Short Synthesis of a Proline Amide Orexin Receptor Antagonist on the Pilot Plant Scale

A three-step fully telescoped synthesis of an <i>N</i>-sulfonyl proline amide, a nonpeptide antagonist of human orexin receptors, is described. The process development from the medicinal chemistry route up to the 240 kg production of <b>1</b> is discussed with a focus on an economical and efficient amide bond formation and identification of a new polymorph. The routes are compared using green metrics

Additional file 1 of Correlation of plasma cell assessment by phenotypic methods and molecular profiles by NGS in patients with plasma cell dyscrasias

Additional file 1. Supplementary tables and figures

presentation_1_Host-Derived Microvesicles Carrying Bacterial Pore-Forming Toxins Deliver Signals to Macrophages: A Novel Mechanism of Shaping Immune Responses.PDF

<p>Bacterial infectious diseases are a leading cause of death. Pore-forming toxins (PFTs) are important virulence factors of Gram-positive pathogens, which disrupt the plasma membrane of host cells and can lead to cell death. Yet, host defense and cell membrane repair mechanisms have been identified: i.e., PFTs can be eliminated from membranes as microvesicles, thus limiting the extent of cell damage. Released into an inflammatory environment, these host-derived PFTs-carrying microvesicles encounter innate immune cells as first-line defenders. This study investigated the impact of microvesicle- or liposome-sequestered PFTs on human macrophage polarization in vitro. We show that microvesicle-sequestered PFTs are phagocytosed by macrophages and induce their polarization into a novel CD14⁺MHCII^lowCD86^low phenotype. Macrophages polarized in this way exhibit an enhanced response to Gram-positive bacterial ligands and a blunted response to Gram-negative ligands. Liposomes, which were recently shown to sequester PFTs and so protect mice from lethal bacterial infections, show the same effect on macrophage polarization in analogy to host-derived microvesicles. This novel type of polarized macrophage exhibits an enhanced response to Gram-positive bacterial ligands. The specific recognition of their cargo might be of advantage in the efficiency of targeted bacterial clearance.</p

Molecular status of diagnosis total AML and sorted stem cell fractions from the same BM.

§<p>signal originating from the bulk of the leukemic blasts has been used for reference. Sorted lymphocytes from the same BM showed no molecular aberrancies.</p><p>^*Aberrant markers with the highest coverage of the CD34+CD38- compartment were used for sorting HSC (aberrant marker negative) and pLSC (aberrant marker positive).</p>¥<p>Shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone-0107587-g001" target="_blank">Figure 1</a>.</p>#<p>For FLT3-ITD+ cases, the percentage of ITD of the total signal is indicated.</p>$<p>Median frequency of HSCs (46.5% of CD34+CD38- compartment) was higher than the median in a larger group of cases (median 18%), since cases in this table had to be selected on clear availability of both HSCs and pLSCs.</p><p>Abbreviations: wt, wild type: no FLT3-ITD or NPM1 peak present; mut: mutation of NPM1 (not quantitative), neg: negative, pos: positive, BM: bone marrow.</p><p>Molecular status of diagnosis total AML and sorted stem cell fractions from the same BM.</p

Prognostic value of frequencies of CD34+CD38- pLSC compartment at follow-up.

<p>This figure shows the Kaplan-Meier analyses for RFS for the CD34+CD38- pLSC compartment at follow up for three consecutive therapy cycles. The optimal cut-off levels were chosen to define pLSC + and pLSC- after 1^st induction cycle (0.0003%,which is 3 pLSCs in 1,000,000 WBC) and after 2^nd induction cycle and consolidation therapy 0.0001% (1 pLSC in 1,000,000 WBC). Results for other cut-offs are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone.0107587.s008" target="_blank">Table S7</a>. After the first induction cycle (B, 71 patients), second induction cycle (C, 77 patients), and after consolidation therapy (D, 48 patients), patients with high pLSC frequency (pLSC+) showed significantly more adverse performance compared with patients with low pLSC frequency (LSC-).</p

Prognostic value of frequencies of pLSC compartments at diagnosis.

