocyte-specific reduction of Smad3 causes progressive articular cartilage degeneration due to imbalanced cartilage matrix synthesis and degradation. In addition to reduced collagen II mRNA expression, Col2-Cre;Smad3fl/fl articular cartilage is severely deficient in collagen II and aggrecan protein, due to excessive MMP-13-mediated proteolysis of these key cartilage matrix constituents. Normally, TGF-β signals through Smad3 to confer a rapid and dynamic repression of Runx2-inducible MMP-13 expression. However, in the absence of Smad3, TGF-β signals through p38 and Runx2 to induce MMP-13 expression. Conclusion This work elucidates a mechanism by which Smad3 mutations in humans and mice cause cartilage degeneration and osteoarthritis. Specifically, Smad3 maintains the balance between cartilage matrix synthesis and degradation by inducing collagen II expression and repressing Runx2-inducible MMP-13 expression. Selective activation of TGF-β signaling through Smad3, rather than p38, may help to restore the balance between matrix synthesis and proteolysis that is lost in osteoarthritis.

show abstract

“…Adenovirus with GFP/Cre-recombinase or GFP were added to the chondrocytes at 400 pfu/cell. Analysis of GFP expressing cells 12 h after infection verified that this protocol yields a 90% infection rate ( Suter et al, 2006 ). All experiments used cells at third passage (after harvest digestion, infection and appropriate media change) at day 2 after initial harvest.…”

Section: Methodsmentioning

confidence: 91%

Smad4 regulates growth plate matrix production and chondrocyte polarity

et al. 2017

View full text Add to dashboard Cite

Smad4 is an intracellular effector of the TGFβ family that has been implicated in Myhre syndrome, a skeletal dysplasia characterized by short stature, brachydactyly and stiff joints. The TGFβ pathway also plays a critical role in the development, organization and proliferation of the growth plate, although the exact mechanisms remain unclear. Skeletal phenotypes in Myhre syndrome overlap with processes regulated by the TGFβ pathway, including organization and proliferation of the growth plate and polarity of the chondrocyte. We used in vitro and in vivo models of Smad4 deficiency in chondrocytes to test the hypothesis that deregulated TGFβ signaling leads to aberrant extracellular matrix production and loss of chondrocyte polarity. Specifically, we evaluated growth plate chondrocyte polarity in tibiae of Col2-Cre+/−;Smad4fl/fl mice and in chondrocyte pellet cultures. In vitro and in vivo, Smad4 deficiency decreased aggrecan expression and increased MMP13 expression. Smad4 deficiency disrupted the balance of cartilage matrix synthesis and degradation, even though the sequential expression of growth plate chondrocyte markers was intact. Chondrocytes in Smad4-deficient growth plates also showed evidence of polarity defects, with impaired proliferation and ability to undergo the characteristic changes in shape, size and orientation as they differentiated from resting to hypertrophic chondrocytes. Therefore, we show that Smad4 controls chondrocyte proliferation, orientation, and hypertrophy and is important in regulating the extracellular matrix composition of the growth plate.

show abstract

Optimized Transduction of Canine Paediatric CD34+ Cells Using an MSCV-based Bicistronic Vector

Cited by 7 publications

References 91 publications

Small-Molecule Inhibitors of the CD40–CD40L Costimulatory Protein–Protein Interaction

Small-Molecule Inhibitors of the CD40–CD40L Costimulatory Protein–Protein Interaction

Chondrocyte‐intrinsic Smad3 represses Runx2‐inducible matrix metalloproteinase 13 expression to maintain articular cartilage and prevent osteoarthritis

Smad4 regulates growth plate matrix production and chondrocyte polarity

Contact Info

Product

Resources

About