This study demonstrates that PLT microparticles formed by cryopreservation are phenotypically distinct from those present before freezing. These differences may be associated with the procoagulant properties of cryopreserved PLTs.
Background: Nutrient-sensing receptors located on enteroendocrine (EEC) cells modulate appetite via detection of luminal contents. Colonic ‘tasting’ of luminal contents may influence changes to appetite observed in obesity and after weight loss induced by bariatric surgery. We assessed the effects of obesity and gastric bypass-induced weight loss on expression of nutrient-sensing G-protein coupled receptors (GPCRs), EEC and enterochromaffin (EC) cells and mucosal innervation. Methods: qPCR and immunohistochemistry were used to study colonic tissue from (a) chow-fed/lean, (b) high-fat fed/obese, (c) Roux-en-Y gastric bypass surgery (RYGB), and (d) calorie restriction-induced weight loss mice. Results: Expression of GPR41, GPR43, GPR40, GPR120, GPR84, GPR119, GPR93 and T1R3 was increased in obese mice. Obesity-induced overexpression of GPR41, 40, 84, and 119 further increased after RYGB whereas GPR120 and T1R3 decreased. RYGB increased TGR5 expression. L-cells, but not EC cells, were increased after RYGB. No differences in mucosal innervation by protein gene product (PGP) 9.5 and GLP-1R-positive nerve fibers were observed. Stimulation of colonic mucosa with GPR41, GPR40, GPR85, GPR119, and TGR5 agonists increased cell activation marker expression. Conclusions: Several nutrient-sensing receptors induced activation of colonic EEC. Profound adaptive changes to the expression of these receptors occur in response to diet and weight loss induced by RYGB or calorie restriction.
Allogeneic hematopoietic stem cell transplant (HSCT) represents a potential curative treatment for a number of life-threatening blood malignancies. The utility of this treatment regimen, however, is limited by a number of serious complications including graft versus host disease, which occurs in approximately half of all transplant patients. Standard-of-care for treating acute GvHD has remained unchanged for several decades and consists of high doses of steroids, which are only effective in approximately 35 percent of the cases. Therefore, the reduction of GvHD represents a large unmet medical need, and new approaches are needed to effectively attenuate GvHD. Here we present a fundamentally novel strategy for potentially reducing GVHD - by modulating donor mobilized peripheral blood cells with small molecules prior to HSCT, a programmed mobilized peripheral blood (mPB) allogeneic graft, with reduced T-cell alloreactivity, can be administered as the hematopoietic cell source for HSCT. To this end, we applied our screening platform to identify a combination of small molecule modulators (FT1050, FT4145) that promote the activation of genes implicated in cell cycle, immune tolerance and anti-viral properties of T cells, as well as in the survival, proliferation and engraftment potential of CD34+ cells. Genome-wide expression analysis of the T-cell compartment of mobilized peripheral blood following treatment with FT1050+FT4145 revealed the induction of genes involved in cell cycle (e.g., CCND1, CCNE1), immune tolerance (e.g., ALDH, AREG) and anti-viral properties (e.g., EFNB2). To further assess the therapeutic impact of ex vivo programming with FT1050 and FT4145, a number of T cell assays to assess T cell phenotype and function were conducted on mPB. Overall, ex vivo programming of mPB resulted in reduced allogeneic T cell responses and was accompanied by reduced capacity of modulated T cells to produce Interferon Gamma (IFN-ɣ). Concomitantly, the ability of the modulated T cells to make Interleukin 4 (IL-4) and 10 (IL-10) was enhanced, suggesting a polarization of these cells towards a less inflammatory functional state. This was further evidenced by increased surface expression of an immune-inhibitory molecule, PD1, and reduced expression of the activation markers 41BB and ICOS. We next examined the potential beneficial role of ex vivo programming with FT1050+FT4145 in a major histocompatibility complex (MHC) mis-matched HSCT mouse model. Briefly, lethally irradiated BALB/c mice received bone marrow and splenocytes from C57BL/6 donor mice pulse treated with vehicle or FT1050+FT4145. Significantly less GvHD, as determined by survival, weight loss, GVHD score (diarrhea, inactivity, hunched posture, ruffled fur, eye lesion, snout swelling/skin integrity), cytokine production and histopathology of GvHD target organs was observed in recipients receiving FT1050+FT4145 treated cells as compared to those receiving vehicle treated cells. In addition, we observed increased levels of donor T regulatory cells (Tregs) in secondary lymphoid organs concomitant with decreased levels of circulating IFN-ɣ in recipients receiving FT1050+FT4145 treated cells. Based on the attenuation of alloreactive T-cell responses in these preclinical studies, we believe our findings provide a compelling scientific basis to support the clinical evaluation of ex vivo programmed mobilized peripheral blood in patients undergoing HSCT for the treatment of hematologic malignancies. Disclosures Levin: Fate Therapeutics, Inc: Employment, Equity Ownership. Shoemaker:Fate Therapeutics Inc: Employment.
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