Obligations of Contracts: Intent and Distortion

<p><b>Introduction:</b> Treosulfan is an alkylating agent that is used for the treatment of ovarian cancer and for conditioning prior to stem cell transplantation. It is a prodrug that is activated non-enzymatically to two active epoxides.</p> <p><b>Objectives:</b> To optimize a protocol for both <i>in vivo</i> samples handling and <i>in vitro</i> drug preparation. Treosulfan stability was tested in biological fluids at different conditions as well as for its cytotoxicity on cell lines.</p> <p><b>Results:</b> Plasma samples can be safely frozen for a short period up to 8 h, however; for longer periods, samples should be acidified. Urine samples and cell culture media can be safely frozen regardless their pH. For <i>in vitro</i> investigations, incubation of treosulfan at 37 °C for 24 h activated 100% of the drug. Whole blood acidification should be avoided for the risk of hemolysis. Finally; treosulfan cytotoxicity on HL-60 cells has increased following pre-incubation for 24 h at 37 °C compared to K562 cell line.</p> <p><b>Conclusion:</b> The stability profiling of treosulfan provided a valuable reference for handling of biological samples for both <i>in vivo</i> and <i>in vitro</i> studies. These results can be utilized for further investigations concerning the drug kinetics and dynamics in addition to the development of new pharmaceutical formulations.</p

Long-Term Stable Mixed Chimerism after Hematopoietic Stem Cell Transplantation in Patients with Non-Malignant Disease, Shall We Be Tolerant?

<div><p>Long-term stable mixed chimerism is a rare and poorly understood phenomenon post hematopoietic stem cell transplantation. This study aims to shed light on whether the two hematopoietic systems in patients with mixed chimerism remain functional. Additionally, we investigate possible immunologic differences in these individuals compared to patients with only donor derived immune cells. Patients with donor and mixed chimerism, at median 10 (5–16) years post-HSCT for non-malignant diseases, were assessed regarding clinical situation and immune system (phenotypical and functional). No difference in long-term outcome was seen in terms of general wellbeing, central phenotypic immune system features (<i>e</i>.<i>g</i>., differentiation status, CD4/CD8 ratio, B and NK-cell frequency) and antibody responses to immunizations. At a median of 10 years post transplantation, patients with mixed chimerism had significantly higher IgG3 and platelet levels. Additionally, these patients had higher NKT-cell levels (CD94+CD8+ and CD56+CD8+) than patients with donor chimerism. In depth phenotypic analysis of patients with mixed chimerism demonstrated recipient-derived fractions in most immune cell lineages (<i>e</i>.<i>g</i>., T-cell, B-cell and NK-cell subsets). Recipient cells were also capable of responding to mitogenic stimulation with production of several cytokines. In conclusion, long-term mixed chimerism did not negatively affect patient wellbeing and long-term outcome. Moreover, recipient-derived immunity may still be functional in these patients, suggesting an active state of tolerance and immunologic dependence on both hematopoietic systems.</p></div

Gene clusters in relation to Cy treatment.

<p>The expression of Cy treatment specific genes at 6(30 h) was normalized to the pre-treatment and divided to the following clusters: Cluster 1 showed highly down-regulated genes throughout the treatment (A). Cluster 2 showed highly up-regulated genes throughout the treatment (B). Cluster 3 showed early up-regulated but later normalized genes (C). Cluster 4 showed moderately up-regulated genes (D).</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

Effect of cyclophosphamide treatment on ANGPTL1 and c-JUN.

<p>The relative expression as measured by qRTPCR (normalized to GAPDH) of disease-related up-regulated genes, ANGPTL1 (A) and c-JUN (B), compared to normal subjects. Cy treatment did not affect the up-regulation of ANGPTL1 and c-JUN.</p

Phenotypic comparison of cellular subsets between patients with mixed and donor chimerism.

