Introduction Analysis of extracellular vesicles (EVs) derived from plasma or cerebrospinal fluid (CSF) has emerged as a promising biomarker platform for therapeutic monitoring in glioblastoma patients. However, the contents of the various subpopulations of EVs in these clinical specimens remain poorly defined. Here we characterize the relative abundance of miRNA species in EVs derived from the serum and cerebrospinal fluid of glioblastoma patients. Methods EVs were isolated from glioblastoma cell lines as well as the plasma and CSF of glioblastoma patients. The microvesicle subpopulation was isolated by pelleting at 10,000×g for 30 min after cellular debris was cleared by a 2,000×g (20 min) spin. The exosome subpopulation was isolated by pelleting the microvesicle supernatant at 120,000×g (120 min). qRT-PCR was performed to examine the distribution of miR-21, miR-103, miR-24, and miR-125. Global miRNA profiling was performed in select glioblastoma CSF samples. Results In plasma and cell line derived EVs, the relative abundance of miRNAs in exosome and microvesicles were highly variable. In some specimens, the majority of the miRNA species were found in exosomes while in other, they were found in microvesicles. In contrast, CSF exosomes were enriched for miRNAs relative to CSF microvesicles. In CSF, there is an average of one molecule of miRNA per 150-25,000 EVs. Conclusion Most EVs derived from clinical biofluids are devoid of miRNA content. The relative distribution of miRNA species in plasma exosomes or microvesicles is unpredictable. In contrast, CSF exosomes are the major EV compartment that harbor miRNAs.

Regulation of cellular metabolism by the citric acid cycle occurs in the mitochondria. However, the citric acid cycle intermediate succinate was shown recently to be a ligand for the G-protein-coupled receptor GPR91. Here, we describe a role for succinate and its receptor in the stimulation of hematopoietic progenitor cell (HPC) growth. GPR91 mRNA and protein expression were detected in human bone marrow CD34+ progenitor cells, as well as in erythroid and megakaryocyte cultures and the erythroleukemic cell line TF-1. Treatment of these cell cultures with succinate resulted in increased proliferation rates. The proliferation response of TF-1 cells was pertussis toxin (PTX)-sensitive, suggesting a role for Gi signaling. Proliferation was also blocked when TF-1 cells were transfected with small interfering RNA specific for GPR91. Succinate stimulated activation of the Erk MAPK pathway and inositol phosphate accumulation in a PTX-sensitive manner. Pretreatment of TF-1 cells with the Erk1/2 kinase (MEK) inhibitor PD98059 blocked the proliferation response. Succinate treatment additionally protected TF-1 cells from cell death induced by serum deprivation. Finally, in vivo administration of succinate was found to elevate the levels of hemoglobin, platelets, and neutrophils in a mouse model of chemotherapy-induced myelosuppression. These results suggest that succinate-GPR91 signaling is capable of promoting HPC development.

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We developed logistic regression models that combine information from the automated CBC and manual 100-cell differential counts to predict bacterial infection. The logistic models were fitted from a case group of 116 patients with proven bacterial infection and a control group of 930 presumably uninfected outpatients. A 4-variable, 15-parameter model, which includes automated absolute neutrophil, manual band, and manual immature granulocyte counts, performed best with a receiver operating characteristic (ROC) curve area of 89%. A more practical 2-variable model including automated absolute neutrophil and manual band counts performed almost as well with an ROC curve area of 86%. The automated neutrophil count-only model is less informative with an ROC curve area of 78%. The combined information from automated and manual differential cell counts more accurately predicts bacterial infection than automated counting alone. Despite these modest improvements, the high cost of manual differential cell counts dictates careful patient selection. The supplemental information gained from manual differential counts is most useful for patients with low to normal neutrophil counts (8,000/microL [8.0 x 10(9)/L] or less). Further studies are indicated to determine the characteristic patient populations deriving maximal benefit from this information.

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