Project description:For the blood contamination studies a CSF pool was made with 1mL CSF free of blood from n=4 patients. The pool was divided into four aliquots. One aliquot was kept as reference CSF without added blood (named “neat” in the raw files), one was spiked with 20 µL blood/mL CSF (2%) (“20S”) and two were spiked with 5 µL blood/mL CSF (0.5%) (named “5S” and “5U”, S=centrifuged, U=not centrifuged). The sample spiked with 2% blood and one of the samples spiked with 0.5% blood were centrifuged at 4C at 400 x g for 10 minutes. In one experiment (BloodContamination_GeLC-MS_comb1-10) the reference CSF (neat), and 0.5% centrifuged (5S) and 2% centrifuged (20S) were protein depleted using the MARS Hu-14 column, separated by SDS-PAGE into ten fractions and in-gel digested. The samples (30 in total) were analysed by LC-MS on an OrbiTrap Velos Pro online coupled to a Dionex Ultimate 3000 nano RSLC system. The data was analysed by the Progenesis LC MS software 2.7 (Nonlinear Dynamics), and the MS/MS spectra were searched against UniProt/SwissProt using the open-source graphical user interface SearchGUI (version 1.7.3), with search engines OMSSA and X!Tandem. PeptideShaker (version 0.14.7) was used to assemble the peptides into proteins. The raw files were named according to sample and fraction, e.g. the first fraction of the reference CSF was called “BK_GeLC_neat_F1”, and the second fraction was called “BK_GeLC_neat_F2”. In the second blood contamination study the reference CSF (neat), and the 0.5% blood spiked samples centrifuged (5S) and not centrifuged (5U) were trypsin digested by in solution protocol and analysed using the same instruments as in the first study. (In the search output file are also the results for 2% blood spiked with and without centrifugation, 20S and 20U, but since the data was not used, the raw files are not distributed). The raw files were named “BK_Insol_FD_X” (X = neat, 5S or 5U). In the third experiment we examined the rostro-caudal gradient (RCG) on CSF in the spinal cord by sampling the 1st, 10th, 16th, 24th, 31st, 38th and 44th mL CSF in volumes of approximately 1 mL of a PSP patient during lumbar puncture. The CSF was centrifuged at 2000 x g for 10 min. We did an iTRAQ discovery study, and to be able to compare all seven RCG points, three related iTRAQ experiments (RCG exp 1, 2 and 3) were done. In each related experiment we included an identical reference which we labeled with the iTRAQ 114 reagent. The reference sample contained equal volumes of the seven RCG points, and was used as the reference in the data analysis. In the experiment we had twelve samples (equal volume) that were digested and labeled with iTRAQ reagents according to the vendor’s manual. The samples were combined into three related experiments as follows: Exp. 1 (common reference, 44th mL, 24th mL and 1st mL), Exp. 2 (common reference, 1st mL, 38th mL and 16th mL), and Exp. 3 (common reference, 10th mL, 44th mL and the 31st mL). The 1st and 44th mL were included twice, since they were expected to be the most different samples. The three combined samples (RCG exp 1, 2 and 3) were fractionated into 21 fractions using mixed mode reversed phase-anion chromatography (MM (RP-AX)). Fractions 1-4 were excluded from LC-MS analysis and the two latest fractions were combined before analysis on an Orbitrap Velos Pro, resulting in 16 fractions per combined sample. The raw files were named according to experiment (RCG 1, 2 or 3) and number of fraction (F4-F19), e.g. the raw file by the name EA_RCG3_F15 is fraction 15 from RCG experiment 3.

Project description:Peripheral blood stem and progenitor cells (PBSC) are widely used for autologous and allogeneic hematopoietic stem cell transplantation. PBSC can be mobilized into the peripheral blood using cytokines, cytotoxic chemotherapy or a combination of both. Granulocyte colony stimulating factor (G-CSF) has become the most commonly administered cytokine for PBSC mobilization because of its high potency and lack of serious toxicity. Recently, a modified form of recombinant human G-CSF has been introduced. This new compound is pegylated G-CSF (Peg-G-CSF) which possesses a substantially longer half-life than the unconjugated drug because of its reduced renal excretion and therefore provides the basis for continuous G-CSF serum-levels after a single injection. The use of Peg-G-CSF in patients who had received a cytotoxic chemotherapy was accompanied by mobilization kinetics different from those observed in patients who had received unconjugated G-CSF. In particular, more rapid leukocyte recovery and occurrence of CD34-positive cells in the peripheral blood was seen. These findings are presumably related to the more even and continuously high serum level of G-CSF maintained by Peg-G-CSF. Using microarray technology and functional assays, we examined whether pegylation of G-CSF and its different pharmacokinetics results in addition to an earlier leucocyte recovery in transcriptional and functional changes in CD34+ cells obtained from patients who had received either G-CSF or Peg-G-CSF following a standard cytotoxic chemotherapy for PBSC mobilisation. CD34+ hematopoietic stem and progenitor cells were isolated immunomagnetically from patients with multiple myeloma during chemotherapy and G-CSF-induced mobilization. Patients received either unconjugated G-CSF (n=9) or polyethylenglykol (PEG)-conjugated G-CSF (n=7). Total RNA was extracted, reversely transcribed, in vitro transcribed and labelled and hybridized to Affymetrix HG Focus Arrays. Following quality control and normalization differentially expressed genes were identified by significance analysis of microarrays (SAM). Comparing both groups 339 genes were significantly differentially expressed (q value <5%; fold change > 1.2). Peg-G-CSF-mobilized CD34+ cells showed an expression pattern of more early progenitor cells as early stem cell markers such as HOX genes were higher expressed and differentiation-associated genes were lower expressed in comparison with G-CSF-mobilized cells. Moreover, Peg-G-CSF-mobilized CD34+ cells had a greater expression level of cell-cycle-promoting genes suggesting a greater cycle activity of these cells. In conclusion, despite the similar active drug component different pharmacokinetics of Peg-G-CSF and G-CSF result in distinct molecular phenotypes reflecting different functional characteristics. Keywords: ordered

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