Could mucin 16 and colony-stimulating factor 2-receptor beta possible graft versus host disease biomarkers? Medical hypotheses.

Graft versus host disease (GVHD) occurs after bone marrow transplantation and is one of the most important causes of death worldwide. Reviews demonstrated GVHD patients with involvement of various tissues and organs, such as salivary glands. The diagnosis of acute GVHD has been the biopsies and the histopathologic evaluation of tissue from an involved organ. These procedures are invasive. Saliva proteins as possible biomarker for GVHD could facilitate the management and diagnosis accuracy. For support the proposed hypotheses, in this pilot study we collected whole saliva samples from patients with undergoing allogeneic hematopoietic cell transplantation (HCT) and from healthy subjects. Samples were collected prospectively between pre-transplant, thirty days, one hundred and, two hundred days after transplant. The proteomic profile was analyzed using SDS-PAGE and LCMS-ESI-IT-TOF mass spectrometry. The relevant personal data, past medical history were also recorded. The most relevant proteins found exclusively in GVHD patients were: CSF2RB, Protocadherin (Pcdh) Fat 2 precursor, protein capicua homolog isoform CIC-S, MUC16 and RGPD8_HUMAN RANBP2. This study aims to conduct an initial evaluation of the possible presence of such biomarkers in saliva from GVHD patients, and suggested a potential application of proteomics analysis as a alternative method to diagnose GVHD.

Potential role of erythropoietin receptors and ligands in attenuating apoptosis and inflammation in critical limb ischemia.

OBJECTIVE: Managing critical limb ischemia (CLI) is challenging. Furthermore, ischemic myopathy prevents good functional outcome after revascularization. Hence, we have focused on limiting the tissue damage rather than angiogenesis, which has traditionally been the motivation to develop nonsurgical treatments for CLI. Erythropoietin (EPO) protects ischemic tissue, and this property may also benefit CLI. The objective of this study was to examine the expression of the tissue-protective EPO receptor complex (EPOR-CD131 [ß-chain of interleukin (IL)-3/IL-5/granulocyte macrophage colony-stimulating factor receptor]) in skeletal muscle obtained from humans with CLI. Because native EPO is thrombogenic, the antiapoptotic and anti-inflammatory effects of a nonhematopoietic helix-B peptide of EPO (ARA 290) were investigated on ischemic myotubes in vitro. METHODS: Tissue was obtained from gastrocnemius muscle of 12 patients undergoing amputation for CLI and from 12 patients without limb ischemia. The expression of EPOR and CD131 was demonstrated by immunohistochemistry and Western blot. A validated in vitro model of myotube ischemia was used in which mature C2C12 myotubes were cultured 6 to 12 hours in a depleted media and gas mixture (20% CO2 and 80% N2). The myotubes were pretreated with EPO or ARA 290 before exposure to simulated ischemia. Apoptosis and cell death were determined by cleaved caspase-3 assay and lactate dehydrogenase release assay. Enzyme-linked immunosorbent assay measured the inflammatory cytokines. RESULTS: EPOR and CD131 were expressed and significantly upregulated in CLI (average optical density [OD] in Western blot [control vs CLI] EPOR, 0.05 U vs 0.1 U; CD131, 0.10 U vs 0.22 U; P < .01). There was colocalization of EPOR and CD131 in the sarcolemma (cell membrane) of the skeletal myofiber. There was no difference in the distribution of colocalization between the CLI and the normal muscle. The ischemic myotubes treated by ARA 290 in vitro had a significantly decreased number of apoptotic cells (ischemia vs ischemia plus ARA 290: 71.1% vs 55.1%; P < .01), cleaved caspase-3 (OD of ischemia vs ischemia plus ARA 290: 0.15 U vs 0.02 U; P < .01), lactate dehydrogenase release (ischemia vs ischemia plus ARA 290: 32.5 U/L vs 21.3 U/L; P < .01), and IL-6 release (OD at 450 nm, ischemia vs ischemia plus ARA 290: 0.18 vs 0.13; P < .01). CONCLUSIONS: This study demonstrates the expression and the upregulation of EPOR and CD131 in CLI and also shows that EPOR and CDI are colocalized in the cell membrane of both ischemic and control muscle fiber. The in vitro experiments demonstrate that ARA 290 decreases inflammation and apoptosis of ischemic myotubes. ARA 290 may potentially be used as adjunctive treatment for CLI.