<p>This figure shows the Kaplan-Meier analyses at diagnosis for the three compartments putatively containing pLSCs: CD34+CD38- (A,B,C), CD34+CD38+ (D) and CD34- (E). Of the 117 patients shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone.0107587.s004" target="_blank">Table S3</a>, for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone-0107587-g005" target="_blank">Figures 5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone-0107587-g006" target="_blank">6</a>, 88 patients were chosen who had at least one follow-up time point. Of these, 70 entered Complete Remission, of whom 53 after the first course, 13 after the second course and 4 at later stages. Eighteen never reached CR. The size (median values) of the CD34+CD38- compartment at diagnosis was significantly (six-fold) higher in patients who did not enter CR (n = 18) compared with patients who did (n = 70): 0.225% of WBC versus 0.036% of WBC (p = 0.041). For CD34+CD38+ and CD34-, there were no significant differences (see text). Cut-off levels were defined to divide the total population into high stem cell frequencies (above cut-off) and low stem cell frequencies (below cut-off). A particular cut-off value was chosen (A, D, E) to ensure approximately equally numbers of patients in the resulting high and low stem cell frequency compartments. Results for other cut-offs for the three pLSC compartments are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone.0107587.s007" target="_blank">Table S6</a>. A–C: CD34+CD38-; D: CD34+CD38+; E: CD34-. A. RFS in remission patients (n = 70) with diagnosis CD34+CD38- cut-off of 0.03%; B. RFS in the same patient group (n = 70), but now with 2 cut-offs (0.005% and 0.1%); C. Event-free survival for all CR and non-CR patients (n = 88); D. RFS in remission patients (n = 70) with CD34+CD38+ cut-off of 25%; E. RFS in remission patients (n = 70) with CD34- cut-off of 3%. All relevant prognostic variables with statistical significance were investigated in a multivariate model. In this multivariate analysis it was found that risk group (according to the HOVON 102 trial) was an independent prognostic factor for OS at diagnosis (p = 0.001). For RFS, both risk group and CD34+CD38- leukemic stem cell load (using a 0.03% cut-off point) were independent prognostic factors at diagnosis (p = 0.017 and p = 0.011, respectively).</p

FSC/SSC position relative to lymphocytes.

¥<p>Shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone-0107587-g001" target="_blank">Figure 1</a>.</p><p>* FSC/SSC values are based on the position of cells in FSC/SSC relative to the</p><p>FSC/SSC position of normal lymphocytes in the same BM sample.</p><p>The molecular status of the sorted cell fractions is summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone-0107587-t001" target="_blank">Table 1</a>.</p><p>FSC/SSC position relative to lymphocytes.</p

Marker negative pLSCs co-exist with marker positive pLSCs and are identified by scatter and CD34/CD45 expression patterns.

<p>CD34+ CD38- cells (patient 372) were identified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone-0107587-g001" target="_blank">figure 1</a>.I and gated for sub-compartments as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone-0107587-g002" target="_blank">figure 2</a>.I. In the stem cell compartment of this AML case, only 11% could be identified as CD19+ (A). When back-gated in FSC/SSC (similar as performed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone-0107587-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone-0107587-g002" target="_blank">2</a>), two different populations were identified based on the position in FSC/SSC: the small CD19+ fraction (events in red in A–D) is characterized by FSC/SSC^high (B), low CD34 expression (C) and high CD45 expression (D). CD19 negative cells (events in green in A–D), apart from a small FSC/SSC^low/CD34^high/CD45^low population of putative HSCs (A–D), contained a large population of cells that, similar to the CD19+ population in A–D, were FSC/SSC^high (B), CD34^low (C), and CD45^high (D). Apart from CD19, CD7 was an aberrant marker: 61% of the cells was CD7+ (E). Upon backgating, CD7+ cells (events in red in E–H), similar to CD19+ cells, were all FSC/SSC^high (F) CD34^low (G) and CD45^high (H). In contrast to CD19, CD7 negative cells (events in green in E–H) now completely consisted of a small FSC/SSC^low (F), CD34^high(G), CD45^low (H) fraction. CD7 thus covered the whole neoplastic CD34+CD38- population and shows perfect discrimination between HSC and pLSC. CD19 expression in the absence of both CD7 expression and other scatter and CD34/CD45 expression parameters would have under-estimated the pLSC in the CD34+CD38- compartment by a factor 5.5 (61%/11%), while the HSCs would have been over-estimated by a factor 2.3 (89%/39%). In this case CD7 was a good marker to compare with the poor CD19 marker; it can be seen, however, that in the absence of CD7 expression, but with the scatter and CD34/CD45 aberrancies present, these would have enabled a complete discrimination between putative HSC and LSC compartments. This patient was identified as NPM1-positieve and FLT3-ITD positive. Other molecular aberrancies were not detected.</p

Multilineage engraftment of CD34/CD38 and scatter-defined putative HSCs.

<p>Unsorted mononuclear cells (MNCs) were injected intravenously and resulted in leukemic engraftment: cells were CD45+ (A) and of myeloid origin (B). In this case, the myeloid cells were positive for the diagnosis of leukemia-associated phenotype (LAP): partly CD33+CD13- (C) and CD11b+ (D). Sorted putative HSCs were injected intrafemorally (details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone.0107587.s006" target="_blank">Table S5</a>). Engrafted CD45+ cells (E), contained both B-cells and myeloid cells (F), and lacked LAP (G,H). Multilineage engraftment of the sorted subpopulations was seen for patient 598 (I), 661 (J), 423 (K), and 928 (L). B-cells and myeloid cells (percentage of CD45+ cells) are in the upper left and lower right corners of the plots, respectively. The AML cells of patients 598 (I) and 661 (J) had an aberrant phenotype at diagnosis that was present in the neoplastic engrafted cells, but absent in the normal cells (not shown).</p

Gating strategy for the CD34+CD38- compartment and identification of pLSCs and HSCs in this compartment. I. Gating of CD34+CD38- AML cells.

<p>Cells were labeled with antibody-fluorochrome combinations as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#s2" target="_blank">Patients, Materials and Methods</a>. Remaining erythrocytes, debris and dead cells are largely excluded in an FSC/SSC plot (A). CD45^dim/SSC^dim blast cells (B) were gated to homogeneity in FSC/SSC plot (C). CD34 positive cells are gated (D) and the CD38- stem cells are gated within this fraction (E). The CD38-negative fraction in D may contain two stem cell populations differing in CD34 expression (details in text). F. Within the CD34+CD38- gate, CD38 is plotted against an aberrant marker (in this case CD19) to indicate presence of putative LSCs (pLSCs) and HSCs. <b>II. Identification of pLSCs and HSCs.</b> This patient (nr 317) was diagnosed with t(8;21). Primary gating was as in I. Sorted CD34+CD38-/CD19+ cells (A, in red) were t(8;21) positive; sorted CD34+CD38-/CD19- cells (A, in green) were t(8;21) negative. These two populations were backgated in FSC/SSC (B,E), CD34/SSC (C,F) and CD45/SSC (D,G) plots. The CD19- cells are shown in the upper panels and the CD19+ cells in the lower panels. Dotted vertical lines (B–D) show that normal CD19- cells are FSC^low (B), CD34^low (C) and slightly lower in CD45 (D), compared to CD19+ cells (E–G). The dotted horizontal line shows that SSC of the normal stem cells (in green) was slightly lower than that of neoplastic stem cells (in red). FISH data are from an example published previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone.0107587-VanRhenen3" target="_blank">[19]</a>. Similar results were found in an additional series of 7 patients (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone-0107587-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107587#pone-0107587-t002" target="_blank">2</a>). FSC and SSC of CD19+ pLSC were factor 1.71 and 1.77 higher than lymphocyte present in the same samples. FSC and SSC of the CD19 negative cells were only 1.08 and 1.20 times lower than lymphocytes.</p