<p>For most cellular subsets no significant differences were observed between 9 mixed chimerism (MC) and 10 donor chimerism (DC) patients (A-C). Asterisks indicate significant <i>P</i>-values (* = <i>P</i> < .05 and ** = <i>P</i> < .01), symbols indicate individual patient levels and horizontal bars in scatter graphs indicate median values of the patient group. (A) The white blood cell (i), platelet (ii) and neutrophil (iii) count in the two patient groups. Platelet counts were higher in MC patients (<i>P =</i> .041). K/mL = 1 000 cells/mL. (B) Radar graphs depicting relative distribution of T, B and NK-cells (i) and T-cell subsets (ii) for DC and MC patient groups. (C) Differentiation status of total T-cells (CD3+), as defined by naïve memory (CCR7+ CD45RO-), central memory (CCR7+ CD45RO+), effector memory (CCR7- CD45RO+) and terminally differentiated memory (CCR7- CD45RO-), was found to be similar between the DC and MC patient groups. (D) Representative FACS plots of potential NKT-cells (CD56+ (i-ii) or CD94+ (iv-v)) gated on CD8+ T-cells. In the corresponding graphs (iii, vi)), individual ratios of the subsets for each group are shown (<i>P =</i> .004 and <i>P =</i> .035 respectively).</p

Comparison of soluble biomarkers between patients with mixed and donor chimerism.

<p>Concentrations of cytokines, IgG and IgG subclasses were determined in the plasma of 9 mixed chimerism (MC) and 10 donor chimerism (DC) patients at median 10 years post-HSCT. Asterisks indicate significant <i>P</i>-values (* = <i>P</i> < .05 and ** = <i>P</i> < .01), symbols indicate individual patient levels and horizontal bars in scatter graphs indicate median values of the patient group. (A) A higher IgG3 concentration was seen in MC patient plasma (<i>P =</i> .027). (C-D) A lower concentration of IL-4 (B), IL-12 (p40) (C) and G-CSF (D) was observed in MC patients (<i>P =</i> .016, <i>P =</i> .003 and <i>P =</i> .022 respectively).</p

Protein expression of molecules involved in lymphocyte signalling between patients with mixed and donor chimerism.

<p>Protein expression of ZAP-70, LCK and actin was assessed in lymphocytes of 9 mixed chimerism (MC) and 10 donor chimerism (DC) patients. Asterisks indicate significant <i>P</i>-values (* = <i>P</i> < .05), symbols indicate individual patient levels and horizontal bars in scatter graphs indicate median values of the patient group. (A) Representative blots displaying 3 DC (UPN 1167, 887 and 1065) and 3 MC patients (UPN 921, 652 and 527). (B) The individual values for LCK and ZAP-70 of all patients, with regards to their respective actin intensity. A difference was observed for ZAP-70 expression (<i>P =</i> .013) between the DC and MC patient groups.</p

The pathways related to each cluster and number of genes involved in each cluster.

<p>Cluster 1 for highly down-regulated genes throughout the treatment (A) included the highest number of genes. The majority of these genes belonged to the immune system and its functions. Cluster 2 for highly up-regulated genes throughout the treatment (B), the majority of these genes are involved in 3 important biological pathways involving cytokine-cytokine receptor interaction, transcriptional misregulation in cancer and hematopoietic cell lineage. Cluster 3 showed early up-regulated but later normalized genes (C), these genes were more related to biological pathways including Jak-STAT and MAPK signalling. Cluster 4 showed moderately up-regulated genes (D), the pathway analysis demonstrated that several pathways including cytokine-cytokine receptor interaction, Jak-STAT signalling pathway and TGF-beta signalling are involved.</p

Heat map of patient gene expression during Cy treatment.

<p>Genes expression for genes specific for Cy before and though the treatment (A). Differential expression of a group of 299 genes was identified as being specific for Cy treatment. After subjecting these genes to hierarchical clustering analysis, 4 clusters of up- and down-regulated genes were identified as: highly down-regulated genes (cluster 1), highly up-regulated genes (cluster 2), early up-regulated but later normalized genes (cluster 3) and moderately up-regulated genes (cluster 4) as seen in B.</p