Interleukin (IL)-3/granulocyte macrophage-colony stimulating factor/IL-5 receptor alpha and beta chains are preferentially expressed in acute myeloid leukaemias with mutated FMS-related tyrosine kinase 3 receptor.

The common beta chain subunit (beta(c)), also known as CDw131, shared by the interleukin-3 (IL-3), granulocytic macrophage colony-stimulating factor (GM-CSF) and IL-5 receptors, is required for high-affinity ligand binding and signal transduction. The present study explored the expression of CDw131 in 105 de novo cases of acute myeloid leukaemia (AML). The levels of CDw131 expression were used to identify two AML subgroups characterized by low (75/105) and high (30/105) expression of this receptor chain. It was observed that (i) the level of CDw131 expression strictly correlated with the level of CD116 (GM-CSFalpha receptor chain) and CD123 (IL-3Ralpha chain); (ii) AMLs with high CDw131 expression were characterized by low CD34 expression and usually high CD11b, CD14 expression; (iii) AMLs with high CDw131 expression frequently co-expressed receptors for angiogenic growth factors (vascular endothelial growth factor R2, Tie-2); (iv) AMLs with high CDw131 expression were more cycling than those with low CDw131 expression; (v) AMLs with high CDw131 frequently displayed Feline Murine Sarcoma (FMS-related) tyrosine kinase 3 (FLT3) internal tandem duplication and constitutively activated Signal Transducer and Activator of Transcription-5 (STAT5). In conclusion, the analysis of the level of CDw131 expression enabled the identification of a subset of AMLs characterized by a high cycling status, the expression of myelo-monocytic markers, mutated FLT3 and the co-expression of receptors for angiogenic growth factors. These findings are of value for the development of new therapeutic strategies for the treatment of these AMLs.

Expression of interleukin-3 receptor subunits on defined subpopulations of acute myeloid leukemia blasts predicts the cytotoxicity of diphtheria toxin interleukin-3 fusion protein against malignant progenitors that engraft in immunodeficient mice.

The interleukin-3 receptor (IL-3R) subunits are overexpressed on acute myeloid leukemia (AML) blasts compared with normal hematopoietic cells and are thus potential targets for novel therapeutic agents. Both fluorescence-activated cell sorter (FACS) analysis and quantitative real-time reverse transcription-polymerase chain reaction (QRT-PCR) were used to quantify expression of the IL-3Ralpha and beta(c) subunits on AML cells. QRT-PCR for both subunits was most predictive of killing of AML colony-forming cells (AML-CFCs) by diphtheria toxin-IL-3 fusion protein (DT(388)IL3). Among 19 patient samples, the relative level of the IL-3Ralpha was higher than the IL-3Rbeta(c) and highest in CD34(+)CD38(-)CD71(-) cells, enriched for candidate leukemia stem cells, compared with cell fractions depleted of such progenitors. Overall, the amount of IL-3Rbeta(c) subunit did not vary among sorted subpopulations. However, expression of both subunits varied by more than 10-fold among different AML samples for all subpopulations studied. The level of IL-3Rbeta(c) expression versus glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (set at 1000) ranged from 0.14 to 13.56 in CD34(+)CD38(-)CD71(-) cells from different samples; this value was correlated (r = .76, P = .05) with the ability of DT(388)IL3 to kill AML progenitors that engraft in beta(2)-microglobin-deficient nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice (n = 7). Thus, quantification of IL-3R subunit expression on AML blasts predicts the effectiveness IL-3R-targeted therapy in killing primitive leukemic progenitors.