Oxidation-Specific Epitopes (OSEs) Dominate the B Cell Response in Murine Polymicrobial Sepsis

In murine abdominal sepsis by colon ascendens stent peritonitis (CASP), a strong increase in serum IgM and IgG antibodies was observed, which reached maximum values 14 days following sepsis induction. The specificity of this antibody response was studied in serum and at the single cell level using a broad panel of bacterial, sepsis-unrelated as well as self-antigens. Whereas an antibacterial IgM/IgG response was rarely observed, studies at the single-cell level revealed that IgM antibodies, in particular, were largely polyreactive. Interestingly, at least 16% of the IgM mAbs and 20% of the IgG mAbs derived from post-septic mice showed specificity for oxidation-specific epitopes (OSEs), which are known targets of the innate/adaptive immune response. This identifies those self-antigens as the main target of B cell responses in sepsis

Minimal residual disease assessment in B-cell precursor acute lymphoblastic leukemia by semi-automated identification of normal hematopoietic cells:A EuroFlow study

Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating &amp; Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data.</p

Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies — a EuroFlow study

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 programme of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA). TS and LS were supported by a Scientific Grant from the Medical University of Silesia Nr. PCN-1-050/K/0/K

Design, synthesis and structure-activity relationship study of wollamide B; a new potential anti TB agent

<div><p>Wollamide B is a cationic antimycobacterial cyclohexapeptide that exhibits activity against <i>Mycobacterium bovis (M</i>. <i>bovis)</i> (IC₅₀ of 3.1 μM). Aiming to define its structural activity relationship (SAR), optimizing potency and pharmacokinetic properties, libraries of analogues were synthesized following a standard Fmoc-based solid phase peptide synthesis approach. The antimycobacterial activities of wollamide B and all the synthesized analogues were tested against <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) H37Rv. Parallely, <i>in vitro</i> drug metabolism and pharmacokinetic (ADME) profiling was done for the synthesized compounds to evaluate their drug likeness. Among the 25 synthesized wollamides five of them showed potent activities with MICs ≤ 3.1 μM and found to be nontoxic against human HepG2 cells up to 100 μM. The results of the <i>in vitro</i> ADME profiling revealed the remarkable plasma stability and very good aqueous solubility of the class in general while the metabolic stability was found to be moderate to low. Of particular note, compounds <b>7c</b> (MIC = 1.1 μM) and <b>13c</b> (0.6 μM) that exhibited good balance of antimycobacterial activity vs. optimal pharmacokinetic properties could be used as a new lead for further development.</p></div

Structure of wollamide B.

<p>Structure of wollamide B.</p

CD20 Expression as a Possible Novel Prognostic Marker in CLL: Application of EuroFlow Standardization Technique and Normalization Procedures in Flow Cytometric Expression Analysis

Background: CD20 expression is a controversial issue regarding response prediction to anti-CD20 therapy in chronic lymphocytic leukemia (CLL). Methods: Median fluorescence intensities (MFIs) of standard fluorescence beads from the daily calibration of flow cytometers according to EuroFlow protocols were used to establish a normalization approach to study CD20 expression on CLL cells. CD20 MFI was retrospectively assessed prior to and during treatment from flow cytometric measurements of peripheral blood in patients with different depths of molecular response in the four phase-II CLL2-BXX trials (BIG; BAG; BIO; BCG; N = 194) administering either Obinutuzumab or Ofatumumab in combination with targeted agents. Results: No significant difference was observed between the normalized and measured MFIs of CD19 and CD20 on CLL cells. During treatment, CD20 expression levels on CLL cells did not significantly differ between the four investigated different treatment schemes, but a strong molecular response to Ofatumumab seemed to correlate with higher CD20 expression prior to therapy. Conclusions: Standardized staining and instrument monitoring enable a robust assessment of longitudinal biological variations of marker expression based on MFI values. Obinutuzumab showed a higher proportion of patients with a strong MRD response independent from initial CD20 expression, whereas high pre-therapeutic CD20 expression levels seem to correlate with a profound response to Ofatumumab

A synthetic scheme that shows the approach used to synthesize the linear precursor of wollamide B using SPPS.

<p>Reagents and conditions: a) Fmoc-L-Leu-OH, DIPEA, DCM, 2 h; b) 20% piperidine/DMF, 10 min (2X); c) Fmoc-L-Trp (Boc)-OH, HATU, DIPEA, NMP, 1 h; d) Fmoc-D-Orn (Boc)-OH, HATU, DIPEA, NMP, 1 h; e) Fmoc-L-Asn (Trt)-OH, HATU, DIPEA, NMP, 1 h; f) Fmoc-L-Val-OH, HATU, DIPEA, NMP, 1 h; g) Fmoc-D-Leu-OH, HATU, DIPEA, NMP, 1 h; h) 20% HFIP/DC, 1 h.</p

Microsomal stabilities of wollamide B analogues.

<p>Microsomal stabilities of wollamide B analogues.</p

Antimycobacterial activities and <i>in vitro</i> ADME profiles of wollamide B (1c).

<p>Antimycobacterial activities and <i>in vitro</i> ADME profiles of wollamide B (1c).</p

The <i>in vitro</i> physicochemical properties of the synthesized wollamide B analogues.

<p>The <i>in vitro</i> physicochemical properties of the synthesized wollamide B analogues.</p