Rat Anti-Human IL-4 Monoclonal antibody, clone MP4-25D2 Functional Grade

Neurodegenerative disorders present a broad group of neurological diseases and remain one of the greatest challenges and burdens to mankind. Maladies like amyotrophic lateral sclerosis, Alzheimer's disease, stroke or spinal cord injury commonly features astroglia involvement (astrogliosis) with signs of inflammation. Regenerative, paracrine and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) could target the above components, thus opening new therapeutic possibilities for regenerative medicine. A special interest should be given to hMSCs derived from the umbilical cord (UC) tissue, due to their origin, properties and lack of ethical paradigms. The aim of this study was to establish standard operating and scale-up good manufacturing practice (GMP) protocols of UC-hMSCs isolation, characterization, expansion and comparison of cells' properties when harvested on T-flasks versus using a large-scale bioreactor system. Human UC-hMSCs, isolated by tissue explant culture technique from Wharton's jelly, were harvested after reaching 75% confluence and cultured using tissue culture flasks. Obtained UC-hMSCs prior/after the cryopreservation and after harvesting in a bioreactor, were fully characterized for "mesenchymness" immunomodulatory, tumorigenicity and genetic stability, senescence and cell-doubling properties, as well as gene expression features. Our study demonstrates an efficient and simple technique for large scale UC-hMSCs expansion. Harvesting of UC-hMSCs' using classic and large scale methods did not alter UC-hMSCs' senescence, genetic stability or in vitro tumorigenicity features. We observed comparable growth and immunomodulatory capacities of fresh, frozen and expanded UC-hMSCs. We found no difference in the ability to differentiate toward adipogenic, osteogenic and chondrogenic lineages between classic and large scale UC-hMSCs expansion methods. Both, methods enabled derivation of genetically stabile cells with typical mesenchymal features. Interestingly, we found significantly increased mRNA expression levels of neural growth factor (NGF) and downregulated insulin growth factor (IGF) in UC-hMSCs cultured in bioreactor, while IL4, IL6, IL8, TGFb and VEGF expression levels remained at the similar levels. A culturing of UC-hMSCs using a large-scale automated closed bioreactor expansion system under the GMP conditions does not alter basic "mesenchymal" features and quality of the cells. Our study has been designed to pave a road toward translation of basic research data known about human UC-MSCs for the future clinical testing in patients with neurological and immunocompromised disorders. An industrial manufacturing of UC-hMSCs next will undergo regulatory approval following advanced therapy medicinal products (ATMP) criteria prior to clinical application and approval to be used in patients.

Rheumatoid arthritis is considered a T-lymphocyte-mediated disease. However, studies have focussed on CD4 T-lymphocytes, ignoring CD8 T-lymphocytes despite the latter being found abundantly in the synovium. Specifically, there is little data of the effect of methotrexate, the gold-standard DMARD, on various CD8 cytokine T-lymphocyte subsets and conflicting data on CD4 subsets. In this prospective study, patients with active rheumatoid arthritis, who were 18 to 65years of age, were treated with methotrexate (up to 25mg per week) for 24weeks. At baseline and 24weeks, frequencies of CD8(+)IFN(+), CD8(+)IL17(+), CD8(+)IL4(+), corresponding CD4 subsets and plasma levels of IFN, IL-12, IL-10, IL-4 and IL-17 were determined by flow cytometry. These are summarised as median (IQR=interquartile range, 25th-75th percentile) and paired data compared using Wilcoxon signed rank test. This study included 67 patients (F/M=4:1) with rheumatoid arthritis, 57 (85%) being RF positive and 20 receiving prednisolone at baseline. Mean (+/- SD) dose of methotrexate at 24weeks was 22.9 +/- 3.0mg per week. On treatment with methotrexate, there was a significant (p=0.04) decline in CD8(+)IFN(+) cells from 37.2 (IQR 19.4-60.2) to 22.7% (IQR 8.5-49.7) and a marginal increase in CD8(+)IL17(+) cells from 0.3 (IQR 0.1-0.6) to 0.4 (IQR 0.2-1.2), p=0.006. There was no significant change in the other subsets. There was also a significant decline in circulating levels of IL-12, IL-10 and IL-17 and marginal increase in IL-4. On evaluating by response, non-responders but not responders had a significant increase in CD8(+)IL17(+) (p=0.01). There is a significant decline of CD8(+)IFN(+) T cells and marginal increase in CD8(+)IL17(+) T cells after methotrexate. Change in Tc1 subset may be mediated through reduction in IL-12 levels.