Inflammatory signals from fatty bone marrow support DNMT3A driven clonal hematopoiesis.

Both fatty bone marrow (FBM) and somatic mutations in hematopoietic stem cells (HSCs), also termed clonal hematopoiesis (CH) accumulate with human aging. However it remains unclear whether FBM can modify the evolution of CH. To address this question, we herein present the interaction between CH and FBM in two preclinical male mouse models: after sub-lethal irradiation or after castration. An adipogenesis inhibitor (PPARγ inhibitor) is used in both models as a control. A significant increase in self-renewal can be detected in both human and rodent DNMT3AMut-HSCs when exposed to FBM. DNMT3AMut-HSCs derived from older mice interacting with FBM have even higher self-renewal in comparison to DNMT3AMut-HSCs derived from younger mice. Single cell RNA-sequencing on rodent HSCs after exposing them to FBM reveal a 6-10 fold increase in DNMT3AMut-HSCs and an activated inflammatory signaling. Cytokine analysis of BM fluid and BM derived adipocytes grown in vitro demonstrates an increased IL-6 levels under FBM conditions. Anti-IL-6 neutralizing antibodies significantly reduce the selective advantage of DNMT3AMut-HSCs exposed to FBM. Overall, paracrine FBM inflammatory signals promote DNMT3A-driven clonal hematopoiesis, which can be inhibited by blocking the IL-6 pathway.

Modulation of MS-like disease by a multi epitope protein is mediated by induction of CD11c+CD11b+Gr1+ myeloid-derived dendritic cells.

Specific neutralization of the pathogenic autoimmune cells is the ultimate goal in therapy of Multiple Sclerosis (MS). However, the pathogenic autoimmunity in MS, can be directed against several major target antigens, and therefore targeting pathogenic T-cells directed against a single target antigen is unlikely to be effective. To overcome this multiplicity and the potential complexity of pathogenic autoreactivities in MS, we have put forward the concept of concomitant multi-antigen/multi-epitope targeting as, a conceivably more effective approach to immunotherapy of MS. We constructed an (Experimental Autoimmune Encephalomeylitis (EAE)/MS-related synthetic human Target Autoantigen Gene (MS-shMultiTAG) designed to encode in tandem only EAE/MS related epitopes of all known encephalitogenic proteins. The MS-related protein product (designated Y-MSPc) was immunofunctional and upon tolerogenic administration, it effectively suppressed and reversed EAE induced by a single encephalitogenic protein. Furthermore, Y-MSPc also fully abrogated the development of "complex EAE" induced by a mixture of five encephalitogenic T-cell lines, each specific for a different encephalitogenic epitope of MBP, MOG, PLP, MOBP and OSP. Strikingly, Y-MSPc was consistently more effective than treatment with the single disease-specific peptide or with the peptide cocktail, both in suppressing the development of "classical" or "complex" EAE and in ameliorating ongoing disease. Overall, the modulation of EAE by Y-MSPc was associated with anergizing the pathogenic autoreactive T-cells, downregulation of Th1/Th17 cytokine secretion and upregulation of TGF-ß secretion. Moreover, we show that both suppression and treatment of ongoing EAE by tolerogenic administration of Y-MSPc is associated also with a remarkable increase in a unique subset of dendritic-cells (DCs), CD11c+CD11b+Gr1+-myeloid derived DCs in both spleen and CNS of treated mice. These DCs, which are with strong immunoregulatory characteristics and are functional in down-modulation of MS-like-disease displayed increased production of IL-4, IL-10 and TGF-ß and low IL-12. Functionally, these myeloid DCs suppress the in-vitro proliferation of myelin-specific T-cells and more importantly, the cells were functional in-vivo, as their adoptive transfer into EAE induced mice resulted in strong suppression of the disease, associated with a remarkable induction of CD4 + FoxP3+ regulatory cells. These results, which highlight the efficacy of "multi-epitope-targeting" agent in induction of functional regulatory CD11c+CD11b+Gr1+myeloid DCs, further indicate the potential role of these DCs in maintaining peripheral tolerance and their involvement in downregulation of MS-like-disease.

Acoustic and optical transduction of BuChE binding to procainamide modified surfaces.

A novel polymer, poly(procainamide), PPA, containing numerous binding sites for cholinesterases was synthesized as a recognition layer for butyryl cholinesterase (BuChE) interaction with the ligand procainamide, utilizing TSM and SPR sensors. The polymer was synthesized by the reaction of methacryloyl chloride and procainamide followed by radical polymerization. Sensor surfaces (Au or SiO(2)) were spin-coated by the polymer solution to form thin layers. Binding of BuChE was found to be sensitive to the drying procedure of the polymer layer. The binding of BuChE to the polymer coated sensors was monitored on-line by following the response of thickness shear mode (TSM) and surface plasmon resonance (SPR) sensors. Binding of BuChE to PPA-coated TSM sensors were shown to follow a Langmuir isotherm giving association constant 3.4x10(6) M(-1).

Aggrecan turnover in human articular cartilage: use of aspartic acid racemization as a marker of molecular age.

Aggrecan is a key component of the cartilage matrix. During aging, many changes occur in its composition and structure; in particular, there is an increase in the proportion of lower molecular weight monomers and of the "free" binding region. An important question has been whether these changes represent alterations in biosynthesis or whether they are due to the accumulation with age of the partially degraded fragments of the originally synthesized large monomer. In the present work we have used an independent tool, viz., the extent of racemization of aspartic acid to study the molecular "age" of different buoyant density fractions of the aggrecan of human articular cartilage, as well as of isolated free binding region and link protein. By measuring the D/LAsp ratio of the different aggrecan species, we were able to establish directly the relative residence times of these molecules in the cartilage matrix and, in combination with compositional and structural analyses, to define their "history" and calculate some of the kinetics constants characterizing their turnover. The value of the turnover constant for the large monomer in fraction A1D1 is 0.206 per year, which corresponds to a half-life of 3.4 years, while the turnover constant for the free binding region is 0.027 per year, which corresponds to a half-life of 25 years. It is thus clear that the rate of formation and turnover of the large monomer is much more rapid than the final degradation of the free binding region fragments, which explains the accumulation of the latter in cartilage during aging.