Involvement of BMP-2, TGF-ß2 and TGF-ß3 Signaling in Initial and Early Stages of Heterotopic Ossification in a rat Experimental Model

This study focused on the localization and expression of bone morphogenetic protein 2 (BMP-2) and different  isoforms of transforming growth factor s (TGF-s1, TGF-s2 and TGF-s3) in the initial and early stages  of heterotopic ossification (HO) employing an animal model mimicking the situation after total hip arthroplasty  (THA). Bone growth was induced in rats using s-tricalcium phosphate implants immersed either in  osteoinductive rhBMP-2 solution or in saline and implanted at the site where the HO is usually expected  to develop after THA. Implants were removed at 3 or 21 days after the operation and handled according to  stereology principles. mRNA expression and protein staining of growth factors in different types of tissues  was determined by in situ hybridization and immunohistochemistry, respectively. After three days, TGF-s3 content in the undifferentiated mesenchymal-like cells in the rhBMP-2 treated  implants was, as assessed by immunohistochemistry, 49.6% higher compared to the saline treated group  (p=0.024). This was also supported by in situ hybridization of mRNA of TGF-s3, which showed stronger  expression in rhBMP-2 treated group. Immunohistochemical investigation showed that after 21 days the  connective tissue in the rhBMP-2 treated implants contained more TGF-s1, TGF-s2 and TGF-s3, compared  to BMP-2 and osteoblasts contained significantly (27.2%) more TGF-s3 compared to TGF-s1 (p=0.045). In  the formed HO the proportion of the TGF-s2 and TGF-s3 producing bone tissue was increased by 32.1% and  47.8% respectively, compared to the TGF-s1 producing bone tissue (p=0.007 and p=0.006) and although  this difference was not so clear at mRNA level, this suggests that TGF-s2 and TGF-s3 signaling seem to play  an important role during initial and early stages of HO formation.

Mast cells differentiated in synovial fluid and resident in osteophytes exalt the inflammatory pathology of osteoarthritis

Introduction: Osteophytes are a prominent feature of osteoarthritis (OA) joints and one of the clinical hallmarks of the disease progression. Research on osteophytes is fragmentary and modes of its contribution to OA pathology are obscure. Aim: To elucidate the role of osteophytes in OA pathology from a perspective of molecular and cellular events. Methods: RNA-seq of fully grown osteophytes, collected from tibial plateau of six OA patients revealed patterns corresponding to active extracellular matrix re-modulation and prominent participation of mast cells. Presence of mast cells was further confirmed by immunohistochemistry, performed on the sections of the osteophytes using anti-tryptase alpha/beta-1 and anti-FC epsilon RI antibodies and the related key up-regulated genes were validated by qRT-PCR. To test the role of OA synovial fluid (SF) in mast cell maturation as proposed by the authors, hematopoietic stem cells (HSCs) and ThP1 cells were cultured in a media supplemented with 10% SF samples, obtained from various grades of OA patients and were monitored using specific cell surface markers by flow cytometry. Proteomics analysis of SF samples was performed to detect additional markers specific to mast cells and inflammation that drive the cell differentiation and maturation. Results: Transcriptomics of osteophytes revealed a significant upregulation of mast cells specific genes such as chymase 1 (CMA1; 5-fold) carboxypeptidase A3 (CPA3; 4-fold), MS4A2/FCERI (FCERI; 4.2-fold) and interleukin 1 receptor-like 1 (IL1RL1; 2.5-fold) indicating their prominent involvement. (In IHC, anti-tryptase alpha/beta-1 and anti- FC epsilon RI-stained active mast cells were seen populated in cartilage, subchondral bone, and trabecular bone.) Based on these outcomes and previous learnings, the authors claim a possibility of mast cells invasion into osteophytes is mediated by SF and present in vitro cell differentiation assay results, wherein ThP1 and HSCs showed differentiation into HLA-DR+/CD206+ and FCERI+ phenotype, respectively, after exposing them to medium containing 10% SF for 9 days. Proteomics analysis of these SF samples showed an accumulation of mast cell-specific inflammatory proteins. Conclusions: RNA-seq analysis followed by IHC study on osteophyte samples showed a population of mast cells resident in them and may further accentuate inflammatory pathology of OA. Besides subchondral bone, the authors propose an alternative passage of mast cells invasion in osteophytes, wherein OA SF was found to be necessary and sufficient for maturation of mast cell precursor into effector cells

Expression of Class II Cytokine Genes in Children’s Skin

Immune regulation of the skin plays an important role in susceptibility and development of illnesses. The aim of our study was to localise the interleukin (IL)-10 family of cytokines, in children’s skin and to determine possible age-related differences in the expression level. The mRNA expression level of IL10, IL19, IL20, IL22, IL24, IL26, IL28B, IL29 and their receptors IL10RA, IL10RB, IL20RA, IL20RB, IL22RA1, IL22RA2, IL28RA was compared in skin biopsies of children and adults and in childrens’ skin cells by quantitative real-time PCR (qRT-PCR). Immunohistochemistry was performed to confirm the qRT-PCR findings. We found age-related differences in the expression of IL10RB, IL20, IL20RA, IL22RA1, IL22RA2, IL26 and IL28RA genes. Cell type-dependent expression of IL10 family cytokines was apparent in the skin. In addition to previously known differences in systemic immunological response of adults and children, the present results reveal differences in immune profile of adult and juvenile skin