Apparent enzyme kinetic parameters for 4-OH-Cy formation by different microsome batches with recombinant <i>CYP2B6*1</i> and different POR/CYP ratios.

<p>* POR measured as Cytochrome c reductase activity as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141979#pone.0141979.t001" target="_blank">Table 1</a></p><p>** <i>V</i>_<i>max</i> is based on 4-OH-Cy formed</p><p><b><i>CL</i></b>_{<b><i>int</i></b>} Calculated as <b><i>V</i></b>_{<b><i>max</i></b>}<b><i>/K</i></b>_{<b><i>m</i></b>}</p><p>Cyclophosphamide at different concentrations was incubated in 2 independent experiments with 3 batches of CYP2B6.1 containing different POR/CYP ratios. The 4-OH-Cy formation, used as a measure of Cy metabolism, was measured using HPLC. The results shown are the averages obtained for each of the three batches. The results show almost proportional increase of Cy intrinsic clearance (i.e. apparent <i>V</i>_<i>max</i>/<i>K</i>_<i>m</i>, with <i>V</i>_<i>max</i> measured from 4-OH-Cy formation) with higher POR/CYP ratios from Batch 1 to Batch 3.</p

Patient characteristics.

<p><b>Abbreviations:</b> ALL: acute lymphoblastic leukemia; AML: acute myeloid leukemia; ATG: antithymocyte globulin; B: B lymphocyte; CLL: chronic lymphoblastic leukemia; CD 34: bone marrow-derived stem cells; CR: complete remission; Cy: cyclophosphamide; Flu: Fludarabine; HSCT: hematopoietic stem cell transplantation; NHL: Non-Hodgkin lymphoma; P: patient; PR: partial remission; T: T lymphocyte; TBI: Total body irradiation;</p><p>^†: survival time. fTBI: fractionated total body irradiation</p><p>Patient characteristics.</p

Effect of fludarabine on <i>POR</i> gene expression.

<p>The gene expression of <i>POR</i> was not significantly changed after treatment with fludarabine.</p

Up-regulation of <i>POR</i> mRNA during Cy conditioning.

<p>The gene expression of <i>POR</i> was significantly (<i>p</i> < 0.001, t-test) up-regulated 24 hours after the first Cy infusion and 6 hours after the second dose of Cy, compared to pre-Cy conditioning. The inter-individual variation in relative POR expression increased from 4.3-fold before Cy conditioning to 9.9-fold before the 2^nd dose of Cy and to 16.5-fold by the end of the treatment. The high variation in up-regulation of <i>POR</i> gene expression during Cy treatment may contribute to the high inter-individual variations in Cy kinetics reported in patients, possibly in part due to different inducibility of the polymorphic forms of <i>POR</i>. Circles in the figure are for four patients with <i>POR*28</i> polymorphism.</p

Characteristics of commercial microsomes containing recombinant human CYP2B6.1 and POR.

<p>Batch 1–3 were microsomes (Bactosomes) from <i>E</i>. <i>Coli</i> containing co-expressed <i>CYP2B6*1</i> and human <i>POR</i>. The POR batch was a control without CYP.</p><p>*Batch 2 also contained a supplement of purified human cytochrome b5</p><p>**P450 concentration (nmol/mL)</p><p>***Protein concentration (mg/mL)</p><p>^#Specific P450 content (pmol/mg protein)</p><p>^##Cytochrome c reductase activity (nmol/min/mg protein) as marker for POR activity</p><p>N/A Not applicable</p><p>Characteristics of commercial microsomes containing recombinant human CYP2B6.1 and POR.</p

Michaelis–Menten curves and Hanes-Woolf plots for 4-OH-Cyclophosphamide enzyme kinetics for different batches of CYP2B6.1.

<p>In 2 independent experiments, Cyclophosphamide (at different concentrations) was incubated with 3 batches of <i>Escherichia coli</i> microsomes containing cDNA-expressed CYP2B6.1 (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141979#pone.0141979.t001" target="_blank">Table 1</a>) with different POR/CYP ratios. The CYP concentration per mg protein and the protein concentration per mL incubation were similar between batches. The 4-OH-Cy formation was measured using HPLC with fluorescence detector. The results shown are the averages obtained with each of the three batches. Fitting the data to Michaelis-Menten kinetics gave an apparent intrinsic clearance, <i>CL</i>_<i>int</i> (<i>V</i>_max/<i>K</i>_m), of 3.1, 25.1, 33.7 μL/min/nmol CYP for Batch 1, Batch 2 and Batch 3, respectively. The intrinsic clearance thus increased with increasing POR/CYP ratio. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141979#pone.0141979.g002" target="_blank">Fig 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141979#pone.0141979.t003" target="_blank">Table 3</a>.</p

Scatter plots showing significantly different ratios (immune modulator concentration in supernatants to medium).

<p>The concentration of immune modulators in all the cultured supernatants (SEA, ConA, IAV, PWM and medium) was analyzed and the ratio of cytokine concentration in each supernatant to medium was calculated. The three groups of healthy controls, pre-dialysis patients and hemodialysis patients were compared. The results from cultured cells with SEA (A), IAV (B) and PWM (C) are shown respectively. Whiskers represent 25-75% interquartile range with the median shown by a line. Kruskal-Wallis test was used for comparison between the three groups. Significant differences between groups were analyzed using the post hoc Multiple Comparisons p values (2-tailed) test. p<0.05 was considered significant.</p

Scatter plots showing cytokines with significantly different plasma concentrations (pg/ml).

<p>The concentration of soluble immune modulators in the plasma of healthy controls, pre-dialysis patients and hemodialysis patients were analyzed and compared. Whiskers represent 25-75% interquartile range with the median shown by a line. Kruskal-Wallis test was used for comparison between the three groups. Significant differences between groups were analyzed using the post hoc Multiple Comparisons p values (2-tailed) test. p<0.05 was considered significant.</p

Flow-cytometric analysis of cell-mediated immune response in activated whole blood.

<p>The FITC-CD45+ resting and activated leukocytes from medium, Pokeweed mitogen-, Concanavalin A-, Influenza A vaccine- and Staphylococcus enterotoxin A-stimulated supernatants were gated according to their size and granularity on forward and side scatter plot (in region R and A respectively). The CD3+ CD4+ cells and CD3+ CD8+ cells were detected within the regions R and A accordingly.</p

Pathways reported in each cluster and genes involved in each of them.

<p>Pathways reported in each cluster and genes involved in each of them.</p