Collagen-mimetic peptide-modifiable hydrogels for articular cartilage regeneration

Regenerative medicine strategies for restoring articular cartilage face significant challenges to recreate the complex and dynamic biochemical and biomechanical functions of native tissues. As an approach to recapitulate the complexity of the extracellular matrix, collagen-mimetic proteins offer a modular template to incorporate bioactive and biodegradable moieties into a single construct. We modified a Streptococcal collagen-like 2 protein with hyaluronic acid (HA) or chondroitin sulfate (CS)-binding peptides and then cross-linked with a matrix metalloproteinase 7 (MMP7)-sensitive peptide to form biodegradable hydrogels. Human mesenchymal stem cells (hMSCs) encapsulated in these hydrogels exhibited improved viability and significantly enhanced chondrogenic differentiation compared to controls that were not functionalized with glycosaminoglycan-binding peptides. Hydrogels functionalized with CS-binding peptides also led to significantly higher MMP7 gene expression and activity while the HA-binding peptides significantly increased chondrogenic differentiation of the hMSCs. Our results highlight the potential of this novel biomaterial to modulate cell-mediated processes and create functional tissue engineered constructs for regenerative medicine applications

Involvement of the Cytokine MIF in the Snail Host Immune Response to the Parasite Schistosoma mansoni

We have identified and characterized a Macrophage Migration Inhibitory Factor (MIF) family member in the Lophotrochozoan invertebrate, Biomphalaria glabrata, the snail intermediate host of the human blood fluke Schistosoma mansoni. In mammals, MIF is a widely expressed pleiotropic cytokine with potent pro-inflammatory properties that controls cell functions such as gene expression, proliferation or apoptosis. Here we show that the MIF protein from B. glabrata (BgMIF) is expressed in circulating immune defense cells (hemocytes) of the snail as well as in the B. glabrata embryonic (Bge) cell line that has hemocyte-like features. Recombinant BgMIF (rBgMIF) induced cell proliferation and inhibited NO-dependent p53-mediated apoptosis in Bge cells. Moreover, knock-down of BgMIF expression in Bge cells interfered with the in vitro encapsulation of S. mansoni sporocysts. Furthermore, the in vivo knock-down of BgMIF prevented the changes in circulating hemocyte populations that occur in response to an infection by S. mansoni miracidia and led to a significant increase in the parasite burden of the snails. These results provide the first functional evidence that a MIF ortholog is involved in an invertebrate immune response towards a parasitic infection and highlight the importance of cytokines in invertebrate-parasite interactions

TLiSA1, a human T lineage-specific activation antigen involved in the differentiation of cytotoxic T lymphocytes and anomalous killer cells from their precursors.

The characteristics of a novel T lineage-specific activation antigen, termed TLiSA1, are described. The antigen was detected with a mouse monoclonal antibody, LeoA1, that was raised against activated human T cells generated in mixed lymphocyte culture (MLC). The antigen became strongly expressed on T cells 48-72 h after stimulation with phytohemagglutinin, and retained expression on MLC-activated T cells after 10 d of culture. The antigen was absent from a range of human T, B, myeloid, fibroblast, and tumour cell lines, but was present on the surface of the interleukin 2 (IL-2)-dependent gibbon cell line MLA-144. Analysis of the antigen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from activated human T cells demonstrated a broad band in the region of 70 kD, whereas precipitates obtained from MLA-144 revealed a single narrow band of 95 kD. The molecule was expressed with a maximum density of 66,000 copies per cell on the surface of MLC-activated T cell blasts, as assessed by Scatchard analysis. TLiSA1 was distinguished from the IL-2 receptor bound by the anti-Tac monoclonal antibody by demonstrating that the antigens did not comodulate or coprecipitate, and by constructing an IL-2-independent human T X T hybrid that expressed the TLiSA1 but not the Tac antigen. MLC with B lymphoblasts was used to generate cytotoxic T lymphocytes (CTL) specific for the stimulating cell, and anomalous killer (AK) cells able to kill melanoma target cells. The presence of LeoA1 or F(ab')2 fragments of the antibody from the beginning of coculture did not affect proliferation in these cultures, but did inhibit the induction of both CTL and AK cells from their precursors. This inhibition of differentiation by LeoA1 was confirmed under conditions of limiting dilution, where it was shown that the antibody reduced the frequency of CTL produced, and greatly (fourfold) reduced the frequency of AK cells generated from their precursors. We discuss the possibility that human CTL may express a differentiation factor receptor that is distinct from the receptor for IL-2

Temporally degradable collagen–mimetic hydrogels tuned to chondrogenesis of human mesenchymal stem cells

Tissue engineering strategies for repairing and regenerating articular cartilage face critical challenges to recapitulate the dynamic and complex biochemical microenvironment of native tissues. One approach to mimic the biochemical complexity of articular cartilage is through the use of recombinant bacterial collagens as they provide a well-defined biological 'blank template' that can be modified to incorporate bioactive and biodegradable peptide sequences within a precisely defined three-dimensional system. We customized the backbone of a Streptococcal collagen-like 2 (Scl2) protein with heparin-binding, integrin-binding, and hyaluronic acid-binding peptide sequences previously shown to modulate chondrogenesis and then cross-linked the recombinant Scl2 protein with a combination of matrix metalloproteinase 7 (MMP7)- and aggrecanase (ADAMTS4)-cleavable peptides at varying ratios to form biodegradable hydrogels with degradation characteristics matching the temporal expression pattern of these enzymes in human mesenchymal stem cells (hMSCs) during chondrogenesis. hMSCs encapsulated within the hydrogels cross-linked with both degradable peptides exhibited enhanced chondrogenic characteristics as demonstrated by gene expression and extracellular matrix deposition compared to the hydrogels cross-linked with a single peptide. Additionally, these combined peptide hydrogels displayed increased MMP7 and ADAMTS4 activities and yet increased compression moduli after 6 weeks, suggesting a positive correlation between the degradation of the hydrogels and the accumulation of matrix by hMSCs undergoing chondrogenesis. Our results suggest that including dual degradation motifs designed to respond to enzymatic activity of hMSCs going through chondrogenic differentiation led to improvements in chondrogenesis. Our hydrogel system demonstrates a bimodal enzymatically degradable biological platform that can mimic native cellular processes in a temporal manner. As such, this novel collagen-mimetic protein, cross-linked via multiple enzymatically degradable peptides, provides a highly adaptable and well defined platform to recapitulate a high degree of biological complexity, which could be applicable to numerous tissue engineering and regenerative medicine applications