Matrix metalloproteinase-9 and cell kinetics during the collection of peripheral blood stem cells by leukapheresis.

The plasma levels of matrix metalloproteinase-9 (MMP-9) were determined during 41 collections of peripheral blood stem cells (PBSC) by standard volume (two blood volumes) leukaphereses (SVL) in 29 donors (7 allogeneic and 22 autologous) mobilized with the granulocyte-colony stimulating factor (G-CSF). The association between MMP-9 levels and cell counts in donor's blood was explored. During the processing of the first blood volume (BV), MMP-9 levels declined on average by 31% and persisted at the same level during the processing of the second BV. During the collection, a slight decline of white blood cells (WBC), polymorphonuclear neutrophils (PMN) and platelets (PLT) in donor's blood was accompanied by a significant drop of CD34+ cells by 37% after 1 BV and by 44% after 2 BV had been processed (p=0.001). The drop of MMP-9 plasma levels showed a loose correlation with the decrease of WBC (r=0.68, p=0.002) and PMN counts (r=0.67, p=0.001). We conclude that the levels of MMP-9 that have been elevated by the mobilization of donors with G-CSF, decrease during the collection of PBSC by 4h SVL. The observed decrease was indirectly related to the drop of WBC and PMN counts, suggesting that certain other factors have an influence on MMP-9 kinetics during PBSC collection.

Vitamin C induced apoptosis in human articular chondrocytes.

Chondrocytes present in articular cartilage survive as a resident cell population throughout the lifespan of the individual organism. However, articular chondrocytes as other cells also undergo apoptosis and there is an ever increasing list of diverse stimuli that can induce this phenomenon in vitro. Our main interest was to investigate potential cytotoxic effects of vitamin C (L-ascorbic acid) on human articular chondrocytes. The present study suggests that vitamin C can induce apoptosis in a cell culture of chondrocytes after 18 h of cultivation. Apoptosis-inducing activity of L-ascorbic acid is dose dependent and significantly affected by the presence of serum. The increased number of vitamin C induced apoptotic cells was associated with DNA fragmentation and morphological changes of the cells.

Expression of TIMP-1 in Pichia pastoris. Selection of an anti-TIMP-1 specific single-chain Fv antibody from a large non-immune library.

To quantitate tissue inhibitor of metalloproteinase (TIMP)-1 in biological samples, a strategy for isolation of monoclonal antibodies was applied that employs a phage-displayed single-chain Fv (scFv). In order to obtain sufficient amounts of TIMP-1 to use as an antigen, high-level expression in Pichia pastoris was achieved under the control of the AOX-1 promotor. Purified protein antigen was then used for panning to achieve enrichment of specific phage from naive scFv library. In five subsequent panning rounds, antibody fragments that display specificity to TIMP-1 were selected. Regions encoding scFv were subcloned into a vector allowing production of scFv-alkaline phosphatase (AP) fusion proteins. Two such conjugates displaying dose-dependent reactivity with TIMP-1 were isolated and characterised, providing the basis for the construction of a TIMP-1 quantitation assay based entirely on recombinant proteins.

An ELISA for the detection of TIMP-1 based on recombinant single chain Fv fusion proteins.

BACKGROUND: Altered serum levels of TIMP-1 are an indicator of various pathological states. To quantitate TIMP-1 in biological samples, we have previously isolated TIMP-1 specific single-chain Fv fragments (scFvs) using phage-display. In the work presented here, we have used these scFvs to establish a TIMP-1 ELISA based exclusively on recombinant scFv fusion proteins. METHODS: Two distinct TIMP-1 specific scFvs were used as the antigen binding components after being genetically fused to the N-termini of two different fusion protein constructs. One fusion protein, comprising a CL domain, serves as a coating reagent, while the second fusion protein with a modified form of bacterial alkaline phosphatase is used as a detection reagent. A double antibody sandwich-ELISA was then established and optimized. RESULTS: An ELISA for the quantitation of tissue inhibitor of metalloproteinase (TIMP)-1, based entirely on recombinant antibody fragments, was developed as an alternative to assays using polyclonal antisera or monoclonal antibodies. Its performance was shown to compare well with a conventional ELISA. Finally, TIMP-1 concentrations in the sera of sixty healthy individuals were determined. CONCLUSION: The assay described here provides a standardized, reliable and readily available means of quantitation of TIMP-1 in a large number of blood samples.

The outcome of autologous chondrocyte transplantation treatment of cartilage lesions in the knee.

Recent results of the clinical outcome of autologous chondrocyte transplantation (ACT) treatment in a group of 28 patients with focal femoral condyle cartilage lesions revealed a correlation trend with the quality of the in vitro cell culture matrix-protein synthesis. No impact of the patients' age and chondrocyte cryopreservation prior to implantation was observed. Further studies are needed to confirm the preliminary